Chemo-enzymatic synthesis of bicyclic 3′-azido- and 3′-amino-nucleosides

Article abstract of DOI:10.1039/c4ra06805j

Conformationally locked 3′-azido-3′-deoxythymidine analogues of T, U, A and C containing a 2′-O,4′-C-methylene linked bicyclic furanose moiety has been efficiently synthesized following a greener chemo-enzymatic convergent route. Thus, one of the two dias

Full text of DOI:10.1039/c4ra06805j

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Chemo-enzymatic synthesis of bicyclic 30-azido-
and 30-amino-nucleosides†
Cite this: RSC Adv., 2014, 4, 37231
a
a
b
a
*
Manish Kumar, Vivek K. Sharma, Carl E. Olsen and Ashok K. Prasad
Conformationally locked 30-azido-30-deoxythymidine analogues of T, U, A and C containing a 20-O,40-C-
methylene linked bicyclic furanose moiety has been efficiently synthesized following a greener chemo-
enzymatic convergent route. Thus, one of the two diastereotopic hydroxyl groups of 3-azido-3-deoxy-
4-C-hydroxymethyl-1,2-O-isopropylidene-a-D-ribofuranose has been regioselectively acetylated using
Novozyme®-435 in quantitative yield. The selective enzymatic acetylation can be carried out with the
same efficiency using Novozyme®-435 for 10 cycles of reaction. The monoacetylated sugar derivative
was converted to bicyclic 30-azidonucleosides in four steps in overall yields of 60 to 68%. It has been
demonstrated that 30-azido-30-deoxy-20-O,40-C-methylenethymidine can easily be converted into
30-amino-30-deoxy-20-O,40-C-methylenethymidine in 95% yield, which is an important monomer for the
synthesis of therapeutically useful sugar-modified oligonucleotides.
Received 8th July 2014
Accepted 8th August 2014
DOI: 10.1039/c4ra06805j
www.rsc.org/advances
i.e. 30-azido-30-deoxy-20-O,40-C-methyleneribonucleosides have
been classically synthesized from 3-azido-3-deoxy-4-C-hydroxy-
Introduction
methyl-1,2-O-isopropylidene-a-D-ribofuranose or its derivatives
in 18–41% yields.6 All these reported methods either suffer from
the poor selectivity between two primary hydroxyl groups of
3-azido ribofuranose sugar or are lengthy due to multiple
protection–deprotection steps. The use of lipases for selective
protection and de-protection of hydroxyl groups as key step in
the total synthesis of important compounds has been well
established.7 Herein, we report an efficient chemo-enzymatic
convergent synthesis of 30-azido-30-deoxy-20-O,40-C-methylene-
ribonucleosides 1a–d and conversation of 30-azido-30-deoxy-20-
O,40-C-methylenethymidine (1a) to corresponding 30-amino
derivative 2 in 60–68% and 64% yields, respectively (Fig. 1).
Various sugar-modied oligonucleotides have been synthesized
with improved pharmaceutical properties but none of the
analogues were widely accepted as a Locked Nucleic Acid
(LNA).1,2 LNA is basically modied RNA in which a methylene
bridge between 20-oxygen and 40-carbon locks the ribose in the
30-endo conformation (Fig. 1). This leads to the signicant
enhancement of their hybridization property with the compli-
mentary DNA/RNA strand, which plays a crucial role in the
development of nucleic acid based therapeutics and
diagnostics.2–4
The 30-amino-30-deoxy-20-O,40-C-methyleneribonucleosides
(30-amino-LNA monomers) are important intermediate for N30-
P-O50 oligonucleotides (ONs) possessing good antisense and
antigene activity.5 The precursor for 30-amino-LNA monomers,
Results and discussion
The starting sugar derivative, 3-azido-3-deoxy-4-C-hydroxymethyl-
1,2-O-isopropylidene-a-D-ribofuranose (3) was prepared from
D-glucose by following literature procedure in 44% overall yield.8
The regioselective acetylation of C-5 hydroxyl over C-4 hydroxy-
methyl group was achieved by incubation of sugar derivative 3
Fig.
nucleosides.
