Regioselective acylation of carbohydrate derivatives using lipases leading to a facile two-step procedure for the separation of some α- and β-glucopyranosides and galactopyranosides

Article abstract of DOI:10.1016/j.tet.2003.11.035

The resolution of α- and β-anomers of glucopyranosides and galactopyranosides was achieved via enzyme-catalysed regioselective acylation. This two-step procedure to prepare pure α- and β-anomers of glycopyranosides would be most useful for the cases where glycosidases are not available or expensive to purchase. From a synthetic viewpoint, the regioselective acylation of glycopyranosides provides access to mono- and di-esters with well-defined substitution patterns.

Full text of DOI:10.1016/j.tet.2003.11.035

Tetrahedron 60 (2004) 927–932
Regioselective acylation of carbohydrate derivatives using lipases
leading to a facile two-step procedure for the separation of some
a- and b-glucopyranosides and galactopyranosides
b
Pedro M. L. Gonc¸alves,a Stanley M. Robertsa and Peter W. H. Wan
,
*
aDepartment of Chemistry, Liverpool University, Crown Street, Liverpool L69 7ZD, UK
bResearch and Development Centre, British American Tobacco, Regents Park Road, Millbrook, Southampton SO15 8TL, UK
Received 12 September 2003; revised 23 October 2003; accepted 13 November 2003
Abstract—The resolution of a- and b-anomers of glucopyranosides and galactopyranosides was achieved via enzyme-catalysed
regioselective acylation. This two-step procedure to prepare pure a- and b-anomers of glycopyranosides would be most useful for the cases
where glycosidases are not available or expensive to purchase. From a synthetic viewpoint, the regioselective acylation of glycopyranosides
provides access to mono- and di-esters with well-defined substitution patterns.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction and background information
by Riva and co-workers.8 The Italian team showed that
a-methyl- and a-benzyl-D-glucopyranoside gave only the
6-acetates 4 and 5 (98% yield) on reaction with vinyl acetate
and Candida antarctica lipase B in THF/pyridine over 20 h.,
while b-methyl-D-glucopyranoside furnished the 3,6-di-
acetate 6 in 97% yield. Under the same conditions
a-methyl-D-galactopyranoside gave a mixture of the
6-acetylated product 7 (74%) and a small amount of the
2,6-diester 8 (24%). In contrast b-methyl-(D)-galacto-
pyranoside afforded the 3,6-diacetate 9 as the major product
(44%) with lesser amounts of the 2,6-diacetate 10 (31%) and
the 6-monoester 11 (24%) after 48 h. Later, it was shown
that the benzyl compound 12 was acylated using trifluoro-
ethyl butanoate in acetone and in the presence of subtilisin
to give the 3,6-diester 13 in 89% yield.9
Enzyme-catalysed esterification of carbohydrates and their
simple derivatives has been studied extensively.1 For
hexapyranoses, the primary hydroxyl group is esterified
preferentially and the preparation of 6-O-acylated carbo-
hydrates and glycopyranosides has been a subject of
continued interest,2 especially in connection with the
synthesis of novel surfactants3 and the manufacture of
antibacterial agents of potential interest to the food
industry.4
Following acylation at the 6-position of glycopyranosides,
one or more of the secondary hydroxyl groups may be
esterified on prolonged reaction.5 It has also been shown
that the configuration of the substituent at the anomeric
position can influence the regioselectivity of the acylation of
the secondary hydroxyl groups. For example, in a study of
the acylation of 4,6-benzylidene glucopyranosides using
Pseudomonas cepacia lipase as catalyst, acetylation of the
a-methoxy compound 1 occurred exclusively at C(2)–OH
while the isomer 2 was acetylated with very high selectivity
at C(3)–OH.6 The ethylthio derivative 3 behaves in a
similar manner and this regioselective reaction has been
used as a key step in the synthesis of the core motif of
asparagine-linked glycoprotein oligosaccharides.7
Of greatest relevance to the present study is the earlier work
Keywords: Regioselective acylation; a- and b-Anomers;
Glycopyranosides.
*
Corresponding author. E-mail address: smrsm@liv.ac.uk
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.11.035

