Selective biocatalytic deacylation studies on furanose triesters: A novel and efficient approach towards bicyclonucleosides

Article abstract of DOI:10.1039/b711455a

Lipozyme TL IM catalyses the deacylation of 4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-(1-methylethylidene)-β-l-threo- pentofuranose to form 3,5-di-O-acyl-4-C-hydroxymethyl-1,2-O-(1-methylethylidene) -α-d-xylo-pentofuranose in a highly selective and

Full text of DOI:10.1039/b711455a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Selective biocatalytic deacylation studies on furanose triesters: a novel and
efficient approach towards bicyclonucleosides†‡
Ashok K. Prasad,*a Neerja Kalra,a,e Yogesh Yadav,a Sunil K. Singh,a Sunil K. Sharma,a Shamkant Patkar,b
Lene Lange,c Carl E. Olsen,d Jesper Wengele and Virinder S. Parmar*a
Received 26th July 2007, Accepted 12th September 2007
First published as an Advance Article on the web 25th September 2007
DOI: 10.1039/b711455a
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Lipozymeꢀ TL IM catalyses the deacylation of 4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-
(1-methylethylidene)-b-L-threo-pentofuranose to form 3,5-di-O-acyl-4-C-hydroxymethyl-1,2-O-
(1-methylethylidene)-a-D-xylo-pentofuranose in a highly selective and efficient manner. The rate of
lipase-catalyzed deacylation of tributanoyl furanose is 2.3 times faster than the rate of deacylation of
the triacetyl furanose derivative. In order to confirm the structure of the lipase-catalyzed deacylated
product, it was converted to a bicyclic sugar derivative, which can be used for the synthesis of bicyclic
nucleosides of importance in the development of novel antisense and antigene oligonucleotides.
Further, it has been established that the monohydroxy product of the lipase-catalyzed reaction is the
result of selective deacylation of the 4-C-acyloxymethyl function in the substrate and not of any acyl
migration process.
yields of the desired products and make the whole process
Introduction
tedious, time-consuming and inefficient.33,34 It is at this juncture
The synthesis of novel nucleoside analogues is gaining im-
that nature’s catalysts, enzymes, come into the picture. Recent
portance because of their applications as key intermediates in
advances in enzyme-assisted organic synthesis have allowed the
the development of antisense and/or antigene oligonucleotides
preparation of structurally well-defined molecules in high yields
to regulate targeted gene expression,2–12 and for their direct
and greater selectivity. The added advantages of the application
utilization as anti-tumor or antiviral compounds.13–17 In re-
of enzymes in organic synthesis are that they work under mild
cent studies for development of ideal and practical antisense
reaction conditions and are often environmentally benign. Among
molecules, oligonucleotide analogues containing non-genetic 2ꢀ,5ꢀ-
the different biocatalytic processes, lipase-catalyzed selective
phosphodiester linkages have been found to be good candidates
acylation/deacylation reactions represent an important class of
due to their RNA-selective hybridization properties and resistance
enzymatic transformations in organic synthesis, which is mainly
towards enzymatic degradation.18–25 A novel class of 2ꢀ,5ꢀ-linked
attributed to the low cost of lipases and their wide tolerance
oligonucleotide analogs containing 3ꢀ-O,4ꢀ-C-methylene bridged
ribonucleosides—i.e. an oxetane-fused ribofuranoside ring system
along with normal 3ꢀ,5ꢀ-linked oligonucleotide analogs containing
2ꢀ-O,4ꢀ-C-methylene bridged ribonucleosides—commonly known
as locked nucleic acids (LNAs), have been known to possess
favorable features towards development of antisense and/or
antigene candidates.26–31
One of the major problems emphasized in the synthesis of
modified nucleosides is the presence of multiple functionalities
of nearly identical reactivity which are difficult to protect and
deprotect selectively.32,33 Further, synthetic routes involving var-
ious protection and/or deprotection steps reduce the overall
towards a variety of reaction conditions and substrates.35,36
Enzymes are being recognized as efficient catalysts for many
of the stereospecific and regioselective reactions necessary for
carbohydrate modifications and nucleoside synthesis.37–47
4-C-Hydroxymethyl-1,2-O-(1-methylethylidene)-b-L-threo-pen-
tofuranose (A) is an important precursor for the synthesis of
different types of bicyclonucleosides, i.e. 3ꢀ-O,5ꢀ-C-methylene
bridged nucleoside B, 3ꢀ-N,4ꢀ-C-methylene bridged nucleoside C
and 2ꢀ-O,4ꢀ-C-methylene bridged 3ꢀ-azido-/3ꢀ-aminonucleoside
D (Scheme 1). For the synthesis of these bicyclonucleosides,
discrimination between the two primary hydroxyl groups of the
trihydroxyfuranose precursor sugar A is highly desired. Chemical
aBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi
110 007, India. E-mail: ashokenzyme@yahoo.com
bNovozymes A/S, Smormosevej 25, DK-2880 Bagsvaerd, Denmark
cDepartment of Molecular Biology, University of Copenhagen, Ole Maaloes
Vej 5, DK-2200 Copenhagen N, Denmark
dUniversity of Copenhagen, Faculty of Life Sciences, Department of Natural
Sciences, DK-1871 Frederiksberg C, Denmark
eNucleic Acid Center, Department of Physics and Chemistry, University of
Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
† A part of the present work was published previously, see Ref. 1.
