Bioconjugates: an introduction

Ferenc Hudecz1,2

1Department of Organic Chemistry, 2Research Group of Peptide Chemistry

Hungarian Academy of Sciences, Eötvös Loránd University Budapest

Conditions

1. 5-7 lectures plus substantial homework

2. Participation at lectures > 70 %

3. Examination: 25-30 min presentation based on literature Website: http://szerves.chem.elte.hu/oktatas/ea/Hudecz/index.htm#biokonjugatumok Email: fhudecz@elte.hu

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 - Limited reactivity (eg. – OH vs. - CHO) - Improved selectivity (e.g. - NH2 vs. - SH)

- Space considerations - Convenient chemistry (e.g. - COOH vs. - NH2)

4. Detection of reactive groups sensitive quantitative quick small sample

5. Conjugation - Chemical synthesis - Enzymatic synthesis (e.g. - NH2 vs. - SH)

- Gene technology

6. Analysis of conjugates Purification Structure determination

Introduction

Transformation

Destructive

Non-destructive

Design of bioconjugates

Why?

What?

With What?

How?

Synthetic antigens or drug targeting

Peptide epitope, drug, reporter molecules

Protein, DNA, liposome

Covalent bond

Introduction

Application of bioconjugates: immunoassays

a) competitive

b) direct

Introduction

Application of bioconjugates: histo- and cytochemistry

Introduction

Application of bioconjugates

Western blotting FACS analysis

Introduction

Application of bioconjugates: DNA detection

Introduction

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

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)

Historical background

2) Structural studies Proteins

Determination of N-terminal amino acid

Sequencing (1956)

Edman, P., Begg, G.: A protein sequenator Eur J Biochem 1 80 (1967)

Determination of amino acid composition

(1960) Moore, S., Stein, W.H.: Chromatographic determination of amino acids … Methods in Enzymol 6 819 (1963)

F

O2N NO2

N

SO2Cl

CH3CH3

= 254 nm s = 360 nm

e = 480 nm

NC

Sop.: -21 oC

O

O

OH

OH

CHO

CHO

s = 340 nm

e = 455 nm

Carbohydrates E. Fischer (1884) Reducing monosaccharides

O

OH

H

OH

OH

OH

OH

NHNH2

fenil-hidrazin

N

OH

H

OH

OH

OH

OH

NH

D-Glükóz Glükóz-1-fenilhidrazon

NHNH2

fenil-hidrazinN

OH

H

OH

OH

OH

N

NH

NH

Glükóz fenil-oszazon

phenyl-hydrazine

D-glucose-1-phenylhydrazone

phenyl-hydrazine

D-glucose–bis–phenylhydrazone (osazone)

Carbohydrates Periodate oxidation to form oxo-function

oxo-group from (secondary) hydroxyl-function

O

OH

O

OH OH

O

O

OH

O O

O O

10 mM Nátrium-perjodát

-D-Mannóz részlet C - C kötés hasadása

poliszacharid láncban aldehid részlet oxidálásávalNaIO4

10 mM Sodium periodate

-D-mannosyl unit of a polysaccaride chain

C – C bond cleavage and oxidation of the OH groups

Nucleic acids Maxam, A.M., Gilbert, W … et al. A new method for

sequencing DNA PNAS 74 560-564 (1977)

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

Smith, L.M. et al. Nature 321 674 (1986)

N

N

N

N

NH2

R

1

2

3

4

56 7

8

9

1

2

3

4

56 7

8

9N

NH

N

N

NH2

O

R

N+

N

N

N

NH2

RCH3

N

NH

N

N+

NH2

O

R

CH3

(CH3)2SO4

a) 0.1 M HCl

b) pH 7, 100 oC

N

N

O

H

O H

NH (CH2)5

O

NH

O

O

H

H

OH

H

H

H

O O

O-

O

P O

O-

O

P OH

O-

O

P

O OOH

O

OH

3 Na+

Structure of ChromaTide fluorescein-12-dUTP

(C-7604)

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 + H

2O H

2OI+ + I-

Tyr His

I

O-

COO-

NH3

+

I

O-

I

NH+

NH

I

COO-

NH3

+

Hnatowich, D.J. et al.:

The preparation and labeling of DTPA-coupled albumin Int J Appl Radiat Isot 33 327-332 (1982)

N

O

NH+ N

O

O

O

O

O

O-

O

NNH

+ N

O-

O

NH OR

O

O-

O-

O

O-

O

R NH2

amin tartalmú molekula

DTPA

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)

NH NH

S

O

COOH

2

4*

D-Biotin

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

Ka = 1015 M-1

op.: 232-233 oC

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

m.p.

