Chemo-enzymatic route to bridged homolyxofuranosyl-pyrimidines

Article abstract of DOI:10.1016/j.carres.2020.108013

Synthesis of 2′-O,5′-C-bridged-β-D-homolyxofuranosyl nucleosides U and T have been achieved starting from diacetone-D-glucose in overall yields 55.7 and 57.1%, respectively. Quantitative regioselective monoacetylation of the lone primary hydroxyl group in

Full text of DOI:10.1016/j.carres.2020.108013

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Chemo-enzymatic route to bridged homolyxofuranosyl-pyrimidines
Sandeep Kumar, Harbansh Singla, Jyotirmoy Maity, Priyanka Mangla, Ashok K.
Prasad
PII:
S0008-6215(20)30109-9
DOI:
https://doi.org/10.1016/j.carres.2020.108013
CAR 108013
Reference:
To appear in: Carbohydrate Research
Received Date: 20 February 2020
Revised Date: 6 April 2020
Accepted Date: 8 April 2020
Please cite this article as: S. Kumar, H. Singla, J. Maity, P. Mangla, A.K. Prasad, Chemo-enzymatic
route to bridged homolyxofuranosyl-pyrimidines, Carbohydrate Research (2020), doi: https://
doi.org/10.1016/j.carres.2020.108013.
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2020 Published by Elsevier Ltd.

Chemo-enzymatic route to bridged homolyxofuranosyl-pyrimidines
a
a
b
a
Sandeep Kumar, Harbansh Singla, Jyotirmoy Maity, Priyanka Mangla
a,
and Ashok K. Prasad *
a
Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi- 110 007, India
b
Department of Chemistry, St. Stephen’s College, University of Delhi, Delhi - 110 007, India
Email: ashokenzyme@gmail.com
Graphical Abstract
Abstract
Synthesis of 2'-O,5'-C-bridged-β-
D
-homolyxofuranosyl nucleosides U and T have been
-glucose in overall yields 55.7 and 57.1%, respectively.
Quantitative regioselective monoacetylation of the lone primary hydroxyl group in trihydroxy
achieved starting from diacetone-
D
nucleoside intermediate, i.e. 3'-O-benzyl-β-D-glucofuranosyl nucleosides mediated by
®
Novozyme -435 has been utilized as the key step in the synthesis of homolyxofuranosyl
nucleosides. The structure of the synthesized 2'-O,5'-C-bridged-β-D-homolyxofuranosyl
1
13
uracil and -thymine has been established on the basis of their spectral (IR, H, C NMR and
HRMS) data analysis and the structure of earlier nucleoside was confirmed by its X-rays
diffraction analysis which revealed that these 2'-O,5'-C-bridged homo-nucleosides are locked
into S-type sugar puckering.
®
Keywords: Chemo-enzymatic pathway, Regioselective monoacetylation, Novozyme -435,
Bridged homolyxofuranosyl nucleosides.
*
Corresponding Author
Ashok K. Prasad: Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi
10007, India; Phone: 00-91-11-27662486; E-mail: ashokenzyme@gmail.com
1
1
. Introduction
Modified nucleosides and their analogues are of much importance due to their immense
potential as key precursors for the synthesis of oligonucleotide based therapeutic agents for

1
,2
RNA / gene targeting. Most of the bicyclic sugar modified nucleosides possess constrained
sugar puckering, which enable them to mimic a DNA or RNA type furanose ring
conformation. The oligonucleotides derived from such nucleosides show high levels of
complementarity with their corresponding DNA / RNA strands together with more stability
towards nucleases. Quite a few sugar modified nucleosides have been found to exhibit
3
excellent anti-tumour or antiviral activities.
Figure 1. Representative bicyclic and homo-nucleosides.
Bicyclic nucleosides 1 and 2, with extra methylene group in the sugar moiety are well known
as locked nucleic acids (LNA) and used for the development of antisense / anti-gene
4
-6
oligonucleotides due to their restricted conformational structures (Figure 1).
Likewise, homonucleoside monomers having an additional methylene group at the C-5' end
7
-10
11,12
as in nucleoside 3
or between C-1' and nucleobase as in nucleoside 4
have established
their significance with diverse biological activities. Extensive modifications have been
carried out in the sugar ring to synthesize homonucleoside analogues, such as 1,3-dioxolane
1
3,14
15,16
17
nucleosides 5,
homo-N,O-nucleosides 6
and bicyclic-N,O-iso-homonucleosides 7.
On the other hand, a 2'-O,5'-C-methylene-linked bicyclic nucleoside 8 was synthesized and
1
8
its conformational studies revealed that it adopted S-type furanose configuration. Two novel
bicyclic nucleotide monomers 9 (5R & 5S) were synthesized with 3'-endo conformational
1
9
restriction, where the sugar moiety had the
D-arabinose conformation. The 9-mer or 14-mer
oligonucleotides with mono-, di- or tri-incorporation of these nucleoside monomers in