‡ Electronic supplementary information (ESI) available: Characterization
data for compounds 2b,c, 3b,c and 9b–d. See DOI: 10.1039/b711455a
Scheme 1 Compound A—a key precursor for the synthesis of various
bicyclonucleosides.
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methods for the manipulation of two such primary hydroxyl
groups in sugar precursors as well as in nucleosides are reported,
but they are non-selective and lead to the formation of isomeric
mixtures. For example, an effort towards selective benzylation of
3-O-benzyl-4-C-hydroxymethyl-1,2-O-(1-methylethylidene)-a-D-
erythro-pentofuranose led to the formation of the desired 3,5-
di-O-benzyl-4-C-hydroxymethylpentofuranose product in 59%
yield only together with other benzylated compounds.48 Similarly,
selective tosylation of one out of the two primary hydroxyl
functions of 2ꢀ,3ꢀ-O-cyclohexylidene-4ꢀ-hydroxymethyluridine
afforded a mixture of 4ꢀ-tosyloxymethylated- and 5ꢀ-O-tosylated
derivatives.49 We have earlier reported the asymmetrization of
the two diastereotopic diols viz. 3-O-benzyl-4-C-hydroxymethyl-
1,2-O-(1-methylethylidene)-b-L-threo-pentofuranose and 3-azido-
3-deoxy-4-C-hydroxymethyl-1,2-O-(1-methylethylidene)-a-D-
erythro-pentofuranose by lipase-catalyzed acylation reactions in
organic solvents as part of our chemosynthetic approach targeted
towards the formation of modified nucleosides.38,40 Recently,
Kumar and Gross50 have used the trihydroxy pentofuranose
derivative A as a suitable scaffold for building well-defined
macromolecules. They used different lipases for the strict control
of the three-dimensional arrangement of substituents on the
carbohydrate core.
(Scheme 2). Based on our experience of biocatalytic acyla-
tion/deacylation reactions, we chose to use the enzymes Candida
antarctica lipase-B immobilized on polyacrylate (Lewatit),
commonly known as Novozyme-435, porcine pancreatic
lipase (PPL), Candida rugosa lipase (CRL), Thermomyces
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lanuginosus lipase immobilized on silica (Lipozymeꢀ TL IM)
and Candida antarctica lipase-B immobilized on accurel (CAL
B–L(A)) for selective deacylation of the triacylated pentofuranose
derivatives 2a–2c in different organic solvents in the presence of
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n-butanol as the acyl acceptor. Lipozymeꢀ TL IM in toluene
was found to be the most efficient biocatalyst for the selective
deacylation of compounds 2a–2c. The other four lipases, i.e.
Novozyme-435, PPL, CRL and CAL B–L(A) did not accept any
of the acylated sugar derivatives as substrates.
In a typical reaction, a solution of 4-C-acetoxymethyl-3,5-di-O-
acetyl-1,2-O-(1-methylethylidene)-b-L-threo-pentofuranose (2a)
in toluene containing a small amount of n-butanol was agitated
with Lipozymeꢀ TL IM in an incubator shaker at 40–42 ◦C. On
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completion of the reaction, as indicated by TLC examination, the
enzyme was filtered off and the solvent removed under reduced
pressure. The crude product thus obtained was passed through a
small silica gel column to afford the pure deacylated compound
3a, with lower Rf value than the starting compound 2a in 98% yield
(Scheme 2, Table 1). The structure of the diacetylated compound
3a was established as 3,5-di-O-acetyl-4-C-hydroxymethyl-1,2-O-
(1-methylethylidene)-a-D-xylo-pentofuranose by a detailed study
In the present study, we have developed a highly efficient chemo-
enzymatic route for the diastereo- and regioselective deacylation
of one out of the three acyloxy groups of 2a–2c (Scheme 2),
derived from two primary and one secondary hydroxyl groups
of the potentially useful triol A, and subsequent reactions on the
novel diacetyl derivative 3a leading to the formation of the bicyclic
nucleoside sugar precursor 8.
1
1
of its IR, H and 13C NMR, HRMS and H NOE data, and
1
comparison of its H NMR spectrum with that of the starting
triacetate 2a.1
Table 1 Diastereoselective deacylation study of 4-C-acyloxymethyl-3,5-
di-O-acyl-1,2-O-(1-methylethylidene)-b-L-threo-pentofuranose 2a–2c and
the mixed esters 4-C-acyloxymethyl-3,5-di-O-acetyl-1,2-O-(1-methyl-
ethylidene)-b-L-threo-pentofuranose 9a–9d catalyzed by LipozymeꢀR TL
IM in toluene in the presence of n-butanola
Results and discussion
The trihydroxy sugar derivative A was synthesized starting from
D-glucose following the modified procedure1 of Youssefyeh et al.
and others51,52 via the furanose derivative 1 (Scheme 2).
