Synthesis of 6-(2-thienyl)purine nucleoside derivatives that form unnatural base pairs with pyridin-2-one nucleosides

Unnatural bases, 2-amino-6-(2-thienyl)purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace the previously developed purine analogue, 2-amino-6-(N,N-dimethylamino)purine, which specifically pairs with pyridin-2-one. These nucleoside derivatives were synthesized via the 6-substitution of 6-iodopurine nucleosides with tributylstannylthiophene or tributylstannylfuran. As compared with 2-amino-6-(N,N-dimethylamino)purine, 2-amino-6-(2-thienyl)purine reduced the interference in the stacking interactions with the neighboring bases in a DNA duplex and improved the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite the unnatural base.     

Synthesis of 6-(2-thienyl)purine nucleoside derivatives toward the expansion of the genetic code.

Unnatural bases specifically pairing with pyridin-2-one, 2-amino-6-(2-thienyl) purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace 2-amino-6-(N,N-dimethylamino)purine. It was expected that these novel purine analogues, as compared with 2-amino-6-(N,N-dimethylamino)purine, might reduce the interference in the stacking interactions with the neighboring bases in a duplex and improve the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite these unnatural bases. The syntheses of these nucleoside derivatives and the DNA fragments were examined.     

Evidence for direct inhibition of de novo purine synthesis in human MCF-7 breast cells as a principal mode of metabolic inhibition by methotrexate

We have investigated the role of dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo purine synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Previous studies have shown that cytotoxic concentrations of MTX that inhibit dihydrofolate reductase produce only minimal depletion of the reduced folate cofactor, 10-formyltetrahydrofolate, required for purine synthesis. At the same time, de novo purine synthesis is totally inhibited. In these studies, we show that 10 microM MTX causes inhibition of purine synthesis at the step of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase, as reflected in a 2-3-fold expansion of the intracellular AICAR pool. The inhibition of purine synthesis coincides with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of AICAR transformylase. When the generation of H2PteGlu is blocked by pretreatment with 50 microM 5-fluorodeoxyuridine (FdUrd), an inhibitor of thymidylate synthase, MTX no longer causes inhibition of purine synthesis. Intermediate levels of H2PteGlu produced in the presence of lower (0.1-10 microM) concentrations of FdUrd led to proportional inhibition of purine biosynthesis, and the exogenous addition of H2PteGlu to breast cells in culture re-established the block in purine synthesis in the presence of FdUrd and MTX. The early phases of inhibition of purine biosynthesis could be ascribed only to H2PteGlu accumulation. MTX polyglutamates, also known to inhibit AICAR transformylase, were present in breast cells only after 6 h of incubation with the parent compounds and were not formed in cells preincubated with FdUrd. The lipid-soluble antifolate trimetrexate, which does not form polyglutamates, produced modest 10-formyltetrahydrofolate depletion, but caused marked H2PteGlu accumulation and a parallel inhibition of purine biosynthesis. This evidence leads to the conclusion that MTX and the lipid-soluble analog trimetrexate cause inhibition of purine biosynthesis through the accumulation of H2PteGlu behind the blocked dihydrofolate reductase reaction.     

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.     

Synthesis of 9-(2-deoxy-beta-D-ribofuranosyl)purine-2-thione

A synthesis of 9-(2-deoxy-beta-D-ribofuranosyl)purine-2-thione was performed by desulfurization of 2'-deoxy-6-thioguanine to give 2-amino-9-(2-deoxy-beta-D-ribofuranosyl)purine, diazotization with chloride replacement to give 2-chloro-9-(2-deoxy-beta-D-ribofuranosyl)purine, and the replacement of chloride with sulfur using thiolacetic acid and deacetylation.     

Studies on extracellular ribonucleases of Ustilago sphaerogena. Characterization of substrate specificity with special reference to purine-specific ribonucleases

1. Ribonuclease U(1) splits only the phosphodiester bonds of guanosine 3'-phosphates in RNA. It may be regarded as a guanyloribonuclease [ribonucleate (guanine nucleotide)-2'-transferase (cyclizing), EC 2.7.7.26] similar to ribonuclease T(1) (Egami, Takahashi & Uchida, 1964). It seems to be identical with the extracellular ribonuclease described by Glitz & Dekker (1963, 1964a,b). 2. Ribonucleases U(2) and U(3) are novel enzymes with a strict specificity. They split the internucleotide bonds between purine 3'-nucleotides and 5'-hydroxy groups of adjacent nucleotides in RNA with the intermediary formation of purine nucleoside 2',3'-(cyclic)-phosphates, which are slowly hydrolysed to purine 3'-nucleotides. So they may be classified as ;puryloribonucleases [ribonucleate (purine nucleotide)-2'-transferase (cyclizing)]'. Double-stranded RNA is scarcely split by ribonucleases U(2) and U(3). 3. Ribonuclease U(4) has no absolute base specificity, and produces the mononucleotides 3'-adenylate, 3'-guanylate, 3'-cytidylate and 3'-uridylate from RNA.     

