WO2012041965A1

WO2012041965A1 - Process for producing dialkylphosphotriesters of nucleosides by enzymatic transesterification and deprotection thereof for producing nucleoside monophosphates

Info

Publication number: WO2012041965A1
Application number: PCT/EP2011/066988
Authority: WO
Inventors: Adolfo Marcelo Iribarren; Elizabeth Sandra Lewkowicz; Lucas Andrés DETTORRE
Original assignee: Universidad Nacional De Quilmes; Iribarren, Claudia, Cristina
Priority date: 2010-09-29
Filing date: 2011-09-29
Publication date: 2012-04-05
Also published as: AR083221A1

Abstract

A process for producing nucleotide prodrugs, more particularly, the invention relates to a process for preparing dialkylphosphotnesters of nucleosides by enzymatic transesterification. The invention further describes deprotection processes for producing nucleoside monophosphates from nucleoside dialkylphosphotnesters.

Description

PROCESS FOR PRODUCING DIALKYLPHOSPHOTRI ESTERS OF NUCLEOSIDES BY ENZYMATIC TRANSESTERIFICATION AND DEPROTECTION THEREOF FOR PRODUCING NUCLEOSIDE MONOPHOSPHATES

FIELD OF THE INVENTION The present invention relates to a process for producing nucleotide prodrugs. More particularly, the invention relates to a process for preparing dialkylphosphotnesters of nucleosides by enzymatic transesterification. The invention further pertains to deprotection processes for producing nucleoside monophosphates from nucleoside dialkylphosphotnesters. BACKGROUND OF THE INVENTION

During the past 25 years, there have been important improvements in antiviral therapies. The potential benefits of a great variety of new compounds and new strategies for treating many of the viral infections have been already studied. Among them, several nucleoside analogs and their respective prodrugs have been synthesized and tested in the treatment of several viral infections such as human immunodeficiency virus (HIV), hepatic B virus (HBV), and herpes simplex virus (HSV), and for the treatment of some cases of leukemia (C. M. Galmarini, J. R. Mackey, C. Dumontet, The Lancet Oncology, 3, 415-429, 2002).

To exert its action, it is essential that the nucleoside is converted intracellular^ to the corresponding triphosphate, being the limiting step the formation of the monophosphate. Therefore, a new approach in the rational drug design aims to the development of nucleotide prodrugs, wherein the negative charges of the phosphate are "masked" with suitable functional groups that are further turned to the parental nucleotide either by chemical or biological means (Nucleoside and nucleotide prodrugs, J.S. Cooperwood, G. Gumina, F. Douglas Boudinot, C:K. Chu in "Recent advances in nucleosides chemistry and chemotherapy" C.K. Chu, Ed., Elsevier, Holanda, 2002). Then, synthesis of phosphotriesters of nucleosides is a promising alternative in developing new products with good bioavailability. (Romanowska, J., Szymanska-Michalak, A., Boryski, J., Stawinski, J.,Kraszewski, A. Bioorganic & Medicinal Chemistry (2009), 17, 3489-3498). This approach, known as the design of prodrugs of nucleotides (or ProTides pro nucleotides), provides two benefits: on one side the enhanced lipophilicity with respect to the parental nucleotide could improve its penetration through cell membranes, and on the other one this strategy makes possible the nucleoside 5'-monophosphate intracellular release thus skipping the first and more difficult phosphorylation step that is carried out by specific kinases in the respective triphosphate anabolism (Poijarvi-Virta, P.; Lonnberg, H. Curr. Med. Chem. 2006, 13, 3441-3465.).

Up to now the synthesis of nucleoside phosphotriesters was carried out by chemical reactions. Bis (S-pivaloil-2-tioethyl)-5-phosphotriester of AraC was synthesized as a prodrug of AraCMP while several dialkylphosphates of 2',3'-dideoxy-3-deazaadenosine demonstrate better anti-HIV properties than the corresponding nucleoside, at greater lipophilicity of the derivative.

W. Szer et al. (Chemical and enzymatic properties of the methyl phosphate esters of certain pyrimidine nucleoside 5'-phosphate, Biokhimiya, 1961 , 25, 840-5) describe a synthesis of dimethylphosphate esters of nucleosides by chemical reaction of corresponding 5'NMP with diazomethane. Phosphotriesterases (PTEs) constitute an enzyme group that catalyzes the stereoselective hydrolysis of organophosphorus triesters compounds. The use of PTEs has been reported for the enzymatic resolution of chiral analog phosphotriesters of sarin and somar, (Li, W.S, Lum, K.T., Chen-Goodspeed, M., Sorgorb, M.A. and Raushel F.M. Bioorg. Med. Chem. (2001), 9, 2083-2091). Further, the use of a biocatalyst for transesterification of paraoxon with 2-phenylethanol in anhydrous organic medium was also reported, (Sode, K., Ohuchi, S., Nakamura, H. and Narita, M., Biotechol. Lett. (1996), 18, 923-926).

The presence of phosphotriesterases has been described along evolutive scale from bacteria to mammals, with exception of insects. The best characterized PTEs are those from Brevundimonas diminuta (formerly known as Pseudomonas diminuta) and Flavobacterium sp., and human and rabbit serum paraoxonases. These enzymes have been associated to degradation of many organophosphorous toxic agents such as nervous gases and agricultural pesticides, (Raushel, F.M. Curr. Opin. Microbiol. (2002), 5, 288-295). Another strategy for improving nucleoside bioavailability consists in the use of more hydrophilic produgs obtained by the introduction of polar or ionic functions, such as phosphates, which are subsequently biodegraded and converted into the corresponding active principle. A typical case is the Fludarabine 5'-phosphate which is the drug generally used for the treatment of follicular lymphoma and chronic lymphocytic leukemia (T. Yamauchi, T. Ueda / J. Chromatogr. B, 799 81-86, 2004).

In addition, nucleoside monophosphates (NMPs), both natural and modified, have other applications. They are precursors of nucleoside-5' triphosphates (NTPs) that are used as substrates in polymerase chain reaction (PCR) assays, that are broadly used both in molecular biology and in diagnosis practice. They are also employed for finding new antiviral or antitumor agents by means of screenings in vitro.

