Biocatalytic route to C-4′-spiro-oxetano-xylofuranosyl pyrimidine nucleosides

Article abstract of DOI:10.1080/10242422.2018.1438416

A facile access to C-4′-spiro-oxetano-xylofuranosyl nucleosides has been demonstrated for the first time through Lipozyme TL IM-mediated regioselective acetylation of one of the primary hydroxyl group over the other primary and secondary hydrox

Full text of DOI:10.1080/10242422.2018.1438416

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Biocatalytic route to C-4′-spiro-oxetano-
xylofuranosyl pyrimidine nucleosides
Pallavi Rungta, Priyanka Mangla, Vinod Khatri, Jyotirmoy Maity & Ashok K.
Prasad
To cite this article: Pallavi Rungta, Priyanka Mangla, Vinod Khatri, Jyotirmoy Maity & Ashok K.
Prasad (2018): Biocatalytic route to C-4′-spiro-oxetano-xylofuranosyl pyrimidine nucleosides,
Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2018.1438416
To link to this article: https://doi.org/10.1080/10242422.2018.1438416
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BIOCATALYSIS AND BIOTRANSFORMATION, 2018
https://doi.org/10.1080/10242422.2018.1438416
RESEARCH ARTICLE
Biocatalytic route to C-40-spiro-oxetano-xylofuranosyl pyrimidine nucleosides
Pallavi Rungta, Priyanka Mangla, Vinod Khatri, Jyotirmoy Maity and Ashok K. Prasad
Department of Chemistry, Bioorganic Laboratory, University of Delhi, Delhi, India
ABSTRACT
ARTICLE HISTORY
A facile access to C-40-spiro-oxetano-xylofuranosyl nucleosides has been demonstrated for the
Received 10 August 2017
Revised 9 January 2018
Accepted 1 February 2018
R
first time through LipozymeV TL IM-mediated regioselective acetylation of one of the primary
hydroxyl group over the other primary and secondary hydroxyl groups in 30-O-benzyl-40-C-
hydroxymethyl-b-D-xylofuranosyl nucleosides. Attempts to optimize a convergent route for these
spironucleosides via selective manipulation of hydroxyl groups in 3-O-benzyl-4-C-hydroxymethyl-
1,2-O-isopropylidene-a-D-xylofuranose were unsuccessful. Nevertheless; the present linear bioca-
talytic route efficiently afforded the C-40-spiro-oxetanoxylofuranosyl nucleosides T and U in 47
and 38% overall yields, respectively, starting from corresponding furanose diol.
KEYWORDS
Biocatalysis; regioselective
acetylation;
spiro-nucleosides
1. Introduction
a nucleoside, attempts have been made to install oxe-
tano-spiro cyclization at C-40-position in xylofuranosyl
nucleosides ((Sharma et al. 2014a), Figure 1) that have
earlier turned out to be futile (Roy et al. 2006). Our
group has earlier demonstrated the synthesis of vari-
ous C-40-spiro-oxetano-ribonucleosides (Sharma et al.
2014c; Kumar et al. 2015, 2017). Kumar et al. optimized
Chemical modifications of the sugar functionality in
nucleosides have continuously reflected its supremacy
for the treatment of cancer and viral infections over
the past 50 years (De Clercq 2011; Prakash 2011; Sofia
et al. 2012; Jordheim et al. 2013; Sharma et al. 2014a,
2014b). Among the studied sugar-modified nucleo-
sides, spironucleosides had only little impact and pro-
gress until the recent finding that these are selective
and potent inhibitors of Hepatitis C Virus (HCV) (Dang
et al. 2014a, 2014b; Du et al. 2014; Jonckers et al.
2010, 2014). The presence of spiro-carbon in nucleo-
sides imparts restriction to the conformational flipping
of furanose ring and thus can modulate or enhance
their selectivity and biological activity. This has
prompted the synthesis of all possible classes of spiro-
nucleosides, e.g. C-10-spiro-(Maza et al. 2009; Pal and
Yashwant 2010 ), C-20-spiro-(Du et al. 2014; Dang et al.
2014b; Jonckers et al. 2010, 2014), C-30-spiro-(Camarasa
et al. 1992; De Castro et al. 2007; Das et al. 2011) and
C-40-spironucleosides (Paquette et al. 2003; Paquette
2004; Paquette et al. 2004; Dong and Paquette 2005;
Dong and Paquette 2006; Roy et al. 2006; Dang et al.
2014a; Sharma et al. 2014c; Kumar et al. 2015; Kumar
et al. 2017); and many of them have shown excellent
antiviral activity (Figure 1). Prominent among them are
the C-40-spironucleosides which display distinct sugar
conformation (Paquette 2004). In order to scrutinize
the presence of the spiro- and the oxetano-systems in
R
NovozymeV-435 mediated regio-selective deacetylation
of C-5 acetoxy group in 5-O-acetyl-4-C-acetyloxy-
methyl-3-azido-1,2-O-isopropylidine-a-D-ribofuranose to
establish a methodology for successful synthesis of
C-40-spiro-oxetano-b-D-ribonucleosides (Kumar et al.
2015). In another venture, Kumar et al. successfully
R
used LipozymeV TL IM-catalyzed acylation on
C-40-hydroxymethyl-b-D-xylofuranosyl nucleosides and
synthesized structurally novel C-40-spiro-oxetano-a-L-
ribonucleosides (Kumar et al. 2017). In the present
communication, we have reported the first synthesis
of C-40-spiro-oxetano-xylofuranosyl nucleosides that is
R
devised by the mediation of LipozymeV TL IM, an
immobilised Thermomyces lanuginosa lipase.
2. Experimental section
Materials. Analytical TLCs were performed on pre-
coated Merck silica gel 60F254 plates; the spots were
detected either using UV light or by charring with 4%
alcoholic sulphuric acid. The optical rotations were
measured on Rudolph autopol II automatic polarimeter
using light of 546 nm wavelegth. The melting points
CONTACT Ashok K. Prasad
ashokenzyme@gmail.com
Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India
Supplemental data for this article can be accessed here.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
P. RUNGTA ET AL.
O
B
(HO)2P
B
B
B
HO
HO
OH
O
O
O
O
O
O
O
O
OH
OH OH
OH
OH
1
2
3
4
Figure 1. Some of the structures of biologically active spironucleosides 1-3 and the targeted C-40-spiro-oxetano-xylofuranosyl
nucleoside 4.
were determined on Buchi M-560 instrument and are
uncorrected. The IR spectra were recorded on a
Perkin-Elmer model 2000 FT-IR spectrometer by mak-
ing KBr disc for solid samples and thin film for oils.
2.1.1.
