One- and Two-Dimensional 1H NMR, Fluorescence, and Molecular Modeling Studies on the Tomaymycin-d(ATGCAT)2Adduct. Evidence for two Covalent Adducts with Opposite Orientations and Stereochemistries at the Covalent Linkage Site

Steve Cheatham, Alan Kook, Laurence H. Hurley, Mary D. Barkley, William Remers

Research output: Contribution to journalArticle

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Abstract

Tomaymycin is a member of the pyrrolo [1,4] benzodiazepine antitumor-antibiotic group that binds covalently to the exocyclic 2-amino group of guanine in DNA. Previous correlation of fluorescence and NMR data suggested that the llR.llaS and the 11S.11aS diastereomers of tomaymycin could bind to DNA in two orientations relative to the covalently modified guanine (Barkley, M. D.; Cheatham, S.; Thurston, D. E.; Hurley, L. H. Biochemistry 1986,25, 3021-3031). We now report on fluorescence, one- and two-dimensional proton NMR, and molecular modeling studies of the tomaymycin-d(ATGCAT)2 adduct, which corroborate these earlier observations. Fluorescence measurements show that there are two species of tomaymycin bound to d(ATGCAT)2, which are tentatively identified as the 11R.11aS and 11S.11aS diastereomers. Two distinct sets of signals for the tomaymycin molecule are present in the proton NMR spectrum of the tomaymycin-d(ATGCAT)2duplex adduct. Two-dimensional correlation spectroscopy (2D-COSY) studies also show connectivities for four cytosine H5-H6 and eight thymine methyl-H6 protons and thus clearly establish the presence of two distinct species of tomaymycin-d(ATGCAT)2adducts in solution. A single scalar 11-11a 1H NMR coupling in the 2D-COSY spectrum is indicative of an adduct species that has an S configuration at the C-ll position. Two-dimensional nuclear Overhauser effect (NOESY) spectra of the tomaymycin- d(ATGCAT)2duplex adduct show that the adducts are relatively nondistortive. In a NOESY experiment, cross-peaks were identified between both the aromatic H9 proton and the ethylidine methyl protons of tomaymycin and two different adenine H2 protons of d(ATGCAT)2. Molecular mechanics calculations with amber show that the two species with the thermodynamically most favorable binding energies are the llR.llaS and llS.llaS isomers with their aromatic rings to the 5’ and 3’ sides of the covalently bound guanine, respectively. The NOEs observed between tomaymycin protons and adenine H2 protons are in accord with molecular modeling studies. Taken together, these results strongly suggest that the two forms of tomaymycin bound to d(ATGCAT)2are the 11S.11aS and 11R.11aS species, oriented with their aromatic rings to the 3’ and 5’ sides, respectively, of the covalently modified guanines.

Original languageEnglish (US)
Pages (from-to)583-590
Number of pages8
JournalJournal of Medicinal Chemistry
Volume31
Issue number3
DOIs
StatePublished - Mar 1 1988

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

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