Besides, some mononucleotides (disodium salts of UMP, CMP, AMP y GMP) are used in food industry mainly for preparing powder milk for children, because they provide similar nucleotide levels to those found in breast milk to strengthen immunity of the baby. The taste of food can be improved with the addition of flavor enhancers. The most used are: table salt, monosodium glutamate (MSG) and 5^'-ribonucleotides like IMP, GMP or the salts thereof. GMP and IMP have the ability to increase the affinity of the MSG receptor site, thus producing a synergic effect that allows the use of less quantity of them to obtain the same effect as a flavor enhancer. The taste Umami, also known as fifth taste because it differs from the classic four tastes, is involved in taste-enhancing activity by means of the enhancers, so for example, the MSG, IMP and GMP combination significantly enhances (p<0.05) the flavor of the dehydrated chicken soup(Carla Gutierrez y Elba Sangronis, Archivos Latinoamericanos de nutricion, 56,3, 2006). In addition it has been found that IMP and GMP act as stimulants for use in aquaculture. The increasing demand for these compounds encourages investing efforts in developing strategies to obtain them in an environmentally clean manner.

The traditional synthetic pathways are not simple. The 5'-phosphorylation of nucleosides can be chemically achieved using chlorophosphates as phosphate donors, (D. V. Yashunsky, A. V. Nicolaev, J. Chem. Soc. Perkin Trans I, 1 195-1 198, 2000); but these reactions show some drawbacks such as formation of higher degree of phosphorylation, the step numbers required and the toxicity of reagents and solvents. The use of enzymes for the regioselective preparation of phosphate esters avoids these limitations. Traditionally, nucleoside monophosphates were enzymatically prepared employing kinases, but this methodology is limited due to the narrow substrate recognition and the requirement of ATP as phosphate donor (H. Mori, A. Lida, T. Fujio, S. Teshiba, Appl. Microbiol. Biotech nol,AQ, 693-698, 1997; US patent No 5,874,272). Barai et al, (Barai VN, Kvach SV, Zinchenko Al, Mikhailopulo IA, Biotechnol Lett. 26(24): 1847-50, 2004) used a nucleoside phosphotransferase from Erwinia herbicola in the presence of Zn²⁺ and p-nitrophenilphosphate for the preparation of different NMPs in yields ranging from 41 to 91 %. More recently, the use of acid phosphatase has been reported for the synthesis of nucleotides. Microbial acid phosphohydrolyses (NSPAs) are non specific enzymes that hydrolyze phosphoesters and phosphoanhydro bonds from a wide range of organic compounds at acid or neutral pH. Employing whole cells or isolated enzymes from enterobacters, the preparation of IMP (K. Ishikawa, Y. Mihara, N. Shimba, N. Ohtso, H. Kawasaki, E-l Suzuki, Y. Asano Protein Eng., 15, 539-543, 2002; US patent 6,987,008) and glucose 6-phosphate (T. Herk, A Hartog, A. Burg, R. Weber, Adv. Synth. Catal, 347, 1 155-1162,2005) was carried out using sodium pirophosphate as a cheap phosphate source.

As far as we know there are no previous reports of NMP enzymatic synthesis from the corresponding phosphotriesters. SUMMARY OF THE INVENTION

The present invention provides a process for producing nucleotide prodrugs. More particularly, the invention relates to a process for preparing nucleoside dialkylphosphotnesters by enzymatic transesterification catalyzed by phosphotriesterase enzymes using nucleosides as nucleophiles and activated phosphotriesters as phosphate donors.

The invention also relates to deprotection processes for producing nucleoside monophosphates from dialkylphosphotnesters.

According to a preferred embodiment of the invention a process for producing a nucleoside monophosphate is provided, the process comprising the steps of:

a) conducting an enzymatic transesterification by reacting a nucleoside with a phosphate donor in the presence of a phosphotriesterase enzyme; b) removing alkyl groups from the nucleoside dialkylphosphotriester obtained in step (a) by reaction with a chemical agent selected from the group of inorganic or organic basic reagents, inorganic or organic acidic reagents, thioalcohols and hydrogenating reagents, or

c) removing alkyl groups from the nucleoside dialkylphosphotriester obtained in step (a) by hydrolysis according to the following steps:

i) incubating the nucleoside dialkylphosphotriester in the presence of a phosphotriesterase enzyme,

ii) incubating the nucleoside alkylphosphodiester of step (i) in the presence of a phosphodiesterase enzyme, and

collecting a nucleoside monophosphate from the reaction mixture.

The invention particularly relates to an enzymatic hydrolysis process for producing nucleoside monophosphates from nucleoside dialkylphosphotriesters, the process comprising the steps of:

collecting a nucleoside monophosphate from the reaction mixture.

The enzymatic hydrolysis according to above steps i) and ii) may be carried out separately or sequentially in one pot. Preferably the steps i) and ii) are carried out sequentially in one pot without isolating the intermediate nucleoside alkylphosphodiester. Alternatively the enzymatic hydrolysis may be carried out in one step using only a phosphotriesterase enzyme according to the teachings of Shim H, Hong SB, Raushel FM (Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase, J. Biol Chem 273 (1998) 17445-50), wherein a hydrolysis of ethyl-4- nitro phenyl phosphate is described. According to a preferred embodiment the source of a phosphotriesterase enzyme for the enzymatic transesterification step of the present invention is Brevundimonas diminuta bacteria, although other sources are also comprised without limiting. Phosphotriesterase enzymes from genetically modified microorganisms are also comprised. The phosphate donors for the purposes of present invention are preferably organophosphorus triesters, preferably alkyl or aryl trisubstituted phosphates, more preferably paraoxon, methylparaoxon, coroxon, chlorpyrifos-oxon, bis β-cyanoethyl p- nitrophenylphosphate, derivatives thereof and the like. The nucleosides used as nucleophile in the process of the invention may be natural or non-natural, containing purinic, pyrimidinic or analogue bases and different sugar moieties including acyclic derivatives and other natural and unnatural sugar analogues . Preferably the nucleoside is selected from the group of inosine, isopropilideninosine, adenosine, guanosine, uridine, cytidine, arabinouridine, arabinoinosine, and 0-2',3'-diacetylinosine. According to a more preferred embodiment a process for producing inosine 5' dimethylphosphotriester is provided by reacting inosine with methyl paraoxon in the presence of a phosphotriesterase enzyme of Brevundimonas diminuta bacteria.

Following similar procedure, uridine 5' dimethylphosphotriester, cytidine 5' dimethylphosphotriester, arabinosine 5' dimethylphosphotriester, 2'3'-0-diacetylinosine 5' dimethylphosphotriester and isopropyliden inosina 5' dimethylphosphotriester may be produced.

According to a preferred embodiment the source of a phosphotriesterase enzyme for the enzymatic hydrolysis of the present invention is Nocardia asteroids CECT 3051 , although other sources are also comprised without limiting. Phosphotriesterase enzymes from genetically modified microorganisms are also comprised.