3-O-Benzyl-1,2-O-isopropylidene-4-C-propa-
noyloxymethyl-5-O-propanoyl-a-D-xylofuranose (7b)
It was obtained as a colourless viscous oil (0.14 g) in
97% yield. Rf ¼ 0.5 (30% ethyl acetate in petroleum
1
The H-,13C- and 2D NMR spectra were recorded on a
28
ether); [a]D ¼ ꢂ48.23 (c 0.1, CHCl3); IR (thin film) ꢀmax
Jeol alpha-400 spectrometer at 400 and 100.6 MHz,
respectively, using TMS as internal standard. The
chemical shift values are on d scale and the coupling
constants (J) are in Hertz. The HRMS analyses were
done on micro TOF-Q instrument from Bruker
Daltonics, Bremen. The single crystal diffraction data
were collected on an X’Calibur single crystal X-ray dif-
fractor having CCD camera [Mo/Ka radiation
(k ¼ 0.71073 Å)]. The T. lanuginosus lipase immobilized
cmꢂ1: 608, 700, 740, 807, 863, 1020, 1072, 1168, 1383,
1423, 1463, 1744, 2943, 2984; 1H NMR (CDCl3, 400 MHz):
d 1.06 (3H, t, J ¼ 7.6 Hz, –OCOCH2CH3), 1.12 (3H, t,
J ¼ 7.6 Hz, –OCOCH2CH3), 1.36 and 1.55 (6H, 2s, 3H
each, –C(CH3)2), 2.24 (2H, q, J ¼ 7.6 Hz, –OCOCH2CH3),
2.33 (2H, q, J ¼ 7.6 Hz, –OCOCH2CH3), 4.04 (1H, s, C-3H),
4.14 (1H, d, J ¼ 11.6 Hz, C-10Ha), 4.22 (2H, s, –OCH2Ph),
4.38 (1H, d, J ¼ 11.6 Hz, C-10Hb), 4.51 (1H, d, J ¼ 12.4 Hz,
C-5Ha), 4.72–4.75 (2H, m, C-2H
& C-5Hb), 5.97
R
on silica (LipozymeV TL IM) was obtained as a gift
(1H, d, J ¼ 4.8 Hz, C-1H), 7.29–7.37 (5H, m, ArH);
13C NMR (CDCl3, 100.6 MHz): d 8.88 and 9.02 (2 X
–OCOCH2CH3), 26.68 and 27.09 (–C(CH3)2), 27.22 and
27.50 (2 X –OCOCH2CH3), 63.08 (C-10), 63.35 (C-5), 72.24
(–OCH2Ph), 83.63 (C-3), 85.37 (C-2), 85.89 (C-4), 105.20
(C-1), 113.42 (–C(CH3)2), 127.69, 128.02, 128.46 and
136.84 (ArC), 173.72 and 173.82 (2 X –OCOC2H5); HR-
ESI-TOF-MS: m/z 440.2279 ([M þ NH4]þ), calcd. for
[C22H30O8þNH4]þ 440.2279.
from Novozyme A/S Denmark. Other enzymes Candida
R
antarctica lipase-B (NovozymeV-435), C. rugosa lipase
(CRL) and porcine pancreatic lipase (PPL) were pur-
chased from Sigma-Aldrich Co. (St. Louis, MO, USA).
The enzyme was dried over P2O5 under vacuum for
24 h prior to use. For all the lipase-mediated reactions
AR grade organic solvents were used, which were pur-
chased from SD Fine-Chem Ltd, Mumbai, India.
2.1.2. 3-O-Benzyl-4-C-butanoyloxymethyl-5-O-buta-
noyl-1,2-O-isopropylidene-a-D-xylofuranose (7c)
2.1. General procedure for the synthesis of
diacylated xylofuranose 7b-c
It was obtained as a colourless viscous oil (0.15 g) in
To a solution of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-
isopropylidene-a-D-xylofuranose (5) (Youssefyeh et al.
1979) (0.10 g, 0.32 mmol) in CH2Cl2 (10 mL), propionic
(6b) or butyric anhydride (6c) (0.71 mmol) and catalytic
amount of DMAP (0.03 mmol) were added. The reac-
tion mixture was stirred at 25 ꢀC for 2–4 h and then
was poured into ice-water. The mixture was extracted
with ethyl acetate (3 ꢁ 50 mL), the combined organic
extract was washed with saturated aq. bicarbonate
solution (2 ꢁ 25 mL), water (1 ꢁ 25 mL) and dried over
anhydrous sodium sulphate. The excess of solvent was
evaporated under reduced pressure and the residue
thus obtained was purified by silica gel column chro-
matography using ethyl acetate in petroleum ether as
eluent to afford the diacylated compounds 7b-c in
quantitative yields.
98% yield. Rf ¼ 0.5 (20% ethyl acetate in petroleum
27
ether); [a]D ¼ ꢂ18.74 (c 0.05, MeOH); IR (thin film)
ꢀmax cmꢂ1: 700, 744, 1016, 1075, 1167, 1379, 1458,
1739, 2876, 2966; 1H NMR (CDCl3, 400 MHz): d 0.88–0.94
(6H, m, 2 X –OCOCH2CH2CH3), 1.36 (3H, s, –C(CH3)2),
1.54–1.65 (7H, m, 2 X –OCOCH2CH2CH3 and –C(CH3)2),
2.19 (2H, t, J ¼ 7.6 Hz, –OCOCH2CH2CH3), 2.29 (2H, t,
J ¼ 7.6 Hz, –OCOCH2CH2CH3), 4.03 (1H, d, J ¼ 1.6 Hz,
C-3H), 4.14 (1H, d, J ¼ 12.0 Hz, C-10Ha), 4.21 (2H, s,
–OCH2Ph), 4.38 (1H, d, J ¼ 11.6 Hz, C-10Hb), 4.51 (1H, d,
J ¼ 11.6 Hz, C-5Ha), 4.72-4.75 (2H, m, C-5Hb & C-2H), 5.98
(1H, d, J ¼ 4.8 Hz, C-1H), 7.29-7.35 (5H, m, ArH); 13C NMR
(CDCl3, 100.6 MHz): d 13.60 (–OCOCH2CH2CH3), 18.21
and 18.34 (2 X –OCOCH2CH2CH3), 26.68 and 27.09
(–C(CH3)2), 35.83 and 36.11 (2 X –OCOCH2CH2CH3),
63.04 (C-10), 63.35 (C-5), 72.27 (–OCH2Ph), 83.74 (C-3),

BIOCATALYSIS AND BIOTRANSFORMATION
3
85.36 (C-2), 85.88 (C-4), 105.22 (C-1), 113.40 (-C(CH3)2),
127.69, 128.02, 128.46 and 136.85 (ArC), 172.90 and
173.01 (2 X –OCOC3H7); HR-ESI-TOF-MS: m/z 468.2586
([M þ NH4]þ), calcd. for [C24H34O8þNH4]þ 468.2592.
The reaction was quenched with cold saturated aq.
solution of sodium bicarbonate (100 mL) and extrac-
tion of the compound was performed with dichloro-
methane (3 ꢁ 100 mL). The combined organic phase
was washed with saturated aq. sodium bicarbonate
solution (2 ꢁ 100 mL), brine solution (2 ꢁ 100 mL) and
was dried over anhydrous sodium sulphate. The excess
of solvent was removed under reduced pressure and
the residue thus obtained was purified by silica gel
column chromatography using methanol in chloroform
as eluent to afford nucleosides 10a-b in 90 and 88%
yields, respectively.
2.2. 4-C-Acetoxymethyl-1,2,5-tri-O-acetyl-3-O-
benzyl-a,b-D-xylofuranose (9a-9b)
Acetic anhydride (4.8 mL, 50.7 mmol) and concentrated
sulphuric acid (0.02 mL, 0.50 mmol) were added to a
stirred solution of compound 7a (Youssefyeh et al.
1979) (2.0 g, 5.07 mmol) in acetic acid (28.9 mL,
507 mmol) at 0 ꢀC and the resulting reaction mixture
was stirred for 6 h at 25 ꢀC. The reaction was quenched
by the addition of cold water (200 mL) and stirring at
25 ꢀC for 30 min. The crude compound was extracted
from the reaction mixture with ethyl acetate
(3 ꢁ 200 mL). The organic layer was washed with satu-
rated aq. sodium bicarbonate solution (3 ꢁ 100 mL),
brine solution (2 ꢁ 100 mL) and then dried over anhyd-
rous sodium sulphate. The excess of solvent was
removed under reduced pressure and the residue thus
obtained was purified by silica gel column chromatog-
raphy using ethyl acetate in petroleum ether as gradi-
ent solvent system to afford an anomeric mixture
(a:b ¼ ca. ꢃ 1:2, based on comparison of integration of
anomeric proton) of 9a-9b (2.16 g) in 97% yield as col-
ourless viscous oil. Rf ¼ 0.4 (20% ethyl acetate in pet-
2.3.1. 40-C-Acetoxymethyl-20,50-di-O-acetyl-30-O-ben-
zyl-b-D-xylofuranosyl thymine (10a)
It was obtained as sticky solid (1.03 g) in 90% yield.
