Process for producing nucleoside derivatives

Abstract: There is disclosed a process for producing a nucleoside derivative of the formula [I]: characterized by contacting 2',3',5'-O-triacylribonucleoside derivative of the formula [II]: with an ester hydrolase: (i) capable of regio-selectively deacylating the acyl group at 5'-O-position in the formula [II] above, and (ii) having an amino acid sequence encoded by a gene which hybridizes to a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 1

Description: FIELD OF THE INVENTION The present invention relates to a process for producing nucleoside derivatives. DESCRIPTION OF THE RELATED ART 2',3'-O-diacylribonucleosides are useful as intermediates for the synthesis of oligosaccharides such as sialic acid-containing oligosaccharides that are useful for pharmaceutical preparations effective for prophylaxis of inflammation, inhibition of metastasis of cancer, prevention of viral infection and so on. Therefore, a convenient process for producing 2', 3'-O-diacylribonucleosides derivative has been desired. US Patent No. 4 055 718 discloses 9- (2-O-Acyl-β-D-arabinofuranosyl) adenine compounds and a method for their production which comprises the enzymatic removal of the 3-O-acyl and 5-O-acyl groups of a 9-(β-D-arabinofuranosyl)adenine ester. Enzymes that are considered suitable for deacylation are enzymes elaborated by microorganisms of the order Actinomycetales, for example, bacteria of the family Mycobacteriaceae, Streptomycetaceae, Actinomycetaceae, Streptosporangiaceae, and Actinoplanaceae. Also considered suitable are enzymes elaborated by fungi imperfecti. As especially suitable are considered strains of Bacillus subtilis. H. K. Singh, et al. disclose in "Enzymatic regioselective deacylation of 2',3',5'-tri-O-acylribonucleosides: Enzymatic synthesis of 2',3'-di-O-acylribonucleosides", Tetrahedron Letters, Vol. 34, No. 33, pp. 5201-5204, the production of 2',3'-di-O-acylribonucleosides out of 2',3',5'-tri-O-acylribonucleosides using Subtilisin, Protease N and PPL in an organic solvent and a phosphate buffer. A. Kamimura, et al. disclose in Jpn. Kokai Tokkyo Koho JP 02 69,191 the deacylation of 3',5'-dihexanoyl-2'-deoxyuridine with lipase or protease to yield 5'-hexanoyl-2'-deoxyuridine. SUMMARY OF THE INVENTION An object of the invention is to provide: a process for producing a nucleoside derivative of the formula [I]: wherein B represents a purine base in which an amino group may be protected or a pyrimidine base in which an amino group may be protected, and R represents an alkyl group having 1 - 20 carbon atoms, in accordance with claim 1. DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention is described below in detail. In the present process, the purine base in which an amino group may be protected for B in formula [I] or [II] includes adenine, guanine, xanthine and hypoxanthine. The pyrimidine base in which an amino group may be protected for B in formula [I] or [II]includes cytosine, thymine, uracil, 5-methylcytosine and 5-oxymethylcytosine. The amino group of the pyrimidine or purine may be protected by an acyl group such as an acetyl group, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, p-methoxybenzoyl, benzoyl, isobutyryl, 2-methylbutyryl group, all of which and other protective groups can be used according to a conventional method as disclosed in "Protective Groups in Organic Synthesis", Theodora W. Greene, Published by John Wiley & Sons,Inc. (1981), the whole content of which is incorporated herein by reference. The alkyl group having 1 - 20 carbon atoms for R in formula [I] or [II] includes methyl group, ethyl group, propyl group, isopropyl group, isobutyl group, butyl group, t-butyl group, wherein "t" means "tertiary", and the same shall apply hereinafter, amyl group, isoamyl group, t-amyl group, n-hexyl, n-octyl, n-nonyl, n-decyl,, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl and n-Icosyl. The 2',3',5'-O-triacylribonucleoside derivative of the formula [II] can be prepared from the corresponding ribonucleoside according to a known method, for example, a method described in Tetrahedron Letters, No. 9, p. 733 (1969). For example, a 2',3',5'-O-triacylribonucleoside derivative can be produced by reacting a carboxylic acid anhydride such as acetic anhydride or a carboxylic acid halide such as acetyl chloride with a ribonucleoside dissolved in pyridine. The carboxylic acid anhydride used here includes, beside acetic anhydride, propionic anhydride, butyric anhydride and valeric anhydride, and the carboxylic acid halide includes, beside acetyl chloride, propionyl chloride, butyryl chloride, acetyl bromide, propionyl bromide and butyryl bromide. Thus produced 2',3',5'-O-triacylribonucleoside derivative can be isolated by a conventional