According to a preferred embodiment the source of a phosphodiesterase enzyme for the purposes of the present invention is phosphodiesterase I from Crotalus atrox although other sources are also comprised without limiting. Phosphodiesterase enzymes from genetically modified microorganisms are also comprised. According to a more preferred embodiment a process for producing isopropylideninosine monophosphate is provided by incubating isopropylideninosine 5'dimethylphosphate by sequential reaction with phosphotriesterase enzyme from Nocardia asteroids bacteria and phosphotriesterase I enzyme from Crotalus atrox bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the metil paraoxon ³¹ P-RMN spectrum. Figure 2 shows the reaction mixture P-RMN spectrum with inosine as nucleophile. The signal at 2.41 ppm corresponds to the phosphorylated product.

Figure 3 shows a HPLC chomatogram of the reaction mixture at cero time; inosine peack (4.43 min) and methylparaoxon peack (19.3 min) are shown.

Figure 4 shows a HPLC chomatogram of the reaction mixture at the 4^th day; inosine peack at 4.2 min and p-nitrophenol peack (17.5 min) are shown; The retention time signal at 6.8 min corresponds to the transesterification product.

Figure 5 shows yields of the enzymatic transesterification process of the invention for several phosphate donor MPO/nucleoside molar ratios.

Figure 6 shows HPLC chromatogram of the reaction mixture at cero time; isoplno peack (15.7 min) and isopDMMIP peack (17.5 min) are shown.

Figure 7 shows HPLC chromatogram of the reaction mixture at 4^th day; isopDMIMP peack (17.6 min), isoplno peack (15.7min) and MeisoIMP peack (12.8 min) are shown.

Figure 8 shows HPLC chromatogram of the reaction mixture at 1 day after addition of phosphodiesterase I enzyme; isopDMIMP peack (17.5 min), isoplno peack (15.7 min), isopMeIMP peack (12.7 min) and isopIMP peack (1 1.9 min)

DETAILED DESCRIPTION OF THE INVENTION

According to a preferred process for obtaining phosphotriesterase enzyme, cultures of Brevundimonas diminuta are run in an appropriate liquid media and incubated at about 30°C for about 48 hours. The biomass is harvested by centrifugation. The pellet thus obtained is resuspended in a pH7 buffer solution and sonicated. The resulting homogenate is centrifuged. The supernatant is discarded and the pellet is washed and resuspended in distilled water, lyophilized and stored at -20°C until use. The thus obtained phosphotriesterase enzyme may be assessed in transesterification reactions using suitable phosphate donors.

According to a preferred embodiment of the invention dialkylphosphotriesters of nucleosides are obtained by enzymatic transesterification catalyzed by phosphotriesterase enzyme using activated phosphotriesters as phosphate donors, preferably organophosphorus triesters compounds. The reaction mixture containing the organophosphorus triester is incubated in the presence of a nucleoside and the enzymatic preparation of a phosphotriesterase enzyme. The solvent of the reaction mixture may be selected from anhydrous organic solvents, preferably dimethylsulfoxide (DMSO) or N,N- dimethylformamide (DMF), being DMF the most preferred solvent. Preferably, molecular sieves are used.

According to a preferred embodiment of the invention nucleoside nucleophiles are compounds of structural formula I which is of the stereochemical configuration:

wherein A, D and G are independently N or CH;

E is N, CH, C-CN, C-N0₂, C-d.₃ alkyl, C-NHCON H₂, C-CONR₁₂Ri₂, C-CSNR₁₂R₁₂, C- COOR₁₂, C-C(=NH)NH₂, C-hydrohy, C-d.3 alkoxy, C-amino, C-d.₄ alkylamino, C-di(d.₄ alkyl)amino, C-halogen; wherein alkyl is unsubstituted or substituted with one to three groups independently selected from halogen, amino, hydroxyl, carboxy and C1.3 alkoxy; J is O or S;

and R₆ are each independently hydrogen, methyl, hydroxymethyl, or fluoromethyl; R2, R3, R1₀ and Rn are independently H, Ci_-4 alkyl, C₂.₆ alkenyl, C₂.₆ alkynyl, OH, Ci_-4 alkoxy, Ci-e alkylcarbonyloxy, aryloxyacarbonyl, SH, Ci_-4 alkylthio, NH₂, Ci_-4 alkylamino, di(Ci-4 alkyl)amino, C₃.₆ cycloalkylamino, , azido, halogen, Ci_-4 alkyl, or CF₃; R₂ and R₃ together with the carbon to which they are attached form a 3- to 6-membered satured or unsaturated monocyclic ring system optionally containing a heteroatom selected from O, S and NC₀-4 alkyl;

R₄ is hydrogen, CF₃, or Ci_-4 alkyl and one of R₃ and R₅ is OH or Ci_-4 alkoxy and the other of R₃ and R₅ is selected from the group consisting of hydrogen, hydroxyl, halogen, Ci_₃ alkyl, trifluoromethyl, Ci_₄ alkoxy, Ci_₄ alkylthio, Ci_₈ alkylcarbonyloxy, aryloxycarbonyl, azido, amino, Ci-₄ alkylamino, and di(Ci_₄ alkyl)amino; or

R₅ is hydrogen, CF₃, or Ci_₄ alkyl and one of R₃ and R₄ is OH or Ci_₄ alkoxy and the other of R₃ and R₄ is selected from the group consisting of hydrogen, hydroxyl, halogen, Ci_₃ alkyl, trifluoromethyl, Ci_₄ alkoxy, Ci_₄ alkylthio, Ci_₈ alkylcarbonyloxy, aryloxy carbonyl, azido, amino, Ci_₄ alkylamino, and di(Ci_-4 alkyl)amino; or

R₄ and R₅ together with the carbon to which they are attached form a 3- to 6-membered satured or unsaturated monocyclic ring system optionally containing a heteroatom selected from O, S and NC₀.₄ alkyl;

R₇ is H, OH, SH, NH₂, Ci_₄ alkylamino, di(Ci_-4 alkyl)amino, C₃.₆ cycloalkylamino, halogen, Ci-4 alkyl, Ci_-4 alkoxy, or CF₃;

R₈ is H, Ci-6 alkyl, C₂-6 alkenyl, C₂-6 alkynyl, Ci_-4 alkylamino, di(Ci_-4 alkyl)amino, CF₃, or halogen;

R₉ is H, halogen, CN, carboxy, Ci_-4 alkyloxycarobnyl, N₃, amino, Ci_₆ alkoxy, Ci_₆ alkylthio, Ci-6 alkylsulfonyl, or (Ci_-4 alkyl)₀.₂ aminomethyl;

Ri₂ is Ci-4 alkyl;

and the acyclic-sugar analogs thereof.

