Biocatalytic deacylation studies on tetra-O-acyl-β-d-xylo furanosyl Nucleosides: Synthesis of xylo-LNA monomers

Article abstract of DOI:10.1021/jo201060t

A Novozyme-435 catalytic methodology has been developed for selective deacylation of one of the acyloxy functions involving a primary -OH group over the other acyloxy functions involving primary and secondary -OH groups in 4′-C-acyloxymethyl-2′,3′,5′-tri-

Full text of DOI:10.1021/jo201060t

NOTE
pubs.acs.org/joc
Biocatalytic Deacylation Studies on Tetra-O-acyl-β-D-xylofuranosyl
Nucleosides: Synthesis of xylo-LNA Monomers
Sunil K. Singh,† Vivek K. Sharma,† Kapil Bohra,† Carl E. Olsen,‡ and Ashok K. Prasad*,†
†Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
‡Faculty of Life Sciences, Department of Natural Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark
S Supporting Information
b
ABSTRACT: A Novozyme-435 catalytic methodology has
been developed for selective deacylation of one of the acyloxy
functions involving a primary ꢀOH group over the other
acyloxy functions involving primary and secondary ꢀOH
groups in 40-C-acyloxymethyl-20,30,50-tri-O-acyl-β-D-xylofura-
nosyl nucleosides. Optimization of the biocatalytic reaction
revealed that tetra-O-butanoyl-β-D-xylofuranosyl nucleosides are the best substrates for the enzyme. The possibility of acyl migration
during enzymatic deacylation reactions has been ruled out by carrying out biocatalytic deacylation reactions on mixed esters of 40-C-
hydroxymethyl-20,30,50-tri-O-acetyl-β-D-xylofuranosyl nucleosides. The developed methodology has been used for the efficient
synthesis of xylo-LNA monomers T, U, A, and C in good yields.
ecently, research has been focused on restricting the con-
R
formation of the furanose ring either in C-20 endo or in C-30
endo conformations, primarily to develop antisense oligonucleo-
tides with striking target affinity and biological stability. This has
resulted in the synthesis of a large number of chemically modified
nucleotides/oligonucleotides, among which the locked nucleic
acid (LNA, I) has attracted extensive attention.1 In this series
fully modified oligonucleotides R-L-ribo-LNA (II) and β-D-xylo-
LNA (III) hybridize to both DNA and RNA with high affinity
(Figure 1). The experiments toward slightly mismatched RNA
targets showed that hybridization of β-D-xylo-configured LNA
toward RNA is selective.2
Figure 1. Structures of LNA, R-L-ribo-LNA, and β-D-xylo-LNA.
Scheme 1. Synthesis and Biocatalytic Deacetylation Studies
on Tetra-O-acetylated Nucleosides 4aꢀda
One of the major problems in the synthesis of LNA and its
diastereomeric analogues is the discrimination between two
primary hydroxyl groups, as in 40-C-acetoxymethyl-20,30,50-tri-O-
acetyl-β-D-xylofuranosyl nucleosides (4), which is the key pre-
cursor for the synthesis of xylo-LNA.3 Herein we report an
environmentally friendly biocatalytic4ꢀ6 one-pot synthesis of 40-
C-hydroxymethyl-20,30,50-tri-O-acyl-β-D-xylofuranosyl nucleosides
from their corresponding peracylated derivatives and conversion
of the synthesized monohydroxy nucleosides into xylo-LNA in an
overall yields of 65ꢀ79%.
a Reaction conditions: (i) Ac2O, DMAP (cat.), CH2Cl2, 25 °C; (ii)
Ac2O, AcOH, H2SO4 (100/10/0.1), 0 °C; (iii) for T, U, and C N,
O-bis(trimethylsilyl)acetamide, trimethylsilyltrifluoromethane sulfo-
nate in acetonitrile, 70ꢀ80 °C, and for A SnCl4 in acetonitrile, 25 °C;
(iv) Novozyme-435, THF, n-butanol, 50 °C.
The trihydroxy furanoside 1 was synthesized from D-glucose
following the procedure described by Moffatt et al.7 Peracetyla-
tion of compound 1 followed by acetolysis of the resulted
triacetate 24c afforded pentaacetoxy furanoside 3 in an overall
yields of 89%. The Vorbr€uggen coupling8 of 3 with thymine,
uracil, adenine, and cytosine afforded the corresponding perace-
tylated nucleosides 4aꢀd in 65ꢀ75% yields (Scheme 1).
In a model biocatalytic reaction, five different lipases,9 viz.
Novozyme-435 or CAL-B, Lipozyme TL IM, Amano PS, CRL,
and PPL, were screened for the selective deacetylation of nucleo-
side 4a in five different organic solvents, i.e. THF, toluene, DIPE,
acetonitrile, and acetone, using n-butanol as the acetyl acceptor at
40, 50, and 60 °C and at 200 rpm in an incubator shaker. Among
the different lipases, Novozyme-435 and Lipozyme TL IM (50% w/w
of the substrate) in THF and toluene at 50 °C showed selectivity
Received: June 3, 2011
Published: August 04, 2011
r
2011 American Chemical Society
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|

The Journal of Organic Chemistry
NOTE
Table 1. Novozyme-435 Catalyzed Deacylation of 4aꢀd,
7aꢀf, and 9aꢀd in THF and n-Butanola
Scheme 2. Novozyme-435 Catalyzed Deacylation Studies on
Peracylated Nucleosides 7aꢀf: Effect of Acyl Chain Length a
entry
substrate
reacn time (h)
product
yield (%)b
1
4a
4b
4c
4d
7a
7b
7c
7d
7e
7f
20
11
14
16
12
8
5a
5b
5c
5d
8a
8b
8c
8d
nrc
nr
87
92
82
93
85
95
81
73
2
3
4
5
6
7
20
22
48
48
10
8
a Reaction conditions: (i) saturated methanolic ammonia, 25 °C; (ii)
(RCO)2O, CH2Cl2, pyridine, 25 °C; (iii) Novozyme-435, THF, n-butanol,
50 °C.
8
9
10
11
12
13
14
9a
9b
9c
9d
5a
5a
5a
nr
85
90
83
Scheme 3. Novozyme-435 Catalyzed Deacylation Reaction
on Mixed Esters 9aꢀd
15
48
a All these reactions, when performed under identical conditions but
without adding Novozyme-435, did not yield any product. b Isola-
ted yield. c nr = no reaction.
and efficiently deacetylated the C100-acetoxy group over other
acetoxyl groups in compound 4a to afford the monodeacetylated
nucleoside 5a in 87% yield (Scheme 1). The rate of the Novozyme-
435 catalyzed deacetylation reaction in THF was much faster than
that of Lipozyme TL IM in THF, whereas in toluene both reactions
were relatively slower as compared tothereactionsinTHF. Therate
of Novozyme-435 catalyzed reactions in other solvents was too slow
to be of any practical application. Other lipases, i.e. Amano PS, CRL,
and PPL, did not show any selectivity during deacetylation reactions
studied on 4a in any of the organic solvents. The progress of the
Novozyme-435 and Lipozyme TL IM catalyzed deacetylation
reactions in THF on nucleoside 4a was monitored by HPLC. It
was observed that the rate of acceleration of the conversion of the
reactant 4a to the product 5a gradually decreases with time.
was found to be the best substrate for selective deacylation
reactions catalyzed by Novozyme-435. The enhancement in the
rate of biocatalytic debutanoylation reaction of 7b was found to
be in accordance with the “Kazlauskas rule”.13 Nucleosides 7e,f
containing benzoyl and p-methoxybenzoyl instead of acyl groups,
were not found to be substrates for Novozyme-435.
