NMR and computational characterization of mitomycin cross-linked to adjacent deoxyguanosines in the minor groove of the d(T-A-C-G-T-A)·d(T-A-C-G-T-A)duplex

David Norman, David Live, Mallika Sastry, Roselyn Lipman, Brian E. Hingerty, Maria Tomasz, Suse Broyde, Dinshaw J. Patel

Research output: Contribution to journalArticle

Abstract

Two-dimensional homonuclear and heteronuclear NMR and minimized potential energy calculations have been combined to define the structure of the antitumor agent mitomycin C (MC) cross-linked to deoxyguanosines on adjacent base pairs in the d(T1-A2-C3-G4-T5-A6)-d(T7-A8-C9-G10-T11-A12) duplex. The majority of the mitomycin and nucleic acid protons in the MC-X 6-mer complex have been assigned from through-bond and through-space two-dimensional proton NMR studies in aqueous solution at 5 and 20 °C. The C3·G10 and G4·C9 base pairs are intact at the cross-link site and stack on each other in the complex. The amino protons of G4 and G10 resonate at 9.36 and 8.87 ppm and exhibit slow exchange with solvent H2O. The NMR experimental data establish that the mitomycin is cross-linked to the DNA through the amino groups of G4 and G10 and is positioned in the minor groove. The conformation of the cross-link site is defined by a set of NOEs between the mitomycin H1″ and H2″ protons and the nucleic acid imino and amino protons of G4 and the H2 proton of A8 and another set of NOEs between the mitomycin geminal H10″ protons and the nucleic acid imino and amino protons of G10 and the H2 proton of A2. Several phosphorus resonances of the d(T-A-C-G-T-A) duplex shift dramaticaly on mitomycin cross-link formation and have been assigned from proton-detected phosphorus-proton two-dimensional correlation experiments. The proton chemical shifts and NOEs establish fraying at the ends of the d(T-A-C-G-T-A) duplex, and this feature is retained on mitomycin cross-link formation. The base-base and base-sugar NOEs exhibit similar patterns for symmetry-related steps on the two nucleic acid strands in the MC-X 6-mer complex, while the proton and phosphorus chemical shifts are dramatically perturbed at the G10-T11 step on cross-link formation. The NMR distance constraints have been included in minimized potential energy computations on the MC-X 6-mer complex. These computations were undertaken with the nonplanar five-membered ring of mitomycin in each of two pucker orientations. The resulting low-energy structures MX1 and MX2 have the mitomycin cross-linked in a widened minor groove with the chromophore ring system in the vicinity of the G10-T11 step on one of the two strands in the duplex. The experimental evidence supports this model since the H1′ of G10 is shifted dramatically upfield and the H1′ proton of Til is shifted dramatically downfield on complex formation. The unconstrained final minimum energy conformations MX1 and MX2, which differ in the pucker of the five-membered ring of mitomycin, exhibit similar torsion angles at the bonds involved in covalent linkage of the antitumor agent to the DNA. However, there are large differences in several nucleic acid backbone torsion angles between conformations MX1 and MX2. A choice between them cannot be currently made since both MX1 and MX2 satisfy the available experiment NMR parameters for the MC-X 6-mer complex in solution.

Original languageEnglish (US)
Pages (from-to)2861-2875
Number of pages15
JournalBiochemistry
Volume29
Issue number11
StatePublished - 1990

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Deoxyguanosine
Mitomycin
Protons
Nuclear magnetic resonance
Nucleic Acids
Phosphorus
Conformations
Chemical shift
Potential energy
deoxythymidylyl-3'-5'-deoxyadenylate
2'-deoxyadenylyl(3'-5')thymidylyl(3'-5')deoxyguanosylyl(3'-5')thymidine
varespladib methyl
Base Pairing
Antineoplastic Agents
Torsional stress
Biomolecular Nuclear Magnetic Resonance
compound A 12
DNA
Chromophores
Sugars

ASJC Scopus subject areas

  • Biochemistry

Cite this

Norman, D., Live, D., Sastry, M., Lipman, R., Hingerty, B. E., Tomasz, M., ... Patel, D. J. (1990). NMR and computational characterization of mitomycin cross-linked to adjacent deoxyguanosines in the minor groove of the d(T-A-C-G-T-A)·d(T-A-C-G-T-A)duplex. Biochemistry, 29(11), 2861-2875.

NMR and computational characterization of mitomycin cross-linked to adjacent deoxyguanosines in the minor groove of the d(T-A-C-G-T-A)·d(T-A-C-G-T-A)duplex. / Norman, David; Live, David; Sastry, Mallika; Lipman, Roselyn; Hingerty, Brian E.; Tomasz, Maria; Broyde, Suse; Patel, Dinshaw J.

In: Biochemistry, Vol. 29, No. 11, 1990, p. 2861-2875.

