Amino Acids Peptides and Proteins

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Published on 31 August 2013 on http://pubs.rsc.org | doi:10.1039/9781849737081-FP001
Amino Acids, Peptides and Proteins
Volume 38
.
Published on 31 August 2013 on http://pubs.rsc.org | doi:10.1039/9781849737081-FP001 View Online
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A Specialist Periodical Report
Amino Acids, Peptides

and Proteins
Volume 38
Editors
Etelka Farkas, University of Debrecen, Hungary
Maxim Ryadnov, National Physical Laboratory and University of Edinburgh, UK
Authors
Gyula Batta, University of Debrecen, Hungary
Clara Brieke, Max Planck Institute for Medical Research, Germany
Linda Jiaying Chan, University of Melbourne, Australia
Max J. Cryle, Max Planck Institute for Medical Research, Germany
Kristina Haslinger, Max Planck Institute for Medical Research, Germany
André Henrion, Physikalisch-Technische Bundesanstalt, Germany
Mohammed Akhter Hossain, University of Melbourne, Australia
Katalin E. Kövér, University of Debrecen, Hungary
Gábor Mezo } , Eötvös Loránd University, Hungary
Hisakazu Mihara, Tokyo Institute of Technology, Japan
Paulina D. Rakowska, National Physical Laboratory, UK
.
Chrishan S. Samuel, University of Melbourne and Monash University, Australia

Frances Separovic, University of Melbourne, Australia
Hiroshi Tsutsumi, Tokyo Institute of Technology, Japan
John D. Wade, University of Melbourne, Australia
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to the chapters online. Please contact sales@rsc.org with proof of
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Thank you
ISBN: 978-1-84973-585-8
ISSN: 1361-5904
DOI: 10.1039/9781849737081
A catalogue record for this book is available from the British Library
& The Royal Society of Chemistry 2014
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Preface
Etelka Farkas and Maxim Ryadnov
DOI: 10.1039/9781849737081-FP005
This volume continues the legacy of the annual report series launched in
1969 and re-launched last year after a five-year break. The main objective of
the series has been to provide a comprehensive review coverage of research

progress, both critical and systematic, in peptide and protein science. Each
volume of the series strives to highlight the most recent findings and de-
velopments in specific research areas and reviewes literature predominantly
published over the last two-three years. Routine revisions of more tradi-
tional concepts in the light of emerging discoveries, and vice versa, consti-
tute an integral part of each chapter, which makes this series unique and
different from other publications and allows keeping pace with the progress
without losing touch with foundations.
The volume starts with an overview of oxidative transformations cata-
lysed by a prominent class of oxidoreductases, cytochromes P450 (Cryle,
Brieke and Haslinger). This chapter builds around the mechanistic, reactive
and structural aspects of cytochrome-mediated oxidation of amino acids
and peptides with functional implications for primary and secondary me-
tabolism including the biosynthesis of important depsi- and glycopeptide
toxins and antibiotics. A stronger focus on the high-resolution structural
elucidation of peptide macrocylisation through cystine formation is made in
the following chapter (Kövér and Batta) which gives an overview of NMR
investigations of complex cystine-knot structures of plant antibiotics,
cyclotides, and cystine ladder motifs of antimicrobial y-defensins, also
.
covering ion channel blockers and other small cysteine-rich proteins. This
chapter is followed by an overview of one of the most biomedically
prominent peptide families – relaxin family peptide hormones (Chan,
Samuel, Separovic, Hossain and Wade). A specific emphasis is made here on
the unique cystine-knot-like structural properties of the peptides that de-
termine their role in fibrotic diseases. The biological function of peptide
sequences is reviewed from the perspective of de novo peptide design
highlighting pros and cons of autonomously folded and self-assembling
backbone motifs (Ryadnov), with the following chapter giving a detailed
overview of nanomaterial applications of peptide self-assembly ranging
from electronic devices to self-assembly-templated mineralization (Tsutsumi
and Mihara). Emerging high-resolution technologies for the character-
isation of peptide and protein structures in native dynamic environments,
such as protein folds and peptidomes, are reviewed in two subsequent
chapters, first, outlining the applicability of peptide distance constraints
derived from mass spectrometry analyses of chemically cross-linked pep-
tides for protein structure elucidation (Henrion), and, second, giving an
overview of recent developments in peptidome analysis and biomarker
discovery, involving endogenous and exogenous peptides, in the context of
mainstream analytical tools and technologies (Rakowska and Ryadnov).
The volume closes with a comprehensive coverage of marketed peptide and
Amino Acids, Pept. Proteins, 2013, 38, v–vi | v

c The Royal Society of Chemistry 2014
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protein pharmaceuticals and progress in the development of emerging

peptide-based drugs (Mez} o). Each chapter is structured around current
trends in the reviewed research area that are compared with more estab-
lished approaches wherever possible, and the authors’ outlook of future
perspectives, either as a separate section or incorporated in the text. All
chapters are written by leading researchers in their subject areas to enable a
worldwide information source of broad appeal to researchers in academia
and industry.
.
vi | Amino Acids, Pept. Proteins, 2013, 38, v–vi

CONTENTS
Cover
The crystal structure of particulate

methane monooxygenase (pMMO)
reveals many unexpected features
including, a trimeric oligomerization
state and three distinct metal

centers. Image reproduced by
permission of Amy Rosenzweig
from Dalton Transactions, 2005.
Preface v
Etelka Farkas and Maxim Ryadnov
Oxidative transformations of amino acids and peptides catalysed by 1

Cytochromes P450
Max J. Cryle, Clara Brieke and Kristina Haslinger
.
1 Introduction 1
2 P450-catalysed oxidative modification of peptides and 8
amino acids
3 Conclusion 31
References 31
NMR investigation of disulfide containing peptides and proteins 37

Katalin E. Köve´r and Gyula Batta
1 Introduction 37
2 General NMR methods 38
3 Disulfide bond connectivities by NMR spectroscopy 38
4 NMR parameters related to structure 40
5 Antimicrobial peptides (AMPs) 40
6 NMR of AMPs 41
7 Peptide-based toxins 45
8 NMR solution structures of other disulfide proteins 51
9 Folding and dynamics of disulfide proteins 53
Acknowledgements 55
References 55
Amino Acids, Pept. Proteins, 2013, 38, vii–ix | vii

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Relaxin and its role in fibrotic diseases 60

Linda Jiaying Chan, Chrishan S. Samuel, Frances Separovic,
Mohammed Akhter Hossain and John D. Wade
1 Human relaxin-2 (Relaxin) 60
2 Structure activity relationship (SAR) study and RXFP1 61
interaction mechanism
3 Fibrosis and wound healing 64
4 Extracellular matrix 66
5 Fibroblasts and myofibroblasts 66
6 Collagen 68
7 Relaxin and its anti-fibrotic effects in fibrotic diseases 70
8 Future perspectives 74
Acknowledgments 75
References 76
Biofunctional peptide design 79

Maxim G. Ryadnov
1 Introduction 79
2 Generic considerations for biofunctional peptide design 79
3 Functional space of de novo peptide design 84
4 Current trends: environment-defined functions 89
5 Current trends: environment-supporting functions 105
References 116
.
Self-assembly of designed peptides and their nanomaterials 122

applications
Hiroshi Tsutsumi and Hisakazu Mihara
1 Introduction 122
2 Self-assembling peptides and nanostructures 122
3 Application of self-assembling peptides as nanomaterials 136
4 Perspective 144
References 145
Integrating chemical cross-linking with mass spectrometric analysis of 151

peptides and proteins
Andre´ Henrion
1 Introduction 151
2 Cross-linkers and cross-linking/MS strategies 152
3 Mass spectrometric fragmentation of cross-linked peptides 160
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References 169
Peptidome analysis: tools and technologies 172

Paulina D. Rakowska and Maxim G. Ryadnov
1 Introduction 172
2 Peptidomes and peptidomics 172
3 Peptidomics tools 176
4 Peptide quantitation 182
5 Data processing and global identification 185
6 Synthetic peptides as exogenous biomarkers 190
8 Further specialist reading 194
References 195
Peptide and protein based pharmaceuticals 203

Gábor Mezo
1 Introduction 203
2 Glatiramer acetate (Copaxones), the highest income 206
producing peptide-based drug used against multiple
sclerosis
3 Peptide-based drugs for the treatment of diabetes, the 209
fastest growing disease worldwide
.
4 Peptide-based drugs for the prevention of thrombosis, 216

the most ‘‘sneaky disease’’
5 Enfuvirtide (Fuzeons), a HIV fusion inhibitor peptide 221
for the treatment of the most life-threatening disease
6 Lucinactant (Surfaxins), a synthetic surfactant for the 223
prevention of the most tragic deaths
7 Gonadotropin-releasing hormone derivatives as the 225
largest peptide-based drug family
8 Medical applications of somatostatin derivatives that 231
have the most receptors
9 Icatibant (Firazyrs), the most effective compound for the 235
treatment of hereditary angioedema
10 Mifamurtide based on the smallest native 238
immunostimulatory peptide
11 Perspectives 239
References 243
Amino Acids, Pept. Proteins, 2013, 38, vii–ix | ix

A short guide to abbreviations and their use
in peptide science
Abbreviations, acronyms and symbolic representations are very much part

of the language of peptide science – in conversational communication as
much as in its literature. They are not only a convenience, either – they enable
the necessary but distracting complexities of long chemical names and tech-
nical terms to be pushed into the background so the wood can be seen among
the trees. Many of the abbreviations in use are so much in currency that they
need no explanation. The main purpose of this editorial is to identify them
and free authors from the hitherto tiresome requirement to define them in
every paper. Those in the tables that follow – which will be updated from time
to time – may in future be used in this Journal without explanation.
All other abbreviations should be defined. Previously published usage should
be followed unless it is manifestly clumsy or inappropriate. Where it is necessary
to devise new abbreviations and symbols, the general principles behind estab-
lished examples should be followed. Thus, new amino-acid symbols should be
of form Abc, with due thought for possible ambiguities (Dap might be obvious
for diaminoproprionic acid, for example, but what about diaminopimelic acid?).
Where alternatives are indicated below, the first is preferred.
Amino Acids
Proteinogenic Amino Acids
Ala Alanine A
Arg Arginine R
Asn Asparagine N
.
Asp Aspartic acid D

Asx Asn or Asp
Cys Cysteine C
Gln Glutamine Q
Glu Glutamic acid E
Glx Gln or Glu
Gly Glycine G
His Histidine H
Ile Isoleucine I
Leu Leucine L
Lys Lysine K
Met Methionine M
Phe Phenylalanine F
Pro Proline P
Ser Serine S
Thr Threonine T
Trp Tryptophan W
Tyr Tyrosine Y
Val Valine V
Copyright & 1999 European Peptide Society and John Wiley & Sons, Ltd. Reproduced with
permission from J. Peptide Sci., 1999, 5, 465–471.
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Other Amino Acids

Aad a-Aminoadipic acid
bAad b-Aminoadipic acid
Abu a-Aminobutyric acid
Aib a-Aminoisobutyric acid; a-methylalanine
bAla b-Alanine; 3-aminopropionic acid (avoid Bal)
Asu a-Aminosuberic acid
Aze Azetidine-2-carboxylic acid

Cha b-cyclohexylalanine
Cit Citrulline; 2-amino-5-ureidovaleric acid
Dha Dehydroalanine (also DAla)
Gla g-Carboxyglutamic acid
Glp pyroglutamic acid; 5-oxoproline (also pGlu)
Hph Homophenylalanine (Hse=homoserine, and so on). Caution
is necessary over the use of the prefix homo in relation to
a-amino-acid names and the symbols for homo-analogues.
When the term first became current, it was applied to ana-
logues in which a side-chain CH2 extension had been intro-
duced. Thus homoserine has a side-chain CH2CH2OH,
homoarginine CH2CH2CH2NHC(¼NH)NH2, and so on.
In such cases, the convention is that a new three-letter symbol
for the analogue is derived from the parent, by taking H for
homo and combining it with the first two characters of the
parental symbol – hence, Hse, Har and so on. Now, however,
there is a considerable literature on b-amino acids which are
analogues of a-amino acids in which a CH2 group has been
inserted between the a-carbon and carboxyl group. These
analogues have also been called homo-analogues, and there
.
are instances for example not only of ‘homophenylalanine’,

NH2CH(CH2CH2Ph)CO2H, abbreviated Hph, but also ‘homo-
phenylalanine’, NH2CH(CH2Ph)CH2CO2H abbreviated Hph.
Further, members of the analogue class with CH2 interpolated
between the a-carbon and the carboxyl group of the parent
a-amino acid structure have been called both ‘a-homo’- and
‘b-homo’. Clearly great care is essential, and abbreviations for
‘homo’ analogues ought to be fully defined on every occasion.
The term ‘b-homo’ seems preferable for backbone extension
(emphasizing as it does that the residue has become a b-amino
acid residue), with abbreviated symbolism as illustrated by bHph
for NH2CH(CH2Ph)CH2CO2H.
Hyl d-Hydroxylysine
Hyp 4-Hydroxyproline
aIle allo-Isoleucine; 2S, 3R in the L-series
Lan Lanthionine; S-(2-amino-2-carboxyethyl)cysteine
MeAla N-Methylalanine (MeVal=N-methylvaline, and so on).
This style should not be used for a-methyl residues, for
which either a separate unique symbol (such as Aib for
a-methylalanine) should be used, or the position of the methyl
Amino Acids, Pept. Proteins, 2013, 38, x–xvii | xi

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group should be made explicit as in aMeTyr for

a-methyltyrosine.
Nle Norleucine; a-aminocaproic acid
Orn Ornithine; 2,5-diaminopentanoic acid
Phg Phenylglycine; 2-aminophenylacetic acid
Pip Pipecolic acid; piperidine-s-carboxylic acid
Sar Sarcosine; N-methylglycine
Sta Statine; (3S, 4S)-4-amino-3-hydroxy-6-methyl-heptanoic acid

Thi b-Thienylalanine
Tic 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid
aThr allo-Threonine; 2S, 3S in the L-series
Thz Thiazolidine-4-carboxylic acid, thiaproline
Xaa Unknown or unspecified (also Aaa)
The three-letter symbols should be used in accord with the IUPAC-IUB
conventions, which have been published in many places (e.g. European J.
Biochem. 1984; 138: 9–37), and which are (May 1999) also available with
other relevant documents at: http://www.chem.qnw.ac.uk/iubmb/iubmb.
html#03
It would be superfluous to attempt to repeat all the detail which can be
found at the above address, and the ramifications are extensive, but a few
remarks focussing on common misuses and confusions may assist. The
three-letter symbol standing alone represents the unmodified intact amino
acid, of the L-configuration unless otherwise stated (but the L-configuration
may be indicated if desired for emphasis: e.g. L-Ala). The same three-letter
symbol, however, also stands for the corresponding amino acid residue.
The symbols can thus be used to represent peptides (e.g. AlaAla or
Ala-Ala=alanylalanine). When nothing is shown attached to either side
.
of the three-letter symbol it is meant to be understood that the amino group

(always understood to be on the left) or carboxyl group is unmodified, but
this can be emphasized, so AlaAla=H-AlaAla-OH. Note however that
indicating free termini by presenting the terminal group in full is wrong;
NH2AlaAlaCO2H implies a hydrazino group at one end and an a-keto acid
derivative at the other. Representation of a free terminal carboxyl group by
writing H on the right is also wrong because that implies a terminal
aldehyde.
Side chains are understood to be unsubstituted if nothing is shown, but a
substituent can be indicated by use of brackets or attachment by a vertical
bond up or down. Thus an O-methylserine residue could be shown as 1, 2, or 3.
Note that the oxygen atom is not shown: it is contained in the three-letter
symbol – showing it, as in Ser(OMe), would imply that a peroxy group was
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present. Bonds up or down should be used only for indicating side-chain

substitution. Confusions may creep in if the three-letter symbols are used
thoughtlessly in representations of cyclic peptides. Consider by way of
example the hypothetical cyclopeptide threonylalanylalanylglutamic acid. It
might be thought that this compound could be economically represented 4.
But this is wrong because the left hand vertical bond implies an ester link
between the two side chains, and strictly speaking if the right hand vertical
bond means anything it means that the two Ala a-carbons are linked by a
CH2CH2 bridge. This objection could be circumvented by writing the
structure as in 5.
But this is now ambiguous because the convention that the symbols are to
be read as having the amino nitrogen to the left cannot be imposed on both
lines. The direction of the peptide bond needs to be shown with an arrow
pointing from CO to N, as in 6.
Actually the simplest representation is on one line, as in 7.

.
Substituents and Protecting Groups

Ac Acetyl
Acm Acetamidomethyl
Adoc 1-Adamantyloxycarbonyl
Alloc Allyloxycarbonyl
Boc t-Butoxycarbonyl
Bom p-Benzyloxymethyl
Bpoc 2-(4-Biphenylyl)isopropoxycarbonyl
Btm Benzylthiomethyl
Bum p-t-Butoxymethyl
Bui i-Butyl
Bun n-Butyl
But t-Butyl
Bz Benzoyl
Bzl Benzyl (also Bn); Bzl(OMe)=4-methoxybenzyl and so on
Cha Cyclohexylammonium salt
Clt 2-Chlorotrityl
Dcha Dicyclohexylammonium salt
Dde 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl
Ddz 2-(3,5-Dimethoxyphenyl)-isopropoxycarbonyl
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Dnp 2,4-Dinitrophenyl
Dpp Diphenylphosphinyl
Et Ethyl
Fmoc 9-Fluorenylmethoxycarbonyl
For Formyl
Mbh 4,4 0 -Dimethoxydiphenylmethyl, 4,4 0 -Dimethoxybenzhydryl
Mbs 4-Methoxybenzenesulphonyl
Me Methyl
Mob 4-Methoxybenzyl
Mtr 2,3,6-Trimethyl,4-methoxybenzenesulphonyl
Nps 2-Nitrophenylsulphenyl
OA11 Allyl ester
OBt 1-Benzotriazolyl ester
OcHx Cyclohexyl ester
ONp 4-Nitrophenyl ester
OPcp Pentachlorophenyl ester
OPfp Pentafluorophenyl ester
OSu Succinimido ester
OTce 2,2,2-Trichloroethyl ester
OTcp 2,4,5-Trichlorophenyl ester
Tmob 2,4,5-Trimethoxybenzyl
Mtt 4-Methyltrityl
Pac Phenacyl, PhCOCH2 (care! Pac also=PhCH2CO)
Ph Phenyl
Pht Phthaloyl
Scm Methoxycarbonylsulphenyl
Pmc 2,2,5,7,8-Pentamethylchroman-6-sulphonyl
Pri i-Propyl
.
Prn n-Propyl
Tfa Trifluoroacetyl
Tos 4-Toluenesulphonyl (also Ts)
Troc 2,2,2-Trichloroethoxycarbonyl
Trt Trityl, triphenylmethyl
Xan 9-Xanthydryl
Z Benzyloxycarbonyl (also Cbz). Z(2C1)=2-chlorobenzyl-
oxycarbonyl and so on
Amino Acid Derivatives
DKP Diketopiperazine
NCA N-Carboxyanhydride
PTH Phenylthiohydantoin
UNCA Urethane N-carboxyanhydride
Reagents and Solvents
BOP 1-Benzotriazolyloxy-tris-dimethylamino-phosphonium
hexafluorophosphate
CDI Carbonyldiimidazole
DBU Diazabicyclo[5.4.0]-undec-7-ene
DCCI Dicyclohexylcarbodiimide (also DCC)
DCHU Dicyclohexylurea (also DCU)
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DCM Dichloromethane
DEAD Diethyl azodicarboxylate (DMAD=the dimethyl analogue)
DIPCI Diisopropylcarbodiimide (also DIC)
DIPEA Diisopropylethylamine (also DIEA)
DMA Dimethylacetamide
DMAP 4-Dimethylaminopyridine
DMF Dimethylformamide
DMS Dimethylsulphide
DMSO Dimethylsulphoxide
DPAA Diphenylphosphoryl azide
EEDQ 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
HATU This is the acronym for the ‘uronium’ coupling reagent
derived from HOAt, which was originally thought to have
the structure 8, the Hexafluorophosphate salt of the O-(7-
Azabenzotriazol-lyl)-Tetramethyl Uronium cation.
In fact this reagent has the isomeric N-oxide structure 9 in the

crystalline state, the unwieldy correct name of which does not
conform logically with the acronym, but the acronym con-
tinues in use.
.
Similarly, the corresponding reagent derived from HOBt has

the firmly attached label HBTU (the tetrafluoroborate salt is
also used: TBTU), despite the fact that it is not actually a
uronium salt.
HMP Hexamethylphosphoric triamide (also HMPA, HMPTA)
HOAt 1-Hydroxy-7-azabenzotriazole
HOBt 1-Hydroxybenzotriazole
HOCt 1-Hydroxy-4-ethoxycarbonyl-1,2,3-triazole
NDMBA N,N 0 -Dimethylbarbituric acid
NMM N-Methylmorpholine
PAM Phenylacetamidomethyl resin
PEG Polyethylene glycol
PtBOP 1-Benzotriazolyloxy-tris-pyrrolidinophosphonium
hexafluorophosphate
SDS Sodium dodecyl sulphate
TBAF Tetrabutylammonium fluoride
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TBTU See remarks under HATU above

TEA Triethylamine
TFA Trifluoroacetic acid
TFE Trifluoroethanol
TFMSA Trifluoromethanesulphonic acid
THF Tetrahydrofuran
WSCI Water soluble carbodiimide: 1-ethyl-3-(3 0 -dimethylamino-
propyl)-carbodiimide hydrochloride (also EDC)

Techniques
CD Circular dichroism
COSY Correlated spectroscopy
CZE Capillary zone electrophoresis
ELISA Enzyme-linked immunosorbent assay
ESI Electrospray ionization
ESR Electron spin resonance
FAB Fast atom bombardment
FT Fourier transform
GLC Gas liquid chromatography
hplc High performance liquid chromatography
IR Infra red
MALDI Matrix-assisted laser desorption ionization
MS Mass spectrometry
NMR Nuclear magnetic resonance
nOe Nuclear Overhauser effect
NOESY Nuclear Overhauser enhanced spectroscopy
ORD Optical rotatory dispersion
PAGE Polyacrylamide gel electrophoresis
.
RIA Radioimmunoassay
ROESY Rotating frame nuclear Overhauser enhanced spectroscopy
RP Reversed phase
SPPS Solid phase peptide synthesis
TLC Thin layer chromatography
TOCSY Total correlation spectroscopy
TOF Time of flight
UV Ultraviolet
Miscellaneous
Ab Antibody
ACE Angiotensin-converting enzyme
ACTH Adrenocorticotropic hormone
Ag Antigen
AIDS Acquired immunodeficiency syndrome
ANP Atrial natriuretic polypeptide
ATP Adenosine triphosphate
BK Bradykinin
BSA Bovine serum albumin
CCK Cholecystokinin
DNA Deoxyribonucleic acid
FSH Follicle stimulating hormone
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GH Growth hormone
HIV Human immunodeficiency virus
LHRH Luteinizing hormone releasing hormone
MAP Multiple antigen peptide
NPY Neuropeptide Y
OT Oxytocin
PTH Parathyroid hormone
QSAR Quantitative structure–activity relationship

RNA Ribonucleic acid
TASP Template-assembled synthetic protein
TRH Thyrotropin releasing hormone
VIP Vasoactive intestinal peptide
VP Vasopressin
J. H. Jones
.
Amino Acids, Pept. Proteins, 2013, 38, x–xvii | xvii

.
Published on 31 August 2013 on http://pubs.rsc.org | doi:10.1039/9781849737081-FP010 View Online
Oxidative transformations of amino acids
and peptides catalysed by Cytochromes
P450w
Max J. Cryle,* Clara Brieke and Kristina Haslinger
DOI: 10.1039/9781849737081-00001
Published on 31 August 2013 on http://pubs.rsc.org | doi:10.1039/9781849737081-00001
1 Introduction
1.1 Oxidation in nature
Enzymatic oxidation is an essential reaction in both primary and secondary
metabolism. Owing to the large variety of substrates and types of reactions
to be performed, a myriad of oxidoreductases has evolved (see Table 1). In
general, the electron transfer is mediated by cofactors that together with the
protein environment create specific redox potentials. These characteristic
potentials determine the kind of reaction that can be catalysed by a certain
class of enzymes. A very abundant and prominent class of oxidoreductases
comprises Cytochrome P450 monooxygenases that will be subject of this
book chapter in the context of oxidation of amino acids and peptides.
1.2 Cytochromes P450

1.2.1 Cytochrome P450 superfamily. In 1962 Cytochromes P450 (P450s)
were described for the first time as coloured pigments in rat liver micro-
somes.9 As described in this publication P450s show a very characteristic
absorption maximum at 450 nm when the protein solution is saturated with
CO and subsequently reduced with dithionite. This maximum was later
shown to be unique for cytochromes bearing a prosthetic, thiolate co-
ordinated low-spin b-type haem.10 The thiolate ligand was subsequently
.
identified as an invariant cysteine residue which lies within a highly con-

served region of the protein. High sequence conservation in proximity to the
haem and in other sections of the protein now allows the facile identification
of new P450s based on predicted open reading frames. In this way more
than eleven thousand genes through all kingdoms of life have been classified
as members of the P450 superfamily and grouped into families and sub-
families based upon their sequence homologies.7
1.2.2 Structure. The first X-ray structure of CYP101A1 (P450CAM;
P450 nomenclature will be given along with the general enzyme name when
available) revealed a characteristic P450 fold that has been shown to be well
conserved amongst the superfamily (Fig. 1).11 The typical structure shows a
high content of a-helices named in letters starting at the N-terminus (A to L)
and a few b-strands. The most prominent helix is the I-helix spanning the
whole molecule and coming in close proximity to the distal plane of the
haem. The F and G helices, as the most mobile parts of the protein, are
supposed to restrict the entry of potential substrates into the active site
Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
E-mail: Max.Cryle@mpimf-heidelberg.mpg.de
w
This work was supported by the Deutsche Forschungsgemeinschaft (Emmy-Noether
Programme).
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Table 1 List of cofactors occurring in enzymatic oxidation reactions. One example of enzyme
or enzyme family is given for each cofactor.
Cofactor Example Reference
Vitamins
Nicotinamide (NAD/NADP) Alcohol oxygenase 1
Flavin (FAD/FMN) Alcohol dehydrogenase 2
Non-heme metal ions
Copper Laccase 3
Selenium Glutathion peroxidase 4
Iron(II) Methane monooxygenase 5
Haem-coordinated Iron
Haem a Cytochrome A 6
Haem b Cytochrome P450 7
Haem c Cytochrome C 8
.
Fig. 1 Crystal structure of P450CAM.11 The canonical fold comprises helices A to K and a few
b-strands. The prosthetic haem is coordinated with the thiol of the highly conserved Cys357 on
its proximal side. The central I-helix spans the whole molecule on the distal side of the haem,
whereas helices F and G restrict substrate entry to the active site.
cavity.12 In the low spin, resting state the ferric haem (Fe(III)) is coordin-
ated to a distal water molecule.
1.2.3 Catalytic cycle. In the catalytic cycle of P450CAM as depicted in
Fig. 2, the entry of the substrate into the active site displaces the distal water
molecule leading to high spin ferric haem (Fe(III)). This is observable as a
shift in absorption in the visible spectrum (decrease of absorption at 420 nm
and increase at 390 nm). Electron transfer from a redox partner leads to
reduction of the haem iron followed by formation of a ferrous dioxy
complex with molecular oxygen (Fe(II)þ-O2). Sequential transfer of a
second electron and a proton lead to formation of Compound 0
(Fe(III)þ-OOH). After a second protonation, the O-O bond is cleaved and
water is released, which leads to the formation of the reactive ferryl-oxo
porphyrin cation radical, Compound I (Fe(IV)þ-O). Following hydrogen
atom abstraction from the substrate, radical recombination leads to the
2 | Amino Acids, Pept. Proteins, 2013, 38, 1–36

Published on 31 August 2013 on http://pubs.rsc.org | doi:10.1039/9781849737081-00001 View Online
Fig. 2 Catalytic cycle of Cytochrome P450s. The axial water ligand of Fe(III) is displaced
upon substrate binding (RH); transfer of a first electron and binding of molecular oxygen forms
a ferrous dioxy complex (Fe(II)þ-O2); sequential protonation and electron transfer lead to
Compound 0 (Fe(III)þ-OOH), followed by a second proton transfer and release of water
forming a ferryl-oxo porphyrin cation radical, Compound I; following hydrogen abstraction
from the substrate, radical recombination leads to the hydroxylated product, which is released
while the P450 relaxes to its resting state.
formation of the hydroxylated product which is released from the active site
while the monooxygenase relaxes to its ferric resting state.13
.
In vitro the proton and electron transfer steps can sometimes be cir-
cumvented by saturating the ferric haem with hydrogen peroxide, a process
called the peroxide shunt pathway. The advantage of this technique is the
simplification of the reaction as neither redox partners nor expensive
cofactors (NADH or NADPH) are required.14
1.2.4 Redox partners. The interplay of P450s with electron transfer
systems is almost as diverse as the reactions catalysed by this machinery.
Hannemann et al. grouped all known P450-redox systems into ten classes
characterised in part by the number of polypeptides involved (one, two or
three) and localisation of the system (cytosolic or ER membrane bound).15
The most well-studied P450, P450CAM, is the prototype of a bacterial
member of class I. Electron transfer occurs between the 2Fe-2S iron-sulfur
cluster of putidaredoxin (Pd) and the P450. An NADH-dependent FAD-
containing ferredoxin reductase, putidaredoxin reductase (PdR), reduces
the oxidised putidaredoxin after each electron transfer to the P450. Mito-
chondrial P450s share the same topology, but many systems are membrane-
associated. Most liver-microsomal P450s, however, belong to class II, where
the redox system is incorporated in a single polypeptide chain (diflavin
reductases). Electrons are transferred from an FMN cofactor via an FAD
cofactor to the P450. These two classes (I and II) are the most important
ones and their members have been studied extensively.

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Despite this, bacterial P450s are often found as ‘‘orphan’’ genes with no
genes coding for potential redox partners in the surrounding regions. Bio-
synthetic gene clusters for glycopeptide antibiotics, for example, contain
several genes coding for P450s but none for potential redox partners.16 In
order to study these P450s the peroxide shunt pathway is applied and/or
heterologous redox systems are tested. However, some P450s show strong
specificity for their redox partners because of protein-protein interactions
and the delicate regulation of redox potentials.17 In the P450CAM catalytic
cycle putidaredoxin reductase (PdR) can be replaced by palustrisredoxin

reductase (PuxR) whereas the same substitution on ferredoxin level doesn’t
allow electron transfer.17 For other P450s, however, the palustrisredoxin/
palustrisredoxin reductase (Pux/PuxR) system has successfully been em-
ployed as heterologous electron donors.18 This indicates the potential to
exploit P450s for biocatalytic processes even where an endogenous redox
system has not been identified.
1.2.5 Reactions. Cytochrome P450 enzymes play very important roles
in all kingdoms of life. On the one hand they are involved in catabolic
processes for the degradation of xenobiotics, such as pharmaceuticals,
pesticides and other potentially detrimental compounds that are taken up
by an organism.15 On the other hand P450s are key players in the bio-
synthesis of many secondary metabolites. In mammals these range from
steroids and vitamins to fatty acids, whereas in prokaryotes many products
are part of biological warfare between individual species20 (e.g. glycopeptide
antibiotics produced by Actinomycetes).19,20 However, there are also ex-
amples of eukaryotes that take advantage of such metabolites in inter- and
intraspecific competition.21 Many of these secondary metabolites are based
on amino acids or peptides that undergo several modifications until they
.
reach their full biological activity. The typical P450 catalysed reactions
within this maturation process are hydroxylation, epoxidation, heteroatom
oxidation or phenolic coupling. In some cases the substrates are free in
solution whereas in other cases they are presented to the monooxygenase by
a carrier protein.
1.3 Secondary metabolites from amino acids and peptides

A wide range of secondary metabolites in prokaryotes and eukaryotes are
derived from amino acids and peptides. In particular, prokaryotic peptide
derivatives have gained great importance as their antibiotic effects are ex-
ploited in clinical applications (penicillin,22 cephalosporin,23 vancomycin,24
bottromycin A225). These peptides can either be of ribosomal (e.g. lanti-
biotics) or of non-ribosomal origin (e.g. glycopeptide antibiotics).
1.3.1 Ribosomal peptides. Secondary metabolites derived from ribo-
somal peptides have a variety of appearances and functions. Their only
common feature is that they are transcribed from genes and translated into
a precursor peptide consisting of leader and core sequence.26 The leader
sequence serves as a guide through maturation of the core peptide by re-
cruiting tailoring enzymes and it mediates the secretion of the product (see
Fig. 3). The most abundant post-translational modifications are listed in
Table 2 and have been reviewed in detail by McIntosh et al.26

Fig. 3 Synthetic pathway of secondary metabolites derived from ribosomal peptides: A. the
precursor peptide is translated by the ribosome bearing an N-terminal leader, a core and an
optional follower sequence; B. the leader sequence directs post-translational modification of the
core peptide to obtain tertiary structure and all biologically important features; C. during se-
cretion of the mature compound, leader and follower sequence are cleaved by specific proteases.
Table 2 Main post-translational modifications of ribosomal peptides grouped into side chain
and main chain modifications. One example is given for each modification.
Modification Example Reference
Side chain
Lanthionine formation Microbisporicine 27
Oxazoline/Thiazoline formation Bottromycin 28
Prenylation ComX 29
.
Disulfide bonding Enterocin A 30

b-Hydroxylation Microbisporicine 27
Halogenation Microbisporicine 27
Alkylation Bottromycin 28
Main chain
Proteolysis
Macrocyclisation SubtilosinA 31
Formylation LacticinQ 32
The mechanism and order of these maturation steps strongly depend on

the substance class. In the discussion we will focus on the maturation of
lantibiotics which involves the actions of P450 enzymes.33
All genes contributing to the biosynthesis of a certain lantibiotic are or-
ganised within a gene cluster starting with lanA, the gene coding for the
precursor peptide. All tailoring enzymes such as the lanthionine synthetase
are encoded downstream of the lanA gene. Lanthionine synthetase is a
homomeric or heteromeric bifunctional complex that catalyses the de-
hydration of serine or threonine and the subsequent addition of the thiol
group of a cysteine.34 This thioether forms the lanthionine bridges that
provide extraordinary rigidity and stability against proteases to the peptides
(see Fig. 7).35 It has been shown for lacticin 481 that basal synthetase

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activity can be detected in the absence of the leader whilst the directionality
of processing from N- to C-terminus is lost.36 Furthermore, leader se-
quences were found to direct synthetase activity even in trans but with de-
creased efficiency compared to the intact precursor peptide.36 Following the
actions of lanthionine synthetase and other tailoring enzymes, the leader
sequence mediates the recognition by cellular secretion systems and is
cleaved from the mature peptide. Most lantibiotics have been shown to be
inactive in presence of the leader. Thus, the leader peptide not only drives
peptide maturation, it also takes part in secretion and self-immunity.33

From a commercial point of view ribosomal peptides are of significant
interest as they are easily modified to generate a range of new bioactive
compounds. Simple exchange of codons in the core sequence can afford
fully functional or even improved compounds as long as the leader re-
mained unchanged.37 Furthermore, a new application utilising the versatile
tool box of naturally occurring post-translational modifications mediated
by leader peptides called leader peptide assisted biosynthesis (LPB) has been
established.38 Target peptides can be expressed in fusion with a helper leader
in order to improve peptide characteristics such as pharmacokinetics,
bioavailability, stability, or conformational uniformity amongst others.39–41
LBP can even be combined with the use of commercially available prote-
ases, ensuring full control over the complete maturation and facilitating
isolation of the final peptide.42
1.3.2 Non-ribosomal peptides. The other group of peptidic secondary
metabolites is of non-ribosomal origin. These peptides are assembled by
large enzyme complexes called Non-Ribosomal Peptide Synthetases
(NRPS). The generic topology of an NRPS is characterised by a modular
organisation where each module specifically incorporates a certain amino
.
acid into the peptide (see Fig. 4). The modules are arranged in one or more
polypeptide chains with the exact number of modules depending on the
system. The minimal module consists of an Adenylation (A), a Peptidyl
Carrier (P or PCP) and a Condensation (C) domain. These modules can be
Fig. 4 Simplified schematic organisation of the non-ribosomal peptide synthetase from the
teicoplanin gene cluster (Actinoplanes teichomyceticus).48 Adenylation (A), Peptidyl Carrier (P),
Condensation (C), Thioesterase (Te) domains are grouped into modules that step-by-step as-
semble a heptapeptide. B, Peptide bond formation between peptide (i) and amino acid (i þ 1) in
the active site of the C-domain; the N-terminus of (i þ 1) attacks the C-terminus of (i) thereby
breaking the thioester and releasing the Pi domain.

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extended by further domains such as Epimerisation (E), Methyltransferase

(Mt), Cyclisation (Cy), Oxidation (O), Reduction (R) or Thioesterase (Te)
domains.43 In the catalytic process the A-domain activates an amino acid by
forming an acyl adenylate with ATP and transfers it onto the 4 0 -phospho-
pantetheinyl (Ppant) arm of the PCP domain, where it forms a reactive
thioester.44 The Ppant group shuttles amino acids from the active site of the
A-domain to the active site of the C-domain. The C-domain bears two
cavities where the amino acids (i) and (i þ 1) can be accommodated.45 In
these positions the free N-terminus of the tethered (i þ 1) amino acid attacks
the Ppant-bound C-terminus of residue (i) leading to peptide bond for-
mation. The nascent peptide chain remains attached to the (i þ 1) Ppant and
is transferred to the active site of the next condensation domain (see Fig. 4).
The final module of the NRPS contains an additional thioesterase domain
that cleaves the thioester bond between the last PCP and the peptide. De-
pending on the system this step yields either a linear or a N-C macrocyclic
peptide.46
The number of additional domains in these modules (E, Mt, Cy etc.)
already suggests that with this system peptides with a range of unusual
amino acids (methylated, Mt-domain; cyclised, Cy-domain) and amino acid
configurations (E-domain) can be produced. Further diversity is introduced
by incorporation of non-proteogenic amino acids like hydroxy-
phenylglycine.47 An array of tailoring enzymes such as methyltransferases,
halogenases and oxygenases modify the peptide backbone to obtain highly
stable and bioactive compounds. Specific transferases attach sugar or fatty
acid decorations depending on subcellular localisation and other required
features as e.g. membrane permeability.
It was shown that the actions of A-domains are highly specific for the side
chains of their substrates49 whereas C-domains are very specific towards the
.
stereochemistry of amino acids.50 These mechanisms ensure the correct

sequence and stereochemistry of the generated peptide. These character-
istics can be exploited for the analysis of newly discovered NRPSs; new
NRPS gene clusters are identified by the high homology of A-domains (up
to 56% sequence similarity) and from the active site residues of A- and C-
domains one can predict the sequence and stereochemistry of the product
peptide with bioinformatic tools.49
The modularity of the NRPS offers a vast platform for biotechnological
applications in order to generate new peptides. The obvious approach,
namely genetic swapping of domains or whole modules, was soon proven to
be difficult:51 only a few examples afforded functional peptides in acceptable
yields.52,53 Possible explanations are the high substrate specificities of the
later synthetic domains, the inactivity of swapped domains due to incorrect
boundary selection or inadequate communication between the domains and
modules. Hahn and Stachelhaus identified short regions (15-25 amino acids)
at the edges of NRPS subunits called communication-mediating domains
(COM).54 These domains are crucial for the assembly of the NRPS and the
communication between the subunits and thereby impose additional com-
plications on domain swapping.
Eppelmann et al. have managed to alter the substrate specificity of
A-domains by site-directed mutagenesis.55 However, this approach is only

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suitable for conservative changes, where few mutations are needed and the
substrate is still tolerated by the downstream domains.
Ribosomal and non-ribosomal peptides are processed by a myriad of
tailoring enzymes contributing to the rich structural and functional diversity
of these secondary metabolites. One important modification is oxidation,
e.g. oxidation of thiazolines, oxidative coupling of aromatic side chains, b-
hydroxylations and epoxidations. These reactions can be catalysed by
members of the P450 superfamily on amino acid and peptide level and will
now be discussed.
2 P450-catalysed oxidative modification of peptides and amino acids

2.1 Hydroxylation
P450-catalysed hydroxylation of amino acids and peptides is mainly found
in secondary metabolic pathways. Gene cluster analyses have identified a
large number of P450s implicated in such reactions, although in many cases
only homology studies have been used for assigning function and still less is
known about the exact substrates. In the biosynthesis of natural products
derived from amino acids P450s often act as tailoring enzymes on PCP-
bound substrates.56 This is especially the case for the b-hydroxylation of
amino acids, which will be discussed below.
2.1.1 b-hydroxylation of PCP-bound amino acids. Hydroxylated amino
acids are widely used intermediates in the biosynthesis of a variety of
complex secondary metabolites. Many substances with antibiotic, antiviral
or antifungal properties are among these natural products and are highly
interesting for biomedical research. Somewhat surprisingly, most P450-
catalysed oxidations on the b-position of amino acids are performed on
carrier protein-bound substrates. The desired amino acid is recognised by
.
the A-domain of an NRPS and loaded onto the PCP domain. The ami-
noacyl-PCP then serves as a substrate for a P450, which catalyses the oxi-
dation of the b-position of the tethered amino acid: this reaction often
proceeds with high stereoselectivity. Following this oxidation, the hydro-
xylated amino acid is released by a thioesterase domain (see Fig. 5) and
further metabolised. b-Hydroxylation of tyrosine, tryptophan, histidine and
valine residues have been found to proceed via this pathway and it is hy-
pothesised that restricting oxidation to amino acids in their PCP-bound
Fig. 5 b-Hydroxylation of PCP-bound amino acids by P450s. A=Adenylation domain;

PCP=Peptidyl Carrier domain; TE=Thioesterase domain.

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form is used to control the fraction of amino acids diverted into secondary
metabolism.57
Aminocoumarin antibiotics. Aminocoumarin antibiotics, including novo-
biocin, clorobiocin, coumermycin A1 or the simocyclinones (Fig. 6) are
produced by certain Streptomyces species and show antiobiotic activity
against Gram-positive bacteria. They effect their antibiotic activity by in-
hibiting DNA replication through binding to bacterial DNA gyrase.57 The
coumarin core of these antibiotics is derived from L-tyrosine, which in the
first biosynthetic step is oxidised to (2S,3R)-b-OH-tyrosine. From the

novobiocin biosynthetic gene cluster of Streptomyces spheroides, novI and
novH have been shown to encode a P450 and a di-domain NRPS consisting
of a tyrosine-selective A domain and a C-terminal PCP domain. In vitro
experiments with these proteins revealed the high substrate-selectivity of
NovH for L-tyrosine and demonstrated the direct oxidation of tyrosine
bound to NovH with NovI (CYP163A1), which exclusively generates
(2S,3R)-b-OH-tyrosine.57 In a divergence from the typical scheme for for-
mation of such hydroxylated amino acids, their cleavage from the PCP
occurs through a subsequent enzymatic transformation rather than
thioesterase-mediated cleavage.58 In the biosynthetic gene clusters of clor-
obiocin, coumermycin and simocyclinone the genes encoding the NovI
homologues CloI (CYP163A2),59 CumD,60 and SimI (CYP163A3)61 have
been identified but not further evaluated. In the biosynthetic pathways of
novobiocin and clorobiocin L-b-hydroxytyrosine serves additionally as
precursor for the prenylated benzoic acid moiety of these antiobiotics.
Glycopeptide antibiotics. L-b-R-hydroxytyrosine is also found in glyco-
peptide antibiotics, such as vancomycin and teicoplanin (see 2.4.1, Fig. 13).
In the biosynthesis of vancomycin, b-hydroxytyrosine is provided by the
interplay of the di-domain NRPS synthase BpsD, the P450 OxyD
.
(CYP146A1) and the thioesterase Bhp. In vitro studies performed with

purified OxyD gave greater insight into substrate binding by the P450.62
OxyD equally accepts PCP-bound tyrosine or phenylalanine, suggesting
that substrate specificity is determined mainly by the respective A-domain
of the NRPS. No binding was observed for the isolated amino acids.62
Amino acid SNAc thioesters – well-established substrate analogues used for
the investigation of NRPS systems63 – showed only unusual, cooperative
binding behavior. An X-ray crystal structure of OxyD revealed a rather
open active site, supporting the fact that the amino acid has to be inserted
into the active site attached to the phosphopantetheinyl linker. Thus, only
PCP-loaded substrates were able to bind to OxyD, with dissociation con-
stants in low micromolar range.62
Nikkomycin antibiotics. Nikkomycins are a group of dipeptidyl nucleo-
side antibiotics produced by Streptomyces tendae Tü901 and Streptomyces
ansochromogenes. They act as strong competitive inhibitors of chitin syn-
thase by mimicking its natural substrate UDP-N-acetylglucosamine thereby
inhibiting growth of insects and fungi.64 They consist of two unnatural
amino acids, hydroxypyridylhomothreonine (HMT) and an N-glycosidi-
cally modified aminohexuronic acid.65 The nucleoside part of nikkomycins
can contain as nucleobase either uracil (nikkomycin J, Z) or 4-formyl-4-
imidazolin-2-one (nikkomycin I, X), whereas L-histidine has been shown to

Published on 31 August 2013 on http://pubs.rsc.org | doi
.
Fig. 6 Structures of natural products derived from b-hydroxyamino acid residues. Groups derived from b-hydroxyamino acids are shown in red; in nikkomycins an
additional moiety is derived from hydroxylation by a P450 is highlighted in blue and surrounded by a dashed line (colour available in electronic version).
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be the biosynthetic precursor for the latter. In S. tendae Tü901 the NRPS
NikP1 selects and loads L-histidine onto its PCP-domain. b-Hydroxylation
of the PCP-bound histidine residue is carried out by P450 NikQ
(CYP162A1), affording (2S,3R)-3-OH-histidine.66 In the nikkomycin gene
cluster of S. ansochromogenes, sanQ is the corresponding P450-encoding
gene (CYP162A2).67 Following P450-catalysed oxidation, the (2S,3R)-3-
OH-histidine is released from NikP1 by the thioesterase NikP2 and further
metabolised in the nikkomycin synthesis pathway. In vitro oxidation studies
using purified proteins verified and highlighted the high substrate selectivity
and stereospecificity achieved by the interplay of NikP1 and NikQ.65
A P450 is also involved in the biosynthetic pathway of the second
unusual amino acid in nikkomycins, HMT. Mutation studies revealed that
the P450s NikF (CYP105K1) in S. tendae Tü901 and SanH (CYP105K2) in
S. ansochromogenes catalyse the aromatic hydroxylation of the pyridyl
residue of this amino acid.66,67 Strains with disrupted nikF/sanH genes
only produced non-hydroxylated pyridylhomothreonine containing
nikkomycins. As mutations in sanI (apparently encoding a ferredoxin) cause
a down-regulation of SanH activity, it has also been suggested that SanI is
the natural redox partner of SanH. The natural redox partners for NikQ
and SanQ have not been identified.
Quinomycin antibiotics. Quinomycins are produced by various species of
Streptomyces and can exhibit highly potent antibacterial, antiviral and/or
antitumor activities. Characteristic for these C2-symmetric, cyclic depsi-
peptides like echinomycin,68 thiochoraline69 or SW-163D,70 are two inter-
calative chromophores, either quinoxaline-2-carboxylic acid (QXC) or
3-hydroxyquinaldic acid (HQA), attached to the peptide core. Both chro-
mophores are derived from L-tryptophan, but two different biosynthetic
pathways for the chromophores were originally suggested. In the pathway
.
initially proposed for HQA the P450 TioI hydroxylates quinaldic acid in the
final step to HQA.69 In contrast, feeding experiments with deuterated
(2S,3S)-b-hydroxytryptophan revealed that stereospecific b-hydroxylation
of PCP-bound L-tryptophan by the TioI-homologue Ecm12 happens at the
beginning of QXC biosynthesis.71 Further investigations have identified
3-hydroxy-L-kynurenine, derived from hydroxytyrosine, as a common
intermediate in biosynthesis of both chromophores, strongly suggesting that
b-hydroxylation of a PCP-bound amino acid by a P450 initiates secondary
metabolite biosynthesis.72,73 In an interesting development for the biological
production of such compounds, the biosynthetic pathways of echino-
mycin74 and SW-163D75 have been re-engineered by Wantanabe and col-
leagues in a plasmid-based system allowing the production of bioactive
natural products in E. coli.
Other examples. There are further examples where P450s have been
identified or suspected to catalyse b-hydroxylations on PCP-bound amino
acid substrates. In the biosynthesis of the bleomycin antibiotic zorbamycin
and the highly potent proteasome inhibitor salinosporamide A,76 slightly
different amino acids are subjected to b-hydroxylation by a P450. In the
former case, gene cluster analysis implicated b-hydroxyvaline as an inter-
mediate synthesised by the interplay of the NRPS ZbmVIIb and the P450
ZbmVIIc,77 while in the latter the P450 SalD (CYP163B1) has been shown

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to hydroxylate the PCP-bound unusual amino acid L-3-cyclohex-2 0 -enyl-

alanine.76,78 In both cases the biosynthetic route is continued by fusion of
the hydroxylated PCP-bound amino acid with the PKS machinery.
2.1.2 Multiple hydroxylations of peptides. Beside b-hydroxylation of

PCP-bound amino acids, P450s are also found to catalyse a variety of
hydroxylation reactions on peptide-derived substrates in secondary me-
tabolism (see Fig. 7). The ribosomally-synthesised, 23 amino acid-long
lantibiotic microbisporicin is produced by the actinomycete Microbispora

corallina. A P450, MibO, has been identified in the microbisporicin gene
cluster and is believed to be responsible for the conversion of a proline
residue into the unusual 4-hydroxyproline and 3,4-dihydroxyproline resi-
dues found in the product peptide.27
The ribosomally-synthesised bottromycin antibiotics79 exhibit activity
against methicillin-resistant Staphylococcus aureus and vancomycin-resist-
ant enterococci by selectively blocking the aminoacyl-tRNA binding site of
bacterial ribosomes. Following identification and analysis of the bot-
tromycin gene cluster from Streptomyces bottropensis, it has been hy-
pothesised that the thiazole moiety, which is important for the biological
activity of several secondary metabolites, is generated by b-hydroxylation of
a cysteine-derived thiazoline ring (Fig. 7 path A). P450 BmbI (highly similar
to CYP283A1) is believed to oxidise the thiazoline moiety and this is followed
by subsequent elimination of water and decarboxylation.28 However, in
another study Müller and colleagues discuss as an alternative thiazole bio-
synthetic route with the oxidative decarboxylation step of cysteine as the first
step, followed by a cyclodehydration step (Fig. 7 path B).80 Therefore, further
data are necessary to identify the correct mechanism.
The antibiotic skyllamycin, a cyclic depsipeptide produced by several
.
strains of Streptomyces, has been identified as a highly potent inhibitor of

the platelet-derived growth factor signaling pathway. Skyllamycin A and B
feature, besides an unusual a-hydroxylated glycine residue, three b-
hydroxylated amino acids ((2S,3S)-b-hydroxyphenylalanine, (2S,3S)-b-
hydroxy-O-methoxytyrosine and (2S,3S)-b-hydroxyleucine). Interestingly,
only one P450 (Sky32, CYP163B3) is responsible for all three b-hydroxy-
lation reactions. This was proven by inactivation of the sky32 gene leading
to the isolation of non-b-hydroxylated skyllamycin from the knockout
strain, which clearly indicates that hydroxylation happens on the peptide
substrate and not before peptide assembly. This was additionally supported
by feeding experiments: hydroxylated amino acids were not able to restore
wild-type peptide production.81 The selectivity of this P450 has also now
been clarified in vitro.136
The natural product himastatin (see Fig. 9) possesses several structural
features derived from different P450-catalysed oxidation reactions. The
P450 HmtN acts as a regio- and stereoselective g-hydroxylase of an unusual
D-piperazic acid residue leading to a monomeric himastatin precursor.82
Bacterial P450s involved in secondary metabolism mostly catalyse only
one reaction. However, in the biosynthesis of the phytotoxic thaxtomin A,
two consecutive hydroxylation reactions (see Fig. 10) are performed by the
P450 TxtC (CYP264A1).83 Remarkably, TxtC modifies two very different

.
Fig. 7 Structures of peptides containing P450-modified residues; hydroxylated amino acids are highlighted. The two proposed mechanisms for thiazole ring formation in
bottromycin biosynthesis are indicated in the box.
Fig. 8 Indolactam biosynthesis and mechanism; structures of lyngbyatoxin and pendolmycin.
sites on the substrate, in terms of both their structure and reactivity: one
reaction is hydroxylation of a tertiary aliphatic carbon on the diketo-
piperazine moiety and the second is the hydroxylation of an aromatic ring.
The mechanistic details of these transformations remain to be elucidated.
2.2 Oxidation of sp2-hydridised carbons

.
P450s are well known to oxidise alkene residues to epoxides: they can also
perform the oxidation of aromatic rings, where the intermediate is generally
believed to be, or at least is most simply rationalised as, a highly reactive
epoxide. Whilst not widely prevalent in the oxidation of amino acids and
peptides by P450s, there are a limited number of examples that centre upon
the apparent epoxidation of tryptophan residues, with resultant interception
of the epoxide by a nitrogen atom within the parent substrate to generate a
new heterocycle in the product. The examples identified here include com-
pounds sharing an indolactam core (Fig. 8), and the cyclic depsipeptides
himastatin and the kutznerides.
Lyngbyatoxin. The best-characterised example of aryl oxidation leading
to bond formation by a P450 is from lyngbyatoxin biosynthesis (Lyngbya
majuscula), where the P450 LtxB (CYP107CC1) is responsible for the for-
mation of the nine membered ring of the (-)-indolactam V moiety.84 The
substrate for this P450 consists of a dipeptide alcohol produced by an NRPS
from N-methyl-L-valine and L-tryptophan. This is released from the NRPS
via reduction to the C-terminal alcohol, catalysed by an NADPH-
dependent reductase. The cyclisation of this dipeptide fragment is then
performed by LtxB, resulting in the attachment of the nitrogen of the
N-methyl-L-valine residue to the indole moiety. The mechanism of this
reaction is most simply rationalised by the epoxidation of the indole ring by

Fig. 9 Himastatin (A) and the kutznerides (B), together with the proposed mechanism for the
.
P450-catalysed formation of the pyrroloindole moiety found in these compounds (C).
LtxB, followed by nucleophilic attack of the N-methyl-L-valine amine to

open the epoxide and subsequent dehydration to restore the aromaticity of
the indole ring.85 The reaction of LtxB with the substrate N-methyl-L-
valine-L-tryptophanol has been performed in vitro with purified enzyme,
where the (-)indolactam V product was identified. Additionally, the speci-
ficity of LtxB for the N-methyl-L-valine residue was explored through the
use of reduced dipeptides with various residues in this position. The results
of turnover experiments showed that LtxB can tolerate a number of ali-
phatic residues in this position: norleucine-, norvaline- and isoleucine-
containing substrates are oxidised at around two-thirds of the efficiency of
the natural substrate and a leucine-containing substrate oxidised at one-
third of the efficiency of the natural substrate. A phenylalanine substitution
at this position abolished activity of LtxB. The differences in oxidation are
somewhat unusual due to the comparable binding strengths reported for the
different reduced dipeptides to LtxB, although slight alterations in the
binding mode for non-native substrates may well be sufficient to prevent
productive enzymatic function. It is also possible that the low efficiency of the
electron transfer system employed exacerbated the differences in product
formation due to low product yields in all cases. One curious feature of LtxB

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is the presence of an N-terminal MbtH domain: these domains of 50–70

amino acids are commonly found encoded in biosynthetic gene clusters
encoding non-ribosomal peptides, although LtxB is the first case of such a
domain fused to a P450.86 The function of MbtH domains has only recently
been identified, where it has been shown that they are important for NRPS-
mediated peptide synthesis through adenylation domain activation and
NRPS di-domain dimerisation.87,88 The possible role of the N-terminal
MbtH domain in LtxB could therefore be to either simply maintain the P450
in close proximity to the NRPS machinery, or it could also be to control the

rate of dipeptide formation dependent upon the presence of the P450.
Methylpendolmycin. The deep-sea bacterium Marinactinospora thermo-
tolerans forms the indolactam-type antibacterial natural products methyl-
pendolmycin and pendolmycin, which share the same core structure as the
lyngbyatoxins discussed above.89 Gene cluster analysis and inactivation has
shown that the P450 encoded by mpnC is responsible for the formation of
the indolactam core. The MpnC protein is highly homologous to LxtB
(44% amino acid identity, 73% similarity) and also possesses an N-terminal
MbtH-domain, albeit one that is slightly shorter than the one found in
LtxB. The major methylpendolmycin product formed in M. thermotolerans
differs in the structure of the reduced dipeptide P450 precursor, which is N-
methyl-L-leucine-L-tryptophanol: this was shown to accumulate when
mpnC was inactivated. From the difference in precursor structure, MpnC
would appear to favour oxidation of dipeptide fragments containing N-Me-
leucine instead of N-methyl-valine, although the selectivity may also be
enforced due to the selectivity of the NRPS module responsible for in-
corporation of this amino acid. In vitro selectivity studies with LtxB confirm
that both substrates are processed by LtxB, although no direct competition
studies are available as of yet.
.
Himastatin. Another example of a P450-catalysed transformation

equivalent to aromatic epoxidation followed by amine addition to afford a
ring closed product is found in the biosynthesis of himastatin in Strepto-
myces himastatinicus.82 Himastatin is a novel antibiotic compound that is
composed of two cyclic depsipeptides joined by a biaryl linkage through a
pyrroloindole moiety. The pyrroloindole, derived from L-tryptophan, has
been shown by gene inactivation studies to be formed by the action of the
P450 encoded by hmtT on the monomer of the cyclic depsipeptide. The
mechanism of formation of the (2R,3aR,8aR)-3a-hydroxyhexahydropyrrolo-
[2,3b]indole 2-carboxylic acid moiety is believed to follow a similar mech-
anism to that of LtxB and MpnC, with initial epoxidation of the tryptophan
indole ring followed by attack of a nitrogen group to open the epoxide and
form the pyrroloindole moiety: the major difference here is that the at-
tacking nitrogen is an amide that links the neighbouring D-threonine resi-
due and originates from the tryptophan itself. This results in the formation
of a five-, rather than a nine-, membered ring as in the case of LtxB/MpnC.
Additionally, there is no elimination of water to restore the aromaticity of
the indole ring: rather the hydroxyl group produced from ring opening of
the epoxide remains in the final pyrroloindole structure.
Kutznerides. The kutzneride antifungal and antibacterial agents are cyclic
depsipeptides with a number of unusual residues isolated from Kutzneria sp.

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90
744. These include a pyrroloindole moiety, albeit with 2-chlorine sub-
stituents upon the 6-membered ring. The similarity of this moiety to that
found in himastatin is striking, and the biosynthesis of the moiety is highly
similar: a P450 encoded by the gene ktzM with high homology to that en-
coded by hmtT (63% sequence identity and 87% sequence similarity) has
been identified in the kutzneride biosynthetic gene cluster. It is thus hy-
pothesised that the synthesis of the dichloropyrroloindole moiety occurs via
the epoxidation of a tryptophan moiety in the cyclic depsipeptide in a
similar manner to that seen in himastatin.
2.3 Heteroatom oxidation and incorporation

Natural products with N-oxidised functionalities are less common. Yet
more uncommon is the implication of P450s in these kinds of oxidation
reactions which are – especially in prokaryotic organisms – more generally
performed by flavin monooxygenases or non-haem iron-dependent dioxy-
genases.91 Nevertheless, some highly interesting examples have been de-
lineated where P450s are involved in N-oxidations of amino acid-derived
natural products.
2.3.1 Cyclic dipeptides
Thaxtomins. The cellulose biosynthesis inhibiting thaxtomin phytotoxins
are produced by plant-pathogenic Streptomyces species. They belong to the
class of cyclic dipeptides (diketopiperazines) which are produced by cyclo-
dipeptide synthases utilising tRNA-loaded amino acids in an ATP-
dependent manner.92 Thaxtomin is biosynthesised by the NRPS-catalysed
condensation of L-4-nitrotryptophan and L-phenylalanine. The non-
proteogenic amino acid L-4-nitrotryptophan is generated by direct nitration
of the indole ring through the actions of P450 TxtE, utilising nitric oxide
.
produced by the NO synthase TxtD (see Fig. 10A). This is highly unusual,
as nitro groups are typically derived from oxidation of an amine. However,
by feeding NO-releasing compounds to a txtD inactivated mutant Challis
and coworkers could show utilisation of NO by TxtE for the oxidative ni-
tration of L-tryptophan.93 Using recombinant TxtE, the regio- and sub-
strate-selectivity of the nitration reaction could be clarified (dissociation
constant for L-tryptophan and TxtE: 60 6 mM) and a mechanism with NO
and O2 as co-substrates has been proposed (see Fig. 10B).93 Thus, in the
biosynthesis of thaxtomin a unique and for P450s very uncommon oxi-
dation reaction type has been realised. TxtE may therefore be an interesting
candidate for expanding the scope of P450-catalysed reactions in bio-
technological applications.
Pulcherriminin. In Bacillus subtilis the cyclic dipeptide cyclo-L-leucyl-L-
leucyl (cLL) is oxidatively transformed by the P450 CypX (CYP134A1) into
pulcherriminic acid, which is a precursor of the extracellular iron chelator
pulcherriminin.94 In vitro experiments with purified CypX revealed that
CypX binds to cLL with a low micromolar affinity (dissociation constant:
24.5 0.5 mM), with similar affinities determined for compounds with larger
hydrophobic side chains.95 It is probable that the high substrate specificity
of the preceding cyclodipeptide synthase in B. subtilis reduces the need for
P450-mediated substrate selectivity. The crystal structure of CypX revealed

.
Fig. 10 Thaxtomin biosynthesis (A) and proposed mechanism93 of TxtE-catalysed nitration (B).
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an interesting alteration of the I-helix, with the alcohol in the typically

highly conserved acid/alcohol pair (responsible for correct protonation of
reactive intermediates) replaced by a proline residue. The role of the missing
alcohol residue may be fulfilled by a highly ordered active site water net-
work or may not be necessary because of an alteration in the mechanism of
oxygen activation due to the oxidative transformations performed by this
P450.
Pulcherriminic acid formation involves a three-step oxidation with trans-
formations of the diketopiperazine nitrogen atoms to the respective N-oxides

and additionally aromatisation of the diketopiperazine ring. Aromatisation is
proposed to occur either via hydroxylation and elimination of water or via an
electron transfer reaction (see Fig. 11). In turnover experiments of cLL with
CypX and a variety of different redox partners two potential singly oxidised
intermediates were observed, showing that different oxidative pathways for
CypX diketopiperazine oxidation potentially exist.95
2.3.2 Oxime formation

Nocardicin A. Nocardicin A, a monocyclic b-lactam antibiotic produced by
the actinomycete Nocardia uniformis features a rare oxime functionality.
Nocardicin A is built up from two hydroxyphenylglycine amino acids that
are attached to the serine-derived b-lactam ring. Further, a homoserinyl side
chain is attached to one of the aromatic side chains.96 Townsend and
colleagues identified NocL as the first prokaryotic P450 able to oxidise an
amine to a syn-oxime and proposed a two-step mechanism via successive
N-hydroxylations.91,97 Following the hydroxylation steps, the neighboring
amide bond could then facilitate elimination of water to generate a nitroso
species and promote tautomerisation to the syn-oxime (see Fig. 11). Intra-
molecular hydrogen-bonding was also invoked to account for the greater
.
abundance of syn-oxime in comparison to anti-oxime.91 Furthermore, in

vitro studies performed by Townsend et al. demonstrated that NocL only
converts nocardicin C to nocardicin A, suggesting the homoserinyl chain is
crucial for NocL activity.97
Cyanogenic glucosides. Cyanogenic glucosides are b-glucosides of amino
acid-derived a-hydroxynitriles. With the exception of some arthropod
clades, these compounds are produced by a large variety of plants and act as
defense compounds by releasing hydrogen cyanide enzymatically when the
organism sustains insect attack.98 In general, the entire biosynthetic path-
way of cyanogenic glucosides is encoded by just three genes, two encoding
membrane-bound P450s and one a UDP-glycosyltransferase. It has been
shown by in vitro experiments that the first P450 (a CYP79) acts as a
multifunctional enzyme, catalysing two sequential N-hydroxylations of the
amine of an amino acid followed by a dehydration/decarboxylation reaction
step, thus producing an aldoxime as a free intermediate. The second P450 (a
CYP71) performs two reactions, first catalysing the dehydration of the
aldoxime to a nitrile and next hydroxylating the Ca-carbon to generate a
cyanohydrin, which is then transformed into the glucoside (Fig. 12).99,100
These cyanogenic glucosides are derived from several aromatic (phenyl-
alanine/tyrosine) as well as aliphatic amino acids (isoleucine/leucine/valine).
All members of the CYP79 family feature some unique amino acid

.
Fig. 11 Possible oxidation pathways of cLL in the formation of pulcherriminic acid catalysed by CypX (A) and oxidation of nocardicin C to nocardicin A catalysed
by NocL (B).
Fig. 12 Biosynthetic pathway of cyanogenic glucoside synthesis exemplified by taxiphyllin

synthesis in Triglochin maritima.100
substitutions resulting in an increase in polar and charged residues in the

.
active site. According to Møller and colleagues these unusual substitutions

may have evolved either due to the unusual catalytic activity of CYP79
enzymes or due to the high hydrophilicity of their amino acid substrates and
intermediates compared to most other P450 substrates.21,98
Interestingly, the biosynthetic pathways to cyanogenic glucosides in
plants and insects proceed using essentially the same intermediates with
highly related enzymes. This is an interesting example of convergent evo-
lution in two different kingdoms.98
2.4 Phenolic and aryl coupling

Of all the classes of chemical transformations affected by P450s upon
amino acids or peptides, the coupling of aromatic side chains has been
the most widely studied both in vivo and in vitro. This is due to the role
that these transformations, catalysed by P450s, play in the biosynthesis of the
glycopeptide antibiotics. These compounds, highly modified heptapeptides
produced via the action of NRPSs, are still in clinical use and provide last-
resort therapeutics with action against Gram-positive bacteria such as
methicilin resistant S. aureus. Additional examples of phenolic and aryl cross
coupling catalysed by P450s have been reported in the biosynthesis of other
NRPS-synthesised natural products (arylomycin A2 and himastatin), in
staurosporine biosynthesis and in the oxidation of cyclic dipeptides.

