Selective Deuteration Labelling of 2′-Deoxyguanosine at the Carbon C-4’ Position

Abstract

Selective incorporation of deuterium within the sugar moiety of nucleosides and oligonucleotides can be used for different purposes including isotopic effect determination in mechanistic studies, massspectrometry fragmentation investigations, nuclear magnetic resonance analyses. We wish to report a simple method which allows the selective deuteration labelling of 2'-deoxyguanosine at the C-4'position through the intermediary of 9-(2-deoxy-B-D-erythropento-1,5-dialdo-114-furanosyllquanine. Heating of aqueous pyridine solution [1:11 of 2′-deoxyguanosine-5′-aldehyde for 1 hr at 60°C leads to a partial epimerisation of carbon C-4' with subsequent formation of 9-(2-deoxy-α-L-threopento-1,5-dialdo-1,4-furanosyl)guanine in 40% yield. A likely intermediate of this reaction appears to be a 5'-enol derivative. Similar treatment of 2′-deoxyguanosine-5′-aldehyde in D₂0-pyridine [1-1] gives after NaBH₄ reduction 60% of 2′-deoxyguanosine which is selectively deuterated at the C-4′ position. The extend of the isotopic labelling was up to 95% as determined by high resolution electron impact mass spectrometry and ¹H NMR analyses. Heating of the aqueous pyridine solution of 2′-deoxyguanosine-5′-aldehyde for a longer period (3–4 hrs) gave rise to two other nucleosides which where assigned as 9-(2-deoxy-α-D-threo-pentofuranosy1)guanine and 9-(2-deoxy-n-L-erythro-pentofuranosyl)guanine. A retro-aldol mechanism appears to be involved in the epimerization reaction which takes place at carbon C-3′.     

Oxidation of Morphine to 2,2′-Bimorphine by Cylindrocarpon didymum

The oxidation of morphine by whole-cell suspensions and cell extracts of Cylindrocarpon didymum gave rise to the formation of 2,2′-bimorphine. The identity of 2,2′-bimorphine was confirmed by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy. C. didymum also displayed activity with the morphine analogs hydromorphone, 6-acetylmorphine, and dihydromorphine, but not codeine or diamorphine, suggesting that a phenolic group at C-3 is essential for activity.The morphine alkaloids are the major alkaloid components of opium, the dried latex material from cut seed capsules of the opium poppy, Papaver somniferum. Of all the alkaloids, the morphine alkaloid group has been studied in most detail, mainly due to the significant therapeutic properties these compounds possess. The morphine alkaloids are narcotic analgesics and are widely used by clinicians for the control of chronic pain. The use of microbial enzymes to provide biological routes for the synthesis of semisynthetic drugs that are difficult to synthesize chemically and as a means of producing new morphine alkaloid derivatives has been the subject of a significant amount of research; this topic has recently been reviewed (3). In recent years, there has been an increasing demand for new morphine alkaloid intermediates for the synthesis of novel semisynthetic drugs, and as part of a study to produce such compounds, we have been exploring fungal transformations of morphine. In this paper, we describe the conversion of morphine to pseudomorphine (2,2′-bimorphine) by Cylindrocarpon didymum 311186.

Biotransformation of morphine.