The problem of acyl migration has been encountered in 1,2-/
1,3-diol systems as well as in partially acylated nucleosides.14,15
Thus, there exists a possibility of formation of 5aꢀd and 8aꢀd
via initial C50 or C30 deacetylation catalyzed by Novozyme-435
and subsequent acyl migration from C100- to C50/C30-positions.
To ascertain the route of formation of compounds 5aꢀd and
8aꢀd during enzyme-catalyzed deacylation reactions on 4aꢀd
and 7aꢀd, the mixed esters 9aꢀd of monodeacetylated nucleo-
sides 5a were synthesized in 89ꢀ94% yields using propanoic,
butanoic, pentanoic, and benzoic anhydrides in CH2Cl2 in the
presence of a catalytic amount of DMAP (Scheme 3).16 The
mixed tetraesters 9aꢀd were then subjected to Novozyme-435
catalyzed deacylation reactions in THF, using n-butanol as an acyl
acceptor and the previously described reaction conditions. It was
observed that the biocatalytic deacylation reaction of the three
mixed esters 9aꢀc resulted in the formation of identical pro-
ducts, i.e. nucleoside 5a in 85, 90, and 83% yields, respectively
(Scheme 3, Table 1). The mixed ester 9d, containing the benzoyl
group, was not found to be a substrate for Novozyme-435. The
results obtained from the deacylation studies on mixed esters
clearly indicated that the formation of monodeacylated product
during biocatalytic deacylation reactions is solely due to the
removal of the acyl group from the C40-acyloxymethyl function
of the starting peracylated compounds.
Other nucleosides 4bꢀd were also subjected to the Novozyme-
435 catalyzed deacetylation reaction in THF using a small amount
of n-butanoltoobtain theexpectedmonodeacetylatedcompounds
5bꢀd in 92, 82, and 93% yields, respectively (Scheme 1, Table 1).
The structures of all tetraacetylated nucleosides 4aꢀd and mono-
deacetylated nucleosides 5aꢀd were unambiguously established
1
on the basis of their IR and H, 13C, COSY, and HSQC NMR
spectra and HRMS data analysis.
To understand the effect of different acyl groups on the catalytic
efficiency of Novozyme-435, six different acylated nucleosides
7aꢀf were synthesized by acylation of 40-C-hydroxymethyl-β-
D-xylofuranosylthymine (6) with propanoic, butanoic, pentanoic,
hexanoic, benzoic, and p-methoxybenzoic anhydrides.10 The
nucleoside 6 in turn was obtained by deacetylation of compound
4a using ammoniacal methanol.11 The acylated nucleosides 7aꢀf
were incubated with Novozyme-435 in THF in the presence of n-
butanol to obtain the monodeacylated compounds 8aꢀd12
(Scheme 2, Table 1).
The results compiled in Table 1 reveal that the rate of the
deacylation reaction decreases on an increase of acyl chain length
from four to six carbons as in entries 6ꢀ8, whereas the rate of the
reaction increases with the increase of acyl chain length from two
to four carbons as in entries 1, 5, and 6. Thus, 40-C-butanoyloxy-
methyl-20,30,50-tri-O-butanoyl-β-D-xylofuranosylnucleoside (7b)
The synthesis of xylo-LNA monomers T, U, and A 11aꢀc
from monodeacetylated nucleosides 5aꢀc was successfully
achieved by tosylation of the C-100 hydroxyl group to produce
10aꢀc followed by deacetylation and concomitant cyclization of
the resultant hydroxyl nucleosides with a large excess of K2CO3
in methanol/water (3/1), in 91ꢀ96% yields (Scheme 4).
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NOTE
Scheme 4. Synthesis of xylo-LNA Monomers 11aꢀc from
Novozyme-435 Catalyzed Monodeacetylated Nucleosides
5aꢀc
6.40 mmol) and thymine (1.21 g, 9.61 mmol)/uracil (1.44 g, 12.82 mmol)
or cytosine (1.07 g, 9.62 mmol) in anhydrous acetonitrile (60 mL), was
added N,O-bis(trimethylsilyl)acetamide (6.4ꢀ11.9 mL) dropwise. The
reaction mixture was stirred at reflux for 1 h and then cooled to 0 °C. To
the cooled reaction mixture was added trimethylsilyltrifluoromethanesulfo-
nate (2.0 mL, 10.90 mmol) dropwise with stirring, and the solution was
heated at 70ꢀ80 °C for 4ꢀ6 h. The reaction was quenched with a cold
saturated aqueous solution of sodium hydrogen carbonate (100 mL), and
extraction was performed with dichloromethane (3 ꢁ 100 mL). The
combined organic phase was washed with saturated solutions of NaHCO3
(2 ꢁ 100 mL) and brine (2 ꢁ 100 mL) and was dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure, and the residue
thus obtained was purified by silica gel column chromatography using
MeOH/CHCl3 as eluent to afford nucleosides 4a,b,d in 65ꢀ72% yields.
1-(40-C-Acetoxymethyl-20,30,50-tri-O-acetyl-β-D-xylofuranosyl)thymine
(4a). Obtained as an off-white solid (2.04 g, 70%). Rf = 0.6 (5% MeOH/
CHCl3). Mp: 130ꢀ132 °C. [R]D23 = ꢀ51.6° (c 0.1, MeOH). 1H NMR
(CDCl3, 400 MHz): δ 1.95 (3H, s), 2.08, 2.09, 2.12, 2.14 (12H, 4s), 4.00
(1H, d, J= 11.8 Hz), 4.25 (2H, d, J=6.6Hz), 4.52(1H, d, J=12.4Hz), 5.39
(1H, t, J = 6.6 Hz), 5.53 (1H, d, J = 5.9 Hz), 6.18 (1H, d, J = 6.6 Hz), 7.37
(1H, s), 8.71 (1H, brs). 13CNMR(CDCl3, 100.6MHz):δ14.4, 22.4, 22.5,
22.8, 64.0, 65.8, 76.7, 79.8, 84.6, 86.6, 114.1, 136.4, 151.9, 164.8, 171.1,
171.4, 171.6, 172.1. IR (thin film) νmax: 3207, 2918, 2849, 1750, 1698, 1459,
1374, 1227, 1107, 1053, 916 cmꢀ1. HR-ESI-TOF-MS: m/z 457.1490
([M + H]+), calcd for [C19H24N2O11 + H]+ 457.1458.
Scheme 5. Synthesis of xylo-LNA-C from Compound 5d and
of xylo-LNA-T from Compound 8ba
a Reaction conditions: (i) Bz2O, DMF, 25 °C; (ii) p-TsCl, pyridine,
25 °C; (iii) K2CO3, MeOH/H2O (3/1), 25 °C.
1-(40-C-Acetoxymethyl-20,30,50-tri-O-acetyl-β-D-xylofuranosyl)uracil
(4b). Obtained as a white sticky solid (1.84 g, 65%). Rf = 0.6 (5% MeOH/
CHCl3). [R]D22 = ꢀ4.3° (c 0.1, MeOH). 1H NMR (CDCl3, 300 MHz):
δ 2.10, 2.17, 2.18, 2.21 (12H, 4s), 4.11 (1H, d, J = 12.0 Hz), 4.31ꢀ4.36
(2H, m), 4.59(1H, d, J =12.0Hz), 5.48 (1H, t, J = 6.0Hz), 5.62 (1H, d, J=
5.8 Hz), 5.91 (1H, d, J = 8.1 Hz), 6.27 (1H, d, J = 6.2 Hz), 7.65 (1H, d, J =
8.1 Hz), 9.81 (1H, br s). 13C NMR (CDCl3, 75.5 MHz): δ 19.2, 19.3,
19.5, 19.6, 61.0, 62.6, 73.9, 76.7, 82.1, 83.9, 102.5, 137.4, 149.2, 161.7,
168.2, 168.5, 168.7, 168.9. IR (thin film): νmax 3206, 2918, 2849, 1750,
1698, 1459, 1374, 1227, 1107, 1053, 916 cmꢀ1. HR-ESI-TOF-MS: m/z
465.1116 ([M + Na]+), calcd for [C18H22N2O11 + Na]+ 465.1116.
