Synthesis, DNA Scission Chemistry, and an Investigation of the Reactive Oxygen Species of Two 2,6-Dimethoxyhydroquinone-3-Mercaptoacetic Acid-Peptide Conjugates

Abstract

This paper reports a continuing study of the use of short-chain peptides as carriers of a potential anti-tumour agent, namely 2,6-dimethoxyhydroquinone–3-mercaptoacetic acid (DMQ–MA). In an effort to carry out anti-cancer drug design, we have synthesized two new peptide–DMQ–MA conjugates: DMQ–MA–Lys(Cbz)–Arg–Arg–OMe and DMQ–MA–Lys(DMQ–MA)–Arg–OMe. These were synthesized by coupling protected amino acids using Pfp/N,N´-dicyclohexylcarbodiimide (Pfp = pentafluorophenol) in solution; the next conjugation was achieved by treatment with the pentafluorophenyl ester of DMQ–MA in dimethylformamide. We have investigated the DNA scission chemistry of these drugs, and the results show that they can cleave pBR322 DNA in concentrations as low as 5–40 M without the addition of hydrogen peroxide or ultraviolet (UV) irradiation. The addition of a catalytic amount of ferrous ions has a significant enhancement on the DNA strand cleavage induced by the drugs: the cleavage degree increases with increasing time. Addition of hydroxyl radical scavengers such as dimethyl sulfoxide, glycerol, etc., significantly inhibits DNA strand cleavage. Oxidants with free-radical character are well known investigators of DNA damage. In order to explore the mechanism of DNA strand cleavage, we also investigated the reactive oxygen species. The active species produced by the drugs were studied using the Rhodamine B (Rhb) assay and electron paramagnetic resonance (EPR) spectroscopy. The results show fast production of the hydroxyl radical, which is trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the characteristic signal of DMPO·OH (1 : 2 : 2 :1) is recorded. DNA binding constants determined by the ethidium bromide assay are approximately 10⁴M^–1. The results of this study show that the degree of DNA scission is not related to the binding constants, nor is it related to the hydroxyl radical intensity data solely, but rather to the ability of the drugs to generate various free radicals.

Manuscript received: 13 June 2001.

Final version: 20 July 2001.