.
Fig. 13 Structures of the glycopeptide antibiotics.

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2.4.1 Oxidative aromatic coupling in glycopeptide antibiotic biosynthesis

Structural classes of glycopeptide antibiotics. A very large number of
different glycopeptide antibiotics have been identified. These have largely
been assigned to one of five classes, which are determined by the cross-
linking state of the amino acid side chains that form the peptide and the
decorating groups found attached to the aglycone.101 From a perspective of
P450-mediated aromatic coupling, there are three main classes: the vanco-
mycin-type (three side chain cross links: AB biaryl coupling, C-O-D
phenolic coupling and D-O-E phenolic coupling); the teicoplanin-type (four

side chain cross links: vancomycin-type plus additional F-O-G phenolic
coupling); and the complestatin-type (two side chain cross links: B-O-D
phenolic coupling and DF biaryl coupling; additional A-O-C phenolic
coupling in some cases).101 In all gene clusters for such compounds iden-
tified to date, there exists a number of P450s implicated in the formation of
such cross links.102 These proteins widely referred to as Oxy proteins
(CYP165 family), exhibit high degrees of similarity across multiple gene
clusters, which has made the assignment of predicted function much more
straightforward. Comparison of the gene clusters of complestatin and the
vancomycin type glycopeptides show a high similarity between P450s en-
coded by oxyB and comJ for example, whilst the other P450 (encoded by
comI) has lower predicted homology to all other Oxy proteins. The product
of comI expression can be assigned to the unusual biaryl tryptophan-
hydroxyphenylglycine cross coupling found in the complestatin-type pep-
tides.103,104 Comparative analysis of the vancomycin- and teicoplanin-types
shows that there is an additional P450 present in the teicoplanin-type gene
clusters that is related to that encoded by oxyA, but more distantly than the
true oxyA gene in the cluster.19,48,105,106 Given the proximity of the add-
itional ring in teicoplanin to that formed by OxyA (see below), the as-
.
signment of this new P450 to that responsible for the F-O-G ring catalysis
becomes somewhat instinctive; this was later confirmed by inactivation
experiments (see below).
Reaction order of glycopeptide antibiotic cross coupling P450s. In the
biosynthesis of the vancomycin type glycopeptides, the P450s encoded by
oxyA, oxyB and oxyC have been shown via inactivation studies in vivo to be
responsible for the D-O-E, C-O-D and AB ring junctions respectively.
Initially, the inactivation of the oxyA gene led to the isolation of hepta-
peptides bearing the C-O-D ring only:107 this could be deduced from an
analysis of the metabolites. Only linear peptides were found when oxyB was
inactivated, indicating that the enzyme encoded by this gene was the first to
act on the peptide and installing the C-O-D ring.47 The oxyC inactivation
experiments showed the presence of peptides bearing both C-O-D and D-O-
E rings, thus allowing the assignment of the AB ring to OxyC and the D-O-
E ring to OxyA. By combining the data from the inactivation of the oxy
genes, the order of the P450s involved in peptide oxidation was revealed as
OxyB, OxyA and finally OxyC.47 Additional gene disruption experiments
hinted that the peptide substrates for the Oxy proteins were still associated
with the NRPS machinery, as disruption of elements of the NRPS caused
the isolation of peptides with only the C-O-D ring installed.108 A compar-
able inactivation study of the four P450s found in the gene cluster encoding

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a teicoplanin-like glycopeptide A47934 was also performed, with the isol-

ation of the peptides produced by the inactivation mutants leading to an
order of P450 action.109 This again showed that the C-O-D ring is installed
first by OxyB, with the remaining order of the last three enzymes not as easy
to deduce as with the vancomycin type systems. The assignment of function
to the P450s was clear however, with the AB ring again installed by OxyC,
the D-O-E ring by OxyA and the F-O-G ring by the new P450 OxyE, which
has its highest homology to OxyA.109
In an elegant in vivo study, Süssmuth and coworkers engineered the

NRPS machinery to incorporate an additional hydroxyphenylglycine (Hpg)
residue into the peptide between the two normal Hpg amino acids and
identified the new biosynthetic products.110 Metabolite analysis indicated
that the NRPS now produced an octapeptide as had been intended, al-
though the C-O-D ring was the only one installed in the peptide product (see
Fig. 14 A). This indicated a somewhat broad peptide substrate tolerance for
OxyB, a finding that has also been confirmed in vitro with shorter peptide
substrates (see below). Despite the lack of in vitro data for OxyC, further
gene inactivation studies have given some insights into the selectivity of this
enzyme for alternative peptide substrates. Using strains where the for-
mation of the final amino acid residue, dihydroxyphenylglycine (Dpg), was
interrupted, feeding of modified Dpg residues (3-hydroxy, 3-methoxy, 3-
hydroxy-5-methoxy, dimethoxy) indicated that these residues could all be
incorporated into peptides that were then fully modified by the Oxy en-
zymes (see Fig. 14 B): this naturally implies that OxyC does not require the
Dpg residue to be absolutely conserved to allow it to perform the AB biaryl
ring closure, but rather that a single meta-oxygen substituent is sufficient for
the enzyme to function.111 An additional in vivo study with relevance for
OxyC showed that the Dpg-knockout strain discussed above would produce
.
very small amounts of a novel vancomycin-like aglycone in the absence of

Dpg: this involved the incorporation of 4-hydroxyphenylglycine as the 7th
amino acid and the formation of a modified AB ring of 13 (rather than the
standard 12) atoms (see Fig. 14 C).108 Whilst this product was inactive as an
antibiotic, it clearly demonstrated that the OxyC enzyme possesses some
flexibility in its requirements for AB ring formation and gives hope for the
future application of such P450s as catalysts in aglycone diversification.
The analysis of the complestatin-type peptide modification has only been
performed by comparison to the other systems. This suggests initial for-
mation of the B-O-D ring (OxyB like ComJ) followed by the (unique)
formation of the DF biaryl system, presumably by the other P450 in the
gene cluster, ComI.103
Structural and functional characterisation of glycopeptide antibiotic cross
coupling P450s. Initial attempts to investigate the Oxy proteins in vitro did
not afford typical P450 binding spectra and the efficiency of enzymatic
turnover was minimal, if present at all. With clues collected from both
in vivo and in vitro sources, it was suggested that the Oxy proteins could well
bind to the peptides not free in solution but rather still bound to an NRPS
carrier protein domain.108 One particular clue was the structural charac-
terisation of the OxyB and OxyC enzymes from the vancomycin gene
cluster: both structures revealed extremely open active sites, which would be

.
Fig. 14 Identified products of in vivo gene knockout and complementation studies of glycopeptide antibiotics with relevance to P450-catalysis: A. Octapeptide product from
NRPS module insertion; B. Heptapeptide products from Dpg knockout strain with precursor supplementation (R1, R2 H or sugars, dependent upon individual examples); C.
Heptapeptide product from Dpg knockout strain with expanded AB ring due to Hpg incorporation.
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16,112
expected for a substrate as large as a peptide-PCP. The same open
active site architecture was later seen also for the teicoplanin F-O-G ring
forming enzyme, OxyE.113,114 The investigation of OxyB from vancomycin
has been undertaken extensively by the Robinson group, who initially
showed excellent turnover of hexapeptide substrates upon both PCP-
domains from the sixth and seventh amino acid incorporation modules
from the corresponding NRPS (see Fig. 4).115,116 PCP-domain seven was the
best behaved in both binding and turnover experiments and the effects of
peptide modification were explored in detail; this included establishing that

OxyB had a wide range of tolerance for alternative peptide lengths, with
peptides loaded on PCP-domain seven and composed of three, five, six and
seven amino acids all being oxidised. Curiously, typical type-I P450 binding
spectra could not be gathered for the PCP-loaded smaller peptide substrates
(Fig. 15 A). The methylation or acetylation of the N-terminus of the peptide
did not appear to effect binding and oxidation of the PCP-peptides by
OxyB. OxyB was also able to bind and oxidise a PCP-loaded peptide
substrate with the incorrect stereochemistry of the tyrosine residue at
position six of the peptide (Fig. 15 B).116–118 The ring formation process
was also shown to require oxygen and recently the effect of substituents
(b-hydroxylation, aromatic chlorination) on the tyrosine residue was also
investigated (Fig. 15 C).117,119
To date, no further in vitro data has been obtained for any other Oxy
protein. Mechanistically, a number of alternatives can be envisaged for
P450-catalysed oxidative aromatic coupling – these either invoke hydrogen
atom abstraction from the phenol group of the aromatic residues by the
highly reactive Compound I species or epoxidation of the aromatic ring
(similar to that invoked for the epoxidation of tryptophan previously dis-
cussed).120 The intermediates following from these initial steps then would
.
be one of the following: a diradical species, a geminal diol or an a-ketoether.

In the final two cases, the phenolic cross-link is already established at this
point of the reaction. Re-establishment of aromaticity or phenolic coupling
of the diradical is then the final step to unify the pathways at the desired
product. The mechanism of oxidative aromatic coupling in the biosynthesis
of glycopeptide antibiotics has been investigated both in vitro by Robinson
and in vivo by the group of Spencer. Use of an atmosphere of 18O2 during
the in vitro oxidation of PCP-peptides by OxyB showed that there was no
incorporation of labeled oxygen into the cross linked peptide products.117
This argues against both mechanisms that invoke a geminal diol, unless the
elimination of water from the diol occurs with complete stereoselectivity.
The in vivo experiments of Spencer used the supplementation of cultures
producing chloroerymomycin with 4-hydroxyphenylglycine (Hpg) that was
labeled with deuterium atoms on the aromatic ring and possessed an 18O
atom in the phenol group.120 They could show that the product aglycone
contained labeled Hpg residues with total retention of the phenolic oxygen,
which indicated that there was no loss of the phenolic oxygen during the
P450-catalysed cross linking reactions. This result was in agreement with the
in vitro experiments for OxyB and indicates that the mechanism for OxyA
and OxyC catalysed coupling follows a similar route with total retention of
the oxygen atoms of the Hpg residues. This also argues against mechanisms

.
Fig. 15 Substrate peptides and products of P450 OxyB as reported by Robinson and co-
workers: A. Demonstrated in vitro peptidyl-PCP substrates of OxyB; B. Peptidyl-PCP substrate
for OxyB with opposite enantiomer of Tyr-6 residue; C. Peptidyl-PCP substrates for OxyB
investigating the effects of substituents of Tyr-6 residue on coupling efficiency.
invoking a geminal diol intermediate, suggesting a mechanism either in-

volving diradical formation or formation of a keto-intermediate. The
mechanisms can be differentiated by the requirements of both phenol rings
to closely approach the haem: the diradical mechanism could well occur
over a longer range through proton-coupled electron transfer (see later) and
would not require both aromatic rings to closely approach the haem, whilst
the keto-mechanism would require that both phenol groups are within

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direct hydrogen abstraction range of the activated haem. Future structural

studies would no doubt be of use to assist in further unraveling the mech-
anism of aromatic coupling in glycopeptide biosynthesis.
2.4.2 Oxidative aromatic coupling in NRPS non-glycopeptide type pep-

tides. Additional examples of P450-catalysed phenolic and aryl coupling in
the biosynthesis of NRPS-produced peptides are found in the cases of
arylcomycin A2 (Streptomyces roseosporus) and himastatin (Streptomyces
hygroscopicus).
Arylomycin A2. Arylomycin A2 (Fig. 16) is a member of the arylomycin
family of antibacterial agents, which exert their activity by targeting type I
signal peptidases – highly conserved and essential proteins located on the
extracellular side of the cytoplasmic membrane.121 The arylomycins are
composed of a hexapeptide backbone modified by a long chain acyl group
on the amino-terminus. The peptide is synthesised by an NRPS, with the
acyl chain also incorporated into the peptide by a starter condensation
domain.122 In contrast to the glycopeptide backbones, the arylomycin
peptide is mainly composed of residues with smaller side chains (Ala, Ser,
Gly) and only two larger aromatic moieties at position four (L-Hpg) and six
(L-Tyr). They form a fused biaryl structure in the final natural product, with
gene disruption studies confirming the candidate for catalysing the aryl
coupling as the P450 encoded by aryC.123 This P450 has a relatively high
amino acid homology to that of the glycopeptide P450 OxyC, which also
catalyses biaryl coupling.122 Additionally, with such a similarity in structure
there is also a high likelihood that the P450 acts upon the NRPS-bound,
rather than the free, peptide. This example is thus the first to extend
glycopeptide-like P450-catalysed aromatic coupling activity to new NRPS-
biosynthetic systems, indicating that yet more examples may well be waiting
.
for discovery by future isolation or gene-sequencing based methods.

Himastatin. Himastatin consists of a cyclic hexadepsipeptide core that
is dimerised via an biaryl linkage at C5 from the tricyclic hexahydro-
pyrroloindole moiety (see Fig. 9).82 The formation of the biaryl crosslink
has been shown by gene inactivation to be performed by the P450 encoded
by hmtS, with the detection of only cyclic hexadepsipeptide monomers in
the knockout strain. HmtS is the first P450 shown to catalyse such a biaryl
coupling in peptide-based systems, although the precedent for such biaryl
cross coupling reactions with identical monomers has already been shown
for the CYP158A1 and CYP158A2 enzymes from Streptomyces
Fig. 16 Arylomycins and P450-catalysed biaryl ring formation.

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124–126
coelicolor. These P450s catalyse the formation of isomers of bifla-
violin and triflaviolin from flaviolin monomers and have been well char-
acterised in vitro. The exact mechanism of the biaryl crosslinking reaction
still remains somewhat unclear however, due to the apparent requirement
for monomer molecules to rearrange in the active site of the P450, as pos-
tulated in current mechanistic proposals.124
2.4.3 Oxidative aromatic coupling of dipeptide substrates
cYY. cYY is a cyclic dipeptide formed from two tyrosine residues via a
cyclodipeptide synthase (CDS) utilising aminoacyl-tRNAs as substrates
(Fig. 17), and is the main cyclic dipeptide produced by Mycobacterium
tuberculosis. In M. tuberculosis, the gene encoding the P450 CYP121A1 is
organised in an operon-like structure with a CDS that was shown to cata-
lyse cYY formation, which in turn led to the investigation of substrate
properties of cYY with CYP121A1.127 As this P450 has been shown to be
essential for viability, there was naturally an interest in characterising this
P450 that is a potential target for azole inhibitors. Belin and coworkers were
able to demonstrate both binding of cYY to CYP121A1 (dissociation
constant: 21.3 3.5 mM) and additionally the P450-catalysed oxidative
transformation of cYY into a biaryl linked product P1, where the crosslink
was established ortho to the tyrosine phenol groups.127 Additionally, they
were able to solve the crystal structure of CYP121A1 with cYY bound in the
active site, which showed the diketopiperazine ring and the distal tyrosine
side chain essentially perpendicular to the haem plane, with the proximal
tyrosine side chain approaching to within 6 Å of the haem iron (the
structure of the unbound form of the enzyme was previously solved by
Munro and co-workers).128 Curiously, aside from Van der Waals contacts,
the interactions of cYY with the protein were limited to one direct hydrogen
.
bonding interaction, and there was surprisingly little alteration to the water
network coordinated to the haem iron. The rearrangement of the molecule
during catalysis would seem to be required from this structure to bring both
phenol groups within an appropriate distance to the haem to allow
hydrogen abstraction; an alternative explanation would be that the sub-
strate bound structure does not represent the final active conformation of
CYP121A1. This interpretation is supported by the fact that there is still a
water molecule bound to the haem iron in the substrate-bound form: the
Fig. 17 cYY and P450-catalysed biaryl ring formation.

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iron-bound water molecule seems to be present in all low temperature ex-

periments but can be displaced in room temperature titrations. One final
alternative would be that the mechanism of such biaryl coupling relies upon
proton-coupled electron transfer from the substrate to the haem via the
water network. This would remove the need for dramatic substrate motion
during the P450 active cycle and may indicate why CYP121A1 is sensitive to
temperature induced spin state changes due to the necessity to maintain
such a water network for activity. Such a mechanism has been invoked for
chromopyrrolic acid coupling via P450 StaP (see below).

In a recent study, the selectivity of CYP121A1 for alternative cyclic di-
peptide substrates has been biochemically and structurally determined.129
The binding properties of a range of cyclic dipeptides and mimics to
CYP121A1 was tested, which revealed that only structures with two aryl
side chains (cYF, cYW, cY-Dopa) displayed binding within the same regime
as the natural cYY substrate. Additionally, CYP121A1-mediated oxidation
of these alternative substrates was either very inefficient (cYF), very un-
specific (cYW) or not able to catalyse the formation of a comparable
phenolic crosslinked product (cY-Dopa). As the cyclodipeptide synthase
associated with CYP121A1 also produces varying amounts of cyclic di-
peptides (including cYF and cYW) in addition to cYY,92 the selectivity of
CYP121A1 for cYY indicates that the phenolic crosslinking of this di-
peptide is the probable role for this enzyme also in vivo.129
Staurosporine. Whilst technically not a dipeptide, the biosynthesis of the
indolocarbazole antitumour agent staurosporine (and the structurally re-
lated rebeccamycin) involves the P450-catalysed biaryl coupling of chromo-
pyrrolic acid, formed ultimately from two molecules of tryptophan via a
two-step enzyme catalysed process.130 The P450 StaP (CYP245A1) catalyses
the biaryl coupling of the indole rings at C5, with the three aglycone
.
products identified from in vitro turnover experiments using StaP shown to

occur via subsequent and rate limiting non-enzymatic processes (dec-
arboxylation and oxidation) from a common intermediate (Fig. 18).131,132
The crystal structures of StaP in both the substrate bound and free forms
have been solved, with the substrate bound form showing three molecules of
chromopyrrolic acid bound to the P450: one in the active site, one some-
what removed from the active site and a third molecule bound at a distant
beta-sheet.133 The molecule bound in the active site appears to be fixed in a
so-called twisted butterfly conformation, with significantly more hydrogen
Fig. 18 Staurosporine and P450-catalysed biaryl ring formation.

View Online
bonding interactions present than was the case with CYP121A1. Add-
itionally, the mechanistic postulate (supported by computational studies) is
that there is no rearrangement of the indole rings during catalysis, but ra-
ther that the enzyme initially forms an indole cation radical via proton
coupled electron transfer (PCET).134,135 This cation can equilibrate to the
distal indole ring, allowing the second PCET to occur from the proximal
indole ring, establishing the diradical species that then undergoes biaryl
coupling. This mechanism is reminiscent of that seen in the case of cyto-
chrome c peroxidase, with the separate ‘‘storage’’ of two oxidising equiva-

lents – one in the iron-oxo species and the other in a tryptophan indole
cation radical.134,135
3 Conclusion
Oxidation of amino acids and peptides is a very common and highly useful
modification in secondary metabolism. On the one hand oxidation reactions
are crucial for conformational maturation and conferring stability and
specific biological characteristics to certain compounds, whilst on the other
hand they diversify the limited range of the ribosomal amino acid code
through post-translational modification. Many of these secondary metab-
olites find biological or medicinal applications as cell culture additives,
antibiotics, antivirals or anti-tumor agents, which makes understanding
their biosynthesis important for exploitation and future modification of
these compounds.
Cytochomes P450 are one of the most versatile superfamily of oxido-
reductases that catalyse natural oxidations. They are found in all kingdoms
of life and perform an array of reactions on a wide range of substrates. Due
to their highly reactive nature, P450s can catalyse very challenging reactions
.
such as C-H bond activations, coupled with high degrees of stereo- and
regiospecificity. Thus, investigating the role of P450s in the oxidation of
amino acids and peptides is not of pure biological or medicinal interest but
it is also target of biotechnological applications in industry. It is clear that
the enormous diversity of secondary metabolites has not been fully explored
to this point and that the discovery and mechanistic investigation of P450s
involved in these biosynthetic pathways has just begun. Thus, further im-
portant and exciting findings in P450-catalysed oxidations of amino acids
and peptides are to be expected.
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NMR investigation of disulfide containing
peptides and proteins
Katalin E. Kövér*a and Gyula Battab
DOI: 10.1039/9781849737081-00037
1 Introduction
We give a short overview of the literature covering a period from 2011 to

2013 concentrating rather to peptides and small proteins and some recent
advances in solution NMR technology devoted to disclosing the structure of
disulfide peptides and proteins. Disulfide peptides and proteins are not only
interesting for their own structural curiosities and biological significance.
They are important models for understanding the dynamic nature of
proteins including folding/unfolding events. Solution NMR is especially
suitable for studying molecular motions in an enormously big timescale,
ranging from ps to days. The analysis and interpretation of 15N-relaxation
for example, has become an everyday practice either by model-free or
reduced spectral density methods. Combining these data with contemporary
extreme long-time (ms range) in-silico molecular dynamics calculations is a
fascinating possibility to better understand the soft or ‘‘fuzzy’’ protein
world. There are so many disulfide peptides and proteins in the databases,
that it is nearly hopeless to cover all, even for a relatively short period of
time. Approximately 10% of the mammalian proteins contain disulfide
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bonds. Apparently, structure-dynamics-folding-function studies are in the

focus of the research of new entities. Parallel to the increasing number of
new disulfide proteins and peptides, capabilities of instrumentation have
been extended significantly. In the PDB database http://www.rcsb.org/pdb/
home/home.do among the 21.000 disulfide containing structures nearly
1900 were solved by NMR, and most of them belong to small proteins (600)
or peptides (130). Among them are knottins (small inhibitors, toxins,
lectins, 272), snake toxin-like (disulfide-rich fold, nearly all beta, 62),
insulin-like (nearly all alpha, 40), defensin-like (disulfide-rich, nearly all
beta, 27), BPTI-like (disulfide-rich alpha þ beta fold, 20), Kazal-type serine
protease inhibitors (16), complement control module/SCR disulfide-rich all
beta (16), other (151). The scope of a recent review1 covered the secreted
human cysteine-rich mini-proteins. Analysis of their molecular targets
showed that these mini-proteins are frequently ligands for G protein- and
enzyme-coupled receptors, transporters, extracellular enzyme inhibitors,
and antimicrobial peptides. As a conclusion, cysteine-frameworks must play
an important role in human biology. Post-translational cleavage or the
formation of disulfide bonds are now being identified in cancer-related
proteins and an opinion was published2 how these allosteric bonds could be
targets for new therapies. The importance of NMR and protein structure in
drug design and application to cyclotides and conotoxins was underlined in
another overview.3 Conotoxins are disulfide-rich peptides from the venoms
a
Egyetem te´r 1, 4032, Debrecen Hungary. E-mail: kover@science.unideb.hu
b
Egyetem te´r 1, 4032, Debrecen Hungary. E-mail: batta@unideb.hu

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of marine cone snails, and they may interact with ion channels, transporters
and other receptor sites. For structure determination of disulfide peptides
and mini-proteins NMR has distinct advantages if compared to X-ray
crystallography.
2 General NMR methods

A still actual review was written on the NMR of unfolded proteins and
protein folding by Dyson and Wright.4 In that review chemical shifts, re-
laxation, NOEs and spin labels, residual dipolar couplings, amide proton
hydrogen exchange, high-pressure NMR, real-time NMR and DOSY
(Diffusion-Ordered SpectroscopY) are dealt with in detail. For the quan-
tification of small cyclic disulfide-rich peptides analytical reverse-phase
high-performance liquid chromatography trace or UV absorbance at
214 nm were suggested besides the simple NMR technique.5 Certain
methods can have high errors (like nanodrop)- except the protocol is
elaborated for a single protein. Due to the widespread problem of ambi-
guities concerning the pattern and conformation of disulfide bonds it has
been suggested6 recently that such clues could be resolved by stereospecific
deuterium labelling of the cysteine beta protons – in addition to uniform 13C
and 2H (except Cys-betas) labelling – reducing thereby the deleterious spin
diffusion effects that hamper the measurement of exact inter-disulfide cross-
relaxation rates in D2O solution. The new method not only gives a way for
disulfide pattern determination, but may allow the determination of the
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conformations around the disulfide bonds as well. Though it must be an

efficient technique, laborious labelling and running of several 13C edited
NOESY-s at different mixing times makes this approach difficult. Alter-
natively, the ‘‘Exact-NOESY’’ may be the preferred technique7 that could
be applicable to determine proton-proton distances through the disulfide
bonds in-spite of the three spin effect caused by the geminal beta protons.
DFT quantum-chemical calculations proved8 that – in agreement with ex-
perimental observations - the chemical-shift ‘‘basins’’ for the oxidized and
reduced cysteine states are clearly separated for reduced-SH 13Cb, but not
for the 13Ca, chemical shifts. So, redox state of cysteines can be safely de-
cided based on Cys 13Cb shifts. A method for identifying cysteine (Cys)
residues with sulfhydryl (SH) groups with slow hydrogen exchange rates
was developed9 in H2O/D2O media. The method utilizes the isotope shifts at
the Cb chemical shifts induced by the deuteration of the SH, NH groups of
[3-13C;3,3-2H2] labelled Cys residues. The fractionation values (F = [XD]/
[XH]) could be determined for both the SH and the amide NH groups of
two Cys residues in EPPIb protein, and gave surprisingly low 0.4–0.5 for
[SD]/[SH] and regular 0.9–1.0 values for [ND]/[NH]. These numbers are
indicative on the microenvironment of the SH groups and may be relevant
for the explanation of enzyme activities. The proton NOE’s of the two
slowly exchanging SH’s could be used as additional structural constraints.
3 Disulfide bond connectivities by NMR spectroscopy

A key issue in the structural characterization of these disulfide-rich peptides
and proteins is the unambiguous assignment of the disulfide-bonds. NMR

Fig. 1 Direct determination of disulfide-bond connectivities using 77Se NMR spectroscopy.

(A) 3D structure of [Sec13, Sec14] analogue of the 37-residue spider toxin k-ACTX-Hv1c.
An enlarged version of the vicinal diselenide bridge is shown on the right with the NMR
active nuclei labelled. (B) natural abundance 1H–77Se HMBC, (C) 77Se-enriched 1H–77Se
HMQC, and (D) 77Se–77Se COSY spectra of the toxin acquired at 500 MHz. (Reprinted with
01/12/2013 14:44:18.
permission from M. Mobli and G. F. King, Toxicon, 2010, 56, 849. Copyright & 2010 by
Elsevier Ltd.)
based determination of disulfide bond connectivities for peptides/proteins

with complex disulfide topologies is a demanding task. The most commonly
used approach relies on detection of intercysteine NOEs. However, this
methodology fails in case of closely packed disulfide bonds, leading to
structures with ambiguous assignment of disulfide network.10 More recently
it has been demonstrated that substitution of sulfur atoms by 77Se with
more favourable magnetic properties makes feasible to determine the di-
selenide through-bond connectivities directly via detecting the inter-residue
vicinal couplings (3J(Se,H)) across the diselenide bond and/or observing the
one-bond Se-Se connectivity (Fig. 1).11,12 It is anticipated that with
improvements in peptide synthesis and recombinant technologies for pro-
ducing selenocysteine (Sec)-mutants of peptides the proposed 77Se-NMR
based methodology will become more generally applicable. Interestingly,
substitution of disulfide by diselenide bonds usually has no significant effect
on biological activity.
Lately Poppe et al. have proposed a new, powerful approach (PADLOC),
based on the combined use of intercysteine NOE pattern and cysteine side
chain conformation obtained from the vicinal proton-proton coupling
constants.10 The pattern of disulfides from the local constraints (PADLOC)
is deduced with the use of Bayesian probability approach. The robustness of
the proposed methodology has been demonstrated by several examples.

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4 NMR parameters related to structure

Disulfide-rich peptides are generally difficult to crystallize therefore nuclear
magnetic resonance (NMR) is by far the most commonly used technique
for studying their structures. The general principles of determining the
structures of peptides and proteins by NMR have been reviewed by several
excellent textbooks and review articles.
In short, NMR structure determination is generally based on a collection
of structural constraints, including inter-proton distances, dihedral angles

and hydrogen bonds, which are next converted to three-dimensional
structural constraints using computational methods.
Chemical shifts are highly sensitive to peptide and protein structure.13
Deviations of the observed chemical shifts from those for random coils, i.e.,
the so-called secondary chemical shifts, are valuable indicators of secondary
structure propensities. Marsh and co-workers developed a method to
evaluate the fraction of a- or b-structures using a combination of the
secondary shifts of different nuclei, referred to the secondary structure
propensity (SSP) score.14
Nuclear Overhauser effects (NOEs) are powerful constraints used for
structure determination of peptides and proteins, providing long-range
distance constraints on the overall topology and short-range distance con-
straints on the local secondary structures. In general, however, only few
NOEs are observed in small, flexible peptides because the polypeptide chain
fluctuates, and conformations with well defined secondary structures are not
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significantly populated.
Although the focus of this concise review is on the structure, and thereby,
to highlight the structural diversity of small disulfide-containing peptide and
proteins, however, it is important to emphasize that NMR can also provide
information about molecular dynamics relevant to binding interactions. In a
current work, NMR relaxation and relaxation dispersion data in combin-
ation with molecular dynamic simulations were used to probe the solution
dynamics over a wide range of time scales (picoseconds to microseconds) of a
40-residue disulfide rich peptide, psalmotoxin-1 isolated from spider venom.
The results of this complementary experimental and theoretical approaches
revealed that the key pharmacophore residues located in a b-hairpin loop
undergo significant motion in solution, thus providing new insights into the
dynamic-driven molecular basis of interaction.15
5 Antimicrobial peptides (AMPs)

Harmful microbial infections in humans are more widespread in the last
decades and they threaten patients suffering from immunosuppressive dis-
eases (e.g. AIDS). In some cases, – when chemotherapy is applied – the
patient’s life cannot be effectively prolonged because of the often lethal
fungal infections. Therefore, development of new antifungal drugs is urgent
and challenging, even because hosts and invading fungi may have similar
cellular, physiological and metabolic properties. Novel antimycotics that
target specific fungi selectively and without side effects are requested.
During the last two decades a broad range of antimicrobial peptides and
proteins (AMPs) from different organisms ranging from bacteria to humans

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have been isolated and characterised. The number of known AMPs with
antifungal activity is around 600. It is well established that NMR
spectroscopy is a favourite tool for disclosing structure and dynamics of
peptides and small proteins.
The increasing bacterial resistance to conventional antibiotics has led to
the need for alternative therapies. With a large spectrum of activity against
bacteria, viruses, protozoa, and cancer cells, high efficiency and low toxicity,
antimicrobial peptides (AMPs) are a very promising alternative to con-
ventional therapies. AMPs are short (typically less than 50 amino acid
residues), cationic and have an amphipathic character, but there are also
hydrophobic peptides and exist in almost all organisms as part of their
innate immune system.16–23 Many of the antimicrobial peptides have
characteristic network of disulfide bonds.
In general, the AMPs present great variability in both sequence and
structure. Based on their secondary structure they can be classified into
three major groups: linear peptides forming a-helical structure in contact
with membranes; disulfide-containing peptides with predominantly b-
structural elements; and linear (or extended) non-a-helix-forming peptides.
Most of the AMP structures known at present were solved by NMR
spectroscopy and are available in the Antimicrobial Peptide Database, APD
(http://aps.unmc.edu/AP/main.php).24,25
Their mode of action – antimicrobial activity – involves their direct or
receptor-mediated interaction with microbial membranes. However, due
to the diversity of antimicrobial peptides, it is not likely that there is one
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unique mechanism for their mode of action. Therefore to improve their

activity and to reduce their toxicity, it is important to understand their
mechanism of action. Powerful biophysical techniques, such as fluor-
escence spectroscopy methodologies, circular dichroism spectroscopy,
atomic force microscopy, nuclear magnetic resonance spectroscopy, in-
frared spectroscopy and surface plasmon resonance provide useful tools
to obtain insights in details of the pertinent peptide-membrane
interactions.23
6 NMR of AMPs
The majority of the antimicrobial peptides are small and flexible, and
therefore are not suitable for crystallization. However, high resolution
NMR spectroscopy serves as an outstanding technique that can be utilized
for structural characterization of antimicrobial peptides in solution or
bound to membrane mimetics.26 In general, standard two-dimensional
proton NMR methods combined with structure calculations provide suf-
ficient information to characterize their high resolution three-dimensional
structures in aqueous solution, in detergent micelles or in membrane mi-
metic organic solvents. NMR studies of antimicrobial peptides have re-
cently been reviewed by Haney and Vogel.26
Many of the antimicrobial peptides which form a b-sheet are cyclic
peptides with the ring structure formed and stabilized by disulfide bonds,
as in the case of the tachyplesins, protegrins, lactoferricin and of the
large group of defensins. The macrocyclic antimicrobial peptides with

Fig. 2 The NMR derived structures of the prototypical Möbius, bracelet and trypsin inhibitor
cyclotides, kalata B1, cycloviolacin O1, and MCoTI-II, respectively show the fold of the dif-
ferent subfamily members. (Reprinted with permission from N. L. Daly, K. J. Rosengren and
D. J. Craik, Advanced Drug Delivery Reviews, 2009, 61, 918. Copyright & 2009 by Elsevier Ltd.)
characteristic cystine knot (CK) motif, such as circulin A and B27,28 and
several other members of the plant-derived cyclotide family form another
fascinating group of disulfide bond containing antimicrobial peptides with
more than 50,000 members.29–31 The unique structural features and range of
biological activities of the head-to-tail cyclized cyclotides with knotted
topology of their three disulfide bonds, referred to as cyclic cystine knot
(CCK) structural motif and their potential in drug development has recently
be reviewed.3,31,32 The NMR derived structures of the prototypical Möbius,
bracelet and trypsin inhibitor cyclotides are shown in Fig. 2.
Recent biophysical studies have established that for different cyclotides a
range of different binding modes seems feasible. The bioactive face com-
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prising of the conserved Glu residue and the adjacent hydrophilic patch is
responsible for the insecticidal activity, while their surface exposed hydro-
phobic patch plays important role in membrane binding.32 A full list of
cyclotide sequences is available on Cybase (http://www.cybase.org.au)
database managed by D. J. Craik and his group at the Institute of
Molecular Bioscience, Brisbane, Australia.
The cystine knot structural motif with one disulfide bond threaded
through the other two forming embedded ring is also present in several
toxins and protease inhibitors and conveys exceptional high rigidity and
stability to these peptides. The KNOTTIN database (http://knottin.
cbs.cnrs.fr) provides comprehensive data on the members of the cystine-
knot structural family.33,34
The naturally stable disulfide-rich scaffolds can be utilized for re-
engineering bioactive peptides, and they have been proved to be invaluable
in the design of peptide-based drugs with enhanced stability and oral ac-
tivity.35 The scheme of the miniprotein scaffold approach to peptide-based
drug design is illustrated in Fig. 3.
Recent data indicate that the cystine-knot scaffold can also be utilized for
generating engineered miniproteins with given binding characteristics to a
broad range of different target proteins and so they are promising candi-
dates for various diagnostic applications.36 The y-defensins, the only known
ribosomally synthesized cyclic peptides in mammals show antimicrobial
activities and their highly conserved structure makes them a promising
scaffold for peptide drugs. Similar to the cyclotides, their solution
structure features a cyclic peptide backbone and three disulfide bonds, but

Fig. 3 Panel A illustrates potential sources of target epitopes, from fragments of proteins,
from bioactive peptides, or from phage display. Panel B depicts a range of disulfide-rich
frameworks, such as SFTI-1, cyclotides and theta-defensins. Panel C shows the bioactive
epitopes grafted into the stable frameworks. (Reprinted with permission from D. J. Craik, D. P.
Fairlie, S. Liras and D. Price, Chem. Biol. Drug Des., 2013, 81, 136. Copyright & 2013 by John
Wiley & Sons).
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Fig. 4 (top) Cyclic cystine ladder (CCL) motif shown in stick representation. (bottom)
Diagrammatic representation illustrating how the CCL could be applied as a stable core for
the development of peptide drugs. White circles indicate how different amino acids
could be used onto the framework. The black line represents how the turn regions could be
expanded or modified. (Reprinted with permission from A. C. Conibear, K. J. Rosengren, P. J.
Harvey and D. J. Craik, Biochemistry, 2012, 51, 9718. Copyright & 2012 by American Chemical
Society).
the y-defensins have their three disulfide bonds arranged differently, in a

cyclic cystine ladder (CCL) motif. The CCL motif (Fig. 4) features an
extended b-sheet region joined by two b-turns and braced by three parallel
disulfide bonds.37 NMR-based structural and dynamic characterization of
three y-defensins, RTD-1, BTD-2, and HTD-2, has shown that all three
peptides have well-defined, highly constrained backbone structures with
relatively flexible side chains exposed to solvent. Although it is not proven

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yet, but it seems to be probable that the concentration-dependent, reversible

self-association observed in all three peptides could play a role in their mode
of action.
The solution structure and oligomerization state of the oxidized form of
human defensin 5 (HD5ox) and its disulfide mutant peptides with broad-
range of antimicrobial and antiviral activity have been assessed by NMR
and analytical ultracentrifugation experiments. The NMR solution
structure of HD5ox exhibits a three-stranded b-sheet with three disulfide
bonds characteristic of a-defensins. The reported extensive biophysical

investigations demonstrate the dynamic and environmentally sensitive
oligomerization behaviour of HD5ox in solution. The propensity of
defensins to self-associate is considered to be important for bacterial
membrane disruption and antiviral activity. The presented results provide
important insights into the structure-activity relationships of defensins and
highlight the importance of both electrostatic and hydrophobic interactions
for their antimicrobial activity.38
The structure and mechanism of action of a new fungal defensin, eurocin
isolated from the fungus Eurotium amstelodami, active against a range of
Gram-positive human pathogens has been recently described. Eurocin
consists of 42 amino acids, forming a cysteine-stabilized a/b-fold (CSab-
motif), similar to other fungal and invertebrate defensin structures published,
and so, it belongs to the group of antimicrobial peptides with b-sheets sta-
bilized by two or more disulfide bonds.39 In vivo and in vitro binding studies
revealed that the eurocin inhibits peptidoglycan biosynthesis of Gram-
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positive bacteria without comprising membrane integrity.

In recent study a novel antifungal peptide EcAMP1 from seeds of
barnyard grass (Echinochloa crus-galli) with strong antifungal action toward
species of the Fusarium genus has been described and characterized.40
EcAMP1 is the first example of AMP with a novel disulfide-stabilized
a-helical hairpin fold (Fig. 5).
Intriguingly, its mode of action differs from other known AMPs, there-
fore, EcAMP1 may present an attractive molecular scaffold for the design of
novel antimicrobials. Similar a-helical hairpin fold stabilized by two di-
sulfide bridges has been found in a novel trypsin inhibitor BWI-2c obtained
from buckwheat (Fagopyrum esculentum) seeds, representing a new family
of protease inhibitors with this rare a-helical fold. It is interesting to men-
tion that EcAMP1 and BWI-2c share little (B20%) amino acid sequence
identity.41 The structure of a small (55 aa) and basic (due to thirteen lysines)
antifungal protein PAF from P. chrysogenum was disclosed42 by NMR.
PAF comprises five b-strands forming two orthogonal b-sheets similar to
the greek key supersecondary structure found in the homologous AFP. 15N
auto-relaxation, NOE and 15N-1H CSA/DD relaxation interference
(cross-correlated cross-relaxation) supported the secondary structure and
model-free analysis suggests a rigid structure on ps/ns timescale. However,
the disulfide pattern could be unequivocally determined neither in AFP,
nor in PAF. Later, chemical synthesis43,44 did help to corroborate the
putative ‘abcabc’ disulfide pattern. According to recent studies,45 the
hydrophobic DewA from Aspergillus nidulans is a highly surface-active
protein that spontaneously self-assembles into amphipathic monolayers at

Fig. 5 Spatial structure of EcAMP1. Ribbon representation of the 20 best CYANA structures
superimposed on the backbone atoms of residues 7–30. Disulfide bridges are displayed as
yellow sticks. (Reprinted with permission from S. B. Nolde, A. A. Vassilevski, E. A. Rogozhin,
N. A. Barinov, T. A. Balashova, O. V. Samsonova, Y. V. Baranov, A. V. Feofanov, T. A.
Egorov, A. S. Arseniev and E. V. Grishin, J. Biol. Chem., 2011, 286, 25145. Copyright & 2011,
by the American Society for Biochemistry and Molecular Biology).
hydrophobic/hydrophilic interfaces. These monolayers comprise fibrils that

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may form amyloid. DewA has four disulfide bonds, exhibits two distinct
conformers in solution that are not exchanging with each other at seconds
NMR time scale and one of them (A) forms dimer. The structure of DewA
shows a sharp separation of charged and uncharged residues on opposing
surfaces of the protein.
The solution structure, backbone dynamics and binding properties of
Aurelin, a 40-residue cationic antimicrobial peptide (CAMP) isolated from
jellyfish Aurelia aurita have been reported. Aurelin represents the first
antimicrobial peptide having a compact globule (ShKT fold – not observed
for any previously identified antimicrobial peptide), featuring one 310-helix
and two a-helical regions cross-linked by three disulfide bonds.46
Structural and functional characterization of a human liver expressed,
40-residue cationic antimicrobial peptide, LEAP-2 with a novel fold
(not observed in other CAMPs) has been presented. The NMR solution
structure is characterized by a compact central core, comprising a b-hairpin
and a 310-helix that are stabilized by two disulfide bonds and an intricate
network of hydrogen bonds. Comprehensive binding studies confirm that
the hydrophobic N-terminal and cationic core regions are essential for
membrane binding. Interestingly, the reduced forms of LEAP-2 show
similar membrane affinity, suggesting that the disulfide bonds are not a
prerequisite for membrane binding.47
7 Peptide-based toxins
An excellent overview of the structural features of disulfide-rich peptide
toxins produced bacteria, plants and animals has recently been reported by

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48
Rosengren et al. The important role of NMR in determining the structural
and dynamic features of a wide range of toxins and in providing insights
into their structure-function relationships has been demonstrated by several
examples.
Ion channel blocker peptide toxins
The inhibition of ion channels (such as potassium, sodium, calcium,
chloride, etc.) by peptide toxins from animal venoms is a subject of broad
interest for its physiological and therapeutic applications. There are several
potential sources of these channel-blocking toxins, including snakes, spi-
ders, scorpions, sea-anemones and cone snails.49
Potassium channel blocker peptide toxins
Potassium channels are involved in the maintenance of resting membrane
potential, control of cardiac and neuronal excitability, neurotransmitters
release, muscle contractility and hormone secretion. Most potassium
channel blocking peptides purified from scorpion venoms contain 30–40
amino acids with three or four disulfide bridges and belong to the well
characterized a-KTx subfamily. This group of short-chain peptides blocks
voltage-gated Kþ channels (Kv) mostly by interacting with the channel
pores.50 Binding is governed by electrostatic interactions between pore
residues in the channel and the positively charged lysine residues in the
peptides.51 In a recent study an efficient Pichia pastoris expression system
has been proposed for production of Kv1.3 channel blockers and other
disulfide-rich venom peptides in quantities large enough facilitating com-
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prehensive structure-function studies.52

The solution structure of a novel 32 residue scorpion peptide, BTK-2
isolated from the venom of red Indian Mesobuthus tamulus, belonging to
the a-KTx subfamily of voltage-gated potassium channel inhibiting
peptides has been determined using heteronuclear NMR methods. The
overall structure of BTK-2 comprises an N-terminal a-helix connected to a
double-stranded anti-parallel b-sheet, forming the well-known, three
disulfide stabilized a–b–b motif found in other a-KTx scorpion toxins. But
interestingly, BTK-2 exhibits a unique structural feature, namely, its
structure lacks the ‘functional dyad’ motif (comprising an aromatic and a
cationic residue) known to be essential for the majority of a-KTx scorpion
toxins.53,54
The recently identified new members of the acidic scorpion toxin family
with unique structural features provide further examples for the occurrence
of diverse scaffolds of peptide toxins with common cysteine framework.55
At this point it is important to note that in general, fine structural al-
terations (i.e. subtle changes in secondary structure elements and/or the
relative orientation of side chains and/or the dynamic properties of the
molecule) may have impact on channel recognition. A comparative docking
protocol in HADDOCK has been developed for the prediction of peptide
selectivity profiles of various a-KTx scorpion toxin families versus Kv1.1,
Kv1.2 and Kv1.3. The authors presume that the proposed docking strategy
can be broadened to arbitrary ion channels of interest with known structure,
especially in cases where a critical binding motif exists to improve the
effectiveness of docking.49 Using exclusively computational methods, a

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mutant, S9R11 of OSK1, a toxin extracted from scorpion venom has been
devised which blocks Kv1.3 with an IC50 of 0.2 pM, one order of magnitude
more potent than the best blocker of Kv1.3 known previously.56 These
studies demonstrate that computational tools alone can be exploited in
designing novel polypeptide blockers of ion channel with improved potency
and selectivity.
b-KTx toxins comprising 61–75 amino acid residues represent another
group of potassium channel toxins. These peptides contain six cysteines
forming three disulfide bridges and present two structural domains: a pu-
tative a-helical N-terminus and a Cys-rich C-terminus with the consensus
signature of CSa/b-motif. Recently, the three-dimensional structure of new
members of the b-KTx subfamily57 present in some scorpion venoms have
been characterized by computational modelling and refined by molecular
dynamic simulations, and their binding mode to Kv1.2 channel has been
assessed by docking simulations.51
Two Kþ channel blocking peptides, k-BUTX-Tt2b and Ts16 belonging to
a new structural group of scorpion venom peptides, isolated from Tityus
venoms have been reported. Based on combined NMR and computational
studies it has been proved that these peptides display an unconventional
disulfide pattern, accompanied by a novel cysteine-stabilized helix-loop-
helix fold (CSa/a). These are the first scorpion venom CSa/a peptides, which
contain three disulfide bonds, and so further exemplifying the structural
versatility and complexity of scorpion peptides.58
Sea anemone venom is also a rich source of peptide toxins59,60 that
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function as modifiers of voltage-gated sodium and/or potassium channels,

or acid-sensing ion channels.61 A novel peptide, APEKTx1 isolated
from the sea anemone Anthopleura elegantissima, containing 63 amino acids
cross-linked by 3 disulfide bonds has been shown to have unique
dual functionality, including trypsin inhibitory activity and potent and
selective inhibition of Kv1.1 channels. Because of this unique feature,
APEKTx1 might be a lead compound in the development of novel ther-
apeutical agents.62
Calcium channel blocker peptide toxins

Conotoxins from marine cone snails have been proven to be potential
therapeutic candidates in neuroscience for the treatment of chronic and
neuropathic pain. The majority of these toxins are small, disulfide-rich
peptides typically containing 10–40 amino acid residues. Due to their
high structural diversity, the conotoxins have been divided in several
superfamilies with characteristic arrangement of cysteine residues. These
superfamilies are further divided into pharmacological families (Fig. 6)
based on their ion channel and receptor targets.63,64
As conotoxins potently interact with a range of ion channels, transporters
and other receptor sites implicated in disease states they are of pharma-
ceutical interest.
The o-conotoxins from the O superfamily represent the largest and
therapeutically most significant voltage gated calcium channel (VGCC)
targeting family. The o-conotoxins are typically basic peptides comprising
24–31 amino acid residues. The six cysteine residues form a cystine knot

Fig. 6 Structures of representative conotoxins from the main pharmacological families.

Disulfide bonds are shown in red. (A) a-Vc1.1, (B) d-TxVIA, (C) k-PVIIA, (D) m-GIIIA,
(E) w-MrIA and (F) o-MVIIA. Positive (blue online), negative (red online) and hydrophobic
(green online) residues are highlighted on the surface profiles. (Reprinted with permission from
01/12/2013 14:44:18.
S. Vink and P. Alewood, Br. J. Pharmacol., 2012, 167, 970. Copyright & 2012 by The British
Pharmacological Society).
motif which conveys thermal, chemical and mechanical stability to the

peptide.63 The first conotoxin-based therapeutic for the treatment of
long-term neuropathic pain is a synthetic version of o-conotoxin MVIIA
(ziconotide, PRIALT).
Recently, further o-conotoxins, CVIE and CVIF, were identified from
C. catus venom glands and were found to be potent, selective and reversible
inhibitors of Cav2.2.65,66 Detailed analysis of structure-activity relationships has
identified critical basic residues and structural motifs essential for VGCC in-
hibition, assisting the design of novel o-conotoxin therapeutics with improved
potency and selectivity and, consequently, with fewer unwanted side effects.
Lately, by means of backbone cyclization, a cyclic analogue of the
a-conotoxin Vc1.1 (Fig. 7) has been developed with improved stability and
oral activity. Remarkably, the engineered cyclic analogue, cVc1.1 proved to
be more than two orders of magnitude more potent than the current leading
drug for neuropathic pain.3,67–70
Kurtoxin, a 63-amino acid polypeptide isolated from the venom of the
South African scorpion Parabuthus transvaalicus has been identified as the
first peptide ligand known to act on Cav3 (T-type) voltage-gated Ca2þ
channels. The NMR structure of kurtoxin closely resembles those of
scorpion a-toxins, characterized by the so-called ‘‘cysteine-stabilized a-helix
and b-sheet (CSab)’’ motif, comprising an a-helix, three b-strands, and
several turns stabilized by four disulfide bonds (Fig. 8).71 Regions of

Fig. 7 Strategy for the cyclization of the a-conotoxin Vc1.1. a) The sequence and the sche-
matic diagram of the 3D structure of Vc1.1 with the classical a-conotoxin fold. The N and C
termini are separated by ca. 12 Å. b) The sequence and the proposed cyclization strategy in
which the N and C termini are joined by a linker sequence of six residues. (Reprinted with
permission from R. J. Clark, J. Jensen, S. T. Nevin, B. P. Callaghan, D. J. Adams and D. J.
Craik, Angew. Chem. Int. Ed., 2010, 49, 6545. Copyright & 2010 by WILEY-VCH Verlag
GmbH & Co. KGaA, Weinheim).
01/12/2013 14:44:18.
Fig. 8 Ribbon structure of kurtoxin illustrating the location of the b-strands (cyan), a-helix
(red and yellow), and disulfide bonds (numbered ball and stick). (Reprinted with permission
from C. W. Lee, C. Bae, J. Lee, J. H. Ryu, H. H. Kim, T. Kohno, K. J. Swartz and J. Il Kim,
Biochemistry, 2012, 51, 1862. Copyright & 2012 by American Chemical Society).
kurtoxin with distinct surface properties which may be accountable for the
selective interactions have been also assigned.
Sodium channel blocker peptide toxins

All known long-chain mammalian scorpion peptides are composed of 59 to
70 amino acid residues and are specific for voltage-gated sodium, Nav
channels. They have a highly conserved scaffold formed by an a-helix and a
three-stranded b-sheet structure, generally stabilized by four disulfide

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72
bonds. The solution structure and structure-activity relationships of na-
tive and recombinant expressed toxin CssII from the venom of the scorpion
Centruroides suffusus suffuses, belonging to the class of b-type scorpion
neurotoxins have been reported. The amino acid residues that could be
crucial for the specificity and affinity for the various Nav isoforms have been
identified, thereby providing useful data for the development of therapeutic
antibodies for clinical treatment of scorpion envenomations.73
Based on two naturally occurring m-conotoxins, KIIIA and BuIIIC, a
series of novel, synthetic, and bioactive compounds, comprising 12-16 amino

acids with only two disulfide bridges has been designed. These miniaturized
peptides potently and selectively block voltage-gated sodium channels (Navs),
providing ideal starting platform for further development of conotoxin-based
therapeutics to treat Nav-related diseases.74 Interestingly, the NMR structure
of the most promising peptide revealed that the peptide adopts a flexible
structure without the typical a-helix observed in m-conotoxins, suggesting that
the a-helix is not strictly essential for the biological activity.
Peptide toxins with insecticidal activity

The inhibitor cystine knot, ICK-fold (or also called as the knottin fold),
consists of a double- or triple stranded antiparallel b-sheet stabilized by
three or more disulfide bonds, forming a b-hairpin and a cystine knot and is
observed only in a few scorpion toxins. In a recent study, the solution
structure of a 36-residue insecticidal peptide toxin, LalT1 isolated from the
venom of the scorpion Liocheles australasiae has been established by
01/12/2013 14:44:18.
heteronuclear NMR spectroscopy using 15N and 13C-labelled LalT1. The

NMR data have confirmed that LalT1 adopts an ICK-like fold which is
stabilized with only two disulfide bonds in contrast to the three-disulfide
inhibitor cystine knot (ICK) motif, suggesting that no additional disulfide
bond is required to adopt the ICK-(like) fold. Based on mutational studies,
two arginine residues, Arg13 and Arg15, forming a basic patch were found to
be important for the insecticidal activity.75 This two-disulfide containing
ICK-like fold has been observed in another insecticidal scorpion peptide,
U1-LITX-Lw1a which shows 92% sequence identity to LalT1.76 The
current work of Smith et al. supports the hypothesis that this unique two-
disulfide stabilized fold (referred also as to disulfide-directed b-hairpin,
DDH fold) is the evolutionary precursor of the ICK motif.76
In a recent study two novel conotoxins, im23a and im23b, from venom of
Conus imperialis, containing a unique cysteine framework, called frame-
work XXIII with an unusual pattern of disulfide bridges (I-II, III-IV, V-VI)
have been identified and characterized. The solution structure of im23a
reveals that this peptide adopts a unique helical hairpin fold, not known for
any previously described conotoxin, thus extending the structural repertoire
of cone snail venom peptides.77
Two novel conotoxins belonging to a new subfamily of the A-superfamily,
identified from Conus pulicarius and Conus tessulatus have been characterized.
Both peptides have four separate cysteines in framework 14 forming two
disulfide bonds with the C1-C3, C2-C4 connectivities identical to that of
a-conotoxins, but with a remarkably different loop spacing pattern
(CX10CX1CX3C) containing four prolines in loop 1. These unique structures

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belonging to the a1-conotoxin subfamily expand the structural diversity

of conotoxins.78 The biological target of these new toxins is not yet identified.
8 NMR solution structures of other disulfide proteins

Small cysteine-rich proteins are found in most living organisms and
play vital roles as hormones, growth factors, ion channel modulators and
enzyme inhibitors in various biological pathways. In a recent review,
structure and function of secreted human cysteine-rich mini-proteins, which

form a unique set of protein frameworks and folds stabilized by intra-
molecular disulfide bonds, have been described and classified, and their
molecular targets have been identified.1 Structure/dynamics of the EF-hand
human apo-S100A1 protein was thoroughly studied by a Polish group79 and
the 15N relaxation dynamics was evaluated using the two-time scale ap-
proach with fully anisotropic reorientation. RcsF plays a role in the regu-
lation of capsule synthesis (rcs) phosphorelay system that is important to
understand the RcsC-D-A/B signalling cascade, which regulates more than
100 genes. It was found80 by solution NMR methods that the disulfide
network of Cys-74/Cys-118 and Cys-109/Cys-124 correlate pairwise with
the activity of RcsF. The authors also monitored the folding stage of RcsF
in vivo in E. coli cells (in-cell NMR) or immediately after cell lysis. The
expressed constructs folded neither in-cell nor post vivo in reducing en-
vironment. Therefore, RcsF needs to be correctly folded in the E. coli
periplasm. Functional role of two interhelical disulfide bonds in human
01/12/2013 14:44:18.
Cox17 protein, a mitochondrial copper chaperone, has been studied.81 It

turned out that the inner disulfide bond Cys-36/Cys-45 stabilizes interhelical
hydrophobic interactions of hCox17 and produces a single conformational
state playing thereby a key role in the CHCH fold, while the outer disulfide
bond ensures the copper binding site environment.
There are peptide agonists of avb6 integrin, a heterodimeric glycoprotein
that is overexpressed in cancer cells. A recent NMR investigation82 of four
21-mer avb6 peptide ligands was carried out with and without tri-
fluoroethanol. The work demonstrates that peptide disulfide bond cyclisa-
tion design changes the structure and dynamics properties of the ligand that
in turn affects the affinity and specificity toward avb6. To prove this, 15N
relaxation data were analyzed both with model-free and reduced spectral
density mapping techniques.
The solution conformation of the 36 residue sex peptide (SP) from
Drosophila melanogaster was determined83 by standard solution NMR
methods. One of its receptors is a G-protein coupled receptor, namely the
sex peptide receptor (SPR). The C-terminal disulfide-bonded loop of SP
populates helical turns or nascent helical structure. SP is a dynamic peptide
that lacks a compact folded structure in solution, though the nascent
structure becomes more evident in water/TFE mixtures.
Using a model peptide it was shown84 that interstrand cystine cross-links
can both stabilize and define the extent of parallel b-sheet secondary
structure in designed peptides and proteins.
Protein maturation in live human cells was observed by monitoring the
redox state of SOD1 using in-cell NMR.85 1H-15N SOFAST-HMQC

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spectra were acquired to detect cysteine signals on human cells co expressing

[15N] cysteine-labelled human superoxide dismutase 1 (SOD1) and copper
chaperone for SOD1 (CCS) in Zn(II)-supplemented medium before and
after incubation with Cu(II).
The role of cysteine residues in heme binding to human heme Oxygenase-
2 was explored86 by the 2D 1H-13C HSQC method. Chemical shifts showed
that none of the cysteines coordinates firmly to the heme iron atom. In the
oxidized state of the apoprotein, heme is bound 2.5-fold more tightly than in
the reduced state.