1
Structure of LNA and bicyclic 30-azido-
&
30-amino- with Novozyme®-435 in the presence of vinyl acetate as acetyl
donor in toluene to obtain 3-azido-5-acetyl-3-deoxy-4-C-hydroxy-
methyl-1,2-O-isopropylidene-a-D-ribofuranose (4) in quantitative
yield (Scheme 1).9 Under optimized conditions, Novozyme®-435
aBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110 007,
India. E-mail: ashokenzyme@gmail.com
bFaculty of Life Sciences, Department of Plant and Environmental Sciences, University
was found efficient and regioselective upto 10 cycles of acetylation
on compound 3.
The tosylation of the lone hydroxyl group in enzymatically
mono-acetylated compound 4 afforded 5-O-acetyl-3-azido-3-
deoxy-4-C-(p-toluenesulphonyloxymethyl)-1,2-O-isopropylidene-
a-D-ribofuranose (5) in 95% yield. The X-ray diffraction analysis
of Copenhagen, DK-1871 Frederiksberg C, Denmark
† Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
of compounds 6a–b, 7a–7d and 1b. CCDC 995894. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c4ra06805j
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oligonucleotides having a N30-P50 phosphoramidate linkage are
well known for showing very high binding affinity with ssDNA
and RNA as well as with dsDNA.6e The selective reduction of
azido group of compound 1a was achieved by Pd–C under
hydrogen atmosphere in ethyl acetate to afford the amino
compound 2 in 95% yield, which is an important precursor for
the synthesis of N30-P50 oligonucleotides (Scheme 1).
The structure of all synthesized compounds, i.e. 1a–d, 2, 3, 4,
5, 6a–b and 7a–d were unambiguously established on the basis
Scheme 1 Chemo-enzymatic synthesis of bicyclic 30-azido- & 30-
amino-nucleosides. Reagents and conditions (i) Novozyme®-435, of their spectral data (1H- and 13C- NMR spectra, IR spectra and
toluene, 25–28 ꢀC, 200 rpm, quantitative yield; (ii) TsCl, Py, 0 ꢀC – rt,
HRMS) analysis. The structure of known compounds 1a, 1c, 1d,
95%; (iii) AcOH–Ac2O–conc. H2SO4 (100 : 10 : 0.1), rt, 94%; (iv)
2, 3, 4 and 5 was further conrmed on the basis of comparison
nucleobase, N,O-bis(trimethylsilyl)acetamide (BSA), TMSOTf, CH3CN
of their physical and spectral data with those reported in the
literature.6,9 The structure of monohydroxy compound
or 1,2-dichloroethane (for ABZ only), 80 ꢀC, 80–88%; (v) 2 M NaOH,
H2O : dioxane (1 : 1), NH4OH (only for 7d), rt, 85–89%; (vi) H2, Pd/C,
EtOAc, rt, 95%.
4
obtained by lipase-mediated selective acetylation of one of the
two primary hydroxyl groups in dihydroxy compound 3 was
conrmed by X-ray crystallographic study of its corresponding
tosyl derivative 5.
of tosylated compound 5 unambiguously established that
Novozyme®-435 exclusively acetylates C-5 hydroxyl over C-4
hydroxymethyl group. The ORTEP diagram of compound 5 is
shown in Fig. 2 as two crystallographically independent units.
