trategies of bioconjugation Homobifunctional Homobifunkciós Zero-length Heterobifunctional Heterobifunkciós Identical functions Azonos funkciós csoportok incs o beépülés insertiona két komponens közé 1. Karbodiimid 1. Carbodiimide Different functions Különböző funkciós csoportok 1. -H 2 H 2 - C 1. H 2 H- 2. -H H- 2. 2. Woodward Woodward's reagens reagent k 2. H- C CH C 3. -CH HC- 3. Fotoreaktív Photoreactive 4. -CH HC- 3. 3. Diimidazole -H 2 5. -H-H 2 H 2 -H- -H 4. xidálószerek 4. xidising agents -CH 6. -H + H- -CH 7. em-kovalens 5. Enzimek 5. Enzymes 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 3.1. Connecting amino- and thiol-functions PDP H 2 H AMID + H H 1 DIZULFID H 1 + b H H 2 DMF H + H H = H, MCC; - 3 - a + Yoshitake,. et al. 101 395 (1979) m-maleimidobenzol- -hydroxysuccinimide ester (MB), 1981 m-maleimidobenzoil--hidroxiszukcinimid észter [MB] (1981) H 1 ph 7 H TIÉTE 1
c H I H 2 H I H IAB AMID Weltman, JK et al. Biotechniques 1 148 (1983) H 1 H 1 H 2 I TIÉTE amide thioether jódecetsav-p-nitrofenilészter iodoacetic acid-p-nitrophenyl ester (1975) 1975
a 3.2. Connecting carboxyl and amino/thiol-functions 2 + - diazoacetic diazoecetsav-nitrofenilészter acid-p-nitrophenyl ester HC 2 H/ H 2 1 + 1 H amide ester észter b + - HC pyridyl-2,2'-dithiobenzyl piridil-2,2'-ditiobenzildiazoecetsav diazoacetate H 1 1 disulphide ester diszulfid észter
a 3.3. Connecting carbonyl and thiol-functions H H 2 3-(2-piridilditio)propionsav hidrazid 3-(2-pyridyldithio)propionic acid hydrazide H H H2 0.1 a-acetát 0.1 M a acetate ph 5.5 5.5 [PDPH] 1 H Zara JJ. Anal Biochem 194 156-162 (1991) + H 1 diszulfid hidrazon disulphide hydrazone b H H 4-(4--maleimidofenil)butánsav H 2 hidrazid 4-(4--Maleimidophenyl)butyric acid hydrazide Chamov M. Et al. J Biol Chem 267 15916 (1992) 1 thioether H H 2 H H tioéter hidrazon 1 hydrazone
Photoactive groups 2 + - Cl CF 3 hν hν hν Cl C CF3 C triplett keton nitrene nitrén karbén 1. Alkyl azide: UV excitation carbene triplett keton 2. Aryl azide: λ = 300 400 nm 3. Acyl azide: reactive in dark too
A. Activation of azide function and its transformation - + H H fenilazid phenyl azide nitrén képzõdés nitrene H 2 aktív hidrogén (C-H) beépülés active hydrogen (C-H) H 2 H gyûrûtagszám növekedés ring expansion dehydroazepine intermedier dehidroazepin intermedier H H aktív hidrogén (-H) beépülés active hydrogen (-H) nucleophile nukleofil reactive hydrogen reaktív hidrogén addíciós reakció addition H
H 2 3 3 3 4,4'-diazidobifenil [DABP] 4,4 -diazodo biphenyl 4-azildobenzoesav -hidroxiszukcinimid észter 4-Azidobenzoic acid [H-ABA, -hydroxysuccinimide HAB] ester Mikkelsen, B et al. J Biol Chem 251 7413 (1976) Ji, T. Anal Biochem 121 286 (1982) CH H 2 H 3 p-azidobenzoesav hidrazid [ABH] p-azidobenzoic acid hydrazide 4 3 4-azidofenil-maleimid [APM] 4-Azidophenyl maleimide H Trommer WE et al. Hoppe-eyleis Z Physiol Chem 356 1455 (1975)
-CH H H 2 ABA H H + - photo activation fotoaktiválás ring gyûrûtágulással expansion H H H H EDC keresztkötött molekula karboxil csoportot tartalmazó vegyület H H H UV fény UV nukleofilt tartalmazó vegyület H amide linkage H amid kötés képzõdés + - H 2