928
P. M. L. Gonc¸alves et al. / Tetrahedron 60 (2004) 927–932
manner, but in a more moderate yield of 36% over the two
steps.
A battery of lipases were inspected for their ability to
catalyse the acylation of ethyl b-glucopyranoside 15.
Immobilised Mucor miehei lipase (Lipozymew) and
immobilised Candida cylindracea B lipase (Novozymew)
were the most efficient catalysts for esterification using
vinyl butyrate in THF. Both enzymes gave the 6-O-butyrate
18 as the first formed product. Further esterification of the
mono-ester 18 with Novozymew in THF at 60 8C gave the
3,6-diester (50%) 19 and the 2,6-diester (30%) 20. In
contrast, prolonged reaction of the b-benzyl compound 14
with vinyl butyrate in THF containing Novozymew afforded
only the 6-O-butanoate 21 (98% isolated yield after a 3 h
reaction), in accord with the earlier results documented by
Riva et al.8
The a-anomers 16 and 17 showed an interesting difference
in reactivity, inasmuch as the a-benzyl anomer 16 reacted
ca. 5£faster than the b-isomer 14 while the a-ethyl
compound 17 reacted ca. 5£slower than the corresponding
b-anomer 15 under our standard esterification conditions. In
fact, similar patterns of a-/b-reactivity for large11 and
small12 substituents at the anomeric position have been
documented previously. The products from the reactions of
16 and 17 were the appropriate 6-O-butanoates 22 and 23.
No further reaction of the latter compounds with vinyl
butanoate and Novozymew could be detected even after
prolonged periods of time. Only when a different enzyme
was employed (namely Pseudomonas cepacia lipase PS-
CII) could further acylation of monoester 22 be achieved.
By employing this enzyme in vinyl butyrate as solvent, an
86% yield of diester 24 was obtained after 3 days. It was
noted that Ps cepacia lipase PS CII did not catalyse
esterification of monoester 21 under these conditions.
In this paper, we extend the studies of Riva et al.8 to show
that the methodology can lead to the separation of a- and
b-glycopyranosides.
2. Results and discussion
Benzyl b-D-glucopyranoside 14 and ethyl b-D-gluco-
pyranoside 15 were synthesised from D-glucose in 43 and
85% yield, respectively, using b-glucosidase from
almonds.10
.
.
From this initial study, it was clear that the different
reactivity patterns could be used to separate mixtures of a-
and b-anomers of glycopyranosides. Thus the mixture of a-
and b-ethyl glucopyranoside, simply prepared from ethanol
and glucose under acidic conditions, was reacted with vinyl
The same enzyme was used selectively to hydrolyse an
anomeric mixture of a- and b-benzyl glucoside to afford the
unreacted a-anomer 16 in 67% yield. The a-ethoxy
compound 17 was prepared from glucose in a similar