31
Rf ¼ 0.3 (2% MeOH in CHCl3); [a]D ¼ ꢂ16.79 (c 0.1,
MeOH); IR (thin film) ꢀmax cmꢂ1: 700, 749, 1041, 1214,
1369, 1455, 1687, 1736, 3027, 3092; 1H NMR (CDCl3,
400 MHz): d 1.83 (3H, s, C-5CH3), 2.02, 2.09 and 2.14 (9H,
3s, 3H each, –OCOCH3), 4.04 (1H, d, J ¼ 2.4 Hz, C-30H),
4.12–4.23 (3H, m, C-50Ha and C-100H2), 4.57 (1H, d,
J ¼ 12.0 Hz, C-50Hb), 4.61 (1H, d, J ¼ 12.0 Hz, –OCHaPh),
4.76 (1H, d, J ¼ 12.0 Hz, –OCHbPh), 5.28 (1H, t, J ¼ 2.8 Hz,
C-20H), 6.25 (1H, d, J ¼ 4.0 Hz, C-10H), 7.28–7.37 (5H, m,
ArH), 7.45 (1H, d, J ¼ 1.6 Hz, C-6H), 8.55 (1H, brs, NH);
13C NMR (CDCl3, 100.6 MHz): d 12.50 (C-5CH3), 20.66,
20.79 and 20.85 (3 X -OCOCH3), 61.92 (C-100), 62.31
(C-50), 72.58 (–OCH2Ph), 80.19 (C-20), 81.00 (C-30), 85.62
(C-40), 87.17 (C-10), 111.93 (C-5), 128.21, 128.50, 128.66
and 135.19 (ArC), 136.21 (C-6), 150.20 (C-2), 163.23 (C-
4), 169.90, 170.13 and 170.20 (3 X –OCOCH3); HR-ESI-
TOF-MS: m/z 505.1824 ([M þ H]þ), calcd. for
[C24H28N2O10þH]þ 505.1817.
27
roleum ether); [a]D ¼ ꢂ37.08 (c 0.1, MeOH); IR (thin
film) ꢀmax cmꢂ1: 700, 745, 1011, 1216, 1371, 1741,
1
2937; H NMR (CDCl3, 400 MHz): d 6.17 (s, C-1Hb), 6.37
(d, J ¼ 4.8 Hz, C-1Ha); 13C NMR (CDCl3, 100.6 MHz): d
20.75, 20.94, 21.03, 21.14, 21.18, 21.21, 21.32, 21.42,
63.39, 63.54, 63.71, 65.60, 72.59, 73.64, 76.74, 77.54,
80.75, 81.45, 82.26, 83.11, 87.14, 92.56, 99.67, 127.84,
128.20, 128.37, 128.42, 128.77, 128.81, 137.26, 137.52,
169.49, 169.87, 169.93, 170.50, 170.58, 170.66, 170.81;
HR-ESI-TOF-MS: m/z 456.1874 ([M þ NH4]þ), calcd. for
[C21H26O10þNH4]þ 456.1864.
2.3.2. 40-C-Acetoxymethyl-20,50-di-O-acetyl-30-O-ben-
zyl-b-D-xylofuranosyl uracil (10b)
It was obtained as sticky solid (0.98 g) in 88% yield. Rf
30
¼ 0.3 (2% MeOH in CHCl3); [a]D ¼ ꢂ7.88 (c 0.1, MeOH);
IR (thin film) ꢀmax cmꢂ1: 753, 1043, 1217, 1374, 1685,
1741, 3028, 3066; 1H NMR (CDCl3, 400 MHz): d 1.98,
2.08, 2.15 (9H, 3s, 3H each, –OCOCH3), 4.02 (1H, d,
J ¼ 2.4 Hz, C-30H), 4.10–4.23 (3H, m, C-50Ha & C-100H2),
4.56 (1H, d, J ¼ 12.0 Hz, C-50Hb), 4.61 (1H, d, J ¼ 12.0 Hz,
–OCHaPh), 4.75 (1H, d, J ¼ 11.6 Hz, –OCHbPh), 5.27 (1H,
t, J ¼ 3.2 Hz, C-20H), 5.76 (1H, d, J ¼ 7.6 Hz, C-5H), 6.23
(1H, d, J ¼ 4.0 Hz, C-10H), 7.28–7.37 (5H, m, ArH), 7.65
(1H, d, J ¼ 8.4 Hz, C-6H), 9.42 (1H, brs, NH); 13C NMR
(CDCl3, 100.6 MHz): d 20.59, 20.74 and 20.80 (3 X
–OCOCH3), 61.80 (C-100), 62.20 (C-50), 72.42 (–OCH2Ph),
80.28 (C-20), 80.58 (C-30), 85.99 (C-40), 87.56 (C-10),
103.25 (C-5), 128.35, 128.48, 128.62 and 136.09 (ArC),
2.3. General procedure for the synthesis of 40-C-
Acetoxymethyl-20,50-di-O-acetyl-30-O-benzyl-b-D-
xylofuranosyl nucleosides 10a-b
To the stirred solution of tetra-O-acetylated sugar
derivative 9a-9b (1.0 g, 2.28 mmol) and thymine/uracil
(3.42 mmol) in anhydrous acetonitrile (40 mL), N,O-bis(-
trimethylsilyl)acetamide (2.2 mL, 9.12 mmol) was added
dropwise. The reaction mixture was stirred at reflux for
1 h, and then cooled to 0 ꢀC. In the cooled reaction
mixture
trimethylsilyltrifluoromethane
sulfonate
(0.702 mL, 3.88 mmol) was added dropwise under stir-
ring and the solution was heated at 70–80 ꢀC for 4 h.

4
P. RUNGTA ET AL.
139.50 (C-6), 150.29 (C-2), 162.99 (C-4), 169.87, 170.09
J ¼ 5.2 Hz, OH), 5.69 (1H, d, J ¼ 8.4 Hz, C-5H), 5.77
(1H, d, J ¼ 5.2 Hz, OH), 5.81 (1H, d, J ¼ 7.6 Hz, C-10H),
7.26–7.36 (5H, m, ArH), 8.07 (1H, d, J ¼ 8.0 Hz, C-6H),
11.32 (1H, brs, NH); 13C NMR (DMSO-d6, 100.6 MHz): d
61.29 (C-100), 62.78 (C-50), 72.02 (-OCH2Ph), 77.44 (C-20),
82.67 (C-30), 85.23 (C-40), 85.80 (C-10), 102.13 (C-5),
127.33, 127.39, 128.20 and 138.57 (ArC), 141.07 (C-6),
150.92 (C-2), 163.14 (C-4); HR-ESI-TOF-MS: m/z
387.1165 ([M þ Na]þ), calcd. for [C17H20N2O7þNa]þ
387.1163.
and 170.20 (3
X –OCOCH3); HR-ESI-TOF-MS: m/z
513.1491 ([M þ Na]þ), calcd. for [C23H26N2O10þNa]þ
513.1480.
