Strategies of bioconjugation Homobifunkciós Homobifunctional
„Zero-length”
Heterobifunkciós Heterobifunctional
Identical functions Azonos funkciós csoportok
Nincs No beépülés insertiona két komponens közé
Different functions Különböző funkciós csoportok
1. Carbodiimide 1. Karbodiimid 1. -NH2
H2N-
2. -SH
HS-
3. -COOH
HOOC-
4. -CHO
OHC-
5. -NH-NH2
H2N-NH-
N
C
N
1. –NH2
2.2.Woodward’ reagens Woodward's reagent k CH C
Diimidazol 3.3.Diimidazole N
N O
2.
HSO
C
HS-
3. 3. Fotoreaktív Photoreactive -NH2
N
6.
4. Oxidising agents 4. Oxidálószerek -SH + HS-
7. Nem-kovalens
5. Enzymes 5. Enzimek
-SH -COOH -CHO 4. Photoaffinity labeling 4. Fotoaffinitás
3. Heterobifunctional reagents
Approach:
selectivity vs. specificity differences between and → two steps
3.1. amino- and thiol-groups 3.2. carboxyl- and thiol/amino-groups 3.3. carbonyl- and thiol-groups 3.4. light sensitive (photoreactive) reagents
a
O
3.1. Connecting amino- and thiol-functions O
N
O
H2N S
S
O
O
O
R R
N
NH
SPDP
S
S
+
N
N O
AMID
HS
R1
O DISZULFID
R
NH
S
+
S
S
N
R1
b O
O O
N
O
O
H O
N
H2N
R
DMF
NH
N
O
O
O H = H,
SMCC;
-
+
O R
HS
pH 7
R1
O O
O
O N
N
O R
O O
O m-Maleimidobenzoil-N-hidroxiszukcinimid m-Maleimidobenzolészter [MBS] (1981)
N-hydroxysuccinimide ester (MBS), 1981
NH TIOÉTER
N O
N O
SO3-Na+
Yoshitake, S. et al. 101 395 (1979)
OH
S
R1
OH
c
O O
O NH
N O
O I
H2N
O
O
R
NH R
SIAB
I
NH
AMID HS
Weltman, JK et al. Biotechniques 1 148 (1983)
R1 O
O NH O O2N
O
R I
jódecetsav-p-nitrofenilészter
iodoacetic acid-p-nitrophenyl ester (1975) 1975
NH TIOÉTER
amide
thioether
S
R1
a
3.2. Connecting carboxyl and amino/thiol-functions O 2N
HOOC
O +
O
R
-
N
O 2N
O
N
O
O
R
+
N
N
O
diazoecetsav-nitrofenilészter diazoacetic acid-p-nitrophenyl ester SH/ H2N O R1
O
NH
R1
R
O
amid amide
észter ester
b N
S S
O
-
+
O
N
N
HOOC
N
R
S
O
S
O
O
O R
piridil-2,2'-ditiobenzildiazoecetsav pyridyl-2,2'-dithiobenzyl diazoacetate
HS R1
R1
S S
O O
O
O R
disulphide diszulfid
ester észter
3.3. Connecting carbonyl and thiol-functions
a
R N
S
S
NH
NH2
O 3-(2-piridilditio)propionsav hidrazid
SH
R
S
S
NH
0.1 M Na-acetát
0.1 M Na acetate pH pH5.5 5.5
+ N
S
O O
3-(2-pyridyldithio)propionic acid hydrazide [PDPH]
R1 H
Zara JJ. Anal Biochem 194 156-162 (1991) R
S
S
NH O
diszulfid
disulphide
b
NH2
O
N
R1
hidrazon hydrazone
O S
R N
R
N
SH
O
O 4-(4-N-maleimidofenil)butánsav hidrazid
O
O
HN
O
NH2
4-(4-N-Maleimidophenyl)butyric acid hydrazide
R1
HN NH2
H O R
S N
Chamov SM. Et al. J Biol Chem 267 15916 (1992)
R1 N
thioether tioéter
NH
O O
hydrazone hidrazon
Photoactive groups
N
O
N2 +
N
-
N
Cl
CF3 O
hν
hν
hν
O N Cl
nitrene nitrén 1. 2. 3.
