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Purine nucleoside synthesis: an efficient method employing nucleoside phosphorylases
An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phos...
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| Published in: | Biochemistry (Easton) 1981-06, Vol.20 (12), p.3615-3621 |
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| Main Authors: | , , |
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| Language: | English |
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| cited_by | cdi_FETCH-LOGICAL-a420t-9f8342b3ea3f600c6c63c8e036c6639f0517a2b17709cee38b1c0338c38c8be73 |
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| container_end_page | 3621 |
| container_issue | 12 |
| container_start_page | 3615 |
| container_title | Biochemistry (Easton) |
| container_volume | 20 |
| creator | Krenitsky, Thomas A Koszalka, George W Tuttle, Joel V |
| description | An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively. |
| doi_str_mv | 10.1021/bi00515a048 |
| format | article |
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Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi00515a048</identifier><identifier>PMID: 6789872</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Escherichia coli - enzymology ; Kinetics ; Methods ; Pentosyltransferases - metabolism ; Purine Nucleosides - chemical synthesis ; Purine-Nucleoside Phosphorylase - isolation & purification ; Purine-Nucleoside Phosphorylase - metabolism ; Substrate Specificity ; Thymidine Phosphorylase - isolation & purification ; Thymidine Phosphorylase - metabolism ; Uridine Phosphorylase - metabolism</subject><ispartof>Biochemistry (Easton), 1981-06, Vol.20 (12), p.3615-3621</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a420t-9f8342b3ea3f600c6c63c8e036c6639f0517a2b17709cee38b1c0338c38c8be73</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi00515a048$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi00515a048$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>315,786,790,27097,27957,27958,57121,57171</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/6789872$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Krenitsky, Thomas A</creatorcontrib><creatorcontrib>Koszalka, George W</creatorcontrib><creatorcontrib>Tuttle, Joel V</creatorcontrib><title>Purine nucleoside synthesis: an efficient method employing nucleoside phosphorylases</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.</description><subject>Escherichia coli - enzymology</subject><subject>Kinetics</subject><subject>Methods</subject><subject>Pentosyltransferases - metabolism</subject><subject>Purine Nucleosides - chemical synthesis</subject><subject>Purine-Nucleoside Phosphorylase - isolation & purification</subject><subject>Purine-Nucleoside Phosphorylase - metabolism</subject><subject>Substrate Specificity</subject><subject>Thymidine Phosphorylase - isolation & purification</subject><subject>Thymidine Phosphorylase - metabolism</subject><subject>Uridine Phosphorylase - metabolism</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1981</creationdate><recordtype>article</recordtype><recordid>eNptkMtLxDAQxoMouj5OnoWe9CDVadM2qTffKyxacMVjSLNTN9rHmmnB_e-N7CIehAyZ4fvNDPMxdhjBWQRxdF5agDRKNSRyg42iNIYwyfN0k40AIAvjPIMdtkv07ssERLLNtjMhcyniEZsWg7MtBu1gauzIzjCgZdvPkSxdBLoNsKqssdj2QYP9vJsF2Czqbmnbt789i3lHPtyy1oS0z7YqXRMerP899nJ3O70eh5On-4fry0mokxj6MK8kT-KSo-ZVBmAyk3EjEbhPMp5X_iih4zISAnKDyGUZGeBcGv9kiYLvsePV3IXrPgekXjWWDNa1brEbSAmepsAT6cHTFWhcR-SwUgtnG-2WKgL146H646Gnj9Zjh7LB2S-7Ns3r4Uq31OPXr6zdh0e4SNW0eFY3j8VV8ZqNVeH5kxWvDan3bnCtN-Xfzd9qCIjZ</recordid><startdate>19810609</startdate><enddate>19810609</enddate><creator>Krenitsky, Thomas A</creator><creator>Koszalka, George W</creator><creator>Tuttle, Joel V</creator><general>American Chemical Society</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19810609</creationdate><title>Purine nucleoside synthesis: an efficient method employing nucleoside phosphorylases</title><author>Krenitsky, Thomas A ; Koszalka, George W ; Tuttle, Joel V</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a420t-9f8342b3ea3f600c6c63c8e036c6639f0517a2b17709cee38b1c0338c38c8be73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1981</creationdate><topic>Escherichia coli - enzymology</topic><topic>Kinetics</topic><topic>Methods</topic><topic>Pentosyltransferases - metabolism</topic><topic>Purine Nucleosides - chemical synthesis</topic><topic>Purine-Nucleoside Phosphorylase - isolation & purification</topic><topic>Purine-Nucleoside Phosphorylase - metabolism</topic><topic>Substrate Specificity</topic><topic>Thymidine Phosphorylase - isolation & purification</topic><topic>Thymidine Phosphorylase - metabolism</topic><topic>Uridine Phosphorylase - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Krenitsky, Thomas A</creatorcontrib><creatorcontrib>Koszalka, George W</creatorcontrib><creatorcontrib>Tuttle, Joel V</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Krenitsky, Thomas A</au><au>Koszalka, George W</au><au>Tuttle, Joel V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purine nucleoside synthesis: an efficient method employing nucleoside phosphorylases</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1981-06-09</date><risdate>1981</risdate><volume>20</volume><issue>12</issue><spage>3615</spage><epage>3621</epage><pages>3615-3621</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><notes>ark:/67375/TPS-DNPBPW6H-P</notes><notes>istex:9764B5304E1B9CABC8D7B1DFAE0B27F4F5F2F9E2</notes><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>6789872</pmid><doi>10.1021/bi00515a048</doi><tpages>7</tpages></addata></record> |
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| identifier | ISSN: 0006-2960 |
| ispartof | Biochemistry (Easton), 1981-06, Vol.20 (12), p.3615-3621 |
| issn | 0006-2960 1520-4995 |
| language | eng |
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| source | American Chemical Society |
| subjects | Escherichia coli - enzymology Kinetics Methods Pentosyltransferases - metabolism Purine Nucleosides - chemical synthesis Purine-Nucleoside Phosphorylase - isolation & purification Purine-Nucleoside Phosphorylase - metabolism Substrate Specificity Thymidine Phosphorylase - isolation & purification Thymidine Phosphorylase - metabolism Uridine Phosphorylase - metabolism |
| title | Purine nucleoside synthesis: an efficient method employing nucleoside phosphorylases |
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