Bioconjugates

Prof. Ferenc Hudecz

Prelude 1) Establishment of protein structure – function

relationship (1925 –

� SolubilitySumner, 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) necessaryfor the protein functionOlcott, 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

� Sequencing (1956)

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

� Aminosav meghatározás (1960)Moore, S., Stein, W.H.: Chromatographic determination of amino acids …

Methods in Enzymol 6 819 (1963)

F

O2N NO2

N

SO2Cl

CH3CH3

λ = 254 nm λs = 360 nm

λe = 480 nm

NC

Sop.: -21 oC O

O

OH

OH

CHO

CHO

λs = 340 nm

λe = 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

β-D-Mannóz részlet C - C kötés hasadása

poliszacharid láncban aldehid részlet oxidálásávalNaIO4

10 mM Sodium periodate

β-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+

NHI

COO-

NH3

+

� Hnatowich, D.J. et al.: The preparation and labeling of DTPA-coupled albumin Int J Appl Radiat Isot 33327-332 (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’-carbonyldiimidazolwhich 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 components2. Covalent linkage

� Approaches1. 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, anhydridebond „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 lightRed lightGreen to red light

D-Luciferin (LH2) Luciferyl-AMP)

Oxyluciferin (keto) Oxyluciferin (enol)

CO2

+ AMP

+ PPi + H+

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

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 chainDTT

B chain A chain

Toxin with A and B subunits

Reduction, free SH groups

Immunotoxin formation with disulphide bridge

N

S

Pyridine-2-thioneλ = 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

Maptioesther 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?