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) T1
contains four disulfide bonds, two of which (C4–C41 and C8–C37) stabilize
the reactive site loop. Intentional single disulfide bond depletion in the form
of C4A/C41A mutant has high inhibitory activity against trypsin, but the
C8A/C37A-T1 mutant is inactive, though both structures are similar to wild
type T1. According to 15N NMR relaxation data analyzed by the model-free
method, the active compound has similar dynamics all over the sequence,
including the reactive-site loop. To the contrary, the inactive mutant ex-
hibits enhanced dynamics at the reactive-site loop at ps/ns timescale.87
Solution structure and dynamics of a monomeric analogue of proinsulin
was investigated88 by heteronuclear NMR. DKP-proinsulin is 8–10-fold
more active than the wild-type. It contains a native-like insulin moiety (A
and B-domains) and a tethered connecting (C) domain. Three evidences
proved that the C-domain is disordered in contrast to the A and B helical
domains: there are no long-range NOE contacts, 15N-1H heteronuclear
01/12/2013 14:44:18.
NOEs are decreased, and the 13Ca, 13Cb secondary chemical shifts are
small. Flexibility explains why proinsulin is reluctant against crystallization.
There are 20 known members of the human protein disulfide isomerase
(PDI) family and their main role is to assist in the formation of the native
disulfide pattern that is essential for the right folding of disulfide proteins. In
a recent structure/dynamics NMR study89 of human ERp27 (endoplasmic
reticulum protein 27.7 kDa, a PDI homologue) extensive interdomain
flexibility was detected by conventional 15N relaxation methods. In con-
clusion, both ERp27 and PDI have considerable conformational flexibility
in solution and known crystal structures possibly captured particular
conformations from an ensemble.
A gene from apple fungus, cin1 (cellophane-induced 1), encodes a
cysteine-rich secreted protein of 523 residues with eight imperfect tandem
repeats of B60 amino acids. The cin1 sequence has no homology to known
proteins. NMR structure of the first two repeat domains of Cin1 (Cin1-
D1D2) has been determined90 and no structural homologues were found.
Each domain forms a helix–loop–helix (HLH) motif as part of a three-helix
bundle, and is stabilized by two intra-domain disulfide bonds. It was shown
by 15N relaxation that the short inter-domain linker has large amplitude
motions that results in reciprocal domain–domain mobility.
The sweet protein brazzein, a member of the Csba family, contains four
disulfide bonds that are responsible for high thermal stability. Changing the
temperature it was shown91 that brazzein undergoes a reversible con-
formational change between 37 and 3 1C with a midpoint about 27 1C that
changes the orientations and side-chain hydrogen bond partners of Tyr8

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and Tyr11. To test the functional significance of this effect, NMR saturation
transfer proved a stronger interaction at 7 1C than at 37 1C between brazzein
and the amino terminal domain of the sweet receptor subunit T1R2. The
low temperature conformation with altered orientations of two loops may
be critical for the sweetness.
9 Folding and dynamics of disulfide proteins

Chang and Ventura edited a recent book92 on the folding of disulfide

proteins including eleven relevant chapters, as follows:
Oxidative Folding: Coupling Conformational Folding and Disulfide
Formation; The Case of Oxidative Folding of Ribonuclease A: Factors
Impacting Fold Maturation of ER-Processed Proteins; Cystine Knot
Folding in Cyclotides; In Vitro Folding of Single/Double Chain Insulins
and Related Proteins; Unfolding and Refolding of Disulfide Proteins Using
the Method Disulfide Scrambling; Oxidative Protein Folding with Small
Molecules; Protein Disulfide Isomerase and the Catalysis of Oxidative
Protein Folding; Allosteric Disulfide Bonds; The Problem of Expression of
Multidisulfide Bonded Recombinant Proteins in E. coli; NMR-Spectro-
scopic Investigation of Disulfide Dynamics in Unfolded States of Proteins;
A Half-Century of Oxidative Folding and Protein Disulphide Formation.
A minireview on the chemical methods for investigating disulfide-coupled
peptide and protein folding has been presented.93 Expressed protein
ligation, a combination of native chemical ligation and an intein-based
01/12/2013 14:44:18.
approach, permit specifically labelled proteins to be produced for protein

folding studies. Regio-selective formation of disulfide bonds using chemical
procedures has also been established. This strategy is very useful for the
study of disulfide-coupled protein folding. Protein folding can be acceler-
ated by using chemicals. According to in-vitro experiments, competition
between physical, conformational folding reactions and chemical, thiol–
disulfide exchange reactions plays a role during folding as summarized in
another minireview.94
The diversity of protein folding mechanism was demonstrated by pre-
senting the divergent folding pathways of two structurally homologous
Kunitz-type protease inhibitors, bovine pancreatic trypsin inhibitor (BPTI)
and tick anticoagulant peptide (TAP).95,96 The two extremes are the
framework model and the hydrophobic collapse model, while the third,
nucleation–condensation model considers the interdependence of secondary
and tertiary structures during folding. A landmark paper was published by
Shaw et al.97 on the millisecond-long molecular dynamics of BPTI and the
detailed results of the calculations are accessible. The Authors disclosed
distinct conformational clusters, and demonstrated that BPTI makes con-
formational excursions between five conformational ‘‘basins’’. On the basis
of the trajectories, the 15N chemical shifts and exchange line broadening was
calculated.98 The results were in agreement with earlier NMR finding99 about
the 10–100 ms timescale isomerisation of the C14-C38 disulfide bond in BPTI.
The modulation of structure and dynamics by disulfide bond formation in
the unfolded states of disulfide depleted hen egg white lysozyme (HEWL)
was studied in detail by Schwalbe.100 The native form of HEWL contains

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two SS bonds in a, one in b-domains and one inter-domain linkage, al-

together four SS bonds (4SS). It was shown earlier by monoclonal anti-
bodies in the 2SSaa mutant that indeed, the a-domain is formed first. Native
b-domains can be detected only in 3SS variants. The fourth SS bond es-
tablishes the inter-domain linkage and structure and these findings were
supported by NMR using 2SS mutants. The a-domain of 2SSa-HEWL
shows a native fold at pH 2.0 in the absence of urea, while 2SSb-HEWL is
unfolded under the same conditions. Schwalbe studied the permanently
trapped early single disulfide (1SS) intermediates, folded during the oxida-
tive refolding of HEWL. They used combined NMR relaxation, SAXS and
CD techniques, and it became apparent that all single disulfide mutants as
well as 0SS-HEWL are mostly unfolded, but not random coil, exhibiting
some a-helix propensity. Compactness of 1SS mutants were compared by
SAXS, DOSY and are in good agreement with the Rg/Rh radii correlation
of flexible meccano simulations. Backbone dynamics of the mutants were
efficiently disclosed using reduced spectral density mapping of 15N relax-
ation data. Two 1SS mutants exhibited very similar dynamics pattern to
unstructured 0SS, while the other three were in between 0SS and the native
4SS structure. Residues with chemical exchange contributions are around
residues close to the disulfide bonds. Rotation around the disulfide bond
can be described as a two-site exchange process due to two main con-
formations around the S-S bond: w3= 901. In summary, there is an
interplay between hydrophobic collapse and disulfide bond formation in
single disulfide mutants of HEWL.
01/12/2013 14:44:18.
Three variants of HEWL including native and partly ester-linked HEWLs

were analyzed by MD calculations, and the predicted NMR observables
were compared to experimental results.101
Japanese authors reinvestigated102 the oxidative folding of reduced HEWL
using the trans-3,4-dihydroxyselenolane oxide reagent DHSox at 5–45 1C
temperatures in the presence of 2M denaturing agent (Urea). 1SS, 2SS, 3SS,
and 4SS intermediates with 1–4 SS linkages were obtained. The major folding
pathways could be adjusted just by changing the temperature.
An excellent review article of structural features, folding, binding and
stability of insulin, a helical rich mini-protein cross-linked by three disulfide
bridges, playing a central role in carbohydrate metabolism have recently
been published.103 NMR played a key role in disclosing the folding pathway
and disulfide pairing of insulin family. This small protein is fully equipped
with the secondary and tertiary structure elements of a functional protein
and as a model it is suitable to study folding intermediates with various
disulfide patterns. Insulin has two chains, A (21aa) and the B (30aa) and
three disulfide bridges (two inter-chain: A7-B7 and A20-B19, and one intra-
chain: A6-A11). The structure, stability and receptor binding of insulin
require the presence of all native disulfide bridges. NMR enabled to disclose
the insulin folding coupled to disulfide pairing, even at early stage. It turned
out that insulin may behave sometimes as a molten-globule. When insulin
binds to its receptor, a conformational change of insulin occurs at the
positions of two structural switches (Fig. 9).
Protein phosphatase of regenerating liver (PRL-1) was used as a model
system to investigate the effects of disulfide bond formation on the protein’s

Fig. 9 Cylinder models of insulin. (A) Structure of an inactive single-chain insulin: Cylinder
model of a 50-residue single-chain insulin analogue in which a peptide bond tethers LysB29 to
GlyA1. Although the crystal structure of this analogue (as a T3Rf3 zinc hexamer) is essentially
identical to that of wild-type insulin, its receptor binding activity is reduced by at least 1000-
fold. (B and C) Cylinder representations of insulin: The ‘‘closed’’ model of insulin (B) and the
proposed model of the active ‘‘open’’ conformation (C) show that partial detachment of the
C-terminal b-strand of the B-chain exposes the side chains of IleA2 and ValA3 (shaded; asterisk
in C) in a recognition a-helix. (Reprinted with permission from by Q. X. Hua, Protein & Cell,
2010, 1, 537. Copyright & 2010, Higher Education Press and Springer-Verlag Berlin
Heidelberg).
stability.104 It was shown that stabilization or destabilization of a protein

can be achieved in the folded state through the collaboration of specific,
local interactions.
01/12/2013 14:44:18.
Acknowledgements
Financial support from TÁMOP-4.2.2/A-11/1/KONV-2012-0025, OTKA
K 105459 and National Excellence Program, Szentágothai János Fellowship
is gratefully acknowledged.
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2643.

Relaxin and its role in fibrotic diseases
Linda Jiaying Chan,a,b Chrishan S. Samuel,a,c Frances Separovic,b
Mohammed Akhter Hossain*a,b and John D. Wade*a,b
DOI: 10.1039/9781849737081-00060
1 Human relaxin-2 (Relaxin)

Relaxin is a 6kDa pleiotropic peptide hormone first discovered for its

reproductive roles. It is a 53 amino acid peptide which has structural
characteristics common to the insulin/relaxin family members, comprising
of two chains and three disulfide bonds. In most mammals, it has long been
regarded as a hormone of pregnancy and is involved in remodeling the
connective tissue of the pelvic ligaments and female reproductive tract to
facilitate delivery of the young. However, relaxin was found to possess
additional roles in several non-reproductive processes which are centered
around its anti-fibrotic,1–3 vasodilatory and cardioprotective effects.4,5
Based on the latter two actions, human relaxin has recently emerged from a
successful Phase III clinical trial for the treatment of patients with acute
heart failure.6
The relaxin hormone is expressed as an inactive pre-prohormone which
encompasses an N-terminus signal sequence and a B-C-A chain structure.
The signal sequence aids the secretion of the pre-prohormone while the
interconnecting C-peptide which links both A- and B-chains facilitates
01/12/2013 14:44:29.
protein folding and the formation of the subsequent three disulfide bridges.
The conversion of the pre-prohormone into the prohormone occurs through
the co-translational loss of the N-terminus signal sequence. Subsequent
enzymatic cleavage in vivo produces the mature active two-chain (A-B)
heterodimeric peptide. The A- and B-chains are covalently linked by two
inter-disulfide bonds made by four conserved cysteine residues, two present
in each of the two chains, respectively. An additional intra-disulfide bond is
formed by two more conserved cysteine residues that are present within the
A-chain (Fig. 1). Human gene-2 (H2) relaxin is the major stored and cir-
culating form of human relaxin (that along with its species equivalent
relaxin peptide) interacts with two of the relaxin family peptide (RXFP)
receptors, RXFP1 and RXFP2 (formerly known as LGR7 and LGR87).
Both RXFP1 and RXFP2 receptors are class C leucine-rich repeat (LRR)-
containing G protein coupled receptors (GPCRs). The large extracellular
domain of these receptors consists of series of LRR strands forming a
parallel b-sheet linked to a LDLa module.8 While its cognate receptor is
RXFP1, relaxin is also able to cross-react with RXFP2, the native receptor
for the related peptide insulin-like peptide 3 (INSL3). The Arg-X-X-X-Arg-
X-X-Ile motif within the relaxin B-chain is essential for primary binding to
the leucine-rich repeats within the receptors that it binds to. A secondary
a
Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria
3010, Australia
b
School of Chemistry, The University of Melbourne, Victoria 3010, Australia
c
Department of Pharmacology, Monash University, Victoria 3800, Australia
E-mail: john.wade@florey.edu.au; akhter.hossain@florey.edu.au

01/12/2013 14:44:29.
Fig. 1 (A) Structural representation of the processing of the relaxin pre-prohormone to

mature hormone states. Conversion of the pre-prohormone into the prohormone state occurs
through the excision of the N-terminus signal sequence. The prohormone undergoes enzymatic
cleavage which involves the excision of the C-peptide resulting in a mature two chain hormone.
(B) Primary structure of the relaxin peptide. (C) Tertiary structure of the relaxin molecule
comprising of the A- and B-chains and the three disulfide bonds.
interaction responsible for receptor activation involves the A-chain of

relaxin and the transmembrane (TM) exoloops of the receptor.8 The potent
anti-fibrotic actions of relaxin are known to be manifested through the
RXFP1 receptor.9
2 Structure activity relationship (SAR) study and RXFP1

interaction mechanism
2.1 Chemical synthesis of relaxin
The two chain, three disulfide bond nature of relaxin presents special
challenges with respect to its chemical synthesis. The first reported syn-
thesis10 of the related peptide, insulin (bovine), used solution phase
synthesis of the two chains as their Cys free thiol forms. Each chain was
then subjected to S-sulfitolysis to aid the handling and purification of
the chains after which these were combined in solution at high pH to

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spontaneously fold and form the three disulfide bonds. This milestone
achievement was followed soon afterwards by a description of the prepar-
ation of human insulin in which the two chains were assembled by Boc-
based solid phase peptide synthesis (SPPS) after which their folding was as
described for bovine insulin. Overall yields of biologically active peptide
were of the order 1% relative to the starting crude B-chain.11 The first re-
ported synthesis of relaxin required modifications to the general insulin
chain folding scheme given the poor solubility of the B-chain even after
S-sulfitolysis. Low yields of active porcine relaxin were obtained after the
chains were dissolved as their free S-thiols in complex buffer mixtures
containing aqueous and organic solvents.12 Over time, it became evident
that this approach would not be satisfactory for the general preparation of
relaxin and its analogues given that yields were always low and highly
variable. Büllesbach and Schwabe then reported the development of an
elegant but complex seven step synthesis strategy for human relaxin based
upon an Fmoc-synthesis of the A-chain and a Boc-SPPS synthesis of each of
the two S-regioselectively protected chains followed by stepwise formation
of the three disulfide bonds via oxidation, thiolysis, and iodolysis.13 Overall
yield of peptide was reported to be approximately 1%. Our laboratory
refined this approach to use wholly Fmoc-based SPPS and a simpler four
step regioselective disulfide bond formation protocol based upon oxidation
of the intra-A-chain disulfide bond, thiolysis between the two chains and
iodolysis.14 Using this approach, the overall yield of synthetic human
relaxin-3 was 10% relative to the crude starting B-chain. This methodology
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has led the successful acquisition of numerous analogues of not only relaxin
but of other members of the insulin superfamily too15–19 and has been
instrumental in enabling comprehensive studies of the structure and activity
relationships of these peptides.
2.2 Primary interaction between relaxin and RXFP1

Chemical peptide synthesis has been successfully employed to prepare
numerous relaxin analogues for detailed structure-function relationship
analysis.20 In addition, site directed mutagenesis studies on RXFP1 have
aided in the identification of key ligand interaction sites.21 Together the
results of these studies have enabled an understanding of the mechanism of
relaxin’s interaction with RXFP1 leading to its potent in vivo activity. The
RXFP1 receptor is currently known to possess high and low affinity binding
sites (also referred to as primary and secondary interaction sites) by which it
interacts with its ligands.22,23 The primary mode of ligand-receptor inter-
action has been extensively studied and well characterized. The binding
cassette (Arg-X-X-X-Arg-X-X-Ile) present within the mid region of the
B-chain is responsible for primary binding interaction of relaxin to the
LRRs of its native receptor RXFP1.24 The corresponding residues in
RXFP1 interacting with the B-chain binding motif were later identified by
mutations of the LRR region of the receptor.25 A pair of acidic residues
(Asp and Glu) within RXFP1 form hydrogen bonds with each conserved
residue (Arg) of the B-chain, while the single isoleucine residue forms
hydrophobic interactions with corresponding hydrophobic cluster (Trp, Ile
and Leu) present in the inner sheets of the LRR. Interestingly, when relaxin

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binds to and activates the RXFP2 receptor, it utilizes a hybrid relaxin/

INSL3 binding site consisting partly of the INSL3-RXFP2 and relaxin-
RXFP1 interactions.26
2.3 Secondary interaction between relaxin and RXFP1

Recent evidence strongly suggests that there is a secondary interaction
between relaxin and RXFP1 which ultimately causes activation and
downstream signaling. Studies involving chimeric peptides and receptors
have provided some insights into the mechanism of this secondary inter-
action. It is now believed that such interactions involve the A-chain of the
ligand and the TM exoloops of the receptor.27
A series of chimeric relaxin peptides were prepared to study the role of the
A-chain in this secondary interaction.28,29 This was conducted using the
B-chain derived from native H2 relaxin in combination with the A-chains
from other insulin/relaxin family members such as H1 and H3 relaxins,
INSL3, INSL5 and insulin. H1A-H2B peptide displayed highest binding
affinity while H3A-H2B retained modest affinity. This could be due to a
compromised overall conformation of the protein leading to reduced
activity. The A-chain replacement with the other INSL/insulin members
resulted in a drastic loss of binding affinity. It is likely due to the fact that
INSL3, INSL5 and insulin are not ligands for RXFP1 and therefore are
not compatible with the secondary binding site of RXFP1. The chimeric
peptides H2A-INSL3B28 and INSL3A-H2B29 exhibited poor or partial
activity at RXFP2. This was surprising given the fact that both INSL3 and
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to a lesser extent, relaxin act as RXFP2 agonists. Interestingly, when the

chimeric peptide H2A-INSL3B was tested against an RXFP2 chimeric
receptor comprising of the TM domain from RXFP1, full activation of the
receptor was observed. These results highlight that the A-chains of relaxin
and INSL3 each play a key role in interacting with the TM exoloops of
RXFP1 or RXFP2 and the mode by which relaxin or INSL3 binds to the
LRRs of RXFP2 is not compatible with the interaction between the A-chain
of these chimeric peptides and TM exoloops of RXFP2.29 Unlike the
INSL3-RXFP2 interaction, the secondary interactions between H2 relaxin
and RXFP1 remain less well characterized and the residues involved in these
interactions have not been fully determined.
To elucidate the secondary interaction between relaxin-RXFP1, Park
et al. have carried out point mutations at the C-terminus of the A-chain.30
Based on analyses of the receptor-activation and receptor-binding activities,
they have identified that Thr16, Lys17 and Phe23 of relaxin as being crucial
residues for the interaction with RXFP1 and RXFP2, and suggested that
these act cooperatively with the well characterized Arg-X-X-X-Arg-X-X-Ile
binding motif in shaping the functional characteristics of H2 relaxin. The
crystal structure of H2 relaxin showed that Lys17 residue is embedded be-
tween the interface of the A-chain and the B-chain and is the only A-chain
residue with its side chain exposed on the same surface with Arg13, Arg17,
and Ile20 of the binding motif. The Thr16 and Phe23 residues are located
opposite to the surface of Lys17 and the Arg-X-X-X-Arg-X-X-Ile motif. It
was hypothesized that primary binding site comprises of B-chain specific
residues (Arg-X-X-X-Arg-X-X-Ile motif) together with one A-chain

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residue, Lys17 that cooperatively interacts with LRRs of RXFP1 and

RXFP2.30 On the other hand, the secondary binding site comprises of at
least two residues Thr16 and Phe23 at the C-terminus of the A-chain where
they interact with the TM exoloop(s), a distinct ligand-binding interface of
RXFP1 and RXFP2.31
Further mutational studies targeting the entire relaxin A-chain were
conducted by Chan et al. in order to elucidate the role of the A-chain in
RXFP1 function.32 It was found that no individual amino acid in the A-
chain dominates in driving the secondary interaction between relaxin and

RXFP1 although some contributions were observed from Tyr3, Leu20
and Phe23. However, these three residues predominantly drive the affinity
and activity of relaxin at RXFP2. When Try3 and Phe3 were mutated or
deleted, they observed complete reduction of both binding and activation at
RXFP2 while retaining the activity of RXFP1. The circular dichroism and
NMR studies suggests that the decreased RXFP2 activity observed from the
alanine-substituted Phe23 residue was due to a structural effect rather than
specific side chain-driven influences. Both Tyr3 and Phe23 are in close
proximity with each other although one is present at the N-terminus and the
other at the C-terminus. It is likely that the hydrophobic or p-p interactions
between the side chains of both aromatic residues are essential for the
maintenance of the native relaxin fold. Thus, the removal or mutation of
either or both Tyr3 and Phe23 residues destabilized the overall structure of
relaxin resulting in the loss of binding affinity and activity.32
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2.4 SAR study resulted in a minimized relaxin analogue

A series of A-chain shortened relaxin analogues were prepared by Hossain
et al. by chemical peptide synthesis to test the influence of the A-chain on
RXFP1 binding and activation.20,33 The removal of the first four to six
amino acids from the N-terminus of the A-chain, H2:A(4-24) and H2:A(7-
24), resulted in a slight reduction of binding affinity. However, there were no
significant differences in the potency of both peptides when compared to
native relaxin. Upon the further truncation of the first eight residues of the
A-chain, H2:A(9-24), there was a marked decrease in potency (10-fold) in
contrast to the binding affinity which was 500-fold lower. Hence, in order to
influence both binding and cAMP responses of native relaxin, the trunca-
tion of the peptide by at least eight residues would cause a prominent effect
on affinity than potency. In a separate study by Hossain et al., the trun-
cation at the termini of both peptide chains revealed that the removal of
residues from both N- and C-termini of the B-chain were still able to retain
potent biological activity.20 Moreover, this ascertains that these regions are
not critical for interactions with RXFP1. Conversely, these truncations
reduce the activity of relaxin at RXFP2 but improve RXFP1 selectivity. The
critical ‘‘active core’’ of native relaxin, H2:A(4–24)B(7–24) although slightly
less potent compared to the native peptide, is still capable of activating the
RXFP1 receptor at nanomolar concentrations.20
3 Fibrosis and wound healing

Fibrosis (progressive scarring) is a universal response to aberrant wound
healing that is initiated by chronic injury and inflammation in several

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1–3,34
organs; and reflective of the limited capacity of these organs to
regenerate post-injury. It is characterized by scar tissue accumulation that is
caused by an excessive build up of extracellular matrix (ECM) components,
mainly fibrillar collagen (types I and III).34,35 This involves an imbalance
between factors that control the rate of matrix synthesis, such as the pro-
fibrotic cytokines angiotensin (Ang) II and transforming growth factor
(TGF)-b vs those that control the rate of matrix degradation, such as the
matrix metalloproteinases (MMPs) which aid in the degradation of collagen
and the endogenous inhibitors of MMPs known as the tissue inhibitors of

MMPs (TIMPs). When the rate of matrix synthesis exceeds that of matrix
degradation, the continual deposition of excessive fibrous connective tissue
in affected organs will result in the disruption of the structural integrity of
normal tissues and ultimately lead to compromised organ function.1–3,34,35
Typical wound healing consists of three dynamic phases: inflammation,
proliferation and remodeling. During the inflammatory phase, a blood clot
composed of fibrin and fibronectin is formed at the site of capillary damage.
Multiple cytokines are released by platelets present within the clot which
contribute to the recruitment of inflammatory cells, fibroblasts and endo-
thelial cells. Active angiogenesis occurs during the proliferative phase whereby
new capillaries are formed to supply nutrients at the site of injury especially to
facilitate fibroblast differentiation.36 A key event in wound healing is the
differentiation of fibroblasts to myofibroblasts which contract and participate
in healing by facilitating wound closure (Fig. 2). Under normal healing con-
ditions, the myofibroblasts present will undergo apoptosis upon full
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Fig. 2 Fibroblast differentiation during injury. The two stage model involves fibroblast dif-
ferentiation into proto-myofibroblast in the presence of mechanical stress. The modulation of
proto-myofibroblasts into differentiated myofibroblasts takes place in the presence of TGF-b1,
ED-A fibronectin and/or a mechanical stimulus. Figure adapted by permission from Macmillan
Publishers Ltd: Nat. Rev. Mol. Cell. Biol. (Tomasek et al.,37 copyright 2002).

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37
epithelialization. Conversely, the persistence of myofibroblast activities
accompanied by elevated stress within the wound region (during aberrant
wound healing) would cause excessive scar formation and tissue fibrosis which
could lead to local immobilization and further implications on function. This
has a significant impact on numerous pathological and clinical outcomes.
4 Extracellular matrix
There are several structural proteins which form the complex mesh network
of the ECM. These include macromolecules such as collagens, fibronectin,
laminin, fibrinogen, vitronectin, thrombospondin, tenascin and proteogly-
cans which aid in directing the organization and composition of the ECM.
During wound healing and fibrosis post-inflammation, massive cell migra-
tion, proliferation, phenotypic differentiation and enhanced biosynthetic
activities occur at the site of repair (Fig. 3). Both wound healing and fibrosis
are two opposite outcomes following post-inflammation where a delicate
balance between both biosynthetic and degradation pathways is essential
for ECM-mediated regulation of intricate processes of repair and fibrosis.
The majority of the ECM components are secreted by fibroblasts present
within connective tissues. The predominant protein being synthesized and
secreted by the ECM is collagen.37
5 Fibroblasts and myofibroblasts

5.1 Scar tissue formation
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Granulation tissue formation occurs as part of the initial response at the

injury site during the process of healing and repair, and is essentially
composed of small vessels, fibroblasts, myofibroblasts and variable amounts
of inflammatory cells.37
Fibroblasts are widely distributed in most tissue types that exhibit
prominent epithelial and microvascular components. They are present in
connective tissues as one of the most abundant cell types. Fibroblasts exist
as spindle-shaped cells which play a crucial role in the regulation of ECM
turnover, wound healing, normal tissue development and homeostasis. At
sites of tissue injury, fibroblasts are recruited, become activated and
undergo differentiation into myofibroblasts. They possess ultra-structures
which are distinct from those of fibroblasts, such as extensive cell-matrix
adhesions, abundant intercellular gap junctions, bundles of contractile
cytoplasmic microfilaments that are related to contractile proteins like
non-muscle myosin which are analogous to stress fibres.38 Myofibroblasts
produce ECM components which help to replace the provisional matrix.
During fibrogenesis, several cytokines and growth factors play a key role
in wound healing. TGF-b, a potent pro-fibrotic stimulus, induces fibroblast
differentiation by acting directly on granulation tissue formation. It also
enhances the production of excessive matrix components, particularly
fibrillar collagens.
5.2 Role of Ang II and TGF-b in fibrosis

There are several profibrotic factors which include cytokines and growth
factors that play a crucial role in promoting fibrogenesis, most notably, Ang

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Fig. 3 Extracellular matrix remodeling by myofibroblasts. (A) Adjacent myofibroblasts attach

to the collagen network. (B) The contraction of myofibroblasts deforms the collagen network.
(C) New matrix components secreted helps to stabilize the contracted network structure. The
process of myofibroblast contraction is repeated causing small incremental collagen-matrix
remodeling leading to tissue contracture. Figure adapted by permission from Macmillan
Publishers Ltd: Nat. Rev. Mol. Cell. Biol. (Tomasek et al.,37 copyright 2002).
II, TGF-b1, and their downstream mediators such as connective tissue

growth factor (CTGF), platelet derived growth factor (PDGF) and endo-
thelin-1 (ET-1) that accelerate the production of ECM and collagen
deposition.

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TGF-b has been identified as one of the key regulators of fibrosis that has
been extensively studied for its role in the ECM.39 It is commonly linked
with the development of fibrosis in several diseases.40 There are three iso-
forms of TGF-b: TGF-b1, TGF-b2 and TGF-b3 of which TGF-b1 is the
predominant form attributed to tissue fibrosis. The key sources of TGF-b1
are circulating monocytes and tissue macrophages. The primary level of
control in macrophages is not governed by TGF-b1 mRNA expression but
instead by the secretion and activation of latent TGF-b1. TGF-b1 is stored
within the cell as a homodimer and is non-covalently bound to a latency-

associated protein (LAP) which renders TGF-b1 inactive. The dissociation
of LAP by several molecules such as plasmin, cathepsins, thrombospondin,
calpain, MMPs aid in the binding of TGF-b1 to its receptors. In the pres-
ence of TGF-b1, the activated transmembrane receptors trigger signaling
intermediates call Smad proteins which modulate important target gene
transcriptions of procollagens I and II.
Ang II is a component of the renin-angiotensin-aldosterone system and
has been found to exhibit profibrotic activity through activation of its type I
(AT1) receptor. In particular, Ang II has been implicated in the develop-
ment of hepatic and renal fibrosis and is primarily responsible for cardiac
fibrosis in hypertensive heart disease.40 Ang II manifests its effect through
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity,
stimulating TGF-b1 production through its AT1 receptor, which in turn
leads to myofibroblast differentiation causing an accumulation of collagen
production. Additionally, Ang II enhances TGF-b1 signalling via increasing
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Smad-2 levels and nuclear translocation of phosphorylated Smad-3, thus

inducing collagen secretion. Since Ang II commonly promotes TGF-b1
production, it is known that both factors are able to act synergistically
through the Ang II-TGF-b1 axis to disrupt normal structure and function
of cardiac tissues causing vascular remodeling.39–41 More recently, it has
been reported that Ang II has implications in dermal fibrosis from a mouse
model where Ang II was found to activate the TGF-b pathway, induce
myofibroblast differentiation and promote inflammation in mouse skin.42
6 Collagen
6.1 Collagen structure
All collagen molecules are modular proteins made up of three polypeptide
chains referred to as alpha chains. The molecular hallmarks of the collagen
structure is an assembly of three parallel polypeptide strands in a left-
handed, polyproline II (PPII)-type helical conformation coil about each
other with a one-residue stagger to create a right-handed triple helix. These
alpha chains are wrapped around each other and look like a rope-like rod
structure (Fig. 4).43–45
To date, there are 42 different polypeptide chains which are encoded by
41 specific genes. In some collagen types, all three alpha chains of the
molecule are identical. However in other types, the collagen molecule is
sometimes made up of two or even three different alpha chains. The triple
helical structure of collagen arises from the unusual abundance of a char-
acteristic triplet repeat motif: (Gly-X-Y)n. The presence of Gly in every

Fig. 4 Collagen structure composed of individual amino acids to collagen fibrils. Three par-
allel polypeptide strands in a left-handed, polyproline II-type (PPII) helical conformation coil
are assembled together to create a right-handed triple helix. The triple helical structure of
collagen arises from the abundance of a characteristic triplet repeat motif: (Gly-X-Y)n. The
amino acids in the X and Y positions are often proline and hydroxyproline, respectively. These
alpha chains are wrapped around each other to form a rope-like rod structure. Collagen fibrils
are made up of several of these rope-like rod structures. Figure adapted from Schulz et al.48 by
01/12/2013 14:44:29.
kind permission from Springer Science and Business Media.
third position of the triple helix is essential for the tight packing of PPII
helices. This is due to the restricted space in the centre of the triple helix
when the three chains come together whereby a larger amino acid will not be
able to fit in. X and Y denote amino acids apart from glycine while n varies
depending on the collagen type and domain. The amino acids in the X and
Y positions of collagen are often proline and hydroxyproline, respectively
whereby the pyrrolidine ring of proline increases stability of the triple helix
while the hydroxyproline residue at the Y position dramatically increases
the thermal stability of the triple helix. It has been shown previously that
only 4(R)-hydroxyproline and not 4(S)-hydroxyproline provides a stabilizer
effect due to the presence and position of the -OH group at the proline ring.
Hydrogen bonds and water bridges within the collagen molecule help to
further stabilize the triple helix.46,47
6.2 Types of collagen

In most animals, collagen constitutes approximately one quarter of the total
protein content as it is the major component in many connective tissues, for
example, skin, tendons, ligaments, cartilage, basement membranes, blood
vessels etc. Collagen is a fibrous protein that exists as structural macro-
molecules of the ECM. During reparative fibrosis, collagen deposition and
accumulation occurs as a result of tissue injury or cell death leading to the
formation of scars. In reactive fibrosis, collagen build-up occurs as a

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consequence of direct stimulation of fibroblasts without cell injury or death.

There are several types of collagen essential for maintaining the structural
integrity or vertebrates and many other multicellular organisms.49
Within the collagen superfamily, there are 28 unique collagen types.
These members can be classified into various subfamilies (a-g) according to
their sequence homologies and similarities in structural organization and
supramolecular assembly.46,47,49 Each individual collagen member is num-
bered with a roman numeral.
(a) Fibrillar collagens – These represent the most abundant products by

connective tissue cells and were the first members of the collagen super-
family to be discovered. Members which are classed under this group are
types I, II, III, V, XI, XXIV and XXVII; with type I collagen being the
predominant isoform of collagen that is present in most tissues, which
determines the maximal shape and volume of the tissues it resides within.
They consist of large triple helical domains of about 1000 amino acids or
330 Gly-X-Y repeats per chain.
(b) Basement membrane collagens – Type IV belongs to this group of
collagen. The collagenous domain of this collagen type is longer that fibril-
forming collagens and comprise of about 1400 amino acids.
(c) Fibril-associated collagens with interrupted triple helices (FACIT
collagens) – These collagens do not form fibrils on their own but attach onto
the surfaces of pre-existing fibrils of fibril-forming collagens. Members of
this class are types IX, XII, XIV, XVI, XIX, XX and XXI and are
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characterized by short triple helical domains interrupted by short non-

collagenous sequences.
(d) Short chain collagens – types VIII and X
(e) Anchoring fibril collagen – Type VII collagen forms fibrils that con-
nect basement membranes to anchoring plaques of type IV collagen and
laminin of the underlying ECM.
(f) Multiple triple helix domains and interruptions (MULTIPLEXINS) –
Types XV and XVIII collagens both possess large N- and C-termini
globular domains and a highly interrupted triple helix. Previous obser-
vations suggest that these collagens may be extensively glycosylated.
(g) Membrane associated collagens with interrupted triple helices (MACIT
collagens) – These are type II transmembrane proteins with the N-terminus
inside the cell. Types XIII, XVII, XXIII and XXV belong to this class and
have a single pass hydrophobic transmembrane domain.
7 Relaxin and its anti-fibrotic effects in fibrotic diseases

Relaxin has demonstrated rapid-occurring, but safe anti-fibrotic actions in
several experimental models of disease, regardless of etiology. Its actions
in various tissues are summarized below:
7.1 Systemic sclerosis/scleroderma

Scleroderma is classified by the presence of fibrotic lesions of the skin,
gastrointestinal tract, lung and arteries. A marked decrease in collagen

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production was observed when dermal fibroblasts, stimulated with TGF-b150

or isolated from scleroderma patients,51 were treated with relaxin. Add-
itionally, Unemori et al. demonstrated reduced collagen accumulation by
recombinant relaxin in two rodent models of dermal fibrosis which was
induced by subcutaneous implantation of polyvinyl sponges in rats or the
capsule formation around osmotic pumps in mice.52 Relaxin-treated rats
were found to have decreased collagen production which correlated to a
decrease in collagen staining. Mice receiving relaxin treatment were found
to have a thinner capsule layer surrounding the osmotic pumps when

compared to the control mice. The capsules belonging to control mice
appeared to have a packed array of parallel collagen fibrils, whereas the
relaxin-treated mice had less abundant collagen fibrils which had random
orientations.
7.2 Cardiac fibrosis

Cardiac fibrosis is a hallmark of heart disease that is characterized by
fibrillar collagen accumulation which occurs after myocyte death, inflam-
mation and stimulation by a series of cytokines, hormones and growth
factors. Structural changes occur to the cardiovascular system due to the
presence of pathological stimuli. The administration of relaxin has several
cardiovascular-related adaptations such as increases in cardiac output,
heart rate, plasma volume, decreases in vascular resistance and blood
pressure.5,53
Previous studies by Samuel et al. have shown that relaxin is able to
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modulate cardiac fibroblast proliferation and production and also reverse

cardiac fibrosis in vivo using two independent murine models of fibrosis.54
It was demonstrated that relaxin was able to inhibit both TGF-b- and Ang
II-stimulated fibroblast differentiation and collagen production by de-
creasing collagen deposition and increasing MMP activity.54 Furthermore,
continuous relaxin infusion was also found to prevent and/or abrogate
cardiac fibrosis in vivo when applied to models of hypertension,55 ischemic
heart disease,56,57 type I diabetic cardiomyopathy58 and isoprenaline-in-
duced cardiac toxicity.32 Consistent with this, male relaxin-deficient mice
underwent elevated levels of collagen deposition in the left ventricle, causing
ventricular diastolic dysfunction and chamber stiffness.59
Relaxin has the ability to induce positive chronotropic effects both in vitro
and in vivo in a number of species.5,53 Additionally, relaxin may possess
inotropic effects in the mammalian heart, which can be abrogated by the Gi
inhibitor, pertussis toxin.60 In experimental heart failure, adenovirus-
mediated gene delivery of relaxin has been used as an effective form of
therapy.61 Systemic adenovirus-mediated delivery of relaxin was utilized for
the treatment of fibrotic cardiomyopathy caused by b2-adrenergic receptor
overexpression in transgenic mice. The outcome of the adenoviral relaxin
gene transfer treatment was a significant reduction in the increased extra-
cellular collagen levels in the left ventricles of treated transgenic mice.
Interestingly, the other chambers of the heart and other organs such as the
kidney and lung remained unaffected, indicating the specific target treat-
ment of relaxin gene therapy.61

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32,54–61
Regardless of disease etiology or its route of administration (via
subcutaneous infusion or viral delivery), the anti-fibrotic properties of
relaxin were found to be associated with its ability to inhibit the pro-fibrotic
actions of TGF-b1 and hence, the down-stream effects of TGF-b1-induced
mesenchymal cell differentiation and ECM/collagen production. Add-
itionally, relaxin was found to increase both collagenase and gelatinase-
induced ECM/collagen degradation and reduce the levels of TIMPs; the net
effects of its actions resulting in a reduction in myocardial stiffness and
improved diastolic function.

Furthermore, when applied to models of ischemic heart disease,56,57 the
ability of relaxin to abrogate fibrosis within the infarct and other regions of
the heart was associated with its ability to promote angiogenesis at both
acute and chronic stages of disease progression and prevent cardiomyocyte
apoptosis during chronic disease.
7.3 Pulmonary fibrosis

A common outcome of pulmonary diseases such as idiopathic lung disease,
asthma and chronic obstructive pulmonary disease is lung fibrosis, which
involves the build-up of collagen deposition caused by an increase in
(myo)fibroblast number. This leads to the loss of normal lung function due
to the disruption of normal lung structure which encompasses a loss in
regular alveolar capillary function and alveolar thickening. In a murine
bleomycin-induced model of interstitial lung fibrosis, relaxin treatment
restored accumulated collagen back to normal levels, when administered
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one week after disease onset.62 Additionally, relaxin was also able to
decrease procollagen and fibronectin overexpression in human lung fibro-
blasts in vitro by modulating the secretion of MMPs and procollagenase
which is crucial for the regulation of collagen degradation.62
In a chronic model of allergic airway disease (AAD), endogenous
relaxin was found to regulate collagen turnover through increased MMP
activity in young mice.63 Furthermore, when older relaxin-deficient mice
with established fibrosis were subjected to ovalbumin-induced AAD, the
absence of relaxin led to more rapidly elevated levels of lung collagen
deposition (fibrosis), epithelial thickening and airway hyperresponsiveness
(AHR; a measure of lung dysfunction), compared to what was measured
in age-matched wild-type mice.64 In line with these findings, exogenous
relaxin treatment was also found to reduce epithelial thickening,
airway fibrosis and AHR when administered to a mouse model of chronic
AAD.65 Moreover, relaxin was shown to inhibit myosin light chain
phosphorylation and lung fibrosis in a mouse model of bleomycin-
induced lung injury, while attenuating the contraction of myofibroblasts
isolated from lungs of patients suffering from idiopathic pulmonary
fibrosis.66
7.4 Hepatic fibrosis

Hepatic stellate cells play a key role during liver injury where they are
activated from a quiescent state. Upon injury, the activated hepatic stellate
cells differentiate to a myofibroblastic phenotype expressing fibrillar colla-
gens and smooth muscle actin where increased proliferation and

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contractility occurs. This leads to the over-production of collagen and the

up-regulation of other ECM components.
In an established in vivo model of induced hepatic fibrosis by carbon
tetrachloride, the administration of relaxin via osmotic mini pumps reduced
collagen accumulation.67 These anti-fibrotic actions of relaxin were found
to be associated with its ability to down-regulate collagen production by
reducing type I collagen protein synthesis and by decreasing TIMP
expression indirectly, thus promoting matrix breakdown, when applied to
activated stellate cell culture in vitro.67 Further evidence has shown that
relaxin increases collagenase activity (MMP-13), decreases smooth muscle
actin levels and TIMP expression (TIMPs 1 and 2) and specifically inhibits
type I collagen production by hepatic stellate cells.68 Acute changes in
hepatic microcirculation and induced morphological changes in sinusoidal
myofibroblastic cells were observed in rats that received relaxin treatment.69
Bennett and colleagues have shown that the expression of RXFP1 is
elevated in hepatic fibrosis and in activated hepatic stellate cells when
compared to the normal liver.70
7.5 Renal fibrosis

A characteristic outcome of renal fibrosis is the activation and proliferation
of interstitial fibroblasts and the extreme production and accumulation of
ECM components which include fibronectin and some collagen types (I, III
and IV) together with a lack of ECM breakdown.71
In established cell lines utilising renal fibroblasts and primary cortical
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fibroblasts, relaxin demonstrated its anti-fibrotic effects by inhibiting TGF-

b-induced fibroblast differentiation, collagen I and fibronectin production
and increased MMP secretion.72,73 The TGF-b1-inhibitory effects of relaxin
were associated with its ability to specifically inhibit the phosphorylation
and nuclear translocalization of Smad2, which led to a reduced ability of
Smad2 to form complexes with Smad3; in the absence of any direct effects
on Smad3, Smad4 and Smad7.9,72
Mookerjee et al. have shown that relaxin interferes with TGF-b1 sig-
nalling through its cognate receptor, RXFP1 and a neuronal nitric oxide
(NO) synthase nNOS-NO-cyclic guanosine monophosphate (cGMP)-
dependent pathway to inhibit Smad2 phosphorylation and thereby prevent
TGF-b1 from stimulating myofibroblast differentiation.9 Likewise, relaxin
was found to signal through this RXFP1-nNOS-NO-cGMP-dependent
pathway to promote collagen degrading MMPs (MMP-1/-13, MMP-2 and
MMP-9).74 Consistent with these findings, endogenous relaxin was found to
be effective in protecting the injured kidney by abrogating the progression
of tubulointerstitial renal fibrosis in vivo;75 while exogenous relaxin has
been found to inhibit fibrosis in experimental model of tubulointerstitial
renal disease,76 chronic papillary necrosis,77 renal mass reduction,78
hypertension55 and crescentic glomerunephritis.79 Its effects were primarily
achieved by suppressing myofibroblast differentiation, cell proliferation and
collagen turnover.
The use of relaxin for the treatment of various fibrotic disorders has made
remarkable progress over the years in different experimental models of
fibrosis. The development of the relaxin knock-out mouse has also

01/12/2013 14:44:29.
Fig. 5 Relaxin mediated signaling pathways via RXFP1. Abbreviations: cAMP, cyclic AMP;
CRE, cAMP response element; CREB, cAMP-responsive element-binding protein 1; eNOS,
endothelial nitric oxide synthase; ERK, extracellular signal-regulated kinase; GR, glucocorti-
coid receptor; GRE, glucocorticoid response element; iNOS, inducible nitric oxide synthase;
NO, nitric oxide; PDE, phosphodiesterase; PKA, protein kinase A; PKB, protein kinase B;
PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; RTK, receptor tyrosine kinase.
Figure adapted by kind permission from Macmillan Publishers Ltd: Nat. Rev. Cardiol.
(Du et al.,5 copyright 2010).
improved our understanding of the protective role that relaxin plays against
age- and injury-related fibrosis and related dysfunction in various organs.80
Some challenges remain and the precise signal transduction mechanisms by
which relaxin abrogate fibrosis and the influence of gender on its effects still
remain unclear. There are several signal transduction pathways activated by
relaxin of which many, but not all, involve the interaction between relaxin
and its native receptor RXFP1 (Fig. 5).5 Relaxin has nonetheless demon-
strated enormous potential for the regulation of fibrogenesis by maintaining
a balance between matrix synthesis and degradation factors (Fig. 6).
8 Future perspectives
Relaxin was first discovered nearly ninety years ago and was long regarded
as solely a hormone of pregnancy. Today it is recognized to be a pleiotropic
hormone with multiple functions, several of which with significant clinical
potential. Chemical peptide synthesis has enabled many studies examining

Fig. 6 Summary of the potent anti-fibrotic effects of relaxin. A balance between matrix
degradation and synthesis factors determine the extent of fibrosis. Relaxin is known to reverse
fibrosis by stimulating MMP-induced matrix degradation and at the same time inhibiting
TIMP expression and matrix synthesis. Furthermore, in many cases, relaxin primarily acts by
inhibiting the pro-fibrotic actions of TGF-b1 and hence, the ability of TGF-b1 to promote
fibroblast proliferation and differentiation into myofibroblast as well as collagen synthesis and
secretion.
01/12/2013 14:44:29.
the structure-function relationship of the relaxin peptide which has, in turn,

led to important insights into relaxin and its interaction with its receptors.
These have also enabled a clear recognition of the hormone’s enormous
potential to treat fibrosis. However for this potential to be realized, relaxin
requires modification to address the longstanding issues that face all peptide
therapeutics, that of short in vivo half lives due to enzymatic cleavage and
rapid renal clearance. With current knowledge of the structural features
that are critical for its function, future work will aim to introduce
chemical modifications to relaxin which will help to improve pharmacoki-
netic and drug-like properties. Together with the development of relaxin
analogues of reduced size, it augurs well for relaxin’s lead into the clinic as
well as making it cheaper and easier to make as a drug compared to the
native hormone.
Acknowledgments
Part of the research reported from the authors’ laboratory was funded by
NHMRC (Australia) project grant 508995 to JDW. CSS is a National
Health & Medical Research Council (NHMRC) of Australia Senior
Research Fellow and JDW is an NHMRC Principal Research Fellow. LJC
was the recipient of the David Hay Postgraduate Writing Up Award from
the University of Melbourne. Studies at the FNI were supported by the
Victorian Government’s Operational Infrastructure Support Program.

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E. N. Unemori and J. P. Iredale, Gut, 2001, 49, 577–583.
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66, 867–874.
69 D. Bani, S. Nistri, S. Quattrone, M. Bigazzi and T. Bani Sacchi, J Endocrinol,
2001, 171, 541–549.
70 R. G. Bennett, S. R. Dalton, K. J. Mahan, M. J. Gentry-Nielsen, F. G. Hamel
and D. J. Tuma, Biochem Pharmacol, 2007, 73, 1033–1040.
71 A. A. Eddy, Pediatr Nephrol, 2000, 15, 290–301.
72 M. H. Heeg, M. J. Koziolek, R. Vasko, L. Schaefer, K. Sharma, G. A. Muller
and F. Strutz, Kidney. Int, 2005, 68, 96–109.
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I. Darby and G. Becker, Nephrol Dial Transplant., 2004, 19, 544–552.
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Samuel, PLoS One, 2012, 7, e42714.
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Becker and C. S. Samuel, Endocrinology, 2007, 148, 660–669.
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Sukhatme, Am J Physiol Renal Physiol, 2003, 285, F59–67.
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2005, 1041, 173–181.

Biofunctional peptide design
Maxim G. Ryadnov
DOI: 10.1039/9781849737081-00079
1 Introduction
This chapter particularly emphasises peptide design as a tool providing
functional peptide structures for biologically relevant applications, and

primarily covers specialist research findings published over the last few
years to the time of its submission. A necessary reference to background
information covers an unlimited timeframe. Prescriptive or de novo design
of polypeptide sequences encoding specific functions found in native sys-
tems is of a primary focus.
The chapter reviews literature sourced from different databases including
Web of Science, RCSB Protein Data Bank and PubMed. Individual sections
are arranged according to targeted functions with structural preferences and
definitions consequently referring to design types. A section describing basic
design principles provides an introduction to the problem of peptide design
and structure-function relationships.
2 Generic considerations for biofunctional peptide design

2.1 Functional space defines sequence space
Over the last two decades considerable progress has been made in the design
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of biofunctional peptide structures as well as novel peptide-based materials,

and predominantly self-assembling systems.1–4 Broadly, all designs to date
can be grouped into two main categories. One would constitute individually
functional or autonomously folded structures, which can include anti-
microbial2 and cell-penetrating sequences,5 peptide switchers,6 structures
targeting protein-protein interactions7 including those serving as folding-
mediated inhibitors;8 and another can represent self-assembling systems,3
which serve specific purposes of providing supramolecular scaffolds for cell
growth and 3D cell culture,2 gene delivery vectors9 and controlled release
systems.10 Other systems that may include peptide aptamers, actuators,
sensors and ligands are out of the scope of this chapter and will not be
discussed. Likewise, constructs assembled from non-natural amino acids
and sequences containing bonds other than peptide bonds (depsipeptides)
are not a subject of this chapter.
Designs that will be described here are meant to highlight de novo peptide
sequences11 that use a rationale allowing to predict and control resulting
structures with respect to a desired function. Because de novo strategies
address specific questions at specific lengthscales generalising biofunctional
designs is admittedly ambitious. However, given that none of the existing
approaches can provide a definite link between structure and function, al-
though exemplified by successes in drug design supported by crystallo-
graphic findings and extensive developments in nanoscale imaging and
National Physical Laboratory, Teddington, TW11 0LW UK. E-mail: max.ryadnov@npl.co.uk;

School of Physics and Astronomy, University of Edinburgh, Mayfield Road, EH9 3JZ UK

Fig. 1 Differenct connectors (loops and turns) direct the packing of an a-helix against different
sides of a b-strand. The connection rules feed into emergent principles of building ideal and
larger motifs, i.e. bab-structure, which is assembled from simpler connected ba- and -ab motifs
(reprinted by permission from Macmillan Publishers Ltd: (B. Höcker, Structural biology: a
toolbox for protein design, Nature, 491, 204), copyright (2012)).
fabrication, a reproducible strategy capable of predicting a mechanistic,

rather then generic, function from polypeptide sequence and strucutre has
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yet to be reported. Therefore, with the available understanding of structure-

function relationships, which is often intuitive as opposed to systemic, the
allowable sequence space of designs discussed in the chapter is limited to the
length of tens of amino-acid residues and primarily supports one folding
element per design. Having such designs free from the constraints of natural
selection (de novo)12 specific biologically active sequences found in nature
are not considered, but instead established amino-acid preferences at both
primary and secondary structure levels are used.
Native sequences can comprise different folding motifs and predicting
sequence-folding pathways for their functional structure is very challenging.
Not that the designs of individual secondary structure motifs, such as an
a-helix or b-sheet, are reproducibly simpler, but these can be demonstrated
as having well defined properties when compared to native analogues, which
in turn can feed into a developing set of fundamental rules.13 Rules that can
describe connections between simpler motifs and more complex (tertiary)
motifs make up a toolbox of design principles that can be applied directly to
functional structures14 (Fig. 1).
2.2 Secondary structure design versus backbone design

As discussed in the previous volume,15 the spatial arrangement of amino-acid
side chains in a peptide sequence is one of the bioactivity determinants. It also
holds true for the synergistic interactions of side chains with the peptide
backbone, the directionality of which can be crucial, but equally amenable to
provide enhanced biological activities. Very often retro and retro-inverso
analogues of the parent peptide16 are used and in many cases as, for example,

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17
for antimicrobial peptides diastereomeric sequences, whether irregular or
alternating, can furnish unique and perhaps more importantly drugable
properties. Partly, the growing interest in the peptide-based drug desing is
justified by that the functional behaviour of secondary structures derived
from designed primary backbones of defined chirality is more predictable and
putatively more exploitable when compared to structures of higher folding
hierarchies – supersecondary, tertiary and quaternary (Fig. 1). Partly, because
stereochemical design can be used to generate self-assembling systems
that are unique to structures assembled from all-L peptide blocks thereby
helping enrich a variety of supramolecular architectures and morphologies3
that can be conducive to the functional mimicry of native macromolecular
assemblies, which are also beginning to find use in biomedical applications.
With stereochemical conversions one can expand not only the repertoire
of possible structures but also the functional space of a given sequence. An
intrinsic limitation for achieving this fully is the lack of native examples.
Iterative approaches are still dominating and functional classes largely
concern non-ribosomal peptides, antibiotics of bacterial origin. Another
matter is the application of relationships between primary and secondary
structures and physico-chemical mechanisms for such structures. Even
though there exist numerous instances of a generic mechanistic action for
particular functional classes (host defence peptides, cell penetrating pep-
tides, integrin antagonists etc.), a view which tends to prevail in the field,
subtle changes in the secondary structure of similar lengths have been re-
ported to dramatically impact on mechanistic and biological activity
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(binding kinetics, active concentrations etc.), while alterations in stereo-

chemistry often prove to give similar results.18 Therefore, an increasingly
popular and efficient strategy is to focus not only on the critical assessment
of protein structure prediction but more on the specific biochemical func-
tion of a given amino-acid sequence and further its biomedical use. Software
packages such as ROSETTA are being developed to aid in applied studies.
Originally the method was developed for de novo protein structure pre-
diction (and by association for de novo design) with a number of predictions
reported with atomic level accuracy better than 2.5 Å. More recently,
ROSETTA methods were used for molecular docking and homology
modeling and applied in the successful designs of novel protein structures,
altered specificity protein-protein and protein-DNA interactions, and
stabilised protein complexes.19 Being developed in this vein the methods are
becoming unified software packages for biofunctional peptide design.
Importantly, the methods provide fairly rapid and convenient assessments
of biomedical hypotheses which are either experimentally impossible or are
prohibitively expensive to test at a given moment, for example, in the
engineering of novel therapeutics using data from genome projects.
However, most, if not all, structure prediction algorithms must first explore
a relevant conformational space, and in the case of design, sequence space,
and then must rank the energy of the resulting structural models that use
systematic sampling strategies for backbone and side-chain degress of
freedom introducing ‘‘rotamers’’. These rotamers in turn capture allowed
combinations between side chain torsion angles and the backbone j and c
angles thereby reducing the amount of conformational space (Fig. 2).19

Fig. 2 De novo folding algorithm ROSETTA. Fragment libraries (upper left) with sequence-
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dependent (j and c) angles capture the local conformational space of a sequence, which folded
by combining different fragments from the libraries through optimization of non-local contacts
(upper right). The low-resolution energy function (lower) smoothes the rough energy surface to
give a deep, broad minimum for the conformation, (reprinted with permission from K. W.
Kaufmann, G. H. Lemmon, S. L. DeLuca, J. H. Sheehan and J. Meiler, Practically useful: what
the Rosetta protein modeling suite can do for you, Biochemistry, 49, 2987. Copyright (2010)
American Chemical Society).
Alternative approaches are also being developed aim to avoid the limi-
tations of native backbones and stereochemical conversions and often
propose radical solutions that offer the mimicry and modulation of con-
ventional peptide backbones in non-canonical arrangements. An efficient
strategy is to develop or expand existing computational design platforms
(i.e. ROSETTA) which provide detailed descriptions of non-canonical
backbones and how these backbones can be applied to potential designs.20
The main argument here is the lack of design tools that can help identify
sequences adopting desired conformations, which in essence is the same
question of enabling structure prediction algorithms. Aside from the use of
non-canonical amino acids and non-canonical protein side chains, non-
canonical backbones are principally non-peptidic oligomers.20
Oligooxopiperazines, oligo-peptoids, b-peptides, hydrogen bond surro-
gate helices and oligosaccharides are all being investigated as alternative
backbones, and computational test protocols for some have been freely
available (see for example, http://rosie.rosettacommons.org). More specific
classes of non-canonical backbones such as poly-N-substituted glycines or
peptoids and foldamers, the main purpose of which may be different from

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Fig. 3 Biofunctional non-canonical peptide backbones. (A) Antimicrobial peptide magainin 2,

originally isolated from a frog Xenopus laevis amide, perpendicular (left) and parallel (right) to
its helix axis (2MAG PDB entry rendered with PyMol); and chemical structures of its oligo-
meric mimetics; helical peptoid (B) and b-amino acid oligomer (C). (C) Helical wheels for the
b-oligomer and magainin 2 amide are shown for comparison. Hydrophobic and cationic
residues in the b-oligomer are indicated by H and þ , a polar angle separating hydrophobic and
polar faces in the magainn wheel is highlighted by the bold line (reproduced with permission
from J. A. Patch and A. E. Barron. Helical peptoid mimics of magainin-2 amide. J. Am. Chem.
Soc., 125, 12092. Copyright (2003) American Chemical Society; reprinted by permission from
Macmillan Publishers Ltd: (E. A. Porter, X. Wang, H. S. Lee, B. Weisblum and S. H. Gellman,
Non-haemolytic beta-amino-acid oligomers, Nature, 404, 565), copyright (2000)).
that of the other oligomeric scaffolds having focused primarily on the

stabilisation of peptides and their conformations, have already gained a
substantial experimental recognition as novel and biologically functional
molecular classes (Fig. 3).21–24

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3 Functional space of de novo peptide design

Ultimately, a de novo design aims at a function, which is linked to a specific
folding motif or to a specific environment which mediates folding. This is a
starting point of assigning sequence to function. As aforementioned,
biofunctional designs may follow one of the two main routes – through
individually (autonomously) folded sequences25–27 and those capable of
supramolecular self-assembly.2,3 Self-assembling structures are of increasing
interest for next generations of nanostructured materials,28,29 whereas au-

tonomous folding remains to be a subject of utmost importance in struc-
tural biology.30 In both cases the aim remains to find novel approaches that
will extend our understanding of protein folding and function and will
broaden the repertoir of biologically functional sequences, but a systemic
approach to rational design is not obvious. Ironically, the types of possible
secondary structures that can be designed are those that already exist in
native biosystems or it is predictable secondary structures that are limited to
those known. Further, the composition of an amino-acid sequence is the
main determinant of structure predictions which makes it the main variable
in specifying function. However, even within these restrictions the choice of
amino acid types is limited to 20 standard amino acids (excl. D-amino acids)
which are grouped into conserved groups of polar, charged and hydro-
phobic side chains. All these limitations direct rather than restrict de novo
designs, but one has yet to systematise generic prediction rules empirically.31
There are at least three reasons for this: one is that functional space is
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directly linked to sequence space and the longer the sequence the more
functional variabilities it contains and hence the less predictable their choice
could be. Another reason is oligomerisation, which in naturally occuring
systems is highly controlled as to the type (homo, hetero), state (number of
monomeric components), component orientation (parallel, anti-parallel)
and extent (length, widths) of oligomerisation, which are compounded by
the morphological complexity of resulting oligomers (quaternary as-
semblies – filaments, spheroids).32–34 Each of these characteristics can be
illustrated by different functional proteins, which prompts a third reason
which is the synthetic availability of desired sequences.
Therefore, from the chemical sciences’ perspective polypeptides are
variations of the same backbone template, which in the context of this
chapter is important for the reasons of
chemical information (e.g. hydrogen bonding, hydrophobic cores and
interfaces) being used to provide functional approximations of designed
sequences thus re-instating the importance of evolving design approaches,
from fine details to generic principles,
and
physical working models relying on basic structural principles to
enable structure-function predictions.
Traditional approaches in soft matter physics seldom go beyond con-
densed phases that explore spatial dimensionality and micro-scale inter-
actions. Symmetry, topology and hydrodynamics are used parameters and

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are equally applicable to liquids, paracrystalline forms and generalised

protein folds. However, the phase approaches do not take into consider-
ation the crucial role of chemical detail in defining function.
A feasible way to address this shortcoming is by accepting a limited
number of ‘‘smoothed’’ secondary structure elements exhibiting well-known
behaviour patterns. For instance, the quasi-equivalence theory can describe
the architecture of any virus,35 but fails to instruct how to construct and
assemble protein subunits, whereas coarse-grained models can help explain
the assembly of protein fibres, but struggle to distinguish between different

morphological types derived from the same secondary structure element.
Proteins and their functional fragments can all be viewed as physical en-
tities. However, what puts them all into one category is the same mode of
their action, which gives rise to approximate functional models.36
3.1 Autonomously folded and self-assembling sequences

In practical terms, de novo design provides a strategy to emulate native
folding elements. Polypeptide sequences are arranged into secondary
structure oligomers, which can be continuous, that is belonging to one
sequence, non-covalent, but monodisperse and autonomously folded, and
supramolecular leading to nanostructured assemblies, which are not ne-
cessarily discrete and uniform.37 Despite the diversity of sequences and
functions they encode there exist relatively few arrangement types, with
each being associated with a specific type of function. This forms the basis
for the classification of polypeptide folds, which help provide a design
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framework for de novo peptides. Therefore, designed sequences that deliver

specific functions are referred to here as functional motifs.
Autonomous motifs
Secondary structure motifs are typically confined into four main classes –
helix, sheet, turn and loop. Other and more specific structures such
as polyproline helices also exist, but are less diverse (Fig. 4). Functional
assignments are common for individual motifs, be they independently
Fig. 4 Secondary structure motifs and their basic sequence patterns (left): a-helix, b-strand,
polyproline (collagen) helix (2ZTA, 1ICO, 1CGD and 2LKW PDB entries rendered with
PyMol); turn structures categorised by the number of peptide bonds between terminal residues
locked in an intra-turn hydrogen bond.

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operating or as a part of a protein. For example, stand-alone a-helices or

b-sheets can be antibiotics, whereas turn and loops are designated receptor
antagonists in folded proteins. Associations of different motifs that give rise
to super-secondary structures are equally functional but tend to be more
stable and specialised.38–40 For example, b-hairpins and helix-loop-helix
motifs act as high-affinity DNA-binding41 and metal-coordinating do-
mains,42 respectively.
The hydrophobic effect plays a major role in biomolecular recognition
and in proteins determines secondary structures and their associations.43

Hydrophobic clusters and interfaces are formed to provide interfacial
contacts within which hydrophobic residues are buried. Created interiors
can be supported by electrostatic interactions provided by side-chain groups
as in cationic peptides that bind to negatively charged bacterial membranes
or in a-helical bundles or coiled coils.43–46 The peptide bond is very polar to
be favoured in a hydrophobic core which is a form of specialised structuring
along the backbone of repeated peptide bonds that order through hydrogen
bonding to neutralise their polar groups. Thus, secondary structures are
local backbone structures in contrast to global protein structures, and fol-
low generic sequence patterns ensuring geometries characteristic of a par-
ticular secondary structure element (a-helix, b-strand) or a specific
hydrogen bond (turn, loop).
For instance, a-helix and b-strand sequences contain inbuilt elemental
repeats which in contiguous folded segments are stabilised by backbone
hydrogen bonding to give rise to 3.6 and 2 residues per turn, respectively.
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For helices the hydrogen bonding is maintained in each i, i þ 4 peptide bond

pair within the same backbone, i.e. between peptide bonds of the same
backbone (Fig. 4). Therefore, these are intra-backbone and allow individual
helices to be of indefinite length. A b-strand is a more extended conform-
ation which cannot be stabilised by hydrogen bonds within the same
backbone and is supported by inter-backbone hydrogen bonding. As a
result, b-strands incorporate into lateral b-sheets, and need not be more
than ten residues in length. An ultimate challenge in designing a-helices
and b-strands is reverse to their geometries, and is to design an individual
b-strand and a homogeneous a-helix oligomer. Given that such structures
are not usually employed by nature, their use or functional relevance cannot
be known and can be viewed as functionally de novo.
Turn- and loop-like structures are different from more extended helices
and b-strands in that their conformational preferences broadly depend on
the sequence length between the two end residues. Turns are very short
motifs that can comprise two (g-turns), three (b-turns) or four (a-turns)
peptide bonds (Fig. 4). Loops are much longer than turns and a rationale
for their preference over turns in many cases is not clear. However, proteins
use both loops and turns for spatial exposure of short signal sequences.
Therefore, backbone closures at the end residues in these structures is the
main factor for function. Free or isolated turns and loops rarely retain
native function because they need to be constrained to form stable
conformations, which otherwise is provided by protein scaffolds that in-
corporate the motifs. Therefore, turns outside their original protein context
are stabilised by covalently fixing a key intra-turn hydrogen bond. The

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caveat is not only to design a stable structure, but also to differentiate be-
tween single, inverse and multiple turns within the same backbone. The role
of turns in protein structure and function is of course broader and includes
their intrinsic ability to reverse polypeptide chains which determines protein
preference for globularity over linearity and the subdivision of turn struc-
tures into several types linked to different globular shapes. Although turns
help design super-secondary structures such as hairpins, both autonomous
and oligomerising, the main interest in turn designs remains due to turns
providing a rich source of potential pharmacophores.

