Reactivity of Damaged Pyrimidines: Formation of a Schiff Base Intermediate at the Glycosidic Bond of Saturated Dihydrouridine

Abstract

DNA glycosylases catalyze the first step of the base excision repair (BER) pathway. The chemistry used by these enzymes for deglycosylation has been largely considered as the chemistry of the oxocarbenium ion, e.g., direct rupture of the C1'-N1 bond resulting in an oxocarbenium ion intermediate. Here we present mechanistic studies revealing the 2'-deoxyribose isomerization and subsequent deglycosylation processes in two pyrimidine lesions: 5,6-dihydro-2'-deoxyuridine (dHdU) and 5,6-dihydrothymidine (dHT), formed via ionizing radiation damage to 2'-deoxycytidine and thymidine, respectively, under anoxic conditions. Acid or heat treatment of these two lesions leads to the production of two pairs of C1' epimers containing a pyranose and a furanose, respectively, indicating that both lesions favor the rupture of the C1'-O4' bond, resulting in a Schiff base intermediate at the N-glycosidic bond. Such a Schiff base intermediate was trapped and characterized by either Pd-catalyzed hydrogenation or thiol-mediated addition reaction. In contrast, in undamaged 2'-deoxyuridine and thymidine, reactions at elevated temperatures lead to the release of nucleobases most likely via the traditional oxocarbenium ion pathway. DFT calculations further support the experimental findings, suggesting that the oxocarbenium ion intermediate is responsible for the deglycosylation process if the integrity of the pyrimidine ring is maintained, while the Schiff base intermediate is preferred if the C5═C6 bond is saturated. Currently, the oxocarbenium ion pathway is indicated to be solely responsible for the deglycosylation in BER enzymes, however our results suggest an alternative Schiff base mechanism which may be responsible for the repair of saturated pyrimidine damages.

Isomerization of 5-Hydroxy-5-methylhydantoin 2′-Deoxynucleoside into α-Furanose, β-Furanose, α-Pyranose, and β-Pyranose Anomers

“…Because a bulky substituent, such as HydT, tends to adopt a low energy equatorial conformation with respect to the pyranose ring, the preferential conformation of HydT-β- pyranose and HydT-α-pyranose are 4 C 1 and 1 C 4 , respectively. This is consistent with the chemical analyses of a number of 2′-deoxyribonucleosides containing modified bases: 5,6-dihydrouracil, N 1 -formamidopyrimidine, N 1 -biuret, and N 1 -urea. ,− Taking the preferential conformation of the sugar ring into account, the structure of products containing a pyranose moiety can be deduced from proton–proton coupling constants. A major distinction between α and β pyranose anomers ( 3a/b and 4a/b ) is the different coupling constants among H1′, H2′, and H2″.…”

“…Intermediate IV can subsequently transform into α and β, and furanose and pyranose anomers ( 1 – 4 ) by nucleophilic attack by either C3′–OH or C5′–OH on either the top side giving α-anomers or the bottom side giving β-anomers (Pathway B; Scheme ). Pathway B was recently proposed to describe the isomerization of 5,6-dihydro-2′-deoxyuridine in aqueous solution . However, protonation of O4′ of 5,6-dihydro-2′-deoxyuridine takes place under rather severe conditions involving treatment with 0.1 M HCl for 72 h to bring the reaction to completion.…”

“…An equal amount of 5R and 5S diastereomers indicates that the hydantoin ring undergoes ring−chain tautomerism with little preference for one or the other diastereomer. At pH 7 and 8, Pathway A (Scheme 1) explains the isomerization of 5R (1a) and 5S (1b) 31 However, protonation of O4′ of 5,6-dihydro-2′-deoxyuridine takes place under rather severe conditions involving treatment with 0.1 M HCl for 72 h to bring the reaction to completion. Isomerization also takes place under neutral conditions, but it requires heating to high temperature (90 °C).…”

“…N 1 -(2-Deoxy-β-D-erythro-pentofuranosyl)-5-hydroxy-5methylhydantoin (the 5R Diastereomer; 1a). 1 H and 13 C data are given in Table 1; proton−proton coupling constants are given in Table 2 (10), 247 (31), 229 (35), 211 (11), 117 (100), 99 (8).…”

A New Cross-Link for an Old Cross-Linking Drug: The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Derived from Abasic Sites in Addition to the Expected Drug-Bridged Cross-Links

“…65 The loss of damaged thymine residues also can give rise to Ap sites with a directly opposing adenine residue. 66 The work described here explored the properties of Ap sites arising from another important cellular process, the depurination of guanine and alkylguanine residues. These events give rise to Ap sites opposed by a cytosine residue.…”