WO2007144168A1

WO2007144168A1 - Enzymatic process for preparation of 5'-monophosphate-nucleotides

Info

Publication number: WO2007144168A1
Application number: PCT/EP2007/005239
Authority: WO
Inventors: Giovanni Cotticelli; Raul Salvetti
Original assignee: Adorkem Technology Spa
Priority date: 2006-06-15
Filing date: 2007-06-14
Publication date: 2007-12-21
Also published as: ITMI20061154A1

Abstract

An enzymatic procedure is described for the preparation of natural and modified nucleotides which are phosphorylated in position 5', starting from corresponding nucleoside precursors, according to the scheme reported below. Formula (I), formula (II). P: pyrimidine bases/purine bases] R=-OH, -H, N3, F, Cl, I, Br; R'=-OH, -H, N3, F,Cl, I, Br; X=CH, S, O. By enzymatic process, it is intended the use of free or immobilised purified enzymes, or enzymes contained in cell pastes or cells.

Description

TITLE

ENZYMATIC PROCESS FOR PREPARATION OF 5 ' -MONOPHOSPHATE-NUCLEOTIDES

DECRIPTION

The present invention concerns a procedure for the preparation of natural and modif ied nucleotides starting from natural or modified nucleosides . The aforesaid compounds are employed in the food and pharmaceutical industries ; among these , fludarabine phosphate , whose formula (A) is reported here below

is an active drug principle employed as anti-cancer agent, in particular in the treatment of chronic lymphocytic leukaemia.

PRIOR ART

The preparation of the nucleotides is described in diverse patent documents and is known in the literature. The phosphorylation reactions are carried out by chemical synthesis, with natural environmental problems due to the use of triethylphosphate, trimethylphosphate with phosphorus oxychloride. Moreover, during the phosphorylation, potential impurities are formed since the phosphorylating agent is not exclusively selective in the position 5', but can react with the other positions in 3', creating other potential impurities.

For example, the document US 4,357,324 describes a phosporylation method for the fludarabine phosphate starting from fludarabine (B) , whose formula is reported here below, with phosphorus oxychloride and trimethylphosphate at 0⁰C; there is hydrolysis with water, formation of the sodium salt and subsequent conversion of the latter into the free acid.

The yields obtainable with the procedure described in this patent are low and hard to reproduce on an industrial scale.

The document US 5,110,919 describes a phosporylation method which foresees the use of phosphorus oxychloride and trimethylphosphate at 0⁰C.

The procedures have the drawback of using chemical reagents with the potential formation of impurities which can be damaging for the organism.

DESCRIPTION OF THE INVENTION

The object of the present invention is that of providing a process for the preparation of 5'- phosphorylated nucleotides, according to the scheme reported here below, which is free of the drawbacks of the prior art processes.

PU: purine bases / analogous purine bases PY: pyrimidine bases / analogous pyrimidine bases

«V -tes priiΥidrid"B/bθE-

R=-CH-H N>,F,O,I,& R=Oi-H ^ F₁Q lB-

X=CH§0

The invention consists of a process for the preparation of 5' -phophorylated nucleotides, and in particular of the fludarabine-5' -monophosphate, by means of the use of regioselective phosphorylating enzymes for the position 5 ' .

The reaction solvent is water, by itself or in a mixture with miscible organic solvents, in the presence or absence of a buffer, the pH being preferably in the range of about 4 to about 8.

The phosphate group can derive from a donor of organic origin, for example ATP, or inorganic origin, such as for example an alkaline salt and/or alkaline earth salt of a phosphoric acid, preferably sodium acid pyrophosphate; enzymes can moreover be used which regenerate the phosphorus donor in the reaction setting. The starting nucleoside does not necessarily have to be anhydrous .

