Methyl 2-azido-2-deoxy-α-D-lyxofuranoside (1a) and methyl 2-azido-2-deoxy-β-D-ribofuranoside (2) were prepared from D-xylose or D-arabinose, respectively. Employing ESR and DFT/B3LYP/6-31G* calculations, we investigated (i) aminyl radical (RNH•) formation and (ii) reaction pathways of RNH•. Prehydrated electron attachment to 1a and 2 at 77 K produced transient azide anion radical (RN3•−) which reacts via rapid N2 loss at 77 K, forming nitrene anion radical (RN•−). Rapid protonation of RN•− at 77 K formed RNH• and −OH. 15N-labeled-1a confirmed this mechanism. Investigations employing in-house synthesized site-specifically deuterated derivatives of 1a (e.g., CH3 (1b), C4 (1c), and C5 (1d)) established that: (a) a facile intramolecular H-atom transfer from C5 to RNH• generated C5• and RNH2. C5• formation had a small deuterium kinetic isotope effect suggesting that this reaction does not occur via direct H-atom abstraction. (b) Subsequently, C5• underwent a facile unimolecular conversion to ring-opened C4• under a reductive environment. Identification of ring-opened C4• intermediate confirms the mechanism of C5′• mediated unaltered base release associated with DNA-strand break. However, for 2, ESR studies established thermally-activated intermolecular H-atom abstraction by RNH• from methyl group at C1. Thus, sugar ring configuration strongly influence site and pathways of RNH• mediated reactions in pentafuranoses.
In
this work, electron-induced site-specific formation of neutral
π-type aminyl radicals (RNH·) and their reactions with
pyrimidine nucleoside analogs azidolabeled at various positions in
the sugar moiety, e.g., at 2′-, 3′-, 4′-, and
5′- sites along with a model compound 3-azido-1-propanol (3AZPrOH),
were investigated. Electron paramagnetic resonance (EPR) studies confirmed
the site and mechanism of RNH· formation via dissociative electron
attachment-mediated loss of N2 and subsequent facile protonation
from the solvent employing the 15N-labeled azido group,
deuterations at specific sites in the sugar and base, and changing
the solvent from H2O to D2O. Reactions of RNH·
were investigated employing EPR by warming these samples from 77 K
to ca. 170 K. RNH· at a primary carbon site (5′-azido-2′,5′-dideoxyuridine,
3AZPrOH) facilely converted to a σ-type iminyl radical (RN·)
via a bimolecular H-atom abstraction forming an α-azidoalkyl
radical. RNH· when at a secondary carbon site (e.g., 2′-azido-2′-deoxyuridine)
underwent bimolecular electrophilic addition to the C5C6 double
bond of a proximate pyrimidine base. Finally, RNH· at tertiary
alkyl carbon (4′-azidocytidine) underwent little reaction.
These results show the influence of the stereochemical and electronic
environment on RNH· reactivity and allow the selection of those
azidonucleosides that would be most effective in augmenting cellular
radiation damage.
Model 3′-azido-3′-deoxynucleosides with thiol or vicinal dithiol substituents at C2′ or C5′ were synthesized to study reactions postulated to occur during inhibition of ribonucleotide reductases by 2′-azido-2′-deoxynucleotides. Esterification of 5′-(tert-butyldiphenylsilyl)-3′-azido-3′-deoxyadenosine and 3′-azido-3′-deoxythymidine (AZT) with 2,3-S-isopropylidene-2,3-dimercaptopropanoic acid or N-Boc-S-trityl-L-cysteine and deprotection gave 3′-azido-3′-deoxy-2′-O-(2,3-dimercaptopropanoyl or cysteinyl)adenosine and the 3′-azido-3′-deoxy-5′-O-(2,3-dimercaptopropanoyl or cysteinyl)thymidine analogs. Density functional calculations predicted that intramolecular reactions between generated thiyl radicals and an azido group on such model compounds would be exothermic by 33.6-41.2 kcal/mol and have low energy barriers of 10.4-13.5 kcal/mol. Reduction of the azido group occurred to give 3′-amino-3′-deoxythymidine, which was postulated to occur with thiyl radicals generated by treatment of 3′-azido-3′-deoxy-5′-O-(2,3-dimercaptopropanoyl)thymidine with 2,2′-azobis-(2-methyl-2-propionamidine) dihydrochloride. Gamma radiolysis of N2O-saturated aqueous solutions of AZT and cysteine produced 3′-amino-3′-deoxythymidine and thymine most likely by both radical and ionic processes.
Dedicated to Professor Antonin Holý on the occasion of his 75th birthday in recognition of his outstanding contributions to the area of nucleic acid chemistry.The 1,4-anhydro-5-deoxy-6-thio-D-ribo-hexofuranitol (1) was prepared from 1,2-O-isopropylidene-α-D-glucose in 10 steps. In a key step treatment of the 1,2-O-isopropylidenehexofuranose derivative with BF 3 /Et 3 SiH effected deacetonization and reductive deoxygenation at carbon 1. Pulse radiolysis experiments with 6-thiohexofuranitol 1 and its disulfide derivative demonstrated formation of the ribosyl-based carbon-centered radical upon generation of 6-thiyl radical in basic medium. The proposed [1,5]-hydrogen shift abstraction with generation of the C3 radical mimics the initial substrate reaction of RNRs. The reversible H-atom transfer has been quantified and was correlated with the computed rate constants for the internal H atom abstraction from C1, C2, C3 and C4 by the thiyl radical. The energy barrier for the H3 and H4 abstractions were calculated to be most favorable with the corresponding barriers of 11.1 and 11.2 kcal/mol, respectively.
This dissertation, written by Thao P. Dang, and entitled Biomimetic Modeling of the Nitrogen-centered radical Postulated to occur during the Inhibition of Ribonucleotide Reductases by 2'-Azido-2'-deoxynucleotides, having been approved in respect to style and intellectual content, is referred to you for judgment.We have read this dissertation and recommend that it be approved.
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