Alkyl azide: UV excitation Aryl azide: λ = 300 – 400 nm Acyl azide: reactive in dark too
C
C CF3
triplett keton
O karbén
carbene
triplett keton
A.
Activation of azide function and its transformation -
N +
N
N
N fenilazid
R
nitrén képzõdés
NH
R
nitrene
phenyl azide
R
H
NH2
aktív hidrogén beépülés active(C-H) hydrogen (C-H)
ringgyûrûtagszám expansion
NH R
növekedés
NH aktív hidrogén (N-H) beépülés
N
dehydroazepine dehidroazepin intermedier intermedier R
NH2
nukleofil nucleophile
R
R
active hydrogen (N-H)
N
H
reaktív hidrogén reactive hydrogen
addíciós reakció
addition N
N
NH R
R
R
NH2 O N3
N3
N3
4,4'-diazidobifenil [DABP]
O
N
O
4-azildobenzoesav N-hidroxiszukcinimid észter HSAB] 4-Azidobenzoic acid[NHS-ABA, N-hydroxysuccinimide ester O
4,4’-diazodo biphenyl
Mikkelsen, RB et al. J Biol Chem 251 7413 (1976)
Ji, T. Anal Biochem 121 286 (1982) SH
CHO O O N
N3 H2N
4
N3
NH
p-azidobenzoesav hidrazid [ABH]
p-azidobenzoic acid hydrazide
O 4-azidofenil-maleimid [APM]
4-Azidophenyl maleimide
Trommer WE et al. Hoppe-Seyleis Z Physiol Chem 356 1455 (1975)
-COOH HO N NH
H2N
O
ASBA
N
+
N
-
N
photo activation fotoaktiválás ring expansion gyûrûtágulással
OH
O HO
R
N
NH O
O
EDC
NH keresztkötött molekula
R
N R
OH karboxil csoportot tartalmazó vegyület
UV fény
UV
O R
NH
nukleofilt tartalmazó vegyület
NH R
O
HO
N
amid kötés képzõdés
N
amide linkage
+ -
N
NH2
HO
NH
B.
Activation of diazo function and its transformation -
N
+
N
λ = 254 / 310 nm O O
N
O
R
F
O
+
R1
F F
NH O
carbene
-
2-diazo-3,3,3-trifluoropropionic acid 2-diazo-3,3,3-trifuorpropionsav p-nitrofenil észter p-nitrophenyl ester [PNP-DTP]
R
NH
R
F F F
keresztkötött molekula
C
F F
O F karbén képzõdés
NH2
primer amint tartalmazó vegyület UV fény H
-
N
OH
reaktív hidrogént tartalmazó vegyület reactive hydrogen
+
N O
+
N
O
-
p-nitrofenol
p- nitrophenol Chowdhry et al. PNAS 73 1406 (1976)
R
NH
F F O
R1
F
C.