1-(40-C-Acetoxymethyl-20,30,50-tri-O-acetyl-β-D-xylofuranosyl)cytosine
(4d). Obtained as a white solid (2.10 g, 72%). Rf = 0.4 (10% MeOH/
CHCl3). Mp: 142ꢀ144 °C. [R]D22 = +13.2° (c 0.1, MeOH). 1H NMR
(CDCl3, 300 MHz): δ2.08, 2.10, 2.13, 2.14 (12H, 4s), 4.08 (1H, d, J =11.9
Hz), 4.30(2H, dd, J=11.7and9.4Hz), 4.50(1H,d, J=11.9Hz), 5.43(1H,
t, J = 5.4 Hz), 5.50 (1H, d, J = 5.2 Hz), 5.70 (1H, br s), 5.89 (1H, d, J = 7.4
Hz), 6.26 (1H, d, J = 5.6 Hz), 7.59 (1H, d, J = 7.5 Hz), 7.80 (1H, br s). 13C
NMR (CDCl3, 75.5 MHz): δ 20.4, 20.5, 20.6, 20.7, 62.1, 63.5, 75.3, 78.5,
83.4, 86.4, 95.7, 139.9, 155.5, 165.6, 169.2, 169.6, 169.8, 170.1. IR (thin
film): νmax 3357, 3188, 1751, 1647, 1601, 1476, 1425, 1370, 1227,
1049 cmꢀ1. HR-ESI-TOF-MS: m/z 442.1490 ([M + H]+), calcd for
[C18H23N3O10+H]+ 442.1462.
Synthesis of 9-(40-C-Acetoxymethyl-20,30,50-tri-O-acetyl-β-
D-xylofuranosyl)adenine (4c). To stirred solutions of penta-O-
acetylated sugar derivatives 3a,b (2.5 g, 6.40 mmol) and adenine (0.87 g,
6.40 mmol) in anhydrous acetonitrile (60 mL) was added SnCl4
(3.2 mL, 27.2 mmol) dropwise. The reaction mixture was stirred at
25 °C for 4 h. The reaction was quenched with a cold saturated aqueous
solution of sodium hydrogen carbonate (100 mL), the reaction mixture
was passed through a Celite pad, and extraction was performed with
ethyl acetate (3 ꢁ 100 mL). The combined organic phase was washed
with saturated aqueous solutions of NaHCO3 (2 ꢁ 100 mL) and brine
(2 ꢁ 100 mL) and was dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure, and the residue was purified by silica
gel column chromatography using MeOH/CHCl3 as eluent to give
nucleoside 4c as a white solid (2.23 g, 75%). Rf = 0.5 (5% MeOH/
CHCl3). Mp: 106ꢀ108 °C. [R]D23 = ꢀ37.1° (c 0.1, MeOH). 1H NMR
(CDCl3, 300 MHz): δ 2.08, 2.11, 2.12, 2.13 (12H, 4s), 4.19 (1H, d,
During the synthesis of 1-(20-O,40-C-methylene-xylofurano-
syl)cytosine (11d; xylo-LNA-C), tosylation of monodeacetylated
nucleoside 5d resulted in the formation of a mixture of products,
perhaps due to involvement of the exocyclic amino group. To
overcome this problem, the cytosine NH2 group in compound 5d
was selectively benzoylated using benzoic anhydride in DMF to
afford nucleoside 12, which on tosylation (to afford compound
10d) and a subsequent one-pot deacetylationꢀcyclization reac-
tion afforded the xylo-LNA-C 11d in 86% yield (Scheme 5).
The debutanoylated nucleoside 8b obtained from Novozyme-435
catalyzed monodebutanoylation of nucleoside 7b was also converted
into xylo-LNA thymine monomer 11a, in an overall yield of 83%
(Scheme 5). The structures of compounds 6, 7aꢀf, 8aꢀd, 9aꢀd,
10aꢀd, 11aꢀd, 12, and 13 have been unambiguously established on
the basis of their IR and 1H and 13C NMR spectra and HRMS data
analysis. The structure of the known compound 11a3a was further
confirmed on the basis of comparison of its spectral data with those
reported in the literature, whereas xylo-LNA monomers U, A, and C
have been synthesized and reported in the literature for the first time.
In summary, highly efficient Novozyme-435 catalyzed biocataly-
tic routes for the selective deacylation of the C-100 acyloxy function
involving a primary ꢀOH group over the other acyloxy functions
involving primary and secondary ꢀOH groups in 40-C-acyloxy-
methyl-20,30,50-tri-O-acyl-β-D-xylofuranosyl nucleosides have been
developed for the first time. The biocatalytic methodology has been
used for the efficient synthesis of xylo-LNA monomers T, U, A, and
C in excellent yields. The developed environmentally friendly
biocatalytic methodology may find application for the synthesis of
xylo-LNA/LNA monomers to be used for the synthesis of modified
oligonucleotides for therapeutic and diagnostic applications.
’ EXPERIMENTAL SECTION
General Procedure for the Synthesis of 1-(40-C-Acetoxy-
methyl-20,30,50-tri-O-acetyl-β-D-xylofuranosyl) Nucleosides 4a,
b,d. To stirred solutions of penta-O-acetylated sugar derivatives 3a,b (2.5 g,
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NOTE
J = 12.0 Hz), 4.33 (2H, s), 4.56 (1H, d, J = 12.0 Hz), 5.65 (1H, d, J = 5.6
Hz), 5.91 (2H, br s), 6.05 (1H, t, J = 5.5 Hz), 6.26 (1H, d, J = 5.8 Hz),
8.10 (1H, s), 8.35 (1H, s). 13C NMR (CDCl3, 75.5 MHz): δ 20.6, 20.7,
20.8, 62.5, 63.8, 76.6, 78.3, 83.8, 84.9, 119.6, 138.4, 149.9, 153.4, 155.6,
169.5, 169.9, 170.2. IR (KBr) νmax: 3300, 2924, 1747, 1640, 1377, 1213,
1041 cmꢀ1. HR-ESI-TOF-MS: m/z 466.1605 ([M + H]+), calcd for
[C19H23N5O9 + H]+ 466.1574.
MeOH/CHCl3). Mp: 132ꢀ134 °C. [R]D25 = ꢀ57.2° (c 0.1, H2O). 1H
NMR (DMSO-d6, 300 MHz): δ 1.78 (3H, s), 3.17 (1H, d, J = 3.5 Hz),
3.41ꢀ3.45 (1H, m), 3.60 (1H, d, J = 3.3 Hz), 4.06ꢀ4.13 (3H, m), 4.61
(1H, br s), 4.84 (1H, br s), 5.25 (1H, br s), 5.48 (1H, d, J = 4.7 Hz), 5.77
(1H, d, J = 4.1 Hz), 7.88 (1H, s), 11.17 (1H, br s). 13C NMR (DMSO-d6,
75.5 MHz): δ 20.3, 69.8, 70.7, 83.4, 86.6, 93.5, 94.1, 117.5, 144.8, 159.0,
171.8. IR (KBr) νmax: 3401, 2929, 1694, 1476, 1265, 1150, 917 cmꢀ1. HR-
ESI-TOF-MS: m/z 311.0822 ([M + Na]+), calcd for [C11H16N2O7 +
Na]+ 311.0850.
