Novel selective biocatalytic deacylation studies on key precursors for bicyclonucleosides

Article abstract of DOI:10.1080/15257770701544302

Immobilized Candida antarctica lipase and Thermomyces lanuginosus lipase catalyze the deacylation of precursors of LNA analogs, 4′-C-acyloxymethyl- 2′,3′,5′-tri-O-acyl-β-L-threo-pentofuranosylthymine and 4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-(1-methylethy

Full text of DOI:10.1080/15257770701544302

Nucleosides, Nucleotides, and Nucleic Acids, 26:1517–1521, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701544302
NOVEL SELECTIVE BIOCATALYTIC DEACYLATION STUDIES ON KEY
PRECURSORS FOR BICYCLONUCLEOSIDES
Ashok K. Prasad and Sunil K. Singh
Bioorganic Laboratory, Department of
2
Chemistry, University of Delhi, Delhi, India
Neerja Kalra
Bioorganic Laboratory, Department of Chemistry, University of Delhi,
2
Delhi, India and Nucleic Acid Center, Department of Chemistry, University of Southern
Denmark, Odense M, Denmark
Nidhi Singhal
Bioorganic Laboratory, Department of Chemistry, University of Delhi,
2
Delhi, India
Jesper Wengel
Nucleic Acid Center, Department of Chemistry, University of Southern
2
Denmark, Odense M, Denmark
Virinder S. Parmar
Bioorganic Laboratory, Department of Chemistry, University of
2
Delhi, Delhi, India
Immobilized Candida antarctica lipase and Thermomyces lanuginosus lipase catalyze
2
the deacylation of precursors of LNA analogs, 4ꢁ-C-acyloxymethyl-2ꢁ,3ꢁ,5ꢁ-tri-O-acyl-β-L-threo-
pentofuranosylthymine and 4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-(1-methylethylidene)-β-L-threo-
pentofuranose, respectively in a highly selective and efficient manner.
Keywords LNA analogs; bicyclonucleosides
INTRODUCTION
The synthesis of novel nucleoside analogues is gaining importance
because of their applications as key intermediates in the development
of antisense and/or antigene oligonucleotides to regulate targeted gene
expression,[1] and for their direct utilization as anti-tumor or antiviral
compounds.[2] A novel class of 3ꢁ,5ꢁ-linked oligonucleotide analogs con-
taining 2ꢁ-O,4ꢁ-C-methylene bridged ribonucleosides, commonly known as
We thank the Danish Natural Science Research Foundation and the Department of Biotechnology
(DBT, New Delhi, India) for financial support to this work. S. K. S. and N. K. thank the Council of
Scientific and Industrial Research (CSIR, New Delhi) for the award of J. R. F. and S. R. F.
Address correspondence to Ashok K. Prasad, Bioorganic Laboratory, Department of Chemistry,
University of Delhi, Delhi 110007, India. E-mail: ashokenzyme@yahoo.com
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A. K. Prasad et al.
FIGURE 1 Bicyclonucleosides and their precursors.
locked nucleic acids (LNAs) have been known to possess favorable features
towards development of antisense and/or antigene candidates.[3] One of
the crucial steps in the synthesis of LNA/LNA analogs is the discrimination
between two primary hydroxy groups of almost identical reactivity in 4/4ꢁ-
hydroxymethylated sugar or nucleoside precursors (Figure 1).
Enzymes are being recognized as efficient catalysts for many of the
stereospecific and regioselective reactions necessary for carbohydrate
modifications and nucleoside synthesis.[4] In the present study, we have
developed a highly efficient enzymatic route for diastereoselective deacyla-
tion of one of the two acyloxy groups involving primary hydroxyl functions
in 4ꢁ-C-acyloxymethyl-2ꢁ,3ꢁ,5ꢁ-tri-O-acyl-β-L-threo-pentofuranosyl thymine
and
4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-(1-methylethylidene)-β-L-threo-
pentofuranose, precursors of bicyclic nucleosides.
DEACYLATION STUDIES ON 4ꢁ-C-ACYLOXYMETHYL-2ꢁ,3ꢁ,5ꢁ-TRI-O-
ACYL-β-L-THREO-PENTOFURANOSYLTHYMINE
The nucleoside 4ꢁ-C-hydroxymethyl-threo-pentofuranosylthymine (1) was
synthesized from triacetylated sugar precursor 4-C-acetoxymethyl-3,5-di-O-
acetyl-1,2-O-(1-methylethylidene)-β-L-threo-pentofuranose[5] in 3 steps in an
overall yield of 64% and quantitatively converted into its peracylates 2a–
2e using corresponding acid anhydride in pyridine (Scheme 1). Three
SCHEME 1

