Chemoenzymatic convergent synthesis of 2'-o,4'-c-methyleneribonucleosides

Article abstract of DOI:10.1021/jo5008338

Novozyme-435-catalyzed efficient regioselective acetylation of one of the two diastereotopic hydroxymethyl functions in 3-O-benzyl-4-C-hydroxymethyl-1,2- O-isopropylidene-α-d-ribofuranose has been achieved. The enzymatic methodology has been successfully

Full text of DOI:10.1021/jo5008338

Note
pubs.acs.org/joc
Chemoenzymatic Convergent Synthesis of
2′‑O,4′‑C‑Methyleneribonucleosides
Vivek K. Sharma,† Manish Kumar,† 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 Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C,
Denmark
S
* Supporting Information
ABSTRACT: Novozyme-435-catalyzed efficient regioselective acetylation of one of the two diastereotopic hydroxymethyl
functions in 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose has been achieved. The enzymatic method-
ology has been successfully utilized for convergent synthesis of bicyclic nucleosides (LNA monomers) T, U, A, and C. Further, it
has been demonstrated that Novozyme-435 can be used for 10 cycles of the acylation reaction without losing selectivity and
efficiency.
he FDA approval of antisense drugs vitravene1 and
kynamro2 has catalyzed the ongoing research on develop-
(Supporting Information SI-Scheme 1).12,13 It is at this juncture
that the selectivity of lipases can be explored for discrimination
between two primary hydroxyl groups in the precursor diol 3.14
We herein report efficient and novel chemoenzymatic synthesis
of bicyclic nucleosides T, U, A, and C via lipase-mediated
regioselective acetylation of diol sugar precursors 3.
T
ment of various types of backbone,3 base,4 and/or sugar-
modified5 antisense oligonucleotides (AONs). Among the
numerous modified nucleic acid analogues, locked nucleic acid
(LNA 2, Figure 1) that has an N-type (C3′-endo) sugar
The diol 3 was synthesized from 1,2,5,6-di-O-isopropylidene-
α-D-allofuranose (5) following a literature procedure,15 which
in turn was synthesized from its gluco-epimer 4 via DMSO/
P2O5 oxidation and selective reduction using NaBH4 (Scheme
1).16 Lipases from different sources, such as Candida antarctica
lipase-B (Novozyme-435), Thermomyces lanuginosus lipase
immobilized on silica (Lipozyme TL IM), Candida rugosa
lipase (CRL), and porcine pancreatic lipase (PPL) were
screened in five sets of organic solvents, such as tetrahydrofuran
(THF), acetonitrile (MeCN), toluene, diisopropyl ether
(DIPE), and acetone using acetic anhydride/vinyl acetate as
acetyl donor at 45 °C and at 200 rpm in an incubator shaker for
the study of selective acetylation of one over the other primary
hydroxyl function in diol 3 (Scheme 1). The solvent
engineering, that is, the change of selectivity of lipases in
different organic solvents, was first demonstrated by Klibanov
et al. in the 1980s.17,18 Since then, the use of lipases in organic
solvents for acylation/deacylation reactions on suitable alcohols
have been well studied to achieve optimum enantio-, regio-, and
chemoselectivity.
Figure 1. Structure of DNA/RNA 1; structural representation of LNA
monomer 2a,b; B = nucleobase.
puckering due to the presence of a 2′-O,4′-C methylene linkage
has shown promising applications in chemical biology.6−9
Presently, more than five LNA-modified oligonucleotides are
under active clinical trials, which demonstrates the commercial
potential of these compounds.10
The linear approach employed for the synthesis of LNA
monomers suffers from poor yields and could not be employed
as a general method for the synthesis of all monomers.11 In the
convergent synthesis, a common glycosyl donor is synthesized
for coupling with different nucleobases.7,12,13 The earlier use of
diol sugar precursor 3-O-benzyl-4-C-hydroxymethyl-1,2-O-iso-
propylidene-α-D-ribofuranose (3) for the synthesis of a
common glycosyl donor is limited because it requires selective
protection of one of the two primary hydroxyl groups.7 To
overcome this limitation, di-O-mesylated derivative of the diol 3
has been employed, but this necessitates an additional two steps
to remove the mesyl function toward the end of the synthesis
Incubation of diol 3 with Novozyme-435 in the above-
mentioned solvents in the presence of vinyl acetate as acetyl
donor led to the formation of a single product with slightly
higher Rf value than the diol, as indicated by TLC/HPLC
Received: April 14, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jo5008338 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
Scheme 1. Synthesis and Regioselective Novozyme-435-Catalyzed Acetylation Studies on Diol 3
examination. On comparison of the rate of acetylation reaction
of diol catalyzed by Novozyme-435, it was observed that the
reaction in DIPE was relatively faster. Further, regioselectivity
was poor when acetic anhydride was used as acetyl donor in
different organic solvents in the presence of the lipase.
Lipozyme TL IM, CRL, and PPL did not accept diol 3 as a
substrate. Based on the screening results, Novozyme-435 in
DIPE in the presence of vinyl acetate was selected for
regioselective acetylation of one of the two primary hydroxyl
groups in sugar diol 3 (Figure 2).
same condition for selective acetylation of diol 3 up to 5 g scale.
The acetylation reaction carried out on compound 3 in the
absence of Novozyme-435 did not yield any product.
The structure of the monoacetylated compound 7 was
confirmed by its chemical transformation to 1,2-O-isopropyl-
idene-3-O,4-C-methylene-α-D-ribofuranose (10) (Scheme 2).