oligonucleotides showed decrease in binding affinity towards complementary DNA and
RNA. Variation of the heterocyclic rings and bicyclic structures associated with these
homonucleoside analogues has enabled researchers to enhance the physiological / biological
properties of oligonucleotides involving them. Further, potential activities of homocytidine
3
a, homouridine 3b and homoarabinofuranosylcytosine 3c have been evaluated against
herpes simplex virus type 1 along with their cytoxicity against HL-60, K-562, U-937 and
7
human LY-PHA cell lines (Figure 1). Similarly, antiviral activity of oxazolidine
homonucleosides 6 were examined against variety of DNA and RNA viruses and these
2
0
nucleosides were found to be non-toxic up to 250 µM concentration.
We have reported the synthesis of several bicyclic and spiro-nucleosides where the bridging
2
1-24
methylene group introduces conformational restriction to the sugar ring of the nucleoside.
Herein, we report a chemo-enzymatic synthesis of bicyclic 5'-homolyxofuranosyl nucleosides
of U and T.
2
. Results and Discussion
It was envisioned that the targeted bridged homo-nucleosides would be synthesized from
diacetone- -glucofuranose because this is an orthogonally protected substrate that will
D
provide tetraacetate intermediate for nucleobase coupling and subsequently produce our
desired nucleoside monomer (Scheme 1).
Scheme 1. Retrosynthetic analysis for synthesis of 2'-O,5'-C-bridged homolyxofuranosyl
nucleosides.
Diacetone-
bromide in DMF in quantitative yield, which on acetolysis with AcOH:Ac O:H SO
4
D-glucofuranose (11) was converted into O-benzylated furanoside 12 using benzyl
2
5
2
2
(100:10:0.1) afforded anomeric mixture of tetraacetate 13a-b (α:β = 1:5, based on integration
1
of anomeric proton in H NMR). The Vorbrüggen nucleobase coupling of tetraacetate 13a-b with
uracil and thymine in presence of N,O-bis(trimethylsilyl)acetamide (BSA) and

trimethylsilyltrifluoromethane sulfonate (TMS-triflate) in acetonitrile afforded 2',5',6'-tri-O-
acetyl-3'-O-benzyl- -glucofuranosyl-uracil (14a) and 2',5',6'-tri-O-acetyl-3'-O-benzyl-β-D-
β
-D
glucofuranosyl-thymine (14b) in 88% and 89% yields, respectively. The complete
deacetylation of the nucleosides 14a and 14b was carried out efficiently with K CO in
2
3
methanol-water (9:1) to obtain trihydroxy nucleosides 15a and 15b in 99% yields (Scheme
).
2
Scheme 2. Synthesis of 3'-O-benzyl-β-D-glucofuranosyl-uracil and -thymine (15a-b).
For the synthesis of targeted bridged homonucleosides the primary hydroxyl group of
trihydroxy nucleosides 15a and 15b needs to be protected, which were achieved by the
mediation of lipase.
®
Two lipases, Novozyme -435 and lipozyme TL IM were screened for selective acetylation of
primary hydroxyl group of nucleosides 15a and 15b in six different organic solvents, such as
toluene, diisopropylether (DIPE), tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-Me-
THF), acetone and acetonitrile using vinyl acetate as acetyl donor in an incubator shaker at 25
®
to 45 °C. It was observed that Novozyme -435 in THF and 2-Me-THF were able to carry out
complete conversion of trihydroxy nucleosides 15a and 15b into monoacetylated nucleosides
1
6a and 16b in 98% and 99% yields, respectively. The reaction time for complete conversion
of 15a and 15b into 16a and 16b in THF was found to be 1 hour, however the same reaction
took 3 hours in 2-Me-THF. Thus, THF was found to be the solvent of choice (Scheme 3).

Scheme 3. Optimized condition for Novozyme-435 catalyzed monoacetylation of nucleosides
5a and 15b.
1
Monoacetylated nucleosides 16a and 16b was converted into bridged homonucleosides 10a
and 10b in three steps (Scheme 4). First, the monoacetylated nucleosides 16a and 16b were
permesylated to afford nucleosides 17a and 17b using methanesulfonyl chloride (MsCl) in
pyridine at 25 °C in 97% yields, which on treatment with 2M aq. NaOH cyclises to afford 3'-
O-benzyl-2'-O,5'-C-bridged-β-D-homolyxofuranosyl nucleosides 18a and 18b in 74% and
7
3% yields, respectively. Finally debenzylation of nucleosides 18a and 18b was carried out
using 20% Pd(OH) -C/HCOOH in a solvent mixture of THF:MeOH (9:1) to afford novel
2
bridged homolyxofuranosyl nucleosides 10a and 10b in 98% and 99% yields, respectively.
Scheme 4. Synthesis of 2'-O,5'-C-bridged-β-D-homolyxofuranosyl nucleosides 10a-10b.
The structures of all the synthesized compounds 12, 13a-b, 14a-b, 15a-b, 16a-b, 17a-b, 18a-
1
13
b, and 10a-b were unambiguously established on the basis of their spectral (IR, H-, C-
1
1
1
13
NMR, H- H COSY NMR, H- C HETCOR NMR, NOESY NMR and HRMS) data
analysis. The structure of known compound 12 was further confirmed by the comparison of
its physical and spectral data with those reported in literature.
The structure of 2'-O,5'-C-bridged-β-D-homolyxofuranosyl nucleoside 10a was further
confirmed by single crystal X-rays diffraction analysis which revealed that sugar puckering

of this molecule is locked into S-type conformation (Figure 2). The conformational analysis
of the furanoside ring was also supported by the dihedral and pseudorotational angle
2
6
calculation.
The crystal structure consisted of two nucleoside monomers with
pseudorotational phase angles (P) 193.74 and 201.13, and two crystal water molecules which
confirmed that the sugar puckering of both the monomeric structures of 10a to be C3'-exo i.e.
S-type conformation. The calculated values of dihedral angles (ν = -4.79, ν = 41.61, ν = -
0
1
2
5
8.23, ν = 56.51, ν = -33.70 and ν = 3.28, ν = 34.91, ν = -56.05, ν = 58.46, ν = -39.84),
3 4 0 1 2 3 4
puckering amplitude (νmax = 59.94 and 60.09), backbone angle (γ = 36.52 and 31.27) as well
as the torsion describing the anomeric bond (χ = -161.00 and -175.40) support the inference.
The detailed crystallographic data of compound 10a have been deposited in the Cambridge
Crystallopraphic Data Centre with CCDC no. 1978207 (Figure 2).
(a)
(b)

