A synthetic route to 3-C-alkyl (or 3-C-phenyl-) 2,3-dideoxy-D-erythro-pentono-1,4-lactones: intermediates in the synthesis of 2(3H)-furanones.

A series of 3-C-alkyl- (and 3-C-phenyl-) 2,3-dideoxy-D-erythro-pentono-1,4-lactones, compounds which are important in the synthesis of modified nucleosides and antibiotic sugars, were synthesized from D-ribonolactone. By a route that proceeded via 5-O-protected D-ribonolactone, 5-O-protected 2,3-dideoxy-D-glycero-pent-2-enono-1,4-lactones were synthesized and reacted with R2CuLi or a complex PhSCu(RMgBr)n to give respectively the 3-C-alkyl or 3-C-phenyl compounds. Details of the preparation of the O-protected intermediates, as well as the selection of the organometallic reagents, are provided.

Characterization of 2',3'-dideoxycytidine diphosphocholine and 2',3'-dideoxycytidine diphosphoethanolamine. Prominent phosphodiester metabolites of the anti-HIV nucleoside 2',3'-dideoxycytidine.

2',3'-Dideoxycytidine (ddCyd) is among the most potent of the anti-human immunodeficiency virus (HIV) agents of the dideoxynucleoside class. Its pharmacologically active metabolite 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) is an effective inhibitor of HIV reverse transcriptase and thus of HIV replication. ddCyd differs, however, from other dideoxynucleoside agents such as 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine in its capacity to generate phosphodiester metabolites (i.e. ddCDP choline and ddCDP ethanolamine). We have synthesized and characterized these two diesters, and established their identity with the metabolites formed in ddCyd-treated Molt-4 cells. Toward this end, the biologically generated metabolites have been isolated on a preparative scale and compared with the synthetic compounds mass spectroscopically, chromatographically, and enzymatically (i.e. their relative susceptibility to the catabolic enzymes alkaline phosphatase and venom phosphodiesterase). The concentration reached by each of these two phosphodiesters within cells can, under certain conditions, equal or exceed that of ddCTP, and their half-times of disappearance are long, indicating that they may serve as depot forms of ddCyd. The possible role of these phosphodiesters in contributing to the unusual toxicity of ddCyd is discussed.

C-methylation occurs during the biosynthesis of heme d1.

The biosynthetic origin of methyl groups in heme d1 isolated from the nitrite reductase cytochrome cd1 was investigated by a stable isotope labeling experiment. Pseudomonas aeruginosa (American Type Culture Collection strain 19429) was grown on a minimal medium supplemented with [13C]methionine. The enzyme was purified, the heme extracted, converted into the free base methyl ester derivative, and purified. 1H NMR and 13C NMR indicated that only the methyl groups attached to C2 and C7 are derived from methionine.

Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine.

The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin.

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.