A mild, efficient and regioselective enzymatic procedure for 5′-O-benzoylation of 2′-deoxynucleosides

Article abstract of DOI:10.1016/j.tetlet.2003.12.098

Lipase from Candida antarctica B catalyzes the selective monobenzoylation at the 5′-hydroxyl group of 2′-deoxynucleosides using vinyl benzoate as acyl transfer reagent in quantitative yields. The industrial suitability of this process via the reclaim and reuse of enzyme and vinyl benzoate has been demonstrated.

Full text of DOI:10.1016/j.tetlet.2003.12.098

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 1709–1712
A mild, efficient and regioselective enzymatic procedure for
50-O-benzoylation of 20-deoxynucleosides
a
a
a
b
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Javier Garcıa, Susana Fernandez, Miguel Ferrero, Yogesh S. Sanghvi
and Vicente Gotora,*
a
ꢀ
ꢀ
ꢀ
ꢀ
Departamento de Quımica Organica e Inorganica, Facultad de Quımica, Universidad de Oviedo, Avenida Julian de Claveria, 8,
33071 Oviedo, Spain
bRasayan Inc., 2802 Crystal Ridge Road, Encinitas, CA 92024-6615, USA
Received 14 November 2003; revised 10 December 2003; accepted 17 December 2003
Abstract—Lipase from Candida antarctica B catalyzes the selective monobenzoylation at the 50-hydroxyl group of 20-deoxynucleo-
sides using vinyl benzoate as acyl transfer reagent in quantitative yields. The industrial suitability of this process via the reclaim and
reuse of enzyme and vinyl benzoate has been demonstrated.
Ó 2003 Elsevier Ltd. All rights reserved.
Antisense oligonucleotides constitute a promising class
of chemotherapeutic agents, which enable selective
inhibition of gene expression.1 As modified oligonucleo-
tides have become a major field of investigation for
chemists, methods for their suitable protection/depro-
tection for the synthesis of nucleoside monomers have
become equally important. Benzoylation remains one of
the most frequently used methods for the protection of
hydroxyl and amino functions in nucleoside and nucleo-
tide chemistry.2 The selective manipulation of hydroxyl
groups over amino groups of nucleobases is an impor-
tant reaction in oligonucleotide synthesis. The classical
method of benzoylation of the hydroxyl group in
nucleosides with benzoyl chloride or benzoic anhydride
provides nonselective reactions. Other mild benzoylating
reagents such as benzotetrazole,3 benzotriazole,4 and
benzoyl cyanide5 are reported for this purpose. How-
ever, lower selectivity has been observed toward the
acylation of different hydroxyl functions. Caddick et al.6
reported the selective protection of primary hydroxyl
groups using microwave irradiation. The method
worked with simple diols containing a primary and a
secondary hydroxyl group, but was unsuccessful in
complex molecules such as glucopyranosides. Recently,
the application of ionic liquids (IL) in nucleoside
chemistry has been investigated.7 Although IL are an
excellent reaction medium compared to conventional
solvents used in nucleoside chemistry, no selectivity has
been observed and the acylation reaction yielded a
mixture of products.
The selective benzoylation of the 50-hydroxyl group of
thymidine has been reported by Mitsunobu et al.8 This
reaction required the use of hexamethylphosphoric
triamide, which is toxic, and the by-product triphenyl-
phosphine oxide can be difficult to remove from the
products. An alternative synthesis was proposed by
Ishido and co-workers, and involved the careful drop-
wise addition (several hours) of a dilute solution of
benzoyl chloride in pyridine to 20-deoxynucleosides.9
Later, Sindona10 reported the regioselective acylation of
20-deoxynucleosides with carboxylic acids after activa-
tion with N,N-bis-(2-oxo-oxazolidin-3-yl)phosphorodi-
amidic chloride, a noncommercial reagent. Moderate
yields of the 50-O-benzoyl derivatives were obtained
after column chromatography.
Enzyme-catalyzed esterification is an alternative to
classical synthetic methods that can overcome these
problems. The potential of enzymes in organic synthesis
is well recognized, in particular when the substrates
possess several functional groups of similar reactivity.11
The chiral nature of enzymes allow selective acylation
and/or deacylation processes on a variety of molecules
avoiding additional chemical protection/deprotection
steps. Recently, Santaniello and co-workers12 reported
the selective benzoylation of diols catalyzed by a suitable
lipase.
* Corresponding author. Tel./fax: +34-98-510-3448; e-mail: vgs@fq.
uniovi.es
0040-4039/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.12.098