Substrate
Reaction time/h
Product
Yield (%)
The trihydroxy sugar derivative A was converted in yields of
80–85% to its triacylated derivatives, 4-C-acetoxymethyl-3,5-
di-O-acetyl-1,2-O-(1-methylethylidene)-b-L-threo-pentofuranose
2a
2b
2c
9a
9b
9c
9d
9
7
4
8.5
8
10
96
3a
98
88
93
95
94
92
0
3b
3c
3a
(2a),
3,5-di-O-propanoyl-1,2-O-(1-methylethylidene)-4-C-pro-
3a
panoyloxymethyl-b-L-threo-pentofuranose (2b) and 4-C-buta-
noyloxymethyl-3,5-di-O-butanoyl-1,2-O-(1-methylethylidene)-b-
L-threo-pentofuranose (2c) using acetic anhydride, propanoic
anhydride and butanoic anhydride, respectively, in the presence
of a catalytic amount of 4-(dimethylamino)pyridine (DMAP)
3a
No Reaction
a All these reactions, when performed under identical conditions but
without adding the lipase LipozymeꢀR TL IM, did not yield any product.
Scheme 2 Synthesis and lipase catalysed selective deacylation studies on compounds 2a–2c. Reagents and conditions: (i) Ac2O, DMAP; (ii) H2/Pd-C,
EtOAc; (iii) K2CO3, MeOH; (iv) excess of (RCO)2O, DMAP; (v) LipozymeꢀR TL IM, toluene, n-butanol.
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The structure of the deacetylated compound obtained in
the lipase-mediated selective deacetylation of 2a was further
analysis. The structure of compound 3a was further confirmed by
its chemical transformation to stereochemically restricted bicyclic
sugar derivative 8. Structures of known compounds mentioned in
Scheme 2 were further confirmed by comparison of their physical
and/or chemical data with those reported in the literature. It
should be noted that though compound 1a is known,38 its physical
and spectral data were not reported earlier. All deacylation
reactions, when performed under identical conditions but without
adding any lipase, did not proceed to any extent.
confirmed
as
3,5-di-O-acetyl-4-C-hydroxymethyl-1,2-O-(1-
methylethylidene)-a-D-xylo-pentofuranose (3a) by its chemical
transformation. Treatment of compound 3a with p-
toluenesulfonyl chloride in the presence of base led to the
tosylation of the free hydroxyl function. The tosylated sugar
derivative on treatment with aqueous methanolic potassium
carbonate formed a diol. Mesylation of this diol afforded a
monomesyl derivative (due to preferential mesylation of the
primary hydroxyl function), which on treatment with sodium
hydride in DMF led to the formation of the novel tricyclic
compound 3-O,4-C-methylene-1,2-O-(1-methylethylidene)-5-O-
(p-toluenesulfonyl)-b-L-arabino-pentofuranose (7), the product of
a series of reactions which are possible only when the hydroxyl
group in compound 3a is at the C-1ꢀ position (Scheme 3). The
tosyloxymethylfuranose 7 was further transformed into the
novel bicyclic compound 5-O-acetyl-3-O,4-C-methylene-1,2-O-
(1-methylethylidene)-b-L-arabino-pentofuranose (8) on treatment
with potassium acetate and 18-crown-6 ether in dioxane. This
series of reactions demonstrates that the compound 3a, obtained
through a highly selective biocatalytic route using a novel silica
gel-supported fungal lipase, can be used as a suitable precursor
for the synthesis of bicyclic nucleosides (Scheme 3).
It has been observed that monoacyl derivatives of certain
1,2-diol systems isomerize by acyl migration, and the problem
of acyl migration has also been reported in partially acylated
nucleosides.53–55 Thus the selectively deacylated 3,5-di-O-acyl-
4-C-hydroxymethyl-1,2-O-(1-methylethylidene)-a-D-xylo-pento-
furanose products 3a–3c may in theory also arise due to
deacylation of other acyloxy functions at C-5/C-3 in 2a–
2c, followed by acyl migration. To confirm that the isolated
compounds 3a–3c are formed in the enzyme-catalyzed reaction
solely due to the deacylation of the 4-C-acyloxymethyl function
and not due to partial or complete acyl moiety migration, the
mixed esters 3-O-acetyl-4-C-acetoxymethyl-1,2-O-(1-methyl-
ethylidene)-5-O-propanoyl-b-L-arabino-pentofuranose (9a), 3-
O-acetyl-4-C-acetoxymethyl-5-O-butanoyl-1,2-O-(1-methylethyl-
idene)-b-L-arabino-pentofuranose (9b), 3-O-acetyl-4-C-acetoxy-
methyl-1,2-O-(1-methylethylidene)-5-O-pentanoyl-b-L-arabino-
pentofuranose (9c) and 3-O-acetyl-4-C-acetoxymethyl-5-O-
benzoyl-1,2-O-(1-methylethylidene)-b-L-arabino-pentofuranose
(9d) were synthesized from 3a using propanoic anhydride,
butanoic anhydride, valeric anhydride or benzoic anhydride in
dichloromethane (DCM) in the presence of a catalytic amount
of DMAP to afford the corresponding mixed triesters in 93–95%
yields (Scheme 4). The mixed triesters 9a–9d were subjected to
