Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/38—Nucleosides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/38—Nucleosides
  • C12P19/385—Pyrimidine nucleosides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/38—Nucleosides
  • C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides

Definitions

• the present invention refers to a process for the selective deprotection of hydroxyl functions of polyesters, in particular of di- or tri-esters, of nucleosides. More particularly, the invention refers to the selective hydrolysis in the positions 3' and/or 5' of 2', 3',5'-tri-O-acylribonucleosides or in the position 3' or 5' of 3',5'-di- O-acyl-2'-deoxyribonucleosides in the presence of a lipase immobilized on a hydrophobic support and in an aqueous solvent to obtain corresponding 2 ',5 '(or 2',3')-di-O-acylribonucleosides, or 2'-O-acylribonucleosides or 5'(or 3')-O-acyl-2'- deoxyribonucleosides respectively.
• the lipase from Candida rugosa is indicated as inactive in the publication by D. I. Roncaglia et al., Biotechnology Letters, 2001, 23, 1439-1443, as it results from the fact that, in the enzymatic hydrolysis of 2',3',5'- tri-O-acetylpurines in a buffer or in mixtures buffer/dioxane or buffer/acetonitrile, it leads to the formation of totally deacetylated products.
• this "one pot" 3',5'-di-O-deacylation also may generally occur with other lipases, such as with lipases from porcine pancreas, from Pseudomonas cepacia or Rhizomucor miehei, immobilized on a hydrophobic support.
• nucleoside base which can be a triazole, imidazole or purine base, and/or on the sugar radical.
• aqueous solvent designates a solvent formed by water, for the most part, and by a water-miscible organic solvent.
• the aqueous solvent in which the hydrolysis is performed consists of water and of a water-miscible solvent selected from the group consisting of polar aprotic solvents such as dimethyl formamide, dimethyl acetamide, dimethyl sulfoxide or, preferably, acetonitrile, wherein water represents at least 60%, practically 60-90%, more advantageously 70-80% and the water-miscible solvent represents 40-10%, more advantageously 30-20%.
• polar aprotic solvents such as dimethyl formamide, dimethyl acetamide, dimethyl sulfoxide or, preferably, acetonitrile
• the pH which may vary from 5 to 9, advantageously from 6.5 to 7.5 and will preferably about 7, is maintained by a buffer such as a buffer formed by alkaline metal phosphates, advantageously a KH 2 PO 4 buffer in a concentration which may vary from 10 and 100 mM and will preferably be of about 25mM, hereinbelow simply referred to as "phosphate buffer".
• a buffer such as a buffer formed by alkaline metal phosphates, advantageously a KH 2 PO 4 buffer in a concentration which may vary from 10 and 100 mM and will preferably be of about 25mM, hereinbelow simply referred to as "phosphate buffer".
• the selective hydrolysis is carried out in phosphate buffer/acetonitrile in a ratio of from 70/30 to 80/20 and at a pH of from 6.5 to 7.5, preferably of about 7, by using a lipase suitably immobilized on a hydrophobic support
• the lipase used as a catalyst for the selective hydrolysis may be anyone of the lipases coming from animal or microbial sources, such as a lipase obtainable from porcine pancreas or from a microorganism, for example from Humicola, Aspergillus, Rhizopus such as Rhizopus arrhizus, Mucor, Rhizomucor such as Rhizomucor miehei, Candida or Pseudomonas.
• Advantageous lipase from animal origin is that from porcine pancreas and advantageous microbial lipases are those obtainable from microorganisms of the genus Candida and of the genus Pseudomonas.
• the lipase from Candida may be obtained from Candida rugosa, Candida antarctica, Candida lipolytica, Candida utilis, that from Candida rugosa being preferred.
• the lipase from Pseudomonas may be obtained from Pseudomonas putida, Pseudomonas pseudoalkaligenes, Pseudomonas alcaligenes, Pseudomonas cepacia or, advantageously, from Pseudomonas fluorescens.