posttreatment such as removal of the solvent by distillation, separation by extraction or chromatography. Specific examples of the 2',3',5'-O-triacylribonucleoside derivative of the formula [II] include: 2',3',5'-O,N 4 -tetraacetylcytidine, 2',3',5'-O-triacetylcytidine, 2',3';5'-O-triacetyl-5-methylcytidine, 2',3',5'-O-triacetyladenosine, 2',3',5'-O-triacetylguanosine, 2',3',5'-O,N 4 -tetraacetylguanosine, 2',3',5'-O-triacetylinosine, 2',3',5'-O-triacetyluridine, 2',3',5'-O,N 4 -tetrapropionylcytidine, 2',3',5'-O-tripropionyluridine and 2',3',5'-O-tributyryladenosine. The present process uses an ester hydrolase (hereinafter referred to as "the present ester hydrolase"): (i) capable of regio-selectively deacylating the acyl group at 5' -o-position in the formula [II] above, and (ii) having an amino acid sequence encoded by a gene which hybridizes to a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 1. The ester hydrolase may be derived from an animal such as pig and human, or derived from a plant such as castor, or derived from a microorganism belonging to Aspergillus, Candida, Fusarium, Geotrichum, Mucol, Nocardia, Penicillium, Rhizopus, Saccharomyces, Achromobacter, Acinetobacter, Alcaligenes, Chromobacterium, Escherichia, Pseudomonas, Sphingomonas, Bacillus; Burkholderia, Moraxella, Lactobacillus, Staphylococcus , Serratia and Yarrowia . In addition, it may be an ester hydrolase produced by a transformant into which a gene for such ester hydrolase is introduced. From the viewpoint of manufacturing cost, enzymes produced by microorganisms are preferred. Examples of the present ester hydrolase include: an ester hydrolase having an amino acid sequence which has homology of 90% or more with an amino acid sequence of SEQ ID NO: 1 and capable of regio-selectively deacylating an acyl group at 5'-O-position of a 2',3',5'-O-triacylribonucleoside derivative of the formula [II], an ester hydrolase having an amino acid sequence in which one or several amino acids are added to, deleted from, modified in or substituted in an amino acid sequence of SEQ ID NO: 1 and capable of regio-selectively deacylating anacyl group at 5'-O-position of a 2',3',5'-O-triacylribonucleoside derivative of the formula [II]; and an ester hydrolase having an amino acid sequence of SEQ ID NO: 1. The term "hybridize" herein refers to the fact that a double-stranded form is produced between a single-stranded DNA and a single-stranded DNA or a single-stranded RNA that is complementary to the former-most when a double-stranded DNA, a single-stranded DNA or/and a single-stranded RNA are mixed and treated for dissociation from a double-stranded DNA to a single-stranded DNA by a heat-treatment under conditions, for example at 95°C for 1 minute, and then treated for radiation of heat under conditions of allowing to stand at room temperature for 1 minute, or treated for dissociation from a double-stranded DNA to a single-stranded DNA by alkali treatment under conditions of 0.5 M NaOH and 1.5 M NaCl and then treated for neutralization under conditions of 0.5 M Tris.HCl (pH 7.0) and 3.0 M NaCl. The term "a gene which hybridizes" refers to a gene having such a complementarity. Specific examples include a gene having a base sequence encoding an amino acid sequence which has a homology of 90% or more with an amino acid sequence of SEQ ID NO: 1. A microorganism (a strain: E . coli JM109/pAL108) which produces an ester hydrolase having an amino acid sequence of SEQ ID NO: 1 has been deposited to the National institute of Bioscience and Human-Technology Agency of Industrial Science and Technology, Ministry of International Trade and Industry, under the Budapest Treaty with an accession number of FERM-BP5739 (date of receipt: November 7, 1996). Said microorganism can be cultured in various media which are used in routine cultivation of general microorganisms and which contain carbon sources, nitrogen sources with optional organic or inorganic salts and so on. The carbon sources include glucose, glycerol, dextrin, sucrose, organic acids, animal or plant oils and molasses. The nitrogen sources include organic or inorganic nitrogen sources and the like such as meat extract, peptone, yeast extract, malt extract, soybean flour, corn steep liquor (Corn steep Liquor), cotton seed flour, dry yeast, casamino acid, sodium nitrate and urea. The organic or inorganic salts include chlorides, sulfates, acetates, carbonates and phosphates of potassium, sodium, magnesium, iron, manganese, cobalt, zinc and so on, specifically, sodium chloride, potassium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, copper sulfate, sodium acetate, calcium carbonate, sodium carbonate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate. The cultivation can be performed according to a conventional