Activation of function with benzophenone moiety O
R1
OH
O
O
N
N
benzofenon-4-maleimid benzophenone-4-maleimide
R
SH
tiol tartalmú molekula
R
O
O
O keresztkötött molekula
C
UV fény O
H
N
Walling C, Gibian MJ, JACS 87 3361 (1965)
S R
O
R1
reaktív hidrogént tartalmazó vegyület reactive hydrogen
O
tioéter kötés képzõdés thioether
S
Conjugates of radioactive isotopes
Biosynthetic approach
Chemical synthesis
15C, 3H, 14C, 32P, 35S
Direct e.g. modification of Tyr/ His residues
Indirect a) Covalent linkage b) Complex formation
Direct incorporation of radioactive isotope Example: 125I, 131I (17 iodo-, 13 bromo-, 6 chloro-, 2 fluoro-isotope t½ > 3 perc)
In vitro 125I long lifetme low energy photon emission (no particle) price 125I, 131I (β-), 123I (γ – 159 keV) In vivo „IMAGING” 18F (97% β+) X-ray or γ-emitting „Single photon emission tomography” (SPECT) „Positron emission tomography” (PET) γ-camera 131I (82Br) In vivo THERAPY Not optimal (high energy: danger)
Characterisctics of the most important radioactive isotopes Sensitivity: 131I > 125I > 14C, 32P, 35S, 3H Atomic number
Izotope
Half life
Decay
Highest particle energy (MeV)
γ-energy (MeV)
H
1
3H
12,33 y
β-
0,0186
-
C
6
11C 14C
20,4 min 5760 y
β+ β-
0,96 0,155
-
N
7
13N
10 min
β+
1,19
-
O
8
15O
2 min
β+
1,73
-
F
9
18F
109,8 min
β+
0,633
-
Na
11
24Na
15,02 h
β-, γ
1,392
2,754 1,369
P
15
32P
14,28 day
β-
1,710
-
S
16
35S
87,2 day
β-
0,167
-
K
19
40K 42K
1,28*109 y 12,36 h
β-, K β-, γ
1,31 3,52 (75%) 1,99 (25%)
1,46 1,525
Ca
20
45Ca
163 day
β-
0,257
-
Cr
24
51Cr
27,7 day
K, e-, γ
0,315 (e-)
0,320
Fe
26
52Fe
8,2 h 44,6 day
β+, γ β-, γ
0,8 1,566
0,5 1,30 1,10
59Fe
Atomic number Co Cu
27 29
Izotope
Half life
Decay
Highest particle energy (MeV)
γ-energy (MeV)
60Co
5,272 y
β-, γ
0,318
1,33 1,17
64Cu
12,74 h
β- (39%) β+ (19%) K (42%) γ (1%)
0,575 0,656 1,34
Ga
31
67Ga
78 h
γ
-
0,185 0,30
Kr
36
85Kr
10,73 y
β-, γ
0,687
0,514
Ru
37
81Rb
4,7 h
β+, γ
0,99
18,65 day
β-, γ
1,78
1,93 0,95 1,078
86Rb
Sr
38
90Sr
29 y
β-
0,546
-
It
39
90Y
64 h
β-, γ
2,29
1,761
Tc
43
99mTc
6,02 h
γ
-
0,140
In
49
111In
67,0 h
γ
-
1,658 h
γ
-
0,173 0,247 0,391
13,3 h 59,7 day 8,04 day
γ K, γ β-, γ
0,606 0,25 0,81
0,16 0,0355 0,364 0,080 0,723
113mIn
I
53
123I 125I 131I
Formation and decay of E
99
42Mo
99MTc
66.7 H e-99M
43Tc
6.03 H γ1
0.1427 0.1405
γ3
γ2 0.0
99
43Tc
2.12X105 Y
eStable 99 Ru 44
(Dillman, LT; Von der Lage, FC: Radionuclide decay schemes and nuclear parameters for use in radiation dose estimates (MIRD Pamphlet No. 10), Society of Nuclear Medicine, 1975)
α-decay [in biological medium: mm] A Y A-4 4 He X + Z Z-2 2 β-decay
e- emission 3
1H2
2He
neutron e- capture 40
3
proton + electron + antineutrino
19K
40
18Ar
electron + proton e+ emission 11
6C5
11
neutron 5B6
proton
+ e- + νe
γ-decay AY*
+ e+ + νe positron + neutrino
AY
+γ
1. Chloramine-T method Greewood, FC et al. Biochem J 89 114 (1963) Wilbur, DS Bioconjugate Chem 3 433 (1992) O H3C
O -
S
+
N
Cl
O
125 -
I
+
+
2H
H3C
(Na125I)
O
His
H3C
S
Tyr
NH +
N H
125
H3C
O
I 125
• 30 s – 30 min • water soluble • pH 7 phosphate 0.05 M
I
-
NH2
+
125
ICl
2. Immobilised chloramine-T (IODO-BEADS) U.S. Patent 4448764 és 4436718 3 mm
polystyrene polisztirol mátrix matrix
• 2 – 5 min • good protein recovery • mild conditions • pH 7.2 – 8.4
O CH
S
-
N
Cl
O
3. IODO-GEN
Fraker, PJ és Speck, JC BBRC 80 849 (1978) O
O Cl
N
N
Cl
Cl
+ Cl
N
N
Cl
O 1,3,5,6-tetraklór-3a,6a-difenilglicouril 1,3,5,6-tetrachloro-3a,6a-diphenyl glycoluril
125 -
I
(Na125I)
+
N
N
H
+
+
H
Cl
N
N O
• non-water soluble • surface adsorbed • speedy • termination by solvent withdrawal
Cl
125
ICl
Enzyme catalysis Marcholonis JJ Biochem J 113 299 (1969)
a) H2O 2
H2O 125
125 -
I2
I
laktoperoxidáz Lactose peroxidase H
laktoperoxidáz H
H2O I
+
+
I
-
+ Tyr His
• pH dependent (pH 6 – 7) • purity of H2O2 !