The detailed crystallographic data of compound 5 has been
deposited in the ESI.†
Acetolysis of compound 5 with acetic acid–acetic anhydride–
sulphuric acid (100 : 10 : 0.1) afforded a mixture of anomeric
azidosugar derivative 6a–b in 94% yield. For the convergent
synthesis of bicyclic 30-azidonucleosides the anomeric mixture
Experimental section
Melting points 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 making KBr disc for
solid samples and thin lm for oils. The 1H- and 13C NMR
spectra were recorded at Jeol alpha-400 spectrometer at 400 and
100.6 MHz, respectively using TMS as internal standard. The
chemical shi values are on d scale and the coupling constants
(J) are in Hz. The mass spectra analyses were done on a
microTOF-Q instrument from Bruker Daltonics, Bremen and
6520 Q-TOF instrument from Agilent Technologies. The optical
rotations were measured on Rudolph Autopol II automatic
polarimeter using light of 589 nm wavelength. Analytical TLCs
were performed on precoated Merck silica-gel 60F254 plates; the
spots were detected either under UV light or by charring with
4% alcoholic H2SO4. Silica gel (100–200 mesh) was used for
column chromatography. The single crystal X-ray diffraction
data was collected on an Oxford Diffraction XCalibur single
crystal X-ray instrument having CCD camera [Cu Ka radiation
(l ¼ 1.54184)] at USIC, University of Delhi, Delhi. The lipase
Novozyme®-435 was obtained as gi from Novozyme A/S
Denmark.
¨
6a–b was used as common glycosyl donor for the Vorbruggen's
coupling reaction10 with different nucleobases, viz. thymine,
uracil, cytosine and 6-N-benzoyladenine in the presence of N,O-
bis(trimethylsilyl)acetamide and trimethylsilyltriuoromethane
sulfonate in acetonitrile or 1,2-dichloroethane (only for 6-N-
benzoyladenine) resulting in the formation of nucleosides 7a–d
in 80–88% yields. Subsequently, deacetylation with concomi-
tant intramolecular 20-O,40-C-cyclization in nucleosides 7a–d
under alkaline condition afforded 30-azido-30-deoxy-20-O,40-C-
methyleneribonucleosides 1a–d in 85–89% yields. The
5-O-Acetoxy-1,2-di-O-acetyl-3-azido-3-deoxy-4-C-(p-
toluenesulphonyloxymethyl)-a,b-D-ribofuranose (6a–b)
Acetic anhydride (12.72 mL, 135.9 mmol) and concentrated
sulphuric acid (0.071 mL, 1.35 mmol) was added to a stirred
solution of compound 5 (6.0 g, 13.5 mmol) in acetic acid (78.04
ꢀ
mL, 1359.1 mmol) at 0 C and mixture was stirred for 5 h. The
reaction was quenched by addition of water (150 mL) and
extracted with chloroform (3 ꢁ 100 mL). The combined organic
layer was washed with bicarbonate solution (2 ꢁ 100 mL), with
cold water (2 ꢁ 100 mL) and then dried over sodium sulphate.
The excess of solvent was removed under reduced pressure, the
residue thus obtained was puried by silica gel column chro-
matography using ethyl acetate in petroleum ether as gradient
Fig. 2 ORTEP diagram of compound 5 drawn in 20% thermal prob-
ability ellipsoids with atomic numbering scheme showing two crys-
tallographically independent units.
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20,50-Di-O-acetyl-30-azido-30-deoxy-40-C-p-toluenesulfonyloxy-
methylcytidine (7c). It was obtained as white solid (1.42 g, 86%).
solvent system to afford an anomeric mixture (a:b ¼ 1 : 5, based
on comparision of integration of anomeric proton) of 6a–b as
colourless oil (6.2 g, 94%). Rf ¼ 0.6 (40% ethyl acetate in
petroleum ether). IR (thin lm) nmax: 2963, 2121, 1752, 1598,
1437, 1369, 1225, 1178, 1096, 1020, 972, 815 and 666 cmꢂ1;1H
NMR (CDCl3, 400 MHz): d 6.34 (J ¼ 4.4 Hz, d, C-1Ha), 6.02 (s, C-
1Hb); Copy of 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3,
100.6 MHz) spectra are given in ESI;† HR-ESI-TOF-MS: m/z
508.0996 ([M + Na]+), calcd for [C19H23N3O10S + Na]+ 508.0996.
