Convenient preparation of S-adenosylhomocysteine and related compounds

The substrate and inhibitory specificity of Escherichia coli adenosylhomocysteine (AdoHcy)/methylthioadenosine (MeSAdo) nucleosidase has been explored with several MeSAdo analogues modified on the sugar moiety at the 2′, 3′ and 5′ positions. Alteration at C3′ or at C2′ and C3′ positions in MeSAdo abolished substrate activity. However, the 2′-deoxy analogue of MeSAdo is effective as a substrate; this result provides evidence against a possible general-base catalysis involving the anchimeric assistance of the 2′-A-hydroxy group and the formation of a 1,2-epoxide as an intermediate in the catalytic process. The results of a study of the interaction of an 8,5′-cyclo analogue of MeSAdo with the enzyme indicate the importance of the glycosidic conformation of the substrate for binding to the active site. The enzyme discriminates against methanol attack from the solvent during catalysis. This implies the participation of an enzyme-directed water nucleophile. A poor solvent kinetic deuterium-isotope effect was measured (0.93) on the V max . Plots of log V max and log (V max /K m ) for MeSAdo as a function of pH values from 5.0 to 8.5 are similar, with two presumably essential ionisable groups for catalysis with apparent pK a values of 5.6 and 8.2, whereas K m is independent of pH. When the 2′-A-hydroxy group of MeSAdo is substituted by fluorine, a significant decrease (28 500-fold) in the V max for enzyme-catalysed hydrolysis of the modified substrate is observed. This result indicates a transition state with a substantial oxocarbenium character. From these data, the reaction mechanism for AdoHcy/MeSAdo nucleosidase is discussed.Keywords : adenosylhomocysteine nucleosidase ; substrate; inhibitor ; catalytic mechanism.S-Adenosylhomocysteine (AdoHcy) is the product of biological transmethylation reactions that utilise S-adenosylmethionine (AdoMet) as a methyl donor [1,2]. In various microorganisms (bacteria and protozoa), this compound and 5′-methylthioadenosine (MeSAdo), formed from adenosylmethionine by several independent pathways [3], are substrates of AdoHcy/ MeSAdo nucleosidase. This enzyme catalyses the cleavage of the metabolites into S-ribosylhomocysteine and methylthioribose, respectively, adenine also being generated by both reactions [4,5].AdoHcy/MeSAdo nucleosidase is involved in a variety of biological processes. In particular it is required for the regeneration of free Hcy from AdoHcy in various prokaryotes [5,6], and plays a significant role in the metabolism of these microorganisms via the regulation of the MeSAdo concentration (a potent inhibitor of spermidine and spermine synthases) [7Ϫ9]. In microorganisms in which MeSAdo phosphorylase is missing, AdoHcy/MeSAdo nucleosidase is essential for methionine salvage [10Ϫ12].Correspondence to G. Guillerm, Laboratoire de Chimie Bioorganique, U.M.R. 6519, U.F.R. Sciences de Reims, Moulin de la Housse, B.P. 1039, F-51687 Reims cedex 2, France Fax: ϩ33 3 26 05 31 66. E-mail: georges.guillerm@univ-reims.fr Abbreviations. AdoHcy, S-adenosylhomocysteine; MeSAdo, 5′-d...

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“…5 Synthesis of substrates. AdoHcy was supplied by Sigma; MeSAdo was prepared in two steps from Ado [19].…”

Section: Methodsmentioning

confidence: 99%

The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli

Allart

Gâtel

Guillerm

et al. 1998

European Journal of Biochemistry

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“…MTR, 5-ethylthioribose (ETR), and 5-isobutylthioribose (iBTR) were synthesized (2,5,18) and tested for antiprotozoal activity (Table 2). For these experinments, cultures of P. falciparum (22), G. lamblia (12) per ml, respectively.…”