C. didymum 311186 was obtained from the International Mycological Institute (Egham, Surrey, United Kingdom). Mycelia were grown in media at pH 7.0 containing (grams per liter) yeast extract (10.0), KH₂PO₄ (10.0), (NH₄)₂SO₄ (5.0), and MgSO₄ (0.5). Trace elements were as described by Rosenberger and Elsden (9). Cultures were incubated at 30°C for 48 h with rotary shaking at 180 rpm. Washed mycelia (typically 0.5 g [wet weight]) were resuspended in 40 ml of medium containing 10 mM morphine (Macfarlan Smith Ltd., Edinburgh, United Kingdom) in 250-ml Erlenmeyer flasks. Samples (0.2 ml) were removed at regular intervals and diluted fivefold in 50 mM phosphoric acid (pH 3.5), to dissolve any insoluble metabolites. Mycelia were removed by centrifugation at 14,000 × g with an MSE Microcentaur microcentrifuge (Patterson Scientific Ltd., Dunstable, United Kingdom). The samples were analyzed by high-performance liquid chromatography (HPLC) with a Waters component system (Millipore Waters UK Ltd., Watford, United Kingdom). The HPLC system consisted of a 600E system controller connected to either a 484 absorbance detector or a model 994 programmable photodiode array detector set to 230 nm, 0 to 1 V full-scale detection. Injections of 50 μl were performed with a WISP 712 autoinjector and data processed with Millennium 2010 software. Separation of samples was achieved with a C₁₈ Spherisorb column (4.6 by 250 mm, 5-μm particle size; Anachem Ltd., Luton, United Kingdom), protected by a guard column of the same packing material with an isocratic solvent system containing 40 mM phosphoric acid buffer (pH 2.5) and acetonitrile in a ratio of 92:8 plus 2 mM pentanesulfonic acid, delivered at a flow rate of 1 ml/min. Analysis of the whole-cell incubation mixture by HPLC showed that morphine was completely removed from the medium after a period of 70 h. No other soluble metabolites were identified by HPLC; however, a white precipitate was found to accumulate in the incubation mixture. Microscopic analysis showed that the precipitate had formed regular cubic crystals. The crystalline material was found to dissolve under mildly acidic conditions, and HPLC analysis after such treatment revealed the stoichiometric conversion of morphine to an unknown compound (Fig. ​(Fig.1)1) that had a retention time that coincided with that of authentic 2,2′-bimorphine, kindly provided by M. McPherson (Macfarlan Smith Ltd.). The compound was analyzed by thin-layer chromatography (TLC) with polyester-backed plates precoated with Polygram Sil G/UV₂₅₄ (Machery-Nagel, Duren, Germany) and developed in ammonia-n-butanol (20/80 [vol/vol]). TLC analysis revealed the appearance of two compounds that were detectable under UV light at 254 nm and with Ludy Tenger reagent (7). Compound 1 had an R_f value of 0.42 corresponding to that of authentic morphine, while compound 2 had an R_f value of 0.25 which coincided with that of authentic 2,2′-bimorphine. 2,2′-Bimorphine shows greatly enhanced fluorescence characteristics, compared to those of morphine, due to extended conjugation (6). Compound 2 fluoresced with a characteristic blue color when the TLC plate was illuminated at 366 nm. Fluorescent excitation and emission spectra of compound 2 dissolved in 50 mM potassium phosphate buffer (pH 7.4) in 1-cm-path-length cuvettes were recorded with a Perkin-Elmer LS 50 B luminescence spectrometer (Perkin-Elmer Ltd., Beaconsfield, United Kingdom). Two principal excitation maxima were found at 280 and 320 nm, with a single emission maximum at 430 nm, typical of authentic 2,2′-bimorphine. Open in a separate windowFIG. 1Accumulation of 2,2′-bimorphine in whole-cell incubations of C. didymum. Whole-cell incubations contained 40 ml of minimal medium, 10 mM morphine, and 0.5 g (wet weight) of mycelia in 250-ml Erlenmeyer flasks. Morphine (•) and 2,2′-bimorphine (○) concentrations were determined by HPLC. The data are means of three replicate incubations.

Identification of 2,2′-bimorphine.

¹H nuclear magnetic resonance spectroscopy of the product was performed at 400 MHz with a Bruker AM-400 spectrometer with tetramethylsilane as an internal standard and D-6 dimethyl sulfoxide as the solvent. The ¹H nuclear magnetic resonance spectrum gave the following signals, which were indistinguishable from those of an authentic sample of 2,2′-bimorphine (5) (for the proton assignments, see Fig. ​Fig.2,2, which gives the 2,2′-bimorphine numbering system): δ H 6.31 (2H, s, 1-H and 1′-H); 5.58 (2H, dd, J = 9.6 and 2.5, 7-H and 7′-H); 5.26 (2H, d, J = 9.6, 8-H and 8′-H); 4.70 (2H, d, J = 5.7, 5-H and 5′-H); 4.10 (2H, dd, J = 5.7 and 2.5, 6-H and 6′-H); 3.29 (2H, dd, J = 6.2 and 2.6, 9-H and 9′-H); 2.91 (2H, d, J = 18.6, 10β-H and 10β′-H); 2.57 (2H, d, J = 2.6, 14-H and 14′-H); 2.50 (2H, dd, J = 12.5 and 3.5, 16β-H and 16β′-H); 2.32 (6H, s, NMe and NMe′); 2.28 (2H, d, J = 12.5, α16-H and α16′-H); 2.23 (2H, dd, J = 18.6 and 6.2, α10-H and α10′-H); 1.99 (2H, dd, J = 11.4 and 3.5, α15-H and α15′-H); 1.68 (2H, d, J = 11.4, β15-H and β15′-H). Open in a separate windowFIG. 2Morphine analogs.The ¹H spectrum agreed with that expected for a symmetrical dimer, and only one aromatic proton signal was observed, instead of the characteristic AB pair of the morphine spectra, suggesting a symmetrical substitution on the aromatic ring. Laser desorption time-of-flight mass spectrometry was performed with a Kompact Maldi III mass spectrometer, and the mass spectrum showed a molecular ion, m/z 569.4, for C₃₄H₃₆N₂O₆.