Thus, the debenzylation of 7 afforded the dihydroxy compound
8, which on selective mesylation of the primary hydroxyl group
followed by the treatment of the resulting mesylated derivative
9 with aqueous NaOH solution resulted into concomitant
intramolecular cyclization to yield the bicyclic compound 10.
The structure of bicyclic compound 10 was unambiguously
confirmed by its spectral (1H and 13C NMR spectra and
HRMS) data analysis and comparison of chemical shift value of
oxetanomethylene protons with the chemical shift value of the
corresponding protons in 3-O,4-C-methyleneribothymidine.19
The synthesis of LNA monomers T, U, A, and C 15a−d was
successfully achieved from monoacetylated compound 7
(Scheme 3). Thus, the common glycosyl donor 12 was
synthesized by tosylation of monoacetylated compound 7
followed by acetolysis of the tosyl derivative 11 in 95% overall
yield. The coupling reaction of compound 12 with nucleobases,
uracil, thymine, 6-N-benzoyladenine, and cytosine under
Vorbruggen’s conditions20 yielded the corresponding nucleo-
̈
sides 13a−d in 71−89% yields. Subsequently, deacetylation
with concomitant intramolecular 2′-O,4′-C-cyclization in
nucleosides 13a−d under alkaline condition afforded 3′-O-
benzyl-2′-O,4′-C-methyleneribonucleosides 14a−d in 89−95%
yields. The debenzylation of compounds 14a−d with palladium
hydroxide afforded the LNA monomers, that is, 2′-O,4′-C-
methyleneuridine (15a), 2′-O,4′-C-methylenethymidine (15b),
2′-O,4′-C-methyleneadenosine (15c), and 2′-O,4′-C-
methylenecytidine (15d) in 81−91% yields (Scheme 3).
The overall yields of LNA monomer synthesis starting from
diol 3 were compared for the developed chemoenzymatic
convergent synthesis to that of the reported classical chemical
methodology (SI-Scheme 1).12,13 The results revealed that the
developed biocatalytic methodology is more efficient in all cases
with remarkable improvement in yields particularly for the
synthesis of LNA-A (SI-Table 1). Since the lipase Novozyme-
435 exclusively acetylates the C-5 hydroxyl group of the sugar
precursor, 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropyl-
idene-α-D-ribofuranose (3), in quantitative yield and it could
Figure 2. Screening of different lipases in organic solvents of varying
polarity at 45 °C for selective acetylation of diol 3. The acetylation
reaction mediated by vinyl acetate in organic solvents did not yield any
product when performed in the absence of Novozyme-435.
In a model reaction, a solution of compound 3 and vinyl
acetate in DIPE was incubated with Novozyme-435 (10% w/w
of 3) at 45 °C and 200 rpm in an incubator shaker, and the
progress of the reaction was monitored on TLC/HPLC. On
completion, the reaction was quenched by filtering off the
enzyme, and solvent was removed under reduced pressure to
afford the crude product, which on stirring with hexane led to
the isolation of pure monoacetylated compound 5-O-acetyl-3-
O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribo-
furanose (7) in quantitative yield. Using optimized conditions,
Novozyme-435 was utilized for 10 cycles for selective
acetylation of 3 and was found to be efficient and equally
regioselective for each cycle. Further, the lipase-mediated
acetylation reaction was successfully scaled up under the
Scheme 2. Chemical Transformation Studies on Novozyme-435-Catalyzed Acetylated Product 7
B
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Note
Scheme 3. Chemoenzymatic Convergent Synthesis of LNA Monomers 15a−d
= 5.2 Hz), 4.12 (1H, d, J = 11.6 Hz), 4.45 (1H, d, J = 8.0 Hz), 4.48
(1H, d, J = 12.0 Hz), 4.65 (1H, d, J = 12.0 Hz), 4.77 (1H, dd, J = 3.6
and 5.2 Hz), 5.70 (1H, d, J = 4.4 Hz), 7.28−7.35 (5H, m); 13C NMR
(DMSO-d6, 100.6 MHz) δ 20.6, 26.2, 26.5, 61.2, 64.5, 71.4, 78.2, 78.3,
85.4, 103.6, 112.5, 127.4, 127.6, 128.2, 137.9, 170.1; HR-ESI-TOF-MS
m/z 375.1403 ([M + Na]+), calcd for [C18H24O7 + Na]+ 375.1414.
5-O-Acetyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-
ribofuranose (8). To a stirred solution of compound 7 (0.50 g, 1.42
mmol) in dry methanol (10 mL) was added Pd−C (0.25 g). The
reaction mixture was stirred for 4 h under H2 atmosphere at 40 °C.
The catalyst was filtered off and washed with methanol. The excess
solvent was removed under reduced pressure, and the crude product
thus obtained was purified by silica gel column chromatography using
methanol in chloroform as gradient solvent system to afford
compound 8 as a white solid (0.35 g, 95%): Rf = 0.3 (5% methanol
be recovered and reused for the multiple cycles of selective
acetylation reaction, the present methodology is greener than
the reported methodologies.12,13
The structure of all the synthesized compounds 3, 5−12,
13a−d, 14a−d, and 15a−d was unambiguously established on
1
1
the basis of their spectral (IR, H and 13C NMR, H−1H
COSY, 1H−13C HMQC, and HRMS) data analysis. The
structures of known compounds 3,15 5,16 6,15 14a−c,12,13 and
15a−d12,13 were further confirmed by the comparison of their
physical and spectral data with those reported in the literature.