Biocatalytic Deacylation Studies
1519
lipases, i.e. Novozyme-435, Candida rugosa lipase (CRL) and Candida antarc-
tica lipase-B immobilized on accurel [CAL-L(A)] were screened for the se-
lective deacylation of the tetraacylated nucleosides 2a–2e in different or-
ganic solvents in the presence of n-butanol as an acyl acceptor. Novozyme-
435 in tetrahydrofuran was found to be the most suitable combination for
the deacylation of nucleosides 2a–2e at 50–55◦C.
In a typical reaction, a solution of 4ꢁ-C-acyloxymethyl-2ꢁ,3ꢁ,5ꢁ-tri-O-acyl-β-
L-threo-pentofuranosylthymine 2a–2e in tetrahydrofuran containing a small
amount of n-butanol was incubated with Novozyme-435 (50% w/w of the
2a–2e) in an incubator shaker at 50–55◦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 deacylated com-
pounds 3a–3e, with lower R f values than the corresponding starting com-
pounds 2a–2e in 73–90% yields (Scheme 1).
The structures of enzymatically deacylated nucleosides 3a–3e was
established as 4ꢁ-C-hydroxymethyl-2ꢁ,3ꢁ,5ꢁ-tri-O-acyl-β-L-threo-pentofuranosyl-
1
thymine on the basis of their IR, H NMR, 13C NMR, HRMS data and
1
by comparison with the H- and 13C NMR spectral data of similar com-
pounds. The structure of 4ꢁ-C-hydroxymethyl-2ꢁ,3ꢁ,5ꢁ-tri-O-acetyl-β-L-threo-
pentofuranosylthymine (3a) was further confirmed by conversion of this
selectively deacetylated compound to the bicyclic nucleoside 5 via tosyla-
tion of its hydroxyl function, followed by hydrolysis of the three acetoxyl
functions with simultaneous cyclisation due to detosylation with the 2ꢁ-
hydroxyl group under aqueous-methanolic potassium carbonate condition
(Scheme 1). Conversion of enzymatically prepared hydroxynucleoside
3a into bicyclic nucleoside 5, unambiguously established the structure
of other selectively deacylated nucleosides 3b–3e as 4ꢁ-C-hydroxymethyl-
pentofuranosylthymine acylates.
DEACYLATION STUDIES ON 4-C-ACYLOXYMETHYL-3,5-DI-O-
ACYL-1,2-O-(1-METHYLETHYLIDENE)-β-L-THREO-
PENTOFURANOSES 8a–8c
The trihydroxy sugar 7 (synthesized starting from D-glucose following
the modified procedure of Youssefyeh et al.[6]) was converted to its triacy-
lated derivatives 8a–8c using acetic anhydride, propanoic anhydride and bu-
tanoic anhydride, respectively in the presence of catalytic amount of DMAP
in 80–85% yields (Scheme 2).[5]
Four lipases, i.e. Candida antarctica lipase-B immobilized on polyacry-
late (Lewatit), commonly known as Novozyme-435, porcine pancreatic li-
pase (PPL), Candida rugosa lipase (CRL), Thermomyces lanuginosus lipase
immobilized on silica (Lipozyme TL IM) and Candida antarctica lipase-B

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A. K. Prasad et al.
SCHEME 2
immobilized on accurel [CAL-L(A)] were screened for the selective deacy-
lation of the triacylated pentofuranose derivatives 8a–8c in different organic
solvents in the presence of n-butanol as the acyl acceptor. Lipozyme TL IM
in toluene was found to be the most suitable combination for the deacyla-
tion of compounds 8a–8c.
In a typical reaction, a solution of 4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-
(1-methylethylidene)-β-L-threo-pentofuranose 8a–8c in toluene containing a
small amount of n-butanol was incubated with Lipozyme TL IM in an in-
cubator shaker at 40–42◦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 deacylated compounds 9a–9c, with
lower R f value than the corresponding starting compounds 8a–8c in 88–
98% yields (Scheme 2).[5]
The structures of enzymatically deacylated compounds 9a–9c were es-
tablished as 3,5-di-O-acyl-4-C-hydroxymethyl-1,2-O-(1-methylethylidene)-α-
1
D-xylo-pentofuranoses on the basis of their IR, H NMR, 13C NMR, HRMS
1
1
and H nOe experiments, and comparison of their H NMR spectrum
with those of the starting triacylates 8a–8c. The structure of one of the
deacylated compound, viz., 9a was further confirmed as 3,5-di-O-acetyl-4-C-
hydroxymethyl-1,2-O-(1-methylethylidene)-α-D-xylo-pentofuranose by chem-
ical transformation as shown in Scheme 3. The formation of the bicyclic
SCHEME 3

Biocatalytic Deacylation Studies
1521
compounds 13 and 14 finally confirmed that the hydroxyl group in com-
pound 9a, and so as in 9b and 9c is at the C-1ꢁ position (Scheme 3).
CONCLUSION
Highly efficient and convenient enzymatic method discovered for the
discrimination between two primary hydroxyl groups of sugar and nucleo-
side precursors, herein may find applications in “green” synthesis[7] of bi-
cyclonucleosides, important precursors for the preparation of antisense or
antigene oligonucleotides.
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