Research output: Contribution to journalArticle

Norman, D, Live, D, Sastry, M, Lipman, R, Hingerty, BE, Tomasz, M, Broyde, S & Patel, DJ 1990, 'NMR and computational characterization of mitomycin cross-linked to adjacent deoxyguanosines in the minor groove of the d(T-A-C-G-T-A)·d(T-A-C-G-T-A)duplex', Biochemistry, vol. 29, no. 11, pp. 2861-2875.
Norman, David ; Live, David ; Sastry, Mallika ; Lipman, Roselyn ; Hingerty, Brian E. ; Tomasz, Maria ; Broyde, Suse ; Patel, Dinshaw J. / NMR and computational characterization of mitomycin cross-linked to adjacent deoxyguanosines in the minor groove of the d(T-A-C-G-T-A)·d(T-A-C-G-T-A)duplex. In: Biochemistry. 1990 ; Vol. 29, No. 11. pp. 2861-2875.
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title = "NMR and computational characterization of mitomycin cross-linked to adjacent deoxyguanosines in the minor groove of the d(T-A-C-G-T-A)·d(T-A-C-G-T-A)duplex",
abstract = "Two-dimensional homonuclear and heteronuclear NMR and minimized potential energy calculations have been combined to define the structure of the antitumor agent mitomycin C (MC) cross-linked to deoxyguanosines on adjacent base pairs in the d(T1-A2-C3-G4-T5-A6)-d(T7-A8-C9-G10-T11-A12) duplex. The majority of the mitomycin and nucleic acid protons in the MC-X 6-mer complex have been assigned from through-bond and through-space two-dimensional proton NMR studies in aqueous solution at 5 and 20 °C. The C3·G10 and G4·C9 base pairs are intact at the cross-link site and stack on each other in the complex. The amino protons of G4 and G10 resonate at 9.36 and 8.87 ppm and exhibit slow exchange with solvent H2O. The NMR experimental data establish that the mitomycin is cross-linked to the DNA through the amino groups of G4 and G10 and is positioned in the minor groove. The conformation of the cross-link site is defined by a set of NOEs between the mitomycin H1″ and H2″ protons and the nucleic acid imino and amino protons of G4 and the H2 proton of A8 and another set of NOEs between the mitomycin geminal H10″ protons and the nucleic acid imino and amino protons of G10 and the H2 proton of A2. Several phosphorus resonances of the d(T-A-C-G-T-A) duplex shift dramaticaly on mitomycin cross-link formation and have been assigned from proton-detected phosphorus-proton two-dimensional correlation experiments. The proton chemical shifts and NOEs establish fraying at the ends of the d(T-A-C-G-T-A) duplex, and this feature is retained on mitomycin cross-link formation. The base-base and base-sugar NOEs exhibit similar patterns for symmetry-related steps on the two nucleic acid strands in the MC-X 6-mer complex, while the proton and phosphorus chemical shifts are dramatically perturbed at the G10-T11 step on cross-link formation. The NMR distance constraints have been included in minimized potential energy computations on the MC-X 6-mer complex. These computations were undertaken with the nonplanar five-membered ring of mitomycin in each of two pucker orientations. The resulting low-energy structures MX1 and MX2 have the mitomycin cross-linked in a widened minor groove with the chromophore ring system in the vicinity of the G10-T11 step on one of the two strands in the duplex. The experimental evidence supports this model since the H1′ of G10 is shifted dramatically upfield and the H1′ proton of Til is shifted dramatically downfield on complex formation. The unconstrained final minimum energy conformations MX1 and MX2, which differ in the pucker of the five-membered ring of mitomycin, exhibit similar torsion angles at the bonds involved in covalent linkage of the antitumor agent to the DNA. However, there are large differences in several nucleic acid backbone torsion angles between conformations MX1 and MX2. A choice between them cannot be currently made since both MX1 and MX2 satisfy the available experiment NMR parameters for the MC-X 6-mer complex in solution.",
author = "David Norman and David Live and Mallika Sastry and Roselyn Lipman and Hingerty, {Brian E.} and Maria Tomasz and Suse Broyde and Patel, {Dinshaw J.}",
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TY - JOUR

T1 - NMR and computational characterization of mitomycin cross-linked to adjacent deoxyguanosines in the minor groove of the d(T-A-C-G-T-A)·d(T-A-C-G-T-A)duplex

AU - Norman, David

AU - Live, David

AU - Sastry, Mallika

AU - Lipman, Roselyn

AU - Hingerty, Brian E.

AU - Tomasz, Maria

AU - Broyde, Suse

AU - Patel, Dinshaw J.