Figure 2: (a) ORTEP diagram of compound 10a drawn in 50% thermal probability ellipsoids
with atomic numbering scheme showing two crystallographically independent units (b)
Exhibition of S-type puckering of furanose ring in nucleoside 10a.
Table 1
Single crystal X-ray diffraction data of compound 10a
Empirical formula
Formula weight
Temperature
C H N O .H O
10 12 2 6 2
274.23
293(2) K
Wavelength
0.71073 A
Monoclinic
P 21
Crystal system
Space group
Unit cell dimension
a = 5.4600(3) A
α = 90 deg.
b = 16.4865(8) A
β = 99.402(4) deg.
c = 13.2042(6) A
γ = 90 deg.
3
Volume
1172.63(10) A
Z
4
3
Density (calculated)
Absorption coefficient
F(000)
1.553 mg/m
0.133 mm
576
-1
Theta range for data collection
3.363 to 25.345 deg.
-6≤h≤6
Index ranges
-
19≤k≤19
15≤l≤15
-
Reflections collected
13110
Independent reflections
Completeness to theta = 25.242
Max. and min. transmission
Refinement method
4289 [R(int) = 0.0645]
99.80 %
0.991 and 0.983
Full-matrix least-squares on F
4289 / 1 / 353
2
Data/restrains/parameters
2
Goodness-of-fit on F
1
.057
Final R indices [1> 2sigma(I)]
R indices (all data)
Absolute structure parameter
Largest diff. peak and hole
CCDC
R1 = 0.0575, wR2 = 0.0932
R1 = 0.1036, wR2 = 0.1187
0.5
-3
0.218 and -0.239 e.A
1978207

Conclusion
A chemo-enzymatic pathway has been developed for the synthesis of 2'-O,5'-C-bridged-β-D-
®
lyxofuranosyl-uracil and -thymine. In a pivotal step of this methodology, Novozyme -435
was used for quantitative regioselective acetylation of one of the three hydroxy functional
group present in the nucleoside. This process provided facile access to 2'-O,5'-C-bridged-β-
lyxofuranosyl-uracil and -thymine with overall yields of 55.7% and 57.1%, respectively
starting from diacetone- -glucose. The synthesized nucleosides were found to be locked into
D-
D
S-type sugar puckering, which is a striking feature of our nucleoside monomer for the
development of therapeutic oligonucleotides.
Experimental
All reagents were purchased from Sigma-Aldrich Chemicals Pvt. Limited, India and from
local commercial sources and were used without any further purification unless otherwise
specified. Melting points were determined on Buchi M-560 instrument and are uncorrected.
The IR spectra of compounds were recorded on Perkin-Elmer model 2000 FT-IR
-1
spectrometer and are expressed as wavenumber (cm ). Specific rotation was measured on
Rudolph Autopol II polarimeter. R values of compounds are reported for analytical TLC
f
using the specified solvents and 0.25 mm silica gel 60 F254 plates that were visualized by UV
irradiation or by charring with 5% alcoholic sulfuric acid solution. Solvents were removed
under reduced pressure using rotary evaporator, followed by further removal of the residual
solvent under high vacuum. Column chromatography was performed on silica gel (100-200
1
13
mesh). The H, C-NMR spectra were recorded on Jeol alpha-400 spectrometer at 400 MHz,
00.6 MHz, respectively by using tetramethylsilane (TMS) as internal standard. The chemical
1
shift values are on δ scale and the coupling constant (J) are in Hz. HRMS analysis was
carried out using Agilent G6530AA LC Q-TOF mass spectrometer using ESI method.
Synthesis of 3-O-benzyl-1,2,5,6-tetra-O-acetyl-α, β-D-glucofuranose (13a-b)
To the stirred solution of 3-O-benzyl-1,2:5,6-O-di-isopropylidene-α-D-glucofuranose (3.5 g,
.98 mmol) in acetic acid (57.12 mL, 998 mmol) at 0° C, acetic anhydride (9.5 ml, 99.8 mmol)
9
and concentrated sulphuric acid (0.053 mL, 0.998 mmol) were added. The reaction mixture was
stirred for 3-4 hour at room temperature. After completion, reaction was quenched by adding
cold water and neutralized by sodium bi-carbonate. The compound was extracted with ethyl
acetate (3 x 100 mL), brine solution (2 x 100 mL) and combined organic layer was dried over

