Introduction to Synthetic Peptides

By Dr John E. Fox
Director, Alta Bioscience

How they are made

Alta Bioscience uses solid phase synthesis to make all of its peptides. Here, the C-terminal
amino acid is anchored to polystyrene based resins and the peptide is grown amino acid by
amino acid towards the amino terminal. When the peptide chain is complete, it is cleaved off
the resin with acid, a process that removes the amino acid side chain protection at the same
time. After removal of the acid, the peptide is ready for QC by HPLC and mass spectrometry.
After satisfactory QC, the peptides are purified by preparative reverse phase HPLC, then
freeze dried, packaged and dispatched.

Aspects of purity
Peptide purity
The purity of all our purified peptides is
determined by reverse phase HPLC. A
wavelength of 215nm is used for the analysis
as this is the optimum for the detection of the
peptide bond and hence detects all peptide
species present.
It should be noted that the purity value obtained
by this method does not include the presence of
any water and trifluoroacetate salt which will be
present in the dried material. Unless specified
in the order, all Alta Bioscience peptides are
supplied with trifluoroacetate as the counter-ion,
acetate or chloride can be supplied on request.
Reverse phase chromatography will remove all
the reagents used in the cleavage process. All
Alta Bioscience peptides that are supplied to a
specified purity will have been through a clean-
up process, even if the crude material exceeds
the requested purity. The laboratory makes
extensive use of capping during synthesis, so
deletion peptides are very rare. However some
truncated material and peptide with modified side
chains could be present.
Net peptide content
All dried peptides will contain a variable
amount of water plus a fixed amount of the
peptide counter-ion, usually trifluoroacetic acid.
Quantitative amino acid analysis is the only
method which enables the net peptide content to
be determined. Here, the amount of each amino
acid is measured after total acid hydrolysis, the
sum total of which gives the amount of peptide
in the product. Typical values for net peptide
content range from 70% – 90% but in extreme
cases can be as low as 20%.

An Introduction to Synthetic Peptides Page 57

Levels of purity
Three levels of purity are offered, >95%, >90%,
>80%, in addition to unpurified material. The
higher the purity, the higher the cost of the
finished product. In general, the >95% purity
is only needed when the peptide is to be used
as an enzyme substrate or in NMR and X-ray
crystallography analysis. It is not necessary to
specify high purity for peptides that are to be
used to raise antibodies.
If a peptide is requested to a set purity, Alta
Bioscience will put it through a purification
process, even though the crude material passes
the HPLC purity specification. All purified
peptides are supplied with HPLC and MS traces.
Salt form of peptides
As peptides are usually purified by HPLC with
acetonitrile gradients and trifluoroacetic acid,
(TFA), as moderator, they exist as their TFA
salts. For most purposes this is not a problem
but when adding peptides to cell cultures, the
TFA can sometimes be toxic. This problem
can be avoided by specifying peptides in either
acetate or chloride salt forms.
The analysis of peptides
Alta Bioscience has the capability to analyse its
product by a wide range of methods.
1. HPLC
High Performance Liquid Chromatography,
HPLC, is the primary method of analysing
peptide purity. Performed typically on a C18
reverse phase column, 4.6mm x 250mm with
300Å pore size silica, using an acetonitrile water
gradient with TFA, as the acidic species.
2. MALDI-TOF
A ‘matrix assisted laser desorption and
ionisation – time of flight’ mass spectrometer is
used to determine the molecular weight of the
peptides. Highly accurate, fast and requiring
small amounts of sample, it is the ideal method
to ascertain that the target peptide has been
Page 58 An Introduction to Synthetic Peptides
synthesised.
3. Amino acid analysis
This technique is primarily used to measure the
net peptide content of a product. The peptide
is acid hydrolysed to its amino acids and these
are quantified after separation by ion exchange
chromatography and detection with ninhydrin.
4. N-terminal sequencing
Amino terminal Edman sequencing can be used
to confirm that the sequence of the amino acids
is correct.
Design and structure of peptides
By convention, peptides are written left to right
with the N-terminus at the left and the C-terminus
at the right. Care must be taken when specifying
modifications. An example of a typical modified
sequence is shown below.
acetyl- KLPSSRY pS AGHLLD -amide
PhosphoSerine is spelled out as pS with spaces
before and after. The words acetyl and amide
are separated from the peptide by spaces and
hyphens. (Don’t forget that both amide and
acetyl spell out a real peptide sequence).
Amino acid classification
The following table gives a general classification
of the amino acids
Acidic, polar Asp, Glu
Basic, polar His, Lys, Arg
Polar uncharged Asn, Cys, Gly, Gln,
Pro, Ser, Thr, Tyr
Nonpolar and Ala, Ile, Leu, Met,
hydrophobic Phe, Trp, Val
Solubility
Solubility, or primarily the lack of it, is the cause
of the majority of problems when working with
peptides. In general, peptides with a large
proportion of nonpolar amino acids will be
difficult to dissolve in aqueous solutions, the O NH2