General Procedure for Novozyme-435 Catalyzed Deacetyla-
tion Reaction on Peracetylated Nucleosides 4aꢀd: Preparation
of Monodeacetylated Nucleosides 5aꢀd. To solutions of tetra-
acetylated nucleosides 4aꢀd (1.09 mmol) in dry THF (30 mL) was added
n-butanol (1.30 mmol), followed by the addition of Novozyme-435 (50%
w/w of the peracetylated nucleosides 4aꢀd). The reaction mixture was
stirred at 50 °C in an incubator shaker, and the progress of the reaction was
monitored periodically by TLC. On completion, the reaction was quenched
by filtering off the Novozyme-435, the solvent was removed under reduced
pressure and the residue thus obtained was purified by silica gel column
chromatography using MeOH/CHCl3 to afford the monodeacetylated
nucleosides 5aꢀd in 82ꢀ93% yields.
General Procedure for the Synthesis of 1-(40-C-acyloxy-
methyl-20,30,50-tri-O-acyl-β-D-xylofuranosyl)thymines 7aꢀf.10
The complete physical and spectral data of acylates 7aꢀf are given below.
1-(40-C-Propanoyloxymethyl-20,30,50-tri-O-propanoyl-β-D-xylofura-
nosyl)thymine (7a). Obtained as a colorless viscous oil (0.8 g, 90%). Rf =
24
1
0.6 (2% MeOH/CHCl3). [R]D = ꢀ28.5° (c 0.1, MeOH). H NMR
(CDCl3, 300 MHz): δ 1.09ꢀ1.25 (12H, m), 1.96 (3H, s), 2.33ꢀ2.47 (8H,
m), 4.02 (1H, d, J = 12.1 Hz), 4.29 (2H, d, J = 3.6 Hz), 4.57 (1H, d, J = 12.1
Hz), 5.45 (1H, t, J = 6.3 Hz), 5.59 (1H, d, J = 6.1 Hz), 6.24 (1H, d, J = 6.5
Hz), 7.43 (1H, s), 9.44 (1H, br s). 13C NMR (CDCl3, 75.5 MHz): δ 7.8,
7.9, 8.08, 8.15, 11.8, 26.26, 26.30, 26.5, 26.7, 61.5, 63.2, 74.2, 76.6, 82.1, 83.5,
111.3, 133.5, 149.6, 162.7, 172.0, 172.4, 172.6. IR (thin film): νmax 3206,
1747, 1696, 1465, 1272, 1152, 1040, 978, 888 cmꢀ1. HR-ESI-TOF-MS:
m/z 535.1902 ([M + Na]+), calcd for [C23H32N2O11 + Na]+ 535.1898.
1-(40-C-Butanoyloxymethyl-20,30,50-tri-O-butanoyl-β-D-xylofuranosyl)
thymine (7b). Obtained as a colorless viscous oil (0.9 g, 92%). Rf = 0.5
(30% EtOAc in petroleum ether). [R]D24 = ꢀ48.4° (c 0.1, CHCl3). HR-
ESI-TOF-MS: m/z 591.2548 ([M + Na]+), calcd for [C27H40N2O11 + Na]+
591.2524.
1-(40-C-Hydroxymethyl-20,30,50-tri-O-acetyl-β-D-xylofuranosyl)thymine
(5a). Obtained as a white sticky solid (0.35 g, 87%). Rf = 0.3 (5% MeOH/
CHCl3). [R]D23 = ꢀ49.5° (c 0.1, MeOH). 1H NMR (CDCl3, 400 MHz):
δ 1.95 (3H, s), 2.08, 2.10, 2.18 (9H, 3s), 3.83 (1H, s), 4.00 (1H, d, J = 12.1
Hz), 4.54 (1H, d, J = 12.1 Hz), 5.43 (1H, t, J = 6.9 Hz), 5.69 (1H, d, J = 5.2
Hz), 6.29 (1H, d, J = 7.0 Hz), 7.48 (1H, s), 9.70 (1H, br s). 13C NMR
(CDCl3, 100.6 MHz): δ 12.7, 20.6, 21.0, 62.7, 63.4, 74.6, 77.2, 83.7, 84.8,
112.4, 134.5, 151.0, 163.6, 169.5, 171.0. IR (thin film): νmax 3447, 2918,
2849, 1749, 1696, 1461, 1376, 1230, 1052 cmꢀ1. HR-ESI-TOF-MS: m/z
437.1169 ([M + Na]+), calcd for [C17H22N2O10 + Na]+ 437.1172.
1-(40-C-Hydroxymethyl-20,30,50-tri-O-acetyl-β-D-xylofuranosyl)uracil
(5b). Obtained as a white solid (0.40 g, 92%). Rf = 0.35 (5% MeOH/
1-(40-C-Pentanoyloxymethyl-20,30,50-tri-O-pentanoyl-β-D-xylofuranosyl)
thymine (7c).Obtained as a colorless viscous oil (0.97 g, 90%). Rf =0.6(20%
EtOAc in petroleum ether). [R]D24 = +14.5° (c 0.1, CHCl3). HR-ESI-TOF-
MS: m/z 647.3158 ([M + Na]+), calcd for [C31H48N2O11 + Na]+ 647.3150.
1-(40-C-Hexanoyloxymethyl-20,30,50-tri-O-hexanoyl-β-D-xylofuranosyl)
thymine (7d). Obtained as a colorless viscous oil (1.11 g, 94%). Rf = 0.5
(10% EtOAc in petroleum ether). [R]D24 = ꢀ15.60° (c 0.1, CHCl3). HR-
23
1
CHCl3). Mp: 116ꢀ118 °C. [R]D = +8.4° (c 0.1, MeOH). H NMR
(CDCl3, 300 MHz): δ 2.09, 2.12, 2.16 (9H, 3s), 3.70 (1H, br s), 3.74 (2H,
br s), 3.98 (1H, d, J = 12.2 Hz), 4.51 (1H, d, J = 12.2 Hz), 5.44 (1H, s),
5.68 (1H, d, J = 6.8 Hz), 5.84 (1H, d, J = 8.1 Hz), 6.24 (1H, d, J = 6.9 Hz),
7.66 (1H, d, J = 8.1 Hz), 9.65 (1H, brs). 13C NMR (CDCl3, 75.5 MHz):δ
19.4, 19.7, 61.4, 62.1, 73.3, 76.2, 82.9, 83.9, 102.7, 137.5, 149.6, 161.8,
168.7, 169.0. IR (thin film): νmax 3447, 2918, 2849, 1749, 1696,
1461, 1376, 1230, 1052, 913 cmꢀ1. HR-ESI-TOF-MS: m/z 423.0999
([M + Na]+), calcd for [C16H20N2O10 + Na]+ 423.1010.
ESI-TOF-MS: m/z 703.3778 ([M + Na]+), calcd for [C35H56N2O11
+
Na]+ 703.3776.
1-(40-C-Benzoyloxymethyl-20,30,50-tri-O-benzoyl-β-D-xylofuranosyl)
thymine (7e). Obtained as an off-white solid (1.13 g, 93%). Rf = 0.7
24
(20% EtOAc in petroleum ether). Mp: 108ꢀ110 °C. [R]D = +6.1°
(c 0.1, CHCl3). HR-ESI-TOF-MS: m/z 727.1893 ([M + Na]+), calcd for
[C39H32N2O11 + Na]+ 727.1898.
