Oxanosine

Oxanosine 3r, 5-amino-3-beta-(d-ribofuranosyl)-3H-imidazo[4,5-d][1,3]oxazine-7-one, was isolated as a novel nucleoside antibiotic in 1981 from Streptomyces capreolus MG265-CF3. Oxanosine became relevant in toxicology in 1996 with the discovery that it is formed in nitrosative guanosine deamination. As part of studies of the mechanism of oxanosine formation, the synthesis was attempted of [7- 18O]oxanosine by enzymatic 16O/18O-exchange with adenosine deaminase (ADA) in analogy to the synthesis of [6- 18O]guanosine from 2-amino-6-chloropurine. Unexpectedly, it was discovered that the incubation of oxanosine 3r with ADA in sodium phosphate buffer (pH = 7.4) results in 1-beta-(d-ribofuranosyl)-5-ureido-1H-imidazole-4-carboxylic acid 4r. The reaction of the 2'-deoxyribose derivative 3d forms 4d in analogy. The reaction products were separated by preparative RP-HPLC and characterized by LC/MS and MS/MS analyses and UV/vis and NMR spectroscopy, and NMR assignments were corroborated by GIAO and GIAO-PCM calculations. Reaction in H2 18O leads to 18O-incorporation at C7. The hydrolysis of 3 to 4 can be rationalized on the basis of the known mode of action of ADA, and an explanation is provided for ADA's accomplishment of the "usual" substitution at C6 of adenosine (addition to the exocyclic bond) and the "lactone hydrolysis" of oxanosine (addition to the endocyclic double bond). The Michaelis-Menten constant of Km = 1.0 (+/-0.2) mM was measured for oxanosine. Implications are discussed for studies of nitrosative deamination of nucleosides, nucleotides, and oligonucleotides.

Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In contrast, the effects of RNS damaged metabolites are less appreciated. 5-Amino-3-β-(d-ribofuranosyl)-3 H-imidazo-[4,5- d][1,3]oxazine-7-one (oxanosine) and its nucleotides are products of guanosine nitrosation. Here we demonstrate that oxanosine monophosphate (OxMP) is a potent reversible competitive inhibitor of IMPDH. The value of Ki varies from 50 to 340 nM among IMPDHs from five different organisms. UV spectroscopy and X-ray crystallography indicate that OxMP forms a ring-opened covalent adduct with the active site Cys (E-OxMP*). Unlike the covalent intermediate of the normal catalytic reaction, E-OxMP* does not hydrolyze, but instead recyclizes to OxMP. IMPDH inhibitors block proliferation and can induce apoptosis, so the inhibition of IMPDH by OxMP presents another potential mechanism for RNS toxicity.

The combined effect against human immunodeficiency virus (HIV) of oxanosine and 2'3'-dideoxyinosine (ddI) has been evaluated by the production of viral particles, the expression of viral antigens on cell surfaces, and the amount of viral genome integrated in the host cells. Oxanosine alone has no effect on HIV replication up to 100 microg/ml, however, in the presence of ddI, oxanosine revealed concentration dependent inhibition against HIV without cytotoxicity.