method for general microorganism, and anyone of solid culture, liquid culture (shaking culture in test tube, reciprocal shaking culture, jar fermenter (Jar Fermenter) culture, fermentation tank culture and the like) is possible. When a jar fermenter is used, it is necessary to introduce sterile air, and usually used aeration condition is about 0.1 - about 2 times the volume of culture/minute. The culturing conditions may adequately be set within a range in which the microorganism can grow, and it is preferred that the culture is conducted, for example, at a culture temperature in a range of about 15°C to about 40°C and a pH of medium from about 6 to about 8. While the culture period varies depending on various culture conditions, about 1 to about 5 days are usually desirable. The present ester hydrolase can be utilized in the reaction in the form of microorganisms or cells containing it or be utilized in the reaction in the form of a crude enzyme, purified enzyme or the like separated from said culture or a tissue containing said ester hydrolase. Methods for such separation include conventional methods in which microorganisms, cells or tissues are crushed by, for example, ultrasonic treatment, trituration with glass beads or alumina, treatment with French press or treatment with enzyme such as lysozyme, treatment with Waring blender, and the crushed product is fractionated by salting out with ammonium sulfate, precipitation with organic solvent or organic polymer such as polyethylene glycol or the like, various chromatographies such as ion-exchange chromatography, hydrophobic chromatography, gel filtration chromatography, affinity chromatography, electrophoresis and so on. These methods can be combined, if necessary. In addition, the present ester hydrolase can be used in the easily separable insolublized form. Insolublization of the enzyme can be accomplished by immobilizing the enzyme by immobilizing methods such as the carrier binding method in which said hydrolase is bound to a carrier by covalent bond, ion bond, adsorption or the like, entrapment method in which said hydrolase is entrapped in a meshwork of high polymer. In the process of the present invention, the present ester hydrolase is contacted with the 2',3',5'-O-triacylribonucleoside derivative of the formula [II], for example, about 15°C to about 60°C, preferably about 25°C to about 40°C. The reaction pH is, for example, about 4 to about 9, preferably about 6 to about 8, and it is preferred to use a pH buffering solution in order to keep the pH value within a suitable range. As the pH buffering solution can be used a pH buffering solution consisting of an aqueous solution of an inorganic salt such as a phosphate buffer or a pH buffering solution consisting of an aqueous solution of an organic base such as trishydroxymethylaminomethane (Tris).HCl buffer. The reaction period is not specifically limited and is usually, for example, about 5 minutes to about 96 hours. In addition, in order to improve solubility of substrate in the reaction solution, a hydrophilic organic solvent that does not inhibit the enzymatic activity can be added to the reaction solution. The hydrophilic organic solvent used for such purpose includes, for example, lower alcohols such as ethyl alcohol, propyl alcohol and the like or dimethylformamide or dioxane or dimethylsulfoxide. After completion of the reaction, 2',3'-O-diacylribonucleoside derivative of the formula [I] can be obtained from the reaction solution, by a conventional procedure including salting out, precipitation by solvent and column chromatography. Specifically, for example, a residue obtainable by evaporating solvent from the reaction solution under reduced pressure can be transferred to silica gel thin layer chromatography to separate the desired product of the reaction. Examples of the 2',3'-O-diacylribonucleoside derivatives of the formula [I] thus produced by the present process include the following compounds. 2',3'-O-diacylribonucleoside derivatives of the formula [I]: 2',3'-O,N 4 -triacetylcytidine, 2',3'-O-diacetylcytidine, 2',3'-O-diacetyl-5-methylcytidine, 2',3'-O-diacetyladenosine, 2',3'-O-diacetylguanosine, 2',3'-O,N 4 -triacetylguanosine, 2',3'-O-diacetylinosine, 2',3'-O-diacetyluridine, 2',3'-O,N 4 -tripropionylcytidine, 2',3'-O-dipropionyluridine, and 2',3'-O-dibutyryladenosine. The present invention will be described in more detail by means of Examples, but the present invention is not limited by these Examples. Example 1 Into a test tube containing 8 ml of a sterilized liquid medium (an aqueous solution containing 10 g/l Bacto Tripton (manufactured by Difco); 5 g/l Bacto Yeast Extract (manufactured by Difco); 5 g/l NaCl; 1 mM isopropyl-β -D-thiogalactopyranoside; 50 µg/ml Ampicillin) was inoculated a