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J. Garcıa et al. / Tetrahedron Letters 45 (2004) 1709–1712
1710
In our ongoing research related to the preparation of
appropriate building blocks for solution phase oligo-
nucleotide synthesis, we have developed the synthesis of
30-and 50-O-levulinyl nucleoside derivatives by enzy-
matic hydrolysis or acylation processes.13 Previously, we
have described the selective enzymatic acylation of
thymidine and 20-deoxyadenosine with oxime esters.14
group and ability to be recycled.13;14 Therefore, thymi-
dine (1a) was dissolved in dry THF under nitrogen and
treated with vinyl benzoate and CAL-B. The reaction
was almost complete in 71 h at 60 °C (Scheme 1). Regio-
selective acylation of the 50-OH group of 1a was
observed furnishing 50-O-benzoylthymidine (2a),8 (entry
1, Table 1) with small (13%) amounts of the starting
material. The same process was repeated with nucleo-
sides 1b–d. The CAL-B maintained the regioselectivity
although low conversions were achieved for cytidine and
guanosine derivatives (entries 2–4, Table 1). The ben-
zoylation of the 30-OH group or the amino group on the
base moiety was not observed indicating the exclusive
selectivity toward the 50-position. No acylation occurred
under identical reaction conditions when CAL-B was
left out. All reactions were carried out with 3 equiv of
vinyl benzoate, a substrate/enzyme ratio of 1:1 (w/w),
and at 0.2 M concentration. In order to reach conver-
sions close to 100%, optimum ratios of acylating agent
and enzyme were established. Thus, acylation of 1a with
5 equiv of vinyl benzoate furnished 2a in quantitative
yield (isolated yield 98%, entry 6, Table 1). Importantly,
pure 2a was isolated via precipitation without a need for
column chromatographic purification. The excess of
vinyl benzoate was recaptured by solvent evaporation
under vacuum from the mother liquor and recycled to
minimize the waste.
The application of acetone oxime benzoate as an acyl-
ating agent for other nucleosides furnished moderate
yields with slow reaction rates. Herein, we report a mild
and efficient procedure for the selective benzoylation of
20-deoxynucleosides through direct enzymatic acylation
with vinyl benzoate, a commercially available reagent.
We selected Candida antarctica lipase B15 (CAL-B), an
immobilized enzyme, due to its well-demonstrated
selectivity in the transesterification of the 50-hydroxyl
O
Ph
O
B
HO
B
CAL-B
O
OH
O
OH
Vinyl benzoate
THF, 60 °C
2a-d
1a-d
Scheme 1. a: B ¼ T; b: B ¼ CBz; c: B ¼ ABz; d: B ¼ Gibu (ibu ¼ iso-
To demonstrate the suitability of the reaction for
industrial applications, both the acylating agent and the
butyryl).
Table 1. Regioselective enzymatic acylation of 20-deoxynucleosides 1 using vinyl benzoate (VB) with CAL-B at 60 °C
Entry
Substrate
VB (eq)
Ratio (1:CAL-B)a Conc. (M)
t (h)
1 (%)b
2 (%)b;c
1
1a
1b
3
3
1:1
1:1
1:1
1:1
1:1
1:1
1:1e
1:1
1:1
1:1
1:1g
1:1h
1:1
1:1
1:2
1:1
1:1
1:1
1:2
1:1
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.1
0.2
0.2
0.2
0.1
0.1
0.1
0.2
0.1
0.2
0.1
0.2
71
98
13
74
2
87 (62)
26
2
3
1c
3
40
96
98 (93)
24
4
1d
3
76
1
5
1ad
1ad
1ad
1a
3
118
72
99 (92)
>99 (98)
>99 (96)
99 (95)
>99 (95)
99 (95)
99 (93)
97 (90)
49
6
5
ND
ND
1
7
5
144
72
8
5f
10
3
9
1bd
1cd
1cd
1cd
1dd
1dd
1dd
1ai
1bi
1ci
1di
1ci
40
23
ND
1
10
11
12
13
14
15
16
17
18
19
20
3
72
1
3
121
120
215
95
3
5
51
ND
ND
ND
2
10
10
5
99 (95)
>99 (89)
>99 (98)
98 (96)
>99 (97)
99
63
68
10
3
32
89
ND
1
10
3
30
ND
>99 (97)
a Ratio w/w.
b Based on 1H NMR and HPLC signal integration.
c Percentages of isolated yields are given in parenthesis.
d 1 g scale of starting nucleoside.
e Recycled enzyme from entry 6.
f Recycled vinyl benzoate from entry 6. Starting nucleoside 0.3 g.
g First run with the recycled enzyme from entry 10.
h Second consecutive run with the recycled enzyme from entries 10 and 11.
i Entries 16–20 are large-scale experiments. Entries 16–19 are 5 g scale of starting nucleoside. Entry 20 is 25 g of starting nucleoside; ND ¼ not
detected under the experimental conditions.