The nucleosides used as nucleophiles in the process of the invention are more preferably selected from inosine, adenosine, guanosine, uridine, cytidine, arabinouridine, arabinoinosine and 2',3'-di-0-acetylinosine, although other nucleosides are also comprised without limiting.

According to a preferred embodiment of the invention the phosphate donors are organophosphorus compounds wherein the compound is selected from:

II wherein the phosphorus atom configuration is R_P or S_P if R_a is different from R and Y, or both in case donor is used as a racemic mixture.

R_a and R are independently CM₀ alkoxy, C2-10 alkenyloxy, C2-10 alkynyloxy, aryloxy, CM₀ alkylthio, C1-4 alkylamino, di(Ci_-4 alkyl)amino, C₃.₆ cycloalkylamino or CM₀ alkyl; wherein alkyl, alkoxy, alkenyloxy and alkynyloxy groups are unsubstituted or substituted with one to six groups independently selected from halogen, nitro, amino, hydroxyl, carboxy, CN, C1-3 alkoxy, C1.3 alkyl or aryl.

X is a heteroatom selected from O and S.

Y is aryloxy or thioaryl, wherein the aryl moiety is an mono or bicyclic aromatic or heterocyclic aromatic system unsubstituted or substituted with one to five groups independently selected from halogen, nitro, cyano, amino, Ci_₄ alkylamino, di(Ci_-4 alkyl)amino, Ci_₆ alkyl, hidroxyl, Ci_₆ alkyloxy, Ci_₆ alkylthio, aryloxy, Ci_₆ alkylcarbonyl, arylcarbonyl, Ci_₆ alkylcarbonyloxy, arylcarbonyloxy, Ci_₆ alkyloxycarbonyl, aryloxycarbonyl or carboxy. More preferably the phosphate donor is selected from paraoxon, methylparaoxon, coroxon, chlorpyrifos-oxon, bis β-cyanoethyl p-nitrophenylphosphate, derivatives thereof and the like.

In principle, the phosphotriesterase used in the present invention may be of any origin. It is noted that phosphotriesterase is originally an enzyme which catalyzes a reaction to hydrolyze phophotriesters to phosphodiester.

A protein having the desired phosphotriesterase activity may be obtained from cells. In accordance with the process of the present invention the cells can be any type of cell capable of performing this reaction. The cell preferably includes those derived from microorganisms. In an especially preferred embodiment, the enzymes used in the process of the present invention is an enzyme derived from a bacterium selected from the genera Brevundimonas, Flavobacterium, Pseudomonas, Agrobacterium, Nocardia, Arthrobacter and Streptomyces. More preferably the cells are from the bacterial species Brevindumonas diminuta and Agrobacterium radiobacter. Phosphotriesterase enzymes from genetically modified microorganisms are also comprised. The enzymes derived from microorganisms, which are used in the present invention for transesterification, are not particularly limited. Other enzymes such as lactonases with phosphotriesterase promiscuity activity may be employed. Enzymes from genetically modified microorganisms are also comprised. According to a preferred embodiment of the invention, the maximum production of phosphotriester product was determined to be obtained after four days of reaction.

In another particular embodiment regardless of the nucleophile used, the higher yields are obtained when using molar ratio Substrate/Nucleophile (S/N) of 1 :10. Moreover, upon comparing ratios S/N 1 : 10, 1 :5 and 1 : 1 , where the initial concentration of phosphate donor (S) is the same, yields are proportional to the concentration of the nucleophile used. On the other hand, by inverting the ratio S/N (S/N 10:1), a remarkable decrease in the reaction yield is verified, which would reflect an enzyme inhibition by the phosphate donor. In an especially preferred embodiment, the phosphotriesterase enzyme used in the first step of hydrolysis, that is the step of hydrolysis from nucleoside dialkylphosphotnester to nucleoside alkylphosphodiester is an enzyme derived from a bacterium selected from the genera Brevundimonas, Flavobacterium, Pseudomonas, Agrobacterium, Nocardia, Arthrobacter and Streptomyces. More preferably the cells are from Nocardia asteroids. Phosphotriesterase enzymes from genetically modified microorganisms are also comprised

In an especially preferred embodiment, the phosphodiesterase enzyme used in the second step of hydrolysis, that is the step of hydrolysis of the nucleoside alkylphosphodiester is an enzyme derived from sources such as general PDE commercial Sources (Sigma): snake venoms (phosphodiesterase I from Bothrop atrox, phosphodiesterase I from Crotalus adamanteus and phosphodiesterase I from Crotalus atrox), bovine (Phosphodiesterase II from bovine spleen), fungi (Nuclease S1 from Aspergillus oryzae, sn-Glycerol-3-phosphocholine Phosphodiesterase from mold), although other sources are also comprised without limiting. More preferably the enzyme is from Crotalus atrox. . Phosphodiesterase enzymes from genetically modified microorganisms are also comprised.

According to the process of the present invention, the enzymes can be used as microorganism whole cells or enzymes isolated from the microorganisms. It means by using a suspension of the microorganisms (i.e. bacteria suspension), or enzymes produced from the microorganisms. Microorganisms or enzymes derived from the microorganisms can be free or immobilized. Additionally, cells dried with acetone, freeze-dried cells, homogeinized cells, cells treated with toluene, surfactants or lysozyme are employed giving desirable results. Preferentially, cells treated by supersonic waves followed by lyophilization are used.

In order to produce the enzyme using the bacteria as mentioned above, the bacteria are cultured in or on conventional culture media. Culture media contain conventional carbon sources, nitrogen sources, inorganic anions, and when required minor organic nutrients such as vitamins and amino acid. The cultivation condition is also not specifically limited, that is, the bacteria are cultured aerobically preferably at a pH of a range from 4 to 9 and a temperature of a range from 25 to 40°C. HPLC analysis was performed using a C18 column with detection at 254 nm in a Beckman chromatograph with manual injection and the following chromatographic methods: for analyzing purity and oxidation of MP and the transesterification reactions with 2-phenylethanol (2PE) as the substrate, a water : acetonitrile 50:50 v/v isocratic may be run, flow 0.9 ml/min may be used; for inosine and arabinoinosine as the substrate; a preferably water : acetonitrile gradient 95:5 to 55:45 v/v may be used, preferably flow 0.9 ml/min may be used; for uridine and cytidine a water : acetonitrile gradient 98:2 to 55:45 v/v may be used, flow 0.9 ml/min may be used; for 2',3'-di-0-acetylinosina as the substrate, a water : acetonitrile gradient 75:25 to 55:45 v/v may be used, flow 0.9 ml/min may be used. Calibration curves were performed for MPO, PNP, uridine and inosine using the above methods at 254nm.

