Characterization of the structure of the anthramycin-d(ATGCAT)2 adduct by NMR and molecular modeling studies. Determination of the stereochemistry of the covalent linkage site, orientation in the minor groove of DNA, and effect on local DNA structure

F. Leslie Boyd, Steven F. Cheatham, William Remers, G. Craig Hill, Laurence Hurley

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Abstract

Anthramycin is a member of the pyrrolo[1,4]benzodiazepine group of antitumor antibiotics. Previous studies have demonstrated that anthramycin binds covalently through N-2 of guanine within the minor groove of DNA, resulting in a relatively nondistortive DNA adduct. From the nuclear Overhauser effect spectroscopy (NOESY) proton NMR spectra of the anthramycin-d(ATGCAT)2 adduct, we have obtained results that unambiguously assign the orientation of the drug molecule in the minor groove of DNA. Four sets of NOE cross-peaks between anthramycin protons and nucleotide protons on either the covalently or the noncovalently modified strands reveal that the drug is specifically oriented with the aromatic ring to the 3′-side of the covalently modified guanine. Unequivocal assignment of the geometry at the site of attachment of anthramycin to d(ATGCAT)2 cannot be made by J-correlated spectroscopy (COSY). However, when combined with the results of modeling with the molecular mechanics program AMBER, an 11S stereochemistry at this site can be confidently predicted. 31P NMR studies show that two of the resonance signals in the anthramycin-d(ATGCAT)2 adduct have moved significantly downfield. Both downfield 31P NMR signals have been assigned by 17O isotopic labeling and 1H-31P two-dimensional J-correlation experiments and shown to correspond to the phosphates on the 5′-sides of the covalently modified deoxyguanine and the deoxycytosine on the opposite strand. Assignment of resonance signals of nonexchangeable base and sugar protons of the anthramycin-d(ATGCAT)2 has been made with two-dimensional Fourier transform NMR methods (COSY and NOESY). Conformational details about the sugar puckers, the glycosidic dihedral angles, and the effect of anthramycin bonding on secondary structure of the duplex have been obtained from the relative intensities of cross-peaks in the two-dimensional NMR spectra in aqueous solution. All of the sugars that are amenable to this analysis possess a conformation consistent with B-type DNA. Molecular mechanics calculations with AMBER are predictive of the orientation and stereochemistry of anthramycin bound to d(ATGCAT)2. The species having an 11S stereochemistry at the covalent bonding site and oriented with the aromatic ring of anthramycin to the 3′-side of the covalently modified guanine of anthramycin-d(ATGCAT)2 appears to be favored over the three other possible species. This is because of the greater intermolecular binding for this species rather than lower helix distortion energies. The molecular modeling is also in accord with the experimentally determined nondistortive nature of the anthramycin-d-(ATGCAT)2 adduct.

Original languageEnglish (US)
Pages (from-to)3279-3289
Number of pages11
JournalJournal of the American Chemical Society
Volume112
Issue number9
StatePublished - Apr 25 1990
Externally publishedYes

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Anthramycin
Stereochemistry
Molecular modeling
DNA
Nuclear magnetic resonance
Protons
Sugars
Spectroscopy
Molecular mechanics
Guanine
Antibiotics
Dihedral angle
Spectrum Analysis
Nucleotides
Labeling
Conformations
Mechanics
Fourier transforms
Phosphates
d(ATGCAT)2

