Bio-catalytic synthesis of unnatural nucleosides possessing a large functional group such as a fluorescent molecule by purine nucleoside phosphorylase

Akihiko Hatano, Hiroyuki Wakana, Nanae Terado, Aoi Kojima, Chisato Nishioka, Yu Iizuka, Takuya Imaizumi, Sanae Uehara

Research output: Contribution to journalArticle

Abstract

Unnatural nucleosides are attracting interest as potential diagnostic tools, medicines, and functional molecules. However, it is difficult to couple unnatural nucleobases to the 1′-position of ribose in high yield and with β-regioselectivity. Purine nucleoside phosphorylase (PNP, EC2.4.2.1) is a metabolic enzyme that catalyses the conversion of inosine to ribose-1α-phosphate and free hypoxanthine in phosphate buffer with 100% α-selectivity. We explored whether PNP can be used to synthesize unnatural nucleosides. PNP catalysed the reaction of thymidine as a ribose donor with purine to produce 2′-deoxynebularine (3, β form) in high conversion (80%). It also catalysed the phosphorolysis of thymidine and introduced a pyrimidine base with a halogen atom substituted at the 5-position into the 1′-position of ribose in moderate yield (52-73%), suggesting that it exhibits loose selectivity. For a bulky purine substrate [e.g., 6-(N,N-di-propylamino)], the yield was lower, but addition of a polar solvent such as dimethyl sulfoxide (DMSO) increased the yield to 74%. PNP also catalysed the reaction between thymidine and uracil possessing a large functional fluorescent group, 5-(coumarin-7-oxyhex-5-yn) uracil (C4U). Conversion to 2′-deoxy-[5-(coumarin-7-oxyhex-5-yn)] uridine (dRC4U) was drastically enhanced by DMSO addition. Docking simulations between dRC4U and E. coli PNP (PDB 3UT6) showed the uracil moiety in the active-site pocket of PNP with the fluorescent moiety at the entrance of the pocket. Thus, the bulky fluorescent moiety has little influence on the coupling reaction. In summary, we have developed an efficient method for producing unnatural nucleosides, including purine derivatives and modified uracil, using PNP.

Original languageEnglish
Pages (from-to)5122-5129
Number of pages8
JournalCatalysis Science and Technology
Volume9
Issue number18
DOIs
Publication statusPublished - 2019 Jan 1

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Purine-Nucleoside Phosphorylase
Uracil
Nucleosides
Functional groups
Ribose
Thymidine
Molecules
Dimethyl sulfoxide
Dimethyl Sulfoxide
Phosphates
Regioselectivity
Inosine
Halogens
Hypoxanthine
Uridine
Bioelectric potentials
Escherichia coli
Medicine
Buffers
Enzymes

ASJC Scopus subject areas

  • Catalysis

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Bio-catalytic synthesis of unnatural nucleosides possessing a large functional group such as a fluorescent molecule by purine nucleoside phosphorylase. / Hatano, Akihiko; Wakana, Hiroyuki; Terado, Nanae; Kojima, Aoi; Nishioka, Chisato; Iizuka, Yu; Imaizumi, Takuya; Uehara, Sanae.

In: Catalysis Science and Technology, Vol. 9, No. 18, 01.01.2019, p. 5122-5129.

Research output: Contribution to journalArticle

Hatano, Akihiko ; Wakana, Hiroyuki ; Terado, Nanae ; Kojima, Aoi ; Nishioka, Chisato ; Iizuka, Yu ; Imaizumi, Takuya ; Uehara, Sanae. / Bio-catalytic synthesis of unnatural nucleosides possessing a large functional group such as a fluorescent molecule by purine nucleoside phosphorylase. In: Catalysis Science and Technology. 2019 ; Vol. 9, No. 18. pp. 5122-5129.
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AU - Wakana, Hiroyuki

AU - Terado, Nanae

AU - Kojima, Aoi

AU - Nishioka, Chisato

AU - Iizuka, Yu

AU - Imaizumi, Takuya

AU - Uehara, Sanae

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