RMN studies were performed using a 500 MHz Bruker spectrometer. ¹ H-RMN and ¹³C- RMN spectra were performed in DMSO-d₆ or CDCI₃. ³¹ P-RMN spectra were carried out analyzing 10% final sample dilutions in DMSO-d₆ as the solvent and using 85% phosphoric acid as the external standard or 10 mM dimethyl-methanophosphonate as the internal standard.

Removal of alkyl groups from dialkylphosphotriesters of nucleosides is preferably applied in the case of alkyl protecting groups that are easily removed by simple chemical reactions. Suitable alkyl groups are C_M0 alkyl, C₂-io alkenyl, C₂-io alkynyl, wherein alkyl, alkenyl and alkynyl groups are substituted with one to six groups independently selected from halogen, nitro, amino, hydroxyl, carboxy, CN, Ci_₃ alkoxy, Ci_₃ alkyl or aryl. More suitable alkyl groups for present chemical deprotection reaction are β-cyanoethyl, trichloroethyl or benzyl, and the like. Removal chemical reaction may be conducted in the presence of an inorganic or organic base such as NH₄OH, organic amines such as methylamine or thialcohols such as thiophenol.

Removal chemical reaction of dialkylphosphotriesters of nucleosides may be conducted in the presence of inorganic or organic acids.

Removal chemical reaction of dialkylphosphotriesters of nucleosides may also be conducted by means of hydrogenating agents such as zinc /acid and the like or by conventional catalytic hydrogenation.

According to the present invention inosine 5- monophosphate was obtained by hydrolysis of inosine 5'-bis(beta-cyanoethyl)phosphate in ammonium hydroxide aqueous solution.

According to the invention the chemical deprotection allows total conversion of the substrate to nucleoside monophosphate product in one step.

The invention also relates to an enzymatic hydrolysis process for producing nucleoside monophosphates from nucleoside dialkylphosphotriesters, the process comprising the steps of:

ii) incubating the nucleoside alkylphosphodiester of step (i) in the presence of a phosphodiesterase enzyme, and collecting a nucleoside monophosphate from the reaction mixture.

The enzymatic hydrolysis according to above steps i) and ii) may be carried out separately or sequentially in one pot. Preferably the steps i) and ii) are carried out sequentially in one pot without isolating the intermediate nucleoside alkyphosphodiester.

Alternatively the enzymatic hydrolysis may be carried out in one step using only a phosphotriesterase enzyme according to the teachings of Shim H, Hong SB, Raushel FM (op.cit).

The nucleoside dialkylphosphotriester substrate used in step i) may be obtained by the enzymatic transesterification process of the present invention or may come from another source. Enzymatic hydrolysis of dialkylphosphotriesters of nucleosides is preferably applied in the case of alkyl protecting groups that are not good leaving groups, preferably selected from C-1 -10 alkyl, C₂-io alkenyl, C₂-io alkynyl, aryl, preferably methyl, ethyl groups and the like, . As mentioned above, in an especially preferred embodiment, the phosphotriesterase enzyme used in the first step of hydrolysis, that is the step of hydrolysis from nucleoside dialkylphosphotriester to nucleoside alkylphosphodiester is an enzyme derived from a bacterium selected from the genera Brevundimonas, Flavobacterium, Pseudomonas, Agrobacterium, Nocardia, Arthrobacter and Streptomyces. More preferably the cells are from Nocardia asteroids.

In an especially preferred embodiment, the phosphodiesterase enzymes used in the second step of hydrolysis, that is the step of hydrolysis of the nucleoside alkylphosphodiester is an enzyme derived from sources such as PDE commercial Sources (Sigma): snake venoms (phosphodiesterase I from Bothrop atrox, phosphodiesterase I from Crotalus adamanteus and phosphodiesterase I from Crotalus atrox), bovine (Phosphodiesterase II from bovine spleen), fungi (Nuclease S1 from Aspergillus oryzae, sn-Glycerol-3-phosphocholine Phosphodiesterase from mold), although other sources are also comprised without limiting. More preferably the enzyme are from Crotalus atrox.. Phosphodiesterase enzymes from genetically modified microorganisms are also comprised.

According to a most preferred embodiment both chemical deprotection and enzymatic hydrolysis are carried out over the substrates obtained from the enzymatic transesterification process of the present invention. It is to be understood that the foregoing conditions are presented for purposes of illustration and description. They are not conceived to be exhaustive or to limit the invention to the precise form herein disclosed.

The following examples illustrate various aspects of the present invention but are not to be construed to unduly limit the invention. Example 1 - Preparation of partially purified phosphotriesterase (PTE) from Brevundimonas diminuta Cultures of B. diminuta (Brevundimonas diminuta CIP71.29) were run in M2 liquid media and incubated at 30°C for 48 hours. The biomass was later harvested by centrifugation at 10000 rpm for 10 minutes at 4°C. The pellet thus obtained from 5.5 L of culture was resuspended in 30 mM potassium phosphate buffer pH7 (5 ml per gram of pellet) and sonicated at a medium power The resulting homogenate was centrifuged at 10000 rpm for 10 minutes at 4°C. The supernatant was discarded and the pellet was washed and resuspended in distilled water. The previous suspension was lyophilized and the lyophilized product obtained (3.8 g) was stored at -20°C until use. This preparation was later assessed in transesterification reactions using methyl paraoxon (Example 3). During the purification procedure, phosphotriesterase activity was analyzed in different cell fractions using paraoxon as the substrate (Example 2).

Example 2 - Determination of phosphotriesterase (PTE) hydrolytic activity

PTE activity test was performed evaluating 1.8 mM paraoxon hydrolysis in TrisHCI buffer 30 mM pH 8.5 and using both B. diminuta whole cells and the enzymatic preparation from Example 1 previously incubated at 4°C for 1 hour in the same buffer. The reaction mixture was incubated at 30°C and samples were taken every 10 minutes. The release of the hydrolysis product p-nitrophenol was analyzed by UV spectrophotometry at 405 nm using 96-well plates. Detection was carried out using a Shimadzu plate reader. One unit of enzyme activity (U) was defined as the amount of enzyme capable of hydrolyzing 1 umol of PO per minute in the described incubation conditions. Simultaneously, the determination of total protein present in the extract was performed by the Bradford assay, using bovine serum albumin (BSA) as a calibration standard. Absorbance was measured at 595 nm in 96-well plates for 200 μΙ of Bradford reagent plus 10 μΙ sample per well post- incubation for 15 minutes using a Shimadzu plate reader. A specific activity of 0.023 U/mg of enzymatic preparation (humid weight) was determined prior to lyophilization and an activity of 0.019 U/mg for the preparation after lyophilization (dry weight). Measured protein content was 0.34 mg total protein per mg of enzymatic preparation.

