Coenzyme Models. 41. On the Unusual Reactivities of N(5)-Hydrogen-Bonded Flavin. An Approach to Regiospecific Flavin Activation through Hydrogen Bonding

A new flavin with hydrogen-bonded N(5) (OHF1: sodium l-hydroxy-7-methylnaphtho[8,7-g]pteridine-9,l 1- (7FL10H)-dione-3-sulfonate) was synthesized. The second-order rate constants (k₂) for the oxidation of NADH model compounds by OHF1 (E_1/2= -0.579 V) were similar to those for the oxidation by 3-methyllumiflavin (MeLFl, E_1/2 = -0.538 V), and the plot for OHFI was included in a linear relation between log k₂ vs. E_1/2(E_1/2 polarographic half-wave potential of flavins). Hence, OHFI acts as a “normal” flavin in this reaction, On the other hand, the pseudo-first-order rate constants (fc/) for the oxidation of thiols by OHFI were greater by 33-645-fold than those for the oxidation by MeLFl, and log k₁' for OHFI deviated to the upper area from the linear log k₁, vs. E_1/2 relationship by more than two log units. The pseudo-first-order rate constant (k_obsd(1)) for the adduct formation of OHFI with S0₃ ^2- was further enhanced (1830-fold relative to that of MeLFl!). The upper deviation from the linear log K_obsd(1) vs. E_1/2 relationship corresponded to 3.61 log units. The OHF1-S0₃ ^2- adduct (λ_max458 nm) further reacted with SO₃ ^2- and finally yielded 8-sulfonated 1,5-dihydro-OHFl. It was suggested on the basis of several experimental data that the intermediate absorbing at 458 nm is the 4a adduct. It was concluded, therefore, that OHFI is “regiospecifically” activated toward reactions involving 4a intermediates such as oxidation of thiols and adduct formation with S0₃ ^2-. This novel finding was ascribed to activation of the 4a position through hydrogen bonding with N(5). Thus, the present study is the first example to support a hypothesis proposed by Massey and Hemmerich that the relative reactivity of C(4a) to N(5) in flavin coenzymes is regulated by the position of hydrogen bonding with flavoapoproteins.