Alteration of purine metabolism by AICA-riboside in human B lymphoblasts.

The effect of 5-amino-4-imidazole-carboximide (AI-CA)-riboside on different pathways of purine metabolism (biosynthesis de novo, salvage pathways, adenosine metabolism, ATP catabolism) was studied in human B lymphoblasts (WI-L2). AICA-Riboside markedly decreased intracellular levels of 5-phosphoribosyl-1-pyrophosphate and in consequence affected purine biosynthesis de novo and purine salvage pathways. AICA-riboside inhibited incorporation of glycine into purine nucleotides, but when formate was used as the precursor of purine biosynthesis de novo, a biphasic effect was observed. The incorporation of formate into purine nucleotides was increased by AICA-riboside at concentrations up to 2 mM but decreased at higher concentrations. Salvage of the purine bases adenine, hypoxanthine, and guanine was markedly inhibited and utilization of extracellular adenosine in B lymphoblasts was reduced by AICA-riboside. AICA-riboside increased ribose 1-phosphate concentrations and increased degradation of prelabeled ATP. No effect on the intracellular levels of orthophosphate was found. Proliferation of WI-L2 lymphoblasts was only slightly affected at concentrations of AICA-riboside below 500 microM but markedly inhibited by higher concentrations.     

Mechanisms of accelerated purine nucleotide synthesis in human fibroblasts with superactive phosphoribosylpyrophosphate synthetases

Concentrations and rates of synthesis of phosphoribosylpyrophosphate (PP-Rib-P) and purine nucleotides were compared in fibroblasts cultured from 5 males with PP-Rib-P synthetase superactivity, 3 normal individuals, and 2 children with severe hypoxanthine-guanine phosphoribosyltransferase deficiency. Although all cell strains with PP-Rib-P synthetase superactivity showed increased PP-Rib-P concentration and generation, increased rates of PP-Rib-P-dependent purine synthetic pathways, and increased purine and pyrimidine nucleoside triphosphate concentrations, two subgroups were discernible. Three fibroblast strains with isolated catalytic defects in PP-Rib-P synthetase showed milder increases in PP-Rib-P concentration (2.5-fold normal) and generation (1.6- to 2.1-fold) and in rates of purine synthesis de novo (1.6- to 2.2-fold) and purine nucleoside triphosphate pools (1.5-fold) than did cells from 2 individuals with combined kinetic defects in PP-Rib-P synthetase, both with purine nucleotide inhibitor-resistance. Values for these processes in the latter two strains were, respectively, 5- to 6-fold, 2.6- to 3.2-fold, 4- to 7-fold, and 1.7- to 2.2-fold those of normal cells. In contrast to cells with catalytic defects, these cells also excreted an abnormally high proportion of labeled purines and resisted purine base-mediated inhibition of PP-Rib-P and purine nucleotide synthesis. Hypoxanthine-guanine phosphoribosyltransferase-deficient cells showed normal regulation of PP-Rib-P synthesis and normal nucleoside triphosphate pools despite increased rates of purine synthesis de novo and of purine excretion. Cells with PP-Rib-P synthetase superactivity thus synthesize purine nucleotides at increased rates as a consequence of increased PP-Rib-P production, despite increased purine nucleotide concentrations. These and additional findings provide evidence that regulation of purine synthesis de novo is effected at both the PP-Rib-P synthetase and amidophosphoribosyltransferase reactions.     