more polar residues that are present, the easier

C
H2
CH 2 C
it will be to dissolve a peptide. Peptides that are O
CH 2 O
C CH2 O + NH 3
acidic, i.e. contain more acidic amino acids than H 2N CH C HN CH C
NH

basic, will be more soluble at higher pH and visa Pep ti

de
P ep
tide

versa, peptides that are overall basic will be most

soluble at lower pH. Figure 1. Mechanism of pyroGlu formation

Length of peptides
Although Alta Bioscience has made some very
long peptides of over 80 amino acids, the solid
phase method essentially has a realistic upper
limit of about 50 amino acids. Above this length,
the high risk of failure tends to make a synthesis
financially uneconomic. As the length increases,
so does the number of impurities that have to
be removed from the target sequence, thus the
absolute purity of the product will be lower. A
longer peptide will also have a higher chance of
containing a sequence region that is difficult to
make.
The ease of synthesis of any peptide is entirely
dependent on its sequence, a difficult sequence
can easily prevent even a short peptide of 10
amino acids being made. Alta Bioscience will
freely give as much help as possible concerning
the viability of a synthesis. Peptides that are
potentially difficult, could cost more to make than
easy ones.
Things to avoid
*
Peptides containing long strings of Valine
or Isoleucine are virtually impossible to
synthesise and work with.
*
A peptide with no charged or polar groups
may be very insoluble.
*
Multiple additions of phospho amino acids
can cause major synthesis and purification
problems. The peptides can be made but
the costs rise steeply with each additional
phospho group.
*
If possible, it is best to avoid cysteine when
designing peptides for raising antibodies.
In proteins, cysteine usually exists as a
disulphide bridge so it would present a very
different shape if presented as the monomer,
as shown in figure 6.
*
These amino acids decrease solubility:-
Trp, Val, Ile, Phe
If in doubt please ask, we are happy to
give advice free of charge

Some sequences can be particularly difficult

and if they can be avoided in some way, the
synthesis will be much easier or even made
possible.
*
N-terminal Gln should be avoided at peptides.
all costs. It is very unstable and rapidly
forms the cyclic pyroglutamic acid as shown
in the illustration. It is best to add either
pyroglutamic acid itself, or include an
acetyl group at the N-terminal Glutamine.
Things to include if possible
*
Proline breaks up beta sheet formation
and although non-polar, helps to solubilise
*
A spacer between a dye or tag and the
rest of the peptide sequence is usually
advantageous. A range of spacers can be
used. Ahx, amino hexanoic acid is a simple,
useful spacer. SGSG is a hydrophilic
sequence designed by Alta Bioscience for
use as a biotin spacer. A range of PEG
spacers are available with varying numbers

An Introduction to Synthetic Peptides Page 59

 of atoms.
*
It is always cheaper to put a dye or tag at the
N-terminus rather than the C-terminus.
*
These amino acids increase solubility:- Lys,
His, Arg, Asp, Glu, Ser, Thr.
Modifications and unnatural amino
acids
There is a huge number of modifications
possible, listed below are the more common
ones. The structures of many of these unusual
amino acids are shown in the accompanying
paper, ‘Table of the amino acids’.
*
Phosphorylated amino acids
Phosphorylated Ser ,Thr and Tyr can be placed
at any specified site in a peptide. However,
multiple incorporations can cause synthesis and
purification problems.
*
Terminus modifications
N-terminal acetyl and C-terminal amides remove
the charges at the ends of a peptide and make it
much more like the parent protein.
*
Methylation
Mono, di and tri methylated Lys, mono and
dimethyl Arg are found in histone proteins,
13C and 15N can be incorporated into peptides
for use in quantitative mass spectrometry. It
is advised to focus on the amino acids with
nonreactive side chains, such as Val and Phe.
The more complex amino acids tend to be
prohibitively expensive, if available at all.
*
Unnatural amino acids
Compounds such as phenylglycine, napthyl
alanine, nor leucine and beta alanine are readily
incorporated into peptides.
*
Spacers
These are used to pull dyes and tags away
from the active site of a peptide, some common
examples are shown here:-
Hydrophobic aminohexanoic acid
Hydrophilic SGSG a short peptide sequence
Hydrophilic PEG, ranging from 9 to 88 atoms
Please let us know if you need
a compound that isn’t in the above
list of modifications
*
Biotin.
O

these methylated amino acids can be easily C

HN NH
incorporated at specific positions. O
CH CH H2 H2
C
*
D amino acids H2 C CH C
C

