TECHNICAL FIELD
The present invention relates to a process for the regioselective acylation of nucleosides.
BACKGROUND
Oligonucleotides involving non-genomic 2',5'-linkages between nucleotide monomers have always been a topic of interest for bioorganic chemists. The discovery of natural 2',5'-oligoadenylates, pppA(2'p5'A)n and their crucial role in activation of RNase L, which is responsible for the degradation of viral dsRNA in mammalian cells is well established. This has elicited a scope for the use of 2',5'-linked oligonucleotides as potent antiviral agents. The unique features of 2',5'-linked oligonucleotides include their ability to bind selectively with complementary RNA and not with DNA togetherwith their resistance towards most of the nucleases.
These advantages suggest wide applicabilities of 2',5'-oligonucleotides such as their use as RNA specific probes and as antisense oligomers. This has concomitantly triggered the search for readily available 3'-O-protected nucleoside precursors to be used further for large scale production of bioactive 2', 5'-oligonucleotides. The selective protection of only one of the various identical functional groups in the nucleosides has always been a fundamental challenge to organic chemists. Such selectivity is highly desirable for making the whole synthetic process more efficient and economical. The conventional methodology of peracylation, followed by subsequent deacylation of the particular
acyloxy functions in the nucleosides is time consuming, tedious and non-selective.
In this aspect, nature's catalysts 'Enzymes' are well recognized alternatives to chemical methods in synthetic organic chemistry with an additional advantage that such enzymes are environment friendly. Recently we have reported efficient and convenient enzymatic methods for the separation of mixtures of furanosyl- and pyranosyl- nucleosides of different pentoses using primary hydroxyl selectivity of Candida antarctica lipase-B.
Lots of work has been reported for selective enzymatic protection of primary over secondary hydroxyl group(s) of the nucleosides. However, little work is known wherein secondary hydroxyl groups of the nucleoside monomers are selectively acylated over primary and/or other secondary hydroxyl groups. Acetonoxime levulinate esters has been used as acylating agents for the secondary hydroxyl protection of 2'-deoxynucleosides. The literature procedures for the synthesis of 2'-O-alkylated ribonucleosides requires selective blocking of 3'- and 5'-hydroxyl groups using TIPDS-Cl2 followed by 2'-O-alkylation and deblocking.
OBJECT AND SUMMARY
The principal object of the present invention is to provide a process for the regioselective acylation of nucleosides wherein secondary hydroxyl groups of the nucleoside monomers are selectively acylated over primary and/or other secondary hydroxyl groups.
Another object of the present invention is to provide a process for the regioselective acylation of nucleosides which is environment friendly.
Still another object of the present invention is to provide a process for the regioselective acylation of nucleosides which is cost effective.
Accordingly, the present invention provides a process for the regioselective acylation of nucleosides, said process comprising :
- preparing solution of a nucleoside, an acylating agent and a lipase;
- subjecting said solution to incubation at a predetermined temperature;
- filtering off the lipase to obtain the product;
DESCRIPTION
The above process relates to the regioselective acylation of nucleosides which involves :
- preparing solution of a nucleoside, an acylating agent and a lipase;
- subjecting said solution to incubation at a predetermined temperature;
- filtering off the enzyme to obtain the product;
(Formula Removed)
The present invention provides the regioselective acylation of C-3' hydroxyl function in thymidine, deoxyadenosine and adenosine by using cheaper and readily available acylating agents in the presence of a novel lipase in moderate to good yields (Scheme 1).
Exceptionally, in the case of 2'-deoxycytidine and cytidine, corresponding N-acylation has been observed instead of O-acylation in excellent yields (Scheme 2). The 3'-OH selectivity of P. aeruginosa lipase has been used for the efficient synthesis of 2'-O-alkyl nucleoside monomers of importance in antisense oligonucleotide synthesis (Scheme 3).
The lipase catalyzed acylation reaction on nucleosides using an acylating agent can be carried out using different lipases, i.e. porcine pancreatic lipase (PPL), Candida antarctica lipase-B immobilized on polyacrylate (Lewatit), commonly known as Novozyme-435, Candida rugosa lipase and Pseudomonas aeruginosa lipase in organic solvents, such as diisopropyl ether, THF, dioxan, DMF and acetonitrile. However, the reaction catalyzed by Pseudomonas aeruginosa lipase in DMF / THF was most efficient and selective over other lipases. Five different acid anhydrides, viz. propanoic (4a), butanoic (4b), pentanoic (4c),
hexanoic (4d) and benzoic anhydride (4e) are found to be suitable for the selective protection of hydroxyl groups in 2'-deoxyadenosine (1) and adenosine (2). However, best results are obtained with butanoic anhydride.
Thus lipase specially Pseudomonas aeruginosa has been found to have remarkable selectivity towards the acylation of 3'-hydroxyl group in nuclosides in the presence of primary and / or secondary hydroxyl group(s), which is otherwise very difficult to achieve by classical chemical methodologies. The developed methodology finds wide application for the synthesis of selectively protected nucloside monomers which are precursors for the synthesis of non-genomic 2',5'-linked-oligonuclotides of biological importance, in particular for the synthesis of antisense and antigene oligonucleotides. Further, biocatalytic direct iV-butyration in case of 2'-deoxycytidine and cytidine has been achieved in excellent yields. Thus an alternative greener methodology has been developed for the synthesis of N-protected nucleosides, which is often required during oligonuclotide synthesis.
EXAMPLES
1. Acylation of adenine and thymine nucleosides (1-3)
In a typical reaction, a solution of 2'-deoxyadenosine (1) / adenosine (2) and butanoic anhydride (4b, 2 equiv.) in DMF was incubated with P. aeruginosa lipase (250 mg / mmol of nucloside) in an incubator shaker at 60 °C. On completion of the reaction as indicated by TLC examination, enzyme was filtered off and the
solvent removed under reduced pressure. The crude product thus obtained was passed through a small silica gel column to afford the pure butanoylated compound 5b / 6b in 78.5 / 59.0 % yield (Scheme 1, Table 1). The structures of the butanoylated compound 5b / 6b was unambiguously established as 3'-Obutanoyl-2'-deoxyadenosine / 3'-Obutanoyladenosine by detailed study of their IR, 1H- & 13C NMR, HRMS spectra and 1H-1H COSY NMR experiments, and comparison of there 1H NMR spectra with those of the corresponding starting nucleoside 1/2.
(Scheme Removed)
SCHEME 1. Selective acylation of 2'-deoxyadenosine (1), adenosine (2) and thymidine (3) using different acid anhydrides in DMF/THF at 60°C.