Nevertheless, basic secondary structure principles can be used to design
artificial sequences and, by association, conformations predictablly. For
example, a sequence with alternating hydrophobic (H) and polar (P)
residues, (HP)n, will give b-strands. Short sequences of the same pattern
can produce turns with the inclusion of glycine or proline residues at turn
points as these residues are known efficient breakers of extended secondary
structures. A polar sequence with hydrophobic residues at every fourth
position, (HPPPHPPP)n, will fold as a helix (Fig. 4). Within a chosen
pattern amino-acid residues can be specified according to their conforma-
tional scoring – for example, glutamines, lysines and leucines favour helices,
whereas tryptophans, threonines and valines exhibit strong preferences for
b-type structures; and functional relevance – for example, lysines, leucines
and tryptophanes are common in membrane-binding sequences, arginines
support intracellular uptake and aspartates and glutamates dominate in
metal coordination sites. Thus, an oligolysine incorporating leucines at
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every fourth position will give an antimicrobial helix.
Self-assembling motifs
For the other category of functional elements, namely self-assembling
motifs, design rules are similar except that a principle emphasis is made on
propagating oligomerisation. The latter is distinguished from mere oligo-
merisation into b-pleated structures or helical bundles, which are meant to
be non-propagating and functionally different. Unlike autonomous (fold-
ing) and oligomerising (folding and oligomerisation) sequences, self-
assembling motifs undergo all three stages, folding, oligomerisation and
propagation, but are meant to become functional only at the final stage.
Oligomerising sequences in some cases can be viewed as the simplest
self-assembling motifs. A necessary requirement is function. Different
approaches used to design self-assembling motifs include those whose as-
sembly modes are fully programmed in linear sequences and those that
make use of auxiliary topological contraints.47–49 Self-assembling motifs
produce a rather limited kit of nanoscale morphologies which typically
include sheets and tape-like structures, fibres and fibrillar matrices, spher-
oids and nanotubes (Fig. 5).3,50 Many of these are proposed as materials for
a variety of uses including biomedical applications. However, most of them
never overcome the hurdle of ‘‘potential’’ use. At least in part this can be
explained by that many of these structures are not soluble in water, they
either precipitate or gel. Although gelation may be appealing for specific
purposes such as tissue engineering or 3D cell culture its use is unlikely to

Fig. 5 Self-assembled peptide materials. Photoluminescent peptide nanotubes assembled with

lanthanide complexes in situ. Nanotube self-assembly and photosensitizer molecules gives en-
hanced lanthanide photoluminescence through a cascaded energy-transfer mechanism (J. Ryu,
S. Y. Lim and C. B. Park, Photoluminescent Peptide Nanotubes, Adv. Mater., 2009, 21, 1577.
Copyright Wiley-VCH Verlag GmbH and Co. KGaA. Reproduced with permission).
extend beyond topical medications or to help formulate a pharmaceutical

product. Designed self-assembled stuctures are often similar to naturally
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occuring analogues associated with nanostructured accumulations or de-

posits that derive from abnormal folding or misfolding and in vivo can
develop into lesions, such as senile plaques, which can lead to undesired
amyloidogenic conditions.51 In this context, self-assembling motifs can in-
stigate pathogenic responses if used in medicine and one should take care to
avoid such corruptive protein templating of de novo design.52
b-structure designs which have dominated the field are known to have a
tendency for aggregation. To design a discrete and stable b-structure
remains one of the major design challenges. A functionally discrete b-
structure has yet to be reported. Little different is the situation with helical
assemblies. Although these are abundant in biosystems ranging from
intermediate fillaments and transmembrane pores to viral core shells,35,53,54
the assembly and maturation of helical designs is difficult to control with
typical products being fibrillar or spherical precipitates. Other approaches
using simpler motifs such as peptide amphiphiles or short hydrophobic
peptides can also give b-structured materials of comparable characteristics
and properties.55 More recent efforts focused on emulating conserved
naturally occuring motifs in the same, native, functional context. The
collagen assembly is a notable example. Its main purpose is to support the
extracellular matrix formation and consequently its role of promoting cell
growth and tissue development. A persistent difficulty in collagen designs is
synthesis, a limitation imposed by the conserved sequence pattern of
glycine, proline and hydroxyproline residues, (Gly-Pro-HyPro)n. Varied
compositions of the pattern in synthetic collagens have been introduced to
enable the assembly of collagen heterotrimers, which in some instances

Fig. 6 Collagen mimetic peptides. (A) Models of electrostatic interactions for Arg-Glu (left)
and Lys-Asp (right) in collagen triple helices. The hydrogen-bond lengths are shown measured
from N to O. In contrast to Arg-Glu interactions, two stable conformers of lysine in Lys-Asp
are formed and enable hydrogen bonding to aspartic acid. (B) Chemical structures of common
amino-acid triplets. (reprinted by permission from Macmillan Publishers Ltd: (L. E. O’Leary,
J. A. Fallas, E. L. Bakota, M. K. Kang and J.D. Hartgerink Multi-hierarchical self-assembly of
a collagen mimetic peptide from triple helix to nanofibre and hydrogel., Nat. Chem., 3, 821),
copyright (2011))
01/12/2013 14:44:38.
prove to produce materials with properties reminiscent of native collagen

materials.56–59
A promising paradigm is the use of oppositely charged strands in collagen
triple helices. This is achieved by introducing cationic (lysines) and anionic
(aspartates) amino-acid residues into collagen amino-acid triplets as shown
in Fig. 6.57 The formation of stable conformers of charged residues and
their accessibility for electrostatic interactions is important for contiguous
electrostatic networks between the strands, which ensures the assembly of
extensive collagen fibrillar structures.
4 Current trends: environment-defined functions

Autonomous sequences and context-dependent folding
The main determinant for specifying the function of an autonomously
folded peptide is its immediate environment, be it a membrane, the active
site of an enzyme, serum matrix or an intracellular organelle.60 Environ-
ments support folding, which for peptides that are smaller and less stable
structures than proteins, is critical. However, environments scarcely dif-
ferentiate between secondary structures, albeit being selective when it con-
cers the relative ratios of hydrophobic and polar residues in the sequence.
Similarly, peptides can adopt structures that are unusual or untypical but
can uniquely fit into a specific environment. For instance, cyclotides – a
class of cyclic peptides originated from plants – have an arrangement of

Fig. 7 NMR structure of a prototypic cyclotide kalata B1 (left, 1N1U PDB entry rendered
with PyMol) and a schematic representation of cyclotides highlighting one disulphide bond
(vertical black) penetrating the ring (gray) formed by the other two disulphides (right, repro-
duced with permission from Ulf Goransson and David J. Craik. Disulfide mapping of the
cyclotide kalata B1, J. Biol. Chem., 2003, 278, 48188–48196).
hydrophobic residues which is distinctive among all other folds.61 Cyclo-

tides are head-to-tail cyclised miniproteins, the backbone of which is folded
back onto itself to surround a tightly packed cystine core (cystine knot).62
Because in this arrangement the core is occupied by disulfide bonds all other
side chains including those of hydrophobic amino acids, which are normally
clustered in peptide cores, are forced to the outside creating thus an inside-
out arrangement (Fig. 7).63 This gives cyclotides extended hydrophobic
surface patches and consequently defines their main function as plant host
defence agents.
Cyclotides represent a structural adaptation towards function. However,
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this is not very common. Other host defence peptides possessing the
same range of activities as cyclotides employ more conventional strategies.1
Being arguably the most diverse class of sequences having related functions
host defence peptides, also and more commonly known as antimicrobial
peptides, are typically amphipathic cationic structures which preferentially
bind to anionic microbial surfaces and function by causing membrane
disruption and lysis in microorganisms.64 Different views exist with regard
to the exact mechanisms of their action, particularly given that there are
antimicrobial sequences that are not membrane active, do not fold, but
translocate across microbial membranes targeting intracellular com-
ponents.1,64 Yet, in design terms preference remains for folding-mediated
sequences that would be able to differentiate between bacterial and mam-
malian membranes.1,44
Antimicrobial peptides are found in all multicellular organisms and their
sequences can be generalised according to secondary and supersecondary
structures they adopt (Fig. 8).1 Synthetic peptides reported to date that can
be hybrids of native sequences or purely artificial sequences are mainly
designed to improve the therapeutic ratio between increased antimicrobial
efficiencies and reduced toxicity.1,44 Many designs are structural simplifi-
cations of native sequences, whereas the usual purpose of de novo designs is
to produce more potent sequences or to probe a specific mechanistic or
design rationale.
Antimicrobial designs provide excellent examples of environmentally
defined functions. Differential membrane binding in conjunction with
amphipathic folding are a priori, though not exhaustive, functional

Fig. 8 Structural classification of antimicrobial peptides; each structure is given as a 16-

dimensional vector coding for the prevalence of backbone torsion angles. (Reprinted by per-
mission from Macmillan Publishers Ltd: Nat. Rev. Drug Discov. (C. D. Fjell, J. A. Hiss, R. E.
Hancock and G. Schneider, Designing antimicrobial peptides: form follows function, 11, 37),
copyright (2011)).
requirements. Peptides of other functional classes can also be represented by

different structural motifs that can be amphipathic and their activities
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can be membrane mediated. Fusogenic peptides constitute one example.

These peptides derive from membrane proximal regions of integral mem-
brane proteins that mediate fusion between viral and cellular membranes (e.g.
protein hemagglutinin of influenza viruses or HIV gp41).65 They share similar
mechanistic patterns of fusion processes, which include binding to and in-
sertion into the target membrane. Fusion or fusogenic peptides are short
amino-acid stretches that act as molecular anchors.66 In contrast to other
domains in fusion proteins that never cease to mutate, these stretches are
highly conserved as they are the only structures that insert into the target
membrane, and have to do this reproducibly over generations.67 To com-
pensate such a lack of sequence plasticity, which is never an issue for anti-
microbial peptides, fusogenic peptides exhibit significant conformational
plasticity under constantly changing membrane environments, compositions
and orientations. This property allows membrane proximal and fusogenic
peptide stretches to respond to the cascades of conformational changes that
occur at different fusion stages, are regulated by external physico-chemical
stimuli (temperature, pH) and may invovle synergistic interactions between
the two end regions of the same protein (Fig. 9).68
Thus, a priori criteria for fusogenic designs can be formulated as inclusive
of an ability to insert into membranes, to remain immersed or anchored and
not to assemble in the lipid bilayers leaving the membrane intact.66–68 A
secondary structure element to meet these requirements would be a kinked
helix that would be able to obliquely sink a halfway into the monolayer
facing the viral envelope.66 This elemental difference in functional expres-
sion is critical when compared with antimicrobial peptides.67

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Fig. 9 Molecular dynamics simulation of synergistic interactions between gp41532–544 and

gp41659–671 fusogenic stretches of HIV gp41. (A) An initial anti-parallel arrangement of the
stretches evolves into (B) a perpendicular arrangement. (C) A hydrophobic interface (light grey
(orange online)) comprising Leu535, Trp666, Leu669, and Trp670 (left), and a complete molecular
surface (dark grey (blue online)) with the interface in the centre (right). (Reproduced from
Ref. 68 with permission from the PCCP Owner Societies).
In marked contrast to fusogenic peptides, antimicrobial peptides do as-

semble in microbial membranes and often with the formation of membrane-
disrupting pores or channels as their functional rationale is not to mediate
membrane fusion, but to damage and destroy membranes, not necessarily

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specifically, but rapidly and within their proteolytic life span.1

Mechanistically related to antimicrobial peptides are bacterial protein
toxins (a-hemolysins) which also assemble in membranes and form pores
whose diameters are just sufficient to induce the rapid discharge of vital
nutrition resources from host cells. In this case and in contrast to the
function of antimicrobial peptides, associated cell death is not a goal but a
consequence of pore formation. To afford such a function protein toxins are
very conserved architectures – a feature they share with the fusogenic do-
mains of fusion proteins,69,70 which is the exact opposite for antimicrobial

peptides that deliberately lack any a priori topological constraints on
assembled structures they can adopt in lipid bilayers.
Therefore, a priori criteria for antimicrobial designs centre around their
ability to assemble at solution-membrane interfaces, within membranes,
but not in solution. Similar to fusogenic peptides, a physical element to be
prescribed to such a feature would also be a helix, but a helix with an ability
to fully imerse into lipid bilayers and oligomerise in membrane environ-
ments, and only in membrane environments.
Indeed, many families of antimicrobial peptides including magainins,
cecropins, bombinins, dermaseptins and others are helical and oligomerising
in membranes.1,44,64 However, amongst more than 1000 antimicrobial pep-
tides known to date the mechanisms of action for only a few have been
studied and, consequently, only a few physical models have been proposed to
describe the mechanistic rationale of antimicrobial peptides. All these mod-
els, which include barrel-stave pore,71 torroidal pore72 and carpet models,73
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differ according to the specific interactions of the peptides with phospholipid

bilayers, but all are thought to involve two distinct peptide-lipid states74 – an
inactive surface-bound S-state and a pore-like insertion I-state (Fig. 10).
Most recently, a pore expansion E-state which follows I-state and pro-
motes cooperative peptide migration in the lipid matrix to the point of
complete membrane removal has been proposed (Fig. 10).75 The E-state
reflects the functional rationale of antimicrobial poration as an adaptation
mechanism for antimicrobial peptides to freely diffuse in lipid bilayers;
that is, without restrictions imposed by local energetics on lateral expan-
sion. To demonstrate this, an archetypal transmembrane sequence was
Fig. 10 Proposed pore expansion mechanism for amphipathic antimicrobial peptides. Anti-
microbial peptides (grey cylinders (blue online)) bind to the surface of the membrane (S-state),
insert into lipid bilayers forming pores (I-state), which can then expand indefinitely (E-state).
(reproduced from P. D. Rakowska et al. Nanoscale imaging reveals laterally expanding anti-
microbial pores in lipid bilayers, Proc. Natl. Acad. Sci. USA, 110, 8918. Copyright (2013)
National Academy of Sciences, U.S.A).

Fig. 11 Antimicrobial peptide insert. (A) Sequence configured onto an a-helical wheel with 3.6
residues per turn showing the clustering of amino acids of same types and i, i þ 7 amino-acid
pairs coloured, different shading (colours online) denote different pairs. (B) Linear sequence
aligned with the repetitive heptad patterns, PPPHPPH (upper) and CNCHNCH (lower), where
P – polar, C – cationic, H – hydrophobic and N – neutral. For clarity i, i þ 3 and i, i þ 4 helical
spacings are shown for N residues only. (C) Schematic representation of a pore edge showing
the thickness of a lipid bilayer matched by the folded peptide. For clarity, only one peptide
01/12/2013 14:44:38.
(grey cylinder= an amphipathic helix (2ZTA PDB entry rendered with PyMoL)) and one
phospholipid per layer (aliphatic chains in light grey, headgroups as spheres (pink online)) are
shown. (Reproduced from P. D. Rakowska et al. Nanoscale imaging reveals laterally
expanding antimicrobial pores in lipid bilayers, Proc. Natl. Acad. Sci. USA, 110, 8918.
Copyright (2013) National Academy of Sciences, U.S.A).
designed to insert into supported lipid bilayers which were then analysed in
great detail using nanoscale imaging.
The design can serve as an example of an environmentally defined sec-
ondary motif whose activity and physical dimensions were precisely tuned
to match those desired. The designed sequence is an experimental model of
helical antimicrobial peptides which uses three i, i þ 7 residue modules,
where i and i þ 7 are of the same type and placed next to one another along
the helix axis characterised by i, i þ 3 and i, i þ 4 helical spacings,76 which
are formed by repetitive heptad patterns, CNCHNCH (C – cationic, H –
hydrophobic and N – neutral).75 This geometry enables the folding of a
contiguous amphipathic helix in phospholipid bilayers (Fig. 11). The im-
mersed helix having 0.54 nm per turn and the thickness of the used bilayer
are matched, both spanning B3.2 nm (Fig. 12).
When imaged by atomic force microscopy, which provided the topo-
graphic detail of peptide-treated bilayers, heterogeneous pores with diam-
eters as large as several microns were observed. The same samples imaged
by nanoscale secondary-ion mass spectrometry, which gave complementary
chemical information regarding peptide content and localisation, revealed
that the interior of the pores was completely free of peptide, while their

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Fig. 12 Nanoscale imaging of antimicrobial poration in supported lipid bilayers. (A) atomic
force topography micrographs. (B) secondary ion mass spectrometry images of 12C14N,
12 15
C N , and 12C15N/12C14N signals from the supported lipid bilayers treated with
isotopically (12C15N) partially labelled peptide. (C) atomic force topography micrographs
taken during incubation with peptide. Colour scale (see inset, 0 min): 3 nm (0–20 min); 9 nm
(30–120 min). (Reproduced from P. D. Rakowska et al. Nanoscale imaging reveals laterally
expanding antimicrobial pores in lipid bilayers, Proc. Natl. Acad. Sci. USA, 110, 8918.
Copyright (2013) National Academy of Sciences, U.S.A).
edges were saturated with peptide whose content increased with increasing
pore sizes. Real time changes in lipid bilayers monitored by atomic force
microscopy in water indicated the beginning of visible pore formation after
10 min of incubation and continued growth in pore sizes and numbers until
two hours at which point lipid was completely removed from the surface.
Moreover, cross sections of pores measured by atomic force microscopy
showed that pore edges and depths precisely matched the size of inserted
folded helices consistent with the design.

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Collectively, these results underpin a new mechanistic basis of pore for-

mation by antimicrobial peptide which can be described in generic terms of
a physicochemical process of cooperative and continuous peptide recruit-
ment in membranes.75
Note should be taken here that an ability of antimicrobial sequences to
porate membranes cannot, though often taken as such, be an a priori re-
quirement for antimicrobial activity. However, poration can help visually
relate established or new mechanistic rationales to function.
Another example to demonstrate poration engineering is the design of

transmembrane pore-forming structures. The choice of a suitable secondary
structure that can enable such an arrangement is entirely driven by the
membrane environment, which imposes specific limitations to design: helix-
forming peptides cannot assemble into hydrogen-bonded barrel structures
and are primarily stabilised by hydrophobic interactions, which in phos-
pholipid bilayers are provided by lipid aliphatic chains, whereas b-strands
can readily form barrel structures through hydrogen bonding without the
support of lipids. Another aspect to consider is whether formed barrels are
sufficiently contiguous to span the lipid bilayers and are progressively
aggregating to cause membrane rupture. A successful approach set out to
build a contiguous network of hydrogen bonds running symmetrically
through a hollow cylinder.77 This geometry gives rise to transient tubular
structures that can disrupt membranes by allowing the passage of water
molecules and ions (Fig. 13).77 Cyclopeptides constructed with alternating
D and L amino acids in a fashion similar to that of naturally occuring
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polypeptide antibiotics, i.e. gramicidins, just achieved this; the peptides

stack on each other with their conformation locked into flat rings that form
hollow nanotubes cemented by an infinite chain of hydrogen bonds. The
alternating arrangement of D- and L-amino acids in the peptide backbone
ensures the positioning of side chains on the outside surface of the assembly
which interact with lipids in the bilayer (Fig. 13).78
D amino acids are commonly used for the design of antimicrobial pep-
tides with improved therapeutic properties. All-D antimicrobial peptides
prove to possess at least similar biological activities but superior enzymatic
stabilities when compared to all-L analogues.79–83 In many instances, all-D
sequences can be useful tools for targeting specific biomolecular processes
where prolonged antimicrobial activity is required. Examples include pro-
apoptotic sequences that trigger mitochondria-dependent apoptosis in
cancer cells and need longer circulation times,84 and antimycobacterial se-
quences as potential anti-tuberculosis agents that act on slowly maturing
mycolate-rich cell walls of Mycobacterium tuberculosis.18
However, sequence conversions from L to D cannot solve issues such as
haemolytic activity which remains to be generic for antimicrobial amphi-
philes and often is associated with peptide amphipathicity. Indeed, all-D
peptides can also form amphipathic structures.79–84 An alternative ap-
proach is to design sequences that lose their ability to fold or do not fold as
amphiphiles, or at least amphipathic folding cannot be detected, but retain
antimicrobial activity.85 This strategy holds promise for decreased haemo-
lytic and cytolytic activities, which otherwise correlate strongly with sec-
ondary structure propensities. Diastereomeric sequences, those designed

Fig. 13 Schematic representation of cyclopeptides assembled into a tubular configuration

inserted into a lipid bilayer. Antiparallel ring stacking with extensive hydrogen-bonding
01/12/2013 14:44:38.
interactions between the peptides are shown. (Reprinted by permission from Macmillan Pub-
lishers Ltd: (M. R. Ghadiri, J. R. Granja and L. K Buehler, Artificial transmembrane ion
channels from self-assembling peptide nanotubes, Nature, 369, 301), copyright (1994)).
with different distributions of D amino acids along the hydrophobic and

hydrophilic faces of L peptides, were shown to be unstructured, less
haemolytic and strongly antimicrobial,86,87 which in conjunction with their
increased stability profiles makes them promising antibiotic candidates.
Most recently, stereochemical variation series of amino acids with hydro-
phobic (Phe, Leu) and cationic (Lys) side chains were used to generate random
sequences.88 Libraries of peptides with controlled chain lengths (20 residues)
and subunit proportions were chemically synthesised using combinatorial
chemistry strategies. Homochiral (L, L or D, D) and heterochiral (L, D or D,
L) binary mixtures were used to reveal that only heterochiral binary mixtures
can achieve selective antibacterial activity (Fig. 14).
Other antimicrobial peptides that can provide reasonable therapeutic
ratios include sequences with alternating Pro and Arg residues,89 the action
of which does not appear to be mediated by membrane binding, or di-, tri-
and tetra-peptide pathogen-associated motifs which are too short to fold
but still bind to microbial membranes.90 In this light, it seems that a pos-
teriori requirements for antimicrobial activity of host defence peptides
concentrate on a combination of membrane activity, binding or folding, and
self-assembly or aggregation in membranes. This is in contrast to fusogenic
peptides for which folding in membrane is sufficient. Using the same
reasoning membrane-independent self-assembly can be expected to define

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Fig. 14 Solid-phase synthesis methodology for the preparation of 20-mer random peptide
polymers from homo- and hetero-chiral binary mixtures, (reprinted with permission from
Z. Hayouka, S. Chakraborty, R. Liu, M. D. Boersma, B. Weisblum and S. H. Gellman,
Interplay among Subunit Identity, Subunit Proportion, Chain Length, and Stereochemistry in
the Activity Profile of Sequence-Random Peptide Mixtures. J. Am. Chem. Soc., doi: 10.1021/
ja406231b. Copyright (2013) American Chemical Society).
different activities of the same sequence, if it were to fold or aggregate au-

tonomously. For instance, it has been shown that membrane-bound pep-
tides can change their orientation on membrane surfaces as a function of
concentration and consequently switch between fusogenic and anti-
microbial mode of action.91
Fusogenic peptides, but not antimicrobial peptides, at physiologically ir-
relevant higher concentrations may assemble into inactive (non-fusogenic)
microscopic morphologies, which is a result of conformational changes and
typically occurs with the formation of b-structured amyloid-like forms.92 This
is the point where the uniqueness of sequence composition loses its role and
functional appeal because amyloid-type aggregates can form by different
01/12/2013 14:44:38.
peptides, proteins or protein fragments, and yet morphologically all are re-
markably similar, and despite that each disease is associated with the amy-
loidogenesis of a unique protein it is a universal phenomenon with a common
biological role.33 In addition, growing evidence suggests that pre-fibrillar or
soluble oligomers are the toxic formations that damage cellular membranes,
but neither pre-fibrillar or fibrillar forms necessarily depend on membrane
binding.93,94 Thus, finding a suitable sequence model is not to reflect the
properties of an independent and hence predictable element, but the variety
of its oligomers. Consequently, sequence composition is much less important.
Therefore, another class of therapeutically relevant and environmentally
defined sequences which do not benefit from membrane-induced switching
or, although being membrane-active, do not interfere with the membrane
structure, can be anticipated. Cell-penetrating peptides may just fit the bill.5
Cell-penetrating peptides, aslo known as protein transduction domains,
attract attention mostly because of their unique ability for intracellular
delivery.5,95 These molecules are relatively short, fewer than fifteen amino
acid residues, and effectively transverse plasma membranes mediating the
delivery of different cargo ranging from small molecules to nanoparticles.
Typical naturally occurring peptides are antennapedia homeodomain of
Drosophila, the transactivator of HIV transcription (TAT) protein, HSV
DNA-binding protein VP22, mastoparan and others (Fig. 15).95,96
The cationic nature of these peptides makes them similar to fusogenic and
antimicrobial peptides. However, in contrast to the other two these mol-
ecules are shorter, predominantly hydrophilic and their structuring does not
appear to occur at any transfection stage suggesting a distinctive and

Fig. 15 Schematic representation of an autonomous pathway for HIV TAT cellular trans-
duction. Cell-penetrating TAT peptide (dark elongated cylinder (blue online)) induces mem-
brane curvature and enters through an induced pore, but large cargo (largest spheres (white
online)) it carries cannot, the peptide then interacts with cytoplasmic actin (chains of spheres
(green online)) promoting cellular uptake of the cargo via endocytosis (Reproduced from A.
Mishra et al., Translocation of HIV TAT peptide and analogues induced by multiplexed
membrane and cytoskeletal interactions, Proc. Natl. Acad. Sci. USA, 108, 16883. Copyright
(2011) National Academy of Sciences, U.S.A).
01/12/2013 14:44:38.
Fig. 16 Heterotrimeric collagen motif incorporating arginine residues (blue) forming a cat-
ionic, cell-penetrating, face on the folded triple helix – the conformation which provides the
motif with a high resistance to proteases. (C. M. Yamazaki , I. Nakase, H. Endo, S. Kishimoto,
Y. Mashiyama, R. Masuda, S. Futaki and T. Koide, Collagen-like cell-penetrating peptides,
Angew. Chem. Int. Ed. Engl., 2013, 52, 5497. Copyright Wiley-VCH Verlag GmbH and Co.
KGaA. Reproduced with permission).
folding-independent mechanism of action, which is believed to invovle

endocytosis, with some sequences capable of targeting nucleus.95–98 Passive
translocation resulting from fairly strong buffering capacities of cell-
penetrating peptides is also acknowledged and directly linked to their
sequence compositions that are of high-density cations which are prone to
protonation at virtually any pH. Indeed, poly- and oligo-arginines, for which
cell membranes are merely a barrier that remain intact and unaffected, are
widely accepted to enable intracellular trafficking and delivery.99 Arginine
oligomers can be used as stand-alone sequences, in which case they remain
susceptible to enzymatic degradation, or can be stabilised and somewhat
ordered when incorporated into pre-folded or self-assembled structures.
For example, the inclusion of arginine residues in collagen motifs, (Gly-
Pro-Arg),100 in all three strands of a collagen triple helix endows them with
enhanced transfection properties and proteolytic stability and by association
can render the construct an efficent gene delivery vector (Fig. 16).

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Although it may be sensible to consider cell penetrating peptides as gene

delivery agents, their own uptake and that of cargo they may carry are not
necessarily the same. In both cases, one barrier to overcome is peptide
endosomes. Oligoarginines as well as other transduction domains become
entrapped in endosomes, cannot easily escape from them and remain sub-
ject to lysosomal degradation.101 Amphipathicity, as in the case of anti-
microbial function, is believed to provide a structurally responsive means to
promote the endocomal escape of cell penetrating peptides into the cyto-
plasm.102 The argument is supported by observations in non-enveloped

viruses that lack lipid components, which otherwise are known to facilitate
the escape, but egress into the cytoplasm by lysing endosomal membranes
with amphipathic helical domains incorporated into their capsid pro-
teins.103 Peptide design allows mimicking this mechanism, which was shown
to efficiently mediate plasmid DNA delivery into primary cells and also
compete with viral infection by counteracting the development of low pH
environment in endosomes during virus internalisation which is necessary
for viral disassembly and the activation of amphipathic capsid domains
(Fig. 17).104
Using the analogy of viral amphipathic domains it should be possible to
design amphipathic sequences capable of active traversal across cell mem-
branes and active nucleic acid transport. A successful approach can be
exemplified by de novo peptides that fold into amphipathic domains which
can penetrate eukaryotic cells (mammalian and yeast), promote DNA
transport (EGFP positive) and undergo differential membrane-induced
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folding; that is, an ability to fold in specific membrane environments and

not in others, which enables them to exhibit both endosomolytic and
antibacterial activities.105 Such single-domain gene transporters follow the
same design principles that are applied to the design of de novo anti-
microbial sequences with additional structural considerations for nucleic
acid complexation, narrower hydrophobic faces and disfavoured formation
of high oligomers (Fig. 18). Importantly, the transporters are designed to
fold only in endosomal membranes bypassing plasma membranes.105
It appears also that amphipathic domains comprising aromatic trypto-
phan and cationic arginine residues and adopting helical conformations in
cell membranes, can form stable complexes with siRNA and promote their
delivery into a variety of cell lines through an endosome-independent
mechanism.106 However, how one can differentiate between endosomal and
endosome-independent mechanisms remains unclear. A reliable rationale
may offer new possibilities for targeted macromolecular delivery (both
nucleic acids and proteins). It is certainly important to understand sequence
preferences for delivery mechanisms including those that aim at intracellular
organelles.97 For example, several reports suggest that short stretches of
cationic residues (lysines, arginines) can facilitate nuclear targeting and
delivery and therefore can be used as nuclear localisation signals. Prefer-
ential nucleolar localisaiton was also observed for peptide sequences derived
from snake toxins and identified by the structure-guided deconstruction of
the native folds.98 These sequences contained short cationic stretches sep-
arated by di-glycinyl spacers. Both the stretches and spacers were found
in the two terminal regions of the same toxin, which are brought in close

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Fig. 17 Targeting endosomal escape by membrane-active peptides. (A) Schematic represen-

tation of the early stages of adenovirus infection cycle, including the binding of the virus to
its receptor followed by virus internalisaiton via endocytosis, active acidification in endosomes
leading to pH-dependent partial disassembly of the virus capsid and escape from endosome
via membrane lysis. (B) a proposed anti-viral mechanim of membrane associating D,L-
cyclopeptides incorporating into endosomal membranes and counter-acting the lowering of pH
inside endosomes via the formation of membrane permeating nanotubes. (Reprinted from Bioorg.
Med. Chem., 13, W. S. Horne, C. M. Wiethoff, C. Cui, K. M. Wilcoxen, M. Amorin, M. R.
Ghadiri and G. R. Nemerow, Antiviral cyclic D,L-alpha-peptides: targeting a general bio-
chemical pathway in virus infections, 5145, Copyright (2005), with permission from Elsevier).
proximity to one another by peptide folding. The ends were spliced together
into a linear sequence thereby arranging cationic residues as cell-penetrating
and nucleus-targeting clusters.98
Mimicking spatial arrangements of bioactive regions from proteins in free
peptides is an efficient strategy for the structural minimisations of different
biofunctional designs. However, of particular importance this is for signal
sequences that are no longer than five amino acids and whose compositions,
as a consequence, are conserved to a single residue. Integrin receptor an-
tagonists are typical examples, the most notable of which is RGD sequence
identified in early 1980s as a abundant cell adhesion motif for extracellular
matrix proteins starting from fibronectin.107 Although many integrins can
recognise RGD, they can also discriminate among different matrix proteins,
which implies that RGD or RGD sequences containing other, flanking,

Fig. 18 A gene transporting (GeT) domain configured into a helical wheel (top left) complexes
with a GFP-encoding plasmid DNA (pEGFP, bottom left), delivers it into human dermal
fibroblasts where GFP is expressed (fluorescence micrograph, right). (Reproduced from B.
Lamarre, J. Ravi and M. G. Ryadnov, GeT peptides: a single-domain approach to gene de-
livery. Chem Commun (Camb), 2011, 47, 9045, with permission from The Royal Society of
Chemistry).
amino acids can adopt distinct conformations.8 Native environments for

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RGD conformations, where they are spatially locked, are folded proteins
that serve as spatial scaffolds to support conformation-dependent recog-
nitions. Therefore, integrin recognition is realised at least at two structural
levels – primary, which is specified by the signal sequence, and secondary,
which is determined by its bioactive conformation. Structural optimisations
of short peptide motifs are then made at both primary and secondary
structure levels. Because peptides are intrinsically susceptible to enzymatic
degradation stabilising peptide backbones is prioritised for creating drug-
like forms.108 These are considered together with the optimisations of side
chains topologies, which often constitute the pharmacophore of a given
motif, and their positional arrangements and interactions with the back-
bone.109 The latter can be critical and can be addressed by comparing
the activities of parent, retro (total inversion of the sequence), inverso (D-
enantiomer of the same sequence), retro-inverse (both) and partial retro-
inverso sequences (Fig. 19).16,108
To meet all these structural requirements a molecular framework allow-
ing to fix the same positional arrangement of side chains in the sequence is
required. In this regard, cyclopeptides in combination with D amino acids
incorporated into specific positions prove to provide an instrumental plat-
form for screening bioactive conformations. Among many designs a se-
lective avb3 integrin inhibitor cyclo(-RGDfV-), also known as cilengitide,
can provide an excellent example.108 Despite its recent failure in phase III
clinical trials as a brain cancer drug,109 the peptide continues being tested
for other treatments and remains one of the most characterised designs in
the literature.110

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Fig. 19 Stereoisomerism in peptide design. (A) Parent peptide (top) and its retro-inverso
(middle) and partial retro-inverso (bottom) analogues. The arrows indicate the direction of the
amide bond, while the orientation of the side chains is similar in all three structures. (B)
Comparison of two different conformations of cilengitide. The peptides have nearly identical
side chain topologies but reversed amide bonds. IC50 values are given as ratios to a standard
(GRGDSPK) (reprinted with permission from J. Wermuth, S. L. Goodman, A. Jonczyk, and
H. Kessler, Stereoisomerism and biological activity of the selective and superactive avb3
integrin inhibitor cyclo(-RGDfV-) and its retro-inverso peptide, J Am Chem Soc., 119, 1328.
Copyright (1997) American Chemical Society).
The peptide was identified in a systemic study of a RGDFV cyclopeptide

series.8,108,111 All retro-inverso isomers, retro isomers and inverso isomers of
the pentapeptide were investigated to confirm that cyclo(-RGDfV-) was not
only a very potent but also a selective inhibitor of the avb3 integrin. The
pentapeptide framework was used as it was shown to provide the most
optimal matching in the orientation of arginyl and aspartyl side chains
necessary to inhibit the receptor. Hexapeptides were found less active
and tetrapeptides could not provide the necessary spacing for obvious
reasons.
The bioactive form of the peptide was believed to be in a b-turn
conformation. Therefore, one amino acid in the pentapeptide was used in
D-configuration. It is known that a D-amino acid in the i þ 1 position can
induce turns with a particular preference for a bII 0 -turn. Such a

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phenomenon can be readily exploited for the investigation of structure-

activity relationships under ‘‘conformational control’’.111 Furthermore,
cyclopeptides show a mixed bII 0 /g-turn arrangement, implying that in the
case of cyclo(-RGDfV-) with D-phenylalanine in the i þ 1 position, the
arginine residue is expected to be in the i þ 3 and a variable amino acid to
allow screening is in position i þ 5 (L-valine in the final peptide), where at
least one hydrophilic and a series of hydrophobic and aromatic amino acids
were used. Furthermore, glycine is also known to facilitate turn formation,
and due to its achirality can serve as a ‘‘D’’-residue, whether used in the
same cyclopentapeptide with or without another D-residue. To achieve the
orientation of the side chains similar to the parent peptide it is in general
advisable to construct retro-inverso peptides.108,110
However, this holds true only if terminal side chains in the sequence are
not critical for binding (for linear sequences) or if they are arranged in a
cyclic system. Unfortunately, the biological activity of retro-inverso pep-
tides is typically different from that of their parent peptides, which may be
explained by at least that peptide bonds, which are specific for interactions
with the receptor in parent peptides, in retro-inverso analogues do not fit
(Fig. 19). Several parent peptides based on the outlined arrangement can be
designed, all of which can be viewed as variants of cyclo(-RGDfV-), which
has a bII 0 /g-turn conformation with D-phenylalanine in the i þ 1 position of
the bII 0 -turn. The g-turn conformation is not well defined and this part of
the backbone can still be flexible. All other parent peptides can have dif-
ferent degrees of flexibility which would be reflected in less pronounced
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hydrogen bonds, whereas their retro-inverso analogues would have inverted

turns (Fig. 20).111
These principles can be applied to other motifs. However, the size of a
cyclopeptide framework depends on the the length of the epitope sequence.
Fig. 20 Schematic representations of parent (P) and their retro-inverso (RI) analogues
of the RGD cyclopentapeptides. The turn-inducing residues are highlighted by a circle and bold
italic. Hydrogen bonds that are less pronounced are indicated by broken arrows (reprinted with
permission from J. Wermuth, S. L. Goodman, A. Jonczyk, and H. Kessler, Stereoisomerism
and biological activity of the selective and superactive avb3 integrin inhibitor cyclo(-RGDfV-)
and its retro-inverso peptide, J. Am. Chem. Soc., 119, 1328. Copyright (1997) American
Chemical Society).

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For example, the conformation of the cell adhesion motif YIGSR identified
in the b1 chain of laminin112 and constrained using cyclic hexapeptide
frameworks113 with one and two D-amino-acid position was found to have
a strong preference for turn conformations. Observed differential responses
in mediating metastatic tumour adhesion and strong affinity to laminin
receptor 1 and associated integrins prompts the rational design of synthetic
disintegrins – receptor antagonists that specifically block cells from binding
to the extracellular matrix.114 Indeed, the peptides have no impact on
proliferation rate or density of tumour cells and do not alter their tumor-
igenicity, suggesting a non-toxic mechanism of action and an efficient
strategy for anticancer therapy.115
5 Current trends: environment-supporting functions

Self-assembling sequences
Sequences that self-assemble do not require specific or supporting en-
vironments, but once assembled impact on the environments, be these live
cells, biological surfaces and interfaces or membranes. Autonomously fol-
ded super-secondary motifs can be viewed as the simplest self-assembling
systems (see 4). They can underpin early folding events without necessarily
being folding intermediates of a compact folded form, and can be relatively
stable, but homogeneous, oligomeric structures with a specific function.
Different types of discrete b-type topologies, helix bundles and mixed
topologies that were described in the previous SPR volume can provide
01/12/2013 14:44:38.
examples.15 Perhaps among the most illustrative examples are the lowest
oligomers such as two-stranded coiled coils – bundles of unconnected
helices. Coiled coils are characterised by heptad repeats (canonical pat-
terns), (HPPHPPP)n, which are designated abcdefg, with a and d being
buried in a hydrophobic interface formed by contiguous hydrophobic seams
contributed by each helix. This packing distinguishes coiled coils from
globular proteins. Because the average spacing of a and d residues along the
sequence (3.5 residues) falls short of one complete turn of an a-helix (3.6
residues), each seam adopts a left-handed twist allowing the association of
helices in the bundle with left-handed helix-crossing angles.116
The native coiled-coil systems are rich in function and can include viral
fusionproteins,117 SNARE complexes,118 membrane skeletal networks119 or
harbor domains of enteric pathogens.120 Each of these relies on the specific
ability of coiled coils to maintain interfacial contacts in a highly cooperative
manner. De novo helical monomers can be designed to incorporate into
such coiled coil interfaces thereby disrupting function.121 This can be il-
lustrated using six-helix bundles – the type preferred by enveloped viruses
such as retroviruses and paramyxoviruses which use it to catalyze fusion
with host cells.117,121 For instance, the trimeric protein gp41, which
assembles into a six-helix bundle and mediates fusion of HIV, adopts a
sequence of large structural re-arrangements driving the fusion.122
Promising fusion inhibitors were designed to incorporate into the assembly
including a gp41 fragment T20 (fuzeon and enfuvirtide) currently used as an
anti-AIDS drug.121 Small molecule drugs also have shown the potential to
disrupt protein-protein interactions but only when it concerns relatively

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small ‘‘hot spots’’. Protein surfaces are much larger and may be extensive
enough to resist inhibition by small molecules which cannot cover extensive
protein-protein contacts. Therefore, stable macromolecular drugs may be
more successful in challenging affinity surfaces across a large protein
interface, which is particularly relevant for fusion proteins.123 Recent at-
tempts using non-canonical backbone oligomers (Fig. 3) proved this to be
the case. Designed a/b-peptides – peptides with a subset of canonical a-
amino acid residues replaced by b-amino acid residues – generated from a
gp41 peptide were found to be potent inhibitors of HIV infection in cell-

based assays.124 A crystal structure resolved for a gp41 complexed with an
a/b-peptide revealed that the a/b-peptide backbone adopted a helical con-
formation, which despite having an extra backbone carbon in each helical
turn, was nearly identical to that of a helical a-peptide. Using PDBe PISA
(Protein Interfaces, Surfaces and Assemblies), it was possible to calculate
that the formation of this complex buries approximately 110 nm of surface
area from gp41 versus 123 nm of surface area from the a/b-peptide. Im-
portantly, key side chains that project from this helical a/b-peptide occupied
the correct sites in the groove displayed by gp41 (Fig. 21A). By changing the
positions of b-amino acid residues in the a/b-peptide combined with partial
a-b and b-a reversions along the sequence a small library of a/b-
analogues was generated to probe contributions to overall affinity from
contacts along the entire gp41-peptide interface.125 However, engineered
electrostatic arrays incorporated by a/b-peptides containing helical i, i þ 3
and i, i þ 4 ionic pairs into the interface were shown to be comparably
01/12/2013 14:44:38.
effective which suggests that complementary acidic and basic residues can
conformationally stabilise the interface thus avoiding entropically costly
ordering of side chains (Fig. 21B).126
Fig. 21 Inhibiting protein interfaces by autonomous self-assembly. (A) Complex between an

a/b-peptide (atomic detail) and a gp41 (gray) (2.8 Å resolution crystal structure; PDB 3O43)
(left), and a close-up of interaction of the Trp-Trp-Ile motif from the peptide with the com-
plementary cleft of the protein (right), (reprinted with permission from L. M. Johnson, W. S.
Horne and S. H. Gellman, Broad distribution of energetically important contacts across an
extended protein interface, J Am Chem Soc., 133, 10038. Copyright (2011) American Chemical
Society). (B) An a/b-peptide sequences with complementary acidic and basis residues forming i,
i þ 3 and i, i þ 4 ionic pairs (upper) and its complex with a gp41 (grey, lower) (2.1 Å resolution
crystal structure; PDB 4DZV), and a close up highlighting charged residues, (reprinted with
permission from L. M. Johnson, D. E. Mortenson, H. G. Yun, W. S. Horne, T. J. Ketas, M.
Lu, J. P. Moore and S. H. Gellman, Enhancement of a-helix mimicry by an a/b-peptide fol-
damer via incorporation of a dense ionic side-chain array, J. Am. Chem. Soc., 134, 7317.

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Complementary ionic arrays are being also explored for the introduction
of antagonistic interfaces by complexing with biofunctional sequences.
Antagonistic anionic helices can co-assemble with helical cationic anti-
microbial peptides whose antimicrobial activity is effectively neutralised
by the formation of inert helical oligomers (Fig. 22A).127 Because helical
antimicrobial peptides share structural similarities with coiled coils, coiled
coil interfacial patterns can be templated on antimicrobial sequences and
their mirror ionic conversions can provide antagonistic sequences able to
regulate or partially neutralise antimicrobial function. These findings are

also important in the context of antimicrobial resistance. The clinical po-
tential of antimicrobial peptides is acknowledged based on that widespread
microbial resistance against them is far too complex and costly for bacteria
to develop. However, it is possible that antagonistic sequences capable of
binding to antimicrobial sequences can be secreted by bacterial cells thus
counteracting host defence responses. Such a counteraction does not have
to be prolonged which is consistent with the functional basis of anti-
microbial peptides as local and short contact time immune regulators.76
Antagonistic mechanisms based on the formation of discrete helical bundles
can also be used for functional switching triggered by external stimuli, e.g.
pH-dependent tumour targeting. In this case, neutral complexes that are
inactive at neutral pH, which provides an environment for normal cell
growth, disassemble and become toxic towards tumours in response to
acidification which develops in tumour cells (Fig. 22B).128
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Fig. 22 Creating regulatory protein interfaces through autonomous self-assembly. (A) De

novo designed sequences acting as antagonists of native antimicrobial peptides (anti-
antimicrobial peptides). Two scenarios of antimicrobial peptide action: (upper) an anti-
microbial peptide binds to microbial membranes and forms membrane-disrupting helical
structures or (lower) when challenged by an anti-antimicrobial peptide it forms a biologically
inert helical complex. L. Ryan, B. Lamarre, T. Diu, J. Ravi, P. J. Judge, A. Temple, M. Carr,
E. Cerasoli, B. Su, H. F. Jenkinson, G. Martyna, J. Crain, A. Watts and M. G. Ryadnov, Anti-
antimicrobial Peptides: folding-mediated host defense antagonists, J Biol Chem., 2013, 288,
20162–20172. & the American Society for Biochemistry and Molecular Biology). (B) An acid-
activated antimicrobial peptide is designed to act in acidic tumour environments and to be
inactive (neutral) at normal pH, (reprinted with permission from J. Song, W. Zhang, M. Kai, J.
Chen, R. Liang, X. Zheng, G. Li, B. Zhang, K. Wang, Y. Zhang, Z. Yang, J. Ni and R. Wang,
Design of an acid-activated antimicrobial peptide for tumor therapy, Mol. Pharm., 10, 2934.

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Low autonomous assemblies designed to embed into lipid bilayers can

induce targeted membrane fusion and can serve as minimised membrane
fusion models. Cholesterol-modified sequences appear to give the most
contolled and efficient fusion, which when used in a liposomal format
provides a means for non-viral or viral-like drug and gene delivery through
induced fusion between liposomal carriers and host cells (Fig. 23A).129,130
01/12/2013 14:44:38.
Fig. 23 Low and medium-range autonomous self-assemblies. (A) Fusogenic liposomes pre-
pared from liposomes mixed with lipidated coiled-coil peptides that following their insertion
into the liposomal membranes and subsequent coiled coil formation induce membrane fusion,
(reprinted with permission from F. Versluis, J. Voskuhl, B. van Kolck, H. Zope, M. Bremmer,
T. Albregtse and A. Kros, In situ modification of plain liposomes with lipidated coiled coil
forming peptides induces membrane fusion, J Am Chem Soc., 135, 8057. Copyright (2013)
American Chemical Society). (B) Pentamer-trimer (upper) and tetramer-trimer (lower) coiled-
coil monomers (left) assembled into icosahedral and octahedral nanoparticles respectively
(right). Disordered loops in the monomers represent immunogenic domain of influenza matrix
protein 2 (reprinted with permission from S. Babapoor, T. Neef, C. Mittelholzer, T. Girshick,
A. Garmendia, H. Shang, M. I. Khan and P. Burkhard, Novel vaccine using nanoparticle
platform to present immunogenic M2e against avian influenza infection, Influenza Res Treat.,
2011, 2011, 126794). (C) Virus-like assembly of lipidated coiled-coil peptides. Lipid tails form
the core of the particle, (A. W. Perriman, D. S. Williams, A. J. Jackson, I. Grillo, J. M.
Koomullil, A. Ghasparian, Koomullil, A. Ghasparian, J. A. Robinson and S. Mann, Synthetic
viruslike particles and hybrid constructs based on lipopeptide self-assembly, Small, 2010, 6,
1191. Copyright Wiley-VCH Verlag GmbH and Co. KGaA. Reproduced with permission).

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Similar concepts are being explored in vaccine designs. In this case, virus-
like particles are assembled from sequences having different oligomerising
modules that direct the assembly of monodisperse nanoparticles. These
sequences can incorporate viral or plasmodium immunogenic epitopes (e.g.
SARS B-cell epitopes) which following the nanoparticle assembly will be
displayed on the surfaces of the nanoparticles (Fig. 23B).131–133
Not only such assemblies are conformationally stable and
morphologically monodisperse, but also they support native epitope con-
formations that are recognised by conformationally specific antibodies. To

better control nanoparticle assemblies coiled-coil folding can be coupled
with micelle formation using lipidated coiled-coil motifs. In this case, lipid
hydrophobic tails cluster together in the particle guaranteeing thus the ex-
posure of immunogenic epitopes on the particle surface (Fig. 23C).134–136
These and similar examples prompt the development of self-assembling
platforms for new vaccine designs in a manner analogous to the use of virus-
based inactivated vaccines with an advantage of creating different vaccines
by using the same structural platform.137 The platform itself however does
not have to be in a nanoparticle format. Other peptide self-assembllies can
be used to modulate adaptive immune responses. Polymer-peptide supra-
molecular assemblies or self-assembled fibrils, which are better known for
their use as cell-supporting extracellular scaffolds, are also being utilised to
elicit controlled immune response using a similar design rationale.138,139
Arguably, however, drug delivery and regenerative medicine are the ap-
plication areas in which the contributions of designed self-assembling motifs
01/12/2013 14:44:38.
are most notable. Progress to date is covered in numerous reviews and

mainly concerns the types of self-assembling structures, their character-
isation and construction principles.3,55 A general tendency is also to use
self-assembling peptide scaffolds and preferentially gels in cell culture to
generate cell encapsulating formulations and induce selective substrate-
supported cell growth and differentiation.140 Approaches are being
developed in the search for new technologies that would allow controlled
cell and tissue patterning or for self-assembly pathways that can lead to
utilisable cellular aggregates in situ.141 It was found, for instance, that by
combining amphiphile assembly with a thermal pathway one can generate
large arrays of aligned nanoscale fibres that can form 2D plaques with a
filamentous texture which can then spontaneously template long-range
alignment of bundled fibres upon cooling. The obtained alignment can be
extended over centimetres in noodle-shaped viscoelastic strings that once
mixed with live cells at physiological temperatures form monodomain gels
comprising aligned cells and filaments.141 It is proposed that such materials
can enable the fabrication of cellular wires with any length and customised
peptide composition (Fig. 24). Other approaches are focused on targeting
specific industrial applications with more research into formulations to help
improve exsiting products, which are strictly biomedical or for the purposes
of regenerative medicine. In one instance a series of recent research revealed
that anti-wrinkle properties of a commercial product Matrixyl are provided
by a specific ingredient – a self-assembling peptide amphiphile which pro-
motes native collagen production in a concentration-dependent manner
(Fig. 25A).142 The peptide assembles into tape-like structures that act as

Fig. 24 Engineered cell alignment on self-assembled peptide filaments. (A) Preferential

alignment of encapsulated human mesenchymal stem cells along the string axis. (B) Calcein-
labelled cells cultured in string. (C) Scanning electron micrographs at different magnifications
of a single cell in a string (zoom out view with arrow indicates alignment direction). (D) A
conductive black string formed by dispersing carbon nanotubes in peptide solutions before
heaing (left) and scanning electron micrograph of aligned nanofibre bundles in the black string
(right). (Reprinted by permission from Macmillan Publishers Ltd: (S. Zhang, M. A. Greenfield,
A. Mata, L. C. Palmer, R. Bitton, J. R. Mantei, C. Aparicio, M. O. de la Cruz and S. I. Stupp,
01/12/2013 14:44:38.
A self-assembly pathway to aligned monodomain gels, Nat. Mater., 9, 594), copyright (2010)).
nano-thin skin adhesives which at critical aggregation concentration

stimulate cell to produce excessive collagen. Examples of applying peptidic
self-assembled gels in tissues and in vivo also exist but remain scarce, which
may be attributed to a more complex nature of biomedical products and
regulatory requirements necessary for their introduction into the market
and to that further developments are needed in terms of converting self-
assembling structures into applied and stress-withstanding materials.3,55
Nonetheless, self-assembled fibrous materials was demonstrated to provide
a permissive environment for adult neurons to initiate axonal extensions
and as a result to regenerate through the site of an acute injury and to
knit the brain tissue together.143,144 The approach, dubbed nano neuro
knitting, showed that regenerated axons can reconnect to target tissues at
the density allowing for the return of functional vision thus providing a
precedent of a nanobiomedical technology as an effective trauma treatment
in vivo (Fig. 25B).143
In general, gelation properties supported by fibrous assembly are con-
sidered most worthy of applications. However, scaffold design at the mo-
lecular level proves to play a critical role in the development of highly
ordered tissues such as a nerve tissue. Indeed, a recently described hybrid
matrix comprising the structural and tensile properties of collagen type I
and the high density epitope display of peptide amphiphile nanofibres was
shown to induce different responses of two major neuronal cellular subtypes
of cerebellar cortex – Granule and Purkinje cells.145 Distinct cellular

01/12/2013 14:44:38.
Fig. 25 Cell-supporting self-assembled fibrous scaffolds for cosmeceutical and biomedical

applications. (A) Collagen production by human dermal fibroblasts in contact with peptide
amphiphile C16-KTTKS used in anti-wrinkle cosmeceutical applications (reprinted with per-
mission from R. R. Jones, V. Castelletto, C. J. Connon and I. W. Hamley, Mol Pharm., 10,
1063. Copyright (2013) American Chemical Society). (B) Composite fluorescence microscopy
images of thirty-day post-lesion cases in a hamster brain – from left to right – a saline control
with a large gap; a peptide-treated animal with the gap completely closed; and a sixty-day post-
peptide-treated animal. (Reproduced from R. G. Ellis-Behnke, Y. X. Liang, S. W. You, D. K.
Tay, S. Zhang, K. F. So and G. E. Schneider, Nano-neuro-knitting: peptide nanofiber scaffold
for brain repair and axon regeneration with functional return of vision, Proc. Natl. Acad. Sci.
USA, 103, 5054. Copyright (2006) National Academy of Sciences, U.S.A). (C) Schematic
representation of collagen type I fibre assembled from a collagen triple helix (left) and a model
of a hybrid nanofibre assembled from peptide amphiphile (PA) and collagen molecules (right)
(upper). Summary of responses of Purkinje cells to the hybrid matrix at various IKVAV-PA
concentrations. (S. Sur, E. T. Pashuck, M. O. Guler, M. Ito, S. I. Stupp and T. Launey, A
hybrid nanofiber matrix to control the survival and maturation of brain neurons, Biomaterials,
33, 545, Copyright (2012), with permission from Elsevier). (D) Schematic representation of
heparin-nucleated nanofibre, in which anionic heparin chains bind to the positively charged
fibre (I). In vivo angiogenesis assay. Rat cornea photographs 10 days after the placement of
various materials at the site indicated by the black arrow (II–V), heparin-nucleated PA
nanofibre with growth factors showing extensive neovascularisation (II), collagen, heparin and
growth factors showing some neovascularisation (III), PA with growth factors (IV) and col-
lagen with growth factors (V), (reprinted with permission from K. Rajangam, H. A. Behanna,
M. J. Hui, X. Han, J. F. Hulvat, J. W. Lomasney and S. I. Stupp, Heparin binding nano-
structures to promote growth of blood vessels, Nano Lett., 9, 2086. Copyright (2006) American
Chemical Society).

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responses resulted from changes in epitope concentrations in assembled

fibres and the adjustment of the epitope number allowed to achieve fine
control over dendrite and axon growth of Purkinje cells, which offers an
ability to guide neuron maturation by empirical tuning of epitope density
(Fig. 25C).145 Applications that focus on those tissues that are often con-
sidered as irreparable use self-assembled fibrous materials, for example, to
regenerate dental pulp or injured spinal cord.56,145–150
Dental pulp is a connective tissue enclosed in mineralised matrices that
together constitute a tooth, it exhibits multiple roles including immune

defence, dentin production and repair, sensory and nutritive functions.
Similar to other technologies, the incorporation of stem cells derived from
dental pulp into a fibrous hydrogel in combination with growth factors was
shown to promote cell proliferation and angiogenesis.56,147 When trans-
planted within dentin cylinders into immunocompromised mice the
hydrogel led to the formation of a vascularised soft connective tissue
morphologically and functionally similar to dental pulp.147
Spinal cord injuries are often associated with post-traumatic inflam-
mation and glial scarring which affect repair and recovery. Peptide nano-
fibre scaffolds can attenuate both these processes thereby facilitating
functional recovery. For example, a direct injection of self-assembled
scaffolds into the spinal cord tissue of rats was found to lead to significant
reductions in post-traumatic apoptosis and inflammation and to significant
tissue preservation.147 Biofunctional scaffolds can be designed to assemble
into nanofibre scaffolds after being injected as a liquid into the spinal
01/12/2013 14:44:38.
cord.145–148 The materials appear to be able to inhibit glial scar formation

and can be used in uninjured sites without behavioural sequela.149 Cellular
changes caused by such a scaffold include reductions in gliosis and cell death
at up to 400 microns from the injection site. However, observed functional
improvements did not seem to lead to full recovery and the relationships
between the observed improvements in behaviour and apparent axon re-
generation were unclear.150
Corneal regeneration is another area of extensive research due to the
potential in vision repair.151 The cornea accounts for two thirds of the total
refractive power of an eye, but corneal opacities can lead to serious vision
impairment and loss. The cornea is built as a multi-layered structure which
is sufficiently strong to withstand significant pressure. Such complexity
matches the need for high vascularisation and information-rich biomaterials
which can support its development. The mere use of self-assembling fibres
is not sufficient and requires a combinatory strategy incorporating growth
factors and anti-coagulants such as heparin (Fig. 25D).152
With the successful use of self-assembled fibrous scaffolds for biological
applications it is sensible to relate their morphological and dimensional
properties to those of native collagen and fibrin matrices.3,140 Indeed, the
exact secondary structure of designer fibres appears to be of little difference
for as long as the fibres can extend microns in length, are relatively ordered
and nanostructured, and can exhibit regular surface patterns and display
biologically relevant signal molecules. Tuning fibrous architectures proves
to give rise to different morphologies.55 However, the usefulness of these
for biology remains to be demonstrated. The same holds true for other

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self-assembling structures including capsule- or nanoparticle-like as-

semblies, which emphasises that spherical nanostructures are strongly prone
to aggregation and in many cases to environmentally-triggered transitions
to fibrillar phases.153 This in turn points to that designed capsule-like
structures are less robust and stable when compared to naturally occuring
systems such as viruses. Yet, factors that direct and discriminate between
different morphological types have yet to be understood. It may seem to be
more straightforward to rectify imperfections of helical designs that are
intrinsically characterised by greater conformational plasticity and the

ability to accommodate internal structural conflicts, which may not be as
accessible in b-structured or collagen systems. Such a preference for helical
assemblies finds particular use in designing sequences that are able to switch
from one conformational state to a thermodynamically more favourable
conformation under a particular external stress.154 However, it is the pre-
dictability power of such designs which is still lacking. Therefore, a practical
stress is being made on assemblies (irrespective of their underpinning
structure) that are accommodative of functional biomolecular modifications
such as supramolecular recruitment of folded proteins or cell adhesion
motifs. This is what to this end makes their performance comparable with
natural analogues, and the focus is on application-oriented designs, for
which structural boundaries are increasingly less defined. Nature provides
ample examples that may not appear as strictly conventional in our
understanding of functional complementarity.
For example, although it is well known that host defence peptides are fast
01/12/2013 14:44:38.
destroyers of microbial membranes their ability to self-assemble or rather a

biological need for this ability has not been a specific subject of research.
Yet, as recently shown human a-defensin 6 – a host defence peptide that
lacks appreciable bactericidal activity, still promotes immune protection
against invasion by enteric bacterial pathogens in vitro and in vivo. The
peptide binds to bacterial surface proteins after which it undergoes ordered
self-assembly with the formation of fibrils and nanoscale nets that surround
and entangle bacteria.155 This finding is unique but not exhaustive of nat-
ural examples, which somewhat are anticipated in designs – self-assembling
antimicrobial agents are represented by fibrillar and network-like systems as
well as nanoparticles that all exhibit comparable activities and mechanisms
of action similar to antimicrobial peptides (Fig. 26).156–159 Thus, primary
peptide amphiphiles comprising cationic cell-penetrating and membrane-
permeating domains can be designed to form core-shell nanoparticles
without adopting any specific conformation.156,157 Conversely, hydrogels
can be produced through the formation of fibrous networks assembled
from designed b-hairpin motifs.158 The hydrogels are antibacterial and
can provide antimicrobial responses in bacteria-contaminated cell culture
(Fig. 26B).159 Same designs were used to exploit poration mechanisms for
anti-cancer activities otherwise typical of antimicrobial peptides, demon-
strating thus the biological versatility of self-assembled b-hairpins.160
Antimicrobial activities of b-structured fibrils should not come as a surprise
given that the structures are related to b-pleated amyloid assemblies which
not only reveal very similar supramolecular properties and characteristics
but are also inherently antibacterial (Fig. 26C).161

Fig. 26 Engineered antimicrobial self-assembling peptide systems. (A) Core-shell nano-

particles (electron micrographs, upper) assembled from an amphiphilic peptide comprising
cholesterol, glycine, arginine and TAT HIV peptide (lower). Assembled particles exhibit broad
antimicrobial properties against bacteria, yeasts and fungi. (Reprinted by permission from
Macmillan Publishers Ltd: (L. Liu, K. Xu, H. Wang, P. K. J. Tan, W. Fan, S. S. Venkatraman,
L. Li and Y.-Y. Yang, Self-assembled cationic peptide nanoparticles as an efficient anti-
microbial agent, Nat. Nanotechnol., 4, 457), copyright (2009)). (B) Fluorescence micrographs
(xy projections) of E. coli incubated on a borosilicate control surface (upper left) and 2 wt%
peptide hydrogel (upper right) after 24 h. Gel is viewed parallel to the z axis. Light grey (green
online) and central grey (red online) indicate live and dead cells, respectively. xy projections of
01/12/2013 14:44:38.
E. coli incubated on a 2 wt% peptide hydrogel surface viewed perpendicular to the z-axis
(lower). Arrows point to the gel-bacterial interface, (reprinted with permission from, D. A.
Salick, J. K. Kretsinger, D. J. Pochan and J. P. Schneider. Inherent antibacterial activity of a
peptide-based beta-hairpin hydrogel, J. Am. Chem. Soc., 129, 14793. Copyright (2007)
American Chemical Society). (C) Amyloid b-hairpin peptides known to form amyloid fibrils
assemble into antimicrobial channels in phospholipid membranes. Antimicrobial peptide
protegrin-1 killing microorganism through a channel-forming mechanism can form extended
amyloid fibrils through a similar hairpin-like mechanism, (reprinted with permission from, B.
L. Kagan, H. Jang, R. Capone, F. Teran Arce, S. Ramachandran, R. Lal and R. Nussinov,
Antimicrobial properties of amyloid peptides, Mol. Pharm., 2, 708. Copyright (2012) American
Chemical Society).
The applications of self-assembling peptides are diverse so are the

directions they take in different areas which are starting to expand to
diagnostics162 and synthetic biology163 or the cell-free production of
membrane-associated proteins such as G-protein coupled receptors which
once stabilised can be used as functional nanobiodevices.164 However,
materials science and nanotechnology provide natural niches for new
technologies which go beyond designs of specific nano-to-microscale
structures and are begining to adapt the ability of self-assembling sequences
to influence and define their environment to their context-dependent fold-
ing, moreover, to adapt functionally and at the nanoscale (Fig. 27).
Such a synergistic or chameleon behaviour is being exploited in designing
hybrid materials in which self-assembling peptides can sense properties of
other nanoscale materials such as carbon nanotubes whose properties are
expressed at the nanoscale. A hybrid design can incorporate a peptide which
would be able to switch from a disordered state to an a-helical state, and

Fig. 27 Peptide self-assembly sensing the surface curvature of carbon nanotubes, (reprinted
with permission from W.-J. Jeong, S.-J. Choi, J. S. Choi and Y.-B. Lim, Chameleon-like Self-
Assembling Peptides for Adaptable Biorecognition Nanohybrids, ACS Nano, ASAP. Copy-
right (2013) American Chemical Society).
vice versa, in a highly cooperative manner, and would adopt very stable,
discrete and thermally-induced structures with high affinities for target
RNA.164
This chapter has highlighted or rather touched certain aspects of current
trends in biofunctional peptide design without in-depth analysis and cov-
erage of all reported systems, which may not be achievable in a single
01/12/2013 14:44:38.
volume. The given overview aims to provide the reader with sufficient in-
formation to help stimulate follow-up reading and potential interest in
contributing to the advancement of the field. The outlined trends are likely
to retain their current pace with emerging technologies enriching the rep-
ertoire of available develops.
It is reasonable to expect that future designs will increasingly use the
combination of computational and experimental approaches. Both aspects
of computational input including prediction algorithms and molecular dy-
namics simulations will continue influence the field. More designs can be
anticipated along the lines of novel peptide-like backbone oligomers and
their folding motifs, also novel but similar to known protein motifs, and
unprecedented self-assembly pathways leading to potentially novel meso-
scopic morphologies. In this context, one can expect that designs based on
conventional secondary structure motifs and structure-function links will
serve as analogy templates for unconventional proof-of-principle structures.
By providing input parameters for computational analysis and predic-
tion, conventional designs adjust specific assemblies and super-secondary
structures whose folding, oligomerisation state and unltimately function can
be tailored and programmed in artificial designed sequences (Fig. 28).165
Other aspects of functional designs as to more detailed mechanisms are
due to the development of more advanced measurement methods and tools.
Nanoscale imaging and super-resolution imaging have already demon-
strated their significant and exploitable potential to advance context-
dependent peptide design75 and the characterisation of native assemblies at
the molecular and sub-cellular scales.166 The commercial development of

Fig. 28 Experimentally confirmed computational design of a self-assembling foldamer. The

designed sequence packs as a b-314 helix which assembles into hexameric coiled coil – the
stoichiometric preference programmed in the sequence, (reprinted with permission from I. V.
Korendovych, Y. H. Kim, A. H. Ryan, J. D. Lear, W. F. DeGrado and S. J. Shandler,
Computational design of a self-assembling b-peptide oligomer, Org. Lett., 12, 5142. Copyright
(2010) American Chemical Society).
functional designs is clearly one of the main avenues pursued. Possibilities

for exploitation are vast, but ultimately depend on the success of clinical
trials which are just starting to take place for self-assembling formu-
lations.167 The main challenges however remain in rational drug-like design,
which targets autonomous structures, and functionally reproducible de-
signer biomaterials, the applications of which may not be limited to ma-
terials science but are defined by the heterogenous nature of self-assembled
01/12/2013 14:44:38.
structures.
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Self-assembly of designed peptides and
their nanomaterials applications
Hiroshi Tsutsumi* and Hisakazu Mihara
DOI: 10.1039/9781849737081-00122
1 Introduction
The designed self-assembling peptides in the literature of 2011, 2012 and

partly 2013 are reviewed in this Chapter. This Chapter focuses on the
current development in the design and the nanomaterial application of self-
assembling peptides. As for the recent applications of self-assembling pep-
tides as biomaterials for cell/tissue engineering and a drug delivery, refer to
the Chapter of ‘‘Self-assembling peptide materials’’ in Amino Acids, Pep-
tides and Proteins: Volume 37.1 All Scientific Papers published from 2011
to 2013 cited in this Chapter have been sourced mainly from the SciFinder
databases on the internet and from scanning a selection of major journals.
2 Self-assembling peptides and nanostructures

2.1 Generals
The phenomenon that molecules spontaneously assemble into special
structures in nano- and micrometer orders with precise recognition each
other is called self-assembly or self-organization. In recent years, molecular
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design to control self-assembly has attracted much attention for a bottom-

up technology to fabricate various nanostructures.2,3 In particular, bio-
molecules such as DNA,4–6 proteins and peptides7–9 are useful molecular
tools as components to produce self-assembling molecules and to fabricate
structured materials in aqueous media. Hydrophobic interaction, electro-
static interaction, hydrogen bonds and Van del Waals interaction are major
driving forces in self-assembly and cooperatively work to construct well-
ordered supramolecular architectures.
Compared to other biomolecules, peptides have various advantages such
as easy synthesis, abundant building blocks including non-natural amino
acids, availability of selective chemical modification and good bio-
compatibility. In addition, designed self-assembling peptides can form a
variety of nanostructures such as fiber, tube, sheet and sphere.7 To date,
many designed self-assembling peptides are developed, and most of these
peptides are roughly divided into b-sheet peptides, a-helix peptides, lipid/
surfactant-like peptides, peptide amphiphiles and short peptides with a
diphenylalanine motif.
2.2 b-Sheet peptides

b-Sheet forming peptides account for a large portion of self-assembling
peptides. Many of self-assembling peptides that form b-sheet structure have
been designed as repeated-sequences of alternating hydrophobic and
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo

Institute of Technology, Nagatsuta-cho 4259 B-40, Midori-ku, Yokohama 226-8501 Japan.
*E-mail: htsutsum@bio.titech.ac.jp

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hydrophilic amino acids, which form two distinct surfaces of hydrophobic

and hydrophilic (Fig. 1). For example, Ac-(AEAEAKAK)2-NH2 (EAK16-
II) was designed as the first self-assembling peptide with b-sheet structure.10
The complementary electrostatic interactions between Lys residues and Glu
residues guide the EAK16-II peptide to an antiparallel b-sheet structure that
forms a hydrophobic face composed of Ala residues. The formed b-sheet
structure is stabilized by the hydrophobic interactions between Ala residues
and the formation of interchain hydrogen-bonding networks, and then
nanofibers composed of a bilayer of extended b-sheets are formed. Add-

itionally, well-networked nanofibers fabricated from enough concentration
of EAK16-II can form hydrogels by trapping water under physiological
condition. To date, many amphipathic b-sheet peptides that provide various
nanostructures have been developed.10–28,30–40 Representative b-sheet-
forming peptides including newly designed peptides are listed in Table 1.
Self-assembling properties of peptides and morphologies of resultant
assembled nanostructures are strongly dependent on peptide sequences.
Thus, the relationship between peptide sequences and assembled nano- and
microstructures has been continuously investigated.
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Fig. 1 Schematic illustration of self-assembly of amphipathic b-sheet peptides.
Table 1 Representative sequences of self-assembling peptides with b-sheet

structures including recently reported ones.
Name Sequence Reference
EAK16-II Ac-AEAEAKAKAEAEAKAK-NH2 10
RADA16 Ac-RADARADARADARADA-NH2 12
KFE8, 12, 16 Ac-(FKFE)n-NH2 (n=2B4) 13, 14
Y9 Ac-YEYKYEYKY-NH2 15
P11-2 (DN1) Ac-QQRFQWQFEQQ-NH2 16, 17
P9-6 Ac-QRLQLQLEQ-NH2 18
P11-8 Ac-QQRFOWOFEQQ-NH2a 19
Q11 Ac-QQKFQFQFEQQ-NH2 20
RU-002 Ac-AIAKEXAIA-NH2b 21
SASL16 SASLSASLSASLSASL-NH2 22
QL Ac-KKQLQLQLQLQLQLKK-NH2 23
MAX1 VKVKVKVKVDPPTKVKVKVKV-NH2 24
VK13 VKVKVKVKVKVKV-NH2 25
a
O=ornithine.
b
X=2-naphthylalanine.

