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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?
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