To see the effect of different acyl moieties, biocatalytic
deacylation study was extended to 3,5-di-O-propanoyl-1,2-O-
(1-methylethylidene)-4-C-propanoyloxymethyl-b-L-threo-pento-
furanose (2b) and 4-C-butanoyloxymethyl-3,5-di-O-butanoyl-1,2-
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O-(1-methylethylidene)-b-L-threo-pentofuranose (2c). Lipozymeꢀ
TL IM-catalyzed deacylation of compounds 2b and 2c afforded
the corresponding diacylated compounds, 3,5-di-O-propanoyl-
4-C-hydroxymethyl-1,2-O-(1-methylethylidene)-a-D-xylo-pento-
furanose (3b) and 3,5-di-O-butanoyl-4-C-hydroxymethyl-1,2-
O-(1-methylethylidene)-a-D-xylo-pentofuranose (3c), respectively
(Scheme 2, Table 1). It was observed that the increase in the chain
length of the acyl moiety increases the rate of lipase-catalyzed
deacylation reaction (Table 1). Thus, the rate of deacylation of
propanoylated sugar derivative 2b is about 1.3 times faster than the
rate of deacylation of acetylated sugar derivative 2a. Accordingly,
the rate of deacylation of butanoylated sugar derivative 2c is about
2.3 and 1.8 times faster than the rate of deacylation of acetylated
and propanoylated sugar derivatives 2a and 2b, respectively. The
structures of all the novel compounds obtained in this study (1b,
2a–2c, 3a–3c and 4–8) were unambiguously established on the
basis of their spectral (IR, 1H and 13C NMR, HRMS and NOE)
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Lipozymeꢀ TM IL-catalyzed deacylation reactions in toluene in
the presence of n-butanol as acyl acceptor. It was observed that
the lipase-catalyzed deacylation of all the mixed esters resulted
in the exclusive formation of 3,5-di-O-acetyl-4-C-hydroxymethyl-
1,2-O-(1-methylethylidene)-a-D-xylo-pentofuranose (3a), except
in the case of deacylation of 3-O-acetyl-4-C-acetoxymethyl-5-O-
benzoyl-1,2-O-(1-methylethylidene)-b-L-arabino-pentofuranose
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(9d), which is not a substrate for Lipozymeꢀ TM IL (Table 1).
The formation of just one product in all the enzyme-catalyzed
reactions showed that it is obtained by selective deacylation
of the 4-C-acyloxymethyl function of 9a–9c only, and not
through deacylation of any other acyloxy function, followed
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by acyl migration. Furthermore, the results of Lipozymeꢀ TM
Scheme 3 Conversion of selectively deacetylated compound 3 into a bicyclic sugar derivative. Reagents and conditions: (i) p-TsCl, pyridine; (ii) K2CO3,
MeOH–H2O; (iii) MsCl, CH2Cl2, Et3N; (iv) NaH, DMF; (v) CH3COOK, dioxane, 18-crown-6 ether.
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Scheme 4 Lipase-catalyzed selective deacylation studies on mixed triacylated pentofuranoses 9a–9d. Reagents and conditions: (i) (RCO)2O, DCM,
DMAP; (ii) LipozymeꢀR TL IM, toluene, n-butanol.
IL-mediated selective deacylation of mixed esters 9a–9c indicates
that the increase in the chain length of the acyloxymethyl function
at C-4 does not alter the selectivity of the lipase.
was removed under reduced pressure, ice-cold water (200 ml) was
added to the residue and the product was extracted with ethyl
acetate (2 × 150 ml). The combined organic phase was washed with
sodium bicarbonate solution (200 ml) and dried over anhydrous
sodium sulfate. The solvent was removed under reduced pressure
and the residue was subjected to column chromatography on silica
gel. The diacetylated furanose 1a was obtained as a colourless oil
(21 g; 82%) using ethyl acetate–petroleum ether (1 : 4) as eluent.
1H NMR (CDCl3, 300 MHz): d 7.35–7.32 (m, 5H), 5.96 (d, J =
4.0 Hz, 1H), 4.75–4.72 (m, 2H), 4.51 (d, J = 11.7 Hz, 1H), 4.37
(d, J = 11.7 Hz, 1H), 4.20–4.04 (m, 4H), 2.04 (s, 3H), 1.96 (s, 3H),
1.54 (s, 3H), 1.35 (s, 3H); 13C NMR (CDCl3, 75.5 Hz): d 169.91,
169.81, 136.56, 128.12, 127.82, 127.69, 127.41, 113.08, 104.80,
85.40, 85.08, 83.37, 71.98, 63.15, 62.73, 26.75, 26.37, 20.50, 20.27;
IR (thin film) 2941, 1747, 1729, 1497, 1455, 1374, 1236, 1164, 1046,
862, 749, 700, 604 cm−1.
Conclusion
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Lipozymeꢀ TL IM discriminated between three ester functions
derived from two primary hydroxyls and a secondary hydroxyl
group in novel sugar derivatives. There are ways to differentiate
between primary and secondary hydroxyl groups, but discrimi-
nation between two primary hydroxyl groups or their derivatives
is in general not possible by purely classical chemical methods.