Herein, an efficient and environment friendly biocatalytic
methodology has been developed for the first time for the
convergent synthesis of LNA monomers T, U, A, and C with
relatively shorter route and with significant improvement in
overall yields. It has also been demonstrated that the selective
enzymatic acetylation reaction on two diastereotopic hydroxy-
methyl functions of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-iso-
propylidene-α-D-ribofuranose can be carried out in multiple-
gram scale. The developed biocatalytic process will be useful for
the commercial synthesis of LNA monomers required in
various applications.
32
in chloroform); mp 54−56 °C; [α]D = +30.46 (c 0.05, MeOH); IR
(thin film) νmax 3467, 2987, 2945, 1739, 1458, 1385, 1246, 1166, 1045,
1
1019, 874 cm−1; H NMR (CDCl3, 400 MHz) δ 1.39 and 1.61 (6H,
2s, 3H each), 2.10 (3H, s), 2.50 (1H, t, J = 6.8 Hz), 3.09 (1H, d, J =
8.4 Hz), 3.82−3.84 (2H, m), 4.14 (1H, d, J = 11.6 Hz), 4.21−4.25
(2H, m), 4.71 (1H, dd, J = 3.6 and 6.4 Hz), 5.86 (1H, d, J = 4.4 Hz);
13C NMR (CDCl3, 100.6 MHz) δ 20.8, 26.3, 26.5, 62.3, 65.8, 72.9,
79.4, 87.1, 104.7, 113.6, 170.7; HR-ESI-TOF-MS m/z 285.0940 ([M +
Na]+), calcd for [C11H18O7 + Na]+ 285.0945.
5-O-Acetyl-1,2-O-isopropylidene-4-C-methanesulfonyloxy-
methyl-α-D-ribofuranose (9). A solution of compound 8 (0.30 g,
1.14 mmol) and methanesulfonyl chloride (0.10 mL, 1.26 mmol) in
anhydrous dichloromethane/pyridine (10 mL, 4:1) was stirred at 0 °C
for 4 h. On completion, the reaction mixture was poured over 10% ice-
cold hydrochloric acid solution and extracted with chloroform (3 ×
100 mL). The combined organic extract was washed with saturated
aqueous sodium bicarbonate solution (2 × 100 mL), dried over
anhydrous sodium sulfate, and the excess solvent was removed under
reduced pressure. The residue thus obtained was purified by silica gel
column chromatography using ethyl acetate in hexane as eluent to
afford the mesylated compound 9 as a white solid (0.32 g, 83%): Rf =
EXPERIMENTAL SECTION
■
Materials. The Candida antarctica lipase-B (CAL-B or Novozyme-
435) immobilized on polyacrylate, Candida rugosa lipase (CRL), and
porcine pancreatic lipase (PPL) were purchased from Sigma-Aldrich
Co. (USA). Theremomyces lanuginosus lipase (Lipozyme TL IM)
immobilized on silica was obtained as a gift from Novozymes Inc.,
Copenhagen, Denmark. All the enzymes were 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 purchased from SD
Fine-Chem Ltd., Mumbai, India.
5-O-Acetyl-3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropyl-
idene-α-D-ribofuranose (7). To a solution of diol 3 (2.0 g, 6.45
mmol) in DIPE (40 mL) was added vinyl acetate (0.64 mL, 7.10
mmol) followed by the addition of Novozyme-435 (10% w/w of 3).
The reaction mixture was stirred at 45 °C in an incubator shaker at
200 rpm, and the progress of the reaction was monitored periodically
by TLC. After 1.5 h, the reaction was quenched by filtering off the
Novozyme-435; the solvent was removed under reduced pressure, and
the residue thus obtained was washed with hexane (2 × 30 mL) and
dried in vacuo to afford the monoacetylated compound 7 as a white
solid (2.24 g; quantitative yield): Rf = 0.3 (40% ethyl acetate in
hexane); mp 55−56 °C; [α]D32 = +93.3 (c 0.1, MeOH); IR (thin film)
νmax 3529, 3032, 2985, 2943, 1742, 1455, 1383, 1313, 1240, 1167,
32
0.5 (5% methanol in chloroform); mp 80−82 °C; [α]D = +15.36 (c
0.05, MeOH); IR (thin film) νmax 3490, 2989, 2941, 1742, 1353, 1239,
1
1174, 1134, 1093, 1024, 991, 967, 874, 828 cm−1; H NMR (CDCl3,
400 MHz) δ 1.37 and 1.64 (6H, 2s, 3H each), 2.08 (3H, s), 2.69 (1H,
brs), 3.09 (3H, s), 4.12 (1H, d, J = 11.6 Hz), 4.20−4.24 (2H, m), 4.47
(1H, d, J = 11.2 Hz), 4.58 (1H, d, J = 11.6 Hz), 4.71 (1H, dd, J = 3.6
and 6.0 Hz), 5.85 (1H, d, J = 3.6 Hz); 13C NMR (CDCl3, 100.6 MHz)
δ 20.8, 26.1, 26.2, 38.0, 64.5, 68.6, 73.1, 79.2, 84.7, 104.7, 114.0, 170.4;
HR-ESI-TOF-MS m/z 363.0711 ([M + Na]+), calcd for [C12H20O9S +
Na]+ 363.0720.