9-(40-C-Hydroxymethyl-20,30,50-tri-O-acetyl-β-D-xylofuranosyl)adenine
(5c). Obtained as a white solid (0.38 g, 82%). Rf = 0.3 (5% MeOH/
CHCl3). Mp: 130ꢀ132 °C. [R]D24 = ꢀ62.3° (c 0.1, MeOH). 1H NMR
(CDCl3, 400 MHz): δ 2.01, 2.10, 2.14 (9H, 3s), 3.80 (2H, br s), 4.12 (1H,
d, J = 12.0 Hz), 4.56 (1H, d, J= 12.0 Hz), 5.87 (1H, d, J= 6.7 Hz), 6.14 (1H,
t, J = 6.6 Hz), 6.28 (1H, d, J = 6.8 Hz), 6.38 (2H, br s), 8.13 (1H, s), 8.27
(1H, s). 13CNMR(CDCl3, 100.6 MHz): δ19.3, 19.4, 19.7, 61.7, 62.0, 74.1,
77.2, 83.8, 85.4, 118.5, 138.6, 148.9, 152.7, 155.3, 169.8, 170.0, 170.2. IR
(thin film): νmax 3352, 2926, 1750, 1646, 1602, 1478, 1429, 1371, 1228,
1051, 908 cmꢀ1. HR-ESI-TOF-MS: m/z 446.1279 ([M + Na]+), calcd for
[C17H21N5O8 + Na]+ 446.1288.
1-(40-C-p-Anisyloxymethyl-20,30,50-tri-O-p-anisyl-β-D-xylofuranosyl)
thymine (7f). Obtained as an off-white solid (1.34 g, 94%). Rf = 0.7 (20%
EtOAc in petroleum ether). Mp: 98ꢀ100 °C. [R]D24 = ꢀ16.9° (c 0.1,
CHCl3). HR-ESI-TOF-MS: m/z 847.2315 ([M + Na]+), calcd for
[C43H40N2O15 + Na]+ 847.2321.
General Procedure for Novozyme-435 Catalyzed Deacyla-
tion Reaction on Acylated Nucleosides 7aꢀf: Preparation of
Monodeacylated Nucleosides 8aꢀd.12 The complete physical
and spectral data of monodeacylated nucleosides 8aꢀd are given below.
1-(40-C-Hydroxymethyl-20,30,50-tri-O-propanoyl-β-D-xylofuranosyl)
thymine (8a). Obtained as a white sticky solid (0.42 g, 85%). Rf = 0.7
1-(40-C-Hydroxymethyl-20,30,50-tri-O-acetyl-β-D-xylofuranosyl)cytosine
(5d). Obtained as a white solid (0.40 g, 93%). Rf = 0.4 (10% MeOH/
23
(5% MeOH/CHCl3). [R]D = ꢀ32.3° (c 0.1, CHCl3). 1H NMR
22
1
CHCl3). Mp: 155ꢀ157 °C. [R]D = ꢀ4.6° (c 0.1, MeOH). H NMR
(CDCl3, 300 MHz): δ 2.01, 2.05, 2.10 (9H, 3s), 3.62ꢀ3.74 (3H, br s), 4.02
(1H, d, J = 12.0 Hz), 4.47 (1H, d, J = 12.0 Hz), 5.45 (1H, t, J = 6.7 Hz), 5.64
(1H, d, J = 6.5 Hz), 5.99 (1H, d, J = 7.4 Hz), 6.30 (1H, d, J = 6.8 Hz), 6.80
(1H, brs), 7.63(1H, d, J=7.5Hz), 8.20(1H, brs). 13CNMR(CDCl3, 75.5
MHz): δ 20.97, 21.0, 21.3, 63.1, 63.5, 75.3, 77.0, 78.3, 85.2, 97.1, 140.3,
156.5, 166.1, 170.3, 170.5, 171.1. IR (thin film): νmax 3451, 3251, 1752,
1698, 1638, 1575, 1373, 1283, 1225, 1087, 687 cmꢀ1. HR-ESI-TOF-MS:
m/z 422.1198 ([M + Na]+), calcd for [C16H21N3O9 + Na]+ 422.1175.
Synthesis of 1-(40-C-hydroxymethyl-β-D-xylofuranosyl)thy-
mine (6).11 Obtained as a white solid (1.70 g, 90%). Rf = 0.35 (25%
(CDCl3, 300 MHz): δ 1.07ꢀ1.24 (9H, m), 1.95 (3H, s), 2.34ꢀ2.45
(6H, m), 3.62 (1H, br s), 3.74 (2H, br s), 3.97 (1H, d, J = 12.1 Hz), 4.53
(1H, d, J = 12.1 Hz), 5.49 (1H, t, J = 6.8 Hz), 5.69 (1H, d, J = 6.7 Hz),
6.31 (1H, d, J = 7.0 Hz), 7.50 (1H, s), 9.48 (1H, br s). 13C NMR (CDCl3,
75.5 MHz): δ 8.6, 8.62, 8.9, 12.5, 27.0, 27.1, 27.5, 62.6, 63.3, 74.4, 77.0, 83.5,
84.7, 112.3, 134.4, 150.8, 163.4, 173.2, 173.6. IR (thin film): νmax 3454,
2960, 1745, 1694, 1469, 1158, 1093, 1039, 930 cmꢀ1. HR-ESI-TOF-MS:
m/z 479.1629 ([M + Na]+), calcd for [C20H28N2O10 + Na]+ 479.1636.
1-(40-C-Hydroxymethyl-20,30,50-tri-O-butanoyl-β-D-xylofuranosyl)
thymine (8b). Obtained as a colorless viscous oil (0.52 g, 95%). Rf = 0.4
7559
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The Journal of Organic Chemistry
NOTE
(50% EtOAc in petroleum ether). [R]D24 = ꢀ38.9° (c 0.1, CHCl3). HR-
9-(40-C-p-Toluenesulfonylmethyl-20,30,50-tri-O-acetyl-R-L-arabino-
furanosyl)adenine (10c). Obtained as a light yellow solid (0.54 g, 93%).
Rf = 0.5 (5% MeOH/CHCl3). Mp: 128ꢀ130 °C. [R]D24 = ꢀ69.6° (c 0.1,
MeOH). 1H NMR (CDCl3, 300 MHz): δ 2.03, 2.05, 2.09 (9H, 3s), 2.43
(3H, s), 4.15 (1H, d, J = 12.1 Hz), 4.25 (2H, br s), 4.52 (1H, d, J = 12.1
Hz), 5.61ꢀ5.63 (1H, m), 5.79 (2H, br s), 6.11ꢀ6.12 (2H, brs), 7.34 (2H,
d, J = 8.1 Hz), 7.83 (2H, d, J = 8.1 Hz), 7.98 (1H, s), 8.35 (1H, s). 13C
NMR (CDCl3, 75.5 MHz): δ 20.51, 20.54, 20.7, 21.7, 62.3, 68.8, 75.2,
76.6, 83.4, 85.3, 119.8, 128.1, 130.0, 132.3, 138.7, 145.4, 149.9, 153.3,
155.5, 169.4, 169.8. IR (KBr) νmax: 3325, 3157, 2926, 1752, 1647, 1600,
1370, 1224, 1177, 1055, 996, 904 cmꢀ1. HR-ESI-TOF-MS: m/z 600.1370
([M + Na]+), calcd for [C24H27N5O10S + Na]+ 600.1371.
ESI-TOF-MS: m/z 521.2097 ([M + Na]+), calcd for [C23H34N2O10
+
Na]+ 521.2106.
1-(40-C-Hydroxymethyl-20,30,50-tri-O-pentanoyl-β-D-xylofuranosyl)
thymine (8c). Obtained as a colorless viscous oil (0.48 g, 81%). Rf = 0.5
(35% EtOAc in petroleum ether). [R]D25 = ꢀ68.3° (c 0.1, CHCl3). HR-
ESI-TOF-MS: m/z 563.2587 ([M + Na]+), calcd for [C26H40N2O10
+
Na]+ 563.2575.
1-(40-C-Hydroxymethyl-20,30,50-tri-O-hexanoyl-β-D-xylofuranosyl)
thymine (8d). Obtained as a colorless viscous oil (0.46 g, 73%). Rf = 0.4
(20% EtOAc in petroleum ether). [R]D25 = ꢀ39.9° (c 0.1, CHCl3). HR-
ESI-TOF-MS: m/z 605.3051 ([M + Na]+), calcd for [C29H46N2O10
+
Na]+ 605.3045.
