Direct regioselective enzymatic acylation of nucleosides: Building-blocks for the solution phase synthesis of oligonucleotides

Article abstract of DOI:10.1081/NCN-120023009

The synthesis of 3′- and 5′-O-levulinyl nucleosidic monomers through enzymatic acylation with acetonoxime levulinate is demonstrated. The acylation process takes place in one-step and use of expensive reagents, such as DMTrCl is avoided. The regioselectiv

Full text of DOI:10.1081/NCN-120023009

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
This article was downloaded by: [Temple University Libraries]
On: 23 November 2014, At: 07:08
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic Acids
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/lncn20
Direct Regioselective Enzymatic Acylation of
Nucleosides: Building-Blocks for the Solution Phase
Synthesis of Oligonucleotides
Javier García a , Susana Fernández a , Miguel Ferrero a c , Yogesh S. Sanghvi b & Vicente
Gotor a
a Departamento de Química Orgánica e Inorgánica , Facultad de Química , Universidad de
Oviedo , Oviedo, Spain
b Manufacturing Process Department , Isis Pharmaceuticals , Carlsbad, California, USA
c Departamento de Química Orgánica e Inorgánica , Facultad de Química , Universidad de
Oviedo , Avenida Julián de Clavería, 8, E-33006, Oviedo, Spain
Published online: 31 Aug 2006.
To cite this article: Javier García , Susana Fernández , Miguel Ferrero , Yogesh S. Sanghvi & Vicente Gotor (2003) Direct
Regioselective Enzymatic Acylation of Nucleosides: Building-Blocks for the Solution Phase Synthesis of Oligonucleotides,
Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1455-1457, DOI: 10.1081/NCN-120023009
To link to this article: http://dx.doi.org/10.1081/NCN-120023009
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1455–1457, 2003
Direct Regioselective Enzymatic Acylation of Nucleosides:
Building-Blocks for the Solution Phase Synthesis
of Oligonucleotides
1
Javier Garcıa, Susana Fernandez, Miguel Ferrero,
1
1,
*
´
´
Yogesh S. Sanghvi,2 and Vicente Gotor1
1
´
´
´
´
Departamento de Quımica Organica e Inorganica, Facultad de Quımica,
Universidad de Oviedo, Oviedo, Spain
2Manufacturing Process Department, Isis Pharmaceuticals,
Carlsbad, California, USA
ABSTRACT
The synthesis of 30- and 50-O-levulinyl nucleosidic monomers through enzymatic
acylation with acetonoxime levulinate is demonstrated. The acylation process
takes place in one-step and use of expensive reagents, such as DMTrCl is avoided.
The regioselectivity of the procedure makes it very convenient for acylated mono-
mers required for solution phase synthesis of oligonucleotides.
INTRODUCTION
The potential of antisense oligonucleotides for the treatment of a variety of dis-
eases, through sequence-specific modulation of gene expression has been well recog-
nized.[1] Chemical modifications in oligonucleotides[2] have resulted in enhanced
´
´
´
*Correspondence: Miguel Ferrero, Departamento de Quımica Organica e Inorganica, Facul-
tad de Quımica, Universidad de Oviedo, Avenida Julian de Claverıa, 8, E-33006 Oviedo,
´
´
´
Spain; Fax: þ34 98 510 3448; E-mail: mferreo@sauron.quimica.uniovi.es.
1455
DOI: 10.1081/NCN-120023009
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
´
Garcıa et al.
1456
nuclease resistance, cellular uptake, and appropriate hybridization to mRNAs or
target genes. With the recent success of various antisense oligonucleotides under-
going human clinical trials and possibility of their commercial launch in the near
future may require very large quantities of therapeutically useful oligonucleotides.
Particularly when multi-kilogram quantities of oligonucleotides are required, solu-
tion-phase synthesis appears to be an alternative method of choice instead of tradi-