1,2-O-Isopropylidene-3-O,4-C-methylene-α-D-ribofuranose
(10). To a stirred solution of compound 9 (0.25 g, 0.73 mmol) in
water/THF (2 mL, 1:1) was added 2 M NaOH (1.5 mL), and reaction
mixture was stirred at rt for 1 h. The reaction mixture was extracted
with ethyl acetate (3 × 100 mL), washed with saturated aqueous
1135, 1104, 1023, 873, 742, 700 cm−1; H NMR (DMSO-d6, 400
MHz) δ 1.26 and 1.48 (6H, 2s, 3H each), 1.94 (3H, s), 3.60−3.64
(1H, m), 3.82−3.87 (1H, m), 3.95 (1H, d, J = 11.6 Hz), 4.03 (1H, d, J
1
C
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Note
sodium bicarbonate solution (2 × 100 mL) and brine solution (2 ×
100 mL), dried over anhydrous sodium sulfate, and concentrated
under reduced pressure. The residue thus obtained was purified by
silica gel column chromatography using ethyl acetate in hexane as
gradient solvent system to give 10 as a colorless viscous oil (0.13 g,
90%): Rf = 0.5 (5% methanol in chloroform); [α]D32 = −39.65 (c 0.05,
MeOH); IR (thin film) νmax 3372, 2922, 2119, 1740, 1372, 1222, 1177,
dropwise trimethylsilyltrifluoromethanesulfonate (0.28 mL, 1.55
mmol) under stirring, and the solution was heated at 70−80 °C for
6−10 h. The reaction was quenched with a cold saturated aqueous
solution of sodium bicarbonate (100 mL), and extraction was
performed with dichloromethane (3 × 100 mL). The combined
organic phase was washed with saturated aqueous sodium bicarbonate
solution (2 × 100 mL) and brine solution (2 × 100 mL) and was dried
over anhydrous sodium sulfate. The excess 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 13a−d in 71−89% yields.
1
1017, 864 cm−1; H NMR (CDCl3, 400 MHz) δ 1.41 and 1.67 (6H,
2s, 3H each), 1.83 (1H, brs), 3.85 (2H, dd, J = 12.4 and 16.8 Hz), 4.56
(1H, d, J = 7.6 Hz), 4.60 (1H, t, J = 4.4 Hz), 4.68 (1H, d, J = 7.2 Hz),
4.89 (1H, d, J = 6.0 Hz), 6.10 (1H, d, J = 4.4 Hz); 13C NMR (CDCl3,
100.6 MHz) δ 26.3, 27.2, 63.2, 78.1, 79.9, 84.7, 90.0, 109.1, 113.6; HR-
ESI-TOF-MS m/z 225.0728 ([M + Na]+), calcd for [C9H14O5 + Na]+
225.0733.
2′,5′-Di-O-acetyl-3′-O-benzyl-4′-C-p-toluenesulfonyl-
oxymethyluridine (13a). It was obtained as a white solid (0.49 g,
32
89%): Rf = 0.3 (5% methanol in chloroform); mp 70−72 °C; [α]D
=
+4.09 (c 0.1, MeOH); IR (thin film) νmax 3199, 3033, 1748, 1697,
5-O-Acetyl-3-O-benzyl-1,2-O-isopropylidene-4-C-p-toluene-
sulfonyloxymethyl-α-D-ribofuranose (11). A solution of com-
pound 7 (2.0 g, 5.68 mmol) and p-toluenesulfonyl chloride (1.29 g,
6.82 mmol) in anhydrous dichloromethane/pyridine (40 mL, 4:1) was
stirred at rt for 6 h. On completion, the reaction mixture was poured
over 10% ice-cold hydrochloric acid solution and extracted with
chloroform (3 × 100 mL). The combined organic extract was washed
with saturated aqueous sodium bicarbonate solution (2 × 100 mL),
dried over anhydrous sodium sulfate, and the excess solvent was
removed under reduced pressure. The residue thus obtained was
coevaporated with toluene (2 × 50 mL) and dried in vacuo to afford
the tosylated compound 11 as a white solid (2.82 g; 98%): Rf = 0.3
1458, 1366, 1228, 1190, 1177, 1109, 1049, 981, 814, 755, 700, 667
1
cm−1; H NMR (CDCl3, 400 MHz) δ 1.96 and 2.06 (6H, 2s, 3H
each), 2.42 (3H, s), 4.13−4.17 (3H, m), 4.30 (1H, d, J = 10.8 Hz),
4.42−4.45 (2H, m), 4.53 (1H, d, J = 11.6 Hz), 5.42−5.44 (1H, m),
5.61 (1H, d, J = 3.6 Hz), 5.71 (1H, dd, J = 1.2 and 8.0 Hz), 7.21−7.33
(8H, m), 7.75 (2H, d, J = 8.0 Hz), 9.39 (1H, brs); 13C NMR (CDCl3,
100.6 MHz) δ 20.6, 20.6, 21.6, 63.7, 67.8, 73.8, 74.5, 84.4, 91.1, 102.8,
128.0, 128.1, 128.3, 128.5, 129.8, 132.5, 136.6, 141.3, 145.1, 149.8,
163.1, 169.8, 169.9; HR-ESI-TOF-MS m/z 625.1468 ([M+ Na]+),
calcd for [C28H30N2O11S + Na]+ 625.1463.
