Consecutive Halogen Transfer between Various Functional Groups Induced by Reaction of Hypohalous Acids: NADH Oxidation by Halogenated Amide Groups

Abstract

Cyclic dipeptides (c-Gly2, c-Ser2, c-Gly-Phe, etc.) were used as simple protein models to investigate the HOCl-induced generation and reactivity of chlorinated amide groups. The pH dependence of the kinetics of amide chlorination reveals that ClO− (not HOCl) is the reactive agent. N-Chlorinated cyclopeptides are stable up to 30 min, they exhibit narrow absorption bands around 215 nm, and they are capable of oxidizing certain biological substrates, the reactivity decreasing in the order GSH > ascorbate > methionine > NADH ⪢ GSSG. The chloroamide is less reactive, but much more selective in its reactions, than HOCl or ClO−; thus, with formation of the chloroamide prolonged oxidative effects, directed toward specific target molecules, can be expected. Chlorination of NADH, yielding a catalytically inactive species (NAD/Cl), was investigated in most detail because it is likely to be an important and highly lethal process. The chloroamide group is far more reactive toward NADH than chloroamines derived from primary amines. Chloronucleotides formed by reaction of ClO− with inosine, GMP, TMP, or UMP are capable of quantitative chlorine transfer to cyclopeptides; however, no chlorine transfer between the amide nitrogen and primary amines is detectable, in either direction. The results presented enable prediction of chlorine transfer cascades induced by HOCl/ClO−, involving nucleotides, peptide amide groups, and final target molecules. Chlorinated NAD(P)H, as a stable terminal product of consecutive chlorine transfer reactions, might be a useful biological marker for assessing the role of HOCl in inflammatory events. Bromination by BrO− of cyclopeptides is more than two orders of magnitude faster than chlorination by ClO−, and the reactivity of bromoamide with NADH exceeds that of chloroamide by more than four orders of magnitude.