SYNTHESIS, DNA-BINDING, AND SEQUENCE SPECIFICITY OF DNA ALKYLATION BYSOME NOVEL CYCLIC PEPTIDE CHLORAMBUCIL CONJUGATES

In an effort to investigate the potential of cyclic peptides as carrie rs for cytotoxic agents, we synthesized four cyclic peptide-chlorambuc il conjugates: cyclo[Lys(CHB)-Lys(CHB)-Gaba-] (peptide Y), cyclo[Lys(C HB)-Gly-Lys(CHB)Gaba-] (peptide A), cyclo[Lys(CHB)-beta-Ala-Lys(CHB)-G aba-] (peptide B) and cyclo[Lys(CHB)-Gaba-Lys(CHB)-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 protectin g group (Z), the conjugation was achieved by reaction with the pentafl uorophenyl ester of chlorambucil (CHB). These cyclic peptides differ f rom one another in ring size and are disubstituted with CHB via the ep silon-amino group of the lysyl residue. The various conjugates were de signed to study the effect of ring size on the mode of DNA binding and alkylation. A DNA-binding assay using lambda-DNA with ethidium bromid e showed that whereas peptide Y and CHB have no observable binding aff inity, the apparent binding constants for peptide A, peptide B and pep tide C on lambda-DNA were determined to be 2.36 x 10(5), 1.27 x 10(5) and 3.50 x 10(5), respectively. Thus, it is suggested that cyclic pept ides bearing aliphatic side chains attached to a ring larger in size t han 14 members would be more favourable as regards augmenting the bind ing affinity. DNase I footprinting showed that no footprinting pattern s were observed for the 253-mer fragment and 117-mer fragment with pep tide A, but two new bands corresponding to G69 and G80 were observed f or the 117-mer fragment. DNA alkylation studies using a piperidine cle avage assay on the 117-mer DNA fragment showed that the sequence selec tivity, judged by reaction intensity observed with peptide A, peptide B and peptide C, was similar to that seen with CHB alone. The selectiv ity of alkylation for both CHB and its peptide derivatives appears to be: 3'-Pur-G-Pyr-5' > 3'-Pyr-G-Pyr-5' > 3'-Pyr-G-Pur-5' = 3'Pur-G-Pur- 5'. However, there are apparent differences in the intensity of alkyla tion by peptides A, B, C and CHB at certain guanine residues.