2′,5′-Di-O-acetyl-3′-O-benzyl-4′-C-p-toluenesulfonyl-
31
oxymethylthymidine (13b). It was obtained as a white solid (0.49 g,
(40% ethyl acetate in hexane); mp 80−81 °C; [α]D = +43.4 (c 0.1,
31
88%): Rf = 0.3 (5% methanol in chloroform); mp 78−80 °C; [α]D
=
MeOH); IR (thin film) νmax 3065, 3031, 2988, 2942, 2876, 1747, 1598,
−2.28 (c 0.1, MeOH); IR (thin film) νmax 3034, 2928, 1750, 1701,
1496, 1455, 1360, 1310, 1292, 1234, 1190, 1177, 1099, 1023, 979, 913,
1
1364, 1227, 1190, 1177, 1112, 1048, 981, 839, 754, 669 cm−1; H
1
874, 839, 816, 753, 700, 666 cm−1; H NMR (CDCl3, 400 MHz) δ
NMR (CDCl3, 400 MHz) δ 1.89 (3H, s), 1.96 and 2.05 (6H, 2s, 3H
each), 2.42 (3H, s), 4.13−4.17 (3H, m), 4.28 (1H, d, J = 11.2 Hz),
4.41−4.45 (2H, m), 4.53 (1H, d, J = 10.8 Hz), 5.42 (1H, dd, J = 4.4
and 6.0 Hz), 5.61 (1H, d, J = 3.6 Hz), 7.00 (1H, s), 7.21−7.33 (7H,
m), 7.75 (2H, d, J = 8.0 Hz), 8.65 (1H, brs); 13C NMR (CDCl3, 100.6
MHz) δ 14.9, 23.1, 23.1, 24.1, 66.3, 70.3, 76.2, 77.0, 86.7, 93.2, 113.8,
130.5, 130.6, 130.8, 131.0, 132.3, 135.0, 139.2, 139.6, 147.5, 152.4,
166.2, 172.3, 172.5; HR-ESI-TOF-MS m/z 639.1616 ([M + Na]+),
calcd for [C29H32N2O11S + Na]+ 639.1619.
1.28 and 1.36 (6H, 2s, 3H each), 1.91 (3H, s), 2.42 (3H, s), 3.96−4.00
(2H, m), 4.18 (1H, d, J = 11.6 Hz), 4.35 (1H, d, J = 11.2 Hz), 4.48−
4.55 (2H, m), 4.58 (1H, dd, J = 3.6 and 4.8 Hz), 4.70 (1H, d, J = 12.0
Hz), 5.69 (1H, d, J = 3.6 Hz), 7.28−7.35 (7H, m), 7.80 (2H, d, J = 8.8
Hz); 13C NMR (CDCl3, 100.6 MHz) δ 20.6, 21.6, 25.8, 26.1, 64.1,
69.1, 72.4, 78.1, 78.8, 83.1, 104.1, 113.7, 127.83, 128.1, 128.2, 128.5,
129.7, 132.8, 136.8, 144.6, 170.1; HR-ESI-TOF-MS m/z 529.1501
([M + Na]+), calcd for [C25H30O9S + Na]+ 529.1503.
1,2,5-Tri-O-acetyl-3-O-benzyl-4-C-p-toluenesulfonyl-
oxymethyl-α,β-D-ribofuranose (12). Acetic anhydride (1.9 mL,
19.8 mmol) and concentrated sulfuric acid (0.01 mL, 0.19 mmol) were
added to a stirred solution of compound 11 (1.0 g, 1.98 mmol) in
acetic acid (11.3 mL, 198 mmol) at 0 °C, and the resulting reaction
mixture was stirred for 6 h at rt. The reaction was quenched by the
addition of cold water (100 mL) and stirred for 30 min at rt and was
extracted with ethyl acetate (3 × 200 mL). The organic layer was
washed with saturated aqueous sodium bicarbonate solution (3 × 100
mL) and brine solution (2 × 100 mL) and then dried over anhydrous
sodium sulfate. The excess solvent was removed under reduced
pressure, and the residue thus obtained was purified by silica gel
column chromatography using ethyl acetate in petroleum ether as
gradient solvent system to afford an anomeric mixture (α:β = ∼1:16,
based on comparison of integration of anomeric proton) of 12 (1.05 g,
97%) as a colorless viscous oil: Rf = 0.3 (40% ethyl acetate in hexane);
[α]D30 = −17.15 (c 0.1, MeOH); IR (thin film) νmax 2926, 1750, 1367,
1219, 1190, 1177, 1096, 1043, 969, 839, 739, 701, 667 cm−1; 1H NMR
(CDCl3, 400 MHz) δ 6.32 (d, J = 4.4 Hz, C-1Hα), 5.96 (s, C-1Hβ);
13C NMR (CDCl3, 100.6 MHz) δ 20.5, 20.6, 21.0, 21.6, 64.5, 68.4,
2′,5′-Di-O-acetyl-3′-O-benzyl-4′-C-p-toluenesulfonyl-
oxymethyl-6-N-benzoyladenosine (13c). It was obtained as a
white solid (0.47 g, 71%): Rf = 0.4 (5% methanol in chloroform); mp
208−210 °C; [α]D32 = +0.95 (c 0.1, MeOH); IR (thin film) νmax 3256,
3065, 3031, 2930, 1748, 1702, 1610, 1584, 1508, 1484, 1455, 1365,
1227, 1190, 1177, 1110, 1097, 1050, 981, 838, 816, 755, 704, 667
1
cm−1; H NMR (CDCl3, 400 MHz) δ 1.77 and 2.08 (6H, 2s, 3H
each), 2.42 (3H, s), 4.12−4.22 (3H, m), 4.41 (1H, d, J = 11.2 Hz),
4.52 (1H, d, J = 11.6 Hz), 4.60 (1H, d, J = 10.8 Hz), 4.81 (1H, d, J =
2.8 Hz), 5.98−6.01 (2H, m), 7.27−7.35 (8H, m), 7.50 (2H, t, J = 7.2
Hz), 7.59 (1H, dd, J = 7.2 and 14.8 Hz), 7.77 (2H, d, J = 8.0 Hz),
8.03−8.08 (3H, m), 8.72 (1H, s); 13C NMR (CDCl3, 100.6 MHz) δ
23.1, 23.2, 24.2, 65.8, 70.4, 76.3, 77.0, 87.2, 90.1, 125.7, 130.6, 130.7,
130.9, 131.1, 131.2, 131.5, 132.4, 135.2, 135.6, 135.7, 139.1, 145.0,
147.7, 152.1, 153.9, 154.3, 167.2, 172.2, 172.4; HR-ESI-TOF-MS m/z
730.2149 ([M + H]+), calcd for [C36H35N5O10S + H]+ 730.2177.
