Spectrometric studies of cytotoxic protoberberine alkaloids binding to double-stranded DNA

Abstract

The noncovalent complexes of five cytotoxic protoberberine alkaloids, that is, berberine, palmatine, jatrorrhizine, coptisine, and berberrubine with several double-stranded oligodeoxynucleotides were systematically investigated by using electrospray ionization mass (ESI-MS) and fluorescence spectrometric methods, with the aim of establishing the structure–activity relationships. ESI-MS spectrometric studies indicated that these five alkaloids showed both 1:1 and 1:2 binding stoichiometries with d(AAGAATTCTT) 2, d(AAGGATCCTT) 2, and d(AAGCATGCTT) 2. Their relative binding affinities toward these three double-stranded DNA were semi-quantitatively evaluated by measuring the ratios of the complex signals ([ds+alkaloid–5H] 4−+[ds+2alkaloid–6H] 4−) to those of the duplexes ([ds-4H] 4−) and also by ESI-MS competitive binding experiments. These experiments established the relative binding affinities of five protoberberine alkaloids in the order of palmatine > jatrorrhizine > coptisine > berberine > berberrubine with d(AAGAATTCTT) 2, palmatine ⩾ coptisine > jatrorrhizine ⩾ berberine > berberrubine with d(AAGGATCCTT) 2 and palmatine > jatrorrhizine ⩾ coptisine > berberine > berberrubine with d(AAGCATGCTT) 2. Significantly, these alkaloids except berberrubine bound to d(AAGGATCCTT) 2 and d(AAGCATGCTT) 2 with the affinities comparable to Hoechst 33258, a typical DNA minor groove binder. The relative binding preferences of berberine, palmatine, and coptisine with these three double-stranded DNA were further quantitatively assessed by their association constants obtained from fluorescence titration experiments. The values revealed the order of relative binding affinities as berberine > coptisine > palmatine with d(AAGAATTCTT) 2 and coptisine > berberine > palmatine with d(AAGGATCCTT) 2 and d(AAGCATGCTT) 2. These results were not in full agreement with those obtained from ESI-MS experiments, maybe due to the different measuring solution conditions. The results from ESI-MS and fluorescence titration experiments indicated that the sequence selectivities of these five alkaloids were not significant and remarkable AT- or GC-rich DNA binding preferences were not obtained, in contrast to the report that berberine binds preferentially to AT-rich DNA. To provide further insight into the sequence selectivities, the association constants of berberine with d(AAGATATCTT) 2, 5′-AAGTAATCTT-3′/5′-AAGATTACTT-3′, d(AAGGGCCCTT) 2, d(AAGGCGCCTT) 2, and 5′-AAGGCCGCTT-3′/5′-AAGCGGCCTT-3′, that is double helical DNA from AT-rich to GC-rich sequences, were further measured by fluorescence titration methods. No significant differences in their association constants were observed, suggesting that berberine showed no remarkable sequence selectivities.