The very efficient and convenient enzymatic method discovered
for the discrimination of primary hydroxyl groups of furanose
derivatives herein may find applications in ‘green’ synthesis of
bicyclonucleosides, important precursors for the preparation of
state-of-the-art antisense or antigene oligonucleotides.
4-C-Acetoxymethyl-5-O-acetyl-1,2-O-(1-methylethylidene)-b-L-
threo-pentofuranose (1b)
Experimental section
To a solution of the benzylated furanose 1a (19.7 g, 50 mmol)
in ethyl acetate (350 ml), 10% Pd/charcoal (4.5 g) was added
and the reaction mixture was degassed with H2 under reduced
pressure. After stirring the contents at RT under an atmosphere
of H2 gas for 28 h, the reaction mixture was passed through a
pad of silica gel and washed with hot chloroform. The combined
filtrate was concentrated under reduced pressure and the residue
thus obtained was subjected to column chromatography over silica
gel using ethyl acetate–petroleum ether (3 : 7) as eluent to afford
General procedures
Reactions were conducted under an atmosphere of nitrogen
when anhydrous solvents were used. Column chromatography
was carried out using silica gel (100–200 mesh). Melting points
were determined using a H2SO4 bath and are uncorrected. The
1H and 13C NMR spectra were recorded on a Bruker Avance
spectrometer at 300 and at 75.5 MHz, respectively. The chemical
shift values are reported as d ppm relative to TMS used as internal
standard and the coupling constants (J) are measured in Hz.
The FAB-HRMS spectra of all the compounds were recorded
on a JEOL JMS-AX505W high-resolution mass spectrometer in
positive mode using the matrix HEDS (bishydroxyethyldisulfide)
doped with sodium acetate. The Candida antarctica lipase (CAL
B or Novozyme-435), Candida antarctica lipase-B immobilized
on accurel, i.e. CAL B–L(A), and Thermomyces lanuginosus
◦
the furanose 1b as a white solid (10.26 g; 67.5%). Mp 74–76 C.
1H NMR (CDCl3, 300 MHz): d 5.92 (d, J = 4.0 Hz, 1H), 5.35 (s,
1H), 4.60 (dd, J = 1.0 and 4.0 Hz, 1H), 4.36 (d, J = 11.5 Hz, 1H),
4.29 (d, J = 11.5 Hz, 1H), 4.27 (d, J = 11.5 Hz, 1H), 4.10 (d, J =
11.5 Hz, 1H), 2.08 (s, 3H), 2.06 (s, 3H), 1.60 (s, 3H), 1.33 (s, 3H);
13C NMR (CDCl3, 75.5 MHz): d 175.52, 175.42, 117.29, 110.22,
92.92, 91.94, 81.21, 68.30, 32.08, 31.68, 26.21, 26.10; HRMS m/z
calculated for [C13H20O8Na]+ 327.1056, observed 327.1083.
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lipase immobilized on silica (Lipozymeꢀ TL IM) were gifts from
Novozymes A/S (Copenhagen, Denmark), whereas C. rugosa
lipase (CRL) and porcine pancreatic lipase (PPL) were purchased
from Sigma Chemical Co. (USA). All the enzymes were dried over
P2O5 under vacuum for 24 h prior to use. Toluene was distilled
over P2O5 and stored over Na wire prior to use.
General procedure for acylation of 4-C-hydroxymethyl-1,2-O-(1-
methylethylidene)-b-L-threo-pentofuranose (A); preparation of
triesters 2a–2c
To a mixture of 4-C-hydroxymethyl-1,2-O-(1-methylethylidene)-
b-L-threo-pentofuranose (A, 1.98 g, 9.0 mmol) and the corres-
ponding acid anhydride (acetic/propanoic/butanoic anhydride,
3.3 equiv.) was added a catalytic amount of DMAP and the
reaction mixture was stirred for 4–5 h at 28 ◦C. On completion
(analytical TLC), the reaction mixture was poured over ice water
and the product triesters were extracted with ethyl acetate (2 ×
60 ml). The combined organic extract was washed with saturated
4-C-Acetoxymethyl-5-O-acetyl-3-O-benzyl-1,2-O-(1-
methylethylidene)-b-L-threo-pentofuranose (1a)38
To a solution of the diol 140 (20.15 g, 65 mmol) in acetic anhydride
(1.1 equiv.) and pyridine (2.0 equiv.) was added a catalytic amount
of 4-N,N-dimethylaminopyridine and the reaction mixture was
stirred at 25–28 ◦C. On completion (analytical TLC), pyridine
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aqueous NaHCO3 (80 ml), dried over anhydrous Na2SO4, and
the solvent was removed under reduced pressure. The residue
thus obtained was purified by column chromatography on silica
gel using ethyl acetate–petroleum ether (1 : 3) as eluent to
afford the triacylated products 2a–2c (80–85%). (See ESI for the
characterization data of 2b,c.‡)
dilute hydrochloric acid (50 ml) and extracted with ethyl acetate
(2 × 50 ml). The combined organic layer was dried (Na2SO4),
evaporated to dryness under reduced pressure and the residue
was subjected to column chromatography on silica gel using ethyl
acetate–petroleum ether (2 : 3) as eluent ◦to afford the furanose
1
4 as a white solid (1.1 g; 92%). Mp 116 C. H NMR (CDCl3,
300 MHz): d 7.80 (d, J = 7.8 Hz, 2H), 7.35 (d, J = 7.8 Hz, 2H),
5.91 (brs, 1H), 5.24 (s, 1H), 4.52 (brs, 1H), 4.26–4.20 (m, 3H), 4.07
(d, J = 11.7 Hz, 1H), 2.44 (s, 3H), 2.08 (s, 3H), 1.95 (s, 3H), 1.39
(s, 3H), 1.26 (s, 3H); 13C NMR (CDCl3, 75.5 MHz): d 170.32,
169.40, 145.44, 133.06, 132.90, 130.26, 128.54, 113.56, 105.85,
85.87, 85.65, 77.41, 67.75, 62.20, 26.61, 26.20, 21.97, 20.92; IR
(KBr) 2991, 1767, 1745, 1600, 1456, 1364, 1242, 1217, 1181, 1100,
1072, 1047, 1013, 977, 923, 843, 816, 726, 667, 556, 541 cm−1;
HRMS m/z calculated for [C20H26O10SNa]+ 481.1144, observed
481.1164.