anhydrous sodium sulphate to afford the crude product. The solvent was removed under
reduced pressure. The crude product thus obtained was purified by column chromatography
using ethyl acetate in petroleum ether as gradient solvent system to afford an anomeric mixture
1
(
α:β = 1:5, based on integration of anomeric proton in H NMR) of 13a-b as colourless oil, 4.2
g in 96% yield, R = 0.38 (20% ethyl acetate in petroleum ether).
f
3
-O-Benzyl-1,2,5,6-tetra-O-acetyl-α, β-
D
-glucofuranose (13a-b)
24
[
α]D = + 37.18 (c 0.1, MeOH). IR (thin film) νmax: 1739, 1435, 1369, 1211, 1045, 881, 742,
-1 1
7
00, 592, 501 cm . H NMR (CDCl , 400 MHz): δ 7.30-7.37 (5H, m), 6.16 (1H, s), 5.35-5.39
3
(1H, m), 5.26 (1H, s), 4.78 (1H, d, J = 12.1 Hz), 4.66 (1H, dd, J = 12.3, 2.3 Hz), 4.56 (1H, d,
J = 12.1 Hz), 4.45 (1H, dd, J = 7.8, 5.1 Hz), 4.12 (1H, dd, J = 12.3, 5.0 Hz) 4.02 (1H, d, J =
13
5
.0 Hz), 2.12 (3H, s), 2.09 (3H, s), 2.05 (3H, s), 1.88 (3H, s); C NMR (CDCl 100.6 MHz):
3,
δ 170.7, 169.6, 169.5, 136.9, 128.5, 128.0, 127.9, 99.3, 80.8, 79.0, 78.5, 71.7, 69.1, 63.0,
+
2
1.1, 20.9, 20.8, 20.7; HR-ESI-TOF-MS: m/z cal. for C H NO [M+NH ] : 456.1864;
2
1
30
10
4
found: 456.1861.
Synthesis of 2',5',6'-tri-O-acetyl-3'-O-benzyl-β-D-glucofuranosyl pyrimidine nucleosides
(14a-b).
To the stirred solution of tetra-O-acetylated sugar derivative 13a-b (1.0 g, 2.28 mmol) and
uracil thymine (3.42 mmol) in anhydrous acetonitrile (40 mL), N,O-
/
bis(trimethylsilyl)acetamide (2.23 mL, 9.12 mmol) was added dropwise. The reaction
mixture was stirred at reflux for one hour, and then cooled to 0°C. In the cooled reaction
mixture trimethylsilyltrifluromethane sulfonate (0.70 mL, 3.88 mmol) was added dropwise
under stirring and the solution was refluxed for 6-8 hours. The reaction was quenched with
cold saturated aq. sodium bicarbonate solution (150 mL) and extracted with ethyl acetate (3 x
1
00 mL). The combined organic layer was washed with saturated aq. sodium bicarbonate
solution (2 x 100 mL), brine solution (2 x 100 mL), and the combined organic layer was dried
over anhydrous sodium sulfate to afford the crude residue. The crude residue was purified by
silica gel column chromatography using ethyl acetate in petroleum ether as eluent to afford
pure nucleosides 14a and 14b in good yields.
2
',5',6'-Tri-O-acetyl-3'-O-benzyl-β-D-glucofuranosyl uracil (14a)
It was obtained as white solid (0.98 g) in 88% yield. R = 0.23 (2% MeOH/chloroform);
f
24
-1
[
α]D = + 51.78 (c 0.1, MeOH); m/p: 112-115 °C; IR (KBr, cm ): 3064, 1739, 1685, 1454,

1
4
6
1
369, 1325, 1215, 1107, 1045, 939, 916, 866, 812, 748, 700, 634, 601, 570, 545, 495, 418,
1
05; H NMR (400 MHz, CDCl ): δ 9.24 (1H, s), 7.56 (1H, d, J = 7.8 Hz), 7.29-7.34 (5H, m),
3
.19 (1H, s), 5.66 (1H, d, J = 7.5 Hz), 5.43 (1H, d, J = 8.1 Hz), 5.17 (1H, s), 4.68 (2H, t, J =
2.0 Hz), 4.54 (1H, d, J = 11.3 Hz), 4.24 (1H, d, J = 7.8 Hz), 4.09 (1H, d, J = 9.6 Hz), 3.90
1
3
(
1H, s), 2.19 (3H, s), 2.08 (3H, s), 1.86 (3H, s); C NMR (100 MHz, CDCl ): δ 170.7, 169.8,
3
1
6
4
69.3, 162.9, 150.2, 140.1, 135.8, 129.0, 128.8, 128.6, 103.0, 88.8, 79.4, 79.3, 78.1, 71.6,
+
7.5, 62.6, 20.9, 20.8; HRMS (ESI): m/z calcd for C H N O [M+H] : 491.1660; found:
23
27
2
10
91.1671.
2
',5',6'-Tri-O-acetyl-3'-O-benzyl-β-D-glucofuranosyl thymine (14b)
It was obtained as white solid (1.02 g) in 89% yield. R = 0.24 (2% MeOH/chloroform);
f
2
4
-1
[
α]D = + 24.67 (c 0.1, MeOH); m/p: 118-120 °C; IR (KBr, cm ): 1741, 1691, 1460, 1371,
1
1
219, 1049, 746, 700, 592, 480; H NMR (400 MHz, CDCl ): δ 8.75 (1H, s), 7.30-7.36 (6H,
3
m), 6.24 (1H, d, J = 1.4 Hz), 5.47-5.51 (1H, m), 5.15 (1H, s), 4.71 (1H, d, J = 11.2 Hz), 4.66
(1H, dd, J = 12.5, 2.2 Hz), 4.55 (1H, d, J = 11.2 Hz), 4.23 (1H, dd, J = 9.2, 3.2 Hz), 4.10
(1H, dd, J = 12.5, 4.4 Hz), 3.91 (1H, d, J = 3.2 Hz), 2.19 (3H, s), 2.09 (3H, s), 1.94 (3H, s),
1
3
1
.68 (3H, s); C NMR (100 MHz, CDCl ): δ 170.7, 169.9, 169.4, 163.6, 150.4, 136.0, 135.9,
3
1
28.7, 128.6, 111.8, 88.4, 79.5, 79.0, 78.8, 71.8, 67.7, 62.7, 20.9, 20.9, 12.4; HRMS (ESI):
+
m/z calcd for C H N O [M+H] : 505.1817; found: 505.1831.
2
4
29
2
10
Synthesis of 3'-O-benzyl-β-D-glucofuranosyl pyrimidines nucleosides 15a-b.
To the stirred solution of compound 14a (1.2 g, 2.44 mmol) or 14b (1.2 g, 2.38 mmol ) in
methanol:water (9:1, 120 mL), K CO (1.01 g, 7.32 mmol for 14a) or (0.98 g, 7.14 mmol
2
3
for 14b) was added at 0 °C and the reaction mixture was kept under stirring at 25 °C for 1h.
On completion of the reaction, solvent was removed under reduced pressure. The residue thus
obtained was purified by column chromatography with a gradient solvent system of methanol
in chloroform to afford trihydroxy nucleosides 15a and 15b in quantitative yields.
3
'-O-Benzyl-β-
D-glucofuranosyl uracil (15a)
It was obtained as white solid (0.88 g) in 99% yield. R = 0.23 (10% MeOH/chloroform);
f
24
-1
[
α]D = + 45.13 (c 0.1, MeOH); m/p: 150-153 °C; IR (KBr, cm ): 3363, 3059, 2939, 1666,
1
1
462, 1394, 1261, 1207, 1070, 1043, 896, 813, 740, 698, 640, 572, 547; H NMR (400 MHz,
DMSO-d ): δ 11.30 (1H, s), 7.56 (1H, d, J = 8.1 Hz), 7.27-7.34 (5H, m), 5.87 (1H, d, J = 4.0
6
Hz), 5.67 (1H, s), 5.46 (1H, dd, J = 8.1, 2.2 Hz), 4.85 (1H, s), 4.60 (2H, q, J = 11.8 Hz), 4.52