2.4. General procedure for the synthesis of
30-O-Benzyl-40-C-hydroxymethyl-b-D-xylofuranosyl
nucleosides 11a-b
To a stirred solution of compound 10a-b (2 mmol) in
methanol (10 mL), methanolic NH3 was added and the
reaction mixture was stirred overnight at 25 ꢀC. The
reaction mixture was evaporated under reduced pres-
sure. The residue thus obtained was purified by silica
gel column chromatography using methanol in chloro-
form as gradient solvent system to give trihydroxynu-
cleosides 11a-b in 96 and 93% yields, respectively.
2.5. General procedure for selective biocatalytic
monoacetylation: Synthesis of monoacetylated
nucleosides 12a-b
To
a
solution of trihydroxy nucleoside 11a-b
(1.8 mmol) in MeCN (10 mL) was added vinyl acetate
R
(1.98 mmol) followed by the addition of LipozymeV TL
2.4.1. 30-O-Benzyl-40-C-hydroxymethyl-b-D-xylofura-
nosyl thymine (11a) (Rajwanshi et al. 1999)
IM (50% w/w). The reaction mixture was stirred at
45 ꢀC in an incubator shaker at 200 rpm. On comple-
tion of the reaction after 1 h, the reaction mixture was
quenched by filtering off the Lipozyme TL IM; the solv-
ent was removed under reduced pressure, and
the residue thus obtained was purified by silica gel
column chromatography using methanol in chloroform
as gradient solvent system to afford the monoacety-
lated nucleosides 12a-b in 90 and 85% yields,
respectively.
It was obtained as sticky white solid (0.72 g) in 96%
31
yield. Rf ¼ 0.4 (10% MeOH in CHCl3); [a]D ¼ ꢂ30.16 (c
0.1, MeOH); IR (thin film) ꢀmax cmꢂ1: 770, 914, 1071,
1
1260, 1460, 1700, 2855, 2923, 3376; H NMR (DMSO-d6,
400 MHz): d 1.75 (3H, s, C-5CH3), 3.39 (2H, s, C-100H2),
3.45 (1H, dd, J ¼ 5.2 and 11.2 Hz, C-50Ha), 3.65 (1H, dd,
J ¼ 4.4 and 11.6 Hz, C-50Hb), 4.13 (1H, d, J ¼ 6.4 Hz, C-
30H), 4.37 (1H, q, J ¼ 6.4 Hz, C-20H), 4.62 (1H, d,
J ¼ 11.6 Hz, –OCHaPh), 4.72 (1H, d, J ¼ 12.8 Hz,
–OCHbPh), 4.78 (1H, t, J ¼ 5.2 Hz, OH), 5.05 (1H, t,
J ¼ 5.2 Hz, OH), 5.75 (1H, d, J ¼ 4.8 Hz, OH), 5.81 (1H, d,
J ¼ 7.6 Hz, C-10H), 7.27–7.37 (5H, m, ArH), 7.89 (1H, s, C-
6H), 11.30 (1H, s, NH); 13C NMR (DMSO-d6, 100.6 MHz):
d 12.29 (C-5CH3), 61.24 (C-100), 62.68 (C-50), 71.91
(-OCH2Ph), 82.83 (C-20), 85.20 (C-30), 85.73 (C-40), 99.50
(C-10), 109.53 (C-5), 127.31, 127.39, 128.19 and 136.75
(ArC), 138.56 (C-6), 150.96 (C-2), 163.76 (C-4); HR-ESI-
TOF-MS: m/z 379.1503 ([M þ H]þ), calcd. for
[C18H22N2O7þH]þ 379.1500.
2.5.1. 40-C-Acetoxymethyl-30-O-benzyl-b-D-xylofura-
nosyl thymine (12a)
It was obtained as white solid (0.68 g) in 90% yield.
Rf ¼ 0.5 (8% MeOH in CHCl3); M. Pt.: 86–88 ꢀC;
[a]D ¼ ꢂ44.84 (c 0.1, MeOH); IR (KBr) ꢀmax cmꢂ1: 699,
31
751, 917, 1044, 1105, 1234, 1376, 1470, 1683, 2927,
3029, 3064; 1H NMR (CDCl3, 400 MHz): d 1.84 (3H, d,
J ¼ 1.2 Hz, C-5CH3), 2.07 (3H, s, –OCOCH3), 2.72 (1H, dd,
J ¼ 5.2 and 9.2 Hz, C-50OH), 3.72 (1H, dd, J ¼ 9.2 and
12.4 Hz, C-50Ha), 3.88 (1H, dd, J ¼ 4.4 and 12.4 Hz,
C-50Hb), 4.11 (2H, q, J ¼ 12.0 Hz, C-100H2), 4.20 (1H, d,
J ¼ 5.2 Hz, C-30H), 4.57 (1H, dd, J ¼ 5.6 and 9.2 Hz, C-
20H), 4.66 (1H, d, J ¼ 11.2 Hz) and 4.89 (1H, d,
J ¼ 12.0 Hz, –OCH2Ph), 4.98 (1H, d, J ¼ 3.6 Hz, C-20OH),
5.96 (1H, d, J ¼ 5.6 Hz, C-10H), 7.32–7.36 (5H, m, ArH),
7.72 (1H, d, J ¼ 1.6 Hz, C-6H), 10.22 (1H, s, NH); 13C
NMR (CDCl3, 100.6 MHz): d 12.50 (C-5CH3), 20.75
(–OCOCH3), 63.36 (C-50), 64.51 (C-100), 72.94 (–OCH2Ph),
80.24 (C-20), 83.84 (C-30), 85.79 (C-40), 88.33 (C-10),
111.08 (C-5), 127.97, 128.30 and 128.63 (ArC), 136.19
(C-6), 136.90 (ArC), 151.62 (C-2), 164.30 (C-4), 170.69
(–OCOCH3); HR-ESI-TOF-MS: m/z 421.1612 ([M þ H]þ),
calcd. for [C20H24N2O8þH]þ 421.1605.
2.4.2. 30-O-Benzyl-40-C-hydroxymethyl-b-D-xylofura-
nosyl uracil (11b)
It was obtained as white solid (0.68 g) in 93% yield.
Rf ¼ 0.4 (10% MeOH in CHCl3); M. Pt.: 143–145 ꢀC;
[a]D ¼ -23.49 (c 0.1, MeOH); IR (KBr) ꢀmax cmꢂ1: 738,
32
811, 1024, 1084, 1156, 1373, 1471, 1694, 2872, 3331;
1H NMR (DMSO-d6, 400 MHz): d 3.34 (2H, d, J ¼ 5.6 Hz,
C-100H2), 3.43 (1H, d, J ¼ 6.0 Hz, C-50Ha), 3.65 (1H, dd,
J ¼ 4.8 and 11.6 Hz, C-50Hb), 4.14 (1H, d, J ¼ 7.2 Hz, C-
30H), 4.36 (1H, q, J ¼ 6.8 Hz, C-20H), 4.62 (1H, d,
J ¼ 16.4 Hz, –OCHaPh), 4.73 (1H, d, J ¼ 11.6 Hz,
–OCHbPh), 4.79 (1H, t, J ¼ 5.2 Hz, OH), 5.05 (1H, t,

BIOCATALYSIS AND BIOTRANSFORMATION
5
(1H, d, J ¼ 10.4 Hz, C-50Hb), 4.58–4.65 (2H, m, –OCHaPh
& C-20), 4.87 (1H, d, J ¼ 11.6 Hz, –OCHbPh), 5.35 (1H,
brs, C-20OH), 6.07 (1H, d, J ¼ 5.6 Hz, C-10H), 7.29–7.36
(5H, m, ArH), 7.56 (1H, d, J ¼ 1.2 Hz, C-6H), 10.63 (1H,
brs, NH); 13C NMR (CDCl3, 100.6 MHz): d 12.16 (C-5CH3),
20.68 (–OCOCH3), 37.47 (–OSO2CH3), 63.48 (C-50), 68.09
(C-100), 72.83 (–OCH2Ph), 79.07 (C-20), 82.77 (C-30), 83.91
(C-40), 88.76 (C-10), 111.64 (C-5), 128.16, 128.34, 128.60
and 135.87 (ArC), 136.80 (C-6), 151.66 (C-2), 164.24 (C-
4), 170.23 (–OCOCH3); HR-ESI-TOF-MS: m/z 499.1390
([M þ H]þ), calcd. for [C21H26N2O10S þ H]þ 499.1381.