2′,5′-Di-O-acetyl-3′-O-benzyl-4′-C-p-toluenesulfonyl-
oxymethylcytidine (13d). It was obtained as a sticky solid (0.43 g,
32
78%): Rf = 0.3 (10% methanol in chloroform); [α]D = +15.44 (c
0.05, MeOH); IR (thin film) νmax 3355, 3206, 3033, 1744, 1663, 1497,
1457, 1366, 1227, 1190, 1177, 1109, 1049, 1031, 981, 839, 815, 756,
668 cm−1; 1H NMR (CDCl3, 400 MHz) δ 1.91 and 2.03 (6H, 2s, 3H
each), 2.40 (3H, s), 4.07−4.10 (2H, m), 4.16 (1H, d, J = 12.4 Hz),
4.32 (1H, d, J = 11.2 Hz), 4.37−4.40 (2H, m), 4.51 (1H, d, J = 11.6
Hz), 5.45 (1H, s), 5.64 (1H, d, J = 2.4 Hz), 6.25 (1H, d, J = 7.6 Hz),
7.18−7.20 (2H, m), 7.27−7.29 (5H, m), 7.52 (1H, d, J = 6.8 Hz), 7.72
(2H, d, J = 8.0 Hz), 8.46 (1H, brs), 9.32 (1H, brs); 13C NMR (CDCl3,
100.6 MHz) δ 20.6, 20.6, 21.6, 63.3, 68.1, 73.9, 74.2, 84.7, 91.4, 96.1,
127.9, 128.1, 128.27, 128.5, 129.9, 132.3, 136.7, 143.6, 145.2, 151.2,
162.4, 169.8, 170.0; HR-ESI-TOF-MS m/z 602.1793 ([M + H]+),
calcd for [C28H31N3O10S + H]+ 602.1803.
73.7, 73.8, 79.1, 83.1, 97.3, 127.8, 128.2, 128.3, 128.5, 129.6, 132.6,
136.6, 144.8, 168.6, 169.3, 170.0; HR-ESI-TOF-MS m/z 573.1389
([M + Na]+), calcd for [C26H30O11S + Na]+ 573.1401.
General Procedure for the Synthesis of 2′,5′-Di-O-acetyl-3′-
O-benzyl-4′-C-p-toluenesulfonyloxymethylribonucleosides
13a−d. To the stirred solution of tri-O-acetylated sugar derivative 12
(0.50 g, 0.91 mmol) and uracil/thymine/cytosine (1.36 mmol) in
anhydrous acetonitrile (MeCN) (20 mL) was added dropwise N,O-
bis(trimethylsilyl)acetamide (0.91 mL, 3.64 mmol). Dichloroethane
was used as solvent for coupling of 6-N-benzoyladenine (1.36 mmol)
instead of acetonitrile. The reaction mixture was stirred at reflux for 1
h and then cooled to 0 °C. In the cooled reaction mixture was added
D
dx.doi.org/10.1021/jo5008338 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
General Procedure for the Synthesis of 3′-O-Benzylated
LNA Monomers 14a−d. To a stirred solution of compounds 13a−d
(0.67 mmol) in water/dioxane (2 mL, 1:1) was added 2 M NaOH (1.5
mL) (+25% NH4OH, 1 mL for 13c only), and reaction mixture was
stirred at rt for 2−8 h. The reaction mixture was neutralized with acetic
acid, and excess solvent was evaporated under reduced pressure. The
residue thus obtained was purified by silica gel column chromatog-
raphy using methanol in chloroform as gradient solvent system to give
14a−d in 89−95% yields, respectively.
78.9, 86.3, 88.9, 100.8, 139.1, 150.2, 163.3; HR-ESI-TOF-MS m/z
279.0598 ([M + Na]+), calcd for [C10H12N2O6 + Na]+ 279.0588.