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J. Garcıa et al. / Tetrahedron Letters 45 (2004) 1709–1712
1711
enzyme were reused for subsequent reactions. The
recycled CAL-B maintained total selectivity toward
acylation of the 50-OH with the exception of the longer
reaction rate (entry 7, Table 1). On the other hand,
benzoylation of 1a with recycled vinyl benzoate gave
identical results compared to the use of fresh acylating
agent (entry 8, Table 1).
the acylation process with total selectivity furnishing the
50-O-benzoylated derivatives in quantitative yields.
In conclusion, we have developed an efficient and
practical green alternative for the syntheses of 50-O-
benzoyl-20-deoxynucleosides 2a–d by selective acylation
of the parent nucleosides catalyzed by Candida antarc-
tica lipase B with vinyl benzoate. The use of THF as a
solvent avoids the tedious work-up of the reaction with
other solvents traditionally used in nucleoside chemistry
such as pyridine or DMF. The easy scalability of the
process and the fact that both the acylating agent and
enzyme can be reclaimed and reused after each reaction
makes the new process atom efficient19 and very
attractive for industrial applications. The protected
monomers 2a–d described in this study are important
raw materials for the synthesis of therapeutically useful
oligonucleotides20 and modified nucleosides.21
For the enzymatic acylation of N-benzoyl-20-deoxycyti-
dine (1b), 10 equiv of vinyl benzoate, which increases
the conversion rate, and more dilute conditions (0.1 M
instead of 0.2 M) to favor the solubility of the starting
nucleoside, were used. Under these conditions, exclusive
formation of 50-O-benzoylated derivative 2b16 was
observed after 40 h (entry 9, Table 1). N-Benzoyl-20-
deoxyadenosine (1c) was found to be more reactive and
only 3 equiv of the vinyl ester were necessary to drive the
reaction to completion and furnish 2c9 in quantitative
yield (99%, entry 10, Table 1). As entries 11 and 12
show, subsequent reactions can be successfully carried
out with the recycled enzyme. In the case of N-isobu-
tyryl-20-deoxyguanosine (1d), a 0.1 M concentration was
used to increase the poor solubility of the starting
material in THF. The best results were obtained when
the reaction was carried out with 10 equiv of vinyl
benzoate with a substrate/enzyme ratio of 1:2 (w/w),
(entries 13–15, Table 1). Thus, N-isobutyryl-50-O-ben-
zoyl-20-deoxyguanosine (2d)9 was isolated in 89% yield.
It is noteworthy that the excellent yields and purity of
2a–d were obtained from 1a–d via a common precipi-
tation protocol.17
Acknowledgements
Financial support from Principado de Asturias (Spain;
Project GE-EXP01-03) and MCYT (Spain; Project
PPQ-2001-2683) is gratefully acknowledged. S.F. also
ꢀ
thanks MCYT (Spain) for a personal grant (Ramon y
Cajal Program).
References and notes
1. (a) Antisense Drug Technology Principles, Strategies and
Applications; Crooke, S. T., Ed.; Marcel Dekker: New
York, 2001; (b) Sanghvi, Y. S. In Comprehensive Natural
Products Chemistry; Kool, E. T., Ed.; Elsevier: Amster-
dam, 1999; Vol. 7; Chapter 7, pp 285–311, and references
cited therein.
2. Greene, T. W.; Wuts, P. G. M. In: Protective Groups in
Organic Synthesis. 3rd ed.; Wiley: New York, 1999; p 173.
3. Stawinski, J.; Hozumi, T.; Narang, S. A. J. Chem. Soc.,
Chem. Commun. 1976, 243–244.
4. Bhat, B.; Sanghvi, Y. S. Tetrahedron Lett. 1997, 38, 8811–
8814.
5. Kumar, V.; Maity, J.; Wang, Z.; Prasad, A. K.; Sanghvi,
Y. S.; Parmar, V. S., in preparation.
6. Caddick, S.; McCarroll, A. J.; Sandham, D. A. Tetrahe-
dron 2001, 57, 6305–6310.
7. Uzagare, M. C.; Sanghvi, Y. S.; Salunkhe, M. M. Green
Chem. 2003, 5, 370–372.
Using the CAL-B benzoylation protocol described
herein, we attempted to benzoylate the 30-OH group of
the 50-O-protected 2c. Interestingly, 2c failed to undergo
a second benzoylation reaction. Additionally, lipase
from Pseudomonas cepacia (PSL-C),18 which has dem-
onstrated high selectivity in the transesterification of the
30-hydroxyl group,13;14 was used as a biocatalyst without
success. In both reactions the starting material was
recovered unchanged. Based on these experimental
results, we postulate that the secondary hydroxyl group
is not accessible for both CAL-B or PSL-C to install a
second benzoyl group in the nucleoside, perhaps due to
the steric hindrance created by the primary benzoyl
group in the vicinity. This would explain our inability to
find 30,50-bis-benzoylated products during the synthesis
of 2.
8. Mitsunobu, O.; Kimura, J.; Fujisawa, Y. Bull. Chem. Soc.
Jpn. 1972, 45, 245–247.
The screening of other acylating agents such as acetone
oxime benzoate gave lower yields and benzoic anhydride
led to nonselective acylation. In place of THF, toluene
was tried as an alternative, cheaper and safer solvent. In
the case of 1d, a substantial amount of starting material
was recovered due to the poor solubility in toluene. For
1a and 1c the reaction took much longer in toluene while
maintaining the regioselectivity observed in THF.
9. Nishino, S.; Yamamoto, H.; Nagato, Y.; Ishido, Y.
Nucleos. Nucleot. 1986, 5, 159–168.
10. Liguori, A.; Perri, E.; Sindona, G.; Uccella, N. Tetrahe-
dron 1988, 44, 229–234.
11. For recent reviews on enzymatic transformations in
nucleosides, see: (a) Ferrero, M.; Gotor, V. Chem. Rev.
2000, 100, 4319–4347; (b) Ferrero, M.; Gotor, V. Monatsh.
Chem. 2000, 131, 585–616.
12. (a) Ciuffreda, P.; Alessandrini, L.; Terraneo, G.; Santan-
iello, E. Tetrahedron: Asymmetry 2003, 14, 3197–3201; (b)
Ciuffreda, P.; Casati, S.; Santaniello, E. Tetrahedron Lett.
2003, 44, 3663–3665.
Next, the large-scale acylation of nucleosides 1a–d was
studied (entries 16–20, Table 1). Experiments were car-
ried out on 5 and 25 g scales of the starting material.
Excellent results were obtained with CAL-B catalyzing
ꢀ
13. (a) Garcıa, J.; Fernandez, S.; Ferrero, M.; Sanghvi, Y. S.;
Gotor, V. J. Org. Chem. 2002, 67, 4513–4519; (b) Garcıa,
ꢀ
ꢀ