The enzymes are preferably used in purified form, both free and immobilised on solid medium. The solid medium can be CLEC (Cross-linked immobilised enzyme) , described for example in Noritomi et al . , Increased thermostability of cross-linked enzyme crystals of subtilisin in organic solvents, Biotechnology Techniques 12:467-469 (1998) and in Margolin, Novel crystalline catalysts, Trends in Biotechnology 14:223- 230 (1996), incorporated here for reference. Or the enzymes can be used in raw form, for example contained in cell pastes or in whole cells. In particular, regioselective phosphorylating enzymes are preferred from strains of Citrobacter amalonaticus, Klebsiella sp. , Klebsiella Planticola, Serratia macescens, Enterobacter aerogenes and Enterobacter gergoviae, or kinases .

The bioreaction is normally conducted at a temperature in the range of 0 ÷ 100° C and its duration is normally in the range of 2-72 hours.

The solid is preferably isolated from the reaction setting by water or water in mixture with organic solvents and the purification can be carried out by crystallisation at different temperatures. Alternatively, the product can be purified on resins and ion exchange material and crystallised from water or from water in organic solvent mixture at different temperatures .

The use of phosphorylating enzymes permits phosphorylating the 5' position of the nucleoside in a selective manner. This process is quite advantageous, since it does not permit the formation of impurities. Moreover, using preferably water as reaction solvent, the ecological and environmental impact is improved. The following examples of the invention are merely illustrative and non-limiting.

Example 1: Synthesis of adenosine 5 ' -monophosphate by means of biocatalysts coming from bacterial strains. The reaction is carried out at 35°C for 40 hours under stirring (200 rpm) in a 0. IM sodium acetate buffer at pH 4 , in a final volume equal to 100 μl . For every ml of solution, the following were added: 20 mg of (74.5mM) adenosine, 58 mg of (0.26M) sodium acid pyrophosphate, 0.2 mg of (ImM) MgSO₄ • 7H₂O and 50 mg of cells (wet weight, strains of Citrobacter amalonaticus, Klebsiella sp. , Klebsiella Planticola, and Serratia macescens) . The reaction was monitored by means of TLC. After 16 hours of reaction, the formation of the product was observed (adenosine 5' -monophosphate) with a conversion of about 50%. It is centrifuged, the pH is brought to 2 and adenosine 5'- monophosphate is obtained.

Example 2: Synthesis of cytidine 5'- monophosphate. The reaction was carried out at 35°C for 72 hours under stirring (200 rpm) in a 0. IM sodium acetate buffer at pH 4 , in a final volume equal to 100 μl .

For every ml of solution the following were added: 74.5 μmols of cytidine, 1 μmol of MgSO₄ -7H₂O, 260 μmols of sodium acid pyrophosphate and 50 mg of cells (wet weight, strains of Klebsiella sp. , Enterobacter aerogenes, Klebsiella Planticola, Serratia macescens and Enterobacter gergoviae) . The formation was observed of the phosphorylation product (cytidine 5'- monophosphate) after 20 hours of reaction. A yield is obtained of 50%. It is centrifuged, the pH is brought to 2 and cytidine 5 ' -monophosphate is obtained. Example 3: Synthesis of guanosine 5^#- monophosphate. The reaction was carried out at 35°C for 72 hours under stirring (200 rpm) in a 0. IM sodium acetate buffer at pH 4 , in a final volume equal to 100 μl . For every ml of solution, the following were added: 74.5 μmols of guanosine, 1 μmol of MgSO₄ -7H₂O, 260 μmol of sodium acid pyrophosphate and 50 mg of cells (wet weight, strains of Klebsiella. sp. , Enterobacter aerogenes, Klebsiella Planticola, Serratia macescens and Enterobacter gergoviae. The formation was observed of the phosphorylation product (guanosine 5'- monophosphate) after 20 hours of reaction. (50% Yield) . It is centrifuged, the pH is brought to 2.2, and guanosine 5'- monophosphate is obtained. Example 4: Synthesis of uridine 5'- monophosphate. The reaction was carried out at 35°C for 72 hours under stirring (200 rpm) in a 0. IM sodium acetate buffer at pH 4, in a final volume equal to 100 μl .