C
C
OH
H2 H2
All the D amino acids can be added at any S

position. Binds irreversibly to streptavidin and is used

*
Analogues extensively in screening assays and to bind
peptide to substrates.
Amino acids with longer or shorter versions of
the side chain length are available. For example, *
Desthiobiotin.
O
homoserine and homoarginine are longer
C
variants of serine and arginine while ornithine
HN NH
and diamino butyric acid are shorter analogues O

of lysine. These are very useful in fine tuning the

CH HC H2 H2
C C C
H3 C C OH
shape of peptides. H2 C
H2
C
H2

*
Isotopes Binds to streptavidin but can be displaced
Amino acids enriched with the stable isotopes by biotin. Useful when you need to get your
peptide out of a binding experiment.

Page 60 An Introduction to Synthetic Peptides

Peptides with Dyes Cross linked peptides

A very wide range of dyes and tags are available, Many bioactive peptides contain several
a short list of the more common ones is shown disulphide bridges. Alta Bioscience has had
here. The accompanying paper, “Introduction to considerable success in the synthesis of these
dyes, labels and tags” describes these more fully. complex compounds.

*
FAM Cyclic with a peptide bond
*
Tamra Either the two ends of a peptide or specific
*
The DyLight™ range of dyes –CO2H and –NH2 residues can be reacted
*
Dansyl to form a peptide bond, resulting in a cyclic
*
NBD compound. Care must be taken in the design of
the peptide for this method to work well.
*
Edans
*
Dabcyl
*
Mca

Cyclic peptides
Alta Bioscience can synthesise both cyclic and
cross linked peptides.

Cyclic disulphide
If a peptide is made with two cysteine residues,
careful oxidation in solution will result in a cyclic
Figure 2. Cyclised with a peptide bond
compound, created as the cysteines bridge to
form their dimer, cystine. This reaction generally Cyclic thioethers
proceeds smoothly with good yield and minimal
These are useful when designing peptide
polymer formation. The bridge can be broken
libraries where the peptide needs to be
under physiological conditions.
presented as a constrained shape. The
cyclisation process proceeeds smoothly, in
good yield. The thioether bond is stable under
physological conditions.

Figure 1. Diagram of a peptide with a disulphide

bridge. Figure 3. Diagram of a thioether cyclic peptide

An Introduction to Synthetic Peptides Page 61

Peptides for raising antibodies
In general, synthetic peptides are too small to
elicit an antibody response, Alta Bioscience
uses two methods to convert its peptides into a
suitable form.
1 MAP peptides
MAP peptides are octomeric molecules with
the peptide chains branching out from a central
poly-lysine core, as shown in figure 4. The eight
peptide chains increase the molecular weight
of the compound sufficiently for it to be easily
recognised as an antigen. It provides an easy
and flexible method for antibody production.
triggers the antibody system, which recognises
the attached peptides. The most popular carrier
protein is keyhole limpet heamocyanin, KLH,
which elicits a strong antibody response and
contains a very large number of lysine residues
which are used to attach the peptide antigen.
This particular approach can be used to attach
the peptide in any orientation, i.e. at either the N
or the C terminus. However, it is not suitable for
any peptide containing cysteine, as that amino
acid is added to the sequence to act as the linker
to the protein.

K
K K
K
K
K
K
K
K
K K
K
K
K

Figure 5. Diagram of a peptide-protein conjugate.

Figure 4. Diagram of an octomeric MAP peptide
It is also possible to make chimeric MAPs with
two different peptides sequences, each forming
four of the chains.
The MAP method however, isn’t suitable for
peptides which come from the C-terminus of a
protein, as that particular amino acid is the one
conjugated to the core peptide and thus not
exposed.
Antigen design considerations
In general, peptides for antibodies will be
hydrophilic and flexible, coming from the exterior
of the parent protein. A hydophilicity plot will
indicate which parts of the protein are likely to
be on the outside of the structure. The Kyte-
Doolittle or the Hopp-Woods algorithms will be
very useful here.

Dialysis through a 2-3kDa membrane is the only
purification method which is required for these
molecules.
2 Peptide – protein conjugates
Here, a synthetic peptide with a free cysteine
residue, is covalently attached to the lysines in a
protein carrier molecule. The size of the protein
Structure predictions can be done with Chou-
Fasman plots. The best source for the data
would be the European Bioinformatics Institute.
Cysteine should be avoided where possible.
The following illustration in figure 6, shows that
a single cysteine would present a very different
shape to the immune system compared with the
disulphide bridged, cystine.

