Synthesis, DNA binding, and sequence specificity of DNA alkylation by some novel cyclic peptide-chlorambucil conjugates
Sheh, L.; Chang, H.W.; Ong, C.W.; Chen, S.L.; Bailly, C.; Linssen, R.C.; Waring, M.J.
Anti-Cancer Drug Design 10(5): 373-388
1995
ISSN/ISBN: 0266-9536 PMID: 7639928 Document Number: 449325
In an effort to investigate the potential of cyclic peptides as carriers for cytotoxic agents, we synthesized four cyclic peptide-chlorambucil conjugates: cyclo(Lys(CHB)-Lys(CHB)-Gaba-) (peptide Y), cyclo(Lys(CHB)-Gly-Lys(CHB)Gaba-) (peptide A), cyclo(Lys(CHB)-beta-Ala-Lys(CHB)-Gaba-) (peptide B) and cyclo(Lys(CHB)-Gaba-Lys(CBB)-Gaba-) (peptide C). The cyclic peptides were synthesized by coupling protected amino acid residues in solution and the subsequent cyclization was performed by the pentafluorophenyl ester method as described previously (Sheh et al., 1990, 1993a,b). After deblocking the lysyl-carbobenzyloxy protecting group (Z), the conjugation was achieved by reaction with the pentafluorophenyl ester of chlorambucil (CHB). These cyclic peptides differ from one another in ring size and are disubstituted with CHB via the epsilon-amino group of the lysyl residue. The various conjugates were designed to study the effect of ring size on the mode of DNA binding and alkylation. A DNA-binding assay using lambda-DNA with ethidium bromide showed that whereas peptide Y and CHB have no observable binding affinity, the apparent binding constants for peptide A, peptide B and peptide C on lambda-DNA were determined to be 2.36 times 10-5, 1.27 times 10-5 and 3.50 times 10-5, respectively. Thus, it is suggested that cyclic peptides bearing aliphatic side chains attached to a ring larger in size than 14 members would be more favourable as regards augmenting the binding affinity. DNase I footprinting showed that no footprinting patterns were observed for the 253-mer fragment and 117-mer fragment with peptide A, but two new bands corresponding to G69 and G80 were observed for the 117-mer fragment. DNA alkylation studies using a piperidine cleavage assay on the 117-mer DNA fragment showed that the sequence selectivity, judged by reaction intensity observed with peptide A, peptide B and peptide C, was similar to that seen with CHB alone. The selectivity of alkylation for both CHB and its peptide derivatives appears to be: 3'-PurG-Pyr-5' gt 3'-Pyr-G-Pyr-5' gt 3'-Pyr-G-Pur-5' = 3'Pur-G-Pur-5'. However, there are apparent differences in the intensity of alkylation by peptides A, B, C and CHB at certain guanine residues.