Biokonjugátumok/

Bioconjugates
Hudecz Ferenc
 Dr. Hudecz Ferenc
ferenc.hudecz@ttk.elte.hu
MSc, PhD, MSc + PhD
II. félévi tanrendjében szereplő hetek
Hónap Hétfő Kedd Szerda Csütörtök Péntek Szombat Vasárnap
2023 március 2
9
16
23
30
április 6
13
20
27
május 4
11
18
25
június 1
8
 Biokonjugátumok: szintézis, jellemzés, alkalmazás; Tárgy kód: bioknjk18em
03.02 03.09 03.16 03.23 03.30 04.06 04.13 04.20 04.27 05.04 05.11 05.18 05.25 06.01 06.08 E-mail

H4LY
Bakos Anna
XZ
Emődi D8N
Nikolett VZY
Jenőfalvi INDT
Ádám HI
GY6K
Lukács Anna
V2
Mogyorósi SJV4
Petra P1
Várnagy G0EF
Erzsébet A1

Bioconjugates Code: 223/KÉM

Basa Bettina CT67
5R
USR5
Ilyés Kinga 4K
Pavela FU7D
Olivér FZ
Sármezey BJX2
Bence CN
András
Salman Saif EPX1
Qahtan YP

MSc órák (magyarul): 03.02, 03.16, 03.23, 04.06, 04.20, 05.04, 05.11 beszámolók: 05.25, 06.01
PhD órák (angolul, English) 03.02, 03.09, 03.30, 04.06, 04.13, 04.27, 05.18 beszámolók: 06.08
Conditions for MSc students
 Conditions for PhD students

1. 5-7 lectures plus substantial homework

2. Participation at lectures > 70 %

3. Examination: 20 min presentation based on literature

Website:
http://szerves.chem.elte.hu/oktatas/ea/Hudecz/index.htm#biokonjugatumok

Email: ferenc.hudecz@ttk.elte.hu
 References
1. Kalia J, Raines RT. Advances in Bioconjugation. Curr Org Chem. 2010
Jan; 14(2): 138–147

2. Aslam M, Dent A. Bioconjugation: Protein Coupling Techniques for the

Biomedical Sciences. London: Macmillan Reference Ltd; 1998.

3. Lundblad RL. Chemical Reagents for Protein Modification. 3rd ed. Boca
Raton, FL: CRC Press; 2005.

4. Hermanson GT. Bioconjugate Techniques. 3rd ed. San Diego, CA: Elsevier
inc; 2013.

5. Bioconjugation Protocols (Ed.: Mark, S.S.) – Methods in Molecular

Biology, Springer, 2011

6. Narain R. Chemistry of Bioconjugates: Synthesis, Characterization, and

Biomedical Applications, J. Wiley, 2014

Bioconjugate Chermistry, American Chemical Society, since 1990

 Outline: Protein bioconjugates
1. Historical background

2. Functional groups of proteins/glycoproteins

N-nucleophiles: -NH2, imidazole, indole, guanidino
S-nucleophiles: -SH, CH2-S-CH3
O-nucleophile: -OH
O/C-nucleophiles: -CHO, -COOH, -CONH2
3. Creation of reactive groups Introduction
- Limited reactivity (eg. – OH vs. - CHO)
- Improved selectivity (e.g. - NH2 vs. - SH)
- Space considerations Transformation
- Convenient chemistry (e.g. - COOH vs. - NH2)

4. Detection of reactive groups Destructive

sensitive
quantitative
quick Non-destructive
small sample
5. Conjugation
- Chemical synthesis
- Enzymatic synthesis (e.g. - NH2 vs. - SH)
- Gene technology
6. Analysis of conjugates
Purification
Structure determination
 Introduction
Design of bioconjugates

Why? Synthetic antigens or drug targeting

What? Peptide epitope, drug, reporter molecules

With What? Protein, DNA, liposome

How? Covalent bond

 Introduction
Application of bioconjugates: immunoassays
a) competitive

b) direct
 Introduction
Application of bioconjugates: histo- and cytochemistry
 Introduction
Application of bioconjugates
FACS analysis Western blotting
 Introduction