PY - 1990

Y1 - 1990

N2 - Two-dimensional homonuclear and heteronuclear NMR and minimized potential energy calculations have been combined to define the structure of the antitumor agent mitomycin C (MC) cross-linked to deoxyguanosines on adjacent base pairs in the d(T1-A2-C3-G4-T5-A6)-d(T7-A8-C9-G10-T11-A12) duplex. The majority of the mitomycin and nucleic acid protons in the MC-X 6-mer complex have been assigned from through-bond and through-space two-dimensional proton NMR studies in aqueous solution at 5 and 20 °C. The C3·G10 and G4·C9 base pairs are intact at the cross-link site and stack on each other in the complex. The amino protons of G4 and G10 resonate at 9.36 and 8.87 ppm and exhibit slow exchange with solvent H2O. The NMR experimental data establish that the mitomycin is cross-linked to the DNA through the amino groups of G4 and G10 and is positioned in the minor groove. The conformation of the cross-link site is defined by a set of NOEs between the mitomycin H1″ and H2″ protons and the nucleic acid imino and amino protons of G4 and the H2 proton of A8 and another set of NOEs between the mitomycin geminal H10″ protons and the nucleic acid imino and amino protons of G10 and the H2 proton of A2. Several phosphorus resonances of the d(T-A-C-G-T-A) duplex shift dramaticaly on mitomycin cross-link formation and have been assigned from proton-detected phosphorus-proton two-dimensional correlation experiments. The proton chemical shifts and NOEs establish fraying at the ends of the d(T-A-C-G-T-A) duplex, and this feature is retained on mitomycin cross-link formation. The base-base and base-sugar NOEs exhibit similar patterns for symmetry-related steps on the two nucleic acid strands in the MC-X 6-mer complex, while the proton and phosphorus chemical shifts are dramatically perturbed at the G10-T11 step on cross-link formation. The NMR distance constraints have been included in minimized potential energy computations on the MC-X 6-mer complex. These computations were undertaken with the nonplanar five-membered ring of mitomycin in each of two pucker orientations. The resulting low-energy structures MX1 and MX2 have the mitomycin cross-linked in a widened minor groove with the chromophore ring system in the vicinity of the G10-T11 step on one of the two strands in the duplex. The experimental evidence supports this model since the H1′ of G10 is shifted dramatically upfield and the H1′ proton of Til is shifted dramatically downfield on complex formation. The unconstrained final minimum energy conformations MX1 and MX2, which differ in the pucker of the five-membered ring of mitomycin, exhibit similar torsion angles at the bonds involved in covalent linkage of the antitumor agent to the DNA. However, there are large differences in several nucleic acid backbone torsion angles between conformations MX1 and MX2. A choice between them cannot be currently made since both MX1 and MX2 satisfy the available experiment NMR parameters for the MC-X 6-mer complex in solution.

AB - Two-dimensional homonuclear and heteronuclear NMR and minimized potential energy calculations have been combined to define the structure of the antitumor agent mitomycin C (MC) cross-linked to deoxyguanosines on adjacent base pairs in the d(T1-A2-C3-G4-T5-A6)-d(T7-A8-C9-G10-T11-A12) duplex. The majority of the mitomycin and nucleic acid protons in the MC-X 6-mer complex have been assigned from through-bond and through-space two-dimensional proton NMR studies in aqueous solution at 5 and 20 °C. The C3·G10 and G4·C9 base pairs are intact at the cross-link site and stack on each other in the complex. The amino protons of G4 and G10 resonate at 9.36 and 8.87 ppm and exhibit slow exchange with solvent H2O. The NMR experimental data establish that the mitomycin is cross-linked to the DNA through the amino groups of G4 and G10 and is positioned in the minor groove. The conformation of the cross-link site is defined by a set of NOEs between the mitomycin H1″ and H2″ protons and the nucleic acid imino and amino protons of G4 and the H2 proton of A8 and another set of NOEs between the mitomycin geminal H10″ protons and the nucleic acid imino and amino protons of G10 and the H2 proton of A2. Several phosphorus resonances of the d(T-A-C-G-T-A) duplex shift dramaticaly on mitomycin cross-link formation and have been assigned from proton-detected phosphorus-proton two-dimensional correlation experiments. The proton chemical shifts and NOEs establish fraying at the ends of the d(T-A-C-G-T-A) duplex, and this feature is retained on mitomycin cross-link formation. The base-base and base-sugar NOEs exhibit similar patterns for symmetry-related steps on the two nucleic acid strands in the MC-X 6-mer complex, while the proton and phosphorus chemical shifts are dramatically perturbed at the G10-T11 step on cross-link formation. The NMR distance constraints have been included in minimized potential energy computations on the MC-X 6-mer complex. These computations were undertaken with the nonplanar five-membered ring of mitomycin in each of two pucker orientations. The resulting low-energy structures MX1 and MX2 have the mitomycin cross-linked in a widened minor groove with the chromophore ring system in the vicinity of the G10-T11 step on one of the two strands in the duplex. The experimental evidence supports this model since the H1′ of G10 is shifted dramatically upfield and the H1′ proton of Til is shifted dramatically downfield on complex formation. The unconstrained final minimum energy conformations MX1 and MX2, which differ in the pucker of the five-membered ring of mitomycin, exhibit similar torsion angles at the bonds involved in covalent linkage of the antitumor agent to the DNA. However, there are large differences in several nucleic acid backbone torsion angles between conformations MX1 and MX2. A choice between them cannot be currently made since both MX1 and MX2 satisfy the available experiment NMR parameters for the MC-X 6-mer complex in solution.

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