2.5.2. 40-C-Acetoxymethyl-30-O-benzyl-b-D-xylofura-
nosyl uracil (12b)
It was obtained as white solid (0.62 g) in 85% yield.
Rf ¼ 0.5 (8% MeOH in CHCl3); M. Pt.: 82–83 ꢀC;
30
[a]D ¼ ꢂ32.41 (c 0.1, MeOH); IR (KBr) ꢀmax cmꢂ1: 699,
742, 841, 1040, 1066, 1260, 1385, 1458, 1687, 2947,
3037, 3408; 1H NMR (CDCl3, 400 MHz): d 2.05 (3H, s,
–OCOCH3), 2.75 (1H, dd, J ¼ 4.8 and 8.4 Hz, C-50OH),
3.66–3.71 (1H, m, C-50Hb), 3.88 (1H, dd, J ¼ 4.0 and
12.6 Hz, C-50Ha), 4.09 (2H, dd, J ¼ 12.0 and 22.2 Hz, C-
100H2), 4.19 (1H, d, J ¼ 6.0 Hz, C-30H), 4.58 (1H, dd,
J ¼ 5.6 and 8.4 Hz, C-20H), 4.64 (1H, d, J ¼ 11.6 Hz) and
4.88 (1H, d, J ¼ 12.0 Hz, –OCH2Ph), 4.96 (1H, d,
J ¼ 2.8 Hz, C-20OH), 5.71 (1H, d, J ¼ 8.0 Hz, C-5H), 5.94
(1H, d, J ¼ 5.6 Hz, C-10H), 7.31–7.35 (5H, m, ArH), 7.95
(1H, d, J ¼ 8.0 Hz, C-6H), 10.37 (1H, s, NH); 13C NMR
(CDCl3, 100.6 MHz): d 20.72 (–OCOCH3), 63.38 (C-50),
64.55 (C-100), 73.00 (–OCH2Ph), 80.51 (C-20), 83.84 (C-30),
86.16 (C-40), 88.82 (C-10), 102.59 (C-5), 128.02, 128.33,
128.64 and 136.86 (ArC), 140.61 (C-6), 151.51 (C-2),
163.95 (C-4), 170.63 (–OCOCH3); HR-ESI-TOF-MS: m/z
407.1454 ([M þ H]þ), calcd. for [C19H22N2O8þH]þ
407.1449.
2.6.2. 40-C-Acetoxymethyl-30-O-benzyl-50-O-methane-
sulfonyl-b-D-xylofuranosyl uracil (13b)
It was obtained as white solid (0.65 g) in 90% yield.
Rf ¼ 0.7 (10% MeOH in CHCl3); M. Pt.: 69–70 ꢀC;
[a]D ¼ ꢂ17.98 (c 0.1, MeOH); IR (KBr) ꢀmax cmꢂ1: 752,
32
1
817, 965, 1045, 1174, 1235, 1355, 1683, 2938, 3029; H
NMR (CDCl3, 400 MHz): d 1.76 (1H, brs, C-20OH), 2.06
(3H, s, –OCOCH3), 2.95 (3H, s, –OSO2CH3), 4.15 (1H, d,
J ¼ 4.0 Hz, C-30H), 4.21 (1H, d, J ¼ 12.0 Hz, C-100Ha), 4.30
(1H, d, J ¼ 11.6 Hz, C-100Hb), 4.37 (1H, d, J ¼ 10.4 Hz, C-
50Ha), 4.48 (1H, d, J ¼ 10.4 Hz, C-50Hb), 4.54 (1H, t,
J ¼ 4.4 Hz, C-20H), 4.60 (1H, d, J ¼ 11.6 Hz, –OCHaPh),
4.82 (1H, d, J ¼ 10.8 Hz, –OCHbPh), 5.70 (1H, d,
J ¼ 8.4 Hz, C-5H), 5.96 (1H, d, J ¼ 4.4 Hz, C-10H),
7.30–7.36 (5H, m, ArH), 7.68 (1H, d, J ¼ 8.0 Hz, C-6H),
10.18 (1H, brs, NH); 13C NMR (CDCl3, 100.6 MHz): d 20.67
(–OCOCH3), 37.54 (–OSO2CH3), 63.09 (C-50), 67.57 (C-100),
72.86 (–OCH2Ph), 79.75 (C-20), 83.06 (C-30), 85.00 (C-40),
90.42 (C-10), 102.78 (C-5), 128.23, 128.43, 128.65 and
136.63 (ArC), 139.92 (C-6), 151.37 (C-2), 163.63 (C-4),
170.17 (–OCOCH3); HR-ESI-TOF-MS: m/z 507.1049
([M þ Na]þ), calcd. for [C20H24N2O10S þ Na]þ 507.1044.
2.6. General procedure for the synthesis of 40-C-
Acetoxymethyl-30-O-benzyl-50-O-methanesulfonyl-
b-D-xylofuranosyl nucleosides 13a-b
A solution of 12a-b (1.5 mmol) and methanesulfonyl
chloride (1.65 mmol) in anhydrous dichloromethane:
pyridine (10 mL, 4:1) was stirred at 25 ꢀC for 4 h. On
completion, the reaction mixture was poured over
10% ice-cold hydrochloric acid solution (100 mL) and
extracted with chloroform (3 ꢁ 100 mL). The combined
organic extract was washed with saturated aq. sodium
bicarbonate solution (2 ꢁ 100 mL) and dried over
anhydrous sodium sulphate, and the excess of solvent
was removed under reduced pressure. The residue
thus obtained was purified by silica gel column chro-
matography using methanol in chloroform as gradient
solvent system to afford the nucleosides 13a-b in 92
and 90% yields, respectively.
2.7. General procedure for the synthesis of 30-O-
Benzyl-C-4'-spiro-xylofuranosyl nucleosides 14a-b
To a stirred solution of compound 13a-b (1.0 mmol) in
dioxane:water (4 mL, 1:1), 2M NaOH (1.5 mL) was
added and the reaction mixture was stirred at 25 ꢀC
for 48 h. The reaction mixture was neutralized with
acetic acid and co-evaporated with toluene under
reduced pressure. The residue thus obtained was puri-
fied by silica gel column chromatography using
methanol in chloroform as gradient solvent system to
give 14a-b in 76 and 70% yields, respectively.
2.6.1. 40-C-Acetoxymethyl-30-O-benzyl-50-O-methane-
sulfonyl-b-D-xylofuranosyl thymine (13a)
It was obtained as white solid (0.69 g) in 92% yield.
Rf ¼ 0.7 (10% MeOH in CHCl3); M. Pt.: 79–81 ꢀC;
32
[a]D ¼ þ10.41 (c 0.05, MeOH); IR (KBr) ꢀmax cmꢂ1
:
2.7.1. 30-O-Benzyl-50-O,40-C-methylene-b-D-xylofura-
nosyl thymine (14a)
1
753, 1047, 1176, 1230, 1357, 1709, 2363, 3451; H NMR
(CDCl3, 400 MHz): d 1.81 (3H, s, C-5CH3), 2.07 (3H, s,
–OCOCH3), 2.95 (3H, s, –OSO2CH3), 4.18–4.24 (3H, m, C-
100H2 & C-30H), 4.37 (1H, d, J ¼ 10.8 Hz, C-50Ha), 4.48
It was obtained as white solid (0.28 g) in 76% yield.