Adaptations for reflection of bioluminescent light in the gas bladder of Leiognathus equulus (Perciformes: Leiognathidae)

The gas bladder of leiognathid fishes functions not only in buoyancy but also in reflection of bioluminescent light from the circumesophageal light organ. Purine distribution, quality (guanine/hypoxanthine ratio), and concentration, as the basis for reflectivity, were assayed enzymatically for different portions of the gas bladder lining of the common leiognathid, Leiognathus equulus (Forskal). For highly reflective areas, the percentage of tissue wet mass and dry mass represented by purine was also determined. The results indicate that total purine content in the reflective areas of the leiognathid bladder was significantly higher than values determined for other similar, shallow water fishes; instead, purine content in these reflective areas was similar to that known for very deep-dwelling fishes, in which heavy purine deposition is correlated with high pressures and high oxygen concentrations in the bladder. In addition, the results show that differential purine distribution within the bladder correlates strikingly with the path of bioluminescent light. The dorsal bladder lining, the primary site of incident luminescence, had extremely high purine concentrations (averaging 2.80 mg/cm2), whereas the secondary reflective surfaces, the lateral (1.81 mg/cm2) and ventral (1.22 mg/cm2), portions, although high in purine content, had concentrations significantly lower than the dorsum. Areas through which light is transmitted, the light organ-bladder interface (0.09 mg/cm2) and the posterior region (0.19 mg/cm2), were greatly reduced in purine content. The enhancement of purine in the reflective portions of the bladder and the correlation of the differential distribution of purines with the path of light indicate that the L. equulus gas bladder is exquisitely adapted to function as a reflector of bioluminescent light.     

Influence of alterations in the purine ring on biological activity of cytokinins

The 1-deaza-, 3-deaza-, 8-aza-1-deaza- and 8-aza-3-deaza-analogs of kinetin and 6-(3-methyl-2-butenylamino)purine and some of their ribosides were synthesized and their growth-promoting activities in the tobacco bioassay were determined and compared with those of the parent compounds. The replacement of nitrogen by carbon in the 1 -position of the purine ring decreases cytokinin activity 15-fold for kinetin and 2-fold for 6-(3-methyl-2-butenylamino)purine (IPA); however, the replacement of nitrogen by carbon in the 3-position decreases the activity 2000 times for kinetin and 1000 times for 6-(3-methyl-2-butenylamino)-purine. The activity of 8-aza-1-deaza-analogs appears to be of the same order of somewhat lower than the corresponding 1-deaza-analogs. The corresponding 8-aza-3-deaza-analogs are less active than kinetin (400 times and 6-(3-methyl-2-butenylamino)purine (40 times). However, they are more active than the corresponding 3-deaza-analogs. The concentration range in which the ribosides show activity is nearly the same as for the corresponding free bases, but the maximum yield of tobacco-callus for the riboside of the 3-deaza-analog of 6-(3-methyl-2-butenylamino)purine is very low.     

Synthesis of 9-(2-Deoxy-β-D-ribofuranosyl)purine-2-thione

Abstract

A synthesis of 9-(2-deoxy-β-D-ribofuranosyl)purine-2-thione was performed by desulfurization of 2′-deoxy-6-thioguanine to give 2-amino-9-(2-deoxy-β-D-ribofuranosyl)purine, diazotization with chloride replacement to give 2-chloro-9-(2-deoxy-β-D-ribofuranosyl)purine, and the replacement of chloride with sulfur using thiolacetic acid and deacetylation.     

Biosynthesis of nucleic acid purines in Mycobacterium tuberculosis H37Rv

1. The biosynthesis of nucleic acid purine in Mycobacterium tuberculosis H(37)R(v) has been studied by using (14)C-labelled precursors. 2. The results indicate that C-2 and C-8 of the purine ring are derived most efficiently from serine and glycine and not from formate. 3. [(14)C]Methionine is not incorporated into the ureide carbon atoms of the purine ring.     

Regulation of purine synthesis de novo in human fibroblasts by purine nucleotides and phosphoribosylpyrophosphate

Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the phosphoribosylpyrophosphate (PP-Rib-P) synthetase reaction or the amidophosphoribosyltransferase (amido PRT) reaction, or both. We studied control of purine synthesis de novo in cultured normal, hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, and PP-Rib-P synthetase-superactive human fibroblasts by measuring concentrations and rates of synthesis of PP-Rib-P and purine nucleotide end products, proposed effectors of regulation, during inhibition of the pathway. Incubation of cells for 90 min with 0.1 mM azaserine, a glutamine antagonist which specifically blocked the pathway at the level of conversion of formylglycinamide ribotide, resulted in a 5-16% decrease in purine nucleoside triphosphate concentrations but no consistent alteration in generation of PP-Rib-P. During this treatment, however, rates of the early steps of the pathway were increased slightly (9-15%) in normal and HGPRT-deficient strains, more markedly (32-60%) in cells with catalytically superactive PP-Rib-P synthetases, and not at all in fibroblasts with purine nucleotide feedback-resistant PP-Rib-P synthetases. In contrast, glutamine deprivation, which inhibited the pathway at the amido PRT reaction, resulted in time-dependent nucleoside triphosphate pool depletion (26-43% decrease at 24 h) accompanied by increased rates of PP-Rib-P generation and, upon readdition of glutamine, substantial increments in rates of purine synthesis de novo. Enhanced PP-Rib-P generation during glutamine deprivation was greatest in cells with regulatory defects in PP-Rib-P synthetase (2-fold), but purine synthesis in these cells was stimulated only 1.4-fold control rates by glutamine readdition. Stimulation of these processes in normal and HGPRT-deficient cells and in cells with PP-Rib-P synthetase catalytic defects was, respectively: 1.5 and 2.0-fold; 1.5 and 1.7-fold; and 1.6 and 4.1-fold. These studies support the following concepts. 1) Rates of purine synthesis de novo are regulated at both the PP-Rib-P synthetase and amido PRT reactions by end products, with the latter reaction more sensitive to small changes in purine nucleotide inhibitor concentrations. 2) PP-Rib-P exerts its role as a major regulator of purine synthetic rate by virtue of its interaction with nucleotide inhibitors to determine the activity of amido PRT. 3) Activation of amido PRT by PP-Rib-P is nearly maximal at base line in fibroblasts with regulatory defects in PP-Rib-P synthetase.     

The importance of methylthio-IMP for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity in Molt F4 human malignant T-lymphoblasts

The importance of methyl-thioIMP (Me-tIMP) formation for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity was studied in Molt F4 cells. Cytotoxicity of Me-MPR is caused by Me-tIMP formation with concomitant inhibition of purine de novo synthesis. Inhibition of purine de novo synthesis resulted in decreased purine nucleotide levels and enhanced 5-phosphoribosyl-1-pyrophosphate (PRPP) levels, with concurrent increased pyrimidine nucleotide levels. The Me-tIMP concentration increased proportionally with the concentration of Me-MPR. High Me-tIMP concentration also caused inhibition of PRPP synthesis. Maximal accumulation of PRPP thus occurred at low Me-MPR concentrations. As little as 0.2 μM Me-MPR resulted already after 2 h in maximal inhibition of formation of adenine and guanine nucleotides, caused by inhibition of purine de novo synthesis by Me-tIMP. Under these circumstances increased intracellular PRPP concentrations could be demonstrated, resulting in increased levels of pyrimidine nucleotides. So, in Molt F4 cells, formation of Me-tIMP form Me-MPR results in cytotoxicity by inhibition of purine de novo synthesis.     

Antisenescent Activity of Natural Cytokinins

The antisenescent activity of naturally occurring cytokinins (bases and ribosides) has been evaluated by measuring chlorophyll retention in detached wheat (Triticum vulgare) leaf segments. 6-(3-Methyl-2-butenylamino)-2-methylthiopurine (ms2ip) was the most active cytokinin followed by 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine (tZ). 6-(4-Hydroxy-3-methyl-cis-2-butenylamino)-9-β-D-ribofuranosylpurine (cZR), 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-2-methylthio-9β-D-ribofuranosylpurine (MstZR), and 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-2-methylthio-9-β-D-ribofuroanosylpurine (mscZR) were essentially inactive. 9-Ribosyl substitution did not affect the activity of tZ, (±)-6-(4-hydroxy-3-methylbutylamino)purine (DHZ), or 6-(3-methyl-2-butenylamino)purine (2ip), but lowered the activity of 6-(o-hydroxybenzylamino)purine (OHBA) and 6-(4-hydroxy-3-methyl-cis-2-butenylamino)purine (cZ). 2-Methylthio substitution increased the activity of 2ip and DHZ, decreased the activity of tZ, and had no effect on the activity of cZ. The activities of the simultaneously substituted 2-methylthio-9-ribosyl compounds are lower than those of their corresponding unsubstituted or 2-methylthio substituted bases with the exception of DHZ. Structure-activity relationships for chlorophyll retention did not parallel many of the relationships found for callus tissue growth stimulation.     

Introduction of Fluorine Into the C8 Position of Purine Nucleosides

Abstract

We have synthesized 2-amino-6,8-difluoro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine (3) from 2-amino-6,8-dichloro-9-(2,3,5-tri-O-acetyl-ß-D-ribofuranosyl)purine (1) in a two-step procedure. The reaction of 3 with anhydrous ammonia in dry 1,2-dimethoxyethane gave 2,8-diamino-6-fluoro-9-(2,3,5-tri-O-acetyl-ß-D-ribofuranosyl)purine (4) in 64.1% yield. Compound 4 was deaminated with t-butylnitrite in tetrahydrofuran to give 2-amino-6-fluoro-9-(2,3,5-tri-O-acetyl-ß-D-ribofuranosyl)purine (6). The ¹H, ¹⁹F, and ¹³C NMR spectral data were determined and evaluated for each of the compounds.     