• said lipase is selected from the group consisting of those obtainable from Candida rugusa or from a microorganism expressing the coding sequence which is present in Candida rugosa or is cloned from it (herein referred to as "Candida Rugosa Lipase”), from Pseudomonas fluorescens or from a microorganism expressing the coding sequence which is present in Pseudomonas fluorescens or is cloned from it (herein referred to as "Pseudomonas Fluorescens Lipase”) and from porcine pancreas or from an animal organism expressing the same coding sequence (or a close homologue thereof) or is cloned from it (herein referred to as "Porcine Pancreatic Lipase”), said lipases being immobilized on a hydrophobic support.
• hydrophobic support designates a matrix containing hydrophobic chemical groups, such as for example alkyl chains or other hydrophobic residues as well as a matrix containing a number of suitably modified, not hydrophobic groups such as, for example, epoxy groups which, appropriately derivatized with reacting groups containing hydrophobic moieties, confer a higher degree of hydrophobicity to the support.
• degree of hydrophobicity means the percent of hydrophobic groups which are present on the surface of the support. As a consequence, two resins having "equal degree of hydrophobicity" will present, on their surface, identical hydrophobic residues or residues endowed with comparable hydrophobic characteristics.
• the immobilization of the lipase is normally made on a solid hydrophobic support, such as, for example on a silicon matrix consisting of an organosilycic compound, namely of a compound containing at least a Si-C bond (US 6,080,402), on a macroporous matrix of silica or silicates (EP 444092), on a matrix consisting of adsorbing, optionally reticulated acrylic resins such as Amberlite ® XAD-8 or Lewatit ® E 2001/85 (EP 529424), of an amphiphilic support containing lipophilic chains (US 5,182,201), on a styrene and divinylbenzene matrix optionally containing epoxy groups such as Lewatit ® R259 K or R 260 K or Diaion ® HP-40, on a polyacrylic resin containing epoxy groups such as FP 4000, on a polymethacrylic resin containing epoxy groups such as Sepabeads ® FP-EP or
• the immobilization may be made on a octyl agarose gel such as Octyl Sepharose ® CL-4B or on polymetacrylate based resin having a butyl character such as Sepabeads ® FP- BU or a octyl character such as Sepabeads ® FP-RPOD which are already totally derivatized with hydrophobic groups, said hydrophobic groups being butyl or decaoctyl chains, respectively.
• a octyl agarose gel such as Octyl Sepharose ® CL-4B
• polymetacrylate based resin having a butyl character such as Sepabeads ® FP- BU or a octyl character such as Sepabeads ® FP-RPOD which are already totally derivatized with hydrophobic groups, said hydrophobic groups being butyl or decaoctyl chains, respectively.
• Preferred selective hydrolysis catalysts are the Candida Rugosa Lipase, the Pseudomonas Fluorescens Lipase and the Porcine Pancreas Lipase, said lipases being immobilized on an octyl agarose gel, in particular on Octyl Sepharose ® CL-4B or on resins presenting comparable hydrophobic groups, namely having a hydrophobicity degree equal or higher than that of said octyl-agarose gel.
• the immobilization on an octyl agarose gel, or on resins wherein comparable hydrophobic groups are present or may be introduced is normally carried out by dissolving the enzyme in phosphate buffer 10-50 mM at the selected pH, advantageously at a pH of from 6.5 to 7.5, preferably in phosphate buffer at a pH of about 7, by adding to the solution the octyl agarose gel washed with the same phosphate buffer at the same pH, by keeping the mixture under stirring at room temperature and by filtering the immobilized enzyme thus obtained.
• the Bradford assay on the filtrate shows that only 5-20% of the enzyme is not immobilized and that the obtained enzyme immobilized on an octyl agarose gel contains 10-100 mg of protein per g of gel or resin.
• the selective hydrolysis is carried out by incubating the 2',3',5'-tri-O- acylribonucleoside or the 3',5'-di-O-acyl-2'-deoxyribonucleoside in an aqueous solvent, as defined hereinabove, at a temperature of from 4 to 40°C, normally at room temperature, for a period of time of from 30 to 75 hours in the presence of the immobilized enzyme or by passing the solution of the starting 2',3',5'-tri-O- acylribonucleoside or 3', 5 '-di-O-acyl-2' -deoxyribonucleoside through a column containing the immobilized enzyme.