General Procedure for the Synthesis of the xylo-LNA Mono-
mers 11aꢀc. Solutions of tosylated nucleosides 10aꢀc (1.0 mmol) and
potassium carbonate (6.0 equiv) in methanol/water (3/1, 15 mL) were
stirred at 25ꢀ28 °C for 8 h. On completion of the reaction (analytical
TLC), the solvent was removed under reduced pressure. The residue thus
obtained was purified by silica gel column chromatography using MeOH/
CHCl3 as eluent to afford nucleosides 11aꢀc in 91ꢀ96% yields.
General Procedure for the Synthesis of Mixed Esters Tetra-
O-acylated Nucleosides 9aꢀd.16 The complete physical and spectral
data of the model mixed ester 9a are given below. The structures of the three
other mixed esters, 1-(40-C-butanoyloxymethyl-20,30,50-tri-O-acetyl-R-L-ar-
abinofuranosyl)thymine (9b), 1-(40-C-pentanoyloxymethyl-20,30,50-tri-O-
acetyl-R-L-arabinofuranosyl)thymine (9c), and 1-(40-C-benzoyloxymethyl-
20,30,50-tri-O-acetyl-R-L-arabinofuranosyl)thymine (9d), were unambigu-
ously established on the basis of their IR, 1H and 13C NMR, and HRMS
data analysis.
1-(20-O,40-C-methylene-xylofuranosyl)thymine (11a).3a Obtained as
a white solid (0.26 g, 96%). Rf = 0.3 (10% MeOH/CHCl3). Mp:
24
122ꢀ124 °C. [R]D = +4.9° (c 0.1, MeOH). The spectral data were
1-(40-C-Propanoyloxymethyl-20,30,50-tri-O-acetyl-R-L-arabinofuranosyl)
thymine (9a). Obtained as a colorless viscous oil (100 mg, 89%). Rf = 0.6
(5% MeOH/CHCl3).[R]D22 =ꢀ70.40° (c0.1, CHCl3).1HNMR(CDCl3,
400 MHz): δ 1.19 (3H, t, J = 7.3 Hz), 1.98 (3H, s), 2.11, 2.17 (9H, 2s), 2.45
(2H, q, J = 7.3 Hz), 4.02 (1H, d, J = 11.8 Hz), 4.28 (2H, d, J = 8.1 Hz), 4.56
(1H, d, J = 11.7 Hz), 5.42 (1H, t, J = 6.6 Hz), 5.58 (1H, d, J = 6.6 Hz), 6.21
(1H, d, J = 6.6 Hz), 7.41 (1H, d, J = 1.5 Hz), 8.95 (1H, br s). 13C NMR
(CDCl3, 100.6 MHz): δ 10.8, 14.6, 22.36, 22.43, 22.7, 29.2, 64.3, 65.8, 77.0,
79.5, 84.8, 86.4, 114.1, 136.3, 152.3, 165.5, 171.3, 171.6, 171.9, 175.4. IR (thin
film): νmax 3216, 2984, 1748, 1464, 1372, 1225, 1057, 896, 756 cmꢀ1. HR-
ESI-TOF-MS: m/z493.1425 ([M + Na]+), calcdfor[C20H26N2O11 +Na]+
493.1429.
found to be identical with those reported in the literature.
1-(20-O,40-C-methylene-xylofuranosyl)uracil (11b). Obtained as a white
solid (0.24 g, 93%). Rf = 0.3 (10% MeOH/CHCl3). Mp: 216ꢀ218 °C.
[R]D23 = +73.7° (c 0.1, MeOH). 1H NMR (DMSO-d6, 300 MHz): δ 3.73
(1H, d, J = 8.2 Hz), 3.83 (2H, m), 3.96 (1H, d, J = 8.2 Hz), 4.08 (1H, s),
4.22 (1H, br s), 4.98 (1H, br s), 5.47ꢀ5.50 (2H, m), 5.66 (1H, br s), 7.69
(1H, d, J = 8.1 Hz), 11.23 (1H, br s). 13C NMR (DMSO-d6, 75.5 MHz): δ
56.0, 71.2, 72.1, 76.8, 87.6, 88.9, 98.0, 140.5, 149.4, 162.7. IR (Nujol) νmax
:
3491, 2922, 1694, 1463, 1305, 1276, 1175, 1098, 1044, 1014, 986, 901,
886 cmꢀ1. HR-ESI-TOF-MS: m/z 279.0591 ([M + Na]+), calcd for
[C10H12N2O6 + Na]+ 279.0588.
9-(20-O,40-C-methylene-xylofuranosyl)adenine (11c). Obtained as a
white solid (0.23 g, 91%). Rf = 0.3 (10% MeOH/CHCl3). Mp:
General Procedure for Novozyme-435 Catalyzed Deace-
tylation Reaction on Mixed Esters 9aꢀd.17
24
1
236ꢀ238 °C. [R]D = ꢀ72.1° (c 0.1, MeOH). H NMR (DMSO-d6,
300 MHz): δ 3.81ꢀ3.84 (3H, m), 4.05 (1H, d, J = 8.1 Hz), 4.18 (1H, s),
4.44 (1H, s), 5.01 (1H, br s), 5.84 (1H, br s), 6.01 (1H, s), 7.20 (2H, br s),
8.13 (1H, s), 8.19 (1H, s). 13C NMR (CDCl3+DMSO-d6, 75.5 MHz): δ
57.7, 73.1, 73.9, 79.2, 87.2, 89.6, 119.2, 140.8, 149.8, 153.3, 156.6. IR (KBr)
νmax: 3332, 3153, 2929, 1665, 1607, 1475, 1419, 1375, 1335, 1301, 1207,
1057, 992, 895, 851, 795 cmꢀ1. HR-ESI-TOF-MS: m/z 280.1028 ([M +
H]+), calcd for [C11H13N5O4 + H]+ 280.1040.
General Procedure for the Synthesis of 10aꢀc.18 The com-
plete physical and spectral data of tosylated nucleosides 10aꢀc are
given below.
1-(40-C-p-Toluenesulfonylmethyl-20,30,50-tri-O-acetyl-R-L-arabino-
furanosyl)thymine (10a). Obtained as a white solid (0.54 g, 95%). Rf =0.5
(5% MeOH/CHCl3). Mp: 155ꢀ156 °C. [R]D23 = ꢀ40.4° (c 0.1, MeOH).
1H NMR (CDCl3, 300 MHz): δ 1.94 (3H, s), 2.08 and 2.09 (9H, 2s), 2.45
(3H, s), 4.01 (1H, d, J = 12.1 Hz), 4.21 (2H, d, J = 3.7 Hz), 4.49 (1H, d, J =
12.1 Hz), 5.40 (1H, t, J = 5.9 Hz), 5.48 (1H, d, J = 5.8 Hz), 6.05 (1H, d, J =
6.0 Hz), 7.29 (1H, s), 7.37 (2H, d, J = 8.0 Hz), 7.82 (2H, d, J = 8.0 Hz), 9.07
(1H, br s). 13C NMR (CDCl3, 75.5 MHz): δ 13.0, 20.8, 20.9, 21.1, 22.0,
62.3, 69.0, 75.5, 76.99, 83.2, 85.8, 112.5, 128.5, 130.4, 132.4, 134.9, 145.8,
150.6, 163.6, 169.6, 169.9, 170.2. IR (thin film): νmax 3207, 2923, 1754, 1693,
1462, 1376, 1216, 1175, 1050, 981 cmꢀ1. HR-ESI-TOF-MS: m/z 591.1261
([M + Na]+), calcd for [C24H28N2O12S + Na]+ 591.1261.