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

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title = "Characterization of the structure of the anthramycin-d(ATGCAT)2 adduct by NMR and molecular modeling studies. Determination of the stereochemistry of the covalent linkage site, orientation in the minor groove of DNA, and effect on local DNA structure",
abstract = "Anthramycin is a member of the pyrrolo[1,4]benzodiazepine group of antitumor antibiotics. Previous studies have demonstrated that anthramycin binds covalently through N-2 of guanine within the minor groove of DNA, resulting in a relatively nondistortive DNA adduct. From the nuclear Overhauser effect spectroscopy (NOESY) proton NMR spectra of the anthramycin-d(ATGCAT)2 adduct, we have obtained results that unambiguously assign the orientation of the drug molecule in the minor groove of DNA. Four sets of NOE cross-peaks between anthramycin protons and nucleotide protons on either the covalently or the noncovalently modified strands reveal that the drug is specifically oriented with the aromatic ring to the 3′-side of the covalently modified guanine. Unequivocal assignment of the geometry at the site of attachment of anthramycin to d(ATGCAT)2 cannot be made by J-correlated spectroscopy (COSY). However, when combined with the results of modeling with the molecular mechanics program AMBER, an 11S stereochemistry at this site can be confidently predicted. 31P NMR studies show that two of the resonance signals in the anthramycin-d(ATGCAT)2 adduct have moved significantly downfield. Both downfield 31P NMR signals have been assigned by 17O isotopic labeling and 1H-31P two-dimensional J-correlation experiments and shown to correspond to the phosphates on the 5′-sides of the covalently modified deoxyguanine and the deoxycytosine on the opposite strand. Assignment of resonance signals of nonexchangeable base and sugar protons of the anthramycin-d(ATGCAT)2 has been made with two-dimensional Fourier transform NMR methods (COSY and NOESY). Conformational details about the sugar puckers, the glycosidic dihedral angles, and the effect of anthramycin bonding on secondary structure of the duplex have been obtained from the relative intensities of cross-peaks in the two-dimensional NMR spectra in aqueous solution. All of the sugars that are amenable to this analysis possess a conformation consistent with B-type DNA. Molecular mechanics calculations with AMBER are predictive of the orientation and stereochemistry of anthramycin bound to d(ATGCAT)2. The species having an 11S stereochemistry at the covalent bonding site and oriented with the aromatic ring of anthramycin to the 3′-side of the covalently modified guanine of anthramycin-d(ATGCAT)2 appears to be favored over the three other possible species. This is because of the greater intermolecular binding for this species rather than lower helix distortion energies. The molecular modeling is also in accord with the experimentally determined nondistortive nature of the anthramycin-d-(ATGCAT)2 adduct.",
author = "Boyd, {F. Leslie} and Cheatham, {Steven F.} and William Remers and Hill, {G. Craig} and Laurence Hurley",
year = "1990",
month = "4",
day = "25",
language = "English (US)",
volume = "112",
pages = "3279--3289",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
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TY - JOUR

T1 - Characterization of the structure of the anthramycin-d(ATGCAT)2 adduct by NMR and molecular modeling studies. Determination of the stereochemistry of the covalent linkage site, orientation in the minor groove of DNA, and effect on local DNA structure

AU - Boyd, F. Leslie

AU - Cheatham, Steven F.