Rf ¼ 0.3 (10% methanol in chloroform); M. Pt.: 94–96 ꢀC; [a]D30
¼
+8.29 (c 0.1, MeOH); 1H NMR (DMSO-d6, 400 MHz): d 7.80 (2H,
d, J ¼ 8.0 Hz), 7.51–7.47 (3H, m), 7.34 (2H, brs), 5.72 (1H, d, J ¼
7.2 Hz), 5.62 (1H, d, J ¼ 4.4 Hz), 5.53 (1H, dd, J ¼ 4.4 and 6.8 Hz),
4.92 (1H, d, J ¼ 6.4 Hz), 4.12–4.07 (4H, m), 2.41 (3H, s), 2.04 (3H,
s) and 1.96 (3H, s); 13C NMR (CDCl3, 100.6 MHz): d 170.1, 169.9,
166.1, 155.1, 145.2, 143.7, 132.5, 129.9, 128.0, 95.4, 93.8, 84.0,
75.1, 67.9, 64.3, 63.5, 21.7, 20.7 and 20.6; IR (thin lm) nmax
:
3344 (br), 2922, 2851, 2121, 1752, 1656, 1483, 1366, 1289, 1225,
1176, 1047, 985, 788 and 667 cmꢂ1; HR-ESI-TOF-MS: m/z
537.1397 ([M + H]+), calcd for [C21H24N6O9S + H]+ 537.1398.
20,50-Di-O-acetyl-30-azido-30-deoxy-40-C-p-toluenesulfonyloxy-
methyl-6-N-benzoyladenosine (7d). To the stirred solution of
compound 6a–b (1.5 g, 3.0 mmol) and 6-N-benzoyladenine
(1.10 g, 4.6 mmol) in anhydrous dichloroethane (20 mL),
N,O-bis(trimethylsilyl)acetamide (4.57 mL, 18.5 mmol) was
added dropwise. The reaction mixture was stirred at reux for 1
General procedure for the synthesis of nucleosides 7a, 7b and 7c
To the stirred solution of compound 6a–b (1.5 g, 3.0 mmol) and
thymine (0.58 g, 4.6 mmol)/uracil (0.51 g, 4.6 mmol) or cytosine
(0.51 g, 4.6 mmol) in anhydrous acetonitrile (30 mL) N,O-bis-
(trimethylsilyl)acetamide (3.05 mL) was added dropwise. The
reaction mixture was stirred at reux for 1 h, and then cooled to
0
ꢀC. In the cooled reaction mixture trimethylsilyltriuoro-
methane sulfonate (0.92 mL, 5.2 mmol) was added dropwise
under stirring and the solution was heated at 70–80 ꢀC for 4–6 h.
The reaction was quenched with a cold saturated aqueous
solution of sodium hydrogen carbonate (100 mL) and the
compound was extracted with chloroform (3 ꢁ 100 mL). The
combined organic phase was washed with saturated aqueous
solutions of NaHCO3 (2 ꢁ 100 mL) and brine (2 ꢁ 50 mL) and
was dried over anhydrous Na2SO4. The excess of solvent was
removed under reduced pressure and the residue thus obtained
was puried by silica gel column chromatography using meth-
anol in chloroform as gradient solvent system to afford nucle-
osides 7a–c in 80–88% yields.