(1H, s), 4.21 (1H, d, J = 3.5 Hz), 4.07 (1H, dd, J = 9.2, 3.1 Hz), 3.91 (1H, s), 3.88 (1H, d, J =
1
3
3
.1), 3.62 (1H, d, J = 11.2 Hz), 3.44 (1H, dd, J = 11.4, 5.4 Hz); C NMR (100 MHz, DMSO-
d6): δ 163.2, 150.4, 140.7, 138.0, 128.2, 127.4, 127.3, 100.8, 91.4, 82.4, 82.2, 77.1, 71.1,
+
6
8.1, 63.7; HRMS (ESI): m/z calcd for C H N O [M+H] : 365.1343; found: 365.1341.
1
7
21
2
7
3
'-O-Benzyl-β-D-glucofuranosyl thymine (15b)
It was obtained as white solid (0.89 g) in 99% yield. R = 0.23 (10% MeOH/chloroform);
f
24
-1
[
α]D = + 40.99 (c 0.1, MeOH); m/p: 94-96 °C; IR (KBr, cm ): 3361, 3034, 2933, 1660,
1
1
465, 1392, 1327, 1261, 1078, 1047, 902, 742, 696, 592, 478; H NMR (400 MHz, DMSO-
d6): δ 11.30 (1H, s), 7.40 (1H, d, J = 1.2 Hz), 7.26-7.34 (5H, m), 5.88 (1H, d, J = 4.0 Hz),
5
.75 (1H, d, J = 0.8 Hz), 4.90 (1H, d, J = 5.9 Hz), 4.58-4.64 (2H, m), 4.55 (1H, t, J = 5.6 Hz),
.23 (1H, d, J = 3.8 Hz), 4.02 (1H, dd, J = 9.2, 3.0 Hz), 3.89 (2H, d, J = 3.0 Hz), 3.61 (1H, d,
4
1
3
J = 11.3 Hz), 3.42 (1H, dt, J = 11.4, 5.8 Hz), 1.47 (3H, d, J = 1.0 Hz); C NMR (100 MHz,
DMSO-d ): δ 163.6, 150.4, 138.0, 136.7, 128.2, 127.4, 108.6, 90.9, 82.5, 81.8, 77.3, 71.0,
6
+
6
3
8.2, 63.7, 12.0; HRMS (ESI): m/z calcd for C H N O [M+H] : 379.1500; found:
18
23
2
7
79.1505.
Synthesis of monoacetylated pyrimidine nucleosides 16a-b.
To a solution of 2',5',6'-trihydroxy-3'-O-benzyl-β-D-glucofuranosyl nucleosides 15a (1.0 g,
2
2
4
.74 mmol) and 15b (1.0 g, 2.64 mmol ) in THF (80 mL) was added vinyl acetate (0.30 mL,
®
.74 mmol for 15a) / (0.29 mL, 3.17 mmol for 15b) followed by addition of Novozyme -
35 (0.5 g, 50% w/w). The reaction mixture was stirred at 45 °C in an incubator shaker at 200
rpm for 50-60 minutes. 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 methanol in chloroform as
gradient solvent system to afford the monoacetylated nucleosides 16a and 16b in quantitative
yields.
6
'-O-Acetyl-3'-O-benzyl-β-D-glucofuranosyl uracil (16a)
It was obtained as white solid (1.09 g) in 98% yield. R = 0.25 (5% MeOH/chloroform);
f
24
-1
[
α]D = + 26.27 (c 0.1, MeOH); m/p: 133-136 °C; IR (KBr, cm ): 3323, 3034, 2947, 2873,
1
7
7
739, 1697, 1674, 1460, 1419, 1386, 1313, 1257, 1228, 1103, 1072, 1043, 972, 900, 831,
1
65, 734, 694, 634, 605, 572, 557, 418; H NMR (400 MHz, DMSO-d ): δ 11.32 (1H, s),
6
.54 (1H, d, J = 8.2 Hz), 7.27-7.35 (5H, m), 5.94 (1H, s), 5.68 (1H, s), 5.46 (1H, d, J = 8.1