Recognition of nucleotide analogs containing the 7,8-dihydro-8-oxo structure by the human MTH1 protein

The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as an important defense system against mutagenesis, carcinogenesis, and cell death induced by oxidized purine nucleotides. We previously reported that the functional groups at the 2- and 6-positions of the purine ring affect the recognition by the human MTH1 protein. 8-Hydroxy-dGTP and 8-hydroxy-dATP are substrates of MTH1, and both have the "7,8-dihydro-8-oxo structure." In this study, three nucleotide analogs containing this motif were examined. A synthetic purine analog containing the 7,8-dihydro-8-oxo structure and the 2-amino function (dJTP) was hydrolyzed to the monophosphate with high efficiency by MTH1. On the other hand, two analogs that lack the two-ring system of their bases [formamidopyrimidine-dGTP (FAPY-dGTP) and 2-OH-dYTP] were poor substrates. FAPY-dGTP is a mixture of conformers and was hydrolyzed more than ten-fold less efficiently than 8-hydroxy-dGTP. These results clarify the effects of the 2-amino group and the two-ring system of the purine base on the recognition by the human MTH1 protein.     

The influence of the exocyclic amino group characteristic of GC base pairs on molecular recognition of specific nucleotide sequences in DNA by Berenil and DAPI

The expedient of preparing homologous DNA samples substituted with inosine for guanosine residues, 2,6-diaminopurine (DAP) for adenine residues, or both, has been used to investigate the role of the purine 2-amino group in determining the preferred binding sites for the drugs berenil [1,3-bis(4-phenylamidinium) triazene] and DAPI (4′,6-diamidino-2-phenyl indole) on DNA. The selectivity of these two minor groove binders for AT-rich sequences is seen to be radically altered in the substituted DNA molecules. Neither berenil nor DAPI bind to DAP-substituted DNA where all purine residues bear a 2-amino group. By contrast, they bind to AT-rich, IC-rich and even mixed sequences of the inosine DNA where all purine residues lack the 2-amino group. With the inosine and DAP double substituted DNA, both berenil and DAPI bind preferentially to IC-rich clusters instead of their canonical tracts endowed with an extra 2-amino group through substitution with DAP. These results establish that the location of the purine 2-amino group represents a critical determinant for recognition of DNA nucleotide sequences by the two drugs. © 1997 John Wiley & Sons, Ltd.     

Synthesis of 9-(3,4-dioxopentyl)hypoxanthine, the first arginine-directed purine derivative: an irreversible inactivator for purine nucleoside phosphorylase

The synthesis of two potential arginine-directed purine-based analogues, 6-chloro-9-(3,4-dioxopentyl)purine (6) and 9-(3,4-dioxopentyl)hypoxanthine (7), is reported. Compound 7 was extensively tested as a potential affinity label of purine nucleoside phosphorylase (EC 2.4.2.1) from human erythrocytes. Evidence that 7 reacted with the catalytic center of purine nucleoside phosphorylase includes the following: (1) time-dependent inactivation of the enzyme by 7 was observed; (2) a plot of the pseudo-first-order rate constant for inactivation of the enzyme vs. concentration of 7 was hyperbolic, characteristic of saturation phenomenon; (3) substrates (Pi, arsenate, inosine) and a competitive inhibitor (formycin B) protected the enzyme from inactivation by 7. Compound 7 was 25 times more effective in inhibiting purine nucleoside phosphorylase than butanedione. Evidence that 7 modified arginine(s) includes the following: (1) when the inactivation was performed in borate, both the rate and the extent of inactivation were enhanced compared to those of the controls run in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer; (2) dialysis of inactivator reversed the inactivation in Tris-HCl but not in borate buffer. All the above evidence combined with the previous demonstration [Jordan, F., & Wu, A. (1978) Arch. Biochem. Biophys. 190, 699-704] that butanedione modified only arginines in purine nucleoside phosphorylases and the results presented here demonstrating the similarities in the behavior of butanedione and 7 imply that compound 7 can be called an arginine-directed affinity label for purine nucleoside phosphorylase.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.