Rf ¼ 0.5 (10% MeOH in CHCl3); M. Pt.: 132–134 ꢀC;

6
P. RUNGTA ET AL.
31
[a]D ¼ ꢂ24.19 (c 0.1, MeOH); IR (KBr) ꢀmax cmꢂ1: 763,
2.8.1. 50-O,40-C-Methylene-b-D-xylofuranosyl thymine
(4a)
968, 1086, 1123, 1270, 1482, 1663, 1690, 2953, 3190,
3411; H NMR (CDCl3, 400 MHz): d 1.66 (3H, s, C-5CH3),
1
It was obtained as white solid (0.10 g) in 90% yield.
4.31 (1H, s, C-30H), 4.50 (1H, s, C-20H), 4.53 (2H, s,
–OCH2Ph), 4.77 (2H, dd, J ¼ 7.2 and 13.2 Hz, C-100H2),
4.94 (1H, d, J ¼ 8.0 Hz, C-50Ha), 5.02 (1H, d, J ¼ 7.6 Hz,
C-50Hb), 5.67 (1H, brs, C-20-OH), 5.87 (1H, s, C-10H),
7.11–7.30 (6H, m, ArH & C-6H), 10.71 (1H, brs, NH); 13C
NMR (CDCl3, 100.6 MHz): d 12.32 (C-5CH3), 72.22
(–OCH2Ph), 76.66 (C-50), 77.32 (C-20), 82.29 (C-100), 83.89
(C-30), 88.64 (C-40), 93.35 (C-10), 109.59 (C-5), 127.80,
128.32 and 128.62 (ArC), 136.53 (C-6), 136.61 (ArC),
150.68 (C-2), 164.72 (C-4); HR-ESI-TOF-MS: m/z
361.1403 ([M þ H]þ), calcd. for [C18H20N2O6þH]þ
361.1394.
Rf ¼ 0.2 (10% MeOH in CHCl3); M. Pt.: 253–255 ꢀC;
31
[a]D ¼ ꢂ39.93 (c 0.1, MeOH); IR (KBr) ꢀmax cmꢂ1: 754,
960, 1065, 1261, 1481, 1689, 2928, 3039, 3408, 3444;
1H NMR (DMSO-d6, 400 MHz): d 1.74 (3H, s, C-5CH3),
4.05 (1H, s, C-30H), 4.14 (1H, s, C-20H), 4.52 (2H, dd,
J ¼ 7.2 and 10.8 Hz, C-100H2), 4.64 (1H, d, J ¼ 7.6 Hz, C-
50Ha), 4.85 (1H, d, J ¼ 7.6 Hz, C-50Hb), 5.68 (1H, d,
J ¼ 2.4 Hz, C-10H), 5.80 (1H, brs, OH), 5.93 (1H, brs, OH),
7.42 (1H, s, C-6H), 11.33 (1H, s, NH); 13C NMR (DMSO-
d6, 100.6 MHz): d 12.29 (C-5CH3), 76.43 (C-50), 76.72 (C-
20), 78.68 (C-30), 80.73 (C-100), 86.25 (C-40), 90.26 (C-10),
108.58 (C-5), 136.98 (C-6), 150.48 (C-2), 163.80 (C-4);
HR-ESI-TOF-MS: m/z 271.0928 ([M þ H]þ), calcd. for
[C11H14N2O6þH]þ 271.0925.
2.7.2. 30-O-Benzyl-50-O,40-C-methylene-b-D-xylofura-
nosyl uracil (14b)
2.8.2. 50-O,40-C-Methylene-b-D-xylofuranosyl uracil (4b)
It was obtained as white solid (0.24 g) in 70% yield.
It was obtained as white solid (0.09 g) in 88% yield.
Rf ¼ 0.5 (10% MeOH in CHCl3); M. Pt.: 71–73 ꢀC;
Rf ¼ 0.2 (10% MeOH in CHCl3); M. Pt.: 203–205 ꢀC;
[a]D ¼ þ3.21 (c 0.1, MeOH); IR (KBr) ꢀmax cmꢂ1: 706,
30
[a]D ¼ ꢂ44.12 (c 0.1, MeOH); IR (KBr) ꢀmax cmꢂ1
:
31
1
762, 969, 1082, 1269, 1458, 1678, 2928, 3207, 3396; H
962, 1075, 1128, 1273, 1470, 1682, 1705, 3018,
3299; 1H NMR (DMSO-d6, 400 MHz): d 4.04-4.05 (1H,
m, C-30H), 4.15 ꢂ 4.17 (1H, m, C-20H), 4.53 (2H, dd,
J ¼ 7.2 and 9.6 Hz, C-100H2), 4.66 (1H, d, J ¼ 8.0 Hz, C-
50Ha), 4.84 (1H, d, J ¼ 7.2 Hz, C-50Hb), 5.57 (1H, dd,
J ¼ 2.0 and 7.6 Hz, C-5H), 5.67 (1H, d, J ¼ 1.6 Hz, C-
10H), 5.83 (1H, d, J ¼ 3.6 Hz, C-20-OH), 5.90 (1H, d,
J ¼ 4.0 Hz, C-30-OH), 7.51 (1H, d, J ¼ 7.6 Hz, C-6H),
11.35 (1H, s, NH); 13C NMR (DMSO-d6, 100.6 MHz): d
76.23 (C-50), 76.67 (C-20), 78.97 (C-30), 80.82 (C-100),
87.03 (C-40), 90.99 (C-10), 100.76 (C-5), 141.26 (C-6),
150.40 (C-2), 163.24 (C-4); HR-ESI-TOF-MS: m/z
257.0768 ([M þ H]þ), calcd. for [C10H12N2O6þH]þ
257.0768.
NMR (CDCl3, 400 MHz): d 4.29 (1H, s, C-30H), 4.48 (1H, s,
C-20H), 4.53 (2H, s, –OCH2Ph), 4.75 (2H, dd, J ¼ 7.6 and
20.4 Hz, C-100H2), 4.94 (2H, dd, J ¼ 7.2 and 24.2 Hz, C-
50H2), 5.43 (1H, brs, OH), 5.56 (1H, dd, J ¼ 1.6 and
8.0 Hz, C-5H), 5.84 (1H, s, C-10H), 7.15–7.17 (2H, m,
ArH), 7.30–7.32 (3H, m, ArH), 7.36 (1H, d, J ¼ 8.4 Hz, C-
6H), 10.64 (1H, brs, NH); 13C NMR (CDCl3, 100.6 MHz): d
72.32 (–OCH2Ph), 76.57 (C-50), 77.25 (C-20), 82.13 (C-100),
83.67 (C-30), 88.81 (C-40), 93.50 (C-10), 101.11 (C-5),
127.98, 128.43, 128.65 and 136.44 (ArC), 140.62 (C-6),
150.71 (C-2), 164.20 (C-4); HR-ESI-TOF-MS: m/z
347.1241 ([M þ H]þ), calcd. for [C17H18N2O6þH]þ
347.1238.