Application of bioconjugates: DNA detection

Ionisation status of proteins as function of pH
Ionisation of aspartic acid
Reactivity and transformation of amino group
Reactivity and transformation of hydroxyl group
Reactivity and transformation of thiol group
 Historical background
1) Establishment of protein structure – function
relationship (1925 –

– Solubility
Sumner, J.B., Graham V.A.: The nature of insoluble
crease Proc Soc Exp Biol Med 22 504 (1925)

– Identification of amino acid side chain(s) necessary

for the protein function
Olcott, H.S., Fraenkel-Conrat, H.:
Specific group reagents for proteins Chem Rev 41 151 (1947)
Herriot, R.M.:
Reactions of native proteins with chemical reagents
Adv Prot Chem 3 161 (1947)
2) Structural studies
– Proteins
• Determination of N-terminal amino acid
H3C CH3
N
F
 = 254 nm s = 360 nm

O 2N NO2 e = 480 nm
SO2Cl
• Sequencing (1956)
Edman, P., Begg, G.: A protein sequenator Eur J Biochem 1 80 (1967)
N O
C o
op.: -21 C
S OH
OH
• Determination of amino acid composition O

(1960) CHO
s = 340 nm
Moore, S., Stein, W.H.: e = 455 nm
Chromatographic determination of amino acids …
Methods in Enzymol 6 819 (1963)
CHO
Determination of the amino acid sequence
1. Determination of the N-terminal
1.1. Sanger reaction

Nobel prize (2),

1958 (insulin) and
1980 (oligonucleotide sequencing
F. Sanger, P. Berg, W. Gilbert)

F. Sanger (1918 - 2013 )

NO 2 NO2
2 F NaHCO3 NH C H CO -

4
+ H2N CH CO -
R
+ HF
O 2N R O 2N

1-fluor-2,4-dinitro benzol

NO 2 NO 2
6 M HCl NH CH COOH
NH CH CO -

R R

O 2N O 2N + amino acids

2,4-dinitrophenyl protein 2,4-dinitrofenil amino acid

=254 nm

Side reaction: Lys e-amino group

1.2. 1-dimetilamino-naftalin-5-szulfonil chlorid (Dansyl-klorid, Hartley, 1963)

H3C CH3
N

+ NH2-L-K-A-D-P-N-R-F-G-A-D-L-COOH
*
O S O
H3C CH3
Cl N
base
pH 7,5-8,0

O S O +HCl
NH-L-K-A-D-P-N-R-F-G-A-D-L-COOH
*

H3C CH3
N
6 M HCl
hydrolisis

O S O

NH-Leu-COOH + K, A, D, P, N, R, F, G-COOH
*
(Dansyl-amino acid)  g=360 nm
 e=480 nm
* Side reaction: Lys e-amino group
2. Determination of the C-terminal
NH2-L-K-A-D-P-N-R-F-G-A-D-L-COOH

H2N - NH2
+ 90o C
20 -100 hrs
+NH -L – CO -NH-NH2,
3
+NH K-CO-NH-NH2.....+NH3-L-COO-
3-

3. Edman degradation (P. Edman, Lund, 1950)

1. step: Reaction with phenylizotiocianate (addition)

N=C=S + NH2 - CH – CO -.....

pH 8-9, 40 oC R

S
II
NH - C - NH - CH – CO -.....
phenylthiocarbamoyl
R derivative
2. step: Hydrolysis (H+/TFA)

PTH
amino acid
3. step: Identification of PTH amino acids
 4. Determination of amino acid composition

1. step - Hydrolysis O O

H2O / 6M HCl
NH2-CH2- C NH2-CH2- C + NH2-CH-COOH
105 oC

NH-CH-COOH OH CH3
Gly-Ala Gly
CH3 Ala

2. step – Separation, derivatization

Ion exchange (cloumn) chromatography, Rockefeller Institute, 1950

Resin: water insoluble polymer with SO3- groups
Detection: nynhidrine reaction ,  = 570 nm
3. step – Derivatization and separation

