New reagents for directed modification of biopolymers: Photoaffinity modification of Tte DNA polymerase

Arylazides N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-beta-alanine (Ia) a nd N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-glycine (Ib) were synthesiz ed and covalently attached to 5-(3-aminopropenyl-1)-dUTP through the amino group to give 5'-triphosphate (IIa) and 5'-triphosphate (IIb), The resultin g azides were subjected to photolysis in aqueous solution. The spectral and photochemical characteristics of azides (I) and (II) imply that their use for the modification of biopolymers holds promise. Compounds (IIa, b) effec tively substituted dTTP in DNA polymerization catalyzed by thermostable DNA polymerase from Thermus thermophilus B-35 (Tte DNA polymerase), Photoaffin ity modification of Tte DNA polymerase was carried out by dTTP analogues (I Ia, b) and by earlier obtained 5-[N-(5-azido-2-nitrobenzoyl)-trans-3-aminop ropenyl-1]deoxyuridine 5'-triphosphate (III) and 5-[N-(4-azido-2,3,5,6-tetr afluorobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine 5'-triphosphate (IV) u sing two variants of labeling. All four dTTP analogues were shown to modify Tte DNA polymerase.