Example 3 - Model reaction of enzymatic transesterification of methyl paraoxon (MPO) in dimethylsulfoxide (DMSO) using phosphotriesterase (PTE)

Transesterification of 10 mM MPO was performed with 100 mM 2-phenyl ethanol (2-PE) (from Sigma) as the nucleophile in 1 ml of anhydrous DMSO, in the presence of 150 mg molecular sieves and using 20 mg of the enzymatic preparation (Example 1). The reaction mixture was incubated for 6 days at 40°C and orbital stirring at 200 rpm. Simultaneously, a reaction mixture was run with 2PE without enzymatic preparation and another one without 2PE but with enzymatic preparation, in the presence of molecular sieves (reaction blanks) and a mixture containing PTE, 10mM MPO and 100mM water in the absence of molecular sieves (hydrolysis blank). Samples were taken every 24 hours and the conversion of MPO to p-nitrophenol was analyzed by HPLC. Additionally, the course of the reaction was monitored by ³¹ P-NMR, analyzing samples taken at time zero and at 3 days after reaction begun. ³¹ P-RMN analysis of the reaction mixture containing 2-PE in the presence of PTE 3 days after reaction began gave place to a spectrum wherein three signals appear (1.96, -3.98 and -4.24 ppm). On the other side, the ³¹ P spectra derived from the reaction blanks at the same time of incubation only show one signal at -3,98 ppm corresponding to MPO. Nevertheless, the same analysis for the mixture containing 100 mM water in DMSO provided a spectrum wherein two signals appear, one at -3.98 ppm and another one at -4.24 ppm, coinciding with two of the signals observed in the spectrum for the enzymatic reaction with 2PE. The signal at -3.98 ppm corresponds to the MPO hydrolysis product, dimethyl phosphate. The signal at 1.96 ppm corresponds to the MPO transesterification product, 0,0'-dimethyl-0"-(2-phenylethyl)phosphate. MPO substrate consumption and PNP formation were verified by HPLC, but the transesterification product could not be detected, possibly due to the low molar absorptivity of the phenyl group at the evaluated wavelength.

Example 4 - Enzymatic transesterification reaction of methyl paraoxon (MPO) using p h os photri esterase (PTE) and nucleosides as nucleophiles Reaction mixtures containing 10 mM MPO were incubated in the presence of 100 mM inosine or uridine, 20 mg of the enzymatic preparation (Example 1) and 150 mg of molecular sieves in 1 ml of anhydrous dimethylsulfoxide (DMSO) or N,N- dimethylformamide (DMF) at 40°C and 200 rpm. Simultaneously, reaction mixtures with the enzymatic preparation were run in the absence of nucleoside, and other reaction mixtures with inosine or uridine in the absence of enzyme (reaction blanks). Samples were taken every 24 hours for 6 days and they were analyzed by HPLC. Similarly, the course of the MPO transesterification reactions was evaluated using other natural and modified nucleosides as the nucleophile: cytidine, adenosine, guanosine, arabinouridine, arabinoinosine and 2',3'-di-0-acetylinosine.

The ³¹ P-RMN spectra for the samples containing nucleosides in the presence of PTE in DMSO after three days of incubation show three signals, two at -3.94 and -4.02ppm, and a third one at lower fields at approximately 2 ppm both for uridine and inosine as nucleophiles. As is shown in Figure 2 a signal at 2.41 ppm arises for the reaction with inosine. A signal at 2.23 ppm arises for the reaction with uridine (spectrum not shown). In none of the blanks this signal around 2 ppm was observed, thus indicating that they correspond to the transesterification products of the substrate triester with these nucleophiles. On the other hand, the production of dimethyl phosphate was verified, which indicates that part of the MPO substrate was hydrolyzed during the course of the reaction.

In turn, the sample analyses by HPLC show that in both cases a peak appears having a retention time 6.8 minutes for inosine 5'-dimethyl monophosphate (Figure 4), and a retention time 1 1.4 minutes for uridine 5'-dimethyl monophosphate (data not shown). These signals have a longer retention time than the nucleoside (4.2 min for inosine) and shorter than MPO (19.3 min, see Figure 3) and the p-nitrophenol by-product (17.5 min). In both cases, the retention times do not coincide with the ones of the nucleobases, which run with shorter retention times than the free nucleosides. Also, none of these signals appears in the samples corresponding to the reaction blanks at that same time, which indicates that they are the phosphorylation products of the respective nucleosides.

After analyzing the behavior of the samples for 6 days by HPLC, the maximum production of phosphotriester product was determined to be obtained after four days of reaction. Based on the assumption that the only chromophore present in these products absorbing at 254 nm is the nitrogenated base, the same calibration curves used in the quantification of the substrate nucleosides were used. Therefore, and in view of the limiting reagent (MPO) consumption, yields for the nucleotidic products after four days of reaction were 81.1 % and 17.6%, for inosine and uridine as substrate, respectively, in DMSO as the reaction solvent. Similarly, upon evaluating the behavior of these reactions with DMF as the solvent for these two nucleophiles, it was verified that the yields after four days of reaction were 96.2 and 38.2 for inosine and uridine, respectively. It can be seen that the yields obtained in this solvent for inosine and uridine are significantly higher with respect to the ones obtained in DMSO. For this reason, further assays were performed in DMF as the solvent.

On the other hand, it was observed by ³¹ P-RMN and HPLC that other nucleosides may act as nucleophiles in the MPO transesterification. Particularly, the presence of phosphorylation products was verified by a signal appearing at the expected field in phosphorus 31 magnetic resonance experiments for the nucleosides cytidine, adenosine, guanosine, arabinoinosine, arabinouridine and the 2',3'-di-0-acetylated inosine derivative, using DMF as the reaction solvent. The final concentrations of the dimethyl NMP derivatives (in rtiM) determined by HPLC for each one of the substrate nucleosides were: 2.51 mM for cytidine, 4.08 mM for arabinoinosine and 5.74 mM for 2',3'-di-0- acetylinosine.

Example 5 - Comparison of the reaction efficiency of different MPO substrate /nucleoside molar ratios (S/N) using phosphotriesterase (PTE)

Reaction mixtures containing MPO and inosine or uridine, 20 mg of the enzymatic preparation and 150 mg of molecular sieves in 1 ml anhydrous DMF, were incubated at 40°C and 200 rpm. Molar ratios S/N and initial absolute concentrations assessed are depicted in Table 1. Samples were taken every 24 hours and they were analyzed by HPLC.