Derivatizations: o-phtalaldehyde
Separation: HPLC,
Detection: g = 360 nm e = 455 nm

Hunkapiller et al. Science, 1984

 Carbohydrates
 E. Fischer (1884) Reducing monosaccharides

O NH
H H2N
N NH
OH H
fenil-hidrazin
HO OH
OH HO
HO
phenyl-hydrazine
OH
HO
HO

HO

D-Glükóz D-glucose-1-phenylhydrazone
Glükóz-1-fenilhidrazon

NH
H2N

N NH phenyl-hydrazine
fenil-hidrazin
H
N
HO NH
OH
HO
Glükóz fenil-oszazon
D-glucose–bis–phenylhydrazone (osazone)
HO
  Carbohydrates
 Periodate oxidation to form oxo-function

HO HO
O
10 mM
10 mM Nátrium-perjodát
Sodium periodate O
O O O O

HO OH O O

-D-Mannóz részlet CC-–CC kötés hasadása

bond cleavage and
-D-mannosyl unit NaIO4
of poliszacharid
a polysaccaride chain
láncban oxidation
aldehid of the
részlet OH groups
oxidálásával

oxo-group from (secondary) hydroxyl-function

 Reaction with N - nucleophile
(Nuleophilic addition + elimination)

Reaction with phenyl-hydrazine

CHO CH N-NHC6H5 CH N-NHC6H5

H OH H OH C N-NHC6H5
C6H5NHNH2 2 C6H5NHNH2 *
HO H HO H HO H
HOAc, HOAc
H OH
- H DO
H OH H OH
2 - H2O
H OH H OH H OH
CH2OH CH2OH CH2OH

+ C6H5NH2 + NH3 + 2 H2O

D-glucose D-glucose D-glucose

phenyl-hydrazone phenylosazon
(yellow)
*the 2nd phenyl-hydrazine reduce
 Nucleic acids
 Maxam, A.M., Gilbert, W … et al. A new method for sequencing DNA
PNAS 74 560-564 (1977)

NH2 O
6 7 6 7
5 N 5
1 N N
1 HN
8 8
2 2
N 4 N9 H2N N 4 N9
3 3
R R

(CH3)2SO4

NH2 O CH3
+
N N a) 0.1 M HCl
N HN
b) pH 7, 100 oC
+ N N
N H2N N
R R
CH3

 Sanger, F. et al. PNAS 74 5463 (1977)

 Smith, L.M. et al. Nature 321 674 (1986)
 Nucleic acids
 Sanger, F. et al. PNAS 74 5463 (1977)
Didezoxy nucleotide incorporation
a) Radioactive isotope (32P)
32
P P O P P P O
O B O B
H H H H
H H 32P
H H
H H H H

b) Fluorophore (labelling,
L. E. Hood, 1986)
 Structure of ChromaTide fluorescein-12-dUTP
(C-7604)
HO O O

OH

O O
O NH
NH (CH2)5
H
N O

O N H
O O O
O P O P O P OH
O - - -
H H O O O
H H +
3 Na
H OH
2) Detection of biopolymers in cell (tissue)
 Labelling proteins with radioactive isotope
 Li, C.H.: Iodination of tyrosine groups in serum albumin and pepsin
JACS 67 1065 (1945)

I131 I3- I 2 + I-

I2 + H2O H2OI+ + I-

Tyr His

COO- COO-
+
H3N +
NH3

+
HN

I N
I I H
- -
I
O O
  Hnatowich, D.J. et al.:
The preparation and labeling of DTPA-coupled albumin Int J Appl Radiat
Isot 33 327-332 (1982)