ꢀ
h, and then cooled to 0 C. To the cooled reaction mixture tri-
methylsilyltriuoromethane sulfonate (2.10 mL, 11.5 mmol)
was added dropwise under stirring and the solution was heated
at 70–80 ꢀC for 10–12 h. The reaction was quenched with a cold
saturated aqueous solution of sodium hydrogen carbonate
(100 mL) and the compound was extracted with chloroform
(3 ꢁ 100 mL). The combined organic phase was washed with
saturated aqueous solutions of NaHCO3 (2 ꢁ 100 mL) and brine
(2 ꢁ 50 mL), and was dried over anhydrous Na2SO4. The excess
of solvent was removed under reduced pressure and the residue
thus obtained was puried by silica gel column chromatog-
raphy using methanol in chloroform as gradient solvent system
to afford nucleoside 7d as yellow solid (1.10 g, 80%). Rf ¼ 0.5
20,50-Di-O-acetyl-30-azido-30-deoxy-40-C-p-toluenesulfonyloxy-
methylthymidine (7a). It was obtained as off white solid (1.50 g,
88%). Rf ¼ 0.5 (5% methanol in chloroform); M. Pt.: 110–112 ꢀC;
[a]2D9 ¼ +15.14 (c 0.1, MeOH); 1H NMR (CDCl3, 400 MHz): d 9.07
(1H, brs), 7.81 (2H, d, J ¼ 8.4 Hz), 7.38 (2H, d, J ¼ 8.4 Hz), 7.01
(1H, s), 5.57–5.60 (2H, m), 4.68 (1H, d, J ¼ 6.0 Hz), 4.36 (1H, d, J
¼ 12.0 Hz), 4.10–4.21 (3H, m), 2.46 (3H, s), 2.16 (3H, s), 2.08 (3H,
s) and 1.92 (3H, s); 13C NMR (CDCl3, 100.6 MHz): d 170.0, 169.8,
163.5, 150.0, 145.3, 137.3, 132.3, 130.0, 128.0, 111.7, 90.8, 83.7,
74.4, 67.5, 64.3, 62.8, 21.7, 20.7, 20.4 and 12.4; IR (thin lm)
nmax: 3199 (br), 2927, 2120, 1696, 1370, 1228, 1049, 998, 815, 732
and 667 cmꢂ1; HR-ESI-TOF-MS: m/z 574.1213 ([M + Na]+), calcd
for [C22H25N5O10S + Na]+ 574.1214.
28
ꢀ
(5% methanol in chloroform); M. Pt.: 90–92 C; [a]D ¼ꢂ14.05
(c 0.1, MeOH); 1H NMR (CDCl3, 400 MHz): d 9.17 (1H, brs), 8.78
(1H, s), 8.05 (1H, s), 8.03 (2H, d, J ¼ 7.2 Hz), 7.82 (2H, d, J ¼ 8.4
Hz), 7.62 (1H, t, J ¼ 7.2 Hz), 7.53 (2H, d, J ¼ 8.0 Hz), 7.37 (2H, d, J
¼ 8.0 Hz), 6.17 (1H, dd, J ¼ 5.2 and 6.4 Hz), 6.05 (1H, d, J ¼ 4.4
Hz), 5.01 (1H, d, J ¼ 6.0 Hz), 4.42–4.14 (4H, m), 2.46 (3H, s), 2.16
(3H, s) and 2.02 (3H, s); 13C NMR (CDCl3, 100.6 MHz): d 169.8,
169.6, 164.6, 152.8, 151.3, 149.8, 145.3, 142.2, 133.3, 132.9,
132.3, 129.9, 128.4, 128.0, 127.9, 123.7, 87.0, 84.1, 74.4, 67.3,
63.7, 62.9, 21.6, 20.6 and 20.3; IR (thin lm) nmax: 3344 (br),
2922, 2851, 2120, 1753, 1599, 1451, 1366, 1219, 1177, 1073, 986,
772, 709 and 668 cmꢂ1; HR-ESI-TOF-MS: m/z 665.1770 ([M +
H]+), calcd for [C29H28N8O9S + H]+ 665.1773.
20,50-Di-O-acetyl-30-azido-30-deoxy-40-C-p-toluenesulfonyloxy-
methyluridine (7b). It was obtained as white solid (0.91 g, 83%).
