One-Pot Two-Step Enzymatic Coupling of Pyrimidine Bases to 2-Deoxy-d-ribose-5-phosphate. A New Strategy in the Synthesis of Stable Isotope Labeled Deoxynucleosides

Abstract

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.

Chemical constituents from Lobelia chinensis and their anti-virus and anti-inflammatory bioactivities

“…Purification of combined fractions 1 and 2 was performed by silica gel chromatography eluted with chloroform and methanol (9:1) to give adenine (18) (1.3 mg) (Wu et al, 2000), L-tyrosine (19) (37.1 mg) (Nord et al, 2004), uridine (20) (2.0 mg) (Chen et al, 1999), gallic acid (21) (0.6 mg) (Wu et al, 1995), Dglucose (22) (337.9 mg) (Shibano et al, 2004), Dfructose (23) (11.5 g) (Shibano et al, 2004), mesoerythritol (24) (1.45 g) (Monrad and Madsen, 2007), glycerol (25) (6.03 g) (Kiuchi et al, 1998), myo-inositol (26) (250.0 mg) (Takahashi et al, 2001), and succinic acid (27) (22.8 mg) (Nord et al, 2004), successively. Silica gel column chromatography of fraction 3 with a mixture of chloroform and methanol (5:1) generated 5-hydroxymethyl-pyrrole-2-carbaldehyde (28) (14.4 mg) (Hiermann et al, 2002), thymidine (29) (1.4 mg) (Ouwerkerk et al, 2002), and protocatechuic acid (30) (1.2 mg) (Wu et al, 1995). Chromatography of fraction 5 on a Sephadex LH-20 column eluted with water and step gradient with methanol followed by preparative TLC purification on silica gel led to the isolation of lobetyolinin (31) (3.2 mg) (Ishimaru et al, 1992), vanillic acid (32) (1.4 mg) (Chen et al, 1999), pcoumaric acid (33) (0.5 mg) (Chiang et al, 2003), methyl vanillate (34) (2.1 mg) (Chen et al, 1999), and caffeic acid (35) (13.2 mg) (Hu et al, 2007).…”

Chemoenzymatic preparation of nucleosides from furanoses

“…In this work, they established that Escherichia coli PPM accepted as substrates deoxyribose 5-phosphate, ribose 5-phosphate and arabinose 5-phosphate but not 2,3-dideoxyribose 5-phosphate; unfortunately no indication of yields or absolute activities were reported. For the particular case of deoxynucleosides, a synthetic use of PPM has been reported by Ouerkerk et al 15 for the preparation of 15 N and 13 C radiolabelled thymidine and 2 0 -deoxyuridine. In the same sense, Shimizu group 16 developed a one-pot microbial synthesis of 2 0 -deoxyribonucleoside in three steps.…”

Synthesis of (ϵ‐13C‐,ϵ‐15N)‐Enriched L‐Lysine − Establishing Schemes for the Preparation of All Possible 13C and 15N Isotopomers of L‐Lysine, L‐Ornithine, and L‐Proline

“…Treatment of -ornithine with the cyanate anion gives citrulline. [14] Earlier, our group prepared all isotopomers of cyanate and prepared site-directed 15 N-and 13 C-enriched urea derivatives from primary amines. [15] The conversion of -ornithine into -arginine with 13 C and 15 N isotopes has also been published.…”

“…[14] Earlier, our group prepared all isotopomers of cyanate and prepared site-directed 15 N-and 13 C-enriched urea derivatives from primary amines. [15] The conversion of -ornithine into -arginine with 13 C and 15 N isotopes has also been published. [16,17] The ability to prepare all 15 N and 13 C isotopomers of the three amino acids ornithine, arginine, and citrulline will be a boon to the metabonomics Scheme 2.…”

“…-lysine (1), -ornithine 2) and -proline (3) In this paper we describe a modular strategy that is able to give access to any site-directed isotopically labeled -lysine, -ornithine and -proline, with the use of commercially available stable isotopically enriched synthons or synthons that are available in any site-directed enriched form by strategies described before by us. [3,4] For the 1 H, 13 C, and 15 N MAS NMR study we need, next to ( 13 C 20 )-11-(Z)-retinal, also (ε- 15 N,ε-13 C)lysine. [5] The one-step synthesis of the protected -glutamate ester by the OЈ Donnell method [6] has been published for the natural abundance materials.…”