O O R NH2 -
O O
amin tartalmú molekula
-
O O O O
N + N N N
O NH O +
NH
O R O -
NH O O O
-
DTPA
O -
O
 Labelling of proteins with biotin
 Bayer, E.A., Wilehek, M.: The use of avidin-biotin complex
Methods Biochem Anal 26 1 (1980)
 Chaiet, L., Wolf, F.J.: The properties of streptavidin, a biotin-
binding protein produced by streptomyces Arch Biochem
Biophys 106 1 (1964)
 Green, N.M.: Avidin Adv Protein Chem 29 85 (1975)
O
2 Ka = 1015 M-1
HN NH
op.: 232-233 oC
m.p.
4
*
S COOH

D-Biotin
[Hexahidro-2-oxo-1H-tieno[3,4-d]imidazol-4-pentánsav]

Hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid; (+)-Biotin

  Labelling of biopolymers with fluorophore
McKinney, R. et al.
Factors affecting the rate of reaction of fluorescein isothiocyanat
with serum proteins J Immunol 93 232 (1964)

-
- O O O
O O O

R NH2
+ O
-
amin tartalmú molekula O

- O
O

amino functional group

HN S
N C
C
S HN
R

Fluoreszcein izotiocianát Tiourea kötés létrehozása

fluorescein isothiocyanate thiourea linkage

2) Affinity chromatography
 Bethell, G.S. et al. A novel method of activation of cross-
linked agarose with 1,1’-carbonyldiimidazol which gives a
matrix for affinity chromatography
J Biol Chem 254 2572 (1979)

CH2OH O
HO
O O O
O O O C NH
O
OH O
CH2 NH
L NH2
CNBr O C NH
CHOH
L
CH2
O
O C N
CH2 O
HO
O O
O O OH
O
OH
 Analysis
(chemical, medical)
Drug research

Proteins:
structure - function

Separation science

Structural
studies Definitions

1. Two or more active components

2. Covalent linkage
 Approaches
1. Size of the partners
 Small – small
 Small – big
 Big – big
2. Type of linkage
 Direct NH2 HOOC
 Indirect

NH2 HOOC

spacer
3. Type of linkage
-
O O O
C NH S NH O P NH
 Acid amide O O

carboxylic acid sulphonic acid phosphoric acid amid

-
O O
 Acid ester C O O P O
O CH3

carboxylic acid phosphoric acid

O O O O O O
 Acid anhydride C C
HO
P P C P
O O O
OH OH

carboxylic acid phosphoric acid mixed

 Hydrazone
O N
C NH
Aldehyde + hydrazine

 Schiff base
C N
Aldehyde + amine
H

 Ether CH2
O
CH2
Alcohol
 Carbamate
O NH
 CH2 C CH2 Alcohol
O

CH2

 Secunder amine NH CH2 Amine

+ aldehyde
(N-glycoside) NH
CH2
+ aryl-halogenide

Isourea
NH NH
 CH2 C CH2
Amine
O

 Isothiourea NH NH
CH2 C CH2
Amine
S
 Thioether
CH2

S CH2 Thiol

S
CH2

 Thioester O
C Thiol
S CH2

 Disulphide CH2
S
S
CH2
Thiol
S CH2
S

 Diazo N
N
Azide

 C–X C I
1. Example: small–small, direct, anhydride bond
„Firefly” luminescence

©nationalgeographic.com,
Radim Schreiber

adenylic acid +

„carboxylic acid” mixed anhydride

 + PPi + H+

D-Luciferin (LH2) Luciferyl-AMP)

CO2

dioxetanone + AMP
oxyluciferin (keto) oxyluciferin (enol)

Green to red light Red light Yellow-Green light

Step a: formation of the enzyme-bound luciferyl adenylate;

Step b: a proton is abstracted from the C-4 carbon of the adenylate by
a basic side chain amino acid residue of luciferase;
Step c: molecular oxygen adds to the newly formed anion;
Step d: a highly reactive dioxetanone intermediate is formed;
Step e: an electronically excited state oxyluciferin molecule, CO2 and red light
emission (λmax = 615 nm) are produced (at pH 6);
Step f: the enolate dianion formed by tautomerization from the exited form of
oxyluciferin and yellow-green light emission (λmax = 560 nm) occurs at pH 8.
http://www.photobiology.info/Branchini2.html
2. Example: small – big, indirect, amide bond
Liposome – hapten conjugate