strain E . coli JM109/pAL108 (Accession number of microorganism: FERM BP-5739) and cultured under reciprocal shaking at 37°C for 8 hours to give a culture solution as a pre-culture. Subsequently, into a 3 liter small fermenter containing 1.5 liter of a sterile medium having the same composition was inoculated 7.5 ml of the pre-culture and the medium was stirred at 37°C at 500 rpm while introducing sterile air at a rate of 0.75 liter per minute. The culture solution obtained after 15 hours was centrifuged to collect cells. The obtained cells were suspended in 50 ml of 100 mM phosphate buffer (pH 7.0) and crushed by ultrasonic treatment. The obtained crushed-cell suspension was centrifuged to give a supernatant as a cell-free extract. To this cell-free extract was dissolved ammonium sulfate to a concentration of 80%, and then the mixture was centrifuged and precipitates were collected. The collected precipitates were suspended in 50 ml of 100 mM phosphate buffer (pH 7.0) and subjected to desalinization treatment by dialysis to give a crude enzyme solution. The obtained crude enzyme solution was freeze-dried to give a powdery crude enzyme sample of the ester hydrolase Subsequently, 9 g of 2',3',5'-O,N 4 -tetraacetylcytidine was dissolved in 9 g of ethyl alcohol in a glass container, 90 ml of 0.2% aqueous Triton X100™ solution was added thereto, 270 ml of 0.2 M concentration phosphate buffer (pH 7.2) was further added, and the mixture was incubated at 30°C while stirring with a magnet rotor. To this was added 20 mg of the above-described crude enzyme sample of the ester hydrolase to commence the enzymatic reaction. 6 Hours after the commencement of the enzymatic reaction, 90 g of anhydrous sodium sulfate was added to and dissolved in the reaction solution, upon which the product precipitated. The precipitates were collected by filtration and air-dried. The obtained solid was charged onto a column packed with silica gel, eluted with a mixed solvent of ethyl acetate and ethyl alcohol (1 : 1 in the volume ratio), and the solvent was stripped off from the obtained eluate under reduced pressure to give 6 g of the product. The product obtained here was confirmed to be an almost pure 2',3'-O,N 4 -triacetylcytidine by 1 H-NMR measurement in heavy water. Example 2 In a 100 ml glass container, 0.37 g of 2',3',5'-o-triacetyluridine was dissolved in 0.5 g of ethyl alcohol, 5 ml of 0.2% aqueous Triton X100 TM solution was added thereto, 15 ml of 0.2 M concentration phosphate buffer (pH 7.2) was further added, and the mixture was incubated at 30°C while stirring with a magnet rotor. To this was added 3 mg of the crude enzyme sample of the ester hydrolase prepared in Example 1 to commence the enzymatic reaction. 6 Hours after the commencement of the enzymatic reaction, 5 g of anhydrous sodium sulfate was added to and dissolved in the reaction solution, and the solvent was stripped off. The obtained solid was extracted with 100 ml of ethyl acetate and the solvent was stripped off from the extract under reduced pressure to give 0.32 g of the product. The product obtained here was confirmed to be an almost pure 2',3'-O-diacetyluridine by 1 H-NMR measurement in heavy water. SEQUENCE LISTING SEQ ID NO: 1 SEQUENCE LENGTH: 393 SEQUENCE TYPE: amino acids TOPOLOGY: linear MOLECULAR TYPE: protein ORIGINAL SOURCE: ORGANISM: Burkholderia cepacia STRAIN: SC-20 SEQUENCE DESCRIPTION:

Claims: (amended) A process for producing a nucleoside derivative of the formula [I]: wherein B represents a purine base in which an amino group may be protected or a pyrimidine base in which an amino group may be protected, and R represents an alkyl group having 1 - 20 carbon atoms, which comprises contacting 2',3',5'-O-triacylribonucleoside derivative of the formula [II] : wherein R and B are the same as defined above, with an ester hydrolase capable of regio-selectively deacylating the acyl group at 5'-O-position in the formula [II] above, and said ester hydrolase has an amino acid sequence: a) represented by SEQ ID NO: 1, b) having 90% or more homology with an amino acid sequence of SEQ ID NO : 1, or c) in which a single amino acid is added to, deleted from, modified in or substituted in the amino acid sequence of SEQ ID NO: 1. The process according to Claim 1, wherein R is a methyl group. The process according to Claim 1, wherein B is uridine or cytidine. The process according to claim 1, 2, or 3, wherein the ester hydrolase is an ester hydrolase that is produced by a strain FERM-BP5739.

Inventors: ISHII TAKESHI, MITSUDA SATOSHI, OHTA HIROMICHI, SUGAI TAKESHI, ISHII, TAKESHI, MITSUDA, SATOSHI, OHTA, HIROMICHI, SUGAI, TAKESHI
Applicants: SUMITOMO CHEMICAL CO, SUMITOMO CHEMICAL COMPANY, LIMITED
Application year: 1998
Main classification: C12P19
IPC codes: C12N9, C12P19