4-C-Acetoxymethyl-3,5-di-O-acetyl-1,2-O-(1-methylethylidene)-
b-L-threo-pentofuranose (2a)
◦
1
Obtained as a white solid (2.6 g; 85%). Mp 45–46 C. H NMR
(CDCl3, 300 MHz): d 5.98 (d, J = 4.1 Hz, 1H), 5.35 (s, 1H), 4.61
(dd, J = 1.0 and 4.1 Hz, 1H), 4.36 (d, J = 11.5 Hz, 1H), 4.29 (d, J =
11.5 Hz, 1H), 4.27 (d, J = 11.6 Hz, 1H), 4.10 (d, J = 11.6 Hz, 1H),
2.10 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.60 (s, 3H), 1.33 (s, 3H);
13C NMR (CDCl3, 75.5 MHz): d 170.60, 170.54, 169.67, 113.95,
105.70, 86.18, 77.78, 63.69, 62.88, 27.19, 26.76, 21.22, 21.16, 21.03;
IR (KBr) 2993, 2948, 1751, 1736, 1461, 1432, 1391, 1373, 1235,
1166, 1117, 1079, 1026, 903, 851 cm−1; HRMS m/z calculated for
[C15H22O9Na]+ 369.1162, observed 369.1165.
4-C-Hydroxymethyl-1,2-O-(1-methylethylidene)-5-O-p-
toluenesulfonyl-b-L-arabino-pentofuranose (5)
To a solution of the furanose 4 (687 mg, 1.5 mmol) in methanol
(20 ml) was added an aqueous solution of potassium carbonate
(414 mg in 10 ml water, 3 mmol), and the reaction was stirred for
1 h at 28 ◦C. On completion (analytical TLC), the reaction mixture
was neutralized with dilute hydrochloric acid and extracted with
ethyl acetate (2 × 40 ml). The combined organic phase was
washed with water (50 ml), dried (Na2SO4), evaporated to dryness
under reduced pressure, and the residue was subjected to column
chromatography on silica gel using ethyl acetate–petroleum ether
(3 : 2) as ◦solvent to afford the diol 5 as a white solid (342 mg; 61%).
Mp 145 C. 1H NMR (CDCl3, 300 MHz): d 7.79 (d, J = 8.2 Hz,
2H), 7.35 (d, J = 8.2 Hz, 2H), 5.95 (d, J = 3.6 Hz, 1H), 4.58 (d,
J = 3.7 Hz, 1H), 4.28 (s, 1H), 4.23 (d, J = 10.0 Hz, 1H), 4.08 (d,
J = 10.0 Hz, 1H), 3.94 (d, J = 11.9 Hz, 1H), 3.73 (d, J = 11.9 Hz,
1H), 2.45 (s, 3H), 1.35 (s, 3H), 1.27 (s, 3H); 13C NMR (CDCl3,
75.5 MHz): d 145.62, 132.73, 130.37, 128.45, 113.07, 105.92, 87.82,
87.56, 78.00, 69.77, 63.26, 26.83, 26.26, 22.02; IR (KBr) 3529,
2975, 2932, 1596, 1451, 1421, 1358, 1261, 1224, 1179, 1068, 1010,
960, 864, 836, 812, 790, 665, 557, 534 cm−1; HRMS m/z calculated
for [C16H22O8SNa]+ 397.0933, observed 397.0957.
