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Identifying the Tautomeric Form of a Deoxyguanosine-Estrogen Quinone Intermediate.

Stack DE - Metabolites (2015)

Bottom Line: This tautomeric form was further verified by use of deuterium labelling of the catechol precursor use to form the estrogen o-quinone.HPLC-MS analysis indicates a reactive intermediate with a m/z of 552.22 consistent with a tautomeric form containing no deuterium.This intermediate is consistent with a reaction mechanism that involves: (1) proton assisted Michael addition; (2) re-aromatization of the estrogen A ring; and (3) glycosidic bond cleavage to form the known estrogen-DNA adduct, 4-OHE₁-1-N7Gua.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Nebraska at Omaha, 6001 Dodge Street, Omaha, NE 68182, USA. dstack@unomaha.edu.

ABSTRACT
Mechanistic insights into the reaction of an estrogen o-quinone with deoxyguanosine has been further investigated using high level density functional calculations in addition to the use of 4-hyroxycatecholestrone (4-OHE₁) regioselectivity labeled with deuterium at the C1-position. Calculations using the M06-2X functional with large basis sets indicate the tautomeric form of an estrogen-DNA adduct present when glycosidic bonds cleavage occurs is comprised of an aromatic A ring structure. This tautomeric form was further verified by use of deuterium labelling of the catechol precursor use to form the estrogen o-quinone. Regioselective deuterium labelling at the C1-position of the estrogen A ring allows discrimination between two tautomeric forms of a reaction intermediate either of which could be present during glycosidic bond cleavage. HPLC-MS analysis indicates a reactive intermediate with a m/z of 552.22 consistent with a tautomeric form containing no deuterium. This intermediate is consistent with a reaction mechanism that involves: (1) proton assisted Michael addition; (2) re-aromatization of the estrogen A ring; and (3) glycosidic bond cleavage to form the known estrogen-DNA adduct, 4-OHE₁-1-N7Gua.

No MeSH data available.


Oxidation of 4-OHE1-1-d to E1-3,4-Q-1-d and subsequent reaction with dG. (A) Reverse phase C-18 HPLC analysis after 1 h (B) Reverse phase C-18 HPLC analysis after 4 h (C) TOF-MS analysis of the early eluting peak in the positive ion mode.
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metabolites-05-00475-f007: Oxidation of 4-OHE1-1-d to E1-3,4-Q-1-d and subsequent reaction with dG. (A) Reverse phase C-18 HPLC analysis after 1 h (B) Reverse phase C-18 HPLC analysis after 4 h (C) TOF-MS analysis of the early eluting peak in the positive ion mode.

Mentions: The recent synthesis of 4-OHE1-1-d provides, after oxidation, an isotopically labelled form of E1-3,4-Q that would produce forms of 1 and 2 with different molecular mass. This allows identification of the reaction intermediate by MS. 4-OHE1-1-d was oxidized in acetonitrile at reduced temperature to form the E1-3,4-Q which was then added to a solution of excess dG in 1:1 acetic acid:water. Analysis of the reaction after 1 h shows an early eluting, polar compound (C18 reverse phase HPLC) that when isolated converts in a unimolecular fashion to the final 4-OHE1-1-N7Gua adduct (Figure 7). This early eluting compound was collected and frozen in liquid nitrogen until a time-of-flight high resolution mass spectrum (TOF-MS) could be performed to determine the mass of this intermediate.


Identifying the Tautomeric Form of a Deoxyguanosine-Estrogen Quinone Intermediate.

Stack DE - Metabolites (2015)

Oxidation of 4-OHE1-1-d to E1-3,4-Q-1-d and subsequent reaction with dG. (A) Reverse phase C-18 HPLC analysis after 1 h (B) Reverse phase C-18 HPLC analysis after 4 h (C) TOF-MS analysis of the early eluting peak in the positive ion mode.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588807&req=5

metabolites-05-00475-f007: Oxidation of 4-OHE1-1-d to E1-3,4-Q-1-d and subsequent reaction with dG. (A) Reverse phase C-18 HPLC analysis after 1 h (B) Reverse phase C-18 HPLC analysis after 4 h (C) TOF-MS analysis of the early eluting peak in the positive ion mode.
Mentions: The recent synthesis of 4-OHE1-1-d provides, after oxidation, an isotopically labelled form of E1-3,4-Q that would produce forms of 1 and 2 with different molecular mass. This allows identification of the reaction intermediate by MS. 4-OHE1-1-d was oxidized in acetonitrile at reduced temperature to form the E1-3,4-Q which was then added to a solution of excess dG in 1:1 acetic acid:water. Analysis of the reaction after 1 h shows an early eluting, polar compound (C18 reverse phase HPLC) that when isolated converts in a unimolecular fashion to the final 4-OHE1-1-N7Gua adduct (Figure 7). This early eluting compound was collected and frozen in liquid nitrogen until a time-of-flight high resolution mass spectrum (TOF-MS) could be performed to determine the mass of this intermediate.

Bottom Line: This tautomeric form was further verified by use of deuterium labelling of the catechol precursor use to form the estrogen o-quinone.HPLC-MS analysis indicates a reactive intermediate with a m/z of 552.22 consistent with a tautomeric form containing no deuterium.This intermediate is consistent with a reaction mechanism that involves: (1) proton assisted Michael addition; (2) re-aromatization of the estrogen A ring; and (3) glycosidic bond cleavage to form the known estrogen-DNA adduct, 4-OHE₁-1-N7Gua.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Nebraska at Omaha, 6001 Dodge Street, Omaha, NE 68182, USA. dstack@unomaha.edu.

ABSTRACT
Mechanistic insights into the reaction of an estrogen o-quinone with deoxyguanosine has been further investigated using high level density functional calculations in addition to the use of 4-hyroxycatecholestrone (4-OHE₁) regioselectivity labeled with deuterium at the C1-position. Calculations using the M06-2X functional with large basis sets indicate the tautomeric form of an estrogen-DNA adduct present when glycosidic bonds cleavage occurs is comprised of an aromatic A ring structure. This tautomeric form was further verified by use of deuterium labelling of the catechol precursor use to form the estrogen o-quinone. Regioselective deuterium labelling at the C1-position of the estrogen A ring allows discrimination between two tautomeric forms of a reaction intermediate either of which could be present during glycosidic bond cleavage. HPLC-MS analysis indicates a reactive intermediate with a m/z of 552.22 consistent with a tautomeric form containing no deuterium. This intermediate is consistent with a reaction mechanism that involves: (1) proton assisted Michael addition; (2) re-aromatization of the estrogen A ring; and (3) glycosidic bond cleavage to form the known estrogen-DNA adduct, 4-OHE₁-1-N7Gua.

No MeSH data available.