2.9. X-ray diffraction studies on 50-O,40-C-
Methylene-b-D-xylofuranosyl thymine (4a)
2.8. General procedure for the synthesis of C-40-
spiro-oxetano-xylofuranosyl nucleosides 4a-b
Single crystals suitable for X-ray diffraction studies
were grown by dissolving 50-O,40-C-methylene-b-D-xylo-
furanosyl thymine (4a) in 5% methanol in chloroform
and allowing slow evaporation of the solution at room
temperature. The X-ray diffraction data was collected
on X’calibur CCD diffractometer with graphite mono-
chromatized Cu/Ka radiation (k ¼ 0.71073 Å) at tem-
perature 298 K. The structure was solved by direct
methods using SHELXS-97, and refined by the full-
matrix least-squares method on F2 (SHELXL-97)
(Sheldrick 2008). All calculations were carried out using
the WinGX package of the crystallographic programs
To a solution of nucleoside 14a-b (0.4 mmol) in anhyd-
rous THF:MeOH (4 mL, 9:1, v/v) was added Pd(OH)2-C
(20 wt%, 0.03 g) and 88% formic acid (0.12 mL,
3.18 mmol). The reaction mixture was refluxed for 1 h
whereupon it was cooled to 25 ꢀC. The catalyst was
carefully filtered off, washed with excess MeOH and
the combined filtrate was concentrated. The crude
product thus obtained was purified by silica gel col-
umn chromatography using methanol in chloroform
as gradient solvent system to afford spiro-nucleosides
4a-b in 90 and 88% yields, respectively.

BIOCATALYSIS AND BIOTRANSFORMATION
7
Figure 2. (a) ORTEP diagram of compound 4a drawn in 20% thermal probability ellipsoids showing atomic numbering scheme.
(b) Preferential S-type sugar ring puckering in compound 4a.
(Farrugia 1999). For the molecular graphics, the pro-
gram DIAMOND-2(Pennington 1999) and Mercury
(Macrae et al. 2006) were used. Molecular structure
was drawn, as given in Figure 2, using ORTEP software.
The selected bond lengths, bond angles, etc. are given
in Table 1.
(Prasad et al. 2007) could provide a key precursor,
4-C-acyloxymethyl-3-O-benzyl-1,2-O-isopropylidene-a-D-
xylofuranose that would lead to the desired spironu-
cleosides on subsequent chemical transformations.
The diacylated xylofuranose 7a-c were synthesized in
quantitative yields by DMAP-catalyzed acylation of
dihydroxy compound 5 with acetic- (6a), propionic-
(6b) and butyric anhydride (6c); the dihydroxy com-
pound 5 in turn was synthesized from D-glucose in
63% yield by following the literature procedure
(Scheme 1) (Youssefyeh et al. 1979). On screening of
different lipases for selective acylation/deacylation of
dihydroxy- and diacyloxy- xylofuranoses 5 and 7a-c,
respectively, in organic solvents of varying polarity
revealed the incapability of various lipases towards
selective acylation/deacylation of these molecules
(Scheme 1).
3. Results and discussion
For the synthesis of targeted C-40-spiro-oxetano-xylo-
nucleosides 4, it was envisaged that either the
selective biocatalytic acylation of C-4-hydroxymethyl
group in 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopro-
pylidene-a-D-xylofuranose (Youssefyeh et al. 1979) (5)
or the selective biocatalytic deacylation of 5-O-acyl
group in corresponding diacyl derivatives 7a-c

8
P. RUNGTA ET AL.
After the discouraging outcome with dihydroxyxylo-
nucleosides 11a-b or deacetylation of 50-O-acetyl
group in 40-C-acetoxymethyl-20,50-di-O-acetyl-30-O-ben-
zyl-b-D-xylofuranosyl nucleosides 10a-b. The desired
diacetylated xylonucleosides 10a-b were synthesized
furanose
5
and diacylated xylofuranoses 7a-c,
we focused our attention to probe selectivity for
biocatalytic acetylation of 40-C-hydroxymethyl group
in
30-O-benzyl-40-C-hydroxymethyl-b-D-xylofuranosyl
from
4-C-acetoxymethyl-5-O-acetyl-3-O-benzyl-1,2-O-
isopropylidene-a-D-xylofuranose (7a) (Scheme 2).
Thus, acetolysis of compound 7a led to the forma-
tion of tetra-O-acetylated glycosyl donor 9a-9b in
Table 1. Crystal data and structure refinement for
compound 4a.
€
97% yield, which on Vorbruggen nucleobase cou-
Empirical formula
Formula weight
Temperature
C11H14N2O6
270.24
298(2) K
0.71073 Å
pling with thymine and uracil in the presence of
N,O-bis(trimethylsilyl)acetamide and trimethylsilyltri-
Wavelength
fluoromethane
sulfonate
(TMS-triflate)
afforded
Crystal system
Space group
Unit cell dimensions
Monoclinic
P 21
40-C-acetoxymethyl-20,50-di-O-acetyl-30-O-benzyl-b-D-xy-
lofuranosyl thymine (10a) and 40-C-acetoxymethyl-
a ¼ 8.8875(11) Å, a ¼ 90ꢀ
b ¼ 6.9418(7) Å, b ¼ 111.685(15)ꢀ
c ¼ 10.3533(13) Å, c ¼ 90ꢀ
593.54(12) Å3
20,50-di-O-acetyl-30-O-benzyl-b-D-xylofuranosyl
uracil
Volume
(10b) in 90 and 88% yields, respectively. The com-
plete deacetylation of nucleosides 10a-b was
effected with MeOH:NH3 to give the corresponding
30-O-benzyl-40-C-hydroxymethyl-b-D-xylofuranosyl thy-
mine (11a) and 30-O-benzyl-40-C-hydroxymethyl-b-D-
xylofuranosyl uracil (11b) in 96 and 93% yields,
respectively (Scheme 2).
Z
2
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to h ¼ 24.99ꢀ
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2r(I)]
R indices (all data)
Absolute structure parameter
Largest diff. peak and hole
CCDC
1.512 mg/m3
0.124 mmꢂ1
284.0
0.20 ꢁ 0.16 ꢁ 0.12 mm3
3.62–25.00ꢀ.
ꢂ10 ꢄ h ꢄ 7, ꢂ7 ꢄ k ꢄ 8, ꢂ12 ꢄ l ꢄ 9
3036
1780 [R(int) ¼ 0.0200]
99.7%
An attempt of selective deacetylation study on tri-
O-acetylated nucleosides 10a-b also led to similar intri-
cate result as with diacylated xylofuranose derivatives
7a-c. However, to establish the optimum reaction con-
ditions, four different lipases, viz. T. lanuginosus lipase
Semi-empirical from equivalents
0.9852 and 0.9755
Full-matrix least-squares on F2
1780/1/185
1.098
R1 ¼ 0.0317, wR2 ¼ 0.0789
R1 ¼ 0.0328, wR2 ¼ 0.0799
0.8(11)
R
immobilized on silica (LipozymeV TL IM), C. antarctica
R
0.159 and ꢂ0.190 e.Åꢂ3
15,26,248
lipase-B immobilized on polyacrylate (NovozymeV-435),
C. rugosa lipase (CRL) and porcine pancreatic lipase
RCOO
HO
Ref. 18
4 steps
OBn
O
OBn
O
(RCO)2O (6a-c)
D-Glucose
O
CH2Cl2, DMAP, rt
quantitative
yield
O
overall yield
63%
HO
RCOO
O
O
5
7a-c
Lipase
Organic Solvent
compds.
R
6-8
RCOOCH=CH2
n-BuOH
CH3
C2H5
C3H7
(8a-c)
a
b
c
inseparable
mixture of
regioisomers
Scheme 1. Synthesis and biocatalytic acylation/deacylation studies on xylofuranose 5 and its diacylated derivatives 7a-c.
