Sequence-specific DNA cleavage by dipeptides disubstituted with chlorambucil and 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid

Two dipeptides, each containing a lysyl. residue,, were disubstitated with chlorambucil. (CLB) and 2',,6-dimethoxyhydroquinone-3-mereaptoacetic acid ( DMQ-MA): DMQ-MA-Lys(CLB)-Gly-NH2 (DM-KCG) and DMQ-MA-beta -Ala-Lys(CLB)-NH2 (DM-BKC). These peptide-drug conjugates were designed to investigate seque nce-specificity of DNA cleavage directed by the proximity effect of the DNA cleavage chromophore (DMQ-MA) situated close to the alkylating agent (CLB) inside a dipeptide moiety. Agarose electrophoresis studies showed that DM- KCG and DM-BKC possess significant DNA nicking activity toward supercoiled DNA whereas CLB and its dipeptide conjugate Boc-Lys(CLB)-Gly-NH2 display li ttle DNA nicking activity. ESR studies of DMQ-MA and DM-KCG both showed fiv e hyperfine signals centered at g = 2.0052 and are assigned to four radical forms at equilibrium, which may give rise to a semiquinone radical respons ible for DNA cleavage. Thermal cleavage studies at 90 degreesC on a 265-mer test DNA fragment showed that. besides alkylation and cleavage at G residu es, reactions with DM-KCG and DM-BKC show a preference for A residues with the sequence pattern: 5'-G-(A)(n)-Pur-3' > 5'-Pyr-(A)(n)-Pyr-3' (where n = 2-4). By contrast, DNA alkylation and cleavage by CLB occurs at most G and A residues with less sequence selectivity than seen with DM-KCG and DM-BKC. Thermal cleavage studies using N7-deazaG and N7-deazaA-substituted DNA sho wed that strong alkylation and cleavage at A residues by DM-KCG and DM-BKC is usually Ranked on the 3' side by a G residue whereas strong cleavage at G residues is flanked by at least one purine residue on either the 5' or 3' side. At 65 degreesC, it is notable that the preferred DNA cleavage by DM- KCG and DM-BKC at A residues is significantly more marked than for G residu es in the 265-mer DNA; the strongest sites of A-specific reaction occur wit hin the sequences 5'-Pyr-(A)(n)-Pyr-3'; 5'-Pur-(A)(n)-G-3' and 5'-Pyr-(A)(n )-G-3'. In pG4 DNA, cleavage by DM-KCG and DM-BKC is much greater than that by CLB at room temperature and at 65 degreesC. It was, also observed that DM-KCG and DM-BKC cleaved at certain pyrimidine residues: C40, T66, C32, T3 4, and C36. These cleavages were also sequence selective since the suscepti ble pyrimidine residues were Ranked by two purine residues on both the 5' a nd 3' sides or by a guanine residue on the 5' side. These findings strongly support the proposal that once the drug molecule is positioned so, as to p ermit alkylation by the CLB moiety, the DMQ-MA moiety is held close to the alkylation site, resulting in markedly enhanced sequence-specific cleavage.