Rf ¼ 0.6 (5% methanol in chloroform); M. Pt.: 86–88 ꢀC; [a]D30
¼
+5.62 (c 0.1, MeOH); 1H NMR (CDCl3, 400 MHz): d 9.24 (1H, brs),
7.81 (2H, d, J ¼ 8.0 Hz), 7.38 (2H, d, J ¼ 8.4 Hz), 7.20 (1H, d, J ¼
8.4 Hz), 5.75 (1H, dd, J ¼ 2.4 and 8.2 Hz), 5.61–5.56 (2H, m), 4.68
General procedure for the synthesis of bicyclic
azidonucleosides 1a–c
(1H, d, J ¼ 6.4 Hz), 4.35 (1H, d, J ¼ 12.0 Hz), 4.20 (1H, d, J ¼ 11.2 To a stirred solution of tosylated nucleosides 7a (0.5 g, 0.91
Hz), 4.16–4.11 (2H, m), 2.47 (3H, s), 2.18 (3H, s) and 2.08 (3H, s); mmol)/7b (0.5 g, 0.93 mmol) or 7c (0.5 g, 0.93 mmol) in
13C NMR (CDCl3, 100.6 MHz): d 170.2, 169.8, 162.8, 149.9, 145.3, dioxane–water (1 : 1, 4 mL) was added 2 M, NaOH (4 mL) and
141.5, 132.3, 129.9, 128.0, 103.2, 91.2, 84.0, 74.5, 67.5, 64.2, 62.8, reaction mixture was stirred at RT for 1–2 hours. On completion
21.7, 20.6 and 20.4; IR (thin lm) nmax: 3188 (br), 2922, 2851, (analytical TLC), the reaction mixture was neutralized with
2121, 1752, 1721, 1692, 1461, 1369, 1225, 1177, 1049, 987, 812, acetic acid, the solvent was removed under reduced pressure.
758 and 667 cmꢂ1; HR-ESI-TOF-MS: m/z 538.1237 ([M + H]+), The residue thus obtained was puried by silica gel column
calcd for [C21H23N5O10S + H]+ 538.1238.
chromatography using methanol in chloroform as gradient
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Table 1 Single crystal X-ray diffraction data of compound 5
reduced pressure and the residue thus obtained was puried by
silica gel column chromatography using methanol in chloro-
form as eluent to afford the locked nucleoside 1d as yellow solid
(0.198 g; 86% yield). HR-ESI-TOF-MS: m/z 305.1104 ([M + H]+),
calcd for [C11H12N8O3 + H]+ 305.1105.
Compound
5
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C
18H23N3O8S
441.45
298(2) K
0.71073 A
Monoclinic
P21
˚
30-Amino-30-deoxy-20-O,40-C-methylenethymidine (2)6e
The reduction of azido group of bicyclic nucleoside 1a (0.2 g,
0.67 mmol) was achieved by 10% Pd–C (0.02 g) in ethyl acetate
solvent (15 mL) under hydrogen atmosphere at RT for 2 hours.
On completion, the reaction was quenched by ltering off the
Pd–C. Excess solvent was removed under reduced pressure and
the residue thus obtained was puried by silica gel column
chromatography using methanol in chloroform as eluent to
afford the bicyclic nucleoside 2 as white solid (0.19 g; 95%
ꢀ
˚
Unit cell dimensions
a ¼ 9.9038(14) A, a ¼ 90
ꢀ
˚
b ¼ 13.785(3) A, b ¼ 101.077(15)
ꢀ
˚
c ¼ 16.1493(3) A, g ¼ 90
3
˚
Volume
2163.7(6) A
Z
4
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
1.355 Mg mꢂ3
0.198 mmꢂ1
928
0.24 ꢁ 0.18 ꢁ 0.14 mm3
2.97 to 25.00ꢀ
yield). HR-ESI-TOF-MS: m/z 270.1093 ([M +
H]+), calcd
[C11H15N3O5 + H]+ 270.1084.