Table 1

MPO Nucleoside

S/N ratio

(mM) (mM)*

10: 1 100 10

5: 1 50 10

1 : 1a** 1 1

1 :1 b 10 10

1 : 1c** 50 50

1 : 1d** 100 100

1 :5 10 50

1 :10 10 100 *Nucleoside: inosine or uridine. **Only inosine was evaluated as the nucleophile. Yields for the enzymatic MPO transesterification reactions with inosine and uridine were studied using several MPO/nucleoside molar ratios in DMF. Table 2 shows molar concentrations obtained after four days of incubation at 40°C for the inosine and uridine phosphorylation products.

Table 2

S/N Ratio Inosine (mM)++ Uridine (mM)++

10: 1 0.34 0.19

5: 1 1.44 0.16

1 :1 a 0.06 —

1 :1 b 1.28 0.24

1 : 1 c 0.10 —

1 :1 d 3.87 —

1 :5 6.20 1.17

1 :10 9.62 3.82

++ molar concentrations of inosine and uridine phosphorylation products

Figure 5 shows the transesterification yields obtained with respect to the limiting agent, using inosine and uridine (Ino and Uri) as the nucleophile, four days after reaction started.

Results in the above Table 2 show that, regardless of the nucleophile used, the higher yields are obtained when using S/N of 1 :10 molar ratio. Moreover, it is observed that, upon comparing S/N ratios 1 :10, 1 :5 and 1 :1 b, where the initial concentration of MPO is the same, yields are proportional to the concentration of the nucleophile used. On the other hand, it can be seen that inverting the ratio (S/N 10: 1), a remarkable decrease in the reaction yield is verified for both nucleosides.

Example 6: Chemical deprotection of inosine 5'-bis(beta-cyanoethyl)phosphate (5'- bisCEIMP)

Ammonium hydroxide 28% aqueous solution (3.5 ml_) was added to 0.1 mmol of 5'- bisCEIMP and the mixture was stirred at 60°C for 6 hours. Reaction course was followed by TLC employing n-propanol: ammonium hydroxide: water 15: 1 1 :2 as developing solvent. The aqueous solution was washed three times with dimethyl ether (30ml_) and later evaporated under vacuum. Total conversion of substrate to inosine 5'-monophospate (IMP in Scheme 1) was confirmed by RP-HPLC employing triethylammonium acetate 100mM pH 7 (TEAA):acetonitrile as mobile phase, retention times: inosine 5'- monophosphate 3.8 min; 5'-bisCEIMP: 15,2 min. Neither inosine nor the phosphodiester derivative was detected in the analyzed samples.

Scheme 1

5'-bisCEMP

Example 7: Preparation of Nocardia asteroides phosphotriesterase containing cells

Cultures of N. asteroids (Nocardia asteroides CECT 3051) were run in M56 liquid media and incubated at 30°C for 72 hours. The biomass was later harvested by centrifugation at 10000 rpm for 10 minutes at 4°C. The pellet thus obtained from 250 ml_ (4.2 g) of culture was resuspended in distilled water (5mL/g biomass) and sonicated at a medium power. The resulting homogenate was centrifuged at 10000 rpm for 10 minutes at 4°C. The supernatant was discarded and the pellet was washed and resuspended in distilled water. The previous suspension was lyophilized and the obtained product (103mg) was store at - 20°C until used. A parallel experiment was run but sonication was not performed. The specific activity of both preparations was determined in aqueous media according to the teachings of Example 2. The specific activity of the lyophilized preparations with or without prior sonication was 0.009 and 0.594 mU/mg, respectively. The non-sonicated preparation was later assessed in hydrolysis reactions using nucleoside dimethyl phosphodiester (Example 9).

Example 8: Enzymatic transesterification reaction of methyl paraoxon (MPO) using phosphotriesterase (PTE) from Brevundimonas diminuta and isopropylidene inosine as nucleophile

Isopropylidene inosine (isoplno) was used as nucleophile in an enzymatic transesterification reaction using phosphotriesterase (PTE) from Brevundimonas diminuta and methyl paraoxon (MPO) as phosphate donor. The reaction conditions were similar to those indicated in Example 4, thus obtaining 2',3'-0-isopropylideneinosine 5'- dimethylphosphate (isopDMIMP in Scheme 2). Example 9: One pot hydrolysis of 2',3'-0-isopropylideneinosine 5'- dimethylphosphate (isopDMIMP) by sequencial reaction with phosphotriesterase (PTE) from Nocardia asteroides and phosphodiesterase I from Crotalus atrox

Reaction mixtures containing 10 mM isopDMIMP (example 8) were incubated in the presence of 10 mg of non-sonicated lyophilized preparation of N. asteroides cells (example 11) in buffer TrisHCI 50 mM pH8,5 at 30°C and 200rpm. Samples were taken every 24 hours and centrifuged at 12000 rpm for 10 minutes to remove enzyme from the medium. Resulting supernatant was diluted ten times in distilled water and further analysis was performed by HPLC in TEAA:acetonitrile. After 4 days, reaction mixture was centrifuged at 12000 rpm for 5 minutes, phosphodiesterase I from Crotalus atrox (50uL of a 0.1 mg/ml_ solution - AE: 0.018U/mg - Sigma) was added to 500 uL of the obtained supernantant and the reaction mixture was incubated for several days at 30°C and 200 rpm. Samples were taken every 24 hours and analyzed by HPLC employing TEAA:acetonitrile as mobile phase. Figure 6 shows a HPLC chromatogram of the reaction mixture at cero time; isoplno peack (rt 15.7) and iospDMMIP peack (rt 17.5) are shown. Figure 7 shows a HPLC chromatogram of the reaction mixture at the 4th day; isopDMIMP peack (rt 17.6), isoplno peack (rt 15.7) and MeisopIMP peack (rt 12.8) are shown. Figure 8 shows a HPLC chromatogram of the reaction mixture at 1 day; isopDMIMP peack (rt 17.5), isoplno peack (rt 15.7), MeisopIMP peack (rt 12.7) and isopIMP (rt 1 1.9) are shown.

Scheme 2

isoplno isopDMIMP MeisopIMP isopIMP

Claims

1. A process for producing a nucleoside dialkylphosphotriester, the process comprising the reaction of a nucleoside with an activated phosphotriester compound as a phosphate donor in the presence of a phosphotriesterase enzyme.

2. The process according to claim 1 , wherein said activated phosphotriester compound is an organophosphorus triester.

3. The process according to claim 2, wherein said organophosphorus triester is selected from the group of paraoxon, methylparaoxon, coroxon, chlorpyrifos-oxon and bis β-cyanoethyl p-nitrophenylphosphate.