B
B
AcO
AcO
AcO
AcO
HO
HO
AcO
AcO
OBn
O
OBn
O
OBn
O
OBn
O
Ac2O, AcOH
H2SO4, 0
97%
OAc
MeOH : NH3
96,93 %
B, BSA, TMSOTf
MeCN, 80
90,88%
°
C
O
°C
OAc
9a-9b
OAc
OH
O
10a-b
11a-b
7a
9a; α-C-1-OAc
9b; β-C-1-OAc
a; B = thymin-1-yl
b
; B = uracil-1-yl
Scheme 2. Synthesis of 30-O-benzyl-40-C-hydroxymethyl-b-D-xylofuranosyl T and U.

BIOCATALYSIS AND BIOTRANSFORMATION
9
(PPL) were screened for selective acetylation of C-40-
hydroxymethyl group over two other hydroxyl group
present in nucleoside 11a against four different sol-
vents, i.e. tetrahydrofuran (THF), acetonitrile (MeCN),
diisopropylether (DIPE) and dioxane at 45 ꢀC and at
200 rpm using vinyl acetate as the acetylating agent.
Notwithstanding, out of four screened lipases,
lone primary hydroxyl group in nucleosides 12a-b
afforded monomesylated compounds 13a-b which on
deacetylation with aq. NaOH in dioxane and
concomitant spirocylization at C-40-position between
40-C-hydroxymethyl and 50-O-mesyl groups led to the
formation of benzylated spironucleosides 14a-b in 70
and 63% yields in two steps, respectively. The deben-
zylation of nucleosides 14a-b with 20% Pd(OH)2-C in
the presence of formic acid afforded the desired
compounds, 50-O,40-C-methylene-b-D-xylofuranosyl thy-
mine (4a) and 50-O,40-C-methylene-b-D-xylofuranosyl
uracil (4b) in 90 and 88% yields, respectively
(Scheme 3, see Supplementary Material). The struc-
ture of one of the synthesized spironucleosides 4a
was confirmed by X-ray diffraction studies on its sin-
gle crystal, which clearly indicated restriction on fura-
nose ring flipping due to the introduction of spiro-
oxetane ring. The presence of spiro-oxetane ring in
the synthesized nucleosides compels the furanose
ring to attain S-type puckering (Figure 2). It is worthy
to mention here that earlier attempts to synthesize
these nucleosides via chemical methodology by
Mandal et al. (Roy et al. 2006) were unsuccessful.
The structures of all the synthesized compounds,
i.e. 4a-b, 5, 7a-c, 9a-9b, 10a-b, 11a-b, 12a-b, 13a-b
and 14a-b were established on the basis of their
R
LipozymeV TL IM in acetonitrile was found to be
selective for the acetylation of 40-C-hydroxymethyl
group in 30-O-benzyl-40-C-hydroxymethyl-b-D-xylofura-
nosyl nucleosides 11a-b to afford the desired
mono-acetylated products, 40-C-acetoxymethyl-30-O-
benzyl-b-D-xylofuranosyl nucleosides 12a-b in 90 and
85% yields, respectively (Scheme 3). Nevertheless,
accomplishment of clean and efficient reactions with
high yield of the products was associated with the
presence of the nucleobase in the molecules. Previous
endeavour for the selective acetylation of furanoside 5
or deacetylation of furanosides 7a-c (Scheme 1) to
afford regioselective product were not successful. This
observation illustrates suitability of substrate molecules
for the enzyme. Structural demarcation between the
substrates dihydroxy xylofuranose 5 and nucleosides
11a-b arises from the downward anomeric and C-2
hydroxy groups, connected by isopropylidene protec-
tion to form a five membered ring (a-configuration at
anomeric position) and the downward C-2 hydroxy
group and upward nucleobase (b-configuration at
anomeric position). This result depicts the requirement
of fittingness of the substrate with the enzyme. When
the substrates are better fitted with enzyme, the
acetylation was carried out regioselectively, whereas
misfit of the substrate led to inseparable mixture of
regioisomers.
spectral (IR, H-, 13C NMR, H-1H COSY, H-13C HMQC
and HRMS) data analysis. The structures of the
known compounds 5, 7a, 11a were further con-
firmed by the comparison of their physical and spec-
tral data with those reported in the literature
(Youssefyeh et al. 1979; Rajwanshi et al. 1999; Prasad
et al. 2007). The structure of 50-O,40-C-methylene-b-D-
xylofuranosyl thymine (4a) was confirmed by its X-
ray data analysis that has been deposited in the
Cambridge Crystallographic Data Centre with CCDC
number 1526248.
1
1
1
The synthesis of targeted C-40-spiro-oxetano-xylo-
furanosyl nucleosides 4a-b was successfully achieved
from nucleosides 12a-b. Thus, mesylation of the
B
B
MsO
AcO
HO
B
HO
OBn
O
OBn
OBn
O
Lipozyme® TL IM
MsCl, CH2Cl2
O
Vinyl acetate, MeCN
HO
Py, 25°C
92, 90%
OH
OH
45°C, 200 rpm
CH3COO
OH
11a-b
13a-b
12a-b
90, 85%
B
B
OH
O
OBn
O
20 % Pd(OH)2-C
aq. NaOH, dioxane
O
O
25°C
76, 70%
HCO2H, THF/MeOH
OH
4a-b
a; B = thymin-1-yl
OH
14a-b
reflux
90, 88%
b
; B = uracil-1-yl
Scheme 3. Biocatalytic route to 50-O,40-C-methylene-b-D-xylofuranosyl T and U 4a-b.

10
P. RUNGTA ET AL.
Das K, Bauman JD, Rim AS, Dharia C, Clark AD Jr., Camarasa
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Butyldimethylsilyl-spiroaminooxathioledioxide-thymine
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Inhibitor-Binding Pocket. J Med Chem 54:2727–2737.
4. Conclusions
In conclusion, novel C-40-spiro-oxetanoxylofuranosyl
thymine and uracil have been synthesized for the
first time following
a biocatalytic pathway. This
R
methodology utilizes LipozymeV-TL IM for the diaster-
eoselective acetylation of one of the two diastereo-
topic hydroxymethyl functions of 30-O-benzyl-40-C-
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hydroxymethyl-b-D-xylofuranosyl
nucleosides.
The
developed ecological method allows an easy access to
these C-40-spiro-xylonucleosides. The structure of one
of the spironucleosides, i.e. 50-O,40-C-methylene-b-D-
xylofuranosyl thymine was further confirmed by the
single crystal X-ray diffraction analysis, which indicates
that the presence of an extra spiro-ring in the xylonu-
cleosides restricts the conformation of the sugar
moiety by locking it in S-type conformation. These
conformationally restricted spironucleosides can serve
as potential drug candidates for viral disease, studies
of which are under progress.
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tane ribonucleosides as novel inhibitors of the HCV NS5B
polymerase. J Med Chem 57:1836–1844.
Acknowledgements
We are thankful to CIF-USIC University of Delhi, Delhi for
providing NMR spectral recording facility. PR, VKS and VK
thank CSIR, and PM thanks UGC for the award of Junior/
Senior Research Fellowships.
Jonckers TH, Lin TI, Buyck C, Lachau-Durand S, Vandyck K,
Van-Hoof S, Vandekerckhove L, Hu L, Berke JM, Vijgen L,
et al. 2010. 20-Deoxy-20-spirocyclopropylcytidine revisited:
a new and selective inhibitor of the hepatitis C virus NS5B
polymerase. J Med Chem 53:8150–8160.
Disclosure statement
No potential conflict of interest was reported by the authors.
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Kumar R, Kumar M, Singh A, Singh N, Maity J, Prasad AK.
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Funding
We are grateful to the University of Delhi for providing finan-
cial support under DU-DST Purse Grant and under scheme
to strengthen research and development.
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