+
NH2 NH3
-
O
O P O
H3C O O
O CH3
O
O
NH2

phosphatidylethanolamine
PE liposzóma Foszfatidil etanolamin
PE liposome
2. Example
+
NH3
O
- tetrapeptide
O P O
++ HOOC
HOOC-GAFA
GAFA O
H3C O O NH
O CH3 CH3
O
O CH3
+
N NH
EDC C CH3
N
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
1-etil-3-(3-dimetil-aminopropil)karbodiimid
H3C
O

NH CO GAFA NH CH3
-
O
O P O
H3C O O + EDC HOH
O CH3
O
NH CO GAFA NHAc
O

NH2

NH CO GAFA NHAc
3. Example: big–big, indirect, disulphide bond
Immunotoxin conjugate

1. step: introduction of protected -SH group(s) of antibody (Ab + SPDP →

amide bond

2. step: creation of free -SH groups of the toxin subunits (cleavage of the
-S-S- bond, DTT)

3. step: free –SH partner reacts with the component having „protected”
SH function (AB-SSP + Toxin-SH → disulphid linkage)
3. Example
 Carlsson, J. et al. Biochem J 173 723 (1978)

N O
+ SPDP aktivált antitest
NH2 O O S N O NH
SPDP modified Ab
S

N OH O S N
Antibodyamino
withcsoportot
free NH 2 group(s)
tartalmazó SPDP

)
S

Cleland
 antitest W. Biochemistry 3 480 (1964
O NHS

A chain + S
DTT

Pyridine-2-thione
Reduction,  = 343 nm
B chain free SH groups B chain A chain

Toxin with
A and B subunits
NH

O S
Immunotoxin formation S

with disulphide bridge

 O O
O

Map
R R
R
S R1 NH R1
O R1 (C, P)
tioesther amide
esther O R3 O
R
R4
NH
OH O R2 O
O
O R4
R
R O O ether carbamate
O
N3
R
R1 NH NH2
R CH2
anhydride hydrazide azide
N N R3 OH
O
oxidation CH2
R diazo
NH N
hydrazone R2
N
O reduction
R2
R2 H
H Schiff base
 Map
R (Ar) R5 NH
R5 NH R
O

secunder amine amide (SO, POH)

R5 NH2 R6 SH

R5 NH R5 NH
R5 N R6 R6 S
NH NH
CH R6 S S R
S R
O R
Schiff-base isourea isothiourea tioether disulphid bridge

1) Introduction of functional groups ? (-COOH, -CHO, -OH, -SH, -NH2)

2) Elimination of functional groups?
 Guidelines for the analysis of papers

1. Description of the scientific problem:

what is the focus of the authors (first or last)?
Aims (short and long term)
Strategy, Tactic

2. What? With what? Why? How?

3. Synthesis of the conjugates

Functional groups involved,
Principle of conjugation, type of reaction, equation of the reaction
Method, coupling reagent
Conditions of the reaction, explanation/interpretation
(e.g. pH, ionic strength, solvent, reaction time, temperature,
metal ion)

4. Analysis Purification methods (principle, reason)

Analysis of homogeneity
Chemical composition, structure determination, identification
Functional analysis of the components of the conjugate
Methods
Long term stability
5. Scientific novelty described in the paper

6. Critical remarks
Suitability of the strategy?
Appropriateness of the synthesis method?
Reproducibility of the synthesis described?

Suitability of the purification methods?

The presence of the product was evidenced?
Side products?

Suitability of the analytical methods?

Are the conclusions correct?

Is the novelty of the results clear?

Structure, composition, clarity of writing, language?

February, 2023 Dr. Ferenc Hudecz