General procedure for lipase-catalyzed selective deacylation of
4-C-acyloxymethyl-3,5-di-O-acyl-1,2-O-(1-methylethylidene)-b-L-
threo-pentofuranose 2a–2c
To a solution of the triacylated furanose 2a–2c (3.0 mmol) in anhy-
drous toluene (30 ml) was added n-butanol (1.2 equiv) followed by
R
the addition of Lipozymeꢀ TL IM (500 mg). The reaction mixture
was stirred with shaking at 40–42 ◦C in an incubator and the
progress of the reaction was monitored periodically by TLC. On
completion, the reaction was quenched by filtering off the lipase,
the solvent was removed under reduced pressure, and the residue
was purified by column chromatography on silica gel using ethyl
acetate–petroleum ether (2 : 3) as eluent to afford the deacylated
products 3a–3c (88–98%). (See ESI for the characterization data
of 3b,c.‡)
3,5-Di-O-acetyl-4-C-hydroxymethyl-1,2-O-(1-methylethylidene)-
a-D-xylo-pentofuranose (3a)
Obtained as a white solid (894 mg; 98%). Mp 95–96 ◦C. 1H NMR
(CDCl3, 300 MHz): d 5.97 (d, J = 4.1 Hz, 1H), 5.29 (s, 1H), 4.64
(dd, J = 0.9 and 4.1 Hz, 1H), 4.24 (d, J = 11.5 Hz, 1H), 4.13 (d, J =
11.5 Hz, 1H), 3.82 (dd, J = 5.43 and 11.91 Hz, 1H), 3.73 (dd, J =
8.26 and 11.91 Hz, 1H), 2.42–2.38 (m, 1H), 2.08 (s, 6H), 1.57 (s,
3H), 1.33 (s, 3H); 13C NMR (CDCl3, 75.5 MHz): d 170.25, 169.50,
113.17, 104.75, 87.73, 85.89, 77.33, 62.46, 62.08, 26.75, 26.22,
20.67, 20.49; IR (KBr) 3439, 2938, 1754, 1718, 1379, 1270, 1216,
1163, 1039, 887 cm−1; HRMS m/z calculated for [C13H20O8Na]+
327.1056, observed 327.1083.
4-C-Methanesulfonyloxy-1,2-O-(1-methylethylidene)-5-O-(p-
toluenesulfonyl)-b-L-arabino-pentofuranose (6)
To a solution of the diol 5 (300 mg, 0.8 mmol) in anhydrous
CH2Cl2 (25 ml), were added triethylamine (121 mg, 1.2 mmol)
◦
and methanesulfonyl chloride (110 mg, 0.96 mmol) at 0 C. The
◦
temperature of the reaction mixture was allowed to attain 28 C
and it was stirred at this temperature for 5 h. On completion
(analytical TLC), the reaction mixture was neutralized with dilute
hydrochloric acid and extracted with CH2Cl2 (2 × 40 ml). The
combined organic phase was washed with water (50 ml), dried
(Na2SO4), evaporated to dryness under reduced pressure, and the
residue was subjected to column chromatography on silica gel
using ethyl acetate–petroleum ether (1 : 1) as eluent to afford the
furanose 6 as an oil (228 mg; 63%). 1H NMR (CDCl3, 300 MHz):
d 7.80 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 5.93 (d,
J = 3.5 Hz, 1H), 4.57 (d, J = 3.9 Hz, 1H), 4.35 (brs, 3H),
4.25–4.16 (m, 2H), 3.01 (s, 3H), 2.45 (s, 3H), 1.36 (s, 3H), 1.26
4-C-Acetoxymethyl-3-O-acetyl-1,2-O-(1-methylethylidene)-5-O-
(p-toluenesulfonyl)-b-L-arabino-pentofuranose (4)
To a solution of 3,5-di-O-acetyl-4-C-hydroxymethyl-1,2-O-(1-
methylethylidene)-a-D-xylo-pentofuranose (3a, 760 mg, 2.5 mmol)
in anhydrous pyridine (25 ml) was added p-toluenesulfonyl
chloride (715 mg, 3.75 mmol) pinchwise during 20 min at 0 ◦C, and
the reaction mixture was stirred for 10 h at 28 ◦C. On completion
(analytical TLC), the reaction mixture was poured into ice-cold
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(s, 3H); 13C NMR (CDCl3, 75.5 MHz): d 145.19, 129.89, 127.99,
112.84, 105.59, 105.03, 86.79, 86.21, 77.30, 67.36, 66.91, 37.29,
29.56, 26.08, 25.63, 21.51; IR (KBr) 3435, 2924, 1376, 1333, 1175,
1036, 1011, 982, 966, 821, 774, 553 cm−1; HRMS m/z calculated
for [C17H24O10S2Na]+ 475.0709, observed 475.0739.
combined organic extract was washed with saturated aqueous
NaHCO3 (2 × 50 ml), dried over anhydrous Na2SO4, and the
solvent was removed under reduced pressure. The residue thus
obtained was purified by column chromatography on silica gel
using ethyl acetate–petroleum ether (1 : 3) as eluent to afford
the pure mixed triesters 9a–9d in 93–95% yields. (See ESI for the
characterization data of 9b–9d.‡)
3-O,4-C-Methylene-1,2-O-(1-methylethylidene)-5-O-(p-
toluenesulfonyl)-b-L-arabino-pentofuranose (7)
3-O-Acetyl-4-C-acetoxymethyl-1,2-O-(1-methylethylidene)-5-O-
propanoyl-b-L-arabino-pentofuranose (9a)
To a solution of the furanose 6 (181 mg, 0.4 mmol) in anhydrous
DMF (8 ml) was added sodium hydride (12 mg, 0.5 mmol) portion-
wise during 10 min at 0 ◦C and the contents were stirred for 12 h
Obtained as a viscous oil (445 mg; 95%). 1H NMR (CDCl3,
300 MHz): d 5.98 (d, J = 4.1 Hz, 1H), 5.36 (s, 1H), 4.62 (d,
J = 3.6 Hz, 1H), 4.33 (d, J = 12.6 Hz, 2H), 4.25 (d, J = 11.7 Hz,
1H), 4.09 (d, J = 11.6 Hz, 1H), 2.36 (q, J = 7.2 Hz, 2H), 2.11 (s,
3H), 2.08 (s, 3H), 1.60 (s, 3H), 1.33 (s, 3H), 1.14 (t, J = 7.5 Hz, 3H);
13C NMR (CDCl3, 75.5 MHz): d 173.90, 170.40, 169.54, 113.67,
105.45, 85.94, 77.50, 63.21, 62.68, 27.52, 26.90, 26.44, 21.00, 20.81,
9.13; IR (thin film) 2993, 2948, 1751, 1736, 1461, 1432, 1391,
1373, 1235, 1166, 1117, 1079, 1026, 903, 851 cm−1; HRMS m/z
calculated for [C16H24O9Na]+ 383.1313, observed 383.1312.