Transformations of morphine analogs by C. didymum.

Whole-cell incubations of C. didymum were challenged with a range of morphine analogs including hydromorphone, 6-acetylmorphine, dihydromorphine, codeine, and diamorphine (see Fig. ​Fig.22 for structures). The incubations contained in 250-ml Erlenmeyer flasks approximately 0.61 g (wet weight) of mycelia and morphine analogs at 5 or 10 mM in a total volume of 40 ml of minimal medium. The flasks were incubated at 30°C with shaking, and samples were removed at intervals for HPLC analysis. Figure ​Figure33 shows that C. didymum was capable of activity with morphine, hydromorphone, 6-acetylmorphine, and dihydromorphine, and precipitates were observed to accumulate. Structural information on these products was not obtained. All of these compounds possess a free phenolic group at C-3 as a common structural feature which is likely to be an essential requirement for activity. This is consistent with the chemical oxidation of morphine to 2,2′-bimorphine, which requires the formation of a phenoxy radical intermediate (1). Open in a separate windowFIG. 3Transformations of morphine analogs by C. didymum. Whole-cell incubations contained 40 ml of minimal medium, 10 mM substrate (5 mM dihydromorphine), and 0.61 g (wet weight) of mycelia in 250-ml Erlenmeyer flasks. Morphine (•), codeine (▵), diamorphine (▴), hydromorphone (○), dihydromorphine (□), and 6-acetylmorphine (■) concentrations were determined by HPLC.

Enzyme activity in cell extracts.

The whole-cell transformation of morphine to 2,2′-bimorphine prompted investigation of subcellular enzyme activity. Cell extract was prepared by the method of Rahim and Sih (8) with the following modifications. Frozen mycelia containing 10 to 14 g (wet weight) of biomass were placed in an ice-cold mortar with an equal weight of acid-washed white quartz sand (50/70 mesh; Sigma Chemical Company, Poole, United Kingdom) and an equal volume of ice-cold potassium phosphate buffer (pH 7.4). The mixture was ground with a pestle for approximately 20 min until it formed a thin paste. The paste was diluted with an equivalent volume of ice-cold buffer, and the sand and cell debris were removed by centrifugation at 20,000 × g for 15 min at 4°C in a Sorvall RC5C centrifuge fitted with an SS34 rotor. Protein was measured by the method of Bradford (2) with the Pierce protein assay reagent according to the manufacturer’s protocol. Typically, protein recoveries of approximately 7 mg of protein/g (wet weight) of cells were obtained. The fluorescent nature of 2,2′-bimorphine enabled the development of a convenient and sensitive enzyme assay. In reaction mixtures which contained potassium phosphate buffer (pH 7.4), morphine (5 mM), and cell extract, activity could be measured spectrofluorimetrically by measuring fluorescence of 2,2′-bimorphine at 440 nm when excited at 330 nm. Cell extract from mycelia harvested after 80 h of incubation with morphine had a specific activity of 0.36 U/mg of protein. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 2,2′-bimorphine from 2 μmol of morphine per min. No activity was observed in control reaction mixtures where the cell extract was replaced with boiled cell extract. Activity was inhibited completely when 0.1 mM azide was added to the reaction mixtures. Interestingly, no activity was observed in cell extract from mycelia that had not been incubated with morphine, which suggests that the activity is inducible. The development of a rapid and sensitive assay should facilitate the purification and characterization of the 2,2′-bimorphine-producing enzyme. 2,2′-Bimorphine has been shown to be a spontaneous reaction product of morphine in aqueous solutions, though the reaction was extremely slow (4). Furthermore, morphine can be oxidized to 2,2′-bimorphine with alkaline ferricyanide, a reaction which is known to proceed via a mesomeric aryloxy free radical, leading to the formation of the dimer (1). However, to the best of our knowledge, this is the first report of the microbial oxidation of morphine to 2,2′-bimorphine.     