2′-O,4′-C-Methylenethymidine (15b). To a solution of nucleo-
side 14b (0.10 g, 0.28 mmol) in anhydrous MeOH (6 mL) were added
Pd(OH)2-C (20 wt %, 0.05 g) and ammonium formate (0.05 g, 0.85
mmol). The reaction mixture was refluxed for 10 min, whereupon it
was cooled to rt. 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 column chromatog-
raphy using methanol in chloroform as gradient solvent system to
afford LNA-T 15b as a white solid (0.06 g, 83%): Rf = 0.2 (10%
3′-O-Benzyl-2′-O,4′-C-methyleneuridine (14a).13 It was ob-
tained as a white solid (220 mg, 95%): Rf = 0.4 (10% MeOH in
CHCl3); mp 216−218 °C (lit.11b mp 217−219 °C); [α]D = +108.7
23
MeOH in CHCl3); mp 194−196 °C (lit.12 mp 196−198 °C); H
1
(c 0.3, MeOH) {Lit11b [α]D = +108.4 (c 0.3, MeOH)}; H NMR
(DMSO-d6, 400 MHz) δ 3.55 (1H, brs), 3.69 (1H, d, J = 8.0 Hz), 3.78
(2H, s), 3.86 (1H, d, J = 8.0 Hz), 3.92 (1H, s), 4.45 (1H, s), 4.60 (2H,
s), 5.48 (1H, d, J = 2.4 Hz), 5.61 (1H, d, J = 8.0 Hz), 7.28−7.35 (5H,
m), 7.73 (1H, dd, J = 2.0 and 8.0 Hz), 11.36 (1H, s); 13C NMR
(DMSO-d6, 100.6 MHz) δ 55.9, 71.1, 71.5, 75.8, 76.4, 86.5, 88.3,
100.9, 127.4, 127.6, 128.2, 137.9, 139.0, 150.0, 163.3; HR-ESI-TOF-
MS m/z 369.1064 ([M + Na]+), calcd for [C17H18N2O6 + Na]+
369.1057.
23
1
NMR (DMSO-d6, 400 MHz) δ 1.77 (3H, s), 3.62 (1H, d, J = 7.2 Hz),
3.76 (2H, d, J = 5.2 Hz), 3.82 (1H, d, J = 7.2 Hz), 3.90 (1H, d, J = 3.6
Hz), 4.11 (1H, s), 5.19 (1H, t, J = 6.0 Hz), 5.41 (1H, s), 5.65 (1H, d, J
= 4.4 Hz), 7.63 (1H, d, J = 1.6 Hz), 10.79 (1H, brs); 13C NMR
(DMSO-d6, 100.6 MHz) δ 12.2, 55.8, 68.5, 70.9, 78.8, 86.1, 88.7,
108.2, 134.7, 149.8, 163.7; HR-ESI-TOF-MS m/z 293.0754 ([M +
Na]+), calcd for [C11H14N2O6 + Na]+ 293.0744.
2′-O,4′-C-Methyleneadenosine (15c).12 To a solution of
nucleoside 14c (0.10 g, 0.27 mmol) in anhydrous EtOH (6 mL)
were added Pd(OH)2-C (20 wt %, 0.03 g) and ammonium formate
(0.09 g, 1.36 mmol). The reaction mixture was refluxed for 1 h,
whereupon it was cooled to rt. 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 column chromatography using methanol in chloroform as gradient
solvent system to afford LNA-A 15c as a white solid (0.07 g, 91%): Rf
= 0.2 (25% MeOH in CHCl3); mp 267−270 °C (lit.12 mp 266−268
°C); 1H NMR (DMSO-d6, 400 MHz) δ 3.75−3.80 (3H, m), 3.92 (1H,
d, J = 8.0 Hz), 4.25 (1H, d, J = 4.4 Hz), 4.40 (1H, s), 5.07 (1H, t, J =
6.0 Hz), 5.70 (1H, d, J = 4.4 Hz), 5.90 (1H, s), 7.33 (2H, brs), 8.14
(1H, s), 8.23 (1H, s); 13C NMR (DMSO-d6, 100.6 MHz) δ 56.7, 69.9,
71.4, 79.2, 85.3, 88.5, 119.0, 137.9, 148.4, 152.7, 156.0; HR-ESI-TOF-
MS m/z 280.1048 ([M + H]+), calcd for [C11H13N5O4 + H]+
280.1040.
3′-O-Benzyl-2′-O,4′-C-methylenethymidine (14b).12 It was
obtained as a white solid (224 mg, 93%): Rf = 0.4 (10% MeOH in
CHCl3); 1H NMR (DMSO-d6, 400 MHz) δ 1.78 (3H, s), 3.69 (1H, d,
J = 7.6 Hz), 3.80 (2H, d, J = 5.2 Hz), 3.86 (1H, d, J = 7.2 Hz), 3.96
(1H, s), 4.44 (1H, s), 4.60 (2H, s), 5.34 (1H, t, J = 4.8 Hz), 5.48 (1H,
s), 7.27−7.33 (5H, m), 7.60 (1H, s), 11.37 (1H, s); 13C NMR
(DMSO-d6, 100.6 MHz) δ 12.3, 55.9, 71.0, 71.5, 75.6, 76.4, 86.3, 88.2,
108.3, 127.3, 127.4, 128.1, 134.6, 137.8, 149.8, 163.8; HR-ESI-TOF-
MS m/z 361.1396 ([M + H]+), calcd for [C18H20N2O6 + H]+
361.1394.
3′-O-Benzyl-2′-O,4′-C-methyleneadenosine (14c).12 It was
obtained as a white solid (220 mg, 89%): Rf = 0.4 (25% MeOH in
CHCl3); mp 217−219 °C (lit.12 mp 218−219.5 °C); 1H NMR
(DMSO-d6, 400 MHz) δ 3.64 (1H, brs), 3.82−3.84 (3H, m), 3.96
(1H, d, J = 8.0 Hz), 4.36 (1H, s), 4.63 (2H, s), 4.74 (1H, s), 5.97 (1H,
s), 7.27−7.31 (5H, m), 7.35 (2H, brs), 8.14 (1H, s), 8.19 (1H, s); 13C
NMR (DMSO-d6, 100.6 MHz) δ 56.7, 71.1, 72.0, 76.9, 77.1, 85.2,
87.9, 119.0, 127.5, 127.6, 128.2, 137.9,, 137.9, 148.6, 152.7, 156.0; HR-
ESI-TOF-MS m/z 370.1520 ([M + H]+), calcd for [C18H19N5O4 +
H]+ 370.1510.