For every ml of solution, the following were added: 74.5 μmols of uridine, 1 μmol of MgSO₄ -7H₂O, 260 μmol of sodium acid pyrophosphate and 50 mg of cells (wet weight, strains of Klebsiella sp and Enterobacter gergoviae) . Samples were taken after 3, 8, 24, 48 and 72 hours by monitoring with TLC. The formation was observed of the phosphorylation product (uridine 5'- monophosphate) after 24 hours of reaction. Example 5: Synthesis of fludarabine 5' -monophosphate through the use of immobilised kinases To a phosphate buffer solution at pH 6 (0.536 1), 0.08 1 are added of a 100 mM ATP solution, 0.128 1 of a 100 mM MgCl₂ solution. 1.8 g of Fludarabine are added and 28 g are added of enzymatic kinase derivative. After 8 hours, the reaction is stopped (94% reaction yield) . It is brought to pH 2, filtered and washed with water. 0.5 g of fludarabine 5 '-phosphate are obtained.

Example 6: Synthesis of fludarabine 5 ' -monophosphate through the use of free purified kinases. In a 50 mM Tris HCl buffer solution at pH 8 (46.4 ml), 2.5 ml were added of a IM solution of MgCl₂ along with 28.6 mg of fludarabine. To the same solution, maintained under mechanical stirring at 37⁰C, 1.37 g of ATP were added. The pH was brought to 9.0 by means of NaOH and the kinase enzyme was added. At the end of the reaction (T= 46 h) , the mixture was poured into a 100 ml single neck flask and the residue was recovered with

2 ml of distilled H₂O. The resulting solution was concentrated at 45°C. The solution becomes turbid, is brought to pH =2 and then left to rest in ice. When the solution is concentrated to about 1/3 the initial volume (pH 2.65), the precipitation of a white solid

(20mg) is observed, corresponding to fludarabine 5'- phosphate.

Claims

1. Process for the preparation of nucleotides of formula

where :

P is a purine or pyrimidine base;

R and R' , equal to or different from each other, are OH, H, N₃, F, Cl, Br or I;

X is CH, S or 0; characterised in that a nucleoside of formula

where P, R, R' and X have the above-listed meanings, is made to react with a phosphorus donor in the presence of a phosphorylating enzyme. Process according to claim 1, characterised in that said nucleotide is selected from among fludarabine 5' -monophosphate, adenosine 5'- monophosphate, cytidine 5' -monophosphate, guanosine 5'- monophosphate, uridine 5'- monophosphate. Process according to claim 1, characterised in that said enzyme comes from strains of Citrobacter amalonaticus, Klebsiella sp. , Klebsiella Planticola, Serratia macescens, Enterobacter aerogenes and Enterobacter gergoviae.

4. Process according to claim 1, characterised in that said enzyme is a kinase.

5. Process according to claim 1, characterised in that said enzyme is used in raw or purified form.

6. Process according to claim 5, characterised in that said enzyme is immobilised on a solid medium.

7. Process according to claim 6, characterised in that said solid medium is CLEC.

8. Process according to claim 5, characterised in that said enzyme is contained in a cell paste or in whole cells.

9. Process according to claim 1, characterised in that said phosphorus donor is organic or inorganic.

10. Process according to claim 9, characterised in that said phosphorus donor is ATP.

11. Process according to claim 9, characterised in that said phosphorus donor is an alkaline salt and/or alkaline earth salt of a phosphoric acid, preferably sodium acid pyrophosphate.

12. Process according to claim 1, characterised in that the reaction solvent is water.

13. Process according to claim 12, characterised in that the water is mixed with an organic solvent .

14. Process according to claim 1, characterised in that the reaction solvent is buffered to a pH preferably in the range of about 4 to about 8.

15. Process according to claim 1, characterised in that it is carried out at a temperature in the range of 0° - 100° C.