• the aqueous solvent consists of 70-90% of 25 mM phosphate buffer at a pH of from 5 to 9, advantageously of from 6.5 to 7.5, preferably of about 7, and 30-10% of acetonitrile.
• the immobilized enzyme is stable and it is able to show and assume important regioselective characteristics.
• the 2'-3'-di-O-acyl-, 2',5'-di-O-acyl- or 2-O- acylribonucleoside or the 3'- or 5 '-O-acyl-2' -deoxyribonucleoside is isolated according to known methods, for example by chromatography, evaporation of the solvent and crystallization of the residue or by freeze drying.
• the product thus isolated consists of a 3'-monodeacylribonucleoside or 5'-monodeacylribonucleoside or 3',5'-di-deacylribonucleoside in which the corresponding 2',3',5'-tri-deacyl- derivative is not detectable at the NMR spectrum at 400 MHz or of a 3'- or 5'- monodeacyl 2 '-deoxyribonucleoside in which the corresponding 3',5'-di-deacyl- derivative is not detectable at the NMR spectrum at 400 MHz.
• the desired product is isolated by freeze drying, it may contain a little amount of starting material which, however, does not influence the use of the desired product as an intermediate for further transformations because the starting material, totally acylated, can be easily removed in the subsequent working operations.
• B represents the radical of a nucleoside base
• Ac is a (C ⁇ -C ⁇ s)acyl radical
• R represents a hydrogen atom or a group OAc, Ac being as defined above.
• Advantageous starting materials are those of formula U, wherein R and Ac are as defined above and B represents the radical of a nucleoside base selected from the group consisting of the radicals having the structures (a)-(d)
• X ⁇ represents hydrogen, a (C ⁇ -C 4 )alkyl group, an aralkyl group or a group Aci, Aci being a (C ⁇ -C 9 )acyl group
• X 2 represents hydrogen or a (C ⁇ -C 4 )alkyl group
• X 3 represents hydrogen, a (C ⁇ -C 4 )alkyl group or a halogen atom
• Y represents an oxygen or sulfur atom.
• the alkyl group may be a saturated, linear or branched-chain aliphatic radical such as ethyl, n-propyl, isopropyl, n-butyl, or, preferably, methyl.
• the aralkyl group is benzyl, optionally substituted with a halogen atom or with a nitro or alkoxy, the ether alkyl group being as defined above.
• the (C ⁇ -C ⁇ g)acyl radical represented by Ac may be a formyl, propanoyl, n- pentanoyl, n-hexanoyl, myristoyl, palmitoyl, stearoyl, benzoyl, phenylacetyl or, preferably, an acetyl or n-butanoyl group.
• the group Aci may be a (C ⁇ -C 9 )acyl such as formyl, propanoyl, phenylpropanoyl, cyclopentylacetyl or, preferably, n-butanoyl, acetyl or phenylacetyl.
• the halogen may be chlorine, bromine, iodine or preferably fluorine. It is understood that Ac means a given (C ⁇ -C ⁇ 8 )acyl radical which is the same in the positions 3', 5' and, if any, 2'.
• interesting starting materials of the process of the present invention are compounds of formula Ha
• Ac is a (C ⁇ -C ⁇ 8 )acyl radical
• R represents a hydrogen atom or a group OAc, Ac being as defined above
• B' represents the radical of a nucleoside base selected from those having the formulas (b), (c) and (d) as defined above.
• the invention provides a process for the mono-deacylation in position 5' or 3' of a nucleoside of formula Ha above, wherein Ac is a (C ⁇ -C ⁇ 8 )acyl radical, R represents a hydrogen atom or a group OAc,
• B' represents the radical of a nucleoside base selected from those having the structures (b), (c) and (d) as defined above, which comprises treating said nucleoside with a lipase immobilized on a hydrophobic support in an aqueous solvent at a pH of from 5 to 9.