ꢂ10 # h # 11,
ꢂ16 # k # 16, ꢂ19 # l # 17
13 901
Reections collected
Independent reections
Completeness to theta ¼ 25.00ꢀ
Max. and min. transmission
Renement method
6745 [R(int) ¼ 0.0420]
X-ray diffraction study of 5-O-acetoxy-3-azido-3-deoxy-4-C-p-
toluenesulphonyloxymethyl-1,2-O-isopropylidene-a-D-
ribofuranose (5)
98.4%
0.9728 and 0.9540
Full-matrix least-squares on F2
6745/1/549
Data/restraints/parameters
Single crystal suitable for X-ray diffraction was grown by dis-
solving compound 5 in THF and allowing it to evaporate slowly
at room temperature. X-Ray diffraction data was collected on an
Oxford Diffraction XCalibur CCD diffractometer with graphite
Goodness-of-t on F2
0.990
Final R indices [I > 2sigma(I)]
R indices (all data)
Absolute structure parameter
Largest diff. peak and hole
CCDC
R1 ¼ 0.0545, wR2 ¼ 0.0992
R1 ¼ 0.0845, wR2 ¼ 0.1118
0.05(8)
ꢂ3
˚
˚
monochromated Cu Ka radiation (l ¼ 1.54184 A) at tempera-
0.190 and ꢂ0.173 e A
995894
ture 298 K. The structure was solved by direct methods using
SHELXS-97 and rened by full-matrix least-squares method on
F2 (SHELXL-97).11 All calculations were carried out using the
WinGX package of the crystallographic programs.12 For the
molecular graphics, the program DIAMOND-2 (ref. 13) and
Mercury14 was used. Molecular structure have been drawn using
ORTEP as soware as given in Fig. 2. The selected bond lengths,
bond angles, etc. are given in Table 1.
solvent system to afford the locked nucleosides T, U and C 1a–c
in 85–89% yields.
30-Azido-30-deoxy-20-O,40-C-methylenethymidine (1a).6b,d,e It
was obtained as white solid (0.237 g, 88%). HR-ESI-TOF-MS: m/z
296.0985 ([M + H]+), calcd for [C11H13N5O5 + H]+ 296.0989.
30-Azido-30-deoxy-20-O,40-C-methyleneuridine (1b). It was
obtained as white solid (0.234 g; 89%). Rf¼0.3 (10% methanol in
chloroform); M. Pt.: 210–212 ꢀC; [a]3D1 ¼ +129.26 (c 0.1, MeOH);
1H NMR (DMSO-d6, 400 MHz): d 11.37 (1H, brs), 7.70 (1H, d, J ¼
8.0 Hz), 5.63 (1H, d, J ¼ 8.0 Hz), 5.49 (1H, s), 5.39 (1H, t, J ¼ 6.0
Hz), 4.53 (1H, s), 4.05 (1H, s) and 3.68–3.78 (4H, m); 13C NMR
(DMSO-d6, 100.6 MHz): d 163.4, 150.1, 139.0, 100.9, 89.3, 86.4,
78.3, 71.2, 59.9 and 55.8; IR (thin lm) nmax: 3384, 2924, 2852,
2371, 2119, 1686, 1458, 1271, 1105, 1055, 1021, 937, 822, 772
and 714 cmꢂ1; HR-ESI-TOF-MS: m/z 282.0833 ([M + H]+), calcd
for [C10H11N5O5 + H]+ 282.0833.
Conclusion
We have successfully demonstrated highly efficient chemo-
enzymatic synthesis of 30-azido-30-deoxy-20-O,40-C-methylene-
ribonucleosides T, U, C and A. There is an enhancement of yield
in chemo-enzymatic synthesis over classical synthesis from 41
to 68% in T, 40 to 64 % in C and 21 to 60 % in case of A. On top
of yield enhancement, the lipase can be recovered and reused
for 10 cycles of regioselective acetylation reaction on diol
precursor. Further, the bicyclic 30-azidonucleosides can be
converted into the corresponding 30-aminonucleosides in high
yield, which is an important monomer for the synthesis of
therapeutically useful sugar-modied oligonucleotides.
30-Azido-30-deoxy-20-O,40-C-methylenecytidine (1c).6a It was
obtained as white solid (0.222 g; 85% yield). HR-ESI-TOF-MS:
m/z 281.0993 ([M + H]+), calcd for [C10H12N6O4 + H]+ 281.0993.
30-Azido-30-deoxy-20-O,40-C-methyleneadenosine (1d).6c To a
stirred solution of tosylated nucleoside 7d (0.5 g, 0.75 mmol) in
dioxane–water (1 : 1, 8 mL) was added 2 M NaOH (8 mL) and
NH4OH (2 mL), and the reaction mixture was stirred at RT for 24
hours. On completion (analytical TLC), the reaction mixture was
neutralized with acetic acid, the solvent was removed under
Acknowledgements
We are grateful to the University of Delhi for providing nancial
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
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recording facility. MK and VKS thank CSIR for the award of
Junior/Senior Research Fellowships.