Hz), 5.32 (1H, s), 4.59 (2H, q, J = 11.7 Hz), 4.29 (1H, dd, J = 11.4, 2.0 Hz), 4.24 (1H, s),
.15 (1H, d, J = 12.8 Hz), 4.10 (1H, dd, J = 9.3, 2.9 Hz), 3.98 (1H, dd, J = 11.4, 5.7 Hz), 3.90
4
1
3
(
1H, d, J = 2.8 Hz), 2.03 (3H, s); C NMR (100 MHz, DMSO-d ): δ 170.5, 163.2, 150.4,
6
1
40.7, 137.9, 128.2, 127.5, 127.4, 100.8, 91.5, 82.3, 82.0, 76.9, 71.1, 66.5, 64.9, 20.8;
+
HRMS (ESI): m/z calcd for C H N O [M+H] : 407.1449; found: 407.1458.
1
9
23
2
8
6
'-O-Acetyl-3'-O-benzyl-β-D-glucofuranosyl thymine (16b)
It was obtained as white solid (1.09 g) in 99% yield. R = 0.26 (5% MeOH/chloroform);
f
24
-1
[
α]D = + 77.90 (c 0.1, MeOH); m/p: 144-146 °C; IR (KBr, cm ): 3414, 3157, 3084, 3005,
2
5
7
1
9
953, 2904, 2827, 1697, 1633, 1487, 1423, 1386, 1240, 1080, 1039, 966, 904, 866, 758, 700,
1
97, 540, 499, 466, 422; H NMR (400 MHz, DMSO-d ): δ 11.32 (1H, s), 7.38 (1H, s),7.28-
6
.33 (5H, m), 5.93 (1H, d, J = 3.8 Hz), 5.75 (1H, s), 5.35 (1H, d, J = 5.9 Hz), 4.60 (2H, q, J =
1.2 Hz), 4.31 (1H, d, J = 11.1 Hz), 4.27 (1H, d, J = 3.3 Hz), 4.15 (1H, s), 4.06 (1H, dd, J =
.3, 2.7 Hz), 3.95 (1H, dd, J = 11.5, 6.3 Hz), 3.91 (1H, d, J = 2.6 Hz), 2.04 (3H, s), 1.50 (3H,
13
s); C NMR (100 MHz, DMSO-d ): δ 170.5, 163.7, 150.4, 137.9, 136.5, 128.2, 127.5, 127.5,
6
1
08.6, 91.1, 82.1, 81.9, 77.0, 71.1, 66.6, 65.0, 20.8, 12.1; HRMS (ESI): m/z calcd for
+
C H N O [M+H] : 421.1605; found: 421.1617.
20
25
2
8
Synthesis of 6'-O-acetyl-3'-O-benzyl-2',5'-di-O-methanesulfonyl-β-D-glucofuranosyl
pyrimidine nucleosides 17a-b.
A solution of 16a (1.0 g, 2.46 mmol) or 16b (1.0 g, 2.37 mmol) and methanesulfonyl chloride
0.48 mL, 6.15 mmol for 16a) and (0.46 mL, 5.92 mmol for 16b) in anhydrous pyridine (10
mL) was stirred at 25 °C for 2 hours. On completion, the reaction mixture was poured over
0% ice cold hydrochloric acid solution (100 mL) to neutralize pyridine and extracted with
(
1
ethyl acetate (3 x 100 mL). The combined organic layer was washed with saturated sodium
bicarbonate solution (200 mL) and dried over sodium sulfate. The excess of solvent was
removed under reduced pressure. The residue thus obtained was purified over silica gel
column chromatography using methanol in chloroform as gradient solvent system to afford
the nucleosides 17a and 17b in pure form.
6
'-O-Acetyl-3'-O-benzyl-2',5'-di-O-methanesulfonyl-β-D-glucofuranosyl uracil (17a)
It was obtained as white solid (1.34 g) in 97% yield. R = 0.31 (5% MeOH/chloroform);
f
24
-1
[
α]D = + 139.11 (c 0.1, MeOH); m/p: 82-85 °C; IR (KBr, cm ): 3030, 2937, 2314, 1741,
1
683, 1456, 1355, 1263, 1226, 1174, 1111, 1049, 1002, 964, 923, 879, 813, 742, 700, 638,

1
6
7
4
4
05, 522, 507, 418; H NMR (400 MHz, CDCl ): δ 9.66 (1H, s), 7.37 (1H, d, J = 8.2 Hz),
3
.27-7.31 (5H, m), 5.92 (1H, s), 5.58 (1H, d, J = 8.0 Hz), 5.37-5.40 (1H, m), 4.97 (1H, s),
.80 (1H, s,), 4.78 (1H, s), 4.50 (1H, d, J = 10.7 Hz), 4.44 (1H, d, J = 9.2 Hz), 4.34 (1H, s),
1
3
.16 (1H, dd, J = 12.8, 5.3 Hz), 3.24 (3H, s), 3.10 (3H, s), 2.15 (3H, s); C NMR (100 MHz,
DMSO-d ): δ 170.5, 163.2, 150.5, 139.3, 136.1, 128.9, 128.8, 128.7, 102.3, 89.9, 83.1, 80.4,
6
+
7
5
9.8, 73.4, 72.9, 63.0, 39.3, 38.7, 20.8; HRMS (ESI): m/z calcd for C H N O S [M+H] :
2
1
27
2
12 2
63.1000; found: 563.1011.
6
'-O-Acetyl-3'-O-benzyl-2',5'-di-O-methanesulfonyl-β-D-glucofuranosyl thymine (17b)
It was obtained as white solid (1.33 g) in 97% yield. R = 0.31 (5% MeOH/chloroform);
f
24
-1
[
α]D = + 82.34 (c 0.1, MeOH); m/p: 96-98 °C; IR (KBr, cm ): 3172, 3030, 2941, 2823,
2
9
314, 1732, 1683, 1645, 1463, 1417, 1355, 1328, 1263, 1240, 1176, 1089, 1041, 1002, 962,
1
18, 833, 792, 758, 702, 624, 586, 549, 518, 501, 422; H NMR (400 MHz, CDCl ): δ 10.01
3
(
1H, s), 7.33 (5H, m), 7.21 (1H, d, J = 1.2 Hz), 6.10 (1H, s), 5.41-5.44 (1H, m), 5.13 (1H, s),
.82 (1H, d, J = 10.6 Hz), 4.78 (1H, d, J = 12.8 Hz), 4.59 (1H, d, J = 10.6 Hz), 4.43 (1H, d, J
2.9 Hz), 4.40 (1H, d, J = 4.3 Hz), 4.16 (1H, dd, J = 12.9, 6.1 Hz), 3.31 (3H, s), 3.12 (3H,
4
=
1
3
s), 2.15 (3H, s), 1.54 (3H, s); C NMR (100 MHz, CDCl ): δ 170.5, 163.5, 151.1, 136.3,
3
1
1
35.4, 128.7, 128.6, 128.5, 111.6, 89.1, 83.5, 79.7, 79.7, 73.1, 72.6, 63.1, 39.3, 38.7, 20.9,
+
2.2; HRMS (ESI): m/z calcd for C H N O S [M+H] : 577.1156; found: 577.1170.
2
2
29
2
12 2
Synthesis
of
3'-O-benzyl-2'-O,5'-C-bridged-β-
D
-homolyxofuranosyl
pyrimidine
nucleosides 18a-b.
To a solution of compound 17a (0.8 g, 1.42 mmol) or 17b (0.8 g, 1.39 mmol) in
dioxane:water (1:1, 20 mL), 2M NaOH (0.8 mL) was added at 0 °C and the reaction mixture
was stirred for 24 h at 25 °C. On completion, acetic acid (10 mL) was added to neutralize the
reaction mixture and co-evaporated with toluene under reduced pressure. The residue thus
obtained was purified by silica gel column chromatography using methanol in chloroform as
gradient solvent system to afford nucleosides 18a and 18b in pure form.
3
'-O-Benzyl-2'-O,5'-C-bridged-β-D-homolyxofuranosyl uracil (18a)
It was obtained as white solid (0.36 g) in 73% yield. R = 0.26 (5% MeOH/chloroform);
f
24
-1
[
α]D = + 265.42 (c 0.1, MeOH); m/p: 145-146 °C ; IR (KBr, cm ): 3334, 3039, 2924, 2812,
1
8
772, 1670, 1620, 1460, 1371, 1315, 1273, 1207, 1178, 1145, 1099, 1041, 1024, 979, 933,
1
94, 850, 812, 752, 700, 636, 590, 555, 470, 420; H NMR (400 MHz, DMSO-d ): δ 11.41
6