• the selective hydrolysis is carried out by incubating the starting nucleoside of formula ⁇ in an aqueous solvent, as defined hereinabove, at a temperature of from 4 to 40°C, normally at room temperature, for a period of time of from 3 to 80, preferably 30 to 75 hours in the presence of the immobilized enzyme or by passing the solution of the starting nucleoside ⁇ through a column containing the immobilized enzyme.
• Advantageous starting materials according to this advantageous embodiment are the compounds of formula Ha, wherein R and Ac are as defined above and B' represents a radical selected from the group consisting of those having the above- defined structures (b), (c) and (d), in which X ⁇ is hydrogen or a (CrC 9 )acyl, X 3 is hydrogen, fluorine or methyl and Y is oxygen.
• Ac is (C ⁇ -C 9 )acyl
• R is a OAc and B' represents a radical of structure (c), in which X 3 is hydrogen or fluorine or methyl and Y is oxygen or
• Ac represents a (d-C 9 )acyl
• R is hydrogen and B' represents a radical of structure (c), in which X 3 is methyl and Y is oxygen.
• Particularly advantageous starting materials according to this advantageous embodiment are those of formula Ha wherein: - R is hydrogen or, preferably, acetyloxy, Ac is acetyl and B' is a radical having the structure (b) wherein X ⁇ is hydrogen, phenylacetyl or acetyl and Y is an oxygen atom; or
• - R is acetyloxy, Ac is acetyl and B' represents a radical having the structure (c), wherein X represents hydrogen or fluorine and Y represents oxygen; or - R is hydrogen, Ac is acetyl and B' represents a radical having the structure (c), wherein X 3 represents methyl and Y represents oxygen; or
• R is hydrogen or, preferably, acetyloxy
• Ac is acetyl
• B represents a radical of structure (d), wherein Xi represents hydrogen, acetyl or phenylacetyl and Y represents oxygen.
• the present invention provides a process for the preparation of a nucleoside of formula la
• R 2 O R wherein B' represents a radical selected from the gr -oup consisting of those having the formulas (b), (c) and (d) as defined above, R represents a hydrogen atom or a group OAc , Ac being a (C ⁇ -C ⁇ s)acyl radical, R] and R 2 represent hydrogen or an Ac radical, Ac being as defined above, one of Rj and R 2 being hydrogen, which comprises treating the corresponding 3',5'-diprotected nucleoside of formula Ha, wherein B', R and Ac are as defined above, with a lipase immobilized on a hydrophobic support in an aqueous solvent at a pH of from 5 to 9.
• the selective hydrolysis is carried out as illustrated above, advantageously at a pH of from 6.5 to 7.5, preferably at a pH of about 7 using a lipase from Candida rugosa as preferred micro-organism of the genus Candida, a lipase from Pseudomonas fluorescens or Pseudomonas cepacia as preferred micro-organism of the genus Pseudomonas or a lipase from porcine pancreas as preferred animal lipase, the aqueous solvent being preferably a phosphate buffer/acetonitrile mixture in the above illustrated ratios and said lipase being immobilized on a gel octyl-agarose or on a Sepabeads resin having a hydrophobicity degree equal to or higher than that of said octyl-agarose gel.
• a lipase from Candida rugosa as preferred micro-organism of the genus Candida
• Y is oxygen
• Y is oxygen
• a lipase from Pseudomonas fluorescens whereby a selective 3'-deacylation occurs, said lipase being immobilized on a hydrophobic support
• - the 3',5'-di-O-acetyl-thymidine [formula Ha in which Ac is acetyl, R is hydrogen and B' is a radical having the structure (c) in which X 3 is methyl and Y is oxygen]
• a lipase from porcine pancreas whereby the selective 3'-deacylation occurs, said lipase being immobilized on a hydrophobic support.
• nucleosides of formula lib are nucleosides of formula lib
• Ac is a (C ⁇ -C ⁇ s)acyl radical
• R represents a hydrogen atom or a group OAc
• B" represents the radical of a nucleoside base having the structure (a) as defined above.