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confidence: 99%

Analogs of 5-methylthioribose, a novel class of antiprotozoal agents

Riscoe

Ferro

Fitchen

1988

Antimicrob Agents Chemother

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Since drug resistance and toxicity limit the use of available antiprotozoal agents, it is important that new drugs be developed as soon as possible. In this study, the method by which several protozoa degrade 5'-methylthioadenosine (MTA) was shown to differ from MTA catabolism in human cells. To exploit this metabolic difference, two analogs of methylthioribose (MTR), an MTA catabolite, were synthesized and found to be cytocidal to Plasmodiumfalciparum, Giardia lamblia, and Ochromonas malkamensis in vitro. In contrast, these analogs had no effect on cultured mammalian cells. Analogs of MTR represent a potential new class of antiprotozoal drugs.Parasitic protozoa cause some of the most prevalent infectious diseases of humans and produce extensive morbidity and mortality around the world (25). For example, over 600 million people are chronically infected with Plasmodium falciparum and at least 1.5 million malaria-related deaths occur each year (4, 15). Despite intensive research efforts, the development of new and effective antiprotozoal drugs has been difficult because of the close metabolic relationship between protozoa and human cells.5'-Deoxy-5'-methylthioadenosine (MTA) is a naturally occurring nucleoside synthesized from S-adenosylmethionine during the production of the polyamines spermidine and spermine (23). Unlike the polyamines, MTA does not normally accumulate in cells but is rapidly degraded by one of two mechanisms (Fig. 1). In mammals (14), MTA is degraded in one step to adenine and 5-methylthioribose-1-phosphate (MTR-1-P) by MTA phosphorylase (Fig. 1, pathway A). In some other organisms (9), however, MTA is catabolized to MTR-1-P in two steps: (i) catabolism to adenine and MTR via MTA nucleosidase and (ii) conversion of MTR to MTR-1-P in an ATP-dependent reaction catalyzed by MTR kinase (Fig. 1, pathway B). In both instances, MTR-1-P is subsequently recycled to methionine through the intermediate a-ketomethylthiobutyric aci'd (1,11,19). Since MTA nucleosidase and MTR kinase are not present in mammalian cells, we hypothesized that these enzymes represent potential targets for the selective killing of organisms which contain them.To test this hypothesis, cell extracts of P. falciparum, Giardia lamblia, and Ochromonas malhamensis were assayed for MTA phosphorylase (14), MTR kinase (9), and MTA nucleosidase (7) activities. 0. malhamensis, a nonpathogenic protozoan, was studied because it can proliferate by using MTR as a sole source of methionine (21), implying that it contains MTR kinase. Enterobacter aerogenes (8) and Persea americana (avocado fruit; 10) were included as positive controls because each contains MTR kinase activity. Extracts derived from human erythrocytes and liver were also tested. Cell extracts from P. falciparum, G. lamblia, and 0. malhamensis contained significant MTA * Corresponding author. nucleosidase and MTR kinase activities (Table 1). MTA phosphorylase was undetectable in these samples, except in the P. falciparum samples, in which low activity, presumably related to residual...

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“…Several methods have been employed for the nucleophilic displacement of leaving groups with thiols at C5' of nucleoside derivatives (1, [22][23][24][25][26]. However, the procedure of Borchardt et al requires the condensation of liquid ammonia (25) and variable yields have been noted.…”

Section: Resultsmentioning

confidence: 99%

“…However, the procedure of Borchardt et al requires the condensation of liquid ammonia (25) and variable yields have been noted. The disulfidelphosphine procedure of Hata and co-workers (26) is conceptually elegant and gives high yields, but the use of tributylphosphine and purification from phosphine oxide by-products is required.…”

Section: Resultsmentioning

confidence: 99%

Nucleic acid related compounds. 66. Improved syntheses of 5′-chloro-5′-deoxy- and 5′-S-aryl(or alkyl)-5′-thionucleosides

Robins¹,

Hansske²,

Wnuk³

et al. 1991

Can. J. Chem.

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Treatment of ribonucleosides with thionyl chloride/pyridine/acetonitrile (0 °C to ambient temperature) resulted in essentially quantitative formation of 5′-chloro-5′-deoxy-2′,3′-O-sulfinylnucleoside derivatives. These diastereomeric sulfite esters underwent deprotection readily with aqueous methanolic ammonia. This gave 5′-chloro-5′-deoxynucleosides without use of the suspected carcinogen, hexamethylphosphoramide (HMPA). Nucleophilic displacement with sodium thiolates in dimethylformamide (−30 °C to ambient temperature) gave 5′-S-aryl(or alkyl)-5′-thionucleosides in high yields. Treatment of ribonucleosides with thionyl chloride/acetonitrile without pyridine followed by aqueous work-up gave diastereomeric 2′,3′-O-sulfinylnucleosides with an unmodified 5′-hydroxyl group. Diagnostic NMR shifts for cyclic sulfite ester stereochemistry are noted. Key words: adenosine, 5′-S-aryl(or alkyl)-5′-thionucleosides, 5′-chloro-5′-deoxynucleosides, uridine, nucleosides.

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Convenient preparation of S-adenosylhomocysteine and related compounds

Cited by 60 publications

References 2 publications

The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli

The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli

Analogs of 5-methylthioribose, a novel class of antiprotozoal agents

Nucleic acid related compounds. 66. Improved syntheses of 5′-chloro-5′-deoxy- and 5′-S-aryl(or alkyl)-5′-thionucleosides

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