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The 16-residue peptide RADA16 (Table 1) is one of often used self-

assembling peptides for cell culture, which assembles into nanofibers and
forms transparent cytocompatible hydrogels under the physiological
aqueous condition.12 Highly transparent hydrogels become useful materials
for applications in the optical and ophthalmological fields. However, there
has not been clear guideline to produce highly transparent hydrogels from
designed self-assembling peptides. Thus, to investigate the parameters ne-
cessary for highly transparent hydrogels, RADA16 analogues with various
net charges were produced by systematic replacement of charged residues to

non-ionic residues (Table 2).26 At least þ 2 or 2 net charges per peptide
and appropriate electrostatic complementarity between peptides were ne-
cessary to form transparent hydrogels. However, the mechanical strength of
transparent hydrogels of 4SA, 9SA and 16SA was weaker than other
hydrogels. Much effort will be necessary to achieve both high transparency
and rigidity of self-assembling peptide hydrogels. As challenges for the
rigidity enhancement, the short peptide motif of GPGGY or GGAGGS
derived from spider fibroin uncrystalline region was elongated at C-
terminus to reinforce the self-assembling ability of RADA16.27 These
peptide motifs are known to contribute to the incomparable elasticity and
hydrophobicity of fibers, and the GPGGY motif effectively enhanced
mechanical strength of RADA16 without any disruption of b-sheet
formation.
The modification of N- and/or C-terminus in amphipathic peptides is
important factor to determine morphologies of nanostructures. As for a
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self-assembling peptide, AIAKEXEIA (X=2-naphthylalanine), the peptide

without any modification at both termini assembled into right-handed
helical fibers (Fig. 2 (a)).28 N-terminus acetylation or C-terminus amidation
induced loosely left-handed twisted ribbon morphology and only plate-like
nanostructures, respectively (Fig. 2 (b) and (c)). The acetylated and
amidated peptide at both N- and C-terminus showed left-handed straight
fibers (Fig. 2 (d)). Numbers of amino acid residues in self-assembling pep-
tides also should be considered from a viewpoint of satisfying all possible
hydrogen bonds and hydrophobic/electrostatic interactions at peptide ends.
For example, in the assembled state of the KFE8 peptide (Table 1), a Phe
residue at N-terminus is thought to be unpaired in the model of antiparallel
b-sheet structure (Fig. 3 (a)).14 It seems that the dangling N-terminal Phe
of KFE8 b-sheet limited assembled structures to left-handed helical
nanoribbons with B8 nm width, because truncation of the N-terminal Phe
residue or the C-terminal Glu residue caused a failure in self-assembly or a
formation of broad nanotapes with B20 nm width, respectively. Therefore,
termini of peptides become exposed on the edge of assembled nanofibers
and may contribute to interfiber interactions.
Highly cationic peptides with alternative hydrophobic/hydrophilic resi-
dues arrangement have been also designed as self-assembling peptides, and
the 20-residue peptide MAX1 (Table 1) is a representative one with þ 9 net
charge. MAX1 does not self-assemble at physiological pH due to strong
electrostatic repulsion between Lys residues, however, MAX1 folds into
b-hairpin structure and assembles into fibrous nanostructures at pH 9.0.24
MAX1 can also self-assemble nanostructures in the presence of salts such as

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Table 2 Sequences of the RADA16 analogue peptides and their properties.
Net charge Secondary

Peptide Sequencea at pH 7 structure Comment Transparency Rigidity
RADA16 Ac-RADARADARADARADA-NH2 0 b-sheet Hydrogel

1SA Ac-RASARADARADARADA-NH2 þ1 b-sheet Turbid solution
2AA Ac-RAAARADARAAARADA-NH2 þ2 b-sheet Hydrogel þþ þ
3NA Ac-RANARADARANARADA-NH2 þ2 b-sheet Hydrogel þ þþþ

4SA Ac-RASARADARASARADA-NH2 þ2 b-sheet Hydrogel þþþ þ
5SA Ac-RASARADARADARASA-NH2 þ2 b-sheet Hydrogel þþ þþþ
6SA Ac-RADARASARASARADA-NH2 þ2 b-sheet Hydrogel þ þþ
7SL Ac-RLSLRLDLRLSLRLDL-NH2 þ2 b-sheet Precipitate
8RA Ac-RARARADARADARADA-NH2 þ2 b-sheet Hydrogel opaque þ
9SA Ac-RASARASARASARADA-NH2 þ3 b-sheet Hydrogel þþþ þ
10SA Ac-RADARADARADASADA-NH2 1 b-sheet Turbid solution
11SA Ac-SADARADASADARADA-NH2 2 b-sheet Turbid solution
12SA Ac-RADASADASADASADA-NH2 3 Random coil Clear solution
13SA Ac-KASAKAEAKASAKAEA-NH2 þ2 b-sheet Hydrogel þþ þ
14SA Ac-SAEAKAEASAEAKAEA-NH2 2 b-sheet Hydrogel þþ þ
15SA Ac-RASARADARADA-NH2 þ1 b-sheet Turbid solution
16SA Ac-RASARADARASA-NH2 þ2 b-sheet Hydrogel þþþ þ
a
Replaced residues are underlined.
Fig. 2 Transmission electron microscope (TEM) images of (a) AIAKEXEIA, (b) Ac-
AIAKEXEIA, (c) AIAKEXEIA-NH2, (d) Ac-AIAKEXEIA-NH2, X=2-naphthylalanine.
From ref. 28. Reprinted with permission from Chemical Society of Japan.
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Fig. 3 Structural models and TEM images of (a) Ac-FKFEFKFE-NH2 and (b) Ac-
FKFEFKF-NH2. From ref. 14. Reprinted with permission from John Wiley and Sons.
NaCl since charged amino acid residues form salt complexes and their
charge are screened. This self-assembling phenomenon triggered by salts
is very useful to prepare injectable hydrogel materials under moderate
conditions. Linear cationic peptides with amphipathic sequences were newly
designed by changing peptide sequence length along with a VK11 peptide or
replacing hydrophobic amino acids (Table 3).25 Designed peptides except

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Table 3 Sequences of linear cationic self-assembling peptides.
Peptide Sequence
VK9 VKVKVKVKV-NH2
VK10 VKVKVKVKVK-NH2
VK11 VKVKVKVKVKV-NH2
VK12 VKVKVKVKVKVK-NH2
VK13 VKVKVKVKVKVKV-NH2
AK13 AKAKAKAKAKAKA-NH2
IK13 IKIKIKIKIKIKI-NH2
LK13 LKLKLKLKLKLKL-NH2
VR13 VRVRVRVRVRVRV-NH2
Table 4 Sequences of self-assembling peptides with aliphatic or aromatic residues.
Scaffold name Sequence
Ac-(XK)4-NH2 VK Ac-VKVKVKVK-NH2
IK Ac-IKIKIKIK-NH2
FK Ac-FKFKFKFK-NH2
F5
FK Ac-F*KF*KF*KF*K-NH2a
Cha-K Ac-ZKZKZKZK-NH2b
Ac-K2(QX)6K2-NH2 QL Ac-KKQLQLQLQLQLQLKK-NH2
QFQL Ac-KKQFQLQFQLQFQLKK-NH2
QF Ac-KKQFQFQFQFQFQFKK-NH2
QW Ac-KKQWQWQWQWQWQWKK-NH2
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QY Ac-KKQYQYQYQYQYQYKK-NH2
a
F*=pentafluorophenylalanine.
b
Z=cyclohexylalanine.
for VK10, VK12 and AK13 successfully formed hydrogels in the presence of
salts (50 mM bis-trispropane buffer containing 150 mM NaCl), and
hydrogel composed of LK13 using cell-culture media (DMEM) showed
good shear-thin recovery property that is suitable for the injectable hydrogel
materials. Interestingly, VK10 and VK12 did not show b-sheet structure
and form hydrogel in the presence of salts, because a positively charged Lys
residue at C-terminus was probably arranged close to N-terminus of paired
peptide. Since AK13 did not assemble, arrangement of charged residues
close to peptide terminus and hydrophobicity of peptides should be con-
sidered to produce stable assembled structures.
The aromatic p-p interaction is considered to be important effect different
from simple hydrophobic interaction of aliphatic amino acids in self-
assembly, since aromatic amino acids seems to play important roles in the
core sequences of amyloid forming peptides.29 To elucidate the effect of
aromatic interaction, several self-assembling peptides were designed based
on the Ac-(XK)4-NH2 sequence30 or the Ac-K2(QX)6K2-NH2 sequence31 by
replacing hydrophobic residues X with amino acids with aliphatic or
aromatic side chains systematically (Table 4). In Ac-(XK)4-NH2 series, the
order of hydrogel rigidity was F5PheWPheWIleWCha, although Cha is
the most hydrophobic residue among used amino acids. Therefore, not the
hydrophobicity but aromatic content strongly affected the rigidity of the

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entangled fibril network. Aromatic amino acids do elicit unique fibril

morphologies and viscoelastic behavior in hydrogels derived from these
fibrils relative to sequences with aliphatic amino acids. In Ac-K2(QX)6K2-
NH2 series, the introduction of Phe residues induced well hydrogelation
property. However, QY and QW formed weaker hydrogels than QFQL and
QF, because QY and QW only assembled into short nanofibers with a
parallel b-sheet conformation probably due to their side chains.
Complement pairs of self-assembling peptides have been developed to
control hydrogelation in situ. Electrostatic complementary design is often

adopted to produce pairs of complementary peptides such as P11-13
(Ac-QQEFEWEFEQQ-NH2)/P11-14 (Ac-QQOFOWOFOQQ-NH2)32 and
AIP-1 (Ac-EFFAAE-NH2)/AIP-2 (Ac-KFFAAK-NH2).33 Since these
peptides have negative or positive net charges in physiological pH
condition, respectively, electrostatic repulsion does not allow each peptide
to self-assemble into nanostructure alone. In the physiological medium,
equimolar mixtures of P11-13/P11-14 or AIP-1/AIP-2 can instantaneously
co-assemble into nanofibers with b-sheet structures and give hydrogels due
to the complementary electrostatic attractive force.
Self-assembling peptides composed of D-amino acids has unique prop-
erties and advantages such as protease resistance. For example, the EAK16
analogue peptide, d-EAK16, composed of all D-amino acids self-assembled
into indistinguishable networked nanofibers from those of l-EAK16, and
form hydrogels.34 In addition, combination of enantiomeric designed
peptides composed of all L- and D-amino acids, respectively can lead mor-
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phological change of assembled structures and enhance the mechanical

properties of hydrogels synergistically. The equimolar mixture of MAX1
and DMAX1, an enantiomer of MAX1, rapidly co-assembled into racemic
nanofibers and formed hydrogels with 4-fold greater rigidity than that of
hydrogels prepared from either pure enantiomer.35 In other case, the ra-
cemic pair of L-Ac-(FKFE)2-NH2 and D-Ac-(FKFE)2-NH2 co-assembled
into flat ribbon, whereas L-Ac-(FKFE)2-NH2 and D-Ac-(FKFE)2-NH2
self-assembled into similar nanofibers left-handed and right-handed helical
fibers with similar diameters (B8.2 nm) and helical pitch (B19 nm), re-
spectively.36 In addition, it was revealed that co-assembly of these enan-
tiomeric self-assembling peptides is more favorable than that of single
stereoisomers of self-assembling peptides thermodynamically. Well-packed
alignment of racemic peptide might be formed within the rippled b-sheets of
the co-fibrils.
Except for nanofibers, toroidal barrel structures can be formed from self-
assembly of chemically modified amphipathic b-sheet peptides.37, 38 In the
designed b-sheet forming peptide, WKWEWYWKWEW-NH2, intro-
duction of branched oligoethyleneglycol dendron unit into the Tyr side
chain induces changes of assembled nanostructures (Fig. 4).38 Whereas the
peptide without modification formed nanofibers, the triethyleneglycol-
modified peptide assembled into discrete nanoring structures with highly
uniform diameter around 11 nm. The nanorings thought to be composed of
a single layer of peptide, with the b-strands oriented perpendicular to the
plane of the nanoring with accompanying moderate curvature. The intro-
duction of appropriate bulky unit can make moderate curvature in the

Fig. 4 Triethyleneglycol-modified peptide, and the model and TEM image of self-assembled
nanorings. From ref 38. Reprinted with permission from John Wiley and Sons.
growth of self-assembled nanofibers resulting in the formation of closed

nanoring structures. Therefore, steric hindrance between peptides in as-
sembled state is an important factor to determine fabricated morphologies.
As mentioned above, a wide variety of self-assembling peptides have been
designed based on b-sheet structures. Although there still need much effort
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to elucidate the relationship between amino acid sequence and assembled

nanostructures, several new findings have been provided about appropriate
net charges, peptide length, hydrophobicity/aromaticity and side chain
packing between assembled peptides to realize efficient fabrication of stable
nano- and microstructures. The modification of peptide termini or side
chains is an important factor affecting morphologies of nanostructures. In
addition, the arrangement of b-sheet self-assembling peptides on template
molecules with rigid structures is also useful to control the assembled
structures.39,40
2.3 a-Helical peptides

a-Helical self-assembling peptides are mainly designed as coiled-coil de novo
designed-proteins with sticky-ends to fabricate elongated fiber assembly
(Fig. 5 (a)). The majority of coiled-coil sequences are usually designed based
on basic heptad repeat (abcdefg) design, which is a pattern of hydrophobic
(h) and polar (p) amino acids, hpphppp. For example, two 28-residue pep-
tides were designed with distinct two parts that can form heterodimeric
coiled-coil structures.41 Coiled-coil dimers are mainly stabilized by the ar-
rangement of hydrophobic Ile and Leu residues at a and d positions and
electrostatic complementarity of oppositely charged Glu and Lys residues at
e and g positions, respectively. A pair of Asn residues was arranged at
hydrophobic face of different, complementary heptads of the two peptides
to favor parallel coiled-coil structures by hydrogen bonding. At first, these
peptides self-assembled into a parallel, sticky-ended coiled-coil dimer via
hydrophobic interactions, electrostatic interactions, the formation of

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hydrogen bond, and Van del Waals interaction. Subsequently, assembled-

dimers were polymerized via sticky-ends, and nanofibers were fabricated.
Recently, the ultrastructure of nanofibers fabricated from self-assembling
coiled-coil pairs was directly imaged using cryotransmission electron
microscopy at approximately 8Å resolution (Fig. 5 (b)).42 To date, various
a-helical self-assembling peptides have been produced continuously.43–49
Binding affinity between self-assembling peptides is one of the important
parameters to control self-assembling process and to stabilize assembled
structures. As for a-helical self-assembling peptides, it is comparatively easy

to control affinities between self-assembling by changing peptide length. For
example, dissociation constants of pairs of coiled-coil forming peptides can
be tuned from micromolar to subnanomolar range by changing length of
coiled-coil peptides from 21 to 28 residues.47
Non-fibrillar nanostructures also can be fabricated using a-helical
coiled-coil peptides with and without sticky-ends. For example, assembled
structures of coiled-coil peptides with sticky-ends are varied from
micrometer-sized fibers to well-defined cyclic oligomers with nanometer
sizes by inserting flexible linker sequence between two a-helical segments.48
On the other hand, self-assembled cage-like particles can be fabricated from
a set of a-helical coiled-coil peptides without sticky-ends.49 Two hetero-
dimeric and homotrimeric coiled-coil bundles were conjugated back to back
to render hexagonal networks and then closed cages with hollow and
unilamellar nanostructures in 100 nm diameter.
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2.4 Lipid/surfactant-like peptides

Lipid- or surfactant-like peptides are a class of self-assembling peptides with
a hydrophilic head group and a hydrophobic tail. In usual cases, one or two
Fig. 5 (a) Schematic illustration of self-assembly of a-helical coiled-coil peptides with sticky-
ends. Embedded Asn residues are expressed as a star mark. (b) High resolution TEM images of
fabricated nanofibers with a cartoon overlay of the postulated assembled peptide structures (C).
Scale bars show 2 mm (A), 50 nm (B) and 10 nm (C), respectively. From ref. 42. Reprinted with
permission from PNAS.

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charged amino acid residues are arranged at N- or C-terminus as a

hydrophilic head group, and 3-6 hydrophobic residues like Ala, Val, Leu
and Ile are used to make a hydrophobic tail. For example, Ac-AAAAAAD
(A6D) and Ac-VVVVVVD (V6D) are first representative lipid-like peptides
that have a negatively charged Asp residue as a hydrophilic head group and
consecutive six Ala or Val residues as a hydrophobic tail, respectively.50
These peptides have similar structures to biological lipid molecules in length
ofB2 nm, the lipophilic tail and the negatively charged head. In aqueous
media, A6D and V6D self-assembled into nanotubes and nanovesicles. To

date, various lipid- and surfactant-like peptides have been designed and
their properties are investigated.51–61
Lipid- and surfactant-like peptides are capable of self-assembly into
various nanostructures including ribbons, tubes and vesicles. However, the
relationship between the peptide design and fabricated morphologies
has not been well understood yet. Nanostructures fabricated from
surfactant-like peptides seem to be mainly determined by hydrophobic and
electrostatic interactions due to their roles as dominant driving forces in
self-assembly. Thus, a series of surfactant-like peptides was systematically
designed based on Ac-X6Kn-NH2 (X6Kn) sequence (X=A, V, L; n=1-5) to
investigate the relationship between hydrophobic/hydrophilic design and
morphologies of assembled nanostructures.58 As shown in Table 5,
morphologies of A6Kn series changed from nanotubes through nanovesicles
to aggregates according to the increase of Lys number. Similar tendency of
morphological change was observed in V6Kn and L6Kn series. Moreover,
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L6K2 self-assembled into nanoribbons with larger width than nanotubes of

L6K3. Thus, surfactant-like peptides with lower critical micelle concen-
tration (CMC) seem to assemble into larger nanostructures.
Many lipid-like peptides have been designed based on the Ac-XmZn motif
(X=nonpolar amino acid, Z=charged amino acid). A unique class of
ultrasmall peptides that have a decreasing gradient of hydrophobicity from
N to C terminus were designed as new lipid-like self-assembling peptides
(Fig. 6).59–61 Several ultrasmall peptides (trimerBhexamer) were designed
Table 5 Sequences, morphologies and critical micelle concentration of X6Kn peptides. From
ref. 58. Reprinted with permission from American Chemical Society.
Peptide Sequence Morphology of self-assemblya CMCb / mM
A6K Ac-AAAAAAK-NH2 nanotubes 2.28

A6K2 Ac-AAAAAAKK-NH2 nanovesicles 7.93
A6K3 Ac-AAAAAAKKK-NH2 aggregates n.d.c
V6K2 Ac-VVVVVVKK-NH2 nanotubes 0.33
V6K3 Ac-VVVVVVKKK-NH2 nanovesicles 0.83
V6K4 Ac-VVVVVVKKKK-NH2 aggregates 4.65
L6K2 Ac-LLLLLLKK-NH2 nanoribbons 0.046
L6K3 Ac-LLLLLLKKK-NH2 nanotubes 0.25
L6K4 Ac-LLLLLLKKKK-NH2 nanovesicles 5.38
L6K5 Ac-LLLLLLKKKKK-NH2 aggregates n.d.c
a
Observed by TEM.
b
Critical micelle concentration.
c
Not detected.

Fig. 6 Illustration of a lipid-like self-assembling peptide and self-assembly process. From

ref. 59. Reprinted with permission from PNAS.
Table 6 Sequence of ultrasmall self-assembling peptides.
Head group Hexamer Pentamer Tetramer Trimer
Asp (D) / Ac-LIVAGD, Ac-ILVAGD, Ac-LIVAD, Ac-IVAD Ac-IVD, Ac-IID

Glu (E) Ac-LIVAAD, Ac-LAVAGD, Ac-LIVGD
Ac-AIVAGD, Ac-LIVAGE
Lys (K) Ac-LIVAGK-NH2
Ser (S) / Ac-LIVAGS, Ac-ILVAGS,
Thr (T) Ac-AIVAGS, Ac-LIVAGT,
Ac-AIVAGT
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Fig. 7 Schematic illustration of self-assembly of peptide amphiphiles.
(Table 6), for example, Ac-LIVAGD and Ac-IVD (named Ac-LD6 and
Ac-ID3) readily self-assembled into well-ordered nanofibrous structures and
formed stable hydrogels. Other ultrasmall peptides also assembled into
various nanostructures such as long helices, straight fibers and hollow
nanospheres. Interestingly, despite of their small size, these peptides showed
secondary structural changes from random coil to a-helical intermediates
terminating cross-b-structures during self-assembly.
2.5 Peptide amphiphiles

Peptide amphiphiles are designed as peptides with a non-polar aliphatic tail
(Fig. 7). First peptide amphiphiles have an ionic peptide part and a long
chain alkyl tail.62 In many peptide amphiphiles, saturated fatty acids such as
a lauric acid (C12), a myristic acid (C14) and a palmitic acid (C16) are
usually used as a hydrophobic tail, but other hydrophobic units such
as oligothiophenes are also available.63,64 Hydrophobic amino acids such as
Val and Ala are arranged next to the fatty acid as a b-sheet forming

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segment, and charged amino acids such as Glu, Lys and Arg are used to
promote solubility of peptide amphiphiles in water. A hydrophobic tail
mainly drives aggregation and peptide segment self-assembles into b-sheet
structures directing charged moiety to outside, therefore, peptide amphi-
philes assemble into cylindrical nanofibers. To date, various peptide
amphiphiles have been produced continuously.63–72
Self-assembly of peptide amphiphiles with a charged head group is usu-
ally triggered by charge screening through pH change or interaction be-
tween oppositely charged molecules that cancels the electrostatic repulsion

between charged peptide amphiphiles. In cases of lauryl-VVAGK-NH2,
lauryl-VVAGD and lauryl-VVAGH-NH2, self-assembling into nanofibers
is triggered by the increase and the decrease of pH or above pH 6.5, re-
spectively, because electrostatic repulsion of each peptide amphiphiles is
neutralized under these conditions.70 Charges on lauryl-VVAGD and
lauryl-VVAGK-NH2 are also screened through interactions with Ca2þ ion
and anionic macromolecules such as DNA and heparin, respectively.
Additionally, lauryl-VVAGD and lauryl-VVAGK-NH2 neutralize each
other, because they are oppositely charged peptide amphiphiles.
Control of interfiber interaction should be considered for application of
self-assembling peptides as bio- and nanomaterials, because the interfiber
interaction is important in the determination of mechanical properties of
nanofibers/hydrogels and in the efficient display of functional units on
nanofibers. For example, lauryl-VVAGERGD, an anionic peptide amphi-
phile, self-assembled into nanofibers and formed hydrogels in the presence
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of CaCl2 or HCl.71 The mechanical strength of peptide amphiphile/CaCl2

hydrogels was higher than that of peptide amphiphile/HCl hydrogels,
because ion bridging formed by calcium ions might enhance interfibrillar
interaction between nanofibers in addition to hydrogen bonding and elec-
trostatic attraction forces. On the other hand, tight interfibrillar interaction
is not effective to display bioactive sequences on the nanofibers. For ex-
ample, there is a possibility that highly hydrophobic epitopes like IKVAV,
promoting neural cell differentiation and neurite outgrowth, form bundled
nanofibers and their binding activity to target receptors may be strongly
restricted. Here, an appropriate electrostatic interaction to control fiber
bundling was investigated using a series of peptide amphiphiles palmitoyl-
VVAA-En-GIKVAV (n=2–4), in which several anionic Glu residues were
inserted between the peptide amphiphile sequence and the IKVAV epi-
tope.72 Peptide amphiphiles with two or three Glu residues form aggregated
fibers such as bundled dimers, however, the peptide amphiphile with four
Glu residues mainly assembles into single cylindrical nanofibers (Fig. 8). In
addition, the peptide amphiphile with four Glu residues can form hydrogel
scaffold with the highest activity to promote neurite outgrowth. Therefore,
the inhibition of nanofiber bundling using electrostatic forces leads the ef-
ficient display of bioactive motifs on nanofibers.
2.6 Diphenylalanine and its derivatives

There is great interest in exploiting minimal self-assembling peptides and
their assembling mechanism, and a diphenylalanine (FF) peptide and its
derivatives have attracted much attention.73–75 Self-assembly of the FF

Fig. 8 TEM images of peptide amphiphiles palmitoyl-VVAA-En-GIKVAV. (a) n=2,

(b) n=3, (c) n=4. Scale bars shows 100 nm. From ref. 72. Reprinted with permission from
John Wiley and Sons.
peptide into nanotubes was found by simple dispersion of concentrated

FF solutions into aqueous solvent.76 The FF peptide is dissolved in
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at high concentration more than
100 mg/mL. Then, FF peptides rapidly self-assemble into ordered nano-
tubes with high-aspect ratio and high-persistence length at a final mM
concentration by dilution into aqueous solution. Here, an aromaticity of the
FF peptide plays an important role to fabricate tubular structures, because
dipeptides of non-aromatic cyclohexyalalanine (Cha), Val, Leu or Ile resi-
dues self-assemble into not nanotubes but spherical aggregates in the same
condition with the FF peptide.77 Other dipeptides, Cha-Phe and Phe-Cha
also do not assemble into clear nanotubes. Therefore, dipeptide structure
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composed of consecutive two Phe residues is important.

Although the FF peptide has a very simple structure, it shows compli-
cated and interesting self-assembly processes. For example, in the growth
chamber condition, the morphology of FF microtubes strongly depends on
the relative humidity (RH) in chamber to the FF peptide concentration
(RH-FF ratios, Fig. 9).78 At first, hexagonal arrangement of FF peptides
grows to the hexagonal nanotubes with oppositely charged ends. Nanotubes
are arranged in an end-to end manner by the dipolar electric field on
themselves. Then, hexagonal hierarchical microtube structures are formed
through side-by-side hexagonal aggregation and end-to-end arrangement.
In this assembly process, initial nanotube numbers are affected by the
relative humidity, because the difference in humidity/FF concentration
ratios induces different contents of crystalline phases, resulting in various
microtubular morphologies.
Several nano- and microstructures of FF peptide and its derivatives are
also fabricated by using various substrates,79–81 tuning of solvent and pH
condition,81,82 co-assembly with FF derivatives,83,84 breath figure method85
and cryogelation.86 Nanofibrous structures and microvesicle structures are
induced on the glass and microporous membrane surfaces after dropping
the pre-assembled FF peptide solutions onto these surfaces.79 When
hydrophilic silicon (Si-OH), anodized aluminum oxide (AAO), phenyl-
modified silicon (phenyl-Si) and AAO (phenyl-AAO) are used as substrates,
tubular and flowerlike/leaflike morphologies are formed on Si-OH/AAO
and phenyl-Si/phenyl-AAO surfaces, respectively (Fig. 10).80 In particular,
the interaction between surface and FF peptides seems to affect assembled

Fig. 9 Field emission-scanning electron microscope images of FF nanotubes/microtubes at

different RH-FF ratios. (a) 0.2, (b) 0.3, (c) 0.5, (d) 0.6–0.9, (e)W1.0. (f) Model structures of the
hexagonal nanotubes with oppositely charged ends. From ref. 78. Reprinted with permission
from American Chemical Society.
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Fig. 10 SEM images of assembled FF structures on Si-OH (a), on phenyl-Si (b) and on
phenyl-AAO (c). Scale bars show 10 mm (a, b) and 5 mm (c). From ref. 80. Reprinted with
permission from American Chemical Society.
nanostructures. Not only surface geometry and surface properties of sub-

strates, solutes and co-solvent in aqueous FF solutions also should be
considerable as important factors to determine self-assembling peptide
nanostructures.
To date, there have been reported many FF peptide derivatives with
9-fluorenylmethyloxycarbonyl (Fmoc) or 2-(naphthalene-2-yl) acetyl (Nap)
units. Fmoc and Nap are very important units, because they mainly
contribute to self-assembly through their hydrophobic and aromatic inter-
actions. As for Phe units, replacement of one or two Phe residues of Fmoc-
FF to Gly residues leads to significant morphology differences probably due
to the decrease of hydrophobicity and aromaticity and the increase of
flexibility.87 Recently, nucleobases88 and glycosides89,90 have been used as
functional units for the construction of FF-based self-assembling peptides.
Nucleobase-FF peptides (nucleopeptides) that have a nucleobase instead of
Fmoc or Nap units can self-assemble into nanofibers and form hydrogels

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88
alone. Moreover, the addition of 10mer of deoxyadenosine A10 to the
hydrogels of a thymine-FF peptide increased hydrogels stability, and
equimolar mixture of complementary nuclepeptides formed stronger
hydrogels than nucleopeptide alone due to their selective hydrogen bonding
and p-p interaction. Glycosylation of the FF peptide (glycopeptides) by
saccharide derivatives such as b-N-acetylglucosamine and a-mannose brings
not only an improvement in solubility of peptides but also morphological
changes such as bundled or elongated fibers and spherical aggregates.89,90
In other cases, both nucleosides and glycosides,91–93 nonsteroidal anti-

inflammatory drugs94,95 and fluorescent metal complexes96 are used to
produce FF-based self-assembling peptides with various functions.
3 Application of self-assembling peptides as nanomaterials

3.1 Generals
Self-assembling peptides can fabricate various nanostructures such as fiber,
tube, sphere and vesicle, and the fabricated peptide materials show inter-
esting structural, optical and electronic properties derived from their
nanostructures. Self-assembled structures have been functionalized as
nanomaterials by selective modification and peptide design. Self-assembling
peptides are also potential scaffolds to construct hybrid nanomaterials for
optical and electronic devices that involve controlled energy, light or charge
transfer. Furthermore, self-assembled nanostructures have much attention
as scaffolds for mineralization of metallic/inorganic nanomaterials, because
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mineralization processes are controlled on functionalized surfaces of

nanostructures.97,98
3.2 Functionalization of self-assembling peptides

Functionalization of nanostructures fabricated from self-assembling pep-
tides is one of important matters to apply them as biomaterials and nano-
materials. There are several approaches to functionalize self-assembling
peptides and their assembled nanostructures.
The use of self-assembling peptides covalently modified with functional
units is one of the most simple and easy accesses to functionalization of
nanostructures. Modified peptide monomers self-assemble alone or together
with parent self-assembling peptides to form the nanostructures displaying
functional units (Fig. 11 (a)). Biotin of an affinity anchor for streptavidin,
bioactive sequences such as a cell adherence-promoting sequence RGD etc.
are often used as functional units and introduced into N and/or C termini
via an appropriate linker sequence to maintain self-assembling ability of
parents.99 In this strategy, the density of functional units on the nanos-
tructures can be easily adjusted by co-assembling of functionalized mono-
mer peptides with parent peptides in the various concentration ratios. In
addition, multi-functionalized materials can be also constructed by the
combinatorial use of several functionalized monomer in a co-assembling
manner.100
A post-assembly functionalization method is also a useful approach to
functionalize nanostructures. For example, enzymatic cross-linking of
peptide nanofibers is performed to stabilize assembled nanostructures with

Fig. 11 Schematic illustration of nanostructure functionalization. (a) Co-assembling of

covalently functionalized self-assembling peptides with parent peptides. (b) Post-assembling
covalent modification by bioorthogonal reaction. (c) Post-assembly binding modification by
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affinity tag to nanofibers.
covalent bond. In the self-assembled state of K2-(SL)6-K2 peptide, plasma

amine oxidase catalyzes oxidation of a primary amine of Lys side chain to
an aldehyde, and subsequent Schiff base formation between the aldehyde
and another amine of Lys can bridge self-assembling peptides. This cross-
linking results in dramatically increased hydrogel stiffness.101 As a similar
strategy, chemical cross-linking between self-assembling peptides in
nanostructures is also performed. Thiol and amine groups were displayed
on co-assembled nanofibers composed of Cys-appended Q11 peptide and
Lys-appended Q11 peptide, and then these self-assembling peptides were
bridged using a heterobifunctional cross-linker with a maleimide and a
succinimidyl ester, leading the stiffening of hydrogels.100
Bioorthogonal reactive tags are also helpful tools to produce functiona-
lized peptide nanomaterials. Small tags such as an azide and an alkene
groups are readily introduced into a-helical self-assembling peptides, and
reactive tags are displayed on the surface of fabricated nanofibers by co-
assembling tagged and non-tagged peptides.102 Then, desired functional
units such as biotin and a fluorescent dye can be introduced onto the
nanostructure surface by bioorthogonal reactions (Fig. 11 (b)). In other
cases, after self-assembly of diphenylalanine derivative with a polymerizable
group, acrylamide, assembled peptide monomers are co-polymerized with a
Ru(II) complex as a catalyst for a Belousov-Zhabotinsky reaction to form
hydrogels with chemical oscillation property.103,104 As described above, the

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post-assembly covalent modification method will become a versatile method

to functionalize self-assembling nanomaterials, because in principle, mul-
tiple functional units corresponding to the number of available bioortho-
gonal reactions can be introduced into the structured nanomaterials with
independent manner after fabrication of nanostructure.
In contrast to the covalent modification methods, a post-assembly
binding method is also used for non-covalent functionalization. In this
method, affinity tags with a selective binding activity to nanofibers act as
anchors to display functional units on the surface of assembled nanofibers

(Fig. 11 (c)). As affinity tags, several binding peptides to nanofiber are
discovered using phage-displayed libraries. For example, nanofibers fabri-
cated from Y9 are functionalized with gold nanoparticles via affinity tag
peptides against nanofibers.105 In similar manner, polyhydroxyalkanoate
(PHA) synthase fused with an affinity tag peptide is immobilized on specific
surface of FI peptide (H-PKFKIIEFEP-OH) nanofibers without significant
activity loss.106 PHA synthase localized on the surface of nanofibers produces
PHA and this brings in situ coating of nanofibers with PHA (Fig. 12). There
appears to be some unique surface structures on self-assembled nano-
structures, and affinity tag peptides screened from phage libraries will be able
to specifically bind to such structures with discrimination.
Regulation of self-assembling process is one of important challenges,
because several applications, for example encapsulation and release of
biomolecules and living cells within hydrogel matrices in a controllable
manner, will be available. Construction of self-assembling peptide materials
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with responsiveness to various stimuli is of efficient approaches. Usually,

pH, ionic strength (salts), temperature, metal/inorganic ions,107–111
enzymatic reaction112–115 and light116 are used as versatile stimuli.
His, Cys107 and Asp108/Glu residues are usually used for design of self-
assembling peptides responding to metal and inorganic ions, since their side
chains can interact with metal/inorganic ions. In addition, iminodiacetic
acid (IDA),109 nitrilotriacetic acid (NTA)110 and catechol111 are also
available as metal/inorganic binding units. In many cases, metal/inorganic
binding units are arranged at termini of self-assembling peptides, and lig-
and-metal/inorganic ion interaction causes neutralization of electrostatic
repulsion and/or bridging of self-assembling peptides via coordination bond
formation. Additionally, disassembly of nanostructures can be triggered by
Fig. 12 Illustration of post-assembly binding of affinity tag-fused PHA synthase to the peptide
nanofiber and in situ coating of the nanofiber with PHA polymer. From ref. 106. Reprinted
with permission from John Wiley and Sons.

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the addition of chelating agents such as ethylenediaminetetraacetic acid

(EDTA).
Enzymes are also available stimuli to control self-assembling process,
because properties of self-assembling peptides drastically change by
enzymatic reactions.112–115 For example, enzymatic phosphorylation/
dephosphorylation reactions of a Ser residue arranged in the sequence of a
peptide amphiphile (KRRASVAGK(lauryl)-NH2) are available to control
assembly/disassembly switching.112 Enzymatic biodegradation functionality
of self-assembling peptides is favorable for cell migration in peptide

hydrogel matrix and replacement by new cell-secreted extracellular mat-
rix.115 The specific cleavage sequences for matrix metalloproteinase-13
(MMP13) were introduced into self-assembling b-hairpin peptides IKV-
KIKVKVDPPTGXKVKIKV-NH2, in which PTG-XKV (X=F, L, I or A)
is an MMP-13 cleavable sequence. Since hydrogels of these peptides were
specifically degraded by MMP-13, SW1353 cells that can be induced to
express MMP-13 migrated through the hydrogels.
Photoregulation of peptide self-assembly has advantages in temporal and
spatial resolution. A photoresponsive azobenzene unit and a photo-
degradable 2-nitrobenzyl group are often used. For example, amphipathic
b-sheet peptides having two trans-azobenzene units at a hydrophobic side
self-assembled into laminated nanofibers and forms hydrogel. Photo-
isomerization of azobenzene units from trans to cis induced drastic change
from fibers to spherical aggregates (Fig. 13).116
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3.3 Self-assembling peptides as scaffolds for hybrid nanomaterials

Carbon nanotube (CNT) is one of attractive nanocarbon materials since
CNTs show unique physical, chemical, optical and electrical properties that
are useful for the construction of nanodevices. However, it is difficult to
disperse CNTs in aqueous media due to their high hydrophobicity. Many of
assembled nanostructures fabricated from self-assembling peptides have
hydrophobic region attributed to their amphipathic properties. Single-
walled CNTs (SWNTs) and multi-walled CNTs (MWNTs) also can be
encapsulated within the self-assembled structures and dispersed in aqueous
media using several self-assembling peptides (Table 7) without the use of
Fig. 13 Nanostructures of photoresponsive self-assembling peptide. (a) trans state. (b) cis
state. From ref. 116.

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Table 7 Self-assembling peptides for the dispersion of carbon nanotubes in water.
Peptide Sequence Reference
EFK16-II H-FEFEFKFKFEFEFKFK-OH 117

EAK16-II H-AEAEAKAKAEAEAKAK-OH 117
(EF)15 H-EFEFEFEFEFEFEFEFEFEFEFEFEFEF-OH 118
(KF)14 H-KFKFKFKFKFKFKFKFKFKFKFKFKFKF-OH 118
(EFEA)7 H-EFEAEFEAEFEAEFEAEFEAEFEAEFEA-OH 118
(KFKA)7 H-KFKAKFKAKFKAKFKAKFKAKFKAKFKA-OH 118

(KFCA)7 H-KFCAKFCAKFCAKFCAKFCAKFCAKFCA-OH 118
(KFKA)6KFCA H-KFKAKFKAKFKAKFKAKFKAKFKAKFCA-OH 118
(KVKA)7 H-KVKAKVKAKVKAKVKAKVKAKVKAKVKA-OH 118
RU-003 Ac-AIEKAXEIA-NH2 a 119
RU-010/021/022 Biotin-(Aeea)n-AIEKAXEIA-NH2 119
RU-020 Ac-AIEKK*XEIA-NH2 b 119
RU-035 Ac-KFEFAXKFE-NH2 119
RU-036 Biotin-(Aeea)n-KFEFAXKFE-NH2 119
RU-037 Ac-KFEFK*XKFE-NH2 119
a
X=2-naphthylalanine.
b
K*=Lys(Biotin-(Aeea)2).
organic solvents and chemical modification of them.117–119 In particular,

nanofiber forming peptides that have Phe residues as hydrophobic residues,
for example, EFK16-II and series of (X1FX2A)7 (X1, X2=E, K, C), can
effectively disperse CNTs in water. Since self-assembling peptides with Ala
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and/or Val residues instead of Phe residues were less effective for CNTs
dispersion, Phe residues may be essential for CNTs dispersion in water
through p-p interaction with CNTs. Some CNTs bound to nanofibers
maintained their optical and electrical properties in addition to stable dis-
persion states after dialysis and re-dispersion ability after lyophilization.
Moreover, dispersed CNTs were decorated with various functional units
such as a biotin tag and fluorescent dyes using covalently functionalized
self-assembling peptides119 or the post-assembly functionalization method
as described at section 3.2.118
The diphenylalanine (FF) peptide is one of promising building blocks to
construct functional nanomaterials, because FF nanotubes/microtubes
show interesting ferroelectric properties such as light-induced ferro-
electricity,120 piezoelectric effect121 and second harmonic generation.122 In
addition, fluorophore-loaded FF microtubes can act as optical wave-
guides.123 Therefore, FF nanotubes/microtubes are expected to be useful
scaffolds to produce highly functionalized nanomaterials. For example,
light-harvesting peptide nanotubes were developed by the integration of
photosynthetic mimicking units of tetrahydroxyphenylprophyrin (THPP) as
a light-harvesting molecule and platinum (Pt) nanoparticle as an electron
separator on FF nanotubes.124 THPP is aligned on the surface of FF
nanotubes accompanying with a J-aggregation formation that was induced
by the electrostatic interaction and the hydrogen bonding. In the presence
of [Cp*Rh-(bpy)H2O]2þ (Cp*=pentamethylcyclopentadiene, bpy=2,2 0 -
bipyridine) as an electron mediator and triethanolamine (TEOA) as an
electron donor, FF/THPP hybrid nanotubes transferred photoinduced

Fig. 14 Schematic illustration of biomimietic photosynthesis system by light-harvesting

peptide nanotubes. M means [Cp*Rh-(bpy)H2O]2þ complex. From ref. 124. Reprinted with
permission from John Wiley and Sons.
electrons from TEOA to [Cp*Rh-(bpy)H2O]2þ such as the reaction center in

natural photosystem I (Fig. 14). Additional decoration of FF/THPP
nanotubes with Pt nanoparticles (nPt) (B30 nm in size) enabled FF/THPP/
nPt hybrid nanotubes to regenerate NADH by visible-light driven manner,
and this NADH regeneration system can be coupled to a redox enzymatic
reaction using glutamate dehydrogenase. In other cases, zinc proto-
porphyrin IX and alkylated zinc phthalocyanine were encapsulated in
nanofibers fabricated from designed peptide amphiphiles, palmitoyl-
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AHLLLKKK125 and lauryl-VVAGH-NH2,126 respectively.

Self-assembling peptide nanofibers and hydrogels also act as useful
scaffolds for optical127–130 and electrochemical sensors.131–135 For example,
FF nanotubes are known as efficient and robust host matrix for lanthanide
complexes, and luminescent lanthanide complexes such as salicylic acid
(SA)/Tb and 1,10-phenanthroline (PHEN)/Eu can be easily immobilized on
FF nanotubes by simple mixing procedure.130 FF/lanthanide complex
nanotubes can selectively detect paraoxan, one of organophosphate
neurotoxins, with luminescence quenching based on the interaction between
paraoxan and lanthanide complexes. In these hybrid materials, FF nano-
tubes significantly enhance the luminescence of incorporated lanthanide
complexes, because FF nanotubes also have fluorescent properties and play
a critical role as a photosensitizer (i.e., antenna).
The collaboration of metal materials and self-assembling peptide ma-
terials can bring interesting phenomena.136,137 For example, a biomimetic
motor system is developed based on a hybrid nanomaterial composed of a
metal-organic framework (MOF) and a self-assembling peptide. Since the
MOF fabricated from Cu(II) ions, 1,4-benzenedicarboxylates and triethy-
lenediamines had a pore of 0.75 nm size, diphenylalanine (DPA, FF) was
incorporated in the MOF as non-assembled monomer (Fig. 15).136 When
the DPA-MOF hybrid was placed onto the aqueous solution containing an
EDTA chelator, partial destruction of the framework of the DPA-MOF
hybrid was triggered, and then stored DPA peptides were slowly released in
a highly isotropic direction. Released DPA peptides self-assembled at the
water/MOF interface and formed hydrophobic domains that decreased the

Fig. 15 Illustration of DPA-MOF motion triggered by partial destruction of the frame. From
ref. 136. Reprinted with permission from Nature Publishing Group.
surface tension of the MOF on the release side. This created a large surface
tension gradient around the MOF, and then, the DPA-MOF motor moved
from lower to higher surface tension side by a strong Marangoni effect. This
artificial motor is expected to miniaturized robotic systems. In other case,
phase transition from gel to sol can be controlled by laser illumination
to hybrid gel materials composed of diphenylalanine peptides and gold
nanoparticles (AuNPs).137 AuNPs with 20 nm size can be embedded in
diphenylalanine organogels without disruption of gel formation. When the
green laser at 532 nm was irradiated to AuNPs-embedded organogels with
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the gelation temperature around 40 1C, organogels quickly became sol due
to photothermal property of AuNPs.
3.4 Self-assembling peptides as scaffolds for biomineralization

nanomaterials
Highly ordered nano- and microstructures define characteristics of metal
and inorganic nanomaterials. Biomineralization is one of the beneficial
phenomena to construct inorganic and organic-inorganic hybrid materials
with highly ordered nano- and microstructures. Therefore, the nanoarchi-
tectures fabricated from self-assembling peptides have been applied as
scaffolds for mineralization of inorganic nanomaterials. To date, various
inorganic nanomaterials such as silica,138–141 hydroxyapatite, semi-
conductor nanocrystals, metal oxides142,143 and metal nanoparticles144–151
are used for mineralization on the surface of supramolecular scaffolds of
self-assembling peptides.
Silica-based nanomaterials with various length and pore sizes have been
expected as platforms for catalysis, sensing, immobilization of enzymes and
drug delivery. For recent examples, nanotubes fabricated from an ultrashort
amphipathic peptide I3K,138 wormlike micelles fabricated from peptide
amphiphile C16-W3K (palmitoyl-WAAAAKAAAAKAAAAKA)139 and
nanofibers composed of the Ac-KFFAAK-NH2 peptide140,141 were used as
scaffolds for the mineralization of silica nanotubes. Deposition of silica
crystals occurred on the surface of these nanotubes or nanofibers by dir-
ecting the sol-gel reaction of tetraethyl orthosilicate as silica precursor,
because these nanostructures displayed many Lys residues that work as

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catalysts for the sol-gel reaction on their surfaces. Then, silica nanotubes
can be isolated under moderate condition of the addition of organic solvents
such as trifluoroethanol139 or methanol140 without calcination usually used,
because these solvents can promote disassembly of peptide nanostructures
by inducing secondary structure change or reducing hydrophobic inter-
action. Moreover, isolated silica nanotubes were functionalized as fluor-
escent sensor materials by loading tetrakis-p-carboxyphenylporphyrin after
removal of nanofiber scaffolds, and constructed fluorescent silica nanotubes
are applied to detect explosive compounds such as trinitrotoluene and

dinitrotoluene.140
Metal oxide nanocrystals are interesting nanomaterials due to their
unique properties such as optical, electrical and magnetic properties. Since
their properties strongly depend on their order structures and sizes in many
cases, self-assembled peptide structures such as nanofibers are used as
scaffolds for the preparation of metal nanomaterials in controllable manner.
For example, the nucleation and the growth of ZnO nanocrystals were
promoted on the surface of nanofibers composed of self-assembling pep-
tides, RU-003 (Ac-AIEKAXEIA-NH2, X=2-naphthylalanine), because
Zn(II) ions were concentrated on nanofiber surface through the binding to
Glu side chains of peptides.142 The precipitated ZnO nanocrystals were
aligned along peptide nanofibers (Fig. 16). On the other hand, different
morphology of ZnO nanocrystals was induced when a RU-027 peptide that
has ZnO-binding sequence (EAHVMHKVAPRP-GGG-AIEKAXEIA-
NH2) was used instead of RU-003. Self-assembled fibers of a peptide
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amphiphile (palmitoyl-AAAAGGGHHHD-OH) were used for the min-

eralization of biomimetic magnetite (Fe3O4) nanocrystals.143 Since His and
Asp residues are expected to form binding sites for both Fe(III) and Fe(II)
on nanofibers of the peptide amphiphile, a larger portion of magnetite
nanocrystals are found on those surfaces.
Metal nanoparticles (NPs) composed of precious metals such as pal-
ladium,144,145 platinum,146 silver147 and gold148–151 are expected as catalysts,
optical probes and antibacterial materials. Their unique characters are
mainly due to quantum size effect and/or specific surface area. Self-
assembling peptides with metal-binding amino acids such as His, Cys and
Asp residues give effective scaffolds to prepare various metal NPs and
control their assembly, because they can fabricate nanostructures with
metal binding sites on their surface. For example, Pd(II) ions were effect-
ively accumulated on peptide nanofibers with Pd(II) binding sites composed
Fig. 16 SEM images of the solid particles obtained (a) in the absence of scaffolds, (b) in the
presence of RU-003 fibers and (c) in the presence of RU-027 fibers. From ref. 142. Reprinted
with permission from American Chemical Society.

Fig. 17 (a) Schematic illustration of PdNPs mineralization on peptide nanofiber scaffolds.

(b) Cross-coupling reaction catalyzed by PdNPs/peptide nanofibers. From ref 144.
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of His residues of a peptide amphiphile, lauryl-VVAGHH, and sub-

sequently Pd nanoparticles (PdNPs) were prepared by the reduction with
L-(þ)-ascorbic acid. Then, repeating accumulation and reduction of Pd(II)
ions on peptide nanofibers resulted in closely-packed one-dimensional
PdNPs (Fig. 17).144 PdNPs/peptide nanofiber materials effectively catalyzed
cross-coupling reactions under environmentally friendly conditions and
were reused several times.
Gold nanoparticles (AuNPs) have been also prepared by using self-
assembling peptides such as b-sheet forming peptides,148,149 surfactant like
peptides150 and peptide amphiphiles.151 The Ac-KFFAAK-NH2 peptide
that is used for the mineralization of silica and titania nanostructures can be
also used as scaffolds for the construction of gold nanostructures.148 After
the adsorption of anionic AuCl4/AuCl2 ions on the surface of positively
charged peptide nanofibers by electrostatic interaction, small AuNPs are
deposited as gold seeds on nanofibers by L-(þ)-ascorbic acid reduction
under optimized condition. Then, 1D gold nanostructures composed of
closely-packed AuNPs are fabricated by a multistep seed-mediated growth
method. The gold-peptide nanowires are highly conductive and the dried
films of them show tunneling-dominated conductance as well as resistive
switching.
4 Perspective
So far, a large variety of self-assembling peptides have been developed using
various structural scaffolds including a b-sheet, an a-helical coiled-coil, a
lipid/surfactant-like motif, a peptide amphiphile with an aliphatic tail and

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a diphenylalanine motif. Nowadays, structure-self-assembly relationship

studies have been continuously performed for better understanding and
precise control of self-assembly. The self-assembling peptides with good
properties and functionalities have been applied to organic-inorganic hybrid
materials in addition to biomedical materials. Moreover, recent computa-
tional approaches to self-assembling phenomena are very notable, because
computational simulations are useful to understand dynamic self-
assembling processes and mechanism152–160 and to predict or corroborate
electronic and structural properties of nanostructures.161,162 Computational

techniques with improved designing works will encourage further devel-
opment of self-assembling peptides and their applications.
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Integrating chemical cross-linking with
mass spectrometric analysis of peptides
and proteins
André Henrion
DOI: 10.1039/9781849737081-00151
1 Introduction
Biomolecular conjugation has been extensively used in molecular engine-
ering and design1,2 including attachments of enzymes, fluorescent labels or
reactive moieties to antibodies or of haptens to proteins or liposomes, and
protein-protein or protein-receptor conjugations.3 Not only has the linking
of different biomolecular building blocks been used to synthesise products
that combine the desired properties of each educt, but from early on it has
also proven useful for the analysis and characterisation of aggregates,
binding interfaces of complexes, and more generally, to reveal topologies of
biological objects at the molecular and atomistic levels. The latter type is the
main focus of this chapter. Information about the size, shape and
arrangement of molecules and their assemblies is inferred from what co-
valent cross-links can be established by a given reagent. Identification of the
connected pairs of reactive sites is the main analytical challenge associated
with this. In the past, as quite a common approach, this was achieved by the
enzymatic or chemical cleavage of cross-linked products and subsequent
17/11/2013 09:06:03.
analysis by liquid chromatography or gel electrophoresis. One typical

example is identification of linked sites in hemoglobin.4 Comparison of
chromatograms or electrophoretic patterns obtained with and without
having applied the cross-linking reagent would reveal which segments on
the biomolecule or aggregate had been left unchanged and which ones had
been involved in cross-links. However, chromatographic fractions (or gel
bands) had to be collected (extracted) and characterized by predominantly
chromatographic methods in order to identify the interconnected
fragments, which are tedious tasks most of the time. Mass spectrometry
(MS), having taken over in this area since mid-nineties,5 owing to its unique
capability of resolving individual species from complex mixtures, enables
unambigous assignments in a much faster and more straightforward man-
ner. Consequently, the number of applications reported has been steadily
strongly growing since then. More recently, different aspects of the subject
have been comprehensively covered in a number of excellent reports by
others.6–10 This chapter provides a further overview of how chemical cross-
linking is being used as an integral capability to the mass spectrometric
analysis of peptides and proteins. The literature coverage is from different
sources (PubMed, Web of Science etc.) and is given as a general intro-
duction to the methodology with several problem-focused experimental
examples.
Physikalisch-Technische Bundesanstalt (PTB), Bundesallee 100, 38116 Braunschweig, Germany.

E-mail: andre.henrion@ptb.de

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2 Cross-linkers and cross-linking/MS strategies

2.1 Workflow and kind of information produced
Components of a generic workflow for cross-linking/mass spectrometry
based measurements of protein structure are schematically shown in Fig. 1.
Despite examples demonstrating the feasibility of the direct top-down an-
alysis of intact cross-linked proteins11–13 the prevailing strategy continues to
be an enzymatic cleavage of a given protein followed by mass spectrometric
identification of the cross-linked peptide products. Quite as in bottom-up

protein identification, information about partial sequences of the inter-
linked peptides is entirely obtained by mass spectrometry. These sequences
provide specific codes, or fingerprints, which can be used both to identify
the ‘parent’-protein(s) and to localize the regions (defined by amino acid
partial sequences) which are close enough to one another to be linked within
the protein or between different proteins in one aggregate.
For this purpose, theoretically, the exact masses of molecular ions might
suffice in many cases. Yet, the extra information as to the sequence tags14
(series of signals coding for partial sequences of amino acids on the peptide),
and cross-linked sites, obtained by additionally monitoring masses of
fragmentation products, has led to exclusive use of single-stage MS just for
screening, routinely followed by MSn-scans to enhance specificity and re-
liability of results.
Extraction of the tags, as well as assignment to amino acid stretches on
proteins, except in very simple cases with only few model proteins in con-
sideration, requires computationally processed algorithms.7 Particularly
17/11/2013 09:06:03.
Fig. 1 An integrated workflow for cross-linking/mass spectrometry measurements. (A) An

outline of a cross-linking/mass spectrometry process. A target complex is cross-linked in so-
lution and is then digested into peptide fragments by trypsin. The peptides are analyzed by
liquid chromatography coupled with high-resolution mass spectrometry (LCMS/MS) to obtain
high-resolution masses and fragment masses (high/high) for cross-linked peptides. The frag-
mentation spectra of all peptides are subjected to a database search to identify cross-linked
peptides. As an optional step, cross-linked peptides can be enriched before their analysis by
LCMS. (B) A typical cross-linker, here bis(sulfosuccinimidyl)glutarate (BS2G), is composed of
two reactive groups on either end separated by a spacer. This cross-linker reacts with primary
amines (lysine side chain, protein N-terminus). Others target thiols (cysteine side chain) or
activate carboxylic acids (aspartate, glutamate, protein C-terminus) for reaction with primary
amines. (C) Reaction of a cross-linker with a primary amine. Part of the cross-linker, the
leaving group, is replaced by the primary amine to form a covalent bond between the spacer
and the amine. In this case, a peptide bond is formed. R can stand for either the rest of the
cross-linker or may contain another protein, if the cross-linker had already reacted on its other
end. Reprinted (adapted) with permission from Rappsilber6 Journal of Structural Biology, 2011,
173, 530. Copyright (2010) Elsevier Inc.

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challenging it is, that the number of possible pairs of connectable sites

(amino acids) that need to be taken into consideration. Such pairs are
increasing with n2-n, where n is the number of reactive (linkable) sites in the
protein (or protein-protein complex) under investigation. The use of MS to
rapid identification of linked sites in a protein complex has been demon-
strated as early as in 1996 by Yang et al.5 Motivation for this work was the
need for a rapid and reliable method for analysis of different crosslinking
products of hemoglobin, engineered with the intended use as blood sub-
stitutes. In the development of these products, a large number of different

cross-linking reagents had to be tested.
Indeed, the pattern of links would change depending on the type of the
linker, particularly regarding its spanning length. The time-of-flight MS of
the resulting products turned out to be an effective alternative to X-ray
crystallography, let alone traditional 2D-gel electrophoresis and liquid
chromatography.
The full scope and potential of integral cross-linking/MS capabilities for
providing important insights into protein 3D-structures was pointed out
somewhat later15 suggesting that the fold of a given protein, exemplified by
bovine basic fibroblast growth factor (FGF-2), could reliably be determined
from cross-linking information acquired. By sequence threading, 20 can-
didate proteins were pre-selected from a database, whose tertiary structures
could be anticipated to be similar to that of FGF-2. The cross-linking ex-
periment would reveal links as shown in Fig. 2. The reagent used, bis(sul-
fosuccinimidyl) suberate, by the maximum through-space distance it can
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span, proves the linked lysine e-amino groups be as close to one another as
11.4 Å, or even closer, which is equivalent to a maximum of about 24 Å
between the corresponding Ca-atoms. Then, the 20 candidate structures
were scored according to the degree of fitting the set of distance constraints
Fig. 2 The 15 non-local through-space distance constraints generated by the chemical cross-
links (yellow dashed lines) superimposed on the average NMR structure of FGF-2 (1BLA). The
14 lysines of FGF-2 are shown in dark grey (red online). Reprinted with permission from
Young et al.,15 Proc. Natl. Acad. Sci. USA, 2000, 97, 5802. Copyright (2000) National Academy
of Sciences of the United States of America.