• the present invention provides a process for the mono- or di-deacylation in the positions 5' and 3' of a 2',3',5'-tri-O- acylribonucleoside of formula E '
• Ac is a (C ⁇ -C ⁇ s)acyl radical and B" represents the radical of a nucleoside base having the structure (a)
• Xi represents hydrogen, a (C ⁇ -C 4 )alkyl group, an aralkyl group or a group
• Aci being a (C]-C 9 )acyl group and X 2 represents hydrogen or a (Cj-C 4 )alkyl group, which comprises treating said compound of formula lib' with a lipase immobilized on a hydrophobic support, in an aqueous solvent at a pH of from 5 to 9.
• the enzymatic hydrolysis is carried out with a lipase isolable from an animal or microbial source, advantageously from porcine pancreas or from Pseudomonas fluorescens, Pseudomonas cepacia, Candida rugosa or Rhizomucor miehei.
• a lipase isolable from an animal or microbial source advantageously from porcine pancreas or from Pseudomonas fluorescens, Pseudomonas cepacia, Candida rugosa or Rhizomucor miehei.
• the obtention of a 5'-deacylation or of a 3',5'-di-deacylation mainly depends on the enzymatic charge and on the incubation time.
• the preferred conditions are as illustrated above, i.e. incubation of the starting nucleoside of formula Ub' in an aqueous solvent, as defined hereinabove, advantageously at a pH of from 6.5 to 7.5, preferably of about 7, at a temperature of from 4 to 40°C, normally at room temperature, for a period of time of from 3 to 80 hours in the presence of the immobilized enzyme or percolation of the solution of the starting nucleoside Ub' through a column containing the immobilized enzyme.
• Particularly advantageous starting materials according to this other embodiment are those of formula Hb' wherein B" represents a radical having the structure (a), in which Ac is a (C]-C 9 )acyl, Xi is hydrogen or Act as defined above and X 2 is hydrogen.
• the N-n- butanoyl-2',3',5'-tri-O-n-butanoyladenosine [formula Ub' in which Ac is n-butanoyl, and B" is a radical having the structure (a) in which X ⁇ is n-butanoyl and X 2 is hydrogen] is used as starting material.
• Said starting material is treated with a lipase selected from the group consisting of those obtainable from porcine pancreas, Pseudomonas fluorescens, Pseudomonas cepacia, Candida rugosa or Rhizomucor miehei, said lipase being immobilized on a hydrophobic support.
• a lipase selected from the group consisting of those obtainable from porcine pancreas, Pseudomonas fluorescens, Pseudomonas cepacia, Candida rugosa or Rhizomucor miehei, said lipase being immobilized on a hydrophobic support.
• the invention provides a process for the preparation of a nucleoside of formula lb
• B" is a radical having the structure (a) in which Xi is n-butanoyl and X 2 is hydrogen, is treated with a lipase immobilized on a hydrophobic support, in particular from Candida rugosa or from Pseudomonas fluorescens, to obtain a corresponding compound of formula lb, in which R 2 is n-butanoyl.
• the product thus obtained may be further submitted to a supplemental hydrolysis with a lipase immobilized on a hydrophobic support, advantageously with a lipase from porcine pancreas, Pseudomonas fluorescens, Pseudomonas cepacia,
• Candida rugosa or Rhizomucor miehei whereby a compound of formula lb, wherein
• R 2 is hydrogen is obtained.
• the selective hydrolysis of N-n-butanoyl-2 , ,3',5'-tri-O-n-butanoyladenosine in the presence of a lipase from Pseudomonas fluorescens preferably gives the N-n-butanoyl-2',3'-di-O- «- butanoyladenosine, while the selective hydrolysis in the presence of lipase from Candida rugosa preferably affords the N-n-butanoyl-2'-O-n-butanoyladenosine
• any lipase from animal or microbial origin immobilized on a hydrophobic support in particular lipases from porcine pancreas, Pseudomonas fluorescens, Pseudomonas cepacia, Candida rugosa or Rhizomucor miehei may generate N-n-butanoyl-2'-O-n-butanoyladenosine starting from N-n-butanoyl-2',3',5'-tri-O-n-butanoyladenosine, the most advantageous conditions for this purpose use a lipase from Candida rugosa immobilized on a hydrophobic support, preferably on octyl-agarose gel in phosphate buffer at a pH of from 5 to 9, advantageously of from 6.5 to 7.5 with an enzyme charge and incubation time sufficient to assure the 3 ',5 '-hydrolysis.
