ALTERATION IN DNA CROSS-LINKING AND SEQUENCE SELECTIVITY OF A SERIES OF AZIRIDINYLBENZOQUINONES AFTER ENZYMATIC REDUCTION BY DT-DIAPHORASE

DT-diaphorase (DTD) mediated reduction of a series of 2,5-bis-substituted-3,6-diaziridinyl-1,4-benzoquinones was found to increase the level of DNA interstrand cross-linking (ISC) formed at neutral pH with an enhancement observed as the pH was decreased to 5.8. The analogues used were symmetrically alkyl-substituted carbamoyl ester analogues of AZQ (DI-D7), 3,6-diaziridinyl-1,4-benzoquinone (DZQ), the 2,5-dimethyl derivative (MeDZQ), and a 2,5-bis[(2-hydroxyethyl)amino] analogue (BZQ). At pH 5.8, the level of DNA ISC induced by enzymatic reduction was as follows: DZQ > MeDZQ >> D1 (methyl) > D3 (n-propyl) > D2 (AZQ; ethyl) > D5 (n-butyl) > D7 (sec-butyl) > D4 (isopropyl) D6 > (isobutyl). A similar trend was observed at pH 7.2. The level of DNA ISC induced by BZQ, which is not a substrate for DTD, was not increased by enzymatic reduction. Dicumarol, a known inhibitor of DTD, was capable of inhibiting the DNA ISC induced by these quinones upon enzymatic reduction. MeDZQ and DZQ reacted with guanines, as measured by Maxam and Gilbert sequencing, with a sequence selectivity similar to that of the nitrogen mustard class of antitumor agents. Enzymatic reduction of DZQ and MeDZQ by DTD was found to alter their sequence-selective alkylation. Reduced DZQ showed enhanced guanine alkylation in 5'-GC-3'sequences and new sites of adenine alkylation in 5'-(A/T)AA-3' sequences. Reduced MeDZQ only showed new sites of adenine alkylation at 5'-(A/T)AA-3' sequences but no enhancement of guanine alkylation. The new sites of adenine alkylation were found to be inhibited in the presence of magnesium and rapidly converted into apurinic sites. Chemical reduction of DZQ by ascorbic acid altered the sequence-selective pattern of alkylation in a manner similar to that observed for enzymatic reduction. Ascorbic acid was unable to reduce MeDZQ. These data show that DTD mediated reduction can effect the sequence selectivity of alkylation and DNA interstrand cross-link induction by aziridinylbenzoquinones but that the alteration in sequence selectivity was compound specific.