b) In situ preparation of H2O2 : immobilised enzymes HO
O HO
Glucose oxidase
OH HO
glükóz
OH
glucose
glükóz oxidáz HO O HO
O HO
oldhatatlan gyöngy immobilizált glükóz oxidázzal és laktoperoxidázzal
OH
δ-glükonolakton
δ-gluconolactone
H2O 2 hidrogén peroxid
hydrogen peroxide 125 -
I
Lactose peroxidase
laktoperoxidáz
I2
reaktív reactive jód
iodine (I+)
N-succinyimidyl 3-(4-hydroxi 5-[125I]iodophenyl) propionate (Bolton and Hunter reagent) OH
OH 125
I
+ CH2 H
C
O
Chloramine -T
125
Na I
incorporation of I CH2
O H
C O
H
N
propionate
OH H2N
I
conjugation O C O
NH NH
125I
N
+
propionate O NH O
CH2
H
O
O jódozott propionát 125I]iodophenyl) N-succinyimidyl 3-(4-hydroxi-5-[ (Bolton és Hunter reagens)
125
C
O
C
H
O N-szukcinimidil 3-(4-hidroxifenil) N-succinyimidyl 3-(4-hydroxi-phenyl) propionát
H
C
O
- labelled125protein I-dal jelölt fehérje
ε-amino of lysine residue O
NH
NH fehérjében levõ lizin ε-amino csoportja jelölendõ
Selection of radionuclide Radioactíve atom
Availability
Price
Half-life
Most important γ energy (keV)
123I
Limited
High
13 h
159
131I
Good
Low
8 days
364
111In
Good
Medium
67 h
173 247
67Ga
Good
Medium
78 h
185 300
99mTc
Good
Low
6h
141
Incorporation of radionuclide by chelator
Application:
1. biodistribution studies (diagnosis, therapy) 2. localization in cells
Priciple:
complex formation high stability indirect method
Types:
1. linear chelator 2. cyclic chelator
Linear chelator O
O
O R
O N
N
+
NH
O O
O O
O N
amint tartalmazó molekula R
DTPA
-
O
NH2
O
O
-
NH
O
N
+
NH O
O O
O
-
amide linkage S
S
C N
O
C
NH
R
NH
-
R
O
O
-
O
N O O
-
DTTA
O
N
O
N
O
-
NH2
O N
-
O
-
O
N O
-
N
O
O
-
-
O tiourea kötés képzõdés
thiourea linkage
-
Linear chelator H3C
OH N
O NH O O N OH N O H2N
O DFA deferoxamin Deferoxamine (DFA)
OH
Cyclic chelator O
-
O
O
-
O
O N
N
N
O -
O
N
-
O
-
O N
1,4,7-triazacyclononaneN,N‘,N''-triacetic acid; (NOTA)
N
-
O O
N
O O
O
-
O
O
-
O
1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid (DOTA)
-
N
N
N
N
O
-
O O
-
O
O
1,4,8,11-tetraazacyclotetradecaneN,N',N'',N'''-tetraacetic acid (TETA)
activityaktivitás (binding) % az eredeti immunológiai maradéka %
The effect of substitution level on immunological reactivity of antibodies
antiserum No 2
Prepared under two different conditions antiserum No 1
number of I125 per protein