Synthesis of 1-(40-C-Hydroxymethyl-20,30,50-tri-O-acetyl-
β-D-xylofuranosyl)-N4-benzoylcytosine (12). Compound 5d
(100 mg, 0.26 mmol) and benzoic anhydride (71 mg, 0.313 mmol) were
dissolved in 5 mL of dry DMF. and the reaction mixture was stirred for 5 h
at 25 °C. After completion of the reaction, the excess DMF was removed
under reduced pressure and the crude product thus obtained was purified
byusing silicagelcolumnchromatographyto yield 12asa whitesolid(115
22
mg, 88%). Rf = 0.6 (10% MeOH/CHCl3). Mp: 96ꢀ98 °C. [R]D
=
ꢀ12.74° (c 0.1, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 2.09, 2.11, 2.17
(9H, 3s), 3.80 (2H, d, J = 2.2 Hz), 4.08 (1H, d, J = 11.7 Hz), 4.58 (1H, d, J
= 11.7 Hz), 5.49 (1H, t, J = 6.6 Hz), 5.67 (1H, d, J = 5.9 Hz), 6.39 (1H, d, J
= 5.8 Hz), 7.50ꢀ7.54 (4H, m), 7.60ꢀ7.62 (1H, m), 7.92 (1H, d, J = 7.3
Hz), 8.14 (1H, d, J = 7.3 Hz). 13C NMR (CDCl3, 100.6 MHz): δ 20.58,
20.63, 20.9, 62.5, 63.2, 75.0, 78.9, 86.0, 97.6, 127.7, 129.9, 132.7, 133.3,
143.4, 162.5, 169.8, 170.0, 170.1. IR (thin film): νmax 3228, 3019, 1753,
1698, 1665, 1620, 1561, 1450, 1431, 1371, 1315, 1241, 1109, 1056, 1003,
914 cmꢀ1. HR-ESI-TOF-MS: m/z 504.1617 ([M + H]+), calcd for
[C23H25N3O10+H]+ 504.1613.
1-(40-C-p-Toluenesulfonylmethyl-20,30,50-tri-O-acetyl-R-L-arabino-
furanosyl)uracil (10b). Obtained as an off-white solid (0.51 g, 92%). Rf =
23
0.5 (5% MeOH/CHCl3). Mp: 136ꢀ138 °C. [R]D = ꢀ34.1° (c 0.1,
MeOH). 1H NMR (CDCl3, 300 MHz): δ 2.04, 2.07, 2.08 (9H, 3s), 2.45
(3H, s), 4.02 (1H, d, J = 12.2 Hz), 4.21 (2H, d, J = 2.4 Hz), 4.45 (1H, d, J =
12.1 Hz), 5.37 (1H, t, J = 5.6 Hz), 5.47 (1H, d, J = 5.4 Hz), 5.79 (1H, d, J =
8.0 Hz), 6.02 (1H, d, J = 5.8 Hz), 7.37 (2H, d, J = 8.1 Hz), 7.47 (1H, d, J =
8.0 Hz), 7.82 (2H, d, J = 8.1 Hz), 9.05 (1H, br s). 13C NMR (CDCl3, 75.5
MHz): δ 22.8, 23.1, 24.0, 64.1, 70.8, 77.3, 80.2, 85.6, 88.3, 105.9, 130.4,
132.4, 134.6, 141.1, 147.9, 152.3, 164.9, 171.6, 171.9, 172.1. IR (thin film):
νmax 3205, 2923, 2854, 1751, 1696, 1460, 1376, 1222, 1176, 1053, 983,
916 cmꢀ1. HR-ESI-TOF-MS: m/z 577.1078 ([M + Na]+), calcd for
[C23H26N2O12S + Na]+ 577.1099.
Synthesis of 1-(40-C-p-Toluenesulfonylmethyl-20,30,50-tri-O-
acetyl-β-D-xylofuranosyl)-N4-benzoylcytosine (10d). Obtained
according to the procedure given for the tosylation of nucleosides 5aꢀc as
7560
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The Journal of Organic Chemistry
NOTE
a white solid in 93% yield. Rf = 0.5 (5% MeOH/CHCl3). Mp: 93ꢀ95 °C.
[R]D22 = ꢀ18.49° (c 0.1, CHCl3). 1H NMR (CDCl3, 400 MHz): δ 2.07,
2.08, 2.10 (9H, 3s), 2.44 (3H, s), 4.08 (1H, d, J = 12.4 Hz), 4.27 (2H, s),
4.55 (1H, d, J = 12.5 Hz), 5.41 (1H, m), 5.48 (1H, d, J = 4.4 Hz), 6.11 (1H,
d, J = 4.4 Hz), 7.37 (2H, d, J = 8.0 Hz), 7.51ꢀ7.53 (2H, m), 7.60ꢀ7.62
(2H, m), 7.83 (2H, d, J = 8.8 Hz), 7.90 (2H, d, J = 7.3 Hz), 7.98 (1H, d, J =
8.0 Hz), 9.04 (1H, br s). 13C NMR (CDCl3, 100.6 MHz): δ 20.4, 20.7,
21.6, 61.4, 67.9, 74.9, 78.9, 84.4, 87.7, 127.6, 128.0, 129.0, 130.0, 133.3,
143.4, 145.4, 162.5, 169.0, 169.4, 169.6. IR (thin film): νmax 3320, 3021,
1755, 1674, 1627, 1557, 1485, 1372, 1313, 1238, 1178, 1096, 1057,
916 cmꢀ1. HR-ESI-TOF-MS: m/z 658.1687 ([M + H]+), calcd for
[C30H31N3O12S + H]+ 658.1701.
Maruyama, A.; Obika, S.; Imanishi, T.; Katayama, T. Biochemistry 2009,
48, 3545–3553.
ꢂ
(2) (a) Rajwanshi, V. K.; Hakansson, A. E.; Dahl, B. M.; Wengel, J.
ꢂ
Chem. Commun. 1999, 1395–1396. (b) Rajwanshi, V. K.; Hakansson,
A. E.; Kumar, R.; Wengel, J. Chem. Commun. 1999, 2073–2074.
ꢂ
(c) Rajwanshi, V. K.; Hakansson, A. E.; Sørensen, M. D.; Pitsch, S.;
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Sharma, V. K.; Olsen, C. E.; Wengel, J.; Parmar, V. S.; Prasad, A. K. J. Org.
Chem. 2010, 75, 7932–7935. (c) Prasad, A. K.; Kalra, N.; Yadav, Y.;
Kumar, R.; Sharma, S. K.; Patkar, S.; Lange, L.; Wengel, J.; Parmar, V. S.
Chem. Commun. 2007, 2616–2617.
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Wang, J. F. Green Chem. 2006, 8, 538–544. (b) Li, N.; Zong, M. H.; Ma,
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Sanghvi, Y.; Ferrero, M.; Gotor, V. J. Org. Chem. 2006, 71, 9765–9771.
(d) Montero, S. M.; Fernandez, S.; Sanghvi, Y. S.; Gotor, V.; Ferrero, M.
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S.; Sanghvi, Y. S.; Detorio, M.; Schinazi, R. F.; Gotor, V.; Ferrero, M.
Bioconjugate Chem. 2010, 21, 2239–2249.