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)

OO-

O

NC

S

O-

O

R NH2

amin tartalmú molekula+

OO-

O

NHC

S

O-

O

NHR

Fluoreszcein izotiocianát Tiourea kötés létrehozása

fluorescein isothiocyanate thiourea linkage

amino functional group

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)

O

OH

OH

OO

CH2OH

O

O

O

O

CH2

CHOH

CH2

O

CH2

O

OH

OH

OOO

O

O

OH

CNBr

C NH

O

O

C NO

L NH2C NHO

NH

L

Drug research

Analysis (chemical, medical)

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

Acid amide

Acid ester

Acid anhydride

C NH

O

S NH

O

O

O P NH

O-

O

O P O

O-

CH3O

C O

O

carboxylic acid sulphonic acid phosphoric acid amid

carboxylic acid phosphoric acid

CO

O

C

O

PO

O

P

OOH

OH

CO

O

P

O

OH

carboxylic acid phosphoric acid mixed

Hydrazone

Schiff base

Ether

C NH

O NAldehyde + hydrazine Aldehyde + amine Alcohol

NC

H

CH2

OCH2

Carbamate

Secunder amine (N-glycoside)

Isourea

Isothiourea

CH2

OC

NH

O

CH2 Alcohol Amine + aldehyde + aryl-halogenide Amine Amine

CH2

NH CH2

NHCH2

CH2

NHC

NH

O

CH2

CH2

NHC

NH

S

CH2

Thioether

Thioester

Disulphide

Diazo

C – X

Thiol Thiol Thiol Azide

CH2

S CH2

SCH2

C

S CH2

O

CH2S

SCH2

SS

CH2

NN

C I

adenylic acid +

„carboxylic acid” mixed anhydride

1. Example: small–small, direct, anhydride bond „Firefly” luminescence

©nationalgeographic.com,

Radim Schreiber

http://www.photobiology.info/Branchini2.html

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.

Yellow-Green light Red light Green to red light

D-Luciferin (LH2) Luciferyl-AMP)

oxyluciferin (keto) oxyluciferin (enol)

CO2

+ AMP

+ PPi + H+

dioxetanone

2. Example: small – big, indirect, amide bond

Liposome – hapten conjugate

NH2

NH2

O

O

O

O

CH3

O

CH3

O

O-

O

P

NH3

+

PE liposzóma Foszfatidil etanolaminphosphatidylethanolamine

PE liposome

2. Example

O

O

O

O

CH3

O

CH3

O

O-

O

P

NH3

+

+ HOOC GAFA

NH

CH3

O

NC

N

CH3

NH+

CH3

CH3

EDC

1-etil-3-(3-dimetil-aminopropil)karbodiimid

O

O

O

O

CH3

O

CH3

O

O-

O

P

NH NH CH3

O

GAFACO

+ EDC HOH

NH CO GAFA NHAc

NH CO GAFA NHAc

NH2

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

HOOC-GAFA +

tetrapeptide

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)

Cleland W. Biochemistry 3 480 (1964)

NH2

+ N O

O S

S

N

O

O

N OH

O

O

NH

O S

S

Namino csoportot tartalmazó SPDP

antitest NHS

SPDP aktivált antitest

NH

O S

S

B chain

A chain DTT

B chain A chain

Toxin with A and B subunits

Reduction, free SH groups

Immunotoxin formation with disulphide bridge

N

S

Pyridine-2-thione = 343 nm

Antibody with free NH2 group(s)

SPDP modified Ab

+

R

O

O

R1 (C, P)

R

S

O

R1

R

NH

O

R1

R

O

O

R1

O

R

NH

O

NH2

R

N3

O

R

NH

O

N

R2

R

OH

O

R2

H

O

R2

H

N

N N

CH2

CH2R

R3 OH

R2 O

R4NH

R4

OR3

O

esther

anhydride hydrazide azide

hydrazone

diazo

Schiff base

ether carbamate

reduction

oxidation

Map tioesther amide

R5 NH2

R5 NH

R (Ar)

R

NHR5

O

CH

NR5NH

R

NHR5

O

NH

R

NHR5

S

Map

Schiff-base isourea isothiourea

secunder amine amide (SO, POH)

R6 SH

R6 S

R6

S R

SR6

tioether disulphid bridge

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

2) Elimination of functional groups?