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J. Garcıa et al. / Tetrahedron Letters 45 (2004) 1709–1712
1712
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J.; Fernandez, S.; Ferrero, M.; Sanghvi, Y. S.; Gotor, V.
Tetrahedron: Asymmetry 2003, 14, 3533–3540.
and the solvents were evaporated under vacuum. The
residue was precipitated in hexane to afford, after filtration
and washing with hexane, the 50-O-benzoyl nucleosides 2
as solids. Vinyl benzoate was recovered by solvent
evaporation under vacuum from the hexane mother
liquors.
14. Moris, F.; Gotor, V. J. Org. Chem. 1993, 58, 653–660.
15. Microbial lipase from Candida antarctica immobilized on
a macroporous acrylic resin (Novozym 435â, 10,000 PLU/
g), Novo Nordisk.
16. Gray, S. H.; Ainsworth, C. F.; Bell, C. L.; Danyluk, S. S.;
MacCoss, M. Nucleos. Nucleot. 1983, 2, 435–452.
18. Microbial lipase from Pseudomonas cepacia immobilized
on ceramic particles (PSL-C, 904 U/g), Amano Pharma-
ceuticals.
19. Trost, B. M. Acc. Chem. Res. 2002, 35, 695–705.
20. Schultz, R. G.; Gryaznov, S. M. Nucleic Acids Res. 1996,
24, 2966–2973.
21. (a) Herdewijn, P.; Van Aerschot, A. Tetrahedron Lett.
1989, 30, 855–858; (b) Herdewijn, P. J. Org. Chem. 1988,
53, 5050–5053.
17. General procedure for the regioselective enzymatic ben-
zoylation of 20-deoxynucleosides: A suspension of 1
(0.4 mmol), vinyl benzoate, and CAL-B in dry THF under
nitrogen was stirred at 60 °C and 250 rpm (orbital shaker)
under the conditions and the reaction time indicated in
Table 1. The reactions were monitored by HPLC. The
enzyme was filtered off and washed with CH2Cl2 and THF