◦
at 28 C. On completion (analytical TLC), the reaction mixture
was poured into ice-cold water (25 ml) and the compound was
extracted with CH2Cl2 (2 × 25 ml). The combined organic layer
was dried over Na2SO4, evaporated to dryness under reduced
pressure, and the residue was subjected to column chromatography
on silica gel using ethyl acetate–petroleum ether (2 : 3) as eluent to
afford the pure furanose derivative 7 as a white solid (91 mg; 64%).
Mp 80–82 ◦C. 1H NMR (CDCl3, 300 MHz): d 7.81 (d, J = 7.9 Hz,
2H), 7.36 (d, J = 7.9 Hz, 2H), 6.19 (s, 1H), 4.98 (s, 1H), 4.67–4.62
(m, 2H), 4.29 (d, J = 7.6 Hz, 1H), 4.19 (brs, 2H), 2.45 (s, 3H),
1.40 (s, 3H), 1.33 (s, 3H); 13C NMR (CDCl3, 75.5 MHz): d 145.61,
132.73, 130.33, 128.47, 115.25, 108.40, 87.55, 86.11, 84.44, 78.70,
68.31, 27.97, 27.03, 22.01; IR (KBr) 2929, 1597, 1454, 1368, 1176,
831 cm−1; HRMS m/z calculated for [C16H20O7SNa]+ 379.0827,
observed 379.0832.
General procedure for lipase-catalyzed selective deacylation of
3-O-acetyl-4-C-acetoxymethyl-5-O-acyl-1,2-O-(1-
methylethylidene)-b-L-arabino-pentofuranose 9a–9d
R
The Lipozymeꢀ TL IM-catalyzed deacylation reaction on mixed
triesters 9a–9d was performed in the same way as that on
compounds 2a–2c. The products formed due to incubation of
triesters 9a–9c were found to be identical to 3,5-di-O-acetyl-4-C-
hydroxymethyl-1,2-O-(1-methylethylidene)-a-D-xylo-pentofura-
nose (3a). Incubation of 3-O-acetyl-4-C-acetoxymethyl-5-O-
benzoyl-1,2-O-(1-methylethylidene)-b-L-arabino-pentofuranose
(9d) did not lead to the formation of any product.
5-O-Acetyl-3-O,4-C-methylene-1,2-O-(1-methylethylidene)-b-L-
arabino-pentofuranose (8)
To a solution of the furanose derivative 7 (71 mg, 0.2 mmol) in
dioxane (8 ml) was added potassium acetate (40 mg, 0.4 mmol) and
18-crown-6 ether (63 mg, 0.24 mmol), and the resulting mixture
was refluxed for 12 h. On completion, the contents were cooled
to RT, poured into ice-cold water (10 ml) and extracted with ethyl
acetate (2 × 20 ml). The combined organic phase was dried over
Na2SO4, evaporated to dryness under reduced pressure, and the
residue was subjected to column chromatography on silica gel
using ethyl acetate–petroleum ether (3 : 7) as eluent to afford
Acknowledgements
We thank the Danish Natural Research Foundation and the
Department of Biotechnology (DBT, New Delhi) for financial
support to this work. NK and SKS thank CSIR (Council of
Scientific and Industrial Research, New Delhi, India) for the award
of senior research fellowships.
1
the acetylated furanose derivative 8 as an oil (35 mg; 71%). H
NMR (CDCl3, 300 MHz) d 6.18 (s, 1H), 4.96 (s, 1H), 4.62–4.59
(m, 2H), 4.32 (d, J = 6.9 Hz, 1H), 4.27–4.21 (m, 2H), 2.04 (s,
3H), 1.46 (s, 3H), 1.30 (s, 3H); 13C NMR (CDCl3, 75.5 MHz):
d 169.54, 113.88, 106.75, 86.52, 85.21, 83.28, 78.13, 62.67, 26.87,
25.76, 19.74; IR (thin film) 2956, 1746, 1372, 1229, 1162, 1094,
1070, 1047, 1012, 961, 890, 850 cm−1; HRMS m/z calculated for
[C11H16O6Na]+ 267.0845, observed 267.0863.
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