Characterization of a Unique Tomaymycin-d(CICGAATTCICG)2 Adduct Containing Two Drug Molecules per Duplex by NMR, Fluorescence, and Molecular Modeling Studies

F. Leslie Boyd, Diana Stewart, William A. Remers, Mary D. Barkley, Laurence H. Hurley

Research output: Contribution to journalArticlepeer-review

51 Scopus citations


Tomaymycin is a member of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. This antibiotic is proposed to react with the exocyclic 2-amino group (N2) of guanine to form a covalent adduct that lies snugly within the minor groove of DNA. While DNA-footprinting experiments using methidiumpropyl-EDTA have revealed the favored bonding sequences for tomaymycin and related drugs on DNA, the stereochemistry at the covalent bonding site (C-11) and orientation in the minor groove were not established by these experiments. In previous studies using a combined fluorescence, high-field NMR, and molecular modeling approach, we have shown that for tomaymycin there are two diastereomeric species (11R and 115) on both calf thymus DNA and d(ATGCAT)2. Although we were able to infer the identity (stereochemistry at C-11 and orientation in the minor groove) of the two species on d(ATGCAT)2 by high-field NMR and fluorescence studies, in combination with molecular mechanics calculations, definitive experimental evidence was lacking. We have designed and synthesized a self-complementary 12-mer [d(CICGAATTCICG)2] based on the Dickerson dodecamer [d(CGCGAATTCGCG)2] that bonds identically two tomaymycin molecules, each having a defined orientation and stereochemistry. Thus the bis(tomaymycin)-12-mer adduct maintains the self-complementarity of the original duplex molecule. Two-dimensional proton J-correlated spectroscopy (COSY) of the bis(tomaymycin)-d(CICGAATTCICG)2 adduct (I = inosine) unequivocally shows that C-11 of tomaymycin covalently bonds through N2 of guanine with an 11 S stereochemistry in the sequence 5′-CGA-3′. Fluorescence studies confirm the “S” stereochemistry at C-11, and two-dimensional proton nuclear Overhauser (NOESY) experiments assign the orientation of the drug molecule in the minor groove of DNA, i.e., with the aromatic ring of tomaymycin to the 3′ side of covalently modified guanine. Molecular modeling experiments with AMBER are consistent with the identification of the species of tomaymycin (115 with 3′ orientation) bound to the 12-mer. This species and the other 115 species are favored over the two 11S species due to a combination of steric and electrostatic interactions. Analysis of two-dimensional COSY and NOESY experiments on the bis(tomaymycin)-d-(CICGAATTCICG)2 adducts reveals minimal effect of covalent bonding on local helix structure. From these experiments the modest but most pronounced distortion is at the deoxyribose attached to the modified guanine and both the phosphate and adjacent deoxyribose to the 5′ side. The distortion of this phosphate between the covalently modified guanine and the 5′ nucleoside is supported by its downfield-shifted phosphorus NMR resonance signal. The discrepancy between the pairs of most energetically favored species of tomaymycin-DNA adducts on d(ATGCAT)2 and the 12-mer is explained by examining individual drug- nucleotide interactions. The results presented in this study together with previous investigations show that the orientation of the drug molecule in the minor groove, and stereochemistry at the covalent linkage site, is dependent upon both the flanking sequence and drug structure. This conclusion mandates caution be used in rationalizing the biochemical and biological effects of P(1,4)B bonding to DNA until precise structural information is established.

Original languageEnglish (US)
Pages (from-to)2387-2403
Number of pages17
Issue number9
StatePublished - Mar 1 1990

ASJC Scopus subject areas

  • Biochemistry


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