New Strategies for Chemical Synthesis of Phosphorodithioates Derived from 2′- or 3′- or 5′-Thionucleosides

Abstract

Various phophorodithioates derived from thionucleosides were synthesis by the reaction anhydronucleosides with phosphorodithioic acids     

Formation of N-(1-Oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine by the Reaction of 2′-Deoxyguanosine with 3-Deoxyglucosone

3-Deoxyglucosone (3DG) has weaker mutagenicity than methylglyoxal by the Ames test. 3DG reacted readily with 2′-deoxyguanosine (dG) in nucleosides. Two major products (G-A and G-B) were isolated and purified from the reaction mixture of 50 mM 3DG and 50 mM dG at 50°C and pH 7.4 for 6d. G-A was identified as N-(1-oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine. G-B was identified as a diastereomer of G-A.     

What Are the Conformational Changes Induced by Hard and Soft Metal Ion Binding to 2′-Deoxyguanosine?

Abstract

The NMR study on the interactions of 2′-dG with Mg²⁺, Zn²⁺ and Hg²⁺ ions in D₂O solution has shown that binding of softer metal ions to N7 shifts N <!—graphic—> S pseudorotational equilibrium slightly towards N-type sugar conformations. There are no detectable changes for the conformational equilibria across C4′-C5′ bond, whereas the population of the syn conformers is slightly increased.     

Improvements to the Chemical Synthesis of Biologically Active RNA Using 2′-O-Fpmp Chemistry

Abstract

The synthetic cycle protocol for the solid phase synthesis of RNA using 5′-O-(DMTr)-2′-O-(Fpmp)-ribonucleoside phosphoramidites is optimised. A simple and reliable two step deprotection procedure is developed to isolate biologically active RNA. It is demonstrated that fully deprotected RNA is completely stable under the deprotection conditions and that it does not undergo internucleotide cleavage and/or migration. Ribozymes and substrate RNAs synthesized using this chemistry were found to be catalytically active.     

Selective targeting of 2′-deoxy-5-fluorouridine to urokinase positive malignant cells in vitro

A urokinase targeting conjugate of 2′-deoxy-5-fluorouridine (5-FUdr) was synthesized and tested for tumor-cell selective cytotoxicity in vitro. The 5-FUdr prodrug 2′-deoxy-5-fluoro-3′-O-(3-carboxypropanoyl)uridine (5-FUdrsuccOH) containing an ester-labile succinate linker was attached to the specific urokinase inhibitor plasminogen activator inhibitor type II (PAI-2) and was found to preferentially kill urokinase-over expressing cancer cells. Up to 7 molecules of 5-FUdr were incorporated per PAI-2 molecule without affecting protein activity. This is the first time a small organic cytotoxin has been conjugated to PAI-2.     

An Improved Protection-Free One-Pot Chemical Synthesis of 2′-Deoxynucleoside-5′-Triphosphates

A facile, straightforward, reliable, and an efficient method for the gram-scale chemical synthesis of both purine deoxynucleotides such as 2 ′-deoxyguanosine-5 ′-triphosphate (dGTP) and 2 ′-deoxyadenosine-5 ′-triphosphate (dATP) and pyrimidine deoxynucleotides such as 2 ′-deoxycytidine-5 ′-triphosphate (dCTP), thymidine-5 ′-triphosphate (TTP), and 2 ′-deoxyuridine-5 ′-triphosphate (dUTP) starting from the corresponding nucleoside is described. This improved “one-pot, three step” Ludwig synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65%–70%).     

