A synthesis of [3′,4′‐13C2]thymidine (1) is described in which [13C2]acetic acid (2) is converted into the nucleoside in twelve steps with 9% overall yield. D‐2‐Deoxyribose‐5‐phosphate aldolase (DERA, EC 4.1.2.4) and triosephosphate isomerase (TPI, EC 5.3.1.1) are used for the stereocontrolled formation of D‐[3,4‐13C2]‐2‐deoxyribose‐5‐phosphate (8) from [2,3‐13C2]dihydroxyacetone monophosphate (DHAP, 7) and acetaldehyde in 80% yield. The route permits the introduction of isotopically enriched carbon atoms at any position or combination of positions in the furanose ring and the product can be coupled with any of the four naturally occurring base moieties.

The H3'-C3'-C4'-H4' torsional angles of two microcrystalline 2'-deoxynucleosides, thymidine and 2'-deoxycytidine.HCl, doubly (13)C-labeled at the C3' and C4' positions of the sugar ring, have been measured by solid-state magic-angle-spinning nuclear magnetic resonance (NMR). A double-quantum heteronuclear local field experiment with frequency-switched Lee-Goldberg homonuclear decoupling was used. The H3'-C3'-C4'-H4' torsional angles were obtained by comparing the experimental curves with numerical simulations, including the two (13)C nuclei, the directly bonded (1)H nuclei, and five remote protons. The H3'-C3'-C4'-H4' angles were converted into sugar pucker angles and compared with crystallographic data. The delta torsional angles determined by solid-state NMR and x-ray crystallography agree within experimental error. Evidence is also obtained that the proton positions may be unreliable in the x-ray structures. This work confirms that double-quantum solid-state NMR is a feasible tool for studying sugar pucker conformations in macromolecular complexes that are unsuitable for solution NMR or crystallography.

The enzymatic synthesis of thymidine from 2-deoxy-D-ribose-5-phosphate is achieved, in a one-pot two-step reaction using phosphoribomutase (PRM) and commercially available thymidine phosphorylase (TP). In the first step the sugar-5-phosphate is enzymatically rearranged to alpha-2-deoxy-D-ribose-1-phosphate. Highly active PRM is easily obtained from genetically modified overproducing E. coli cells (12,000 units/84 mg protein) and is used without further purification. In the second step thymine is coupled to the sugar-1-phosphate. The thermodynamically unfavorable equilibrium is shifted to the product by addition of MnCl(2) to precipitate inorganic phosphate. In this way the overall yield of the beta-anomeric pure nucleoside increases from 14 to 60%. In contrast to uracil, cytosine is not accepted by TP as a substrate. Therefore, 2'-deoxy-cytidine is obtained by functional group transformations of the enzymatically prepared 2'-deoxy-uridine. The method has been demonstrated by the synthesis of [2',5'-(13)C(2)]- and [1',2',5'-(13)C(3)]thymidine as well as [1',2',5'-(13)C(3)]2'-deoxyuridine and [3',4'-(13)C(2)]2'-deoxycytidine. In addition the nucleoside bases thymine and uracil are tetralabeled at the (1,3-(15)N(2),2,4-(13)C(2))-atomic positions. All compounds are prepared without any scrambling or dilution of the labeled material and are thus obtained with a very high isotope enrichment (96-99%). In combination with the methods that have been developed earlier it is concluded that each of the (13)C- and (15)N-positions and combination of positions of the pyrimidine deoxynucleosides can be efficiently labeled starting from commercially available and highly (13)C- or (15)N-enriched formaldehyde, acetaldehyde, acetic acid, potassium cyanide, methylamine hydrochloride, and ammonia.

Enzymatic trans-N-glycosylation has been selected as the method of choice by which to couple [ 13 C 1 ]-adenine to [ 13 C 3 ]-2-deoxy-D-ribose. The enzymatic pentosylation of the labelled adenine base was achieved in a two-step/one-pot reaction, starting from thymidine labelled in the sugar ring, but not at the thymine base. The efficiency of this thymidine phosphorylase catalysed (TP-catalysed) and purine nucleoside phosphorylase catalysed (PNP-catalysed) transamination reaction was demonstrated by a high yield (91%) and stereochemical purity of the obtained [1Ј,2Ј,5Ј,2-13 C 4 ]-2Ј-deoxy-D-

UV-Vis,1 H NMR and EPR (with spin-trapping) spectroscopy, MS and product studies have been used to study the reactions of dihydrazide and monohydrazide esters of oxalic acid in aqueous base (pH 10.5). In the absence of dioxygen, the dihydrazides are stable although the monohydrazide esters are rapidly hydrolysed in a reaction that gives monohydrazide oxalate anions, but not aryl radicals. However, in contrast both types of compound are rapidly degraded by dioxygen to give aryl radicals. The mechanisms involve an initial oxidation of the hydrazide anion by dioxygen followed by hydrolysis to give the corresponding aryl diazene anion. Further oxidation and loss of nitrogen yields aryl radicals. A mechanistic reaction scheme is proposed to account for the role of dioxygen, the fate of the aryl radicals and the products formed. The scope of these reactions as a non-photochemical source of aryl radicals under mild conditions is considered.