Fig. 3 Reconstruction of a complex between S. cerevisiae RNA polymerase II and tran-

scription initiation factor TFIIF using distance information obtained from cross-linking/MS
experiments. TFIIF is represented by its dimerization domain as shown in the upper left part.
(Adapted from Chen et al.,19 EMBO J, 2010, 29, 717, with kind permission through Creative
Commons Attribution License.)
in analogous positions of the molecule (as taken from their known NMR-
crystal structures). Three out of the four highest ranking structures were
members of the b-trefoil fold family. This assignment of FGF-2 was
confirmed by the structural data. The promise envisaged by the authors
is that cross-linking/MS will develop into a high-throughput alternative
to the characterization of protein structure by X-ray crystallography and
NMR, thus offering an option to keep pace with a growing number of
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discovered novel proteins, being applicable to low amounts of sample, at the

same time, even in presence of biological matrices and obviating the need to
tedious efforts by crystallization.
When applied to protein complexes, cross-linking experiments allow for
the spotting of which proteins are involved as well as for localizing the
interfacing regions. This time, the proteins associated with one another are
revealed by the interlinked peptides (revealed by sequence tags), which are
coding for different proteins identifiable by a database search. This type of
information is key for the understanding of biological processes on the
molecular and a single amino-acid residue level.
Next to two-hybrid methods,16 predominantly, immunoaffinity purifi-
cation techniques have been used to extract the protein(s) under investi-
gation together with their interacting partners. However, difficulties are
often encountered that hamper unambiguous assignments, particularly, if it
comes down to the capturing of less stable and transient interactions. Cross-
linking/MS methodologies prove to be a way forward in these situ-
ations.17,18 For a example, Figure 3 shows how inter-protein cross-linking
information can be used to elucidate topologies of specific protein-protein
interactions and complexes.
2.2 Standard cross-linking strategies

Cross-linking chemistries have been known long since and their protocols
have been optimized, with most of the standard reagents being com-
mercially available in a ready-to-use format. A classic book on Bioconjugate
Techniques by Hermanson3 is a well-proven compendium regarding classical

Fig. 4 Reagents representing different cross-linking strategies: DSS, DSP and DMS, 1,2 and 3,
interlink primary amines (lysines, N-termini), DPDPB and BMH, 4 and 5, react by connecting
thiols (cysteines), BASED, 6, photoreacts with nucleophilic groups, mainly amines, and EDC,
7, induces an amide bond formation between carboxylate groups and primary amines. Di-
sulphide-containing linkers, 2 and 6, enable the option of cleaving the link afterwards, either by
chemical reduction or by dissociation during MS-analysis.
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strategies; however, for a summary of more recent progress in the devel-

opment of cross-linking chemistry tailored to particular needs with cross-
linking/MS workflows, the reader is recommended to refer to more recent
reviews.20
Some common principles used in the design and application of cross-
linkers shall be stressed upon with the help of the example reagents shown in
Fig. 4.
The first two, disuccinimidyl suberate, DSS (1) and dithiobis(succinimi-
dylpropionate), DSP (2), are di-carboxylic acids activated at both ends by
esterification with N-hydroxysuccinimid (NHS).
Therefore, this type of cross-linking reagents is referred to as homo-
bifunctional NHS-esters. On the nucleophilic attack by primary amines, i.e.
effectively by reaction with e-amines of lysine side chains and unprotected
a-amines of protein N-termini, the NHS groups will leave, and linkers will
be built into the protein, forming respective amide bonds. The reaction is
virtually specific to primary amines as partners; products formed with other
nucleophiles such as side-chains of serines, tyrosines and cysteines do not
persist but slowly hydrolyze.3 Although serine- and tyrosine-based esters
could be detected by MS shortly after formation.21 In addition to specificity,
the amide bond is as stable as necessary to survive the enzymatic cleavage
and extraction of the linker-reacted fragments in sample preparations for
MS-analysis. Bifunctional amine-reactive cross-linking reagents as DSS
may react with one end of the protein but fail to do so with the other end;
hydrolysis as competing reaction pathway results in a carboxylic ‘dead-end’
linked to the protein/peptide, as shown in Fig. 8. There are a number of

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other homo-bifunctional NHS esters available, that are different from each
other as to the spanning length and polarity. Fairly short ones are a tartrate
(DST, not shown in the figure) with 6.4 Å nominal spacer length, DSG
(glutarate): 7.7 Å, DSS: 11.4 Å, DSP [dithiobis(succinimidylpropionate)]:
12 Å, and up to BS(PEG)9 [NHS-CO-(CH2-CHO)9-CO-NHS]: 35.8 Å.
DMS, dimethyl suberimidate (-dihydrochloride), (3), represents another
class of homo-bifunctional amine-reactive linkers. Specificity of these imi-
doesters is very much the same, but in contrast to NHS-activated carboxylic
acids there are two positive charges introduced with each cross-link formed,
thus making up for the two charges that are at the same time lost at the
protein- a- or e-amino groups. This is postulated to optimally preserve the
protein fold during cross-linking as the overall-charge is not changed, nei-
ther the microenvironment where the link is introduced.3 Apart from this, it
is expected that higher charge states will result in a fragment-ion series with
higher coverage of sequences in collision-activated-dissociation (CAD) MS
analysis of cross-linked peptides.
DPDPB, 1,4-di-[3 0 -(2 0 -pyridyldithio)propionamido]butane, (4), and
BMH, bismaleimidohexane, (5), are examples for thiol- (cysteine-) reactive
linking reagents which can be used as alternatives to lysine-lysine cross-
linking. The attack of thiols at the disulfide-sites of DPDPB results in release
of pyridine-2-thione and formation of R-S-S-linker-S-S-R species, where R-S-
stands for cysteine. Maleimides, on the other hand, are attacked at one of the
double-bonded ring-carbon atoms making a (ring-) C-S-R type of addition to
the double bond. A caveat to be kept in mind with cysteine-cysteine cross-
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linking is that often the existing protein fold requires natural cysteine-cysteine
bonds to be preserved and the insertion of linkers may distort relations and
even could suggest distances to be longer than what is true.22
Photo-induced cross-linking is another useful strategy: Reagents as
BASED, bis-[b-(4-azidosalicylamido)ethyl]disulfide (6) start reacting only
on irradiation with UV-light allowing for control over which stage of the
experiment to initiate linking. The azido-groups, by loss of nitrogen, turn
into nitrenes as reactive intermediates, which further react by insertion of
the aryl-N moiety, formed at the both ends of the linker, into C-H or het-
eroatom-H bonds if in reach. Competitively, the nitrogen becomes ring-
inserted expanding the ring to the (seven-membered) dehydroazepine,
which, as a keteneimine can add nucleophilic compounds as H2NR, which
thus makes the link to appropriate sites within the protein in another way.
Next to aryl azides, alkyl- and trifluoromethylphenyl diazirines and
benzophenone are often discussed as photo-reactive groups applicable as
building blocks for cross-linking reagents.23 Generally, specificity with
photo-induced cross-linking tends to be much lower than with the
aforementioned reagents developed to link lysines or cysteines. This,
however, is to the advantage of reactivity, and the use of hetero-bifunctional
reagents that are designed to first selectively react with the protein by an
appropriate group at one end of the linker and then to find whatever second
partner-site on the protein is in proximity to photo-link to, which is an
interesting hybrid strategy.20
EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (hydrochloride),
(7), is a hetero-bifunctional linker connecting carboxylate (glutamate,

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aspartate, unprotected C-terminus) with lysines or N-terminus. It is the

most popular one out of a series of carbodiimides which are all zero-length
cross-linkers, in that, effectively, they just mediate the formation of an
amide bond between the protein carboxylate- and primary amine moieties,
but do integrate into the protein. Links of the type, by nature, will be scarce.
However, where they are detected they are of particular value as they can
provide through-space-distance information of low uncertainty (high
resolution).
An important design principle with chemical cross-linkers consists in

foreseeing sites that can optionally be cleaved afterwards. DSP (2), DPDPB
(4) and BASED (6) are representative chemistries of this type (Fig. 4).
These reagents contain disulphide (-S-S-) motifs that are chemically
cleavable by reductive agents. Among the diversity of purposes this can
serve the facilitated MS-sequencing of the individual peptide chains is a
strong argument to do so. With cleavable linkers, in a preliminary run, the
molecular mass of the linked species might be ascertained, while in another
run the individual peptides, modified by respective linker fragments, might
be sequenced with a better chance than before with obtaining abundant
ion series. It is equally worth mentioning that disulphide bonds can be
cleaved by the way of electron capture dissociation (ECD) during MS/MS
analysis24 thus providing an alternative to chemical reduction.
Obviously, the series of linkers of different maximal spanning lengths can
serve as rulers on the molecular level narrowing down the distance of re-
active residues in that protein, depending on which of them still makes the
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link and which one is the first to fail. Reliability of this information is
crucial, especially for the strategies of fold identification by threading,
homology assignment and computer modeling of novel proteins using close
fitting to known folds, as exemplified by Fig. 2 for FGF-2. Where precision
is required, the researcher, rather than using the nominal spacer lengths
provided by manufacturers, may want to refer to data acquired by Green
et al.25 based on stochastic dynamics calculations, considering ranges,
average distances, and maximal spans reported for 32 of the most often used
(classical) cross-linkers.
2.3 Cross-linking optimized for mass spectrometric analysis

One of the main challenges in cross-linking/MS workflows (Fig. 1) consists
in the recovery and mass spectrometric identification of peptides and linking
sites. Typically, the amount of a cross-linking reagent is kept low compared
to the protein(s) investigated, so as to generate only few links per molecule,
avoiding distortion of the native protein fold. As a consequence, generally,
the linked species need to be detected and analyzed well distinguishing them
from an excess of unmodified peptides. Knowledge about distinct behavior
of cross-linked peptides in MS, and in particular features that reveal the
presence of the linker, are important in this respect. Therefore, a brief
summary about the mass spectrometric fragmentation of linker-modified
peptides will be given in Section 3.
At the same time, much progress has been made in the development of
novel cross-linkers and analytical techniques capable of addressing this
problem. One set of strategies is based on the use of isotopically labeled

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cross-linkers along with the natural isotopic substances so as to provoke

defined shifts to higher masses (m/z) revealing which MS-signals can be
attributed to linker-modified peptides.22,26–29 The idea may be illustrated by
the example shown in Fig. 5. An equimolar mixture of ethylene glycol
bis(succinimidylsuccinate), EGS, with deuterated EGS (D12-EGS) was used
as a cross-linking reagent. MALDI-MS analysis gave signal doublets for the
molecular ions which are by 12.075 Da apart from one another, the intensity
ratio reflecting the molar ratio of both linker forms. The effect, that the
heavy form of the linker is producing a kind of an ‘echo’ in a precisely

defined mass distance from the natural signal, is helpful for to understand
the localization of the linking sites. The peptide shown in the Figure, was
originally present in an internally linked form, i.e. D1 and K3 were
connected by EGS/D12-EGS. As the fragmentation of such Type-1 linking
products (for classification, refer to Fig. 8) is easier if it is presented in a
ring-opened form, the link chemically was cleaved resulting in the peptide as
shown in Fig. 5. In the y-ion series, generated by collision-activated
dissociation (CAD), y6 is the first one, showing a signal doublet of appro-
priate mass difference (4.004 Da, this time) indicating lysine K3 to be one of
the two linking sites (as opposed to K8).
This is confirmed by the evaluation of the b-ion series as well, where b2 is
featured by a doublet of 4.022 Da mass difference, which is expected, while
an increase to 8.039 Da occurs for the b3-doublet. In order to establish the
compatibility of the isotope-labeling strategy with high-throughput sample
analysis, methods for the automated linker-specific processing of MS-data
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are being developed such as the ICC-CLASS software suite.30
Fig. 5 Fragment-ion spectrum acquired for a peptide which had been crosslinked using
ethylene glycol bis(succinimidylsuccinate), EGS, in a 1:1 mixture of the natural and deuterated
versions of the reagent. In order to facilitate fragmentation, the original intrapeptide-EGS-link
between D1 and K3 had been hydrolyzed leaving the modifications shown. (Adapted from
Petrotchenko et al.,28 Mol. Cell. Proteomics, 2005, 4, 1167 with permission from The American
Society for Biochemistry and Molecular Biology, Inc., Copyright (2005).)

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Another way of inducing characteristic isotopic patterns to be observed

with all those peptides that have been modified by the linker, consists in
running the enzymatic cleavage of the cross-linked protein with two ali-
quots, one using 18O-labeled water as solvent and the other one with natural
water. The labeled water causes a shift by 4.008 Da to be observed with
every C-terminal carboxylic group resulting after peptide-bond cleavage, as
two oxygen atoms are replaced each time.31 It was demonstrated that this
not only works with trypsin as a protease, but also with Lys-C.32 Therefore,
inter-peptide links (Type 2 in Fig. 8), as they have two C-termini, can be
distinguished from all the rest by a shift of 8.016 Da.33 The approach is
generic and irrespective of what linking reagent used. Rather than com-
paring two different aliquots, 18O-labeled and natural water can be mixed,
which can result in characteristic and predictable isotopic patterns revealing
the types of linking products.34 Similarly and more recently, it has been
shown that a generally applicable workflow based on 18O-incorporation can
allow the discovery of unknown structural details of a stressed IgG2
antibody.35
A completely different approach for spotting cross-linked species in
complex protein digests is to interrogate the masses of ions that do not
contain information about peptide sequences but do disclose the presence of
the linker. Fig. 12 demonstrates a set of such ions (Subsection 3.5). In this
example, the signals are produced by mass spectrometric fragmentation of
DSS-modified ions. Monitoring these m/z-values in the second MS-stage,
while allowing for a wide mass window in the first MS, indicates all species
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of interest from a liquid-chromatography (LC) run by respective peaks.

This method principle has been enhanced by appropriate design of new
cross-linking reagents; for instance, a benzylated bifunctional NHS-ester,
N-benzyl-iminodiacetoyloxysuccinimid (BID) was constructed to readily
release a benzyl cation that can be monitored at m/z 91.36
To optimally tailor reagents to suit cross-linking/MS applications a class
of linkers has been introduced that combines several advantageous features
at a time (Fig. 6).37 Two sites in one linker are chosen such that they are
more labile than most of the peptide bonds and will predominantly be
cleaved by mass spectrometric fragmentation. The cleavage of these bonds
results in the release of a reporter fragment.
Fig. 6 Conceptual modular design of novel cross-linkers. Reprinted with permission from
Tang et al.,37 Anal. Chem., 2005, 77, 311. Copyright (2005) American Chemical Society.

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Individual peptide-ions produced by the cleavage of the linker can be then

fragmented following a further collisional activation in an MS/MS/MS (MS3)
experiment which will provide sequence tags for their identification. Finally, a
moiety supporting the straightforward and effective extraction and clean-up
of linked peptides prior to MS-analysis, e.g. an affinity tag, can be built in.
Modularity of this design principle allows for the adaptation of linker
properties to the requirements of a particular experiment, which is illus-
trated by Fig. 7.38 The linkers (known as protein-interaction reporters, PIR)
were all synthesized based on Fmoc-solid phase peptide synthesis, using

appropriately substituted amino acid building blocks to introduce different
features. PIR-1 and 2, by virtue of their biotin moiety, enable very efficient
capturing from complex matrices such as cell lysates. PIR-2, which features
a photo-cleavable site, can be released from the used biotin beads simply by
irradiation, thus obviating the need of traditional elution which often results
in poor recovery. In PIR-3, the attempt was made to optimize the low-en-
ergy fragmentation properties of the molecule, so as to tune properties of
the reporter ions. Virtues of the technology are apparent from applications
to studies in biological environments. For example, it was possible
to identify a set of protein-protein interactions and respective binding
interfaces in Shewanella oneidensis bacterial cells,39 and by using a small
group, -NO2, as a reporter to design a cross-linker that is less bulky than
ones used in other designs.40 Also, with the latter design, an affinity tag is
not present, but just an alkyne functional group, which is coupled to the
affinity reagent (biotin-azide) only after the completion of cross-linking of
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the protein(s) and enzymatic cleavage. The nitro group is released as a

neutral molecule on fragmentation. Therefore, the reporter has to be
monitored during the MS analysis by neutral-loss scans with characteristic
mass differences of 45.99 Da. Recently, a detection of protein complexes in
HEK 293 cells using a similar type of linker chemistry has been reported.41
3 Mass spectrometric fragmentation of cross-linked peptides

Low-energy fragmentation patterns of protonated peptides have com-
prehensively been characterized42–45 since the availability of ionization
methods that are capable of efficiently generating and transferring the type
of ions to MS analysers.46,43 Collision-activated dissociation, CAD (also:
CID, collision-induced fragmentation), based on the conversion of the ion’s
translational energy, by collisions with inert gas, into vibrational energy up
to the level of bond cleavage, has soon become a standard technique to
achieve fragmentation, particularly in proteomics applications. Knowing
commonalities and differences in the behaviour of unmodified (tryptic)
peptides compared to those found modified after chemical cross-linking
may facilitate in the prediction of MS-signals that can be used to identify the
linker-reacted species from the background of unchanged peptides, manu-
ally or in an automatised manner. There have been a few studies aiming at
this, which shed particular light on the reaction products of bifunctional
lysine-reactive cross-linkers. These are summarized in the following Sub-
sections (3.2 to 3.5). Then, in Subsection 3.6, an alternative type of frag-
mentation technique, based on transfer of thermal electrons to the target

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Fig. 7 The chemical structures of three new PIRs. Low-energy MS/MS cleavable bonds are
indicated by bold lines. In PIR-2, the photocleavable bond is indicated by a dashed line. After
cleavage of photocleavable bond, the reactive part of the PIR-2 will be attached to the cross-
linked peptide, and the remainder will be on the avidin beads. Reprinted with permission from
Chowdhury et al.,38 Anal. Chem., 2006, 78, 8183. Copyright (2006) American Chemical Society.

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ion, electron-capture dissociation (ECD) and electron-transfer dissociation

(ETD), are briefly discussed, as these hold promise for providing infor-
mation on peptide structure which to a certain extent is complementary to
the information obtained with the help of CAD.47,48
3.1 Types of cross-linking products and nomenclature

Generally, ions resulting from the gas phase dissociation (fragmentation) of
energetically activated peptides are termed according to the site of cleavage
relative to the peptide bond:49,50(i) Ca-C, (ii) CO-NH bond itself, and
(iii) N-Ca; either as an, bn, cn if the ion contains the N-terminus of the
precursor peptide, and xn, yn, zn, if it contains the C-terminus. The subscript
n specifies the number of amino acids (by side chains) contained, counted
starting from the N- or C-terminus, respectively. If a second backbone bond
is additionally broken, then an ‘internal’ ion formed is denoted by com-
bining letters for the cleavages involved, i.e. b3y4, where three residues lost
are counted from the N-terminus and four from the other end. See also,
however, the notation originally proposed by Roepstorff and Fohlmann,51
where this should be (B3Y4’)2 for an internal acylium ion consisting of two
residues retained from an originally five amino acids long precursor.
Classes of molecular species that may result from reactions between
proteins and bifunctional cross-linkers and subsequent enzymatic cleavage
are outlined in Fig. 8. Different terms have been in use for these products.
To obviate confusion, Schilling et al.52 have introduced the nomenclature
shown in the Figure. Same principles, as mentionned above, are applicable
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for the denomination of fragment-ions of cross-linker-reacted peptides.

Distinction of peptide chains in Type 2 species is made using greek sub-
scripts, e.g. y3ab4b indicates the ion that comes from an y3-cleavage on the
(longer) a-chain, next to b4-cleavage on the b-part.
3.2 CAD-patterns of dead-end (Type 0) products

The fragmentation of a series of peptides like (Ac )AAAAKAAAAAR and
their products with different bicarboxylic-acid based linkers (DSG, DST,
DSS, DSP) have been compared by Gaucher et al.53 The doubly charged
unmodified peptide, in agreement with earlier results,54 predominantly
Fig. 8 (a) Classification of cross-linked peptides into Type 0, Type 1, and Type 2 outcomes.
(b) One can also extend this nomenclature to encompass combinations of these outcomes, such
as Type 0,1, Type 1,1, etc., for cases of multiple crosslinking and/or modification events. In the
latter cases, chain length or mass (a W b) and sequence position (N to C-terminus) determine
the order of the two numbers that designate the type of crosslink. Reproduced from Schilling
et al.,52 J. Am. Soc. Mass Spectrom., 2003, 14, 834 with kind permission from Springer Science
and Business Media. Copyright (2003) American Society for Mass Spectrometry.

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cleaves at the peptide bond right next to the lysine, C-terminal, and the y6-
fragment, i.e., Hþ[H2N-AAAAAR], accounts for the most intense peak in
the spectrum. Dead-end modifications do not principally change this, apart
from other cleavage points catching up in the likelihood, and a more intense
series of both b- and y-ions being observed in addition to the y6-base peak.
This can be attributed to the reduced basicity at the lysine side chain by the
kind of modification, which renders the second proton more mobile over the
whole sequence, while the first one is still sequestered at the arginine, such
that there are other amide bonds that are more likely to be cleaved as well.53
By analogy in the fragmentation behaviour, the same algorithms as used
for the sequence-based prediction of signals for standard peptides are
applicable to dead-end crosslinking products by solely using an increment in
mass for all ions containing the Type-0 modified lysine; with the model
peptide as given above, this would be: for nZ5 with an-, bn- and cn-ions, and
n Z 6 with xn-, yn- and zn-ions.
3.3 CAD-patterns of intra-peptide (Type 1) linking products
Fragment-ion spectra for two model peptides with different proportions
as to the number of within- and off-cycle amino acids are shown in Fig. 9.
The off-cycle parts give rise to the series of b- and y-ions, quite as with linear
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Fig. 9 (a) Deconvoluted tandem mass spectrum of a Type 1 DSS crosslinked model peptide.
(b) Deconvoluted tandem mass spectrum of Type 1 DSS crosslinked (peptide) 6. Each series of
equivalent markings (e.g. filled triangle, filled square, open circle, and open triangle) indicates
the loss of Alan (where n=1 up to 5) from the parent structure (e.g. M þ H, b9, y9, y9/y8/a4)
within the Ala4-Ala8 sequence. Reproduced from Gaucher et al.,53 J. Am. Soc. Mass Spectrom.,
2006, 17, 395 with kind permission from Springer Science and Business Media. Copyright
(2006) American Society for Mass Spectrometry.

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peptides, just featuring gaps (missing series ions) in the cyclic region. So, for
instance: b2, b3, b4 (Fig. 9a), and then continued: b7 to b11; as well as: y3, y4,
and resumed: y7 to y11, if starting from the other terminus. This is basically
the same with the second peptide (Fig. 9b), just the off-cycle part being
much shorter here. Apparently, if these ion series are long enough they can
provide sequence tags for identification of that peptide and distinction from
other products in the mixture. At the same time, the ions confining the gap
reveal the cross-linker bridged stretch of amino acids, provided they can
reliably be seen and identified.

Information about the in-cycle sequence of amino acids is less readily
obtained. A single backbone-bond cleavage does not produce a mass dif-
ference useful in that respect. However, some details may be understood
from series of internal ions, which arise if two or more bonds succesively are
cleaved, e.g. y9y8a4(a5, . . . , a8) labeled by triangles in Fig. 9b. As this
requires more energy for activation, this type of fragment ions is less
abundant and sometimes difficult to see and, therefore, to identify in the
background noise. A way around this consists of the opening of the cycle
enzymatically or chemically, which converts the molecule into an inter-
peptide-linked structure displaying ion series that are easier to interpret, as
shown for an example in Fig. 10.
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Fig. 10 ESI-MS/MS spectra of two cross-linked peptides encompassing the same sequence,
Lys-39 and Lys-53, from cytochrome c. (a) Type 1 peptide K39TGQAPGFS-YTDANKNK55
with crosslink between Lys-39 and Lys-53 with precusror ion [M þ 3H]3þ at m/z 655.703þ and
(b) same peptide digested with chymotrypsin to yield the Type 2 peptide with a-chain Ser(47)-
Lys(55) and b-chain Lys(39)-Phe(46) with precursor ion [M þ 3H]3þ at m/z 661.643þ. A col-
lision energy of 36 eV was used for both precursor ions. Reproduced from Schilling et al.,52
J. Am. Soc. Mass Spectrom., 2003, 14, 834 with kind permission from Springer Science and
Business Media. Copyright (2003) American Society for Mass Spectrometry.

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In addition to backbone peptide bonds, cleavage of lysine-e-amino/

cross-linker-amide bonds can also be observed too as a part of fragmen-
tation pathways.55 So, for instance, with K1GCREVTK2NDLR (DSS-
linked K1 and K2), having cleaved away NDLR, further fragmentation of
K1GCREVTK2 COþ (b8) would result in either (i) cleavage of the peptide
bond left-hand side of K2 or (ii) release of the linker moiety from the side
chain of K2:
(i) K2 linker K1GCREVT COþ (a8b7)

(ii) linker K1GCREVTK2 COþ (‘b8’)
Both ions, now being transformed into linear structures, by a third bond
cleavage produced a series of signals that carry sequence information, as de-
sired, namely a y-series by fragmentation of a8b7, and a b-series originating
from ‘b8’. Here again, as with Type-0 products, masses can be predicted for a
series of ions by simply treating the linker moiety (or K linker, respectively) as a
constant modification (in mass) to lysine. The ions of type (i), being K2-
immonium ions ( þ substituent), are subject to the same type of re-arrangement
as known with pure lysine immonium ions,56 resulting in the elimination of
ammonia. Therefore, in predicting masses for the ion series, a further loss of 17
Da should be allowed for. Indeed, in this example (a8-17)b7yn ions, rather than
a8b7yn, are dominating the spectrum. Likewise, such a loss of ammonia from
cross-linker modified (lysine-) immonium ions was observed by others.52,53,57,58
3.4 CAD-patterns of interpeptide (Type 2) linking products

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Interlinked peptides, the most informative type of cross-linking products,

reveal the identity of peptides and cross-linking sites by different types of
fragments all contributing complementary information (Fig. 11): (i) b- and
Fig. 11 Product ion spectrum for two DSS-interlinked peptides. [M þ 3H]3þ was selected for
fragmentation. Reproduced (adapted) with permission from Santos et al.,58 J. Mass Spectrom.,
2011, 46, 742. Copyright (2011) John Wiley & Sons.

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y-fragment ions in good abundance for both of the linked peptides, (ii)
internal ions with both cleavages occuring on the same chain, as b4ay7a, or,
(iii) inter-chain internal ions (there is none shown in the Figure, but b4ay3b
would be a hypothetical example), (iv) immonium ions such as {K}BBa
or {K}BBb and (v) cleavage products of the lysine-linker amide bond, aþ
and bþ. Inter-chain internal ions, though less abundant, may enhance re-
liability with the identification of Type-2 cross-linking products.52 It has
been observed, that fragmentation of triply charged precursor ions would
yield more abundant signals, while the doubly charged ions added further
information as dissociating along different pathways,53 typically resulting in
inter-chain internal ions in better yields.57
3.5 Linker fragmentation and reporter fragments

As pointed out in Subsections 3.3 and 3.4, the scission of the lysine e-amino/
cross-linker is often observed as a fragmentation pathway competing with the
backbone bond cleavage of peptides. For denominating ions resulting from
such a side-chain cleavage, it is customary to count atoms along the linker
‘backbone’ up to the site of cleavage. Additionally, the peptide is indicated,
which is part of the ion.52 For instance, it would be La13 with the ion at m/z
1148.6 in Fig. 11, as it includes five C-atoms along the lysine-side chain plus
eight linker C-atoms, where ‘L’ stands for ‘linker-backbone cleavage’.
In Fig. 12, different fragment ions are shown, which have been suggested
as generic reporters for the presence of DSS-reacted peptides. Extracting the
respective signals from LC/MS-MS runs may considerably facilitate in the
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filtering out of the cross-linked peptides, while excluding the non-modified

peptides from further consideration. These fragment signals are of general
applicability as they comprise only lysine modified by the linker, and do not
depend, therefore, on the particular sequence of the precursor peptide(s).
Lysine is assumed to be arranged in a six-membered ring configuration,52
see 2-amino-piperidyl moiety in 3, and, most abundantly, to be subject to
the additional elimination of NH3 (as 1,2 and 4). Acquisition of a signal
identifying 1 (m/z 240.160) indicates the presence of a Type-0 peptide.52,59
One or more out of 2-4, on the other hand, if seen as fragment ions, report
the presence of either an intra-peptide (Type 1) or an inter-peptide (Type 2)
cross-link.55,58,59
Fig. 12 Reporter (fragment-) ions for DSS reacted peptides.

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High-resolution precursor ion scans with these signals have been dem-
onstrated to provide an effective way of spotting cross-linked peptides and
making preliminary assignments as to their type.60 It may be assumed that
respective ions derived from the homologuous bifunctional carboxylic acids
would similarly be applicable as reporters, if such reagents are used as cross-
linkers.
3.6 ECD & ETD

Electron-capture dissociation, ECD,61 is provoked by the attachment of
thermal (‘soft’) electrons to multiply protonated peptides in an ion-electron
cascade of reactions,62 rather than by directly imparting vibrational energy
to the ion, as in the case of CAD. Typically, N–Ca bonds are cleaved after
reduction of a proton and transfer of the resulting hydrogen radical to a
carbonyl group:24
R0CONHR þ H $ R0 C ðOHÞNHR ! R0 CðOHÞ¼NH þ R
Therefore, c- and z-fragments are predominantly produced this way.

Figures of merit with ECD are: (i) the mechanism requiring transfer of
Hd to the CO–NH motif does not depend as much on the particular amino
acid pattern, in contrast to CAD, which does. In many instances, therefore,
ion series are more extended and promise a better readability of sequences,47
as well as identification of linked sites; (ii) weakly bonded side-chain
modifications (phosphorylations, glycosylations, palmitoylations) that are
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mostly preserved with ECD but are often lost in CAD.

The original technique, ECD61 requires that the experimental conditions
are maintained which can be put into effect in FT-ICR instruments, but are
difficult to realize in quadrupole ion traps, quadrupole time-of-flight- and
similar types of instruments. A later development, electron transfer dissoci-
ation, ETD,63 has extended applicability to radio frequency operated linear
quadrupole ion trap instruments and rendered amenable the fragmentation
technique to many more laboratories, thus. With this set-up, anthracene-
anions are generated which, after being transferred into the trap which is
storing the peptide precursor cation, act as electron-donors. For an illus-
tration of the technique, a comparison of CAD-results to ETD is shown in
Fig. 13, which highlights an interpeptide cross-linking product of ubiquitin.
ECD and ETD are reported to yield a more complete ion series, the higher
the charge state is of the precursor ion. However, as in this and other similar
examples, the information provided at least partly is complementary, rather
than superior, to what can be acquired using CAD, and to a good extent, by
overlap, can serve to back up results obtained by CAD. With modern
instrumentation, both options are available and are of similar use.
The notion of exploring protein structures by identification which peptide
pairs are in linker-length vicinity to one another has been used for decades.
Within the same timeframe, the availability of mass spectrometry for an-
alysis has turned it from a niche technology to a powerful tool with wide

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Fig. 13 CID and ETD of the 5þ charged cross-linked peptide. CID (a) and ETD (b) of the 5þ
charged cross-linked peptide, IQDKEGIPPDQQR–TLTGKTITLEVEPSDTIENVK where K
is the cross-linked residue. (a) Although the molecular weight (4431 Da) of the cross-linked
peptide is relatively large for fragmentation by CID, good coverage of b- and y-type ions is
observed. The reporter ion at m/z 525.3 is also observed with acceptable yield. (b) The high
charge state of the cross-linked peptide yields abundant c- and z-type ions in the ETD spectrum.
This shows the potential for the general use of CID and ETD for efficient sequencing of cross-
linked peptides. Reprinted with permission from Sohn et al.,41 Anal. Chem., 2012, 84, 2662.
Copyright (2012) American Chemical Society.
recognition, applicability and use. Audaciously, it might be speculated that,

the focus of proteomics research will shift from the high-throughput
identification and comparison of expression levels to the broad-band
characterization of interfacial biomolecular interactions which are enabled
by developing cross-linking/MS technologies. In this regard, the former can
eventually translate into a routine investigation, which is done as a matter of
course for each sample, whereas the latter would be used for the elucidation
of more complex and dynamic structures, up to dynamic macromolecular
self-assemblies, at the single residue level of resolution.
Mass spectrometry appears to be vastly matured with respect to the needs
of the area, particularly so with the latest generation of high-resolution
hybrid instruments such as Q-ToF, LTQ-FTICR and LTQ-Orbitrap, which
match necessary requirements for selectivity and peptide-sequencing

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capabilities, enhanced by the recent generations of dissociation (fragmen-

tation) methods including (higher energy-) C-trap dissociation, electron
capture- (ECD) and electron transfer (ETD) dissociation, and equally im-
portantly, for robustness and routine ease of use. Further efforts are re-
quired for the development of software tools for data extraction from
spectra and the identification of created links (not addressed in this Chapter,
but see Leitner et al.7 for a discussion) so as to catch up with the types of
information available from the latest trends in cross-linker design. This is
particularly so for the mapping of protein-protein interactions, which re-

quires to improve the coverage of all interfaces and contacting sites, which
in turn increasingly necessitates the application of linkers (e.g. formalde-
hyde, diazirine-based photo-cross-linkers) which can react in more a broad-
band way with many potential functional groups,18 resulting in much more
complex product mass spectra than those obtained with merely amine- or
thiol-reactive reagents.
Eventually, it takes little to anticipate the continued expansion and di-
versification of the set of cross-linking reagents available to fit with different
types of samples and investigations – as can be exemplified by the evaluation
of photoreactive amino acids that can be incorporated into living cells
and can link to suitable neighboring groups on irradiation64 or by using
complex forming metals such as cis-[Pt(NH3)2Cl2]65 as alternative candidate
cross-linkers.
17/11/2013 09:06:03.
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Peptidome analysis: tools and technologies
Paulina D. Rakowska and Maxim G. Ryadnov*
DOI: 10.1039/9781849737081-00172
1 Introduction
In the post-genomic landscape of highly specialised ‘–omics’ strategies that
are being developed to provide molecularly traceable links between biology

and pathology at the cellular and systems level, peptidome analysis has
transformed into an information-rich and self-sufficient area, which by as-
sociation has been coined peptidomics. The area encompasses comprehen-
sive and systematic qualitative and quantitative analyses of peptides in
different biological media ranging from biological fluids to cellular extracts.
The very definition of peptidome is to be agreed upon. However, by con-
vention, peptidome is referred to as an array of peptides present in a par-
ticular sampled pool or degradome of biomarkers (cells, plasma, and
cerebrospinal fluid). A clear distinction is made regarding the nature of the
biomarkers, which are endogenous and are of low molecular weight, hence
peptides. These include peptidic factors, hormones and metabolic products
that can originate from protein precursors. Peptidomic profiles obtained for
different conditions and diseases are extremely varied and require similarly
varied tools, the development and optimisation of which are continuing
from the simplest biochemical methods to advanced multidimensional
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separations and bioinformatics-empowered mass spectrometry. This report

is presented in a form of an overview which highlights mainstream ten-
dencies exemplified by recent developments in peptidome analysis from the
perspective of analytical tools and technologies using the literature sourced
from relevant databases including Web of Science, RCSB Protein Data
Bank and PubMed.
2 Peptidomes and peptidomics

2.1 Endogenous peptide biomarkers and pools
Endogenous peptide biomarkers are peptides, which as defined by the
IUPAC-IUB typically contain up to 50 amino-acid residues and do not
exceed molecular weights of 8 kDa.1 These seeming restrictions are sufficient
to provide one of the most abundant classes of specialised biomarkers,
which could broadly be grouped into (i) protein metabolites resulting from
degraded protein precursors or protein digests2–5 and (ii) peptide secondary
metabolites or non-ribosomal peptides (e.g. depsipeptides) incorporating
non-proteinogenic amino acids and modifications.6–8
Tissues are normal sources of such biomarkers. A specific tissue has a
limited set of peptide biomarkers whose content conforms to a relative
specificity of proteolysis. The proteolysis of a given set will give rise to a
large group of peptides defined as a ‘‘tissue-specific peptide pool’’.9,10 The
pools of healthy tissues, cells and biological fluids in their normal state are
National Physical Laboratory, Teddington, TW11 0LW, UK. E-mail: max.ryadnov@npl.co.uk;

School of Physics and Astronomy, University of Edinburgh, Mayfield Road, EH9 3JZ, UK.

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fairly conserved matrices displaying no significant variations. Peptide pools

obtained from related samples originating from different species are gen-
erally very similar in composition. However, peptide pools obtained from
different tissues of the same organism can vary significantly.11 Considerable
evidence exists to suggest that an extensive number of components from a
given pool can reveal a noticeable activity both in vitro and in vivo. This in
turn points to regulatory roles of peptides in biological systems, be it a cell
or an organism. There may hardly be a process that does not involve or
could not be influenced by a peptide.12 A predominant function of peptide

pools is attributed to the controlling of long-term biochemical processes and
to the maintenance of homeostasis, for tissues.9,13 Also, the peptide com-
position undergoes changes as a result of tissue pathology, stress impact,
treatment with drugs or genetic modifications. Based on the above, the
understanding of effects to the peptide pools, caused by malfunctioning
biological systems becomes extremely vital. As a result, the biological po-
tential of peptide biomarkers attracts an increasing attention in academia
and industry and prompts the continuous development of new strategies
and tools to enable peptidomic analysis of specific biological matrices.
Indeed, endogenous peptides can provide excellent biomarkers, even
when their biological activity is unambiguous or, conversely, not apparent.
Being mere products of proteolytic degradation peptides may be indicative
of a particular pathology or a developing process, whilst being highly spe-
cialised molecules they can possess different functions at different levels of
molecular recognition and organismal development. For example, anti-
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microbial peptides (AMPs), which are evolutionarily conserved host defence

effector molecules, can not only recognise and kill microorganisms but also
elicit immunomodulatory effects and serve as stand-alone protein trans-
duction domains. AMPs have been shown to play inhibitory and mediatory
roles in combating and containing various disorders ranging from atopic
dermatitis,14,15 inflammations16–18 and wound healing19,20 to auto-
immunity21,22 and cancers.23–26
2.2 Peptidomic versus proteomics

As peptides play a central role in physiological processes, there is a strong
need for the continuous development of technologies, which would enable
comprehensive analyses of the entire peptidomes of cells, tissues and, in
principle, whole organisms. Peptidomics is strongly related to proteomic
studies, where protein analyses mostly rely on obtaining peptide fragments,
their analysis and sequencing.27,28 However, in comparison to proteomics
and even taking into account the steadily growing effort in the detection and
discovery of peptide biomarkers and consequently numerous projects in the
field, peptidome analysis remains in its infancy. This could be demonstrated
by comparing the number of records returned by PubMed: in excess of 3200
articles for ‘peptidom*’ and over 63000 hits for ‘proteom*’ (Fig. 1).
Though peptidomics and proteomics share many of the scientific ob-
jectives, technologies and techniques, there are several key differences
beteween these fields. Those differences result from the size of peptides,
origin, specific modifications and activity. Therefore, the same methods,
which are well established to ‘‘mine’’ the proteome, unless altered to the

Fig. 1 Number of articles published during the past decade, containing peptidom* and pro-
teom* in their titles. Source: total searches (07/2013) in PubMed (http://www.ncbi.nlm.nih.gov/
pubmed).
analysis of naturally occurring peptides, often cannot be easily applied for

peptidomes. For example, most of proteomic studies use proteolytic en-
zymes such as trypsin to obtain well defined fragments that can be sub-
sequently analysed by mass spectrometry (MS). In peptidomic studies
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digestive enzymes are scarcely employed as this is the native form of the
peptides that is of scientific interest and the size already allows the use of
mass spectrometry. Sample preparation for peptidomic studies can be more
straightforward without proteolysis; however, data analysis can be more
difficult in this case, especially for the analysis of larger peptides and those
with multiple post translational modifications or those that contain unusual
amino acids. This can influence not only the sensitivity of MS experiments
but also affect prediction methods and the understanding of fragmentation
patterns.
2.3 Peptidomic processing

One of the difficulties in analysing complex media comes from wide dynamic
concentration ranges. For example, the number of proteins and peptides
identified in human plasma has expanded into thousands over the past few
years but W90% of the total protein mass in plasma is accounted for by
fewer than 30 major proteins, while the richest information is thought to be
contained in low molecular weight/low abundance fractions. Protein and
peptide concentrations can vary from millimolar for albumin to femtomolar
for the least abundant species. Very often some of the most important
analytes are present at very low levels and masked by high abundance
biomolecules.29–31
The isolation of peptides from such a mixture can prove to be difficult and
almost always requires a combination of several techniques to result in a
homogenous sample. In order to overcome the challenges related to bio-
logical sample complexity, several strategies, forming multi-step workflows,

Fig. 2 Flowchart of an exemplary peptidomic process: tissue sample, after homogenisation

01/12/2013 14:45:06.
and initial clean-up, is processed by nano-LC MS. Mass spectra are acquired continuously
during the LC run and mass spectrometer software uses the MS spectrum to decide on peak
selection for fragmentation and MS/MS analysis. The obtained fragmentation spectra are
converted to peak lists which are then used for query of a database. Reprinted with permission
from G. Baggerman, P. Verleyen, E. Clynen, J. Huybrechts, A. De Loof and L. Schoofs,
Journal of Chromatography B2004, 803, 3–1644; Copyright & 2004, Elsevier.
have been developed and combined together. An example is given in Fig. 2,

which shows major techniques including sample depletion to remove most
abunô˜dant proteins, such as albumin and immunoglobulins that can
interfere with the detection of a less abundant species, and sample frac-
tionation and purification by chromatographic separations to reduce
complexity prior to follow-up detection and identification by mass spec-
trometry supported by computational methods.
2.4 Sample separation and concentration

Size-based separations are occasionally employed for the separation of
desirable peptide biomarkers or biomarker series. There are reports on
successful approaches based on methods such as IEF, GE or even SEC,32–35
which, however, are not exclusive of drawbacks such as the amount of
analyte necessary, but usually relatively high, for successful separations.
Also, the nature of this kind of separations makes them efficient mainly
for higher molecular weight components (>5 kDa).36 Therefore, peptidome
analyses are based, most often, on gel-free approaches. There are many
traditional procedures, resulting in peptide-rich extracts, such as

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37–39 40
ultrafiltration, the use of trifluoroacetic acid and organic solvent ex-
traction.41,42 The choice of a particular purification protocol needs to be
carefully thought through and tailored to the specific target molecule.43
HPLC techniques are introduced at the later stages following initial pre-
cipitation, clarification and preliminary separations.
2.5 LC-MS interfaces

The extracted and enriched peptide pools are next introduced to liquid
chromatography separations often using a multidimensional set-up fol-

lowed by MS detection and analysis.
MS techniques can be applied to almost any kind of charged molecules
including proteins, lipids, nucleotides and amino acids. However, to be
sufficiently sensitive, they require relatively pure samples in certain buffer
compositions. Any salt-containing buffers used to pre-fractionate complex
samples can promote ionization backgrounds, which can make data inter-
pretation difficult, if not impossible. Therefore, an obvious choice for a
preceding MS analysis is a chromatographic step using an MS-compatible
technique such as reversed-phased liquid chromatography (RP-LC). RP-LC
allows setting elution gradients using organic solvents such as acetonitrile,
which is fast evaporating and promotes analyte ionisation, hence is suitable
for the following MS analysis.
For more complex samples, such as cellular extracts, additional pre-
fractionation steps prior to the RP-LC separation prove to be necessary. Ion
exchange chromatography (IEX) presents a conventional approach, which
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enables, in the first instance, separations based on pI values. The ionic

sample fractions consisting of a cationic species and an anionic species can be
retained by the oppositely charged stationary phase of the column and eluted
with the increasing concentration of a similarly charged species, displacing
the analyte ions from the stationary phase. Because IEX separations solely
rely on charge differences, which often results in poor chromatographic
resolution, peptides appearing in multiple fractions are re-run by RP-LC
providing thus a two-dimensional chromatographic approach.45–47
Multi-dimensional chromatographic approaches are also utilized. Sam-
ples can be fractionated based on properties other than charge and
hydrophobicity. Anything from immunoaffinity to chemical separations can
be successfully applied, i.e. MS preceding SEC analysis,33 2D-Hilic MS,48
EX-RP MS49 or two-dimensional 2D-HPLC run at basic pH (first dimen-
sion) followed by acidic pH (second dimension).50
3 Peptidomics tools
3.1 Nanoparticle-enabled approaches
With a growing demand for high-throughput methods, application of
nanoparticles as a preparative step or when interfaced with an MS analysis
has a great exploitation potential to bring up new technologies with ap-
plications in diagnostics and personalised medicine.
Nanoparticles are nanoscale materials with surfaces that can be readily
modified to create highly selective adsorbing probes. This enables new ap-
proaches to detect and concentrate peptides from biological fluids,51 by

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fractionation of complex samples and the capturing of a specific biomarker.

Size-dependent fractionations enabled by mesoporous silica particles52 or
nanoparticles coated with a porous material53 have been developed. Nano-
particle surfaces can also be coated with ionic functional groups to extract
only oppositely charged analytes,54 for instance, weak cation exchanger
magnetic beads used for selective enrichment of low molecular weight pep-
tides. Magnetic beads55 are perhaps the most promising and appear to be
popular among various types of separation particles. Not only they can be
quickly and easily separated from the sample solution but MS analysis
(MALDI-ToF) can be directly performed on the beads, which allows for the
efficient and sensitive detection of peptides that can be successfully employed
to quantify the cellular uptake of cell penetrating peptides.56
Following these and similar approaches, after the removal of nano-
particles, the depleted fraction of the sample becomes easier to analyse.
Also, if analytes remain bound to nanoparticles, they can be removed with
an eluent, which, however, can be omitted as in the case of magnetic
nanoparticles where the presence of nanoparticles not only imposes no
interference on the analysis but can also improve single-to-noise ratios for
the analytes (Fig. 3).57–66
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Fig. 3 Schematic representation of nanoparticle-based capturing of analytes from complex

sample: (a) nanoparticle with selective surface properties; (b) nanoparticle conjugated with a
capturing agent. Reprinted with permission from H. N. Shah, C. Chilton, L. Rajakaruna, T.
Gaulton, G. Hallas, H. Atanassov, G. Khoder, P. D. Rakowska, E. Cerasoli and S. E. Gharbia,
in Mass Spectrometry for Microbial Proteomics, John Wiley & Sons, Ltd, Editon edn., 2010,
pp. 1–34.64 Copyright & 2010 John Wiley & Sons, Ltd [Colour image available on-line].

Fig. 4 Schematic representation of melittin–NP interaction. Contributions of hydrophobic

(light arrow) and electrostatic (dark arrow) interactions between melittin and NPs 7 (a), 6 (b), 2
(c), and 5 (d). Contributions of polymer density to interactions between melittin and NPs 9 (e)
and 10 (f). Reprinted with permission from Y. Hoshino, T. Urakami, T. Kodama, H. Koide, N.
Oku, Y. Okahata and K. J. Shea, Small, 2009, 5, 1562–1568. Copyright & 2009 Wiley-VCH
Verlag GmbH & Co. KGaA, Weinheim.
Approaches to target particular biomarkers aim at the functionalisation

of nanoscale materials with affinity agents. A variety of covalent and non-
covalent chemistries for the derivatisation of nanoparticles with proteins
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and peptides has been reported.61,64 However, this area remains to be

challenging due to the incompatibilities between biomolecules and nano-
structures. It is necessary to preserve the biological function of the attached
capturing agent. However, current progress is encouraging with successful
methods being developed.62 A good example of an efficient harvesting
platform is a well-established bio-barcode assay based on the targeting and
capturing of soluble peptide biomarkers for Alzheimer’s disease from a
cerebrospinal fluid (CSF), by antibody-functionalised nanoparticles.63
Nanoscale systems for a specific peptide binding can be designed using
functionalised polymer particles possessing high affinity for biomacromo-
lecular toxins (melittins) with simultaneous neutralization of their toxic
effects. Co-polymeric nanoparticles constructed from different functional
monomers were shown to be able to capture and neutralize the toxicity of
melittin even in a complex biological milieu (Fig. 4).65
3.2 Peptide sequencing and identification by mass spectrometry

Mass spectrometric techniques are well suited to peptidomic studies as they
allow obtaining accurate and reliable information, often in a high-
throughput manner. The techniques enable the detection of low-molecular-
weight peptides at very low concentrations with adequate resolution and
sensitivity which makes them a useful tool for peptidome profiling.
For the identification of peptides, individual HPLC fractions are intro-
duced for mass spectrometric analyses. Soft ionisation detection
methods (MALDI) that rely on the precise measurement of the peptide
mass are preferred. Tandem mass spectrometry, i.e. nESI-TOF-MS/MS or

Fig. 5 Schematic representation of a tandem mass spectrometry experiment. Following

chromatographic fractionation, sample is introduced to a mass spectrometer where it is ionised.
The resulting ions are detected by first mass analyser; CID of the ions takes place in the col-
lision cell where energy is transformed through collision with neutral molecules (gas) resulting
in the fragmentation of a parent ion to ions, which are then detected by a second mass analyser.
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[Colour image available on-line]
MALDI-TOF/TOF-MS, can provide fragment spectra of the chosen parent

ion, which is used to identify a corresponding peptide sequence (Fig. 5).
Traditionally, peptide sequencing is carried out by the Edman degrad-
ation method66 manually or automatically. Though well-established and
reliable, the technique is characterised by several drawbacks, for example,
when the N-terminus of a peptide is not accessible, or samples used are short
of the material to be sequenced. In addition, it can be problematic to
analyse a mixture of compounds of similar physico-chemical properties that
cannot be easily separated. In recent years mass spectrometry has been
developed as an alternative, now well-established and widely used for
peptide sequencing.67 Peptide identification is achieved through fragmen-
tation by various techniques, such as collision induced dissociation
(CID),68,69 electron capture dissociation (ECD)70,71 or electron transfer
dissociation (ETD).72,73
CID is the most common fragmentation method, according to which a
usually protonated peptide precursor ion is selected and subjected to col-
lision with non-reactive gas molecules such as argon. The impact leads to
the fragmentation of the peptide and the generated fragment ions are mass
analysed. All the fragments in mass spectra relate to the selected parent ion.
If the instrumentation allows performing sub-sequential CID steps the
process can be repeated on any fragment ion so that the elemental com-
position of the peptide fragment can be resolved. There is a substantial
knowledge in terms of fragmentation mechanisms, which allows predicting

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types of fragment ions and their patterns depending on mass spectrometer

specifications. Hence, many tools have been developed to analyse peptide
product ion spectra in a computerised fashion. Collected spectra are com-
pared to the theoretical, in silico performed fragmentation of compounds
contained in databases and best cross-correlating candidates are matched.
Nevertheless, sequencing is an intrinsically difficult process. The spectra
obtained by fragmentation can return only a partial sequence tag as the
cleavage does not occur at every peptide bond, and overall can be difficult to
interpret due to the modifications of amino-acid residues, ion adducts and

the same (Ile/Leu) or very similar (Lys/Gln or Phe/oxidised Met) amino-
acid masses.74
3.3 Tandem mass spectrometry

Different types of tandem mass spectrometry approaches can be used for the
analysis of peptides and proteins.75,76 The most common type, product ion
scanning, is used mainly to elucidate the amino acid sequence of a specific
peptide. Here, the first mass analyser filters ions for one specified precursor
ion, which then undergoes fragmentation (such as CID) in a collision cell.
The created fragment ions (product ions) are further analysed by a second
mass analyser. In the precursor ion scanning mode,77 another approache, it
is the second mass analyser which is set to sieve specified fragment ions and
the first mass analyser detects only those compounds which give that
fragment ion. This type of analysis can be used to detect specific functional
groups or modifications such as carbohydrate adduct,78,79 phosphoryl-
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ation80 or carbonylation.81 In the third approach, neutral loss scanning,

both analysers work in a synhronised manner and are set to detect a neutral
loss of a specific mass. The detected mass difference relates to a fragment
which have been detached from a peptide in the collision cell. Therefore,
simirally to the precursor ion scanning, this method is used to detect those
molecules which contain specific functional groups, such as phosphopep-
tides through the loss of phosphoric acid or glycopeptides due to the loss of
glycan moieties.82–84 Finally, the multiple reaction monitoring method, also
known as selected reaction monitoring, the first and second mass analysers
are set to detect specified precursor ions and product ions that are char-
acteristic for that precursor, respectively. This approach requires the
knowledge of the fragmentation properties of selected parent ions but is
highly sensitive and enables the targeted detection of analytes in complex
biological samples. When combined with isotope dilution it also alows their
absolute quantitation.85–88
Arguably, one of the main advantages of tandem mass spectrometry
procedures is their ability to select a specific macromolecular assembly and
by inducing its dissociation into subunits, which would follow a sequential
pathway, their ability to allow for the full reconstruction of the assembly
under question.89 Therefore, the methodology is often used, and increas-
ingly so for progressively larger and more complex assemblies, to highlight
differences in the dissociation pathways of related macromolecular as-
semblies, which are the direct consequences of changes in local and global
oligomeric organisation. Such a capability can prove to be indispensable for
studying complex and dynamic peptidomes that undergo complex assembly

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Fig. 6 MS-monitored dissociation of a M. jannaschii small heat shock protein, MjHSP16.5.

(A) A contour plot shows that the 24-mers dissociate into highly charged monomers at low m/z
and lowly charged oligomers (23-, 22- and 21-mers) at high m/z; monomers and oligomers are not
on the same scale. (B) Plotting the intensity of the 24-mers (circles, green online), 23-mers (triangles,
blue online), 22-mers (squares, purple online) and 21-mers (diamonds, red online) shows the se-
quential loss of subunits. (C) A delineated dissociation scheme showing the average charge states of
the different species observed. (Reprinted from Chem Biol, 13, J. L. Benesch, J. A. Aquilina, B. T.
Ruotolo, F. Sobott and C.V. Robinson, Tandem mass spectrometry reveals the quaternary or-
ganization of macromolecular assemblies, 597, Copyright (2006), with permission from Elsevier).
or misfolding-aggregation processes, each of which can have biological roles

or abnormal implications, e.g. intracellular machinery or amyloidogenesis.
Fig. 6 highlights a representative study of ‘‘stripped oligomers’’ for a
small heat shock protein from a thermophilic archaea Methanococcus jan-
naschii, MjHSP16.5, which comprises 24 monomeric subunits. The se-
quential loss of the subunits is followed and controlled by MS (Fig. 6A,B),
thus allowing for the full characterisation of oligomeric species at different
stages of the assembly (Fig. 6C). More specifically, the onset of dissociation
into monomeric subunits and 23-mers occurs at one collision energy (70 V),
which continues at slight energy increases when 22-mers are formed. At the
highest collision energy, 21-mers start being observed (Fig. 6A). This al-
together indicates that the multiple loss of subunits during CID is not a
concerted disassembly process but sequential. This and other similar ex-
amples support the application of tandem MS for establishing the stoichi-
ometry of macromolecular assemblies, which is being now widely applied in

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combination with more traditional high resolution approaches. By re-

moving highly charged monomers from the assemblies, sequentially, tan-
dem MS data can be used to precisely interpret both the global oligomeric
organisation and unambiguous stoichiometry and key interactions between
adjacent subunits enabling thus the elucidation of the local structural
organisation.89
4 Peptide quantitation
4.1 Choice between absolute and relative quantitation strategies
Since mass spectrometry has become an essential tool in peptidomics for
detection, identification and characterisation, it has also become the main
tool for quantitative peptide analyses. Quantitative approaches can be
divided into two broad categories, those that provide absolute and relative
quantitation and those that are label-based and label-free.90,91
The strategies of relative quantitation92–94 are based on the comparison
between the levels of individual peptides in a sample with those of an
identical sample, in which the same compounds have been modified. In a
typical experiment, peptides from two experimental samples are isotopically
labelled, totally or at specific amino acid positions. Those two samples that
contain the equal amounts of peptides are combined and analysed by, for
example, LC-MS. Properties of the individual, differently labelled species
coming from both samples remain identical, and hence they co-elute and are
detected by MS at the same time. The intensities of corresponding heavy
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and light peptide ions are then compared for concentration differences.
4.2 Label-based methods

There are several methods enabling the use of peptide labels including:
a) Metabolic labelling, the most common version of which may be stable-
isotope labelling in cell culture (SILAC). This method involves in-vivo in-
corporation, during cell growth and division, of a stably labelled amino acid
into the proteome/peptidome of the cells, which are added to the cell culture
media.95–97 To carry out the relative quantitation control cells grown in
regular media are used. The main advantage of this method lies in its
relatively simple protocol and the minimal manipulation of the probed
systems. Nevertheless, this method is almost only appropriate to cell sam-
ples and may not be applicable to those that are sensitive towards changes in
culture medium composition.
b) Chemical labelling can be applied to samples, which cannot be pro-
cessed with metabolic labelling such as tissues or biological fluids, or when
short experimental time is necessary. Many strategies involving chemical or
enzymatic stable isotopic labelling have been developed, such as enzymatic
labelling with 18O, where tryptic digest is performed in water containing 18O
and another in water with 16O. The mechanism of trypsin action in-
corporates the oxygen atoms at the C-termini of freshly digested pep-
tides.98,99 This method however may not be suitable to study endogenous
peptides or when the trypsin use is not appropriate. Stable isotope dime-
thylation is another example of chemical labelling, which involves the la-
belling of primary amines with deuterated methyl groups by formaldehyde

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100
in deuterated water. ICAT (Isotope Coded Affinity Tag) is the most
utilised of the cysteine tagging methods.101,102 This strategy requires a
label composed of three parts: an isotopically labelled (deuterated) linker,
biotin group and a cysteine reactant. Once peptides are labeled, they are
subjected to column chromatography with immobilized avidin or streptavidin,
then biotin enables separation and enrichment and analysys of cysteine-
containing peptides. This method is applicable to these spieces only.
c) Isobaric tagging with reagents such as iTRAQ,103–105 requires analysis
with tandem mass spectrometry as isobaric tags have identical masses and
chemical properties, therfore there is no distingushment during the LC and
MS runs. During MS/MS experiment, in the collision cell, the tags are
cleaved from the peptides. The peptide ions are sequenced and the tags are
quantified. This allows the simultaneous determination of both the identity
of the analytes and their relative abundance. Because the MS/MS is set up
only for the tag detection unlabeled peptides are not quantified.
4.3 Label-free methods

Label-free quantitation methods are used for both relative and absolute
quantitation.93,106,107 These methods are also well suited for high-
throughput screening and biomarker discovery experiments. They do not
require control or differently labelled samples. Samples are independently
prepared and analysed by LC-MS or LC-MS/MS. Nevertheless, the lack of
controls imposes carefully planned, controlled and reproducible processes
to avoid experimental variations.
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In instances where ion signal intensity correlates with peptide concen-

tration, as for example in electrospray ionisation experiments, the quanti-
tation of peptides can be performed taking into account ion intensity. These
quantitative approaches rely on LC-MS measurements and the relative
levels of peptides between analysed samples can be directly estimated from
the peak intensities. Using spectral counts is another type of label-free
relative quantitation. In this method a tandem-MS set up is normally re-
quired which involves the comparison of the sum of MS/MS spectra ori-
ginating from a peptide of interest across multiple samples, which correlates
with the analyte abundance.
Absolute quantitation108–110 is also possible by a label-free approach but
requires the preparation and incorporation of internal standards – iso-
topically labelled, heavy analogues of the probed peptide, which are spiked
at known concentrations into the sample (Fig. 7).
The spiked sample is then subjected to the processing by LC-MS. Though
having different masses, chemically the peptides are identical, which allows
their simultaneous co-elution and analysis by MS. The abundance of the
target peptide ion is compared to the abundance of its isotopically labelled
analogue in the analysed sample and the quantitation is performed with the
use of a prior-built standard curve.
One of the neat examples reported to date is an isotope dilution approach
used for the accurate quantification of the cellular uptake of cell penetration
peptides (CPPs).56 This method employed magnetic beads for peptide
harvesting which was followed by direct peptide analysis by MALDI MS.
Quantification was achieved using a deuterated analogue of the analysed

Fig. 7 The first isotope of the signals is identified for both [1H]peptide (m/z=2,100.2) and
[2H]peptide including the component with eight deuteriums (m/z=2,108.2) and the non–
completely deuterated components (m/z=2,105.2, 2,106.2 and 2,107.2). Signals including all
isotopes as delimited in the figure are taken into account in the quantification. Reprinted with
permission from F. Burlina, S. Sagan, G. Bolbach and G. Chassaing, Nat Protoc, 2006, 1, 200–
205. Copyright & 2006 Nature Publishing Group. [Colour image available on-line]
CPP as an internal standard (Fig. 7). The method can be used in a generic
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format and allows not only the determination of the intact internalised
peptide but also the identification of intracellular digests. Several different
peptides can also be analysed during a single procedure which makes it
possible for the analysis of multi-component systems, such as drug or gene
delivery non-viral vectors containing protein- or peptide-based cargoes.
Intracellular stability of internalised components is another avenue for such
methods, however, this may provide only a relative quantitation insufficient
for comparative analysis of different carries and cargoes used for intracel-
lular delivery but sufficient for their pre-selection.56
Although it would be ideal to always be able to perform absolute peptide
quantitation, relative quantitation approaches are used more often. There
are several reasons for that, one of which is that absolute quantitation is
mainly limited to the analysis of one analyte at a time and does not allow
monitoring changes in the presence, which can be abundant, of other
compounds or components in one sample simultaneously. This can be ad-
dressed by developing methods that are analogoues to the one descibed
above. However, such developments can be time consuming and in most
cases would have to be carried out with the help of isotopically labelled
reference molecules which can be expensive and not easily amenable for
other systems if one aims at developing high throughput screening assays. In
terms of peptidome analyses, the same labelled peptide standard can be
adapted for different peptide pools containing a variety of peptides of
similar molecular weights. However, specialist quantitation workflows re-
quire specilist labelling strategies, the tendecy which will prevail. An over-
view of quantitation workflows is schematically shown in Fig. 8.