4. The process according to claim 1 wherein the nucleoside is selected from the group of inosine, isopropiliden inosine, adenosine, guanosine, uridine, cytidine, arabinouridine, arabinoinosine, and 2',3'-di-0-acetylinosine.

5. The process according to claim 1 , wherein the phosphotriesterase enzyme is an enzyme derived from a bacterium selected from the genera Brevundimonas, Flavobacterium, Pseudomonas, Agrobacterium, Nocardia, Arthrobacter and Streptomyces.

6. The process according to claim 5, wherein the phosphotriesterase enzyme is an enzyme derived from the bacterial species Brevundimonas diminuta and Agrobacterium radiobacter.

7. The process according to claim 1 , wherein the nucleoside dialkylphosphotriester is inosine 5'- dimethyl monophosphate.

8. The process according to claim 1 , wherein the nucleoside dialkylphosphotriester is uridine 5'-dimethyl monophosphate.

9. The process according to claim 1 , wherein the nucleoside dialkylphosphotriester is isopropyliden inosine 5'-dimethyl monophosphate.

10. The process according to claim 1 , wherein the reaction is carried out in an organic solvent selected from anhydrous dimethylsulfoxide (DMSO) or anhydrous N,N- dimethylformamide (DMF).

1 1. The process according to claim 1 , wherein the phosphate donor/nucleoside molar ratio is selected in the range from 10: 1 to 1 : 10.

12. A process for producing a nucleoside monophosphate monoester, the process comprising the steps of:

a) conducting an enzymatic transesterification according to claim 1 , by reacting a nucleoside with an activated phosphotriester compound as a phosphate donor in the presence of a phosphotriesterase enzyme,

b) removing alkyl groups from the nucleoside dialkylphosphotnester obtained in step (a) by reaction with a chemical agent selected from the group of inorganic or organic basic reagents, inorganic or organic acidic reagents, thioalcohols and hydrogenating reagents, or

c) removing alkyl groups from the nucleoside dialkylphosphotnester obtained in step (a) by hydrolysis according to the following steps:

i) incubating the nucleoside dialkylphosphotnester in the presence of a phosphotriesterase enzyme,

collecting a nucleoside monophosphate from the reaction mixture.

13. The process according to claim 12, wherein said activated phosphotriester compound is an organophosphorus triester.

14. The process according to claim 13, wherein said organophosphorus triester is selected from the group of paraoxon, methylparaoxon, coroxon, chlorpyrifos-oxon and bis β-cyanoethyl p-nitrophenylphosphate.

15. The process according to claim 12 wherein the nucleoside in step a) is selected from the group of inosine, isopropyliden inosine, adenosine, guanosine, uridine, cytidine, arabinouridine, arabinoinosine, and 2',3'-di-0-acetylinosine.

16. The process according to claim 12, wherein the phosphotriesterase enzyme is an enzyme derived from a bacterium selected from the genera Brevundimonas, Flavobacterium, Pseudomonas, Agrobacterium, Nocardia, Arthrobacter and Streptomyces.

17. The process according to claim 16 wherein the phosphotriesterase enzyme is an enzyme derived from the bacterial species Brevindimonas diminuta and Agrobacterium radiobacter.

18. The process according to claim 12, wherein the reaction in step a) is carried out in an organic solvent selected from anhydrous dimethylsulfoxide (DMSO) or anhydrous N,N- dimethylformamide (DMF).

19. The process according to claim 12, wherein the phosphate donor/nucleoside molar ratio is selected in the range from 10: 1 to 1 : 10.

20. The process according to claim 12, wherein the removal chemical agent is an aqueous solution of NH₄OH.

21. The process according to claim 12, wherein the alkyl groups in steps b) and c) are selected from CM₀ alkyl, C₂-io alkenyl, and C₂-io alkynyl, wherein alkyl, alkenyl and alkynyl groups are optionally substituted with one to six groups independently selected from halogen, nitro, amino, hydroxyl, carboxyl, CN, Ci_₃ alkoxyl, Ci_₃ alkyl or aryl.

22. A process according to claim 12, wherein the source of phosphotriesterase enzyme used in step i) is an enzyme derived from a bacterium selected from the genera Brevundimonas, Flavobacterium, Pseudomonas, Agrobacterium, Nocardia, Arthrobacter and Streptomyces.

23. A process according to claim 22, wherein the source of phosphotriesterase enzyme used in step i) is an enzyme derived from Nocardia asteroides bacteria.

24. A process according to claim 12, wherein the phosphodiesterase enzyme used in step i) is an enzyme derived from phosphodiesterase I from Bothrop atrox, phosphodiesterase I from Crotalus adamanteus and phosphodiesterase I from Crotalus atrox), Phosphodiesterase II from bovine spleen, Nuclease S1 from Aspergillus oryzae, and sn-Glycerol-3-phosphocholine Phosphodiesterase from mold.

25. A process according to claim 12, wherein the steps i) and ii) are carried out either separately or sequentially in one pot, without isolating the intermediate nucleoside alkylphosphodiester.

26. A process for producing a nucleoside monophosphate monoester, the process comprising removing alkyl groups from a nucleoside dialkylphosphotnester by hydrolysis according to the following steps:

collecting a nucleoside monophosphate from the reaction mixture.

27. The process according to claim 26, wherein the alkyl groups are selected from C-1 -10 alkyl, C₂-io alkenyl, and C₂-io alkynyl, wherein alkyl, alkenyl and alkynyl groups are optionally substituted with one to six groups independently selected from halogen, nitro, amino, hydroxyl, carboxyl, CN, Ci_₃ alkoxyl, Ci_₃ alkyl or aryl.

28. A process according to claim 26, wherein the source of phosphotriesterase enzyme used in step i) is an enzyme derived from a bacterium selected from the genera Brevundimonas, Flavobacterium, Pseudomonas, Agrobacterium, Nocardia, Arthrobacter and Streptomyces.

29. A process according to claim 28, wherein the source of phosphotriesterase enzyme used in step i) is an enzyme derived from Nocardia asteroides bacteria.

30. A process according to claim 26, wherein the phosphodiesterase enzyme used in step i) is an enzyme derived from phosphodiesterase I from Bothrop atrox, phosphodiesterase I from Crotalus adamanteus and phosphodiesterase I from Crotalus atrox), Phosphodiesterase II from bovine spleen, Nuclease S1 from Aspergillus oryzae, and sn-Glycerol-3-phosphocholine Phosphodiesterase from mold.

31. A process according to claim 26, wherein the steps i) and ii) are carried out either separately or sequentially in one pot, without isolating the intermediate nucleoside alkylphosphodiester.