tional solid-phase synthesis.[3] The key building blocks for the solution-phase
oligonucleotide synthesis are 30- and=or 50-O-levulinyl nucleosidic monomers.[4]
The levulinyl group is frequently chosen because it is stable to coupling conditions
and can be selectively cleaved, without adversely impacting other protecting groups
in the molecule. Until recently, the preparation of these building blocks has been car-
ried out through multiple protection=deprotection steps. For the manipulation of
protecting groups, application of biocatalysts in organic synthesis has become an
attractive alternative to the conventional chemical methods.[5]
Recently, we have reported a short and convenient synthesis of 30- and 50-
O-levulinylnucleosides from the corresponding 30,50-di-O-levulinyl derivatives by
regioselective enzymatic hydrolysis, avoiding several tedious chemical protection=
deprotection steps.[4] During hydrolysis process, base-protected cytidine and adeno-
sine derivatives were not good substrates for the enzyme Candida antarctica lipase
B (CAL-B), which afforded the 30-O-levulinylnucleosides. To overcome this limita-
tion of our earlier work, herein we report for the first time a regioselective enzymatic
acylation of various base-protected nucleosides with acetonoxime levulinate using
biocatalysts.
RESULTS AND DISCUSSION
For the enzymatic acylation, acetonoxime levulinate was used as acylating agent
due to higher selectivity compared with levulinic anhydride. CAL-B catalyzes the
Scheme 1.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Enzymatic Acylation of Nucleosides
1457
acylation of the 50-OH of nucleosides 1 and 2 with excellent regioselectivity affording
50-O-levulinyl derivatives 4 and 6, respectively, in high yields (Sch. 1). In contrast to
the hydrolysis process[4] base-protected cytidine and adenosine derivatives were
adequate substrates for CAL-B during acylation. Nevertheless, when Pseudomonas
cepacia lipase (PSL-C) is employed, 30-OH is acylated with high selectivity furnis-
hing 30-O-levulinylnucleosides 3 and 5. As observed with the hydrolysis process,
the selectivity of PSL-C was lost when acylation of base unprotected or 20-O-methyl
nucleosides was attempted. Furthermore, PSL-C did not shown selectivity during
acylation of guanosine derivatives. Such nucleosides could be prepared by enzymatic
hydrolysis, where CAL-B hydrolyzed selectively the 50-O-levulinyl group to afford
the 30-O-levulinyl derivatives.
In summary, we have developed an efficient method for the synthesis of 30- and
50-O-levulinyl protected nucleosides through regioselective enzymatic acylation of
the parent nucleosides. It is noteworthy that preparation requires a single step,
enzymes can be recycled several cycles, and expensive reagent such as DMTrCl is
eliminated. These advantages make the acylation procedure very convenient from
an industrial point-of-view. In addition, base-protected cytidine and adenosine deri-
vatives, not available through the hydrolysis process, were now accessible. Both
hydrolysis and acylation procedures are complementary to each other and offer an
excellent alternative to the conventional method. We are in process of scaling-up
these reactions for large-scale applications.
REFERENCES
1. For a recent monograph on this topic see: Antisense Drug Technology Principles,
Strategies and Applications; Crooke, S.T. Ed.; Marcel Dekker: New York, 2001.
2. Sanghvi, Y.S. Chapter 7. In Comprehensive Natural Products Chemistry; Kool,
E.T., Ed.; Elsevier: Amsterdam, 1999; Vol. 7, 285–311.
3. Sanghvi, Y.S. Org. Process R&D 2000, 4, 168–169.
´
´
4. Garcıa, J.; Fernandez, S.; Ferrero, M.; Sanghvi, Y.S.; Gotor, V. J. Org. Chem.
2002, 67, 4513–4519.
5. a) Ferrero, M.; Gotor, V. Chem. Rev. 2000, 100; 4319–4347; b) Ferrero, M.;
Gotor, V. Monatsh. Chem. 2000, 131, 585–616.