• N-n-butanoyl-2',3',5'-tri-O-n-butanoyladenosine prepared by dissolving the calculated amount (for example 246 mg) of N-n-butanoyl-2',3',5'-tri- O-n-butanoyladenosine, synthesized e.g.
• the N-n-butanoyl-2'-O-n-butanoyladenosine is purified by chromatographic separation by using an eluting mixture in gradient from CH 2 C1 2 100 to CH 2 Cl 2 -MeOH 90-10.
• anhydride and the halides preferably the chloride, even though mixed anhydrides and active esters may be successfully used.
• tertiary organic bases for example pyridine, dimethylaminopyridine, methylmorpholine, triethylamine and the like may be used. Acylation may be carried out for example according to the general method described by A. Matsuda et al. in
• said compounds can be obtained by reaction of tetra-acylribose or triacyl-2-deoxyribose with the selected base according to Scheme 2 as described by G. Gosselin et al. in Journal Medicinal Chemistry, 1987, 30, 982-991.
• Scheme 2 wherein R and B are as defined above and Ac preferably represents formyl or acetyl.
• the corresponding starting materials of Scheme 1 are known in the literature or can be easily prepared by known methods.
• Analogously, the starting compounds of Scheme 2 are known in the literature or can be easily prepared by known methods.
• the process of the present invention allows the selective preparation on industrial scale of ribonucleoside derivatives of formula la wherein only one of the sugar hydroxy groups is free while the others are O-acylated or, in the case of 2'- deoxynucleoside derivatives, only one of the sugar hydroxy groups is free while the other is O-acylated.
• the compounds of formula la thus obtained are useful intermediates in the preparation of ribonucleoside or 2'-deoxyribonucleoside derivatives by conversion of the free hydroxy group into a corresponding leaving group which allow the replacement of the hydroxy group for example by hydrogen or by another functional group.
• the compounds of formula la obtained by hydrolysis catalyzed by a lipase which is selective for one of the groups 3' or 5' may be submitted to a subsequent hydrolysis catalyzed by the other lipase in order to remove also the other acyl group in the position 5' or 3', respectively.
• This method may be applied to anyone of the products of formula I, but it is particularly useful when R represent a (C ⁇ -C ⁇ g)acyloxy radical because it allows the preparation of ribonucleoside derivatives in which the hydroxy groups in the 3' an 5' are free while that in position 2' is O-acylated.
• the present invention provides the use of compounds of formula l compounds of formula la'
• R and B' are as defined above, by enzymatic hydrolysis.
• compounds of formula la wherein B is as defined above, one of Ri and R 2 represents hydrogen and the other represents a group Ac as defined above and R represents a group AcO, Ac being as defined above, are used as starting materials for the enzymatic hydrolysis.
• the compounds N-n-butanoyl-2',3'-di-O-n-butanoyladenosine and N-n- butanoyl-2'-O-n-butanoyladenosine are novel and represent a further object of the present invention.
• These compounds are useful intermediates in the synthesis of sodium bucladesine, i.e. of the sodium salt of the cyclic phosphate of N-n-butanoyl-2'-O-n- butanoyladenosine which can easily prepared from said N-n-butanoyl-2'-O-n- butanoyladenosine according to known methods.
• said lipase immobilized on a hydrophobic support is selected from the group consisting of lipases from Candida rugosa, lipases from Pseudomonas fluorescens and lipase from porcine pancreas
• said aqueous solvent is phosphate buffer/acetonitrile in a ratio of from 70/30 to 90/10 at a pH of about 7
• said hydrophobic support is selected from the group consisting of octyl-agarose gel and Sepabead resins having a hydrophobicity equal or higher than that of said octyl- agarose gel.
• the derivative thus obtained consisting of the resin whereon the enzyme is immobilized, is washed with the minimal amount of water (5 ml).