(Table Removed)
Table 1. Acylation studies on nucleosides 1-3, 8 and 9 in the
presence of Pseudomonas aeruginosa lipase at 60 °C with different acid anhydrides 4a-4e
Similar result was observed when thymidine 3 was incubated with P. aeruginosa lipase in THF in the presence of butanoic anhydride leading to the formation of 3'-O-butanoylated thymidine in 89.0 % yield (Scheme 1). The butanoylation of 3'-hydroxyl group in each of the nucleosides 1, 2 and 3 was clearly indicated by down field shifts of the C-3 proton by 0.97, 0.93 and 0.96 ppm in the corresponding products 5b, 6b and 7, respectively. In order to further confirm selective enzymatic acylation of C-3' OH, 2'-O-butanoylated adenosine was synthesized using known chemical procedure and spectral data of this compound was compared with the data of enzymatically butanoylated product 6b; they were entirely different. All these reactions, when performed under identical conditions but without adding the lipase did not yield any product.
2. Acylation of cytosine nucleosides (8-9)
(Scheme Removed)
Scheme 2. Selective N-acylation of 2'-deoxycytidine (8) and cytidine (9) using butanoic anhydride (4b) in THF:H2O (5:1) at 60 °C
In case of 2'-deoxycytidine (8) and cytidine (9), the N-butanoylated product was obtained in excellent yields instead of O-butanoylated product (Scheme 2, Table 1). The formation of N-butanoylated 2'-deoxycytidine and cytidine was clearly indicated by the disappearence of -NH2 signals resonating at D 7.17 and 7.25 Hz for 2H each in the 1H NMR spectra of compounds 8 and 9, respectively; instead appearance of new peaks and 10.81 Hz of 1H each in the spectra of products 10 and 11, respectively was
observed. The appreciable downfield movement in the chemical shift values of C-4 and C-5 protons in butanoylated 2-deoxycytidine and cytidine with respect to their corresponding peakes in the starting compounds 8 and 9 also supports the selective N-butanoylation. This result is of particular interest since the direct and selective N-acylation in compounds 8 and 9 avoids the multi-step conventional methods which require transient protection / deprotection using TMS-C1 (Scheme 2). In some cases use of TIPDS-Cl2, a highly expensive bis-protecting reagent has also been reported for the synthesis of N-acylated nucleosides.
The butanolyation reaction on 2'-deoxycytidine and cytidine was carried out in THF:H2O (5:1) as solvent, since
3. Synthesis of 5'-O-(4,4-dimethoxytrityl)-2-O-methyl adenosine (17)
The one-step selective enzymatic methodology of synthesizing 3-O-butanoyl derivative of nucleosides makes the overall synthesis of a variety of desirable antisense oligonucleotides involving 2',5'-linkages, efficient and economical. This methodology has been used for the convenient, economical and efficient synthesis of 2-O-alkylated ribonucleosides of importance
(Scheme Removed)
Scheme 3. Alternative chemoenzymatic approach for the synthesis of 5'-O-DMT-2'-O-methyl adenosine (17)

We Claim,
1. A process for the regioselective acylation of nucleosides,
said process comprising :
- preparing solution of a nucleoside, an acylating agent and a lipase;
- subjecting said solution to incubation at a predetermined temperature;
- filtering off the lipase to obtain the product;

2. The process as claimed in claim 1, wherein said acylating agent is an acid anhydride, acid chloride or active ester.
3. The process as claimed in claim 2, wherein said acid anhydride is selected from the group consisiting of propanoic, butanoic, pentanoic, hexanoic or benzoic anhydride.
4. The process as claimed in claim 3, wherein said acid anhydride is preferably butanoic anhydride.
5. The process as claimed in claim 1, wherein said lipase is a mammalian or microbial lipase.
6. The process as claimed in claim 5, wherein said lipase is selected from the group consisting of porcine pancreatic lipase, Candida antarctica lipase-B, Candida rugosa lipase or Pseudomonas aeruginosa lipase.
7. The process as claimed in claim 6, wherein said lipase is preferably Pseudomonas aeruginosa lipase.
8. The process as claimed in claim 1, wherein the process is carried out at a temperature ranging between 50-70°C.
9. The process as claimed in claim 1, wherein said process is carried out in a solvent.
10. The process as claimed in claim 9, wherein said solvent is an organic or aqueous-organic solvent.
11. The process as claimed in any of the claims 1-10 for use in the preparation of 2',5'-linked oligonucleotides and or 2'-O-alkylated 3',5'-linked oligonucleotides.
12. The process for the regio selective acylation of nucleosides substantially as herein described with reference to foregoing examples.