V. K. Rajwanshi, C. Nielsen and J. Wengel, J. Chem. Soc.,
Perkin Trans. 1, 2000, 3610; (e) S. Obika, J. Andoh,
T. Sugimoto, K. Miyashita and T. Imanishi, Tetrahedron
Lett., 1999, 40, 6465.
7 (a) V. K. Sharma, M. Kumar, C. E. Olsen and A. K. Prasad, J.
Org. Chem., 2014, 79, 6336, DOI: 10.1021/jo5008338; (b)
J. Tulla-Puche, M. Gongora-Benitez, N. Bayo-Puxan,
A. M. Francesch, C. Cuevas and F. Albericio, Angew. Chem.,
2013, 125, 5838; (c) S. K. Singh, V. K. Sharma, K. Bohra,
C. E. Olsen and A. K. Prasad, J. Org. Chem., 2011, 76, 7556;
(d) J. A. Carr and K. S. Bisht, Org. Lett., 2004, 6, 3297.
8 S. A. Surzhykov and A. A. Krayevsky, Nucleosides Nucleotides,
1994, 13, 2283.
References
1 (a) E. S. Hildebrandt-Eriksen, V. Aarup, R. Persson,
H. F. Hansen, M. E. Munk and H. Orum, Nucleic Acid Ther.,
2012, 22, 152; (b) T. P. Prakash, Chem. Biodiversity, 2011, 8,
1616.
2 J. K. Watts, Chem. Commun., 2013, 49, 5618.
3 (a) V. K. Sharma, P. Rungta and A. K. Prasad, RSC Adv., 2014,
4, 16618; (b) J. Wengel, Acc. Chem. Res., 1999, 32, 301.
4 L. F. Gebert, M. A. Rebhan, S. E. Crivelli, R. Denzler,
M. Stoffel and J. Hall, Nucleic Acids Res., 2014, 42, 609.
5 (a) T. Koch, Curr. Phys. Chem., 2013, 3, 55; (b) S. Obika,
S. M. A. Rahman, B. Song, M. Onoda, M. Koizumi,
K. Morita and T. Imanishi, Bioorg. Med. Chem., 2008, 16,
9230; (c) T. Imanishi and K. Satoshi, Jpn. Kokai Tokkyo
Koho, JP 2002255990 A 20020911, 2002; (d) T. Imanishi and
K. Satoshi, PCT Int. Appl., WO 2001007455 A1 20010201,
2001.
9 A. K. Prasad, S. Roy, R. Kumar, N. Kalra, J. Wengel,
C. E. Olsen, A. L. Cholli, L. A. Samuelson, J. Kumar,
A. C. Watterson, R. A. Gross and V. S. Parmar, Tetrahedron,
2003, 59, 1333.
¨
10 H. Vorbruggen, I. M. Lagoja and P. Herdewijn, Curr. Protoc.
Nucleic Acid Chem., 2007, 1, 13.
11 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr.,
2008, A64, 112.
6 (a) V. K. Sharma, M. Kumar, V. Maikhuri and A. K. Prasad,
Trends Carbohydr. Res., 2014, 6(2), 1; (b) V. K. Sharma,
S. K. Singh, K. Bohra, L. Chandrashekhar, V. Khatri,
C. E. Olsen and A. K. Prasad, Nucleosides, Nucleotides
Nucleic Acids, 2013, 32, 256; (c) T. Bryld, M. H. Sorensen,
P. Nielsen, T. Koch, C. Nielsen and J. Wengel, J. Chem.
Soc., Perkin Trans. 1, 2002, 1655; (d) A. G. Olsen,
12 L. J. Farrugia, J. Appl. Crystallogr., 1999, 32, 837.
13 W. T. Pennington, J. Appl. Crystallogr., 1999, 32, 1028.
14 C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock,
G. P. Shields, R. Taylor, M. Towler and J. Van de Streek, J.
Appl. Crystallogr., 2006, 39, 453.
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