The Stability of Trisubstituted Internucleotide Bond in the Presence of the Vicinal 2′-Hydroxyl. Chemical Synthesis of Uridyl(2′-phosphate)-(3′-5′)-uridine

Abstract

The effect of eleven different phosphoryl center protecting groups on the stability of trisubstituted internucleotide bond of the dimers (1a-k), in the presence of the vicinal 2′-hydroxyl, was examined. It has been found that electronic properties of the phosphoryl center protecting groups are essential for the reactivity of the trisubstituted internucleotide bond. Those observations were applied to the chemical synthesis of the uridyl(2′-phosphate)-(3′-5′)-uridine, a useful model for further pre-tRNA splicing studies.     

Nucleosides. 145. Synthesis of 2,5′-Anhydro-2-Thiouridine and its Conversion to 3′-O-Acetyl-2,2′-Anhydro-5′-Chloro-5′-Deoxy-2-Thiouridine. Studies Directed Toward the Synthesis of 2′-Deoxy-2′-Substituted arabino Nucleosides (7)

Abstract

In an attempt to introduce a substituent at C-2′ in the “up” arabino configuration directly by nucleophilic displacement reaction of a preformed pyrimidine ribonucleoside, we synthesized 2,5′-anhydro-5′-deoxy-2-thiouridine (6) in three steps from uridine. Compound 6 was converted into the 3′-O-acetyl derivative 7. Upon treatment of 7 with triflyl chloride in methylene chloride in the presence of triethylamine and p-dimethylaminopyridine, 2,2′-anhydro-1-(3-O-acetyl-5-chloro-2,5-dideoxy-β-D-arabinofuranosyl)-2-thiouracil (9) was obtained as the only isolable product. Obviously, the intermediate 3′-O-acetyl-2,5′-anhydro-2′-O-triflyl-2-thiouridine (8) was attacked by the chloride nucleophile at C-5′ first giving the 2′-O-triflyl-2-thiouridine intermediate from which 9 was formed by intramolecular nucleopilic reaction.     

Adenylyl-(5′, 2′)-adenosine 5′-Phosphate ((2′-5′) pA-A) in Crude Crystals of 5′-Adenylic Acid Isolated from Nuclease P1 (Penicillium Nuclease) Digest of Technical Grade Yeast RNA

Adenylyl (5′,2′)-adenosine 5′-phosphate ((2′-5′)pA-A) was detected in crude crystals of 5′-AMP prepared from Penicillium nuclease (nuclease P₁) digest of a technical grade yeast RNA. While (3′–5′)A-A was split by nuclease P₁, spleen phosphodiesterase, snake venom phosphodiesterase or alkali, (2′–5′)A-A was not split by a usual level of nuclease P₁ or spleen phosphodiesterase. Nuclease P₁ digests of 12 preparations of technical grade yeast RNA tested were confirmed to contain (2′–5′)pA-A. Its content was about 1 to 2% of the AMP component of each RNA preparation. As poly(A) was degraded completely by the Penicillium enzyme into 5′-AMP without formation of any appreciable amount of (2′–5′)pA-A, the technical grade RNA is supposed to contain 2–5′ phosphodiester linkages in addition to 3′–5′ major linkages.     

Detection of Oxidation Products of 5-Methyl-2′-Deoxycytidine in Arabidopsis DNA

Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-2′-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5-HmdC, 5-formyl-2′-deoxycytidine (5-FodC), 5-carboxyl-2′-deoxycytidine (5-CadC), 5-hydroxymethyl-2′-deoxyuridine (5-HmdU), and the (5′S) diastereomer of 8,5′-cyclo-2′-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 10⁶ nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tet-family dioxygenase enzymes in Arabidopsis.     

Selective 4′-O-methylglycosylation of the pentahydroxy-flavonoid quercetin by Beauveria bassiana ATCC 7159

Biotransformation of the pentahydroxy-flavonoid natural product, quercetin, by Beauveria bassiana ATCC 7159 afforded a new derivative, quercetin-4'-O-methyl-7-O-β-D-glucopyranoside, in 87% isolated yield suggesting that glucosylation of the substrate occurred with high selectivity at C–7-OH out of the five hydroxyl groups. Most of the product was isolated from the mycelium and the filtrate of the culture medium did not show any catalytic activity. The mycelium is capable of performing this biotransformation when suspended in buffers of pH 2.1 and 7.2, suggesting that intracellular enzymes are involved and that they are active at a wide range of extracellular pH.     