B. Activation of diazo function and its transformation λ = 254 / 310 nm + 2-diazo-3,3,3-trifluoropropionic acid 2-diazo-3,3,3-trifuorpropionsav p-nitrophenyl ester p-nitrofenil észter [PP-DTP] - H 2 - + primer amint tartalmazó vegyület F F F carbene H C karbén képzõdés UV fény F F F H 1 F F F keresztkötött molekula + - H p-nitrofenol p- nitrophenol H - + F F F H 1 reaktív hidrogént reactive tartalmazó hydrogen vegyület Chowdhry et al. PA 73 1406 (1976)
C. Activation of function with benzophenone moiety 1 H benzophenone-4-maleimide benzofenon-4-maleimid C keresztkötött molekula H tiol tartalmú molekula UV fény H 1 reaktív hidrogént tartalmazó reactive vegyület hydrogen tioéter kötés thioether képzõdés Walling C, Gibian MJ, JAC 87 3361 (1965)
Conjugates of radioactive isotopes Biosynthetic approach 15 C, 3 H, 14 C, 32 P, 35 Direct Chemical synthesis Indirect e.g. modification of Tyr/ His residues a) Covalent linkage b) Complex formation
Direct incorporation of radioactive isotope Example: 125 I, 131 I (17 iodo-, 13 bromo-, 6 chloro-, 2 fluoro-isotope t ½ > 3 perc) In vitro long lifetme low energy photon emission (no particle) price In vivo IMAGIG X-ray or γ-emitting ingle photon emission tomography (PECT) Positron emission tomography (PET) γ-camera In vivo THEAPY 125 I ot optimal (high energy: danger) 125 I, 131 I (β-), 123 I (γ 159 kev) 18 F (97% β+) 131 I ( 82 Br)
Characterisctics of the most important radioactive isotopes ensitivity: 131 I > 125 I > 14 C, 32 P, 35, 3 H Atomic number Izotope Half life Decay Highest particle energy (MeV) γ-energy (MeV) H 1 3 H 12,33 y β - 0,0186 - C 6 11 C 14 C 20,4 min 5760 y β + 0,96 β - 0,155 - - 7 13 10 min β + 1,19-8 15 2 min β + 1,73 - F 9 18 F 109,8 min β + 0,633 - a 11 24 a 15,02 h β -, γ 1,392 2,754 1,369 P 15 32 P 14,28 day β - 1,710-16 35 87,2 day β - 0,167 - K 19 40 K 42 K 1,28 * 10 9 y 12,36 h β -, K β -, γ 1,31 3,52 (75%) 1,99 (25%) 1,46 1,525 Ca 20 45 Ca 163 day β - 0,257 - Cr 24 51 Cr 27,7 day K, e -, γ 0,315 (e - ) 0,320 Fe 26 52 Fe 59 Fe 8,2 h 44,6 day β +, γ β -, γ 0,8 1,566 0,5 1,30 1,10
Atomic number Izotope Half life Decay Highest particle energy (MeV) γ-energy (MeV) Co 27 60 Co 5,272 y β -, γ 0,318 1,33 1,17 Cu 29 64 Cu 12,74 h β - (39%) β + (19%) K (42%) γ (1%) 0,575 0,656 1,34 Ga 31 67 Ga 78 h γ - 0,185 0,30 Kr 36 85 Kr 10,73 y β -, γ 0,687 0,514 u 37 81 b 86 b 4,7 h 18,65 day β +, γ β -, γ 0,99 1,78 1,93 0,95 1,078 r 38 90 r 29 y β - 0,546 - It 39 90 Y 64 h β -, γ 2,29 1,761 Tc 43 99m Tc 6,02 h γ - 0,140 In 49 111 In 113m In 67,0 h 1,658 h γ γ - - 0,173 0,247 0,391 I 53 123 I 125 I 131 I 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
Formation and decay of 99M Tc E 99 42 Mo 66.7 H e - 99 M43 Tc 6.03 H 0.1427 γ 1 0.1405 γ 3 γ 2 99 43 Tc 2.12X10 5 Y 0.0 e - table 99 44 u (Dillman, LT; Von der Lage, FC: adionuclide decay schemes and nuclear parameters for use in radiation dose estimates (MID Pamphlet o. 10), ociety of uclear Medicine, 1975)
