Efficient synthesis of nucleosides

Details Efficient synthesis of nucleosides Efficient synthesis of nucleosides

1999-07-12

2001-11-13

Afremova, Vera (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

82

Reexamination Certificate

active

06316228

ABSTRACT:

The present invention relates to the efficient and general synthesis of nucleosides, including thymidine, with whole bacterial cells.
BACKGROUND OF THE INVENTION
Nucleoside phosphorylases, which can be found in bacteria and other organisms, catalyze the reversible phosphorolysis of nucleosides and the transferase reaction involving purine and pyrimidine bases. Purine and pyrimidine nucleoside phosphorylases have been isolated from a number of bacterial cells. Utagawa et al.,
FEBS Lett.
109: 261-63 (1980); Utagawa, et al.,
Agri. Biol. Chem.
49: 3239-46 (1985); Engelbrecht, et al.,
J. Biol. Chem.
244: 6228-32 (1969); Jensen,
Eur. J. Biochem.
61: 377-86 (1976); Jensen, et al.,
Eur. J. Biochem.
51: 253-65 (1975). For example, uridine phosphorylase, thymidine phosphorylase and purine nucleoside phosphorylase have been purified from
Escherichia coli
. Krenitsky et al.,
Biochemistry
20: 3615-21 (1981); Schwartz, et al.,
Eur. J. Biochem.
21: 191-98(1971). Additionally, a thermostable purine nucleoside phosphorylase enzyme has been isolated from
Bacillus stearothermophilus
. Saunders, et al.,
J. Biol. Chem.
244. 3691-97 (1969); Hori et al.,
Agric. Biol. Chem.
53: 2205-2210 (1989).
These nucleoside phosphorylases possess fairly broad substrate specificity and have been utilized in the synthesis of natural and modified ribonucleoside analogs. Shirae et al.,
Agric. Biol. Chem.
52: 1499-1504 (1988); Hennen et al.,
J. Org. Chem.
54:4692-95 (1989); Hori et al.,
Agric. Biol. Chem.
55: 1071-1074 (1991); Hori et al.,
J. Biotech.
17: 121-131 (1991); Hori et al.,
Biosci. Biotech. Biochem.
56: 580-582 (1992). There are fewer examples of the use of whole cells in the synthesis of nucleosides, particularly 2′-deoxynucleosides. Morisawa et al.,
Tetrahedron Lett.
21: 479-482 (1980); Holy et al.,
Nucleic Acid Research Symposia
18:69-72 (1987). Morisawa et al. used a combination of
Enterobacter aerogene
cells and chemical steps to synthesize arabinofuranosyl purine nucleosides. Holy et al. used
E. coli
cells and 2′-deoxyuridine as substrate to synthesize 2′-deoxy purine and pyrimidine nucleosides, but
E. coli
cannot be used above physiological temperatures.
Several nucleosides have important diagnostic and therapeutic uses. For example, the natural 2′-deoxynucleoside, thymidine, is a precursor for the synthesis of a number of important anti-HIV active nucleosides such as 3′-deoxy-3′azidothymidine (AZT) and dideoxydidehydrothymidine (d4T) and various potential antiviral compounds. Thymidine and other deoxynucleosides (both natural and non-natural) are precursors of certain oligomers referred to as “anti-sense oligonucleotides,” many of which are being investigated as drugs against viruses, tumors and bacterial infections.
Yet thymidine and other deoxynucleosides are relatively expensive and difficult to obtain by known approaches. For example, prior to the present invention, thymidine was produced by the hydrolysis of DNA derived from natural sources, like milt, but there are limitations using this approach. Dekker et al.,
J. Chem. Soc.
947-55 (1953). Chemical methods also are available for the synthesis of thymidine, but these involve complex, multi-step methodologies.
For example, an early chemical synthesis of thymidine involves a multi-step procedure which gave very low overall yields of thymidine (<5%). Shaw et al.,
Proc. Chem. Soc.
81-82 (1958); Shaw et al.,
J. Chem. Soc.
50-55 (1959). A more direct chemical method involves coupling of suitably protected thymine with an &agr;-chloro-2-deoxy sugar. Hubbard et al.,
Nucleic Acids Res.
12: 6827-36 (1984). While the coupling step proceeds in good yields, the desired &bgr;-thymidine derivative is produced together with its &agr;-isomer and separation of the desired &bgr;-isomer requires extensive chromatography. In addition, suitably protected bases have to be prepared for the coupling. Also, the &agr;-chloro sugar for the coupling reaction is unstable and has to be carefully prepared and handled. Thus, the overall yield of thymidine by this method is low. A multi-step synthesis of thymidine from D-xylose also has been reported. Rao et al.,