(1H, s), 7.80 (1H, d, J = 8.0 Hz), 7.31-7.38 (5H, m), 5.77 (1H, s), 5.62 (1H, d, J = 8.1 Hz),
4
.80 (1H, t, J = 5.4 Hz), 4.71 (1H, s), 4.67 (1H, s), 4.61 (2H, q, J = 11.9 Hz), 4.41 (1H, s),
1
3
4
.03 (1H, t, J = 6.1 Hz), 3.61 (1H, dt, J = 11.7, 6.0 Hz), 3.45 (1H, dt, J = 11.1, 5.6 Hz); C
NMR (100 MHz, DMSO-d ): δ 163.2, 150.3, 140.2, 137.5, 128.4, 127.7, 127.6, 100.5, 87.5,
6
+
8
5.2, 79.2, 78.3, 76.1, 71.2, 60.3; HRMS (ESI): m/z calcd for C H N O [M+H] :
1
7
19
2
6
3
47.1238; found: 347.1246.
3
'-O-Benzyl-2'-O, 5'-C-bridged-β-D-homolyxofuranosyl thymine (18b)
It was obtained as white solid (0.37 g) in 74% yield. R = 0.28 (5% MeOH/chloroform);
f
24
-1
[
α]D = + 211.22 (c 0.1, MeOH); m/p: 155-157 °C ; IR (KBr, cm ): 3437, 3026, 2918, 2821,
1
680, 1463, 1371, 1319, 1269, 1205, 1151, 1105, 1070, 1024, 852, 732, 694, 594, 559, 486;
1
H NMR (400 MHz, DMSO-d ): δ 11.40 (1H, s), 7.65 (1H, s),7.31-7.40 (5H, m), 5.75 (1H,
6
s), 4.78 (1H, s), 4.71 (1H, d, J = 2.0 Hz), 4.66 (1H, s), 4.61 (2H, q, J = 11.8 Hz), 4.41 (1H, d,
J = 2.0 Hz), 4.08 (1H, t, J = 6.3 Hz), 3.61 (1H, dt, J = 10.6, 5.4 Hz), 3.46 (1H, dd, J = 10.5,
1
3
5
.0 Hz), 1.82 (3H, s); C NMR (100 MHz, DMSO-d ): δ 163.9, 150.2, 137.5, 135.5, 128.4,
6
1
27.7, 127.6, 108.0, 87.5, 85.1, 79.2, 78.3, 76.2, 71.2, 60.3, 12.3; HRMS (ESI): m/z calcd for
+
C H N O [M+H] : 361.1394; found: 361.1408.
18
21
2
6
Synthesis of 2'-O,5'-C-bridged-β-D-homolyxofuranosyl pyrimidine nucleosides 10a-b.
To a solution of compound 18a (0.2 g, 0.58 mmol) or 18b (0.2 g, 0.55 mmol) in anhydrous
THF:MeOH (9:1, 10 mL) was added Pd(OH) -C (20 wt%, 0.04 g) and 88% formic acid (0.16
2
mL, 4.4 mmol). The reaction mixture was then refluxed for 10 min. After completion of
reaction checked by TLC, catalyst was carefully filtered off and was washed with excess of
MeOH. The combined filtrate was then concentrated under reduced pressure. The crude
product obtained was purified on silica gel column chromatography with a gradient solvent
system of methanol in chloroform to obtain bridged nucleosides 10a and 10b in pure form.
2
'-O,5'-C-Bridged-β-D-homolyxofuranosyl uracil (10a)
It was obtained as white solid (0.15 g) in 99% yield. R = 0.32 (10% MeOH/chloroform);
f
24
-1
[
α]D = + 330.94 (c 0.1, MeOH); m/p: 221-223 °C; IR (KBr, cm ): 3153, 3034, 2927, 2816,
1
8
770, 1676, 1620, 1462, 1398, 1363, 1313, 1269, 1203, 1139, 1093, 1041, 1018, 935, 893,
1
52, 810, 754, 696, 632, 553, 520, 460, 426; H NMR (400 MHz, DMSO-d ): δ 11.38 (1H,
6
s), 7.78 (1H, d, J = 8.1 Hz), 5.95 (1H, s), 5.74 (1H, s), 5.61 (1H, d, J = 8.1 Hz), 4.95 (1H, s),
.45 (1H, d, J = 2.2 Hz), 4.39 (1H, d, J = 2.1 Hz), 4.37 (1H, s), 3.98 (1H, t, J = 5.8 Hz), 3.65
4