α-decay [in biological medium: mm] A Z Y A-4 Z-2 X + 4 2He β-decay e - emission 3 1 H 2 3 2 He + e - + ν e neutron e - capture 40 19 K 40 18 Ar proton + electron + antineutrino electron + proton e + emission neutron 11 6 C 5 11 5 B 6 + e + + ν e γ-decay A Y * proton A Y + γ positron + neutrino
1. Chloramine-T method Greewood, FC et al. Biochem J 89 114 (1963) Wilbur, D Bioconjugate Chem 3 433 (1992) H 3 C - Cl + 125 I - + 2 H + H 3 C H 2 (a 125 I) 125 + I Cl His Tyr H 3 C + H H 125 I 30 s 30 min water soluble ph 7 phosphate 0.05 M H 3 C - 125 I
2. Immobilised chloramine-t (ID-BEAD) U.. Patent 4448764 és 4436718 polystyrene polisztirol mátrix matrix 3 mm CH - Cl 2 5 min good protein recovery mild conditions ph 7.2 8.4 3. ID-GE Fraker, PJ és peck, JC BBC 80 849 (1978) Cl Cl Cl Cl + 125 I - + H + (a 125 I) Cl Cl H Cl 125 + I Cl 1,3,5,6-tetrachloro-3a,6a-diphenyl 1,3,5,6-tetraklór-3a,6a-difenilglicouril glycoluril non-water soluble surface adsorbed speedy termination by solvent withdrawal
Enzyme catalysis Marcholonis JJ Biochem J 113 299 (1969) a) H 2 2 H 2 125 I - 125 I2 H 2 I + + I - laktoperoxidáz Lactose peroxidase H laktoperoxidáz H + Tyr His ph dependent (ph 6 7) purity of H 2 2!
b) In situ preparation of H 2 2 : immobilised enzymes H H H Glucose oxidase H H glükóz glucose glükóz oxidáz H H oldhatatlan gyöngy immobilizált glükóz oxidázzal és laktoperoxidázzal H 2 2 hidrogén peroxid H H δ-glükonolakton δ-gluconolactone hydrogen peroxide 125 I - Lactose peroxidase laktoperoxidáz I 2 reaktív jód reactive iodine (I + )
-succinyimidyl 3-(4-hydroxi 5-[ 125 I]iodophenyl) propionate (Bolton and Hunter reagent) H H CH 2 C H C + a 125 I Chloramine -T incorporation of I 125 I H H CH 2 C H C -szukcinimidil 3-(4-hidroxifenil) propionát -succinyimidyl 3-(4-hydroxi-phenyl) propionate 125 I H conjugation H 2 jódozott propionát (Bolton és Hunter reagens) -succinyimidyl 3-(4-hydroxi-5-[ 125 I]iodophenyl) propionate + H CH 2 H C C H H 125 I - labelled protein 125 I-dal jelölt fehérje H H fehérjében levõ lizin ε-amino csoportja jelölendõ ε-amino of lysine residue H
election of radionuclide adioactíve atom Availability Price Half-life Most important γ energy (kev) 123 I Limited High 13 h 159 131 I Good Low 8 days 364 111 In Good Medium 67 h 173 247 67 Ga Good Medium 78 h 185 300 99m Tc Good Low 6 h 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 - H 2 - - H + DTPA amint tartalmazó molekula H - H + - amide linkage C C H - H - H 2 - - - DTTA - - - tiourea kötés képzõdés thiourea linkage
Linear chelator C H 3 H H H H H 2 DFA deferoxamin Deferoxamine (DFA)
Cyclic chelator - - - - - - 1,4,7-triazacyclononane-,,''-triacetic acid; (TA) - - 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (DTA) - - - 1,4,8,11-tetraazacyclotetradecane-,','','''-tetraacetic acid (TETA)
The effect of substitution level on immunological reactivity of antibodies az eredeti immunológiai activity aktivitás (binding) maradéka % % antiserum o 1 antiserum o 2 Prepared under two different conditions number of I 125 per protein