Studies on the inhibition of hepatic lipogenesis by N6,O2′-dibutyryl adenosine 3′,5′-monophosphate

Fatty acid synthesis by isolated liver cells is dependent upon the availability of lactate and pyruvate. A lag in fatty acid synthesis is explained by time being required for lactate and pyruvate to accumulate to maximum concentrations in the incubation medium. The initial rate of fatty acid synthesis is not linear with cell concentration, being disproportionately greater at higher cell concentrations because optimal lactate and pyruvate concentrations are established in the medium more rapidly. The accumulation of lactate and pyruvate is inhibited markedly by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. This accounts in part for the inhibition of fatty acid synthesis caused by this cyclic nucleotide. Other sites of action are apparent, however, because exogenous lactate plus pyruvate only partially relieves the inhibition. The profile of metabolic intermediates suggests that N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate inhibits the conversion of glycogen to pyruvate and lactate by decreasing the effectiveness of phosphofructokinase and pyruvate kinase.     

Expanding the clinical relevance of the 5′-nucleotidase cN-II/NT5C2

Purine metabolism is depending on a large amount of enzymes to ensure cellular homeostasis. Among these enzymes, we have been interested in the 5′-nucleotidase cN-II and its role in cancer biology and in response of cancer cells to treatments. This protein has been cited and studied in a large number of papers published during the last decade for its involvement in non-cancerous pathologies such as hereditary spastic paraplegia, schizophrenia, and blood pressure regulation. Here, we review these articles in order to give an overview of the recently discovered clinical relevance of cN-II.     

Synthesis of Oligodeoxynucleotides Containing the C-Nucleoside and 2′- Deoxy-2′-Fluoro-ara-Nucleoside Moieties by the H-Phosphonate Method.1,2

Abstract

A module type, computer-controlled, multipurpose synthesizer displaying a novel device for the transport of liquids, was constructed and used in the synthesis of oligomers containing some C-nucleosides and 2′-deoxy-2′-fluoro-ara-nucleoside moieties. H-Phosphonate method was applied in terms of a further adjustment of construction features of the synthesizer versus chemistry of the process. Results of preliminary studies on the effects of the modified nucleosides on the stability of duplexes showed a clear tendency of destabilization of duplexes in the case of C-nucleosides while fluorinated nucleosides in most cases stabilize the formed duplexes.     

Synthesis of 2′,3′-Didehydro-2′,3′-dideoxyisoinosine and Oxidation of Fluorescent 2-Hydroxypurine Nucleosides by Xanthine Oxidase

Abstract

The syntheses of 2′,3′-didehydro-2′,3′-dideoxyisoinosine (d4isoI, 4) as well as 7-deaza-2′,3′-didehydro-2′,3′-dideoxyisoinosine (d4c₇isoI, 5) are described. Compounds 4 and 5 show both strong fluorescence. Compound 4 is oxidized by xanthine oxidase to give the corresponding xanthine 2′,3′-dideoxy-2′,3′-didehydronucleosides. A preparative chemo-enzymatic synthesis of 2′-deoxyxanthosine (3) is described.     

Formation of an Unexpected 2-Deoxy-α-D-Threo-Pentofuranosyl Azide by Reaction of O2,3′-Anhydro-5′-O-trityl-2′-deoxycytidine with Lithium Azide

Abstract

Reaction of 0²,3′-anhydro-5′-0-trityl-2′-deoxycytidine (1) with LiN₃s in DMF resulted in the formation of 1-(3-azido-2,3-dideoxy-5-0-trityl-β-D-erythro-pentofuranosyl) cytosine (2) and 3-0-(4-amino-1,3-pyrimidin-2-yl)-5-0-trityl-2-deoxy-α-D-threo-pentofuranosyl azide (3) (2:3 = 1:1) in 88% yield. Compound 3 was deprotected with 80% aqueous AcOH yielding 4     

Conversion of 5′-inosinic acid to inosine by Streptomyces aureus

The production of inosine by microbial conversion of 5′-inosinic acid (IMP) was investigated. Among the various strains of Streptomyces and Bacillus tested, Streptomyces aureus NCIB 9803 was selected for the microbial conversion process due to its high IMP-degrading activity. A maximum conversion yield of 0.43 (86% of the theoretical value) was obtained when IMP was added to the culture medium at 24 h. Kinetic studies with [8-¹⁴C] IMP showed that the difference from the theoretical values mainly attributable to the uptake of inosine by S. aureus.