AU - Remers, William

AU - Hill, G. Craig

AU - Hurley, Laurence

PY - 1990/4/25

Y1 - 1990/4/25

N2 - Anthramycin is a member of the pyrrolo[1,4]benzodiazepine group of antitumor antibiotics. Previous studies have demonstrated that anthramycin binds covalently through N-2 of guanine within the minor groove of DNA, resulting in a relatively nondistortive DNA adduct. From the nuclear Overhauser effect spectroscopy (NOESY) proton NMR spectra of the anthramycin-d(ATGCAT)2 adduct, we have obtained results that unambiguously assign the orientation of the drug molecule in the minor groove of DNA. Four sets of NOE cross-peaks between anthramycin protons and nucleotide protons on either the covalently or the noncovalently modified strands reveal that the drug is specifically oriented with the aromatic ring to the 3′-side of the covalently modified guanine. Unequivocal assignment of the geometry at the site of attachment of anthramycin to d(ATGCAT)2 cannot be made by J-correlated spectroscopy (COSY). However, when combined with the results of modeling with the molecular mechanics program AMBER, an 11S stereochemistry at this site can be confidently predicted. 31P NMR studies show that two of the resonance signals in the anthramycin-d(ATGCAT)2 adduct have moved significantly downfield. Both downfield 31P NMR signals have been assigned by 17O isotopic labeling and 1H-31P two-dimensional J-correlation experiments and shown to correspond to the phosphates on the 5′-sides of the covalently modified deoxyguanine and the deoxycytosine on the opposite strand. Assignment of resonance signals of nonexchangeable base and sugar protons of the anthramycin-d(ATGCAT)2 has been made with two-dimensional Fourier transform NMR methods (COSY and NOESY). Conformational details about the sugar puckers, the glycosidic dihedral angles, and the effect of anthramycin bonding on secondary structure of the duplex have been obtained from the relative intensities of cross-peaks in the two-dimensional NMR spectra in aqueous solution. All of the sugars that are amenable to this analysis possess a conformation consistent with B-type DNA. Molecular mechanics calculations with AMBER are predictive of the orientation and stereochemistry of anthramycin bound to d(ATGCAT)2. The species having an 11S stereochemistry at the covalent bonding site and oriented with the aromatic ring of anthramycin to the 3′-side of the covalently modified guanine of anthramycin-d(ATGCAT)2 appears to be favored over the three other possible species. This is because of the greater intermolecular binding for this species rather than lower helix distortion energies. The molecular modeling is also in accord with the experimentally determined nondistortive nature of the anthramycin-d-(ATGCAT)2 adduct.

AB - Anthramycin is a member of the pyrrolo[1,4]benzodiazepine group of antitumor antibiotics. Previous studies have demonstrated that anthramycin binds covalently through N-2 of guanine within the minor groove of DNA, resulting in a relatively nondistortive DNA adduct. From the nuclear Overhauser effect spectroscopy (NOESY) proton NMR spectra of the anthramycin-d(ATGCAT)2 adduct, we have obtained results that unambiguously assign the orientation of the drug molecule in the minor groove of DNA. Four sets of NOE cross-peaks between anthramycin protons and nucleotide protons on either the covalently or the noncovalently modified strands reveal that the drug is specifically oriented with the aromatic ring to the 3′-side of the covalently modified guanine. Unequivocal assignment of the geometry at the site of attachment of anthramycin to d(ATGCAT)2 cannot be made by J-correlated spectroscopy (COSY). However, when combined with the results of modeling with the molecular mechanics program AMBER, an 11S stereochemistry at this site can be confidently predicted. 31P NMR studies show that two of the resonance signals in the anthramycin-d(ATGCAT)2 adduct have moved significantly downfield. Both downfield 31P NMR signals have been assigned by 17O isotopic labeling and 1H-31P two-dimensional J-correlation experiments and shown to correspond to the phosphates on the 5′-sides of the covalently modified deoxyguanine and the deoxycytosine on the opposite strand. Assignment of resonance signals of nonexchangeable base and sugar protons of the anthramycin-d(ATGCAT)2 has been made with two-dimensional Fourier transform NMR methods (COSY and NOESY). Conformational details about the sugar puckers, the glycosidic dihedral angles, and the effect of anthramycin bonding on secondary structure of the duplex have been obtained from the relative intensities of cross-peaks in the two-dimensional NMR spectra in aqueous solution. All of the sugars that are amenable to this analysis possess a conformation consistent with B-type DNA. Molecular mechanics calculations with AMBER are predictive of the orientation and stereochemistry of anthramycin bound to d(ATGCAT)2. The species having an 11S stereochemistry at the covalent bonding site and oriented with the aromatic ring of anthramycin to the 3′-side of the covalently modified guanine of anthramycin-d(ATGCAT)2 appears to be favored over the three other possible species. This is because of the greater intermolecular binding for this species rather than lower helix distortion energies. The molecular modeling is also in accord with the experimentally determined nondistortive nature of the anthramycin-d-(ATGCAT)2 adduct.

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