• the lipase from Candida rugosa immobilized on Octyl Sepharose ® CL-4B, containing 42 mg of protein per gram of gel is obtained in an immobilization yield, calculated as amount of immobilized enzyme, equal to 80% (Bradford assay) and, calculated as expressed activity, equal to about 86%.
• the lipase from Pseudomonas fluorescens immobilized on Octyl Sepharose ® CL-4B, containing 42 mg of protein per gram of gel is obtained in an immobilization yield, calculated as the amount of immobilized enzyme, equal to about 99% (Bradford assay) and, calculated as expressed activity, equal to about 56%.
• gel octyl agarose Octyl Sepharose ® CL-4B, Pharmacia Biotech
• the lipase from Rhizomucor miehei immobilized on Sepabeads FP-RPOD, containing 42 mg of protein per gram of resin is obtained in an immobilization yield, calculated as amount of immobilized enzyme, equal to about 39% (Bradford assay) and, calculated as expressed activity, equal to about 450%, value due to the hyperactivation of the enzyme.
• an assay has been made in order to compare the specific activity of the enzyme immobilized on hydrophobic supports (gel octyl-agarose, Butyl-Sepabeads, Decaoctyl-Sepabeads) with that of the enzyme in free form (free enzyme) or immobilized on a non hydrophobic resin (Amberlite, Eupergit C).
• EXAMPLE 2 2 ',5 '-di-O-acetyluridine
• EXAMPLE 1 By operating as described in EXAMPLE 1, to the 25 mM solution of 2',3',5'-tri-O- acetyluridine prepared as described in PREPARATION VI, 2 g of lipase from Pseudomonas fluorescens immobilized on gel octyl agarose as described in PREPARATION II are added, then the mixture is treated as in EXAMPLE 1. After a 64-hour incubation, the 2',5'-di-O-acetyluridine is obtained in a 85% yield.
• the 2',5'-di-O-acetyluridine thus obtained is isolated by chromatographic separation from the starting product by using an eluting mixture in gradient from CH 2 C1 2 100 to CH 2 Cl 2 -MeOH 90-10.
• the pure product thus obtained is characterized by NMR (OH at ⁇ 5.42) and the attribution of the deacetylated position is confirmed by Cosy bidimensional NMR.
• the 5'-O-acetylthymidine thus obtained is isolated by chromatographic separation from the starting product by using an eluting mixture in gradient from CH 2 C1 2 100 to CH 2 Cl 2 -MeOH 90-10.
• the pure product thus obtained is characterized by NMR and the attribution of the deacetylated position is confirmed by Cosy bidimensional NMR.
• EXAMPLES 4-6 2 ',3 '-Di-O-acetyl-5-fluorouridine
• a 25 mM solution of 2',3',5'-tri-O- acetyl-5-fluorouridine prepared by dissolving 191 mg of 2',3',5'-tri-O-acetyl-5- fluorouridine, obtained as described in PREPARATION VI, in 20 ml of 25 mM phosphate buffer at pH 7 containing 30% of CH 3 CN, 2 g of lipase from Candida rugosa or Pseudomonas cepacia or Pseudomonas fluorescens immobilized on gel octyl agarose as described in PREPARATION I, IV and U, respectively, are added, then the mixture is treated as in EXAMPLE 1.
• the 2',3'-di-O-acetyl-5-fluorouridine is obtained in a 56% yield (EXAMPLE 4).
• the 2',3'-di-O-acetyl-5-fluorouridine is obtained in a 49% (EXAMPLE 5) and 60% (EXAMPLE 6) yield respectively
• the 2',3'-di-O-acetyl-5- fluorouridine thus obtained is isolated by chromatographic separation from the starting product by using an eluting mixture in gradient from CH 2 C1 2 100 to CH 2 C1 2 - MeOH 90-10.
• N-n-butanoyl-2 ',3 '-di-O-n- butanoyladenosine thus obtained is isolated by chromatographic separation from the starting product by using an eluting mixture in gradient from CH 2 C1 2 100 to CH 2 C1 2 - MeOH 90-10.
• the pure product thus obtained is characterized by NMR (OH at ⁇ 5.76 ppm) and the attribution of the deacetylated position is confirmed by Cosy bidimensional NMR.

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