Page 62 An Introduction to Synthetic Peptides


Cys S--S Cys Cys SH
Figure 6. Differences in shape between the
disulphide bridge in a protein and a linear
peptide.
Peptides for micro arrays
Virtually any type of sequence can be printed
onto a micro array. To reduce steric hindrance
effects, it is helpful to specify a spacer such as
Ahx or a PEG between the peptide sequence
and any biotin which is used to anchor the
peptide onto the array slide. The biotin is
usually added at the N-terminus but there are no
synthesis difficulties in having either a C-terminal
biotin or it anywhere along the peptide chain.
If a cysteine is being used as the linker amino
acid for binding to maleimide surfaces, then the
array peptide must not contain any sequence
cysteines. If two Cys residues were present,
there would be no control over which of them
would act as the linker.
It is advisable to specify the linker group to be
at the N-terminal of the peptide. The synthesis
proceeds C to N with capping, so only the full
length peptide would contain the linker. All failure
sequences would be washed away and take no
part in the binding.
An Introduction to Synthetic Peptides Page 63
Handling peptides
Storage
Alta Bioscience supplies all its peptides as freeze
dried materials and these can be regarded as
stable compounds for shipping purposes. For
long term storage however, it is recommended
to store them in a deep freeze at -20°C. When
taking them out of the freezer, it is important
to allow the bottles/vials to warm up to room
temperature before opening the container. This
is because peptides are often hygroscopic and it
avoids condensation of atmospheric water on the
peptide.
Peptides in solution can degrade, primarily due
to oxidation of Cys, Met and Trp residues but
they are also susceptible to attack by microbes,
so it is advised to store solutions at -20°C when
not in use. It is difficult to predict the storage
life of a peptide as it is highly dependent on its
amino acid content and sequence.
Dissolving peptides
This can be a very difficult operation.
*
Always try to use volatile materials such as
dilute acetic acid and ammonia solutions
when first dissolving an unknown peptide. If
everything fails, the buffers can be removed
by lyophilisation and the dissolution attempted
again.
*
If the peptide is acidic, i.e. contains more
Asp and Glu residues than His, Lys or Arg,
then first attempt to dissolve the peptide in
dilute ammonia solution, e.g. 0.5%
ammonium hydroxide. Do not use this
method if your peptide has disulphide
bridges, the high pH may cause them to
unfold.
*
If the peptide is basic, i.e. contains and
excess of His, Lys and Arg groups, then
try and dissolve the peptide in something like
10% acetic acid.
*
DMSO is a very good solvent and has the
advantage of being tolerated by cells, it is
 however difficult to remove by drying. Add a References
small amount of a high purity grade DMSO
to the stock peptide solution until it dissolves. The original paper
Once dissolved, water or buffer solution can 1. Merrifield R. B. ‘Solid Phase Peptide
be added very slowly to dilute the DMSO Synthesis’. J. Am. Chem. Soc. 85, 2149 (1963)
content. Stop the water addition if the peptide
starts to precipitate out. DMSO isn’t suitable Recent reviews
for peptides containing single Cys as it
2. Cheng W., White P. D. ‘Fmoc Solid Phase
promotes disulphide bridge formation.
Peptide Synthesis: A Practical Approach’ Oxford
*
Gentle warming and sonication are University Press, 2000
useful tactics in getting peptides to dissolve.
3. Albericio F,. Kates S. A. ‘Solid-Phase
*
Peptides originating from the transmembrane
Synthesis: A Practical Guide’ CRC Press, 2000
regions of proteins will certainly be difficult to
dissolve.

© Copyright by Alta Bioscience

July 2009
Reproduction forbidden without permission

Alta Bioscience is a leading manufacturing laboratory providing analysis and synthesis of DNA, proteins and other biochemical
molecules to clients world-wide. Founded in 1973 at the University of Birmingham, England, we offer a well established and
comprehensive range of synthetic, sequencing and analytical methodologies, which are available to academia and commercial
clients. The following internationally recognised accreditations position Alta Bioscience amongst the few laboratories world-
wide working to such high standards. ISO 9001:2008 Quality management system for the laboratory as a whole, and ISO
17025:2005 Technical competence in amino acid analysis and protein sequencing. The Investors in People accreditation
reflects our commitment to staff development.
This publication is one of a series presenting answers to questions frequently asked by established researchers, as well as
those new to their field. Should you have a question which is not dealt with, or if you find an item lacking clarity, we invite you to
bring it to our attention by sending an email to altabios@bham.ac.uk

Page 64 An Introduction to Synthetic Peptides