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Fig. 8 Overview of quantitation workflows. The points, at which peptides are labelled, or at
which isotopically labelled analogues are used, are indicated by blue and red (heavy). Metabolic
labelling takes place in vivo. [Colour image available on-line]
5 Data processing and global identification

5.1 Differential peptide display
Differential Peptide Display technology (DPDt; BioVisioN) is a workflow,
often utilised for separating, detecting, and identifying peptides derived
from complex samples such as cell and tissue extracts and biological fluids.
It has applications for biomarker discovery and detection of pathological

Fig. 9 An example of peptidomics process. A) Samples are separated RP-HPLC (A) and
eluting peptides are collected into fractions, which are then analysed by MALDI-TOF-MS (B).
The mass spectra of all fractions are combined into an in silico two-dimensional display of
peptide masses (C). Mass spectra of individual samples can be correlated and compared with
other sets against data obtained from different sets, which can result in the visualization and
detection of differences in the resultant peptide display (D). Chosen peaks from individual
HPLC fractions can be then identified (E). Reprinted with permission from H. Tammen, T.
01/12/2013 14:45:06.
Möhring, M. Kellmann, A. Pich, H. H. Kreipe and R. Hess, Clinical Chemistry, 2004, 50, 545–
551. Copyright American Association for Clinical Chemistry. [Colour image available on-line]
states of a biological system, and allows for the detection of differentially

expressed and occurring species between analysed samples.111–116
Samples, separated by means of liquid chromatography, are collected
into fractions, which are then subjected to MS analysis. Mass spectra of all
fractions are combined together in a two-dimensional plot, on which one
dimension represents LC elution time and the second dimension does mass-
to-charge ratios of the detected peptides. Mass spectra of individual samples
can be compared or correlated against control samples and then visualised,
aiding in the detection of dissimilarities in the resultant display (Fig. 9).112
5.2 Bioinformatics and peptide identification by database searching

In parallel to the advances in proteomics and peptidomics a repertoire of
computational methodologies and tools is extensively being developed.
Naturally, many of proteomic tools can be used for peptidomics. Never-
theless, certain bioinformatics tools are specifically tailored for peptidomic
analyses. More comprehensive reviews of available technologies can be
found elsewhere.117
A peptide is identified by a database search. This approach is based on
comparison of the acquired mass spectra, or spectra resulting from frag-
mentation of a parent ion, with the theoretical spectra from a database such
as Mascot118 or SeQuest,119,120 and is predicted for each listed protein or
peptide.

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Several factors make a peptidome search a complex process.

Amongst others post-translational modifications (PTMs) stand out as a
serious problem particularly using available search engines (Mascot). The
programs cannot identify the peptide sought unless amino acid modifi-
cations are pre-specified. Although current search engines provide the
user with the option of including possible modifications to the search
parameters, a considerable awareness of commonly occurring and possible
PTMs to the amino acid residues is pivotal to enable the proper inter-
pretation of mass spectra, which often necessitates the manual interpret-

ation of the results. Some of the PTMs are known to occur in vivo as a
result of natural biological processes. PTMs such as phosphorylation of
serine and threonine residues or the amidation of C-termini are com-
mon.121–125 Modifications known to derive from peptide extraction and
analysis, methionine oxidation, are also frequent.126 A current tendency is
the development of novel algorithms and search engines that will aid in the
sequencing of peptides with unknown modifications for unrestricted
identification.127
Most often, for those endogenous biomarkers, which are metabolite
products of precursor proteins, genome sequencing has identified previously
unknown enzymes, the function and specificity of at least half of which
remains unclear.128,129 In comparison to proteomic studies, for which the
routine application of trypsin and few more well defined proteolytic di-
gestion protocols allow obtaining short, highly regular patterns, there are
no specific cleavage enzymes, which could be applied to peptidomic analyses
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in a similar manner. The considerable amount of modifications in bioactive

peptides130 significantly hinders the identification process. In addition, the
quantity of in vivo peptides, which is often very low, makes peptide iden-
tification less straightforward unless a strong experimental support has been
applied beforehand.
To address these issues and improve the database search, several methods
are proposed to improve existing approaches. A range of software is
available for the application of different cleavage patterns or custom
cleavage algorithms. Some of the peptidomic driven databases, such as
PeptideDB or SwePep, enable searches using a variety of peptide charac-
teristics including sequence length and composition as well as the appli-
cation of fixed and variable PTMs. One major issue, however, which limits
the use of peptide databases remains – the identification of the peptides is
not deemed possible unless representative spectra have been deposited in the
database library.
To demonstrate the variety of bioinformatic tools analiable a represen-
tative list of selected databases and other instumental programs is repre-
sented in the following table.
Many other useful tools are also available to assist in silico prediction
methods, such as NeuroPep designed to help predict peptide cleavage sites
(http://neuroproteomics.scs.uiuc.edu/neuropred.html), Unimod – a peptide
modifications database (http://www.unimod.org), a Database on Demand
(http://www.ebi.ac.uk/pride/dod) or collections of proteomics tools at
http://www.proteomecommons.org166 and ProteinProspector (prospector.
ucsf.edu).

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Name Link and references

Databases of endogenous, biologically active and antimicrobial peptides
AMPer http://marray.cmdr.ubc.ca/cgi-bin/amp.pl131
ANTIMIC http://research.i2r.a-star.edu.sg/Templar/DB/ANTIMIC132
APD http://aps.unmc.edu/AP/main.php133,134
ArachnoServer http://www.arachnoserver.org/mainMenu.html135
ATDB http://protchem.hunnu.edu.cn/toxin136,137
BACTIBASE http://bactibase.pfba-lab-tun.org138,139
BAGEL2 http://bagel2.molgenrug.nl140
BioPD http://biopd.bjmu.edu.cn141
CAMP http://www.bicnirrh.res.in/antimicrobial/142
ConoServer http://www.conoserver.org/143
Defensins http://defensins.bii.a-star.edu.sg/144
knowledgebase
Hmrbase http://crdd.osdd.net/raghava/hmrbase/145
NORINE http://bioinfo.lifl.fr/norine146
PepX http://pepx.switchlab.org
PhytAMP http://phytamp.pfba-lab-tun.org/main.php147
RAPD http://faculty.ist.unomaha.edu/chen/rapd/index.php148
Peptidome databases
SwePep http://www.swepep.org149
EROP-Moscow http://erop.inbi.ras.ru150
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PeptideDB http://www.peptides.be151
Peptidome http://www.peptidome.jp152
PepBank http://pepbank.mgh.harvard.edu
SpectraST http://www.peptideatlas.org/spectrast141,153
X!Hunter http://h201.thegpm.org/tandem/thegpm_hunter.html154
NIST Libraries http://peptide.nist.gov
PRIDE http://www.ebi.ac.uk/pride155
BiblioSpec http://proteome.gs.washington.edu/software/bibliospec156
De novo sequencing tools
PepNovo http://proteomics.ucsd.edu/Software/PepNovo.html157
DirecTag http://fenchurch.mc.vanderbilt.edu158
Peaks http://www.bioinfor.com159
MS-Blast http://genetics.bwh.harvard.edu/msblast160
Spider http://www.bioinfor.com/peaks/features/spider.html161
InsPecT http://proteomics.ucsd.edu162
IggyPep http://www.iggypep.org163
MS-Dictionary http://proteomics.ucsd.edu164
Search engines
X!Tandem http://www.thegpm.org165
MS-Fit http://prospector.ucsf.edu
Mascot http://www.matrixscience.com118
Sequest http://fields.scripps.edu/sequest/index.html119,120

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5.3 Relation to other ‘- omics’ in biomarker search

Different pathology states and conditions, including those of neurological
and cardiovascular diseases,167,168 cancers and diabetes can be identified
using corresponding peptide biomarker profiles.166,167,169,170 The presence
of circulating peptides in biological samples, variations in the concentration
of normally occurring species or certain post-translational modifications,
can serve as an indicator of a pathological state. Genetic variations, gene
expression profiles, metabolites, proteins, or lipids can also constitute bio-
markers. From that, the multidisciplinary aptitudes and applications of

various relevant technologies are necessary for the discovery and validation
of biomarkers. Together with genome sequencing and the emergence of
high-throughput analytical technologies an area of systems biology appears
to provide complementary strategies for the elucidation of complex pepti-
domes (Fig. 10).
Systems biology is an inter-disciplinary field which seeks to explain bio-
logical processes using frameworks of complex interactions describing
biochemical and cellular modules within an individual cell or an organism.
More specialist strategies developed around optical and super-resolution
microscopy, microfluidic approaches or thermophoresis which combine
molecular biophysics capabilities, hence often branded in the field as
systems biophysics, focus on the trials of short-time disequilibrium
01/12/2013 14:45:06.
Fig. 10 Schematic representation of an ‘‘-omics’’ technologies landscape including examples

of corresponding analytical subjects and applied methods. DNA (genomics) undergoes tran-
scription to mRNA (transcriptomics), which is next and translated into protein (proteomics).
Proteins assist catalytic reactions resulting in metabolites (metabolomics), glycoproteins and
carbohydrates (glycomics), and various lipids (lipidomics). Reprinted with permission from
R. Q. Wu, X. F. Zhao, Z. Y. Wang, M. Zhou and Q. M. Chen, Journal of Dental Research,
2011, 90, 561–572; Copyright & 2011, International & American Associations for Dental Re-
search. [Colour image available on-line]

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settings that can be used to modulate autonomous molecular evolution.

In turn, ‘‘-omics’’ technologies, which encompass genomics, tran-
scriptomics, proteomics, metabolomics, lipidomics, glycomics and many
others, existing and emerging, make it possible to collect and analyse
comprehensive data sets on biomolecular and biological processes as in-
tricate or as autonomously restricted as the need be (Fig. 10).
Peptidomics is increasingly seen as a complementary branch of the other
‘‘-omics’’ approaches and systems biology. With intrinsic similarities with
proteomics, peptidomics is more used as a bridging discipline between

proteomics through degradomics to metabolomics, even though endogen-
ous peptides are not necessarily associated with proteome degradation. For
other related ‘‘-omics’’ approaches, in case of peptidoglycans and lipopep-
tides, which can fall under glycomics and lipidomics, respectively, the em-
phasis is equally split between the variabilities of glycans and lipids and
peptide sequence compositions. It is therefore common to relate different
‘‘-omics’’ capabilities with the appropriate weighing of specialist infor-
mation obtained by a specific discipline.
Systems biology, and its branching disciplines, i.e. systems biophysics, may
just address this by integrating complex data from multiple experimental
sources. Starting from the understanding of the genetic information through
the variations in the code, gene expression, nucleic acid-protein binding, to
molecule-molecule interactions, the networks of relations and dependence
can be built, which would enable a deeper understanding of the underlying
molecular biology of a given physiological state or a process.169,170
01/12/2013 14:45:06.
6 Synthetic peptides as exogenous biomarkers

Peptides can be seen and utilised as unique and powerful chemical tools. It is
not only due to the natural diversity of amino acid building blocks, but also
due to the different types of modifications that are possible at an individual
residue level.171 This allows for site specific modifications including labelling
and chemical depletion approaches, which is further facilitated by the
smaller sequence space of peptides when compared with proteins and is also
compounded by higher order structures and intricate networks of tertiary
contacts up to macromolecular oligomeric assemblies. However, peptides
can exhibit distinct biological activities in the context of fully folded pro-
teins and to an equal extent as autonomous structures. The relative lack of
their sequence and structural diversity is compensated by their synthetic
accessibility, which nowadays proves to be indispensable in creating what
has become traditional diagnostic platforms such as peptide micro-
arrays172,173 and less conventional technologies such as electrochemical
probes. Ferrocenyl-based probes developed for the characterisation of
autoantibodies as biomarkers of multiple sclerosis can serve as an ex-
ample.174 Here synthetic peptides are not targeted biomarkers as it would be
for endogenous peptides but rather an auxiliary means for the detection and
capture of other biomarkers, be these proteins, lipids or carbohydrates.
Given that capturing peptides act as baits or epitopes and are usually dis-
played on surfaces or used as conjugates with molecular reporters, their role
in peptidome analyses is that of an exogenous biomarker – a known entity

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with known specific function, deliberately introduced into a peptidome to

target and reveal a molecular mechanism or endogenous biomarker or
to collect and help isolate the latter. Uses are various and include targeted
drug and gene delivery,175,176 antimicrobials, neurodegenerative and
neuroendocrinology research, regenerative medicine and synthetic bio-
logy.174,175 The main challenge is constant and concerns with our ability to
design peptides rationally in order to ensure control over their physico-
chemical and biological properties.
Arguably, one of the most efficient approaches is to emulate naturally

occurring motifs using and extending the available set of the first design
principles linking peptide structure and function. Further structural ma-
nipulations require further or refined rules which are being provided
through intuitive and empirical designs as well as computational analyses.
Targeting specific function determines the nature and structure of a given
design, but often it is the delineation of closely related functions that require
subtle tuning in the sequence, backbone or side chains. For example, generic
peptide sequences can be designed to provide cell penetrating and gene
delivery properties thus promoting the uptake and intracellular release of
DNA and enabling expression of target proteins,176 and can be tailored for
differential membrane binding enabling related functions of endosomal
release and antimicrobial responses.177
Endogenous antimicrobial peptides that are often used as biomarkers in
peptidomics (wound healing monitoring, inflammatory responses) present
an evolutionarily conserved class of the host defence effector molecules.
01/12/2013 14:45:06.
Apart from being promising candidates for next-generation antimicrobials

in the light of emerging antimicrobial resistance the peptides are extensively
being used as structural templates for mechanistic and structural studies at
the cellular and systems level providing thus a convenient interfaces between
systems biology and peptidome analysis. More recently, high resolution
imaging techniques are being employed to address fundamental questions of
peptide interactions with their targets in real time and with nanoscale pre-
cision. De novo design and nanoscale imaging used in combination were
shown to reveal mechanisms that could not be understood using more
traditional approaches used in structural biology including NMR and
crystallography. For example, by modulating peptide-lipid interactions
using archetypal de novo sequences and supported lipid bilayers it was
possible to provide the first evidence of antimicrobial pore expansion
mechanism.177 In a sense, such research can serve as an example of an ex-
ogenous peptidome analysis with the help of isotopically labelled anti-
microbial biomarkers and a combination of high resolution imaging
approaches, namely atomic force microscopy (AFM) and nanoscale sec-
ondary ion mass spectrometry (NanoSIMS). Detailed topography of pep-
tide-treated membranes obtained by AFM indicating pore-like changes in
membranes after peptide treatment was complemented by chemical imaging
(NanoSIMS) performed on the same samples which showed precise peptide
localization in the membranes and predominant peptide accumulation in
the pore edges (Fig. 11A).
Somewhat reverse to this approach is the use of de novo peptides as
exogenous biomarkers targeting native antimicrobial peptides. These

Fig. 11 Examples of exogenous peptide biomarkers. (A) Antimicrobial pores in phospholipid

bilayers imaged by nanoscale secondary ion mass spectrometry (left) and atomic force mi-
croscopy (right). (B) Two scenarios of antimicrobial peptide action: an antimicrobial peptide
binds to microbial membranes and forms membrane-disrupting helical structures (left) or when
challenged by an anti-antimicrobial peptide it forms a biologically inert helical complex (right).
01/12/2013 14:45:06.
[Colour image available on-line]
peptides cannot bind to membrane surfaces, microbial or mammalian, and

are anionic in contrast to cationic antimicrobial peptides, which allows them
to bundle with the antimicrobial counterparts into functionally inert
coiled-coil hetero-oligomers (Fig. 11B).180 In this format, the peptides act as
antagonists of antimicrobial peptides or anti-antimicrobial peptides and can
be used as exogenous biomarkers for capturing endogenous antimicrobial
biomarkers. Furthermore, by designing and using such systems one can
expand proteome analysis to the elucidation of antimicrobial resistance at
the level of specific peptide-lipid interactions with correlative studies of
resistant and wild-type bacterial membranes in situ, in vitro and potentially
in vivo. In therapeutic terms, such antimicrobial ‘‘switches’’ can be used as
antimicrobial regulators or immunomodulators thus aiding in a better
control over antimicrobial treatments. Importantly, by probing potential
molecular avenues of antimicrobial resistance, that is, those that have yet to
be found in nature, peptidome analysis takes a proactive role in the search
of resistance mechanisms from the bottom up, from relatively simple
scenarios, such as the expression of neutralising sequences, to more complex
routes leading to membrane modifications or re-building.178
The better understanding of peptidome content and analysis together with
advances in associated techniques, physicochemical strategies and bioin-
formatics provides an important insight into the nature and role of peptide

01/12/2013 14:45:06.
Fig. 12 A schematic representation of a degradomic-peptidomic analysis strategy used to

identify human blood peptidome. The analysis is preceded by SEC-HPLC isolation and sep-
aration of the peptidome components to deplete the 12 most abundant proteins by affinity
chromatography followed by flow-through fractions separated by size exclusion chroma-
tography (SEC) for species cut off by o20 kDa (upper). The SEC-isolated components are then
separated using HPLC with a separation peak capacity of B300 and identified by correlated
with mass spectrometry analysis (lower), (reprinted with permission from Y. Shen, T. Liu, N.
Tolić, B. O. Petritis, R. Zhao, R. J. Moore, S. O. Purvine, D. G. Camp and R. D. Smith,
Strategy for degradomic-peptidomic analysis of human blood plasma, J Proteome Res., 9, 2339.
Copyright (2010) American Chemical Society.179
biomarkers in complex biological systems. It can also lead to the creation of

new peptide-based technologies with a reach beyond traditional pepti-
domics applications. Yet as outlined in this chapter, strategies that are re-
quired to address applied and fundamental questions in peptidome research
are complex and cannot be put under a restricting set of terms and defin-
itions. Therefore, new and most efficient approaches and technologies tend
to incorporate multiple methods and are often proposed as logical chains of
co-merged, other, measurement and identification capabilities to enable
global analyses.
Combinatory degradomic-peptidomic179 or proteomic-degradomic-
peptidomic180 approaches are examples of emerging strategies that prove to

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be instrumental in deciphering peptidomes as complex as membrane pro-

teomes, otherwise considered intractable, pancreas post mortem degra-
domes,181 human blood plasma, which are highly masked by high
abundance proteins,179 and detailed blood peptidome profiles of aggressive
cancers.180 The main basis of such combinatory strategies is the provision of
a degradome, which is treated as a separate sub-proteome subjected to
proteolysis, and subsequent measurements (degradomic/proteomic) carried
out using both peptides and peptidome peptides to provide detailed infor-
mation on degradome substrates, peptidome and tryptic peptide and

retrospectively proteome protein (Fig. 12).
Considering the development of mainstream trends in peptidomic ap-
plications, it is hard to see a dominating source or medium for peptide
pools. Biological fluids are as ever popular and combine the relative ease of
manipulation and handling with the amount of information one can extract
through a single set of analyses. Blood, serum, urine and cerebrospinal fluid
are traditionally used for diagnostic and forensic purposes. A closer at-
tention is paid to the progress in saliva-based proteomics, which is seen as a
promising alternative to other fluid-based approaches, thanks to its con-
venience, low cost and non-invasiveness – characteristics that are equally
attractive to personalised medicine.182 This is not surprising given recent
progress in direct and indirect diagnostic tools based on saliva analyses
including point-of-care devices. Furthermore, judging by saliva constituents
and their concentrations it is possible to trace diagnostically relevant levels
of drugs and hormones and relate these to those in blood due to dynamic
01/12/2013 14:45:06.
content exchange between saliva and blood. Contemporary developments in

the peptidomics of tissues and cells, which find use in the diagnosis of
complex diseases including cancers, focus on the quantitative approaches
that aim to relate relative peptide levels in different samples as a function of
enzymatic activity. Experimental manipulations with increasing proteolysis
(enzyme overexpression) or decreasing proteolysis (enzyme inhibition) ac-
company these studies and can also be used to investigate substrate speci-
ficity of purified proteases. In the diagnostics of neurodegenerative diseases
peptidomics strategies are developed to discover new and intermediate
biomarkers as well as new pathways from their inert monomeric forms to
toxic pre-fibrillar oligomers, which may shed light on the intrinsic devel-
opment and environmental factors of these diseases. All in all, peptidomics
remains a burgeoning field which holds strong promise for powerful ana-
lytical approaches prompting the elucidation of the molecular origin of
diseases which can help identify new therapeutic cures at the most relevant
time- and length scales of molecular recognition.
8 Further specialist reading

The topic of peptidomics is admittedly very broad. Since it has never been
covered in the series before this chapter gives an overview introduction of
the subject focused primarily on the approach itself rather than on appli-
cation examples. Peptides can provide an application focus which is in-
evitably very broad and can reflect different disciplines including
diagnostics, nutrition, forensics, synthetic biology, therapeutics and many

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others. Also, the variety and complexity of technologies, both analytical and
computational, applied to study endogenous peptides create separate topics
themselves. Exogenous peptides are of lesser emphasis in the literature,
primarily due to molecular diagnostics approaches and nanoscale technol-
ogies that are developed in parallel and inter-relate with peptidomics at the
point of mutual applications – the tendency which can be anticipated to
develop further. A number of excellent specialist reviews providing both
broader and more focused accounts of the developments are available in the
literature and recommended for further reading, with a small fraction of

them being listed below.117,183–187
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01/12/2013 14:45:06.

Peptide and protein based pharmaceuticals
Gábor Mezo
DOI: 10.1039/9781849737081-00203
1 Introduction
The mainstream of the drug development in the 20th century was based on
compounds obeying the ‘rule-of-five’ (also known as Lipinski’s ‘rule-of-five’

or Pfizer’s ‘rule-of-five’), which provided criteria for oral bioavailability
according to molecular weight (o500 Da), lipophilicity and H-bond for-
mation capability of the potential drugs.1,2 The drug development including
ligand-, mechanism- or receptor-based design was mainly generated
from small molecule natural products, either by screening or rational design
processes.3 However, in spite of the benefits of these small molecules (cost,
conformational restriction, membrane permeability, metabolic stability,
oral bioavailability), the conventional small molecule drugs usually have
reduced target selectivity, which may cause significant side-effects in
humans.4
In the latter part of the last century, the emerge of drugs with improved
selectivity and of some new molecular biological tools like recombinant
protein expression, better protein purification protocols and analytical tools
led to the development of protein-based therapeutics. These ‘biologics’
(molecular weight over 5000 Da) have high potency and selectivity, leading
01/12/2013 14:45:16.
to fewer side-effects, which is probably the main drawback of small mol-

ecule drugs.4 However, their poor membrane permeability and metabolic
instability prevent the oral administration, which is the most convenient and
effective dosage due to the observed poor patient compliance. They typically
require injection (via subcutaneous, intramuscular or intravenous routes) or
intranasal delivery. Nevertheless, humanized antibodies or other soluble
proteins have become an extremely successful class of therapeutics,5 both
economically and in targeting certain diseases, and have increased quota of
income in the drug market (protein drug market B55 billion USD in
2011).4,6 Among the best sold antibody and protein drugs, there are com-
pounds for the treatment of rheumatoid arthritis (adalimumab (Humiras),
etanercept (Enbrels), infliximab (Remicades)), cancer (rituximab
(Rituxans) for non-Hodgkin’s B-cell lymphoma, bevacizumab (Avastins)
for colorectal cancer and trastuzumab (Herceptins) for breast cancer),
myelosuppression (pegfilgrastim, Neulastas), renal anemia (epoetin alfa,
Epogens) and multiple sclerosis (interferon beta-1a, Avonexs). Recently, a
monoclonal antibody was introduced for anticancer drug targeting. In
brentuximab vedotin (Adcertist), the chimeric monoclonal antibody
brentuximab (which targets the cell-membrane protein CD30) is attached to
three to five units of the antimitotic agent monomethyl auristatin E through
a self-imolative peptide spacer.
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös Loránd

University Budapest Hungary. E-mail: gmezo@elte.hu

View Online
It is predicted that in the 21st century there will be more and more new
drug investments that violates the ‘rule-of-five’ and will fill the gap between
the molecular weight of small molecule drugs and proteins, combining their
potential advantages. Peptides containing 5–50 residues are suitable for
these purposes and they have received increased interest in the current drug
therapies.4 Advantages of peptide-based pharmaceuticals are the high po-
tency and selectivity as well as their low accumulation in tissues; thus, they
have potentially lower toxicity than the small drug molecules. Furthermore,
the high chemical and biological diversity of peptides provide a broad range
of targets, which could represent a basis for personalized medication.7 The
recent results of genomics and proteomics give a hand for these purposes.6,8
Further benefits of peptides over proteins and antibodies are the lower
manufacturing costs, longer shelf stability, higher activity per mass unit,
better organ and tumor penetration and reduced potential for interaction
with the immune-system.5 However, the faster spread of peptide-based
drugs in medical applications is still partly restricted by their low systemic
stability, high clearance, poor membrane permeability, negligible activity
when administered orally and their high manufacture costs.4 Nevertheless,
the number of peptide-based drugs entering the clinical trials has continu-
ously been increasing during the last decades, too. While in the 1970s only
1.2 molecules per year were introduced, their number was elevated to 4.6/
year in 1980s, 9.7/year in 1990s and 16.8/year in 2000s. Between 2009 and
2011 US Food and Drug Administration (FDA) approved 76 new ther-
apeutics, among them 18 new biologics and 8 (10.4%) peptide-based drugs
01/12/2013 14:45:16.
(peptides and peptidomimetics) (Fig. 1).9 Only in 2012 (a ‘‘Big Year’’ for
novel drug approvals, when 40 drugs were approved), seven peptide-based
pharmaceutics (lucinactant, pasireotide, peginesatide, carfilzomib, linaclo-
tid, teduglutide and exenatide Bydureon) were approved (Fig. 1). The
peptides that have recently entered clinical studies were mainly developed
for the treatment of cancer (18%) or metabolic disorders such as diabetes,
obesity and osteoporosis (17%).10
Fig. 1 The type and number of approved drugs between 2009 and 2011, and in 2012.

View Online
Table 1 Predicted incomes from the peptide drug market in 2015.
Expected income of peptide-based

pharmaceutics in 2015 Generic names of the compounds
W2 billion USD glatiramer, insulin glargin, insulin lispro, insulin aspart

0.8–1.3 billion USD liraglutid, leuprolid, goserelin, teriparatide, octreotide,
exenatide (Bydureon), insulin detemir
0.3–0.6 billion USD bivalirudin, romiplostin, triptorelin, desmopressin,
lanreotide, exenatide (Byetta)
0.1–0.2 billion USD icatibant, calcitonin, eptifibatide, pramlintide

0.04–0.09 billion USD thymalfasin, enfuvirtide, ganirelix, degarelix, cetrorelix
0.02–0.03 billion USD surfaxin, mifamurtide, tesamorelin
Currently, there are more than 70 approved peptide drugs9 on the market,
generating an annual sale close to USD 20 billion in 2012,11 which is ex-
pected to increase over USD 25 billion till 2018 (the numbers reported in
publications are usually different, since the borderline between the groups of
biologics, protein-, and peptide-based pharmaceuticals or peptidomimetics
is not very well defined). The highest incomes were produced by glatiramer
acetate (Copaxones; USD 4 billion) used for sclerosis multiplex, insulin
derivatives (glargin; Lantuss, aspart; NovoLogs, lispro; Humologs, 3, 2.1
and 1.6 billion USD, respectively) followed by two gonadotropin-releasing
hormone (GnRH) derivatives leuprolide acetate (Luprons; USD 1.5
billion) and goserelin acetate (Zoladexs; USD 1.1 billion) and a somatos-
01/12/2013 14:45:16.
tatin derivative octreotide acetate (Sandostatins; USD 1.3 billion), all used
for hormone therapy of cancer or other hormonal disorders. Although
peptide drugs represent only 2% of all drug product sales, the rate is in-
creasing dramatically with a current annual growth rate between 7.5–10%.
The predicted incomes from the peptide drug market in 2015 are sum-
marized in Table 1.
There are more than 140 peptide-based drug candidates in clinical and
another 500–600 in pre-clinical development.12,13 This predicts a promising
future for the marketing of innovative synthetic therapeutic peptides in the
coming years. This is supported by the concept that a drug can not be ‘orally
available’, which has become more and more accepted, and by the new
synthetic strategies (effective large scale solid phase peptide synthesis, re-
combinant DNA techniques or biocatalysis and cell-free expression pro-
cedures) that improve the productivity, as well as by the development of
alternative routes of administration.5 Furthermore, investigation of long-
lasting peptide-based drugs using new stabilization strategies and smart
linkers that exhibit stability towards blood proteases but provides an ap-
propriate intracellular lability will lead to higher bioavailability of peptide-
based drugs.14
It is not a goal of this book chapter to present all peptide- and protein-
based drugs (below 10 kDa molecular weight). The chapter will especially
focus on the leading second and new generation peptide-based pharma-
ceuticals (Table 2), presenting their structure and therapeutic applications.
The majority of the second and new generation peptide-based pharma-
ceuticals are more effective and/or enzymatic stable derivatives of the first

View Online
Table 2 Second and new generation of peptide-based pharmaceutics.
GnRH analogs: buserelin, deslorelin, goserelin, histrelin, leuprolid, nafarelin, triptorelin,

lecirelin, cetrorelix, ganirelix, abarelix, degarelix;
Somatostatin analogs: octreotide, lanreotide, vapreotide, pasireotide;
Vasopressin analogs: terlipressin, felypressin, desmopressin, lypressin, ornipressin, pitressin;
Oxitocin analogs and inhibitors: carbetocin, atosiban;
Peptide drugs for diabetes: insulin lispro, insulin aspart, insulin glulisine, insulin glargine,
insulin detemir, insulin degludec, exenatide, liraglutide, pramlintide;
Peptide drugs with antithrombotic effect: lepirudin, desirudin, eptifibatide, bivalirudin;

Enzyme inhibitors: enalapril, lisinopril (ACE inhibitors), linaclotide, carfilzomib, romidepsin,
boceprevir, telaprevir (antiviral agents);
Peptide drugs for hereditary angioedema: icatibant, ecallantide;
Antibiotics, antibacterial agents: daptomycin, telavancin;
Adjuvants, immunomodulatory agents: glatiramer (multiple sclerosis), mifamurtide
(osteosarcoma), thymalfasin (T-cell production);
Peptide drugs for osteoporosis: salmon calcitonin, teriparatide;
Others: ziconotide (pain relief), enfuvirtide (HIV fusion inhibition), lucinactant (surfactant),
tesamorelin (GHRH analog), peginesatide (anemia), teduglutide (GLP-2 analog).
The peptide-based pharmaceuticals in italic will be discussed in this chapter.
generation ones, which are usually hormone peptides, and used especially
for cancer treatment or in case of metabolic disorders. However, among
the recently approved peptide-based drugs there are compounds for a
broad range of indications. Due to the page number limitation, it is not
possible to present in detail the diseases treated with the appropriate
01/12/2013 14:45:16.
peptide-based drugs; however, the references may provide a good starting

point to the readers who are interested in this topic. It has to be mentioned
that from the discovery of an active compound until it reaches the market, it
takes long time and further studies and developments with them are usually
continued afterwards. This large time period can be seen in the list of
references.
2 Glatiramer acetate (Copaxones), the highest income producing

peptide-based drug used against multiple sclerosis
Glatiramer acetate (GA) denoted as Cop1 (Copolymer 1) was originally
developed in M. Sela’s laboratory at Weizmann Institute of Science,
Rehovot, Israel.15 Glatiramer acetate is a synthetic analogue of the multiple
sclerosis (MS)-associated antigen, myelin basic protein (MBP). This poly-
cationic copolymer showed protection against experimental allergic en-
cephalomyelitis (EAE).16 It was composed of alanine, glutamic acid, lysine
and tyrosine, in a residue molar ratio of approximately 6 : 2 : 5 : 1, re-
spectively, and had an average molecular weight of 23 kDa. The copolymer
was built up by random polymerization using the mixture of N-carboxy-
anhydrides (NCAs) of the appropriate amino acid derivatives. The side
chain of glutamic acid was protected as benzyl ester, while the e-amino
group of lysine was trifluoroacetylated. The polymerization was carried out
at ambient temperature in anhydrous dioxane (18 g Tyr-NCA, 50 g Ala
NCA, 35 g Glu(OBzl)-NCA, and 83 g Lys(Tfa)-NCA in 3.5 L dioxane) with
0.01–0.02% diethylamine (DEA) as initiator (Scheme 1). The benzyl group

Scheme 1 Synthesis of glatiramer acetate by polymerization using amino acid N-carboxy-

anhydride (NCA) derivatives.
01/12/2013 14:45:16.
was removed with 33% HBr in glacial acetic acid, followed by the cleavage
of trifluoracetyl group from lysine with 1 M piperidine/water solution. The
copolymer was dialysed against 0.3% acetic acid, followed by freeze drying.
Later on it was shown that the removal of polymers with a molecular weight
over 40 kDa was important to get a non-toxic compound. Therefore, the
preferred average molecular weight of the copolymer is 7000 2000 Da,
while the amino acid composition should be in a range Ala (4.1–5.8) : Glu
(1.4–1.8) : Lys (3.2–4.2) : Tyr (1.0).17
Glatiramer acetate is approved in the US (1996), the EU (2000) and many
other countries for reducing the frequency of relapses in patients with re-
lapsing-remitting multiple sclerosis (RRMS), but not for the treatment of
patients with primary or secondary progressive MS.18 Glatiramer acetate
(Copaxones) is marketed by Teva Pharmaceuticals. It is administered by
sc. injection of 20 mg daily.
Multiple sclerosis (also known as disseminated sclerosis or encephalo-
myelitis disseminate) is an inflammatory (mainly autoimmune) disease of
the central nervous system (CNS), which causes the damage of the fatty
myelin sheaths around the axons of the brain and spinal cord.19 Usually,
MS occurs in young adults (20–40 years old, and it is more common in
women) as a result of a combination of genetic, environmental and in-
fectious factors. About 0.002–0.150% of the population may suffer from
this illness. It is not easy to detect MS, because neurological symptoms and
signs detected in other neurological diseases can appear at MS patients as

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well. Up to now, MS is not curable; therefore, the main goal of the treat-
ment is to slow the progression of the disease and to improve the quality of
life.20,21 The magnetic resonance imaging (MRI) technique is now available
for the detection of the early neurological disorders related to clinically
isolated syndrome (CIS) that might be caused by an inflammatory demye-
linating illness.22,23 Thus, glatiramer acetate is also approved for the pre-
ventive treatment of patients presenting the first clinical (neurological)
episode with high risk of developing MS (30 to 70% of persons experiencing
CIS later develop MS).24

Glatiramer acetate does not directly suppress the MS-associated
inflammation in CNS, because it does not cross the blood-brain barrier.25,26
Its mechanism of action is still not fully understood in patients, but it is
assumed to act via immunomodulation of pathways involved in the
pathogenesis of MS.27 GA binds with high affinity to major histocomp-
ability complex (MHC) class II molecules on MBP-recognizing antigen
presenting cells.26 The GA/MHC complex binds to the receptors on T
helper cells inducing secretion of anti-inflammatory cytokines such as IL-4,
IL-5 and IL-10, which down-regulates the CNS-based inflammation.28,29
An overview of the key pharmacological effects of GA was published by N.
J. Carter and G. M. Keating.23 Recently, the role of natural killer (NK) and
dendritic cells (DCs) in MS and their influence by GA were also demon-
strated.30,31 The potential application in the treatment of other demyeli-
nating diseases like epilepsy was also indicated.32 The evidence of
neuroprotection with GA was presented in a phase III, multicenter, ran-
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domized, placebo-controlled PreCISe trial in patients with CIS. Patients

receiving GA showed an improvement in brain neuroaxonal integrity.33 GA
is in Phase I clinical trial for Dry Age-Related Macular Degeneration
(AMD), and it showed protection in mouse models against cerebral malaria
as well.34
In comparison with other immunomodulatory drugs used for RRMS
(IFNb-1a, IFNb-1b), GA seems to be associated with a lower cost and
better outcomes in a long term treatment.35 GA also shows significantly
lower influenza-like illness side effects, but the injection site-related re-
actions (pain, irritation etc.) are higher in case of GA.24,36,37 The later ones
disappear usually within 30 min. GA was superior to IFN in reducing the
risk of exacerbation as well, and there was no clinical benefits of the com-
bination of the two compounds.38 A 15-year long tolerability study of GA
using sc. 20 mg daily treatment has recently been completed.39
More recently, mitoxantrone and some antibodies (e.g. natalizumab)
have also become available for the treatment of RRMS. Despite their effi-
cacy, these disease-modifying drugs are not suitable for a long-time treat-
ment and during pregnancy due to their higher toxic side effects compared
to GA.40,41
In conclusion, though CIS and RRMS are not very common diseases
worldwide, Copaxones may remain the highest income producing peptide-
based drug in the near future, because of its effectiveness and the necessary
continuous daily treatment for years. Furthermore, glatiramer acetate
shows that polymer compounds without an exact composition might be also
potential drug candidates.

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3 Peptide-based drugs for the treatment of diabetes, the fastest growing

disease worldwide
3.1 Diabetes mellitus
There are two types of diabetes mellitus: one of them is caused by the lack of
insulin production (type 1 diabetes; formerly insulin-dependent diabetes),
whereas the other one is caused by reduced insulin production or insulin
resistance (type 2 diabetes; formerly noninsulin-dependent diabetes). Type 1
diabetes is developed by the autoimmune destruction of insulin-producing

pancreatic b-cells in islets of Langerhans.42 This autoimmune disease might
have genetic, viral, chemical or environmental origin.43 The incidence of
type 1 diabetes is quite high in Scandinavia, about 35 per 100,000 people,
and in the US, while it is marginal in Japan or China, affecting about 1 m of
the population. The rates of type 2 diabetes, which is a metabolic disorder,
have dramatically increased in the last 50 years. In 2010, there were ca. 285
million people affected by the disease (B90% of diabetes cases). It is be-
lieved that obesity is the primary cause of type 2 diabetes in people who are
genetically predisposed to the disease.44 It is expected that the number of
patients with type 2 diabetes will increase to 439 million (7.7% of the world
population) by 2030. According to a newer estimation, the incidence will be
even higher by that time, indicating that diabetes affects the population
faster than expected.45
In both cases, the sugar level in blood and urine is increased (hyper-
glycemia and glycosuria) and may cause serious complications in time (e.g.
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stroke, heart disease, blindness, kidney failure, or amputation) in the ab-

sence of diabetes treatment. More than 4 million diabetes-related deaths
that are associated with these complications occur every year.
3.2 Insulin replacement therapy

Before the discovery of insulin in 1921, type 1 diabetes was a fatal disease
because of the developed diabetic ketoacidosis in the late stages of the
disease process.46 Insulin is a peptide hormone (Fig. 2) produced by b-cells
in pancreas and plays a central role in the regulation of the carbohydrate
and fat metabolism in the body. In the nature, the disulfide bridges are
formed by the folding of preproinsulin, the primary translational product
that is converted to proinsulin by signal peptidases. The bioactive hormone
is produced by the removal of the connecting peptide (C-peptide) sequence
between A- and B-chain afterwards (Fig. 2).47 The presence of the C-peptide
in patients provides a possibility to measure endogenous insulin production
and to distinguish type 1 and type 2 diabetes. Insulin is an asymmetric dimer
peptide containing 51 amino acid residues (A chain 21 amino acids and B
chain 30 amino acids) that are connected by two intermolecular disulfide
bridges. Furthermore, there is an intramolecular disulfide bridge within the
A chain.
Therapeutic insulin, as a first generation peptide-based drug, is used for
lowering the blood glucose level in case of type 1 diabetes. Earlier, isolated
bovine or porcine insulins were used for the treatment of type 1 diabetes
(they differ in 3 (Ala8(A-chain), Val10(A-chain) and Ala30(B-chain)) and
1 (Ala30(B-chain)) amino acid residues from human insulin, respectively).

Fig. 2 Structure of proinsulin and insulin.

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In spite of the significant improvement of purification techniques,48 these

types of insulin are still immunogenic in some patients.49 Therefore, the
most common insulins used nowadays are biosynthetic products produced
by recombinant DNA technology. In this case, the A- and B-chains of
human insulin are separately produced in E. coli, followed by the formation
of disulfide bridges.50 The first such insulin was marketed by Eli Lilly in
1982 with the brand name Humulins. It has to be mentioned that
Humulins was the first medication produced by modern genetic engin-
eering techniques. However, the treatment with produced insulin differs
from the pharmacokinetic effect of the endogenous insulin. Therefore, the
main side effect (hypoglycemia) that can occur by over-dosing might be
more severe in case of the application of Humulins compared to the isol-
ated insulins. The developed hypoglycemia may result in memory lapse, loss
of concentration, convulsion or even slip into the coma. Thus, the main
challenges in finding the appropriate clinical treatment for diabetes are the
mode of administration, the right dose and timing, as well as the selection of
proper insulin preparation based on speed of onset and duration of action.
3.3 Insulin analogs

The native insulins become active within 30 min and exert the activity for
about 5–8 hours. The reason of the delayed effect is the hexamer formation
of insulins that prevents the fast entry of the bioactive monomer form into
the blood. The C-terminal part of the B chain is critical for the stability of
the hexamers. The drawback of the long development and short duration of

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Table 3 Insulin derivatives obtained by modifications of human insulin sequence.
Human insulin A-chain: GIVEQCCTSICSLYQLNYCN

Human insulin B-chain: FVNQHLCSHLVEALYLVCGERGFFYTPKT
Insulin lispro B-chain: FVNQHLCSHLVEALYLVCGERGFFYTKPT
Insulin aspart B-chain: FVNQHLCSHLVEALYLVCGERGFFYTDKT
Insulin glulisine B-chain: FVKQHLCSHLVEALYLVCGERGFFYTPET
Insulin glargine A-chain: GIVEQCCTSICSLYQLNYCG
Insulin glargine B-chain: FVNQHLCSHLVEALYLVCGERGFFYTPKTRR

Insulin detemir B-chain: FVNQHLCSHLVEALYLVCGERGFFYTPK (C13H27CO-)
Insulin degludec B-chain: FVNQHLCSHLVEALYLVCGERGFFYTPK
(HOOC-C14H28CO-cE)
action (e.g., treatment before food intake, multiple injections per day)
prompt the researchers to develop fast-acting and long-acting insulin de-
rivatives (Table 3).46,51 The fast-acting insulin analogs (lispro, aspart and
glulisine) show the effect in 5–15 min after sc. injection and the activity lasts
for 3–4 hours. These analogs could be administered right before the meals. In
contrast, the long-acting analogs (glargine, detemir, degludec) start working
within 1–2 hours and continue to be active for about 24 hours without major
peaks or dips of the activity. The perfect timing of these analogs is not so
important and one or two injections per day are enough to control the blood
glucose level. These compounds are also used for type 2 diabetes as well.
Insulin lispro marketed by Eli Lilly as Humalogs was approved by FDA
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in 1996. In this insulin analog, the proline at position B28 and lysine at
position B29 are reversed (Table 3), blocking the formation of insulin
dimers and hexamers, which results in an immediate bioactive monomer
after injection. Proline in position B28 is replaced by aspartic acid (Table 3)
in insuline aspart introduced by Novo Nordisk with the trade name
NovoLogs. The change of the charge of the C-terminal end of the B chain
also prevents the hexamer formation. Insulin aspart was approved by FDA
in 2000. Sanofi-Aventis developed insulin glulisine, in which Asn in position
B3 was replaced by Lys, while in position B29 Lys was changed to glutamic
acid, respectively (Table 3). The trade name of this insulin analog is
Apidras and it was approved by FDA in 2004. These compounds may also
be used for extreme hyperglycemia by iv. administration.
In case of long-acting basal insulin derivatives, the applied modifications
at the C-terminal end of B-chain increase the stability of the formed
hexamers or the non-covalent interactions of insulin with human serum
albumin (HSA) that result in slow release of the bioactive monomer form of
the hormone peptide. In insulin glargine, in addition to the change of Asn at
A21 position to Gly, two additional Arg were attached to the C-terminus of
Thr at position B30 (Table 3). This modification shifts the isoelectric point
of the peptide from pH 5.4 to 6.7, making the molecule more soluble at an
acidic pH. After sc. injection of the acidic solution, the primarily formed
hexamers aggregate further at physiological pH. From this depot, insuline
glargine is continuously released. Insulin glargine was introduced by Sanofi-
Aventis with the trade name Lantuss and it was approved by FDA for
clinical use in 2000.

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s
In insulin detemir marketed by Novo Nordisk as Levemir , the Thr from
the C-terminus of B-chain was removed and the side chain of the new C-
terminal amino acid lysine was acylated with myristic acid (Table 3). There
are five binding sites of myristic acid to HSA; thus, the fatty acid modified
insulin binds quickly to HSA in blood. From the complex, the insulin dis-
sociates slowly afterwards. This insulin derivative was approved by FDA in
2006.
Recently, ultralong-acting basal insulin analog, insulin degludec was de-
veloped by Novo Nordisk with the trade name Tresibas. Its action lasts up
to 40 hours; therefore, it is enough to inject it three-times a week to control
the blood glucose level. The modification in this derivative is similar to that
in insulin detemir, but the side chain of Lys at position B29 is acylated with
hexadecanedioic acid through a g-L glutamyl spacer (Table 3). The fatty
acid chain helps the formation of multi-hexamers, producing a depot after
sc. injection. Tresibas might be marketed in 2015–2016 after completion of
additional cardiac safety studies requested by FDA.
3.4 Increntin-based therapy of type 2 diabetes with glucagon-like peptide-1

(GLP-1) derivatives
Glucagon-like peptide-1 (GLP-1) is a native peptide hormone containing 30
amino acid residues derived from a prohormone (proglucagon consisting of
180 amino acid residues). From the prohormone, several other smaller
peptides (glucagon, GLP-2, oxyntomodulin and glicentin) with hormonal
effect are released (Fig. 3). Proglucagon is expressed and produced in
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pancreatic a-cells within islets Langerhans and also in intestinal endocrine L

cells.52 It is worth mentioning that glucagon has an opposite effect as insulin,
namely it increases the blood glucose level by converting the stored glycogen
to glucose in the liver. Therefore, glucagon and insulin are part of a feedback
system that keeps the glucose level in blood stable. Glucagon consists of
29 amino acids (HSQGTFTSDYSKYLDSRRAQDFVQWLMNT)53 and
binds to glucagon receptor (G-protein-coupled receptor), followed by the
Fig. 3 Peptides released from proglucagon with influence on blood glucose level.

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GLP-1(7-37): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
Exenatid (Exendin-4): HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-amide
Liraglutide: HAEGTFTSDVSSYLEGQAAK((C15H31CO-γE)EFIAWLVRGRG
Lixisenatide: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSK6-amide
Scheme 2 Sequences of peptide pharmaceutics derived from GLP-1 (the sequence identity
with GLP-1 is marked in bold letters).
activation of adenylate cylase resulting in cAMP production. It is used as a

first generation peptide-based drug for hypoglycemia.

GLP-1 is released in the gut by several enzymes from the proglucagon.54
Its bioactive forms are GLP-1(7-37) and GLP-1(7-36)-amide.55 GLP-1 is a
potent antihyperglycemic hormone by increasing insulin secretion in a
glucose-dependent manner as well as decreasing glucagon secretion from
pancreas.56 Because of the mechanism of action, GLP-1 would be an ideal
drug for the treatment of type 2 diabetes. However, GLP-1 has a half-life of
2–3 min, being rapidly degraded by dipeptidylpeptidase IV (DPP IV). Thus,
intensive research was conducted in the last decades to develop compounds
with similar activity but higher resistance against enzymatic degradation.
There are at least 6 GLP-1 analogs (functional or sequence) that have
reached the market (exenatide, liraglutide) or are in Clinical Trial (e.g.
lixisenatide, albiglutide, taspoglutide).57
Exendin-4 was the first discovered peptide that showed similar functional
properties with GLP-1. It was identified in the venom of Glia monster
(Heloderma suspectum).58,59 Exendin-4 containing 39 amino acid residues
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shares 53% amino acid homology with the full-length GLP-1 (Scheme 2)
and acts as GLP-1 receptor agonist. The second amino acid from the N-
terminus is Gly instead of Ala in exendin-4, it is much more resistant against
DPP IV, which plays a central role in the degradation of GLP-1. The half-
life of exendin-4 is about 3-4 hours in the circulation.60 Similarly to GLP-1,
exendin-4 stimulates insulin- and inhibits glucagon secretion; furthermore,
promotes b-cell proliferation and protects against their apoptosis and in-
hibits gastric emptying.59
Exenatide (Exendin-4) (a new generation peptide-based drug) was ap-
proved by FDA in 2005 for patients whose type 2 diabetes was not well
controlled by other medication. The polypeptide manufactured by chemical
synthesis and marketed as Byettas (a short-acting formulation) and
Bydureons (a long-acting formula) by Amylin Pharmaceuticals. Byettas is
administered twice daily (sc. injection), 60 min before the first and last meal.
It raises insulin levels quickly and the effects on blood sugar diminish in 6–8
hours. Bydureons, which was approved in 2012, requires only once-week
injection. The advantage of Bydureons is the long effect, but Byettas
shows better control of blood glucose level. Recently, some benefits of once-
weekly exenatide treatment of patients with type 2 diabetes over insulin
glargine and oral glucose lowering medication were shown (e.g. lack of
weight gain or hypoglycemia during the treatment).61 The meta-analysis of
the exenatide treatment also suggested satisfactory effects on lipid profile
and blood pressure.62 However, some gastrointestinal side effects (vomiting,
diarrhea, reflux) were also observed, but they were less frequent in case of
the treatment with once-weekly exenatide. There have been reports of acute

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pancreatitis associated with the use of exenatide. Therefore, the treatment

should be discontinued if pancreatitis is suspected.
Liraglutide (NN2211) is a novel GLP-1 receptor agonist with 97% se-
quence homology with the native GLP-1 (Arg34, Lys26(N-e-(g-Glu(N-a-
hexadecanoyl)))-GLP-1[7-37], Scheme 2).63 The sequence modification and
the incorporation of palmitic acid enhance the enzymatic stability of lir-
aglutide by self-association (micelle like heptamer) and binding to HSA that
delay absorption and make it suitable for once-daily dosing.64 Its plasma
half-life is about 13 hours; thus, it is a longer acting GLP-1 receptor agonist

than the Byettas form of exenatide, but with similar blood glucose con-
trolling properties. The advantages and the mechanism of action are rather
the same as in case of exenatide, but liraglutide shows negligible risk of
hypoglycemia (even lower than exenatide). Liraglutide was approved by
FDA and European Medicines Agency (EMA) in 2009 and marketed by
Novo Nordisk under the brand name Victozas. In 2012, in a petition of
public citizens, FDA was asked to withdraw liraglutide from the market
because of the risk of medullary thyroid cancer (MTC). In contrast to
animal studies, there are no consistent data supporting this side effect in
humans. However, further studies are required to exclude any relationship
between liraglutide and MTC.65,66 It is expected that in a few years lir-
aglutide might be the best sold synthetic peptide-based drug if the associ-
ation with MTC will not be confirmed.
Lixisenatide (ZP10A) is a once-daily GLP-1 receptor agonist based on
exendin-4. Proline is excluded at position 38 in exendin-4 and the C-term-
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inal is elongated with an amidated hexalysine chain (Sheme 2).67 It was

developed at Zealand Pharma in the early 2000s and the global rights were
licenced to Sanofi-Aventis for further development for clinical trials in 2003.
The half-life of lixisenatide is 2-4 hours and it shows all benefits of GLP-1
receptor agonists, although its binding affinity to GLP-1 receptor is a bit
lower than the other’s (IC50: 1.4 nM compared to GLP-1: 0.35 nM, exe-
natide: 0.55 nM and liraglutide: 0.11 nM).68 If it is approved, lixisenatide
will be the third GLP-1 receptor agonist to enter the market, with the name
Lyxumias.69
It is worth mentioning that lixisenatide, similarly to exenatide and lir-
aglutide, can cross the blood brain barrier (BBB) and they have neuro-
protective properties.70 GLP-1 receptors were found on neurons in the
human brain.71 This may be important, since type 2 diabetes through in-
sulin failure represents a risk factor for Alzheimer’s disease. Considering
that GLP-1 receptor agonists affect blood glucose level only in hyper-
glycemia,72 they might efficiently be used for neurodegenerative diseases in
non-diabetic people, too.
Albiglutide is a long lasting GLP-1 analog under investigation (Phase III)
by GlaxoSmithKline for the treatment of type 2 diabetes.73,74 Albiglutide is
a GLP-1 mimetic generated by genetic fusion of a dipeptidyl peptidase-IV-
resistant GLP-1 dimer (a 30 amino acid sequence of human GLP-1[7-36] in
which Ala is replaced by Gly in position 2 of the peptide sequence and it
binds to HSA.75 This GLP-1 mimetic has a half-life of about 6–8 days,
providing a biweekly or weekly administration, which might reduce the
discomfort and inconvenience of daily or more frequent injections. In

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addition to this benefit, albiglutide reduces myocardial infarct size and

improves post-ischemic cardiac function, which is associated with enhanced
myocardial glucose uptake.76
Taspoglutide is a human GLP-1[7-36]-amide analog in which Ala8 and
Gly35 (the second amino acids from both termini) are substituted by Aib (a-
aminoisobutyric acid). These changes result in a fully resistant GLP-1
analog to DPP IV, providing a much longer half-life of about 10 hours
compared with the parent peptide GLP-1[7-36]-NH2.77 It was co-developed
by Ipsen and Hoffman-La Roche. In September 2010, Roche halted the late
stage clinical trials due to a high rate of adverse reactions (e.g., gastro-
intestinal side effects). Further efficacy and safety studies of the once-weekly
GLP-1 analog taspoglutide are in progress.78,79
3.5 Amylin analog in the treatment of diabetes mellitus

Amylin, also called diabetes-associated peptide or IAPP (islet amyloid
polypeptide), is produced by pancreatic b-cells in the islets of Langerhans
and co-secreted with insulin in a ratio of approximately 1 : 100. The 19-mer
oligopeptide containing a disulfide bridge between cysteines in positions 2
and 7 (Scheme 3) was discovered in humans in 1987 by Cooper et al.80
Amylin inhibits the food intake and the appearance of nutrient, especially
glucose in blood. Thus, it synergistically acts with insulin in controlling the
blood sugar level.81 However, amylin is an insoluble peptide even at a low
concentration and forms stable fibres.82 The fibers exert toxic effects and
induce cell apoptosis in pancreas. In contrast, the native amylin analog in
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rat and mouse does not have the ability to form fibers. The sequences of the
two amylin derivatives differ in six amino acids. Among them, there are
three proline substitutions in positions 25, 28 and 29 (Scheme 3). These
prolines as structure-breaking residues make this amylin derivatives unable
to form fibers. This observation led to the idea to replace the amino acids in
human amylin by proline residues for the development of a soluble amylin
derivative (Scheme 3) that can be used as a potential peptide-based drug for
the treatment of diabetes.83 This new soluble amylin derivative Pramlintide
(AC137) was developed by Amylin Pharmaceuticals (now a wholly owned
subsidiary of Bristol Myers-Squibb) and it was approved by FDA for
supplementary treatment with insulin analogs for both type 1 and type 2
diabetes mellitus in 2005. Except insulin analogs, pramlintide acetate
(Symalins) is the only drug approved by FDA to lower blood glucose level
in type 1 diabetes.84 It is also used as a supplement to insulin and it
synergistically increases the effect of insulin to reduce proprandial hyper-
glycemia.85 However, insulin alters the chemical properties of pramlintide.
Therefore, they should not be mixed in the same syringe. In addition,
pramlintide reduces the glucagon secretion that results in lower hepatic
Amylin: NH2-Kc(CNTATC)ATQRLANFLVHSSNNFGAILSSTNVGSNTY-CONH2
Rat amylin: NH2-Kc(CNTATC)ATQRLANFLVRSSNNLGPVLPPTNVGSNTY-CONH2
Pramlintide: NH2-Kc(CNTATC)ATQRLANFLVHSSNNFGPILPPTNVGSNTY-CONH2
Scheme 3 Sequences of human- and rat amylin and of peptide-based drug pramlintide.