2′-O,4′-C-Methylenecytidine (15d). A mixture of compound
14d (0.25 g, 0.72 mmol), 20% Pd(OH)2-C (0.12 g), and ammonium
formate (0.14 g, 2.17 mmol) in methanol (4 mL) was refluxed for 10
min. 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 column chromatography using
methanol in chloroform as gradient solvent system to afford LNA-C
15d as a white solid (0.15 g, 81%): Rf = 0.2 (25% MeOH in CHCl3);
mp 274−276 °C (lit.12 mp 275−278 °C); Rf = 0.2 (25% MeOH in
CHCl3); 1H NMR (DMSO-d6, 400 MHz) δ 3.62 (1H, d, J = 7.2 Hz),
3.72 (2H, d, J = 5.2 Hz), 3.79−3.81 (2H, m), 4.07 (1H, s), 5.13 (1H, t,
J = 5.2 Hz), 5.37 (1H, s), 5.64 (1H, d, J = 4.4 Hz), 5.74 (1H, d, J = 7.6
Hz), 7.12 (1H, brs), 7.23 (1H, brs), 7.73 (1H, d, J = 7.6 Hz); 13C
NMR (DMSO-d6, 100.6 MHz) δ 56.1, 68.4, 71.0, 79.0, 86.9, 88.6,
93.4, 139.9, 154.8, 165.8; HR-ESI-TOF-MS m/z 278.0757 ([M +
Na]+), calcd for [C10H13N3O5 + Na]+ 278.0747.
3′-O-Benzyl-2′-O,4′-C-methylenecytidine (14d). It was ob-
tained as a white solid (217 mg, 94%): Rf = 0.4 (25% methanol in
32
chloroform); mp 76−78 °C; [α]D = +112.89 (c 0.1, MeOH); IR
(KBr) νmax 3357, 2945, 1645, 1482, 1396, 1282, 1152, 1087, 1048, 938,
789, 699 cm−1; 1H NMR (DMSO-d6, 400 MHz) δ 3.70 (1H, d, J = 7.2
Hz), 3.79 (2H, s), 3.84−3.86 (2H, m), 4.41 (1H, s), 4.56 (2H, dd, J =
11.6 and 16.8 Hz), 5.26 (1H, brs), 5.48 (1H, s), 5.73 (1H, d, J = 7.2
Hz), 7.18 (1H, brs), 7.21 (1H, brs), 7.27−7.34 (5H, m), 7.72 (1H, d, J
= 7.6 Hz); 13C NMR (DMSO-d6, 100.6 MHz) δ 56.1, 70.9, 71.4, 75.5,
76.4, 86.9, 87.9, 93.3, 127.3, 127.4, 128.1, 137.7, 139.6, 154.6, 165.7;
HR-ESI-TOF-MS m/z 346.1390 ([M + H]+), calcd for [C17H19N3O5
+ H]+ 346.1397.
2′-O,4′-C-Methyleneuridine (15a).13 To a solution of nucleoside
14a (0.10 g, 0.29 mmol) in anhydrous THF/MeOH (6 mL, 9:1, v/v)
were added Pd(OH)2-C (20 wt %, 0.02 g) and 88% formic acid (0.09
mL, 2.31 mmol). The reaction mixture was refluxed for 3 h,
whereupon it was cooled to rt. 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 column chromatography using methanol in chloroform as gradient
solvent system to afford LNA-U 15a as a white solid (0.07 g, 91%): Rf
= 0.2 (10% MeOH in CHCl3); mp 242−244 °C (lit.11b mp 239−243
ASSOCIATED CONTENT
■
S
* Supporting Information
SI-Scheme 1, SI-Table 1, and H and 13C NMR spectra of
1
compounds 7−12, 13a−d, 14a−d, and 15a−d. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
°C); [α]D = +91.6 (c 0.3, MeOH) {lit.11b [α]D = +92.2 (c 0.3,
MeOH)}; 1H NMR (DMSO-d6, 400 MHz) δ 3.61 (1H, d, J = 8.4 Hz),
3.72 (2H, d, J = 4.8 Hz), 3.80 (1H, d, J = 7.2 Hz), 3.84 (1H, d, J = 4.4
Hz), 4.12 (1H, s), 5.13 (1H, t, J = 5.2 Hz), 5.40 (1H, s), 5.60 (1H, dd,
2.4 and J = 8.0 Hz), 5.66 (1H, d, J = 4.4 Hz), 7.73 (1H, d, J = 8.0 Hz),
11.34 (1H, s); 13C NMR (DMSO-d6, 100.6 MHz) δ 55.9, 68.6, 71.0,
23
23
■
Corresponding Author
*Phone: +91-9818131666. E-mail: ashokenzyme@yahoo.com.
Notes
The authors declare no competing financial interest.
E
dx.doi.org/10.1021/jo5008338 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
ACKNOWLEDGMENTS
■
We are grateful to the University of Delhi for providing
financial support under DU-DST Purse Grant and under
scheme to strengthen research and development. We are also
thankful to CIF-USIC University of Delhi, Delhi, for providing
NMR spectral recording facility. V.K.S. and M.K. thank CSIR
for the award of Junior/Senior Research Fellowships.
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