Proc. Ind. Acad. Sci.
(
Chem. Sci.
) 106:1415-21 (1994). However, the overall yield in this case is only 24% and the synthesis involves the use of some difficult to handle reagents (e.g. SnCl
4
). Morisawa et al.,
Tetrahedron Lett.
21: 479-482 (1980) used a combination of
Enterobacter aerogene
cells and chemical steps to synthesize a few arabinofuranosyl purine nucleosides. However, the yield in the enzymatic step alone was merely 34% and only arabinofuranosyl purine nucleosides were synthesized. Holi et al.,
Nucleic Acid Research Symposia
18: 69-72 (1987) used
E. coli
cells and only 2′-deoxyuridine as substrate to synthesize only 2′-deoxy purine and pyrimidine nucleosides. Holi et al. reported that the % conversion to thymidine was 67% in their synthesis. The disadvantage of the approach of Holi et al. include the use of 2′-deoxyuridine, which is expensive relative to other substrates. Additionally, their reliance upon
E. coli
limits the reaction temperature, which leads to slower reaction kinetics and decreased solubilities of reactants and products.
Accordingly, there is a need for improved methods for producing thymidine and other deoxynucleosides that have broad range applications, including the production of other natural and non-natural nucleosides. Due to the societal importance of these nucleosides, there is needed methods capable of being undertaken on an industrial scale. These needs have been unresolved until the advent of the present invention.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods and compositions, including cultures, to produce nucleosides.
It is another object to produce nucleosides from nucleoside precursors and sugar moiety donors.
It is still another object to produce nucleosides with cells, including bacterial cells.
In accomplishing these and other objects, there are provided, in accordance with one aspect of the present invention, methods for producing a nucleoside, comprising the steps of contacting a nucleoside precursor and a sugar moiety donor with a whole cell containing a nucleoside phosphorylase in order to produce the nucleoside, and obtaining the nucleoside from the cell. Xanthine oxidase and the like can be present at this stage, which can shift the reaction equilibrium to the product side.
The nucleoside precursor can be a purine or pyrimidine base and the sugar moiety donor can comprise a ribose, a deoxyribose (including 2-deoxyribose), or other sugar of choice. The nucleoside phosphorylase can be a purine nucleoside phosphorylase or a pyrimidine nucleoside phosphorylase, depending upon the sugar moiety donor. The cell can be any type of cell capable of performing the reactions. The cell can be a bacterial cell, such as
Bacillus stearothermophilus
cells. In accordance with one embodiment, the base is thymine, the sugar moiety donor is 2′-deoxyinosine, and the nucleoside phosphorylase is a purine nucleoside phosphorylase. 2′-Deoxyinosine can be produced in situ from the reaction of adenosine deaminase on 2′-deoxyadenosine.
Depending upon the starting compounds, both natural and non-natural nucleosides can be produced according to the invention. Anticancer nucleosides, such as 5-fluorouracildeoxyriboside (5-FUDR), 5-azacytidine, cytosine arabinoside, arabino-5-azacytidine (Ara-AC) can is be obtained according to the invention. Moreover, nucleoside precursors of therapeutically useful compounds, as the anti-HIV compounds, AZT and dideoxydidehydrothymidine (d4T), can be produced according to the invention.
The invention can employ cytosine, thymine or uracil as a pyrimidine base or adenine and guanine as a purine base, 2′-deoxyinosine as a sugar moiety donor, and purine nucleoside phosphorylase. A pyrimidine nucleoside phosphorylase can be used where the sugar moiety donor is a pyrimidine nucleoside,

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