Synthesis of 1-(20-O,40-C-Methylene-xylofuranosyl)cytosine
(11d). Obtained according to the procedure given for the synthesis of
11aꢀc as a white solid in 86% yield. Rf = 0.3 (30% MeOH/CHCl3). Mp:
200ꢀ203 °C dec. [R]D19 = +25.44° (c 0.1, MeOH). 1H NMR (DMSO-d6,
400 MHz): δ 3.71 (1H, d, J = 8.0 Hz), 3.77 (1H, d, J = 11.7 Hz), 3.84 (1H,
d, J = 11.7 Hz), 3.95 (1H, d, J = 8.1 Hz), 4.03 (1H, d, J = 2.2 Hz), 4.15 (1H,
d, J = 2.2 Hz), 5.47 (1H, s), 5.61 (1H, d, J = 8.0 Hz), 6.96 (1H, br s), 7.07
(1H, br s), 7.67 (1H, d, J = 8.1 Hz). 13C NMR (DMSO-d6, 100.6 MHz): δ
56.8, 72.2, 72.8, 77.4, 88.8, 89.0, 91.6, 142.0, 155.1, 165.8. IR (KBr): νmax
3405, 1645, 1494, 1196, 1050, 994, 790 cmꢀ1. HR-ESI-TOF-MS: m/z
278.0740 ([M + Na]+), calcd for [C10H13N3O5 + Na]+ 278.0747.
Synthesis of Tosylated Compound 13. Obtained according to
the procedure given for the tosylation of nucleosides 5aꢀc as a white solid
in93% yield. Rf = 0.5 (30% EtOAc inpetroleum ether). Mp: 102ꢀ104 °C.
22
1
[R]D = ꢀ55.04° (c 0.1, CHCl3). H NMR (CDCl3, 400 MHz): δ
0.87ꢀ0.94 (9H, m), 1.55ꢀ1.67 (6H, m), 1.92 (3H, s), 2.24ꢀ2.30 (6H,
m), 2.43 (3H, s), 3.93 (1H, d, J = 11.7 Hz), 4.12ꢀ4.20 (2H, m), 4.50 (1H,
d, J = 11.7 Hz), 5.37 (1H, m), 5.46 (1H, d, J = 5.9 Hz), 6.03 (1H, d, J = 6.6
Hz), 7.30ꢀ7.35 (3H, m), 7.79 (2H, d, J = 8.8 Hz), 8.49 (1H, br s). 13C
NMR (CDCl3, 100.6 MHz): δ 15.0, 15.7, 15.8, 15.9, 20.3, 20.4, 20.5, 24.0,
37.79, 37.85, 38.3, 64.1, 70.9, 77.0, 79.7, 84.9, 87.2, 114.4, 130.3, 132.3,
134.4, 136.8, 147.8, 152.4, 165.6, 174.2, 174.6, 174.8. IR (thin film): νmax
3200, 2967, 2877, 1749, 1697, 1459, 1368, 1250, 1190, 1177, 1096, 987,
814 cmꢀ1. HR-ESI-TOF-MS: m/z 675.2184 ([M + Na]+), calcd for
[C30H40N2O12S + Na]+ 675.2194.
(7) Youssefyeh, R. D.; Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem.
1979, 44, 1301–1309.
(8) Vorb€uggen, H.; Lagoja, I. M.; Herdewijn, P. Synthesis of Ribonu-
cleosides by Condensation using Trimethylsilyl Triflate. Current Protocols in
Nucleic Acid Chemistry 2007, 27:1.13.1ꢀ1.13.16.
(9) Lipase Novozyme-435 and Lipozyme TL IM were obtained as
gifts from Novozyme A/S Denmark. CRL and PPL was purchased from
Sigma-Aldrich Chemical Co., and Amano PS was purchased from
Amano Pharmaceuticals.
(10) Peracylated nucleosides 7aꢀf were synthesized by addition of
pyridine (10 mL) to a mixture of tetrahydroxy nucleoside 6 (1.74 mmol)
and the corresponding acid anhydride (propanoic, butanoic, valeric,
hexanoic, benzoic, or p-anisyl anhydride, 7.66 mmol), followed by
stirring of the reaction mixture at 25 °C for 4ꢀ5 h in 90ꢀ94% yields.
(11) Compound 6 was synthesized by stirring a solution of the
corresponding peracetylated analogue 4a (6.57 mmol) in half-saturated
methanolic ammonia (60 mL) at 25 °C for 10 h, followed by purification
with the help of CC using MeOH/CHCl3 as eluent in 90% yield.
(12) The procedure used for the Novozyme-435 catalyzed deacetyla-
tion of peracetylated nucleosides 4aꢀd was followed for the deacylation of
peracylated nucleosides 7aꢀf to afford 8aꢀd in 73ꢀ95% yields. Incuba-
tion of nucleosides 7e,f with Novozyme-435 did not yield any product.
(13) Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia,
L. A. J. Org. Chem. 1991, 56, 2656–2665.
Preparation of xylo-LNA-T 11a from Compound 13. Obtained
from the tosylated compound 13 according to the procedure given for the
synthesis of xylo-LNA monomers 11aꢀc as a white solid in 90% yield.
’ ASSOCIATED CONTENT
Supporting Information. Figures giving 1H and 13C
S
b
NMR spectra of compounds 3, 4aꢀd, 5aꢀd, 6, 7aꢀf, 8aꢀd,
9aꢀd, 10aꢀd, 11aꢀd, 12, and 13. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(14) (a) Doerschuk, A. P. J. Am. Chem. Soc. 1952, 74, 4202–4203.
(b) Ness, R. K.; Fletcher, H. G. J. Am. Chem. Soc. 1956, 78, 4710–4714.
(c) Kupchan, M.; Slade, P.; Young, R. J.; Milne, G. W. A. Tetrahedron
1962, 18, 499–506.
Corresponding Author
*Tel: 00-91-11-27662486. E-mail: ashokenzyme@yahoo.com.
(15) (a) Liu, K.-C.; Nozaki, K.; Wong, C.-H. Biocatal. Biotransform.
1990, 3, 169–177. (b) Edin, M.; Steinreiber, J.; Backvall., J.-E. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 5761–5766.
(16) To a solution of compound 5a (0.24 mmol) in 5 mL of dichlo-
romethane was added the corresponding acid anhydride (propanoic,
butanoic, pentanoic, or benzoic anhydride, 0.29 mmol) in the presence of
a catalytic amount of DMAP, and the reaction mixture was stirred at 25 °C
to afford compounds 9aꢀd in 89ꢀ94% yields after aqueous workup and
CC purification using MeOH/CHCl3.
’ ACKNOWLEDGMENT
We are grateful to the University of Delhi (DU-DST Purse Grant)
and Department of Biotechnology (DBT, New Delhi, India) for the
financial support. S.K.S., V.K.S., and K.B. thank the CSIR, New Delhi,
India, for the award of Junior/Senior Research Fellowships.
’ REFERENCES
(1) (a) Wengel, J. Acc. Chem. Res. 1999, 32, 301–310. (b) Kaur, H.;
Babu, B. R.; Maiti, S. Chem. Rev. 2007, 107, 4672–4697. (c) Torigoe, H.;
(17) The procedure used for the Novozyme-435 catalyzed deacetyla-
tionofperacetylatednucleosides 4aꢀd was followedfor thedeacylation of
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The Journal of Organic Chemistry
NOTE
mixed esters 9aꢀd to afford the identical product 5a in 85, 90, and 93%
yields, respectively, in all cases except9d. Incubationof nucleoside 9d with
Novozyme-435 did not yield any product.
(18) Solutions of compounds 5aꢀc (1.0 mmol) and p-toluenesul-
fonyl chloride (1.5 mmol) in anhydrous pyridine (10 mL) were stirred at
25ꢀ28 °C for 10 h. On completion the reaction mixture was poured
over 10% ice-cold hydrochloric acid solution and the product was
extracted with chloroform (2 ꢁ 100 mL). The combined organic extract
was washed with saturated aqueous NaHCO3 (2 ꢁ 100 mL) and dried
over anhydrous Na2SO4, and the solvent was removed under reduced
pressure. The residue thus obtained was purified by silica gel column
chromatography using MeOH/CHCl3 as eluent to afford the tosylated
nucleosides 10aꢀc in 92ꢀ95% yields.
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