13
(
1H, dd, J = 11.5, 6.1 Hz), 3.50 (1H, dd, J = 11.5, 5.5 Hz); C NMR (100 MHz, DMSO-d ):
6
δ 163.3, 150.4, 140.2, 100.4, 87.6, 85.2, 80.1, 78.5, 72.3, 60.3; HRMS (ESI): m/z calcd for
+
C H N O [M+H] : 257.0768; found: 257.0761.
10
13
2
6
2
'-O,5'-C-Bridged-β-D-homolyxofuranosyl thymine (10b)
It was obtained as white solid (0.15 g) in 98% yield. R = 0.31 (10% MeOH/chloroform);
f
24
-1
[
α]D = + 140.99 (c 0.1, MeOH); m/p: 225-228 °C; IR (KBr, cm ): 3396, 3035, 2821, 1676,
1
5
5
2
=
469, 1359, 1271, 1205, 1143, 1099, 1043, 1014, 983, 947, 894, 831, 756, 732, 692, 609,
1
74, 491, 420; H NMR (400 MHz, DMSO-d ): δ 11.37 (1H, s), 7.63 (1H, d, J = 1.1 Hz),
6
.96 (1H, s), 5.71 (1H, d, J = 0.9 Hz), 4.95 (1H, s), 4.45 (1H, d, J = 2.3 Hz), 4.39 (1H, d, J =
.2 Hz), 4.36 (1H, s), 4.02 (1H, t, J = 5.8 Hz), 3.66 (1H, dd, J = 11.5, 6.1 Hz), 3.51 (1H, dd, J
1
3
11.5, 5.5 Hz), 1.81 (3H, d, J = 0.9 Hz); C NMR (100 MHz, DMSO-d ): δ 163.9, 150.3,
6
1
35.5, 107.9, 87.5, 85.0, 80.1, 78.5, 72.2, 60.3, 12.3; HRMS (ESI): m/z calcd for C H N O
11 15 2 6
+
[
M+H] : 271.0925; found: 271.0935.
Notes
The authors declare no competing financial interest.
Acknowledgement
We are grateful to University of Delhi for providing financial support under DU-DST Purse
grant to strengthen research and development. We are thankful to CIF-USIC, University of
Delhi, for providing the NMR spectral and HRMS recording facilities. S. K. thanks CSIR,
New Delhi for award of SPM Research Fellowship [File No. 09/045(0269)/2018-EMR-1]. H.
S. thanks CSIR, New Delhi for the award of Junior Research Fellowship.
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1
2
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89-598.
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944-1954.
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0. Yu, J.; Sahu, P. K.; Kim, G.; Qu, S.; Chol, Y.; Song, J.; Lee, S. K.; Noh, M.; Park, S.;
Jeong, L.S. Future Med. Chem., 2015, 7, 1643-1655.
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Zhang, X.; Leese, D.; Glen, R.; Hart, J.; Forrest, N.; Guyot, T. Tetrahedron Lett.,
2
011, 52, 6415-6419. (b) Winkley, M. W. Carbohy. Res. 1973, 31, 245-254.
1
1
2. Balo, C.; Fernandez, F.; Lens, E.; Lopez, C. Chem. Pharm. Bull., 1998, 46, 687-689.
3. Mikhailov, S. N.; Efimtseva, E. V.; Meshkov, S. V.;Kern, E. R. Nucleosides and
Nucleotides, 1992, 13, 615-623.
1
4. Efimtseva, E. V.; Mikhailov, S. N.; Meshkov, S. V.; Lonnberg, H. Acta Chemika
Scandinavica 1992, 46, 1122-1126.
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1
1
1
1
5. Chiacchio, U.; Saita, M. G.; Crispino, L.; Gumina, G.; Mangiafico, S.; Pistara, V.;
Romeo, G.; Pipero, A.; Clercq, E. D., Tetrahedron 2006, 62, 1171-1181.
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Romeo, R.; Procopio, A.; Romeo, G. Tetrahedron 2004, 60, 441-448.
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231-5240.
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2

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0. Lysakowska, M.; Balzarini, J.; Piotrowska, D. G. Arch Pharm. Chem. Life Sci. 2014,
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1. Sharma, V. K.; Kumar, M.; Olsen, C. E.; Prasad, A. K. J. Org. Chem. 2014, 79, 6336-
341.
6
2
2. Mangla, P.; Rungta, P.; Maikhuri, V. K.; Shivani; Prasad, A. K. Trends in Carbohy.
Res. 2017, 9, 34-42.
2
3. Mangla, P.; Maity, J.; Rungta, P.; Verma, V.; Prasad, A. K. ChemistrySelect 2019, 4,
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4. (a) Sharma, V. K.; Kumar, M.; Sharma, D.; Olsen, C. E.; Prasad, A. K. J. Org. Chem.
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2
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9033.
6. Pseudorotational parameters were calculated by using the following
website:https:/cactus.nci.nih.gov/prosit/.

Highlights:
1
2
. 2'-O,5'-C-Bridged-β-
synthesized.
. X-rays diffraction analysis revealed that 2'-O,5'-C-bridged homo-nucleosides are
locked into S-type sugar puckering.
. Environment friendly regioselective monoacetylation carried out by Novozyme -435.
. Overall yields for synthesis of 2'-O,5'-C-bridged-β-
D-homolyxofuranosyl nucleosides U and T have been
®
3
4
D-homolyxofuranosyl nucleosides
U and T, starting from diacetone-D-glucose are 55.7 and 57.1%, respectively.

Notes
The authors declare no competing financial interest