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glucose production. This effect may contribute to the reduction of the

required insulin dosage.86
3.6 Teduglutide derived from GLP-2, a peptide fragment of proglucagon

Teduglutide, a GLP-2 analog, is applied not for the treatment of diabetes
mellitus. However, due to its origin, it is worth giving a short overview on
this peptide pharmaceutical in the next paragraph.
Glucagon-like peptide-2 (GLP-2) is a 33 amino acid peptide with
the sequence HADGSFSDEMNTILDNLAARDFINWLIQTKITD in

humans. GLP-2 is produced by proteolytic cleavage of proglucagon in in-
testinal endocrine L cells and in the central nervous system. It is co-secreted
with GLP-1 upon food intake. There are different effects of GLP-2 in
humans, including intestinal growth, enhancement of the intestinal func-
tion, reduction in bone breakdown and neuroprotection. In a novel ap-
proved peptide-based drug, teduglutide, alanine in position 2 of GLP-2 was
replaced by glycine to increase the stability of the peptide against DPP IV,
similarly to the GLP-1 derivatives. This modification can increase the bio-
logical half-life from 7 min to 2–3 h. Teduglutide was approved by FDA in
2012 under the brand name Gattexs (NPS Pharmaceuticals, Inc.) for the
treatment of short bowel syndrome. It is marketed as Revestives in Europe
by Nycomed. More information about teduglutide can be found in P. B.
Jeppensen’s publication.87
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4 Peptide-based drugs for the prevention of thrombosis, the most ‘‘sneaky

disease’’
Platelets or thrombocytes (nucleus free cells) represent a remarkable mam-
malian adaptation that is required for human survival by preventing bleeding.
Normally, platelet activation and coagulation do not occur in an intact blood
cell. However, after injury of the blood vessel wall, platelet plug formation is
induced by the adherence of the platelets to subendothelial collagen.88 During
the coagulation process, platelet and fibrin containing clot is formed, it covers
the blood vessel wall and stops bleeding. Therefore, coagulation (thrombo-
genesis) is an important part of haemostasis. Nevertheless, disorders of
thrombogenesis can lead to a high risk of bleeding (hemorrhage) in case of
e.g. low number of platelets (thrombocytopenia) or obstructive clotting
(thrombosis), resulting in events such as stroke, myocardial infarction, pul-
monary embolism or the blockage of blood vessels in other parts of the
body.89 It is worth mentioning that thrombocytopenia can be treated with a
recombinant fusion protein, romiplostim (Nplates, developed and marketed
by Amgen) that was approved by FDA in 2003. It contains two identical
single-chain subunits of a human immunoglobulin IgG1 Fc domain co-
valently linked at the C-terminus to a peptide containing two thrombopoietin
receptor-binding domains.90 However, this therapeutical recombinant pro-
tein will not be discussed among the peptide-based drugs in this chapter.
The coagulation process, which leads to haemostasis, involves a complex
set of enzymatic reactions involving approximately 30 different proteins.
At the end of the coagulation cascade, fibrinogen (a soluble protein) is
converted into insoluble strands of fibrin by thrombin (a serine protease),

Fig. 4 Coagulation cascade.
which, together with platelets, forms a stable thrombus (blood clot)

(Fig. 4).91 Thrombin plays a central role in the generation of thrombus. It
activates different factors in the cascade (factor V, VIII, and IX) that are
involved in generating more thrombin. Furthermore, it also activates factor
XIII, which is involved in fibrin cross-linking and clot stabilization. Because
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of the complexity of coagulation cascade, there are many factors that in-
fluence the process of thrombogenesis.
Most of the inhibitors that have an influence on the coagulation process
are small organic compounds. Only a few peptide-based drugs have been
developed and approved for this purpose. They are used as anticoagulants
to prevent the unwanted thrombus formation and for the treatment of
e.g. venous thromboembolism (VTE), heparin-induced thrombocytopenia
(HIT), acute coronary syndromes (ACS). One of the mechanisms of action
is the blockade of GPIIb/IIIa receptor (aIIbb3 integrin receptor) on the
surface of platelets, which plays a key role in platelet aggregation. Eptifi-
batide, a cyclic heptapeptide derivative, is an antiplatelet drug that an-
tagonizes the platelet aggregation via GPIIb/IIIa receptor.92 The second
category of peptide-based drugs is represented by the direct thrombin in-
hibitors (DTIs).93 These recombinant hirudin derivatives (lepirudin and
desirudin) and a shorter synthetic analog, bivalirudin, have a dual action by
blocking both the enzyme active site and the exosite 1 domain (fibrin
binding site) on thrombin. DTIs can inhibit both soluble thrombin and fi-
brin-bound thrombin and they do not require a cofactor such as antith-
rombin to exert their effect.94
4.1 Eptifibatide, the GPIIb/IIIa receptor antagonist peptide

Eptifibatide is a cyclic heptapeptide derivative in which the C-terminal
cysteine is connected through a disulfide bridge to the 3-mercapto-propionic
acid (des-amino-cysteine) acylated N-terminus (c[S-CH2-CH2-CO-homoArg-
Gly-Asp-Trp-Pro-Cys]-NH2) (Fig. 5). This RGD peptide mimetic is derived

Fig. 5 Structure of eptifibatide and its platelet aggregation inhibitory effect.
from a protein found in the venom of the southeastern pygmy rattlesnake

(Sistrus miliarius barbouri). Eptifibatide was originally developed by R. M.
Scarborough and D. Phillips at COR Therapeutics, Inc., in San Francisco.95
The company was acquired by Millennium Pharmaceuticals, Inc. in 2001 and
it markets the drug under the trade name of Integrilins. Integrilin is the third
inhibitor of GPIIb/IIIa (GP: glycoprotein) that was approved in 1998 by
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FDA and widely accepted by the global market after the specific antibody
abciximab and the non-peptide RGD-mimetic tirofiban.
Eptifibatide is recommended for the treatment (iv. only) of acute coron-
ary syndrome and to reduce acute ischemic events (e.g. myocardial in-
farction) after precutaneous coronary intervention. It is highly selective for
the GPIIb/IIIa receptor. However, its low binding affinity (dissociation
constant of 120 nM) to the receptor causes rapid dissociation from the re-
ceptor.96 Therefore, eptifibatide reversible inhibits the platelet aggregation.
Its plasma half-life is about 1–1.5 hours and the restoration of normal
haemostatic function after cessation of the treatment with eptifibatide oc-
curs within 3 to 4 hours.97 The clearance of the drug occurs primarily via
renal route. The drug is usually applied together with aspirin or clopidogrel
(antiplatelet agents) and heparin (either low molecular weight or unfrac-
tionated), which is an indirect thrombin inhibitor. The recommended dose
for adults is 180 mg/kg body weight over 1–2 min immediately after diag-
nosis, followed by continuous iv. infusion of 2 mg/kg body weight per mi-
nute until the end of the treatment (max. 72 hours). The main risk of
eptifibatide application is the hemorrhage, which is much lower in case of
bivalirudin.
It is worth mentioning, that another drug tirofiban ((S)-2-(butylsulfon-
amino)-3-(4-[4-(piperidin-4-yl)butoxy]phenyl)propanoic acid; Aggrastats,
Medicure Pharma) that was approved by FDA in 1999 has identical target
receptor and similar activity as eptifibatide. It is a tyrosine analog modified
at its N-terminus and on the side chain and it is based on the anticoagulant
found in the venom of the saw-scaled viper Echis carinatus. Though such

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type of compounds is called as peptidomimetic in many publications, but it

will not be discussed in this book chapter similarly to other amino acid
derivatives.
4.2 Direct thrombin inhibitor peptides

John Berry Haycraft discovered in 1884 that the European leech (Hirudo
medicinalis) secreted a powerful anticoagulant, which was named hirudin.
Its structure was fully determined in 1976. Hirudin contains 65 amino acid
residues, with three disulfide bridges at the compact N-terminal domain,
and the hydroxyl group of tyrosine in position 63 is sulfonated. Hirudin is
the most potent natural inhibitor of thrombin. However, it is difficult to
isolate a large amount of hirudin from natural sources. Therefore, re-
combinant biotechnology in Saccharomyces cerevisiae has been developed
for the production of hirudin like proteins that can be used as anticoagulant
pharmaceutical products.98 One of them is lepirudin (Refludans), which
differs from hirudin at the N-terminus (Ile is replaced by Leu) and the Tyr
does not contain a sulfate group at position 63, while desirudin (Revascs or
Iprivasks) is an unsulfonated hirudin. The amino acid sequences of lepir-
udin and desirudin are the follows:
1
Leu/Ile-Thr-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-
16
Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu-
31
Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu-Gly-Thr-
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46
Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
61
Glu-Glu-Tyr-Leu-Gln65
The disulfide bridges are between the cysteines (1-2, 3-5, 4-6 according to the
order of cysteines in the sequence). The underlined sequence is used for the
shorter synthetic analog, bivalirudin.
The recombinant hirudins have 10 times weaker affinity compared with
the parent protein; however, they are still the most potent thrombin in-
hibitors.99 The plasma half-life of the compounds is 60 min in case of iv.
administration and 120 min for sc. injection, respectively.100 Lepirudin is
licensed for the treatment of thrombosis, complicating heparin-induced
thrombocytopenia (HIT). Desirudin is used in case or for prevention of
deep vein thrombosis (DVT) and pulmonary embolism (PE). Significant
limitations for the use of recombinant hirudins are the narrow therapeutic
window and their potential to increase bleeding events.98 Furthermore,
antihirudin antibodies may be produced in patients and can rarely lead to
anaphylaxis after repeating the treatment. Therefore, it seems that the
shorter synthetic peptide bivalirudin might be a better choice for the
treatment.
Bivalirudin (Hirulog-1) is a shorter synthetic analog of hirudin, containing
two binding sequences from its C-terminus connected with a tetraglycine
spacer. The sequence of the specific and reversible direct thrombin inhibitor
20-mer peptide is as follows: D-Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-
Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu. The peptide was originally developed
by J. M. Maraganore et al. at Biogen in 1990.101 Compared with the

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recombinant hirudin derivatives (Asp in position 53) mentioned above, in

Hirulog-l (D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(53-64) (HVl)), the
Asn in position 9 is derived from another hirudin isomer. Bivalirudin was
approved by FDA in 2000 and it is manufactured and marketed by the
Medicines Company under the trade name Angiomaxs.
Bivalirudin inhibits the thrombus formation as well as the clot propa-
gation in a concentration-dependent manner, both in vitro and in vivo. The
Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu sequence binds to the
fibrin(ogen) binding site exosite-1, while D-Phe-Pro-Arg-Pro sequence

binds to the active site of thrombin (Fig. 6). It is interesting to note, that
this sequence is derived from a tripeptide aldehyde (D-Phe-Pro-Arg-H,
GYKI-14166) developed by Sándor Bajusz in Hungary at the late 1970s.102
This was further developed to increase its enzymatic stability (D-MePhe-
Pro-Arg-H, GYKI-14766)103 which reached the Phase III Clinical Trial at
Eli Lilly under the name efegatran. Since then, the synthetic thrombin in-
hibitors have received a generic name ending in ‘‘gatran’’. Thrombin cleaves
the Arg-Pro bond slowly, followed by the recovery of thrombin active site,
resulting in a reversible inhibitory effect. Though the thrombin inhibitory
activity of bivalirudin is 800– and 80–100 times weaker than that of hirudin
and the recombinant hirudin analogs, respectively, the significantly lower
side effects observed in case of the treatment with bivaluridin makes it an
attractive anticoagulant agent.92,104 In contrast to the recombinant hirudin
analogs, clinically significant antibody formation in response to bivalirudin
monotherapy has not been observed. However, bivalirudin cross-reacts with
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antilepirudin antibodies that might cause anaphylaxis in bivalirudin treated

patients who were previously medicated with lepirudin.105 The half-life of
bivaluridin is 25–30 min, which is much lower than that of recombinant
hirudin derivatives or the compounds used for heparin þ GPIIb/IIIa
treatment; therefore, the incidence of acute bleeding complication is sig-
nificantly lower in this case.92,106
Fig. 6 Effect of direct thrombin inhibitors

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Originally, FDA approved the application of bivalirudin for the treatment

of unstable or post-infarct angina in patients undergoing percutaneous
transluminal coronary angioplasty (PTCA).107 In 2005, the approval was
expanded to the application of bivalirudin in a combination with GPIIb/IIIa
inhibitors for patients undergoing elective or urgent percutaneous coronary
intervention (PCI).108 Bivalirudin is also indicated for patients with or at risk
of HIT and with myocardial infarction undergoing PCI.109,110 The usually
applied dose for patients undergoing PCI is 0.75 mg/kg (iv. bolus injection),
followed by 1.75 mg/kg/h for 4 hours (iv. infusion). The drug cleared mainly
by proteolytic cleavage and hepatic metabolism, but 20% of the dose is
renally eliminated.111 Coagulation times return to baseline approximately 1 h
following cessation of bivalirudin administration.
Recently, it has been shown that bivalirudin is an effective inhibitor of the
interaction of thrombin with protease-activated receptors PAR1 and PAR4,
suppressing both PAR1- and PAR4-dependent platelet activation during
PCI.106 This fact, together with its reducing effect on the platelet surface
expression of P-selectin, might cause the greater inhibitory efficiency of
bivalirudin compared with heparin plus eptifabide on thrombin-induced
platelet activation.112,113
5 Enfuvirtide (Fuzeons), a HIV fusion inhibitor peptide for the treatment

of the most life-threatening disease
The oligopeptide enfuvirtide, consisting of 36 amino acid residues (originally
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called T20), is an anti-human immunodeficiency virus (HIV) drug. It was

developed at Duke University in the middle of 1990s by researchers who
established a pharmaceutical company known as Trimeris.114,115 The de-
velopment was completed in a partnership with Hoffman-La Roche and
enfuvirtide was approved by FDA in 2003 as the first HIV fusion/entry
inhibitor, a new class of antiretroviral drugs. The drug is marketed now
under the brand name Fuzeons by Roche. Fuzeons is used for the treat-
ment of HIV/AIDS patients who fail to respond to the other currently used
antiretroviral drugs.116
The acquired immunodeficiency syndrome (AIDS) caused by HIV is still
the most life-threatening disease. More than 60 million people have been
infected by HIV worldwide, and more than 40% of the infected persons died
so far.117 The lack of an efficient vaccine against HIV makes its prevention
difficult. Therefore, the only effective way to treat persons infected with HIV
is the antiretroviral therapy, which uses synthetic drugs to prevent the
development of AIDS.118 Among the currently used B30 drugs there are
integrase-, protease-, fusion- and reverse transcriptase inhibitors.119
The HIV infection is initiated by the recognition and binding of HIV-1 to
the CD-4 receptor of the host T-cells. First, the viral envelop surface subunit
gp120 binds to CD-4 and then to the co-receptor (CCR5 or CXCR4), fol-
lowed by the conformational changes of gp41 which is non-covalently as-
sociated to gp120.120 The gp41 has three important functional regions. The
fusion peptide (FP, residues 512–527) inserts into the target cell membrane
after conformational changes. Through the interaction of the N-terminal
heptad repeats (NHR, residues 542–592) and the C-terminal heptad repeats

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(CHR, residues 623–663) a six-helix bundle (6-HB) core is formed, which

brings the viral and target cell membrane in an appropriate distance for
fusion. The interaction of NHR and CHR peptide sequences in the for-
mation of 6-HB led to the idea that some peptide fragments from NHR or
CHR may inhibit the fusion through the binding to their counterpart se-
quences.121 In the 1990s, several C-peptides (derived from CHR) exerting
HIV-1 inhibitory effect in nM range were discovered. One of them was T20
(residues 638–673, Fig. 7), which significantly decreased the viral load and
increased CD-4 cell counts after 24 weeks of treatment in a clinical trial,

and became the first HIV-1 fusion inhibitor with the generic name
enfuvirtide.122,123
Fuzeons is applied in sc. injection twice daily using 90 mg enfuvirtide per
injection. Usually, the injection site reactions and other hypersensitivity
reactions are mild to moderate, but occasionally may be severe. For in-
stance, increased incidence of pneumonia was observed; however, it is un-
clear whether it is related to the use of enfuvirtide. A one year therapy in the
US costs ca. 25000 USD. In addition to the side effects, costs and relatively
fast degradation (half-life is about 3.8 hours) of T20, the main problem of
enfuvirtide treatment is that T20 could rapidly induce HIV-1 variants with
strong drug resistance in patients.124
It has recently been shown that T20 does not contain the pocket-binding
domain (PBD) in its sequence; therefore, it does not efficiently inhibit the
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Fig. 7 Schematic representation of HIV-1 gp41 with the cell fusion mechanism, as well as the
sequences of NHR and CHR and designed small therapeutic peptides.

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development of stable 6-HBs and block the formation of fusogenic

core.125,126 T20 may interact with N-peptides in NHR to form unstable or
insoluble complexes. Furthermore, it binds to gp120 and to the lipid
membrane of the host cells. Thus, new C-peptides containing the PBD were
selected for further studies. One of the best compounds is C34 (Fig. 7),
which binds to the binding cavity of NHR and shows much higher potency,
longer half-life and better drug resistance than T20.127 This peptide, called
sifuvirtide, is located in the 628–661 domain of CHR (partially overlapped
with T20) and it is not only highly active against T20-resistant HIV strains,
but also shows significantly lower injection site reactions (7% instead of
98% in case of T20).128 Up to now, it has not been shown that C34 can
induce drug resistant mutation in the gp41 binding pocket.129 Sifuvirtide is
in Phase II Clinical Trial in China and might be the next generation of
fusion inhibitors as peptide-based drug.130
Because T20 and C34 bind to different sites of NHR and inhibit the
HIV-1 fusion in different ways, the combined application of these fusion
inhibitors might lead to synergistic effects resulting in a potential new
therapeutic strategy for the HIV infection or AIDS.128,131 Furthermore, the
conjugates of small molecules with inhibitory effect (binding to the hydro-
phobic pocket of NHR) with C-peptides (e.g. P26, a part of C34, Fig. 7)
might provide efficient peptide-based drug delivery systems with anti-
retroviral effect in the near future.132
6 Lucinactant (Surfaxins), a synthetic surfactant for the prevention of

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the most tragic deaths

Respiratory distress syndrome (RDS) is the leading cause of neonatal
morbidity and mortality in premature infants.133 This syndrome is caused
by a lack of endogenous surfactant production in the lungs. Surfactant
complex and its individual components (lipids, proteins and glycoproteins)
decrease surface tension, which helps to prevent the collapse of the terminal
air-spaces (the future site of alveolar development) throughout the normal
cycle of inhalation and exhalation.134 Surfactant replacement therapy was
established as a safe and effective treatment in the 1990s and has become
the standard of care for these newborns. Surfactant products are either
protein-free synthetic phospholipid compounds or animal-derived lung
preparations. Currently, about 90,000 infants a year receive treatment with
one of the commercially available animal-derived surfactants.135 The higher
efficacy of the animal derived products compared with the synthetic protein-
free drugs indicated the importance of the surfactant proteins in the treat-
ment.136 Of the four known surfactant proteins (SP), the hydrophobic SP-B
and SP-C have a critical role in stabilizing the phospholipid monolayer and
in enhancing the ability of phospholipids to lower the surface tension. The
deficiency in SP-B production may cause the fatal form of respiratory
failure shortly after birth, while the disturbance of SP-C production might
lead to the development of chronic lung disease in early adulthood.137
Lucinactant is a new synthetic surfactant containing a pulmonary sur-
factant-associated protein B mimic that was approved by FDA in 2012.
Lucinactant was developed at the Scripps Research Institue by Charles

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138
Cochrane’s group in the 1990s. It was then developed as a marketed drug
by Discovery Laboratories of Warrington, PA and sell under the trade
name Surfaxins. Lucinactant contains phospholipids (1,2-dipalmitoyl-sn-
glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphogly-
cerol as their sodium salt and palmitic acid) and the peptide sinapultide
(KL4) acetate (Scheme 4). The basis of the sequence for the 21-mer helical
KL4 (KLLLLKLLLLKLLLLKLLLLK) was the charge distribution and the
hydrophilic/hydrophobic ratio of the amino acid sequence of the C-terminal
domain of SP-B (SP-B59-80: DTLLGRMLPQLVCRLVLRCSMD).139 Sina-

pultide mimics the action of human SP-B, but it is more resistant to in-
activation by serum proteases and reactive oxygen species than the natural
occurring SP-B.140
Clinical trials indicated that lucinactant is superior to protein free syn-
thetic surfactant preparations and at least as effective as the animal-derived
surfactant in reducing morbidity and mortality from RDS.135 In addition to
the higher resistance of lucinactant to inactivation, improved overall lung
function was observed, potentially leading to lower rates of reintubation.140
Furthermore, the synthetic lucinactant results in lower risk of infection and
inflammation compared to its animal derived counterparts, which are
presently the standard treatment for RDS.133
The pharmacoeconomic modeling also suggests the benefit of lucinactant
treatment compared to the other surfactant replacement therapy.137 The
advantage of aerosolized lucinactant over diluted Surfaxins lavage as
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Scheme 4 Components of lucinactant (Surfaxins).

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potential alternative to intratracheas surfactant replacement therapy was

also indicated.141
Lucinactant is applied for the prevention or treatment of several other
conditions like meconium aspiration syndrome and bronchopulmonary
dysplasia in newborn and premature infants, as well as acute respiratory
distress syndrome in adults. It seems that after the unusual long approval
process (because of challenges in manufacturing and some ethical criticism),
Surfaxins as an orphan drug (i.e., a pharmaceutical agent that has been
developed specifically to treat a rare medical condition) will become an

important member of the peptide-based drug family.
7 Gonadotropin-releasing hormone derivatives as the largest peptide-based

drug family
7.1 Gonadotropin-releasing hormone
The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH),
also known as luteinizing hormone-releasing hormone (LH-RH), was dis-
covered in 1971 by Andrew V. Schally and his colleagues.142 The structure
of GnRH (Glp-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2; where Glp is
pyroglutamic acid) was elucidated by the research groups of the 1977 Nobel
Laureates Andrew V. Schally and Roger Guillemin.143,144 In mammals, the
linear decapeptide end-product is produced from a 92-amino acid pre-
prohormone in the preoptic anterior hypothalamus. The primary biological
function of GnRH is the regulation of the gonadal activity by stimulating
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the release of gonadotropic hormones, luteinizing hormone (LH) and fol-

licle stimulating hormone (FSH), from the anterior pituitary gland that act
on the female and male gonads to trigger the gametogenesis, as well as the
synthesis and release of sex steroids (androgens, estrogens). Therefore, this
neurohormone plays a central role in vertebrate reproduction.145 GnRH is
secreted in a pulsatile fashion by the hypothalamic neurons (low-frequency
GnRH pulses lead to FSH release, whereas high-frequency GnRH pulses
stimulate the LH release)146 and it has a short half-life in vivo of 2–5
minutes.147 Continuous exposure to GnRH results in the suppression of
gonadotropin and sex steroid secretion due to the desensitization of gon-
adotropic cells and the down-regulation of pituitary GnRH receptors
(GnRH-R, a seven-transmembrane G-protein-coupled receptor).148 The
observed selective chemical castration is a medical approach to therapy of
hormone-dependent tumors such as prostate and breast tumors, benign
prostatic hyperplasia (BPH), endometriosis and female infertility.149 Since the
discovery of the mammalian GnRH (human GnRH or GnRH-I), 22 further
natural isoforms have been identified and several thousand synthetic GnRH
derivatives have been developed.150 GnRH, with the generic name of gona-
dorelin diacetate tetrahydrate (Cystarelins, Merial Ltd.), as a first generation
peptide-based drug was approved by FDA (U S Food and Drug Adminis-
tration) for veterinary medical applications (for the reproductive perform-
ance of cows with ovarian cysts) in 1978. During the last 35 years, about a
dozen GnRH agonists (second generation) and antagonists (new generation)
have found therapeutic applications (both veterinary and human), providing
the largest family of peptide-based drugs.

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7.2 GnRH agonists as pharmaceutics

By modifying the Gly6 and Gly10 in human GnRH, superagonist derivatives
can be produced. Gly6 is replaced by D-amino acid derivatives with bulky
apolaric side chain (Leu, Trp, Ser(tBu), His(Bzl), a-tert-butyl-glycine (Tle)),
while Pro9-Gly10-NH2 is modified as Pro-NHCH2CH3 (ethylamide; EA) or
Pro-Azagly-NH2.151 These modifications provide highly effective GnRH
agonists (Table 4), which are potent drugs for several hormone dependent
diseases like breast, ovarian and prostate cancers, endometriosis, benign
prostatic hyperplasia and uterine fibroids. In all cases, the continuous

treatment with superagonist GnRH derivatives can lead to decreased serum
levels of sex steroid hormones resulting in significant remission of the dis-
ease. GnRH agonists are also efficiently used for central precocious puberty
in children by reducing the testosterone (male) or estrogen (female) level.152
The third application of GnRH agonists in humans is the prevention of LH
surge in in vitro fertilization (IVF).153 The in vitro fertilization process in-
volves controlled ovarian stimulation (COS) to enhance the follicular
growth. In fact, high LH levels have a negative effect in the IVF; therefore,
the reduction of bioactive LH in the blood is required.154 Without the ap-
plication of GnRH analogs, LH surges occur in B20% of the stimulated
IVF patients.155 Some of the GnRH agonists are used for all these very
different purposes mentioned above. However, the applied doses may be
different depending on the hormone levels necessary to be reached.156
Nevertheless, GnRH agonists exert an initial stimulatory effect (flare-up),
before leading to the desensitization of gonadotropic cells and a decrease in
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the number of GnRH-R on the cell membrane (down-regulation) followed

by the reduction of LH and FSH release.151 The blockade of the production
of gonadotropins results in the inhibition of sex steroid production. The
complete inhibition can be reached in 15–20 days, causing a subsequent
delay in the therapeutic benefit.157 The temporary increase of sex steroids
(e.g., testosterone surge) can exacerbate the hormone-sensitive cancers and
may cause catastrophic flare events such as spinal-cord compression and
urinary-tract obstruction. This clinical flare should be avoided especially in
the case of cancer metastasis.158
The GnRH agonist analogs used either as veterinary pharmaceuticals
(lecirelin, deslorein) or in humans (buserelin, goserelin, leuprolide, triptor-
elin, histrelin, nafarelin) are summarized in Table 4. In human medication,
the most frequently used compounds are goserelin,159 leuprolide160 and
triptorelin.161 The peptides were developed in the middle of 1970s and they
reached the market in the late ‘80s and early ‘90s. Since then, mainly the
formulation was improved to develop long acting derivatives of these
peptide-based drugs. For instance, in case of leuprolide, a microsphere-
based depot formulation is used for Luprons, from which the drug release
occurs in a biphasic manner. In the first phase, the product is released within
a relatively short time from the surface of the spheres, which functions as a
higher initial dose to reach quickly an effective plasma concentration.162 In
the second phase, the microspheres are ‘‘digested’’ during the next months
(the 1–6 months length depends on the composition of the microspheres)
providing the release of a maintenance dose for constant plasma concen-
trations over time. Eligards, on the other hand, is a gel-based formulation

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Table 4 Summary of GnRH agonists as peptide pharmaceuticals.
GnRH agonist drugs Sequences and applications
gonadorelin diacetate tetrahydrate Glp-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

Cystorelins, Merial Ltd. 0.1 mg/cow intramuscular injection
Gonadorelin hydrochloride Veterinary pharmaceutical for treatment of cystic
Factrels, Zoetis Inc. ovaries in cattle
Buserelin acetate Glp-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-EA
Suprefacts, Sanofi-Aventis sc. injection 0.5 mg 3x daily for 7 days (initial)

Cinnafacts, CinnaGen (generic) 0.2 mg once daily (maintenance) for cancer
Depot 6.3 mg or 9.45 mg sc. implant for 8 or 12 weeks
Treatment of hormone dependent prostate cancer
Goserelin acetate Glp-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-
Zoladexs, AstraZeneca Azagly-NH2
3.6 mg (1 month) or 10.8 mg (3 months) depot
sc. implantation
Treatment of hormone dependent prostate and breast
cancers, endometriosis, uterine fibroids, central pre-
cocious puberty and in in vitro fertilization
Histrelin acetate Glp-His-Trp-Ser-Tyr-D-His(Ns-Bzl)-Leu-Arg-Pro-EA

Vantass, Orion Pharma Ltd. 50 mg sc. implant/year for prostate cancer
Suprelin LAs, 50 mg sc. implant/year for treatment of central
Endo Pharmaceuticals Solutions Inc. precocious puberty in children
Leuprolide (Leuprorelin) acetate Glp-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-EA
Luprons, Abbott Laboratories 1 mg/day sc. single injection or depot sc. 7.5 mg/
Eligards, Sanofi-Aventis month, 22.5 mg every 3 months, 30 mg every 4
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months or 45 mg every 6 months for cancer treat-

ment. A lower dose is used for endometriosis, uterine
fibroids, central precocious puberty.
Nafarelin acetate Glp-His-Trp-Ser-Tyr-D-2-Nal-Leu-Arg-Pro-Gly-NH2
Synarels, Searle (now part of Pfizer) Nasal spray (2 mg/mL)
Treatment of endometriosis, uterine fibroids, central
precocious puberty and in in vitro fertilization
Triptorelin acetate or pamoate Glp-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2
Decapeptyls SR, Ipsen sc. injection 0.5 mg once daily for 7 days (initial)
Gonapeptyl Depots, 0.1 mg once daily (maintenance) for cancer
Ferring Pharmaceuticals Treatment of hormone dependent prostate and breast
cancers, endometriosis, uterine fibroids, central pre-
cocious puberty and in in vitro fertilization
Lecirelin acetate Glp-His-Trp-Ser-Tyr-D-Tle-Leu-Arg-Pro-EA
Dalmarelins, Biopeptide Co. Inc. Veterinary pharmaceutical, both for the treatment
of conditions such as ovarian cysts and for the
improvement of conception rates in cows,
mares and rabbits
Deslorein acetate Glp-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-EA
Ovuplants, Peptech Ltd. Veterinary pharmaceutical to induce ovulation in
mares as part of the artificial insemination process
Suprerolins, Peptech Ltd. Long or short term suppression of reproductive
functions of dogs and cats
delivered by using a biodegradable polymer of D,L-lactide-co-glycolide

dissolved in N-methyl-2-pyrolidone.163 In this delivery system, the polymer
and the lyophilized peptide drug must be mixed less than 30 minutes before

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164
injection. It is injected in a liquid form that condenses into a solid sub-
cutaneous depot, which releases the drug over time.165
A high number of publications provide overviews on their efficacy and
appropriate applications for different hormonal diseases. In recent studies,
the benefits of the six-months depot formulation in the treatment of prostate
cancer have been reported.166,167
7.3 GnRH antagonists as pharmaceutics

The most promising GnRH antagonists discussed here belong to the new
generation of peptide-based drugs and contain Ac-D-Nal-D-Cpa-D-Pal-Ser
N-terminal sequence (Nal: 2-naphtylalanine; Cpa: 4-chlorophenylalanine;
Pal: 3-pyridylalanine), Leu in position 7, and Pro-D-Ala-amide at the C-
terminus. In positions 5, 6, and 8, a large variety of amino acid derivatives
(mainly non-natural ones) have been substituted (Scheme 5). The benefit of
the application of GnRH antagonists over agonists is the immediate de-
crease of gonadotropin as well as sex steroid hormone levels through the
rapid but reversible blockade of GnRH receptors without any flare ef-
fect.151,156 GnRH antagonists can be used in all application area of GnRH
agonists, including endometriosis, leiomyoma and breast cancer in women,
BPH and prostate cancer in men and central precocious puberty in children.
However, the best clinical evidence has been in in vitro fertilization and
prostate cancer.149 At present, four GnRH antagonists – cetrorelix, abar-
elix, ganirelix and degarelix, in their acetate form – are marketed.
Cetrorelix (Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-
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NH2, where Cit is citrulline; Scheme 5) was developed by S. Bajusz in

A. V. Schally’s laboratory in 1988 and it was approved in Europe in 1999
under the trade name Cetrotides (ASTA Medica AG, later Aeterna
Zentaris GmbH, Frankfurt, Germany) as the first GnRH antagonist to be
marketed.168 Cetrotides is marketed by Merck Serono worldwide except
Japan, where it is sold by Shinogo and Nippon Kayaku for IVF. Cetrorelix
could be administered in its physiological acceptable soluble acetate salt or
pamoate suspension. A long acting depot system was developed by the
complexation of cetrorelix with polyglutamic acid.156 The success of this
compound resulted from its lower toxic side effects (e.g., oedematous re-
action, cyanosis and respiratory depression) that were observed in case of
earlier developed GnRH antagonists.
In case of IVF, the minimal effective dose of cetrorelix that could prevent
premature LH surges and necessary to obtain good quality oocytes was
0.25 mg/day. However, it has recently been reported that there was no
significant difference in the LH surge when either 0.25 mg/day or 0.125 mg/
day doses were applied.169 Intensive studies are running to compare the
efficacy of cetrorelix and other GnRH antagonists with GnRH agonists for
assisted reproductive techniques. There are more and more results showing
that the clinical pregnancy rate is significantly higher and miscarriage rate is
significantly lower in case of GnRH antagonist administration vs. GnRH
agonists. Therefore, the cost per pregnancy in case of applying GnRH an-
tagonists is lower.170 However, until the breakthrough in the application of
GnRH antagonists instead of GnRH agonists, further optimization of the
treatment protocols and comparative studies are required.171–173

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Scheme 5 Structure of GnRH antagonists used as peptide drugs.

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Cetrorelix has been tested in various diseases of reproductive organs like

uterine myoma, BPH and ovarian and prostate cancers. Positive effects were
detected in all cases after optimization of the doses and treatment schedules.156
It has to be mentioned that cetrorelix, similarly to other GnRH analogs,
has not only an indirect antitumor activity through the hormone cascade re-
sulting in chemical castration, but also it shows a direct tumor growth in-
hibitory effect through GnRH receptors that are highly expressed on cancer
cells. It was also indicated that GnRH antagonists act as agonists on tumor
cells.174
Ganirelix (Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-hArg(Et2) -Leu-hArg(Et2) -
Pro-D-Ala-NH2, where hArg(Et2) = N6-[bis(ethylamino)methylene]-lysyl;
Scheme 5) was developed by J. J. Nestor et al. as a GnRH antagonist with
low histamine-releasing activity, in the early 1990s.175 Ganirelix was ap-
proved by FDA in 1999 and it is marketed by Organon International as
Antagons. Ganirelix has longer half-life than cetrorelix (13 h and 5–10 h,
respectively) after a single dose administration. Ganirelix is used as a fer-
tility treatment drug for women. Specifically, it is used to prevent premature
ovulation in women undergoing fertility treatment involving ovarian
hyperstimulation that causes the ovaries to produce multiple eggs. Ac-
cording to the optimization studies, it seems that 125 mg/day dose is pre-
ferred over 250 mg/day, because the later one increased early pregnancy loss
in some women by marked suppression of LH level.176,177
Abarelix (Ac-D-Nal-D-Cpa-D-Pal-Ser-N-MeTyr-D-Asn-Leu-Lys(iPr)-
Pro-D-Ala-NH2; Scheme 5) was developed by T. Cook and W. F.
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Sheridan.178 It has higher antitumor activity than cetrorelix, since it efficiently

decreases not only the LH but also the FSH secretion, which may stimulate
the proliferation of human androgen-resistant prostate cancer. However,
abarelix showed some significant side effects during clinical trials (e.g.,
bronchitis, fever, hot-flushes, headache, decrease of libido); therefore, in 2003
FDA approved its application under the brand name Plenaxiss only for
patients with advanced, symptomatic prostate cancer who could not have or
who refused other treatments. In 2005, however, abarelix was withdrawn
voluntarily by the marketing company Praecis Pharmaceuticals, due to oc-
curred systemic allergic side effects. In contrast, abarelix was approved in
Germany that time.156 Nevertheless, studies with abarelix continued, because
of its high activity on immediate testosterone suppression. Recently, abarelix
has been used for a new treatment paradigm for prostate cancer, in which
abarelix initiation therapy is followed by GnRH agonist treatment.179
Degarelix (Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(Hor)-D-Aph(Cba)-Leu-
Lys(iPr)-Pro-D-Ala-NH2, where Aph(Hor) is 4-[2,6-dioxohexahydropyrimidin-
4(S)-ylcarboxamido]-L-phenylalanyl and Aph(Cba) is 4-ureido-phenylalanyl;
Scheme 5) was developed by G. Jiang et al. at around the turn of the new
century.180 It was approved in 2008 in the USA and in 2009 in Europe for
the treatment of advanced prostate cancer. Degarelix is produced by
Ferring Pharmaceuticals and sold under the trade name Firmagons. The
benefits of degarelix over the GnRH antagonists described before are its
higher solubility (no gel formation at higher concentration) by prevention of
b-structure formation with carbamoyl type side chains, and lower histamine
release (EC50 values for cetrorelix, ganirelix, abarelix and degarelix were 1.3,

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181
11, 100 and 170 mg/ml, respectively). Though the binding affinity of
degarelix to GnRH receptor is not significantly different from that obtained
with the other antagonists, degarelix shows increased duration of action.
Using a dose of 2 mg/kg sc. administration abolished the plasma testo-
sterone level (below castration level) till the day 50. It is hypothesized that
this ability of degarelix is derived from the strong structure stabilization by
intermolecular H-bonds between the peptide and its receptor.181 As the
most potent GnRH antagonist for prostate cancer, degarelix is intensively
used in comparative studies with GnRH agonists.182,183 These data indicate

benefits not only in the activity but also in the cost-effectiveness of
degarelix.184,185
8 Medical applications of somatostatin derivatives that have the most

receptors
8.1 Somatostatin and somatostatin receptors
Somatostatin (also known as growth hormone-inhibiting hormone (GHIH)
or somatotropin release-inhibiting factor (SRIF)) is a cyclic neuropeptide
containing a disulfide bridge between the cysteine residues in positions 3 and
14. Somatostatin was discovered in the early 1970s by R. Guillemin and
coworkers, who also determined its structure.186,187 The native somatostatin
(SST) has two molecular forms, SST-28 and SST-14, containing 28 or 14
amino acid residues, respectively, produced by proteolysis of a precursor
molecule. The hormone is expressed in high concentrations in brain, liver,
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lungs, pancreas, thyroid, gastrointestinal tract, and adrenal gland. It

mainly acts as an inhibitor of exocrine and endocrine secretions on target
organs (e.g. SST suppresses growth hormone (GH), prolactin (PL),
thyroid stimulating hormone (TSH), adrenocorticotrop hormone (ACTH)
production from pituitary glands, insulin and glucacon secretion from
pancreas and several gastrointestinal peptides). Moreover, the synthesis of
many growth factors (IGF, EGF, VEGF, FGF) is also inhibited by
SST.188,189 Furthermore, there are five subtypes (six subtypes, if sst2A and
sst2B are separately counted) of SST receptors (sst1-5 belong to the
superfamily of GPCRs)190 that allow SST to possess the wide range of
biological functions. The relationship of SST receptor subtypes and hor-
mone secretion in brain and gastrointestinal tract as well as other biological
functions are summarized in a review article by F. Barbieri et al.191 Fur-
thermore, an overview of the overexpressed SST receptor subtypes on
different types of cancer cells was also published.192 The binding affinity of
SST-14 to all somatostatin receptors is in the range of 0.1–2 nM (IC50).191
However, the main limitation in the in vivo application of native SST is its
very short plasma half-life (2–3 min). The main enzymatic cleavage sites are
shown in Scheme 6.192 During the development of more stable analogs, the
L-Trp in position 8 was replaced by the D-isomer and a D-amino acid was
placed at the N-terminus of the shortened peptide chain. Furthermore, a
disulfide bridge was formed between cysteines that were incorporated in-
stead of phenylalanines in positions 6 and 11 in the native sequence. The
main synthetic analogs used as peptide-based pharmaceuticals are octreo-
tide, lanreotide, pasireotide and vapreotide (as acetate salts) (Scheme 6).

Scheme 6 Structures of somatostatin (SST 14) with the main enzymatic cleavage sites and of
somatostatin derivatives applied as peptide pharmaceuticals.
Table 5 Binding affinities (IC50 nM) of native and clinically used synthetic analogs for SST
receptor subtypes.191
Ligands sst1 sst2 sst3 sst4 sst5
Endogenous
SST-14 0.1–2.3 0.2–1.3 0.3–1.6 0.3–1.8 0.2–0.9
SST-28 0.1–2.2 0.2–4.1 0.3–6.1 0.3–7.2 0.1–0.4
Synthetic
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Octreotide >1000 0.4–2.1 4.4–34.5 >1000 5.6–32

Lanreotide >1000 0.5–1.8 43–107 >1000 0.6–14
Pasireotide 9.3 1.0 1.5 >100 0.2
Vapreotide >1000 0.2–5.4 31 45 0.7
The binding affinities of these compounds to the SST receptors are pre-
sented in Table 5. It is worth mentioning that some radiolabelled analogs
are used as diagnostic tools.193
8.2 Somatostatin derivatives as pharmaceutics

Octreotide (SMS 201-995: H-D-Phe-c(Cys-Phe-D-Trp-Lys-Thr-Cys)-
Thr(ol)), an octapeptide that mimics the natural somatostatin, was first
synthesized by W. Bauer in the early 1980s.194 Octreotide (injection) was
approved by FDA in 1988 and it is marketed under the brand name San-
dostatins by Novartis Pharmaceuticals (previously Sandoz, before it was
unified with Ciba-Geigy to form Novartis). Ten years later, its long-acting
release/repeatable formulation Sandostatin LARs was also approved for
the treatment of acromegaly, gigantism, thyrotropinoma (mainly caused by
pituitary tumors also called pituitary adenoma), and for relief of symptoms
caused by advanced neuroendocrine tumors (NETs) including gastro-
intestinal tract, lung and pancreas tumors.195–197
Lanreotide (BIM-23014: H-D-Nal-c(Cys-Tyr-D-Trp-Lys-Val-Cys)-Thr-
NH2, where Nal is 3-(2-naphthyl)-alanine) was developed by D. H. Coy
et al. in 1988.198 It is manufactured by Ipsen and marketed under the brand
name Somatulines. It was approved by FDA in 2007 in two formulations: a

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sustained release formulation Somatuline LA which is injected im. every 10

or 14 days and an extended release formulation Somatuline Autogels (UK)
or Somatuline Depots (US). The indications of lanreotide are the same as in
case of octreotide and used especially for the treatment of acromegaly and
neuroendocrine tumors.199–201
Similarly to GnRH derivatives, the somatostatin analogs exert their
antineoplastic action in a direct or indirect way.201 The direct activities are
mediated through the SST receptors on tumor cells, inducing antimytotic
and apoptotic effects. The mitosis inhibition is mediated by sst2 and sst5,
resulting in the cell cycle arrest. Apoptosis is induced by sst3 or by the in-
hibition of IGF1, a potent antiapoptotic hormone. In case of the indirect
mechanism, the inhibition of growth factors (IGF, EGF) and angiogenesis
(VEGF) as well as their immunomodulating activity play a key role in the
antiproliferative effect. The antitumor effects of SST analogs highly depend
on the tumor types and receptor subtypes expressed on the cell membrane.
There are numerous clinical trials with octreotide and the data on lanreotide
is continuously increasing. Up to now, no significant differences between the
efficacies of the two compounds could be concluded. Furthermore, the
complete or partial remission of tumors could be detected only in a few
cases; however, they can stabilize or at least slow the progression of the
disease. The results are summarized in recent publications.192,199,202,203 The
safety dose optimization of octreotide LAR and lanreotide Autogel in
patients with acromegaly and neuroendocrine tumors has been reviewed by
W. H. Ludlam and L. Anthony.204
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Recently, the FDA has approved extended dosing intervals for lanreotide
(120 mg dose in every 6 or 8 weeks). However, the pharmacodynamics of
octreotide do not allow an extended dosing interval and the FDA has
approved an interval of 4 weeks for this medicament.205,206 It is worth
mentioning that the pharmacoeconomic aspects and the easy application
in clinical use show some benefits in case of lanreotide compared to
octreotide.206,207
Pasireotide (SOM230: c[2-amino-ethyl-carbamoyl-oxy-Pro-Phg-D-Trp-
Lys-Tyr(Bzl)-Phe], a homodetic ‘‘head-to-tail’’ cyclic peptide, where Phg is
phenylglycine), as a multireceptor somatostatin analog, was developed by
C. Burns et al. at Novartis Pharma AG in the 2000s.208 This somatostatin
derivative has much longer elimination half-life (23 hours) than octreotide
(2 hours) or lanreotide (4–5 hours). Furthermore, it efficiently binds to 4 sst
(sst1-3 and sst5) and except sst2, pasireotide has significantly higher binding
affinity to the other 3 receptors than octreotide and lanreotide. This fact
provides its special therapeutic application. Pasireotide was approved by
FDA for the treatment of Cushing’s disease in 2012. It is marketed by
Novartis Pharma AG under the brand name Signifors.
Cushing’s disease is caused by an adrenocorticotropin (ACTH)-pro-
ducing adenoma that secrets excess ACTH, which in turn stimulates the
adrenal glands leading to the chronic excess of cortisol production.209 This
disease is a rare endocrine disorder with an incidence of 1-2 new cases per
million population per year.210 However, the chronic glucocorticoid excess
leads to considerable morbidity and the not adequately treated disease may
cause death.211 Prolonged hypercortisolism produces a range of signs and

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symptoms, including central obesity, diabetes, hypertension, muscle weak-

ness, depression and osteoporosis.212 Patients who are ineligible for surgery
or have had unsuccessful surgery are candidates for drug therapy. For the
drug treatment there are glucocorticoid receptor antagonists, adrenal-
targeting drugs and pituitary-targeting drugs. Among the latest ones,
pasireotide was the first approved for the treatment of Cushing’s disease.
The reason that pasireotide can be used for the treatment of Cushing’s
disease is that corticotroph tumor cells frequently overexpress sst5, whereas
other receptor subtypes are usually expressed at a lower level.213 The

treatment with pasireotide decreased the ACTH secretion and normalized
the glucocorticoid level. Furthermore, it significantly suppresses the cell
proliferation of corticotroph tumors by decreasing the GH and IGF1 levels.
The reduction of adenoma size was demonstrated by MRI.214 It has also
been observed that most patients who do not respond to pasireotide in the
first two months do not respond at a later time point as well. In this case, an
immediate change of the therapy is necessary.213 Because of the broad
spectrum of activity of somatostatin analogs, the careful monitoring of the
patients during the therapy is needed. The adverse effects are summarized
by K. McKeage.213 The most severe side effects are hyperglycemia and
diabetes mellitus. Pasireotide shows marked suppression of insulin and
GLP-1 secretion and only a mild suppression of glucagon secretion.215,216
This disadvantage might be partly overcome by co-administering an anti-
hyperglycemic drug. The best results have been observed in case of lir-
aglutide.217 The usually applied dose of pasireotide is 600 or 900 mg twice
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daily. However, it should be decreased when significant hyperglycemia syn-

dromes are detected. It has to be mentioned that the tolerability profile of
pasireotide is similar to octreotide and lanreotide, pasireotide being associ-
ated with higher incidence of hyperglycemia.215 The increased risk of arterial
thrombosis was also shown in case of patients with Cushing’s disease. During
the pasireotide treatment, a slightly decrease in antithrombin and thrombin-
activatable fibrinolysis inhibitor levels as well as a transient increase in
plasminogen activator inhibitor type 1 levels were observed.218 Other par-
ameters of coagulation and fibrinolysis did not significantly change.
The case events for comparison of pasireotide with other compounds
developed for the treatment of Cushing’s disease are not sufficient (more
data is available for pasireotide). It seems that pasireotide resulted in long
term urinary free cortisol excretion in 16–29% of the patients.209 Therefore,
studies in which pasireotide is used in combination therapy with other pi-
tuitary-targeting or adrenal-targeting drugs are in progress to improve the
efficacy of the medication of Cushing’s disease.209,219
Recently, a long acting formulation of pasireotide (pasireotide LAR) was
developed and studied in case of neuroendocrine tumors.220,221 The 20, 40
or 60 mg dose was administered once every 28 days for 3 months. Pasir-
eotide LAR seems to be a useful tool in the medication of Nelson’s syn-
drome (rapid enlargement of a pituitary adenoma that occurs after the
removal of both adrenal glands) as well.222
The history of the fourth somatostatin analog on the market is not as
successful as the previous ones. Vapreotide (RC-160: H-D-Phe-c[Cys-Tyr-
D-Trp-Lys-Val-Cys]-Trp-NH2) was developed in A. V. Schally’s

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223
laboratory. It efficiently binds to sst2 and sst5 and has much higher
metabolic stability than the parent hormone peptide. Debiopharma received
the right to develop it for the European market, while H3 Pharma tried to
bring it to the Market in North America under the trade name Sanvars.
The Sanvar Immediate Release form has got the orphan drug status in the
US for the treatment of acute esophageal variceal bleeding (EVB) in pa-
tients with cirrhotic liver disease and AIDS-related diarrhea in 2003. The
results demonstrating the efficacy of vapreotide in case of EVB were pub-
lished by B. E. Fortune et al.224 However, more data was required for the
approval. Finally, in 2009, FDA decided not to approve the drug for
medical use. Therefore, the compound is approved only in a few countries
(e.g. France, Mexico). It seems that this somatostatin analog is not a subject
for further studies. Only a couple of manuscripts have been published in the
last years, reporting the application of vapreotide for different pur-
poses.225,226 Its radioiodinated version has recently been used for the visu-
alization of sst2.227
8.3 Somatostatin derivatives as targeting moieties for diagnosis and

radiotherapy
The high density of somatostatin receptors on human neuroendocrine
tumors has allowed the development of somatostatin receptor scintigraphy
for tumor imaging as well as somatostatin receptor-targeted radio-
therapy.190 An 111In labeled octreotide derivative 111In-DTPA-octreotide
(111In-Pentetreotide; where DTPA is diethylene triamine pentaacetic acid
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used for the complexation of In) has become the most widely used tracer for
somatostatin receptor scintigraphy.228 It has been approved by FDA in
1994 and it is produced and sold by Mallinckrodt Medicals under the brand
name OctreoScans. New tracers including 68Ga and 99mTc derivatives,
showing a different and/or wider affinity and also a broader spectrum of
applications than tumor diagnosis, are now available and radiolabeling has
been obtained both with gamma and positron emitters that are summarized
in several publications.193,229 In case of somatostatin receptor-targeted
radiotherapy, 90Y-DOTA-Tyr3-octreotide (90Y-DOTATOC, Onaltas,
BioMedica Life Sciences S.A.) and 177Lu-DOTA-Tyr3-octreotate (177Lu-
DOTATATE) were approved for clinical trials (Scheme 7). Octreotate
has free carboxyl C-terminus instead of alcohol in case of octreotide.
The radiocharacteristics of the isotopes are as follows: 90Y (b-energy: 2.27
MeV; b-range: 12 mm; T1/2: 64 hrs) and 177Lu (b-energy: 0.49 MeV; b-range:
2 mm; T1/2: 6.7 days).230,231
9 Icatibant (Firazyrs), the most effective compound for the treatment of

hereditary angioedema
Hereditary angioedema (HEA) is a rare (ca. 2–3 individuals/100000), but
unpredictable and sometimes life-threatening condidion.232,233 It is char-
acterized by acute attacks of edema with multiple localization (e.g. face,
upper airways, extremities, genitals, gastrointestinal tract). Episodes of la-
ryngeal edema are the least frequent, but are the primary cause of mortality
associated with HEA.232 The pathophysiological background of the disease

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Scheme 7 Examples for radiolabelled somatostatin derivatives.
is represented by the decreased or dysfunctional production of C1 inhibitor

enzyme (C1-INH). C1-INH normally inhibits various protease systems like
complement, coagulant and kallikrein pathways.232,234 The lack of its direct
inhibitory effect on kallikrein activity results in the uncontrolled release of
bradykinin. This nonapeptide hormone (Arg-Pro-Pro-Gly-Phe-Ser-Pro-
Phe-Arg) binds to Bradykinin type 2 (B2) receptors on endothelial cells,
causing increased vascular permeability (e.g. edema and swelling), vaso-
dilation (e.g. hypotension), and contraction of nonvascular smooth muscle
(e.g. cramps, pain). Various treatment options for acute attacks or pre-
vention (prophylaxis) of HEA are available on the market, including plasma
derived (Berinerts, Cinryzes, and Cetors) and recombinant (Rhucins,
Ruconestt) C1 inhibitors, kallikrein inhibitor (ecallantide, Kalbitors, a
60 amino acid containing polypeptide with three disulfide bridges
between cysteines in order 1-6, 2-4, and 3-5 (EAMHSFCAFKADDGP-
CRAAHPRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD)
developed from a Kunitz domain through phage display to mimic anti-
bodies inhibiting kallikrein) and a B2 receptor antagonist (icabitant).232 In
this chapter, only the latter one will be discussed.
The B2 receptor antagonist peptidomimetic (Hoe 140: D-Arg-[Hyp3,
Thi5,D-Tic7,Oic8]-bradykinin, Scheme 8) as a potent and long acting
bradykinin antagonist was developed at Hoechst AG in the early
1990s.235,236 In addition to D-Arg, further non-proteinogenic amino acids

Scheme 8 Structure of icatibant.
(Hyp: hydroxyprolyl; Thi: 3-(2-thienyl)alanyl; Tic: 1,2,3,4-tetra-

hydroisoquinolin-3-yl-carbonyl; Oic: (3aS,7aS)-octahydroindol-2-yl-car-
bonyl) provide higher stability compared to the native hormone. The
compound with a generic name icatibant has received an orphan drug status
and was approved by the European Commission in 2008 and by FDA in
2011 for the treatment but not for prophylaxis (due to its fairly short half-
life) of HEA. Icatibant acetate is marketed by Jerini AG (now in cooper-
ation with Shire Ltd.) with the trade name Firazyrs.
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It has been shown that among bradykinin antagonists, icatibant has

the highest binding affinity to B2 receptor, which is similar to the activity
of the native hormone. Furthermore, it is selective for B2, while there is
no significant binding (100 times lower) to Bradykinin type 1 receptor.232 The
effect and tolerability of sc. or iv. treatment with icatibant at a dose of 0.1 to
1 mg/kg has been demonstrated. Since bradykinin has potential cardiopro-
tective properties, icatibant could impair the cardiac function and decrease
the coronary blood flow.237,238 Therefore, patients with heart disease should
carefully be monitored during the treatment. Furthermore, the application of
icatibant in patients with acute cardiac or brain ischemia is not recom-
mended.232,238 Due to the short duration of action, the hypertension, which
might be caused by bradykinin antagonists, is not a long-term risk factor.
Because icatibant could decrease the antihypertensive effect of ACE (angio-
tensin-converting-enzyme) inhibitors (some of them belong to the second
generation peptide-based drugs, e.g. enalapril (Vasotecs); N-[(1S)-1-(ethoxy-
carbonyl)-3-phenylpropyl]-L-alanyl-L-proline, lisinopril (Prinivils); Na[(1S)-1-
carboxy-3-phenylpropyl]-L-lysyl-L-proline), the co-treatment with these
compounds is not recommended.239
One of the main benefits of icatibant over other drugs used for HEA is
that icatibant can be used as a self-medication (sc.) at home. This is highly
important, since acute attacks of HEA are unpredictable and can be po-
tentially life-threatening if the appropriate treatment is not timely. The self-
administration in time could reduce the mortality associated with this
disease.240 Furthermore, HEA attacks treated within 1 hour of attack onset
resolved significantly faster than those treated after 1 hour (median 1.7

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233
hours versus 6 hours, respectively). The equivalent cost profile supports
also the use of icatibant compared to other drugs used for the treatment of
HEA.241,242
In addition to HEA, there are other types of bradykinin-mediated
angioedemas that can be treated with icatibant. A number of case reports
have recently been published, demonstrating the efficacy of icatibant in
these areas as well.243–245
10 Mifamurtide based on the smallest native immunostimulatory peptide

Muramyl dipeptide (MDP) is the smallest natural occurring immunosti-
mulatory component found in both Gram-positive and Gram-negative
bacterial cell walls. However, MDP was identified as being too pyrogenic
and arthrogenic for pharmaceutical applications in humans.246,247 The ef-
forts to develop less pyrogenic derivatives led to the discovery of muramyl
tripeptide phosphatidyl ethanolamine (MTP-PE, Scheme 9) at Ciba-Geigy
(now Novartis) in the early 1980s. Later on, its liposome-encapsuled for-
mulation has been prepared (L-MTP-PE), which is called mifamurtide.
Mifamurtide is 10-fold less toxic than the free MTP-PE. Mifamurtide was
granted an orphan drug status by FDA in 2001 and European Medicines
Agency (EMA) in 2004. While the drug was finally approved in Europe in
2009, it was denied by FDA in 2007. The companies that own mifamurtide
have been changed several times and now Takeda Pharmaceutical Company
Ltd has the right to sell it. The original brand name Mepacts was also
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changed to Junovans. Mifamurtide is indicated for postoperative treatment

of high-grade, resectable, non-metastatic osteosarcoma in children, ado-
lescents and young adults, aged between 2 and 30 years. It is used in
combination with chemotherapeutic agents, doxorubicin and methotrexate,
with or without cisplatin and ifosfamide.
Osteosarcoma is the most common primary malignant bone tumor; it is a
rare disease affecting less than 3 individuals in 100000 per year. It usually
arises in the metaphyses of long bones in children and adolescents, having
Scheme 9 Structure of MTP-PE.

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higher incidence in these ages (1–11/100000 new cases per year in the age
group 15–19 years).248,249
Osteosarcoma derived metastasis (mainly in the lung and other bones)
can clinically be detected only at 20–25% of the patients, but most of the
patients have undetectable micrometastatic disease.250 In addition to the
surgery, the above mentioned chemotherapeutic agents are used for the
treatment of osteosarcoma. With the combination of two or more com-
pounds, 60–70% of patients with localized disease show event-free survival
at 3–5 years, while this rate is only 20–30% in case of patients with me-
tastasis.250 It seems that the effectiveness of the treatment with cytostatic
drugs have reached a plateau and it cannot further be improved by other
drug combinations.251 However, a significant improvement was observed
when mifamurtide was combined with one of the cytostatic drugs (doxor-
ubicin, methotrexate, cisplatin, cyclophosphamide) or in appropriate com-
binations, especially in metastatic cases (ca. 10–20% higher survival).252–254
Interestingly, a combination chemotherapy using doxorubicin and cyclo-
phosphamide suppress the positive effect of mifamurtide.252
Though the mechanism of action is not fully understood, the animal tests
clearly indicate that L-MTP-PE is effective against the micrometastasis of
osteosarcoma, but not against bulky disease. Most likely, the adjuvant effect
of mifamurtide is based on the stimulation of monocytes and macrophages
that selectively phagocytosed L-MTP-PE. The liposomal vesicles slowly
degraded lead to the release of MTP-PE into the cytosol. The higher efficacy
of mifamurtide over the free MTP-PE can be explained by the longer re-
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tention in the target cells.250 The released MTP-PE like MDP specifically
binds to nucleotide-binding oligomerization domain 2 (NOD2) receptor
and NOD-like receptor P3 (NLRP3), resulting in the activation of NF-kB,
MAPKs and inflammasome, as well as proinflammatory cytokines such as
TNF-a, IL-6, IL-1b.246,247 The activation of monocyte-mediated cytotoxic
activity was detected in almost 90% of the patients, which took for up to
96 h following the mifamurtide infusion. The treatment schedule is 1-hour
infusion of 2 mg/m2 mifamurtide twice weekly for 12 weeks, followed by
once weekly administration for an additional 12 weeks. Mifamurtide is well
tolerated even the released cytokines may cause inflammation, fever, etc.255
The net positive effect of the mifamurtide treatment in patients with osteo-
sarcoma is clearly demonstrated, suggesting its use in the frontline therapy.
11 Perspectives
It is assumed that the ratio of small molecule-based drugs approved for
clinical applications will decrease continuously. In contrast, the peptide-
and protein-based drugs, in particular the short synthetic and long-acting
ones are quickly increasing in the global drug market. The reason for that is
the high selectivity, low accumulation in tissues and other benefits of pep-
tides and proteins.11 Furthermore, the new synthetic approaches are
dropping the production costs of peptides, especially in large scale. In
addition, appropriate peptide formulations (microspheres, liposomes) pro-
vide slower drug release and elimination, resulting in longer effect. The drug
formulation of peptides will remain one of the main topics in the

View Online
development of peptide pharmaceuticals. However, the oral administration,

as the most convenient route of treatment, is still the most challenging one
for peptide drugs. The solution to this problem will represent a real
breakthrough in the application of peptide-based pharmaceuticals.
In order to increase the stability of small peptides, they could be modified
by cyclization, substitution with non-native amino acids including D- and
b-amino acids or by replacement of amide bonds between the amino acids.
The combination of this structural elements leads to the development of
peptidomimetics. Recently, a growing number of peptidomimetics has been

approved for medication and the tendency to develop mimetics as ther-
apeutics is continuously rising. As enzyme inhibitors or compounds dis-
rupting the cell membranes, they are mainly applied as antibacterial or
antiviral agents. However, the question is which structures of peptidomi-
metics belong to the class of peptide-based drugs (e.g. telaprevir (Inciveks)
and boceprevir (VictrelisTM) used against hepatitis C virus; Scheme 10).9
Some criteria were given by R. Kharb et al., who classified the four types of
peptidomimetics in pseudopeptides, functional-, topographical- and non-
peptide mimetics.256
Almost half of the marketed drugs target G protein-coupled receptors
(GPCRs). Some of these receptors are highly expressed in certain diseased
cells/tissues (e.g. cancer cells) compared to the normal/healthy ones. These
differences may provide possibilities for selective drug targeting. For this
purpose, drugs are attached to peptides that specifically bind to the highly
expressed receptors and deliver the drugs into the cells by receptor mediated
01/12/2013 14:45:16.
way. The perspective of such type of peptide – drug conjugates that increase
the selectivity and reduce the toxic side effects of the drugs is significant.
About 20 peptide-drug conjugates are in clinical trials, particularly for
targeted cancer therapy. One of the most promising conjugate is the NGR-
hTNFa (Arenegyrs), in which the cytokine tumor necrosis factor-a is fused
to a cyclic CNGRC pentapeptide as homing device (Scheme 11).257 The
conjugate was granted as orphan drug by the European Commission in
2008 for the treatment of malignant pleural mesothelioma. Since then, the
antitumor activity of the conjugate was shown on many tumor types
Scheme 10 Schematic structure of telaprevir and boceprevir.

Scheme 11 Schematic structure of NGR-hTNFa and cilengitide.
(e.g. colon cancer and non-small cell lung cancer). Peptides with Asn-Gly-
Arg (NGR) motif recognize CD13 membrane bound receptors that are
highly expressed on newly formed blood vessels on cancer cells. It has to be
noted that NGR sequence containing peptides can easily be deamidated
leading to the formation of isoAsp-Gly-Arg through succinimide ring
closure. However, similarly to RGD peptides, the isoAsp-Gly-Arg motif
containing peptides can recognize integrin receptors that are involved in
tumor vascularization, as well. Thus, the conjugates with NGR sequence
01/12/2013 14:45:16.
might have dual site of action that should be intensively studied.257,258 It is

worth mentioning that a cyclic RGD analog (c[RGDf(NMe)V]; cilengitide
(Centrics), Scheme 11) is in clinical studies as the first anti-angiogenic small
peptide drug candidate.259 Some disappointing results on glioblastoma
bearing patients have recently been published, but further trials are carried
out on other types of cancer. Nevertheless, the author believes that cyclic
RGD peptide-based drug delivery systems will become therapeutics in the
future. In these drug delivery systems, the cyclic RGD peptide is directly
attached to the drug (e.g. paclitaxel) or it is incorporated into the surface of
nanoparticles that contain the targeted drug.260,261 Further anti-angiogenic
peptides that are in clinlical and preclinical phases have been summarized in
a review article.262
Peptide conjugates based on hormone peptides as homing devices (es-
pecially GnRH and somatostatin analogs with own antiproliferative effect)
are very promising in targeted tumor therapy. D-Lys6-GnRH-I is conju-
gated with doxorubicin through glutaric acid linker, providing a labile ester
linkage that allows the free drug release. The Zoptarelin Doxorubicin
(AEZS-108, AN-152) is in Phase III Clinical Trials for endometrial cancer
and in Phase II for ovarian, prostate, bladder and breast cancers
(Scheme 12).263,264 Intensive research is carried out for the evaluation of the
antitumor effect of the compound on other types of cancer (e.g. pancreatic
cancer, glioblastoma).265,266 In another promising GnRH conjugate, which
is in preclinical studies, disorazol Z, a novel natural compound isolated from
myxobacterium Soranglium cellulosum with outstanding cytotoxic activity,
is attached to D-Lys6-GnRH-I (AEZS-125). Cytotoxic drug containing

Scheme 12 Schematic structure of Zoptarelin Doxorubicin.
Scheme 13 Schematic structure of zı́conotide.
somatostatin derivatives (AEZS-124, AN-162) are also available,267 but it

seems that the radiolabelled somatostatin analogs are more promising for
01/12/2013 14:45:16.
targeted radiotherapy of cancer.193 Further possibilities for targeted drug

delivery with peptides and antibodies have recently been reviewed.10,268
The application of peptide delivery vectors using cell penetrating peptides
(CPPs) is also a promising area of peptide-based drug development. The
ability of CPPs to cross the plasma membranes has been used for various
applications, including the delivery of bioactive molecules (nucleic acids,
short interfering RNA (siRNA), antisense oligonucleotides, plasmids,
proteins, drugs) to inhibit disease-producing cellular mechanisms. Recent
review articles have provided an overview on this topic, including preclinical
and clinical studies.269,270 Considering that the CCPs do not have high se-
lectivity for different cells, it is important that the cargo compound attached
to CPP have specific targets in the diseased cells.
The number of bioactive peptides isolated from marine organisms con-
tinues to grow. Marine peptides have medical relevance, including an ability
to interact with a variety of ion channels. Furthermore, there are marine
peptides with antitumor, antimicrobial, antiviral, anticoagulant and anti-
freeze properties.271–274 Therefore, one can say that marine peptides might
be a ‘‘gold mine’’ for peptide-based drugs. The marine peptides may be used
in therapy in their native form or peptidomimetics can be developed from
their structural elements. One compound, o-conotoxin peptide (Ziconotide,
SNX-111; Prialts, Scheme 13) derived from Conus magnus (‘‘Cone Snail’’)
was approved by FDA in 2004 for the treatment of chronic pain.
Ziconotide acts as a selective N-type voltage-gated calcium channel
blocker. This action inhibits the release of pro-nociceptive neurochemicals
like glutamate, calcitonin gene-related peptide, and substance P in the brain

View Online
and spinal cord, resulting in pain relief. It is expected that the importance of
marine drugs will increase in the future and ziconitide will be followed by
other marine drugs.
Antibiotic resistance is increasing at a rate that far exceeds the pace of the
development of new drugs. Antimicrobial peptides (AMPs) can be isolated
not only from marine origins but also from other natural sources. Although
AMPs possess considerable benefits as a new generation antibiotics, their
clinical applications still have some limitations such as potential toxicity,
susceptibility to proteases and high cost of peptide production. In order to

overcome these drawbacks, extensive efforts are made on the design of short
peptides with non-natural amino acids or peptidomimetics retaining anti-
microbial activities. Thus, the development of antimicrobial peptides as
peptide-based pharmaceuticals remains a hot topic and interest will be raised
as pathogens become resistant against conventional antibiotics.275,276 The
status of antimicrobial peptides in clinical trials is summarized by J. L. Fox.277
The tendency to extend the scale of activity of peptide-based pharma-
ceuticals will continue. However, the drug market will have an influence on
the success of individual drugs. Therefore, it is believed that the largest
increase in the number of peptide and protein pharmaceuticals can be ex-
pected in the area of diseases (e.g. diabetes, cancer) that result in the highest
income for the pharmaceutical industry.
All together, the principles of drug development are changing. The ratio
of peptide- and protein-based pharmaceuticals among the approved drugs
elevates rapidly. This fact may result in the inspiration of peptide and
01/12/2013 14:45:16.
protein research and increase interest of the pharmaceutical industry in

peptide- and protein-based drugs.
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Published on 31 August 2013 on http://pubs.rsc.org | doi:10.1039/9781849737081-FP001

Amino Acids, Peptides and Proteins

A Specialist Periodical Report

Amino Acids, Peptides

Chrishan S. Samuel, University of Melbourne and Monash University, Australia

& The Royal Society of Chemistry 2014

All rights reserved

in accordance with the terms of licences issued by the Copyright Licensing

Published by The Royal Society of Chemistry,

Registered Charity Number 207890

For further information see our web site at www.rsc.org

the series has been to provide a comprehensive review coverage of research

Amino Acids, Pept. Proteins, 2013, 38, v–vi | v

protein pharmaceuticals and progress in the development of emerging

vi | Amino Acids, Pept. Proteins, 2013, 38, v–vi

The crystal structure of particulate

state and three distinct metal

Oxidative transformations of amino acids and peptides catalysed by 1

NMR investigation of disulﬁde containing peptides and proteins 37

Amino Acids, Pept. Proteins, 2013, 38, vii–ix | vii

Relaxin and its role in ﬁbrotic diseases 60

Biofunctional peptide design 79

Self-assembly of designed peptides and their nanomaterials 122

Integrating chemical cross-linking with mass spectrometric analysis of 151

viii | Amino Acids, Pept. Proteins, 2013, 38, vii–ix

4 Future perspectives 167

Peptidome analysis: tools and technologies 172

Peptide and protein based pharmaceuticals 203

4 Peptide-based drugs for the prevention of thrombosis, 216

Amino Acids, Pept. Proteins, 2013, 38, vii–ix | ix

Abbreviations, acronyms and symbolic representations are very much part

Asp Aspartic acid D

x | Amino Acids, Pept. Proteins, 2013, 38, x–xvii

Other Amino Acids

Aze Azetidine-2-carboxylic acid

are instances for example not only of ‘homophenylalanine’,

Amino Acids, Pept. Proteins, 2013, 38, x–xvii | xi

group should be made explicit as in aMeTyr for

Sta Statine; (3S, 4S)-4-amino-3-hydroxy-6-methyl-heptanoic acid

of the three-letter symbol it is meant to be understood that the amino group

xii | Amino Acids, Pept. Proteins, 2013, 38, x–xvii

present. Bonds up or down should be used only for indicating side-chain

Actually the simplest representation is on one line, as in 7.

Substituents and Protecting Groups

Amino Acids, Pept. Proteins, 2013, 38, x–xvii | xiii

xiv | Amino Acids, Pept. Proteins, 2013, 38, x–xvii

In fact this reagent has the isomeric N-oxide structure 9 in the

Similarly, the corresponding reagent derived from HOBt has

Amino Acids, Pept. Proteins, 2013, 38, x–xvii | xv

TBTU See remarks under HATU above

propyl)-carbodiimide hydrochloride (also EDC)

xvi | Amino Acids, Pept. Proteins, 2013, 38, x–xvii

QSAR Quantitative structure–activity relationship

Amino Acids, Pept. Proteins, 2013, 38, x–xvii | xvii

1.2 Cytochromes P450

identiﬁed as an invariant cysteine residue which lies within a highly con-

Amino Acids, Pept. Proteins, 2013, 38, 1–36 | 1

Cofactor Example Reference