P. M. L. Gonc¸alves et al. / Tetrahedron 60 (2004) 927–932
929
butyrate in THF at 60 8C in the presence of Novozymew for
3. Experimental
a period of 4 days. The diesters 19 and 20 (31%) were
separated from the monoester 23 (48%). Methanolysis of the
diesters gave ethyl b-D-glucopyranoside 15 while similar
treatment of the mono-ester gave the a-anomer 17 in
quantitative yield.
3.1. General
All reactions were monitored by thin layer chromatography,
which was performed on 200–250 mm thickness Merck
silica gel plates (60F254). Compounds were detected by
ultraviolet light or by staining with ceric ammonium
molybdate solution followed by heating. Column chroma-
tography was performed on Merck-60 silica gel (230–240
mesh). Melting points were recorded on a Reichert
instrument and are uncorrected. Elemental analyses were
performed on a Carlo Erba Elemental Analyser model 1106.
IR spectra were recorded on a Perkin–Elmer 883 spectro-
A combination of Novozymew and Ps. cepacia lipase PS
CII could be used to effect a separation of a- and b-benzyl
(D)-glucopyranosides. A mixture of the a- and b-benzyl
anomers was reacted with the two enzymes in vinyl butyrate
for 4 days. The monoester 21 (36%) was readily separated
from the diester 24 (28%) by column chromatography.
Base-catalysed methanolysis of these esters gave pure
b-benzyl-(D)-glucopyranoside and pure a-benzyl-D-gluco-
pyranoside, respectively, in quantitative yields.
photometer. H and 13C NMR spectra were recorded as
1
solutions in deuteriated solvents (Aldrich, Fluorochem) on
AVANCE 400 MHz or Varian Gemini300 instruments. 1H-
and 13C-spectra were referenced using TMS as internal
standard. Chemical shifts (d) are quoted in parts per million
(ppm) and the coupling constants (J) in Hertz (Hz). The
following abbreviations are used to describe the multi-
plicity: s, singlet; d, doublet; t, triplet; tt, triplet of triplets; q,
quartet; dd, doublet of doublets; ddd, doublet of doublets of
doublets; dt, doublet of triplets; m, multiplet; br, broad.
Optical rotations [a]TD (concentration in g/100 cm3, solvent)
were recorded on an Optical Activity A1000 polarimeter at
589 nm, where T is the temperature in 8C. Low resolution CI
mass spectra were measured on a Fisons TRIO 1000
spectrometer. Accurate mass spectra were obtained on a VG
Analytical 7070E double focussing magnetic mass spec-
trometer. b-Glucosidase from almonds (EC number
3.2.1.21) and Novozymew 435 (EC number 3.1.1.3) were
purchased from Sigma-Aldrich. Lipase PS-C II was
purchased from Amano Pharmaceuticals. All solvents and
reagents were purchased and used directly from commercial
suppliers without purification.
It was also clear that this three-step procedure to prepare
glycopyranosides would be more useful for the cases where
glycosidases are not available, or are expensive to purchase.
Thus, we extended the study to galactosides because, while
b-benzyl-D-galactoside 26 is available by a modified
13
Konigs–Knorr glycosidation procedure, the correspond-
¨
ing a-anomer 27 is not easy to access. Standard Fischer
derivatisation of galactose gives a mixture of 26 and 27.
Incubation of this mixture with Novozymew in THF
containing vinyl butyrate over 7.5 h gave the mono ester
28 (46%) and the diester 29 (23%) which were readily
separated by chromatography over silica. Methanolysis of
the mono ester 28 gave benzyl a-D-galactoside 27 in 91%
yield.
3.1.1. Ethyl b-D-glucopyranoside (15). D-Glucose (0.81 g,
4.48 mmol) was added to a solution of water (2 cm3) in
ethanol (18 cm3), and heated to 50 8C, with stirring. After
10 min b-glucosidase from almonds (0.11 g) was added to
the reaction mixture which was stirred for 3 days. The
solution was filtered through Celitew and washed with
ethanol (20 cm3). The excess of solvent was removed under
reduced pressure yielding a yellow crude product, which
was chromatographed over silica with chloroform–metha-
nol (9:1) yielding compound 15 as a white solid (0.79 g,
85%); mp 82–84 8C (lit.14 98–100 8C). (Found: C, 45.88;
H, 7.80. C8H16O6 requires C, 46.15; H, 7.75%); [a]2D3 228.5
(c 1.0 in MeOH); 1H NMR (D2O): d 1.11 (3H, t, J¼7.1 Hz,
CH3), 3.12 (1H, t, J¼9.1 Hz), 3.25 (1H, t, J¼9.1 Hz), 3.31–
3.38 (2H, m, CH2), 3.56–3.64 (2H, m), 3.79 (1H, d,
J¼12.1 Hz), 3.84 (1H, t, J¼8.1 Hz,), 4.34 (1H, d,
J¼8.1 Hz,); 13C NMR (D2O) d 14.7, 61.2, 66.6, 70.1,
73.6, 76.3, 76.3, 102.3; m/z (CI) [MþNH4]þ226 (100%).
.
It is also obvious, but noteworthy from a synthetic
viewpoint, that selectively esterified glucose or galactose
derivatives are available from some of the compounds in the
above portfolio by hydrogenation to remove the anomeric
benzyl group. For example, the ester 24 afforded the 2,6-
diester 25 (75% yield) on reduction, using palladium on
charcoal (10%) as the catalyst.
3.1.2. Ethyl 6-O-butyryl b-D-glucopyranoside (18).
Novozymew 435 (0.42 g) was added to a solution of ethyl
b-D-glucopyranoside 15 (0.42 g, 2.01 mmol) in vinyl
butyrate (0.52 cm3, 4.1 mmol) and THF (10 cm3). The
reaction mixture was stirred for 1 h at 60 8C, after which
time the enzyme was filtered off and washed with THF
(10 cm3). The excess THF was evaporated under reduced
This work provides further evidence that the regioselective
acylation of glycopyranosides can provide access to mono-
and di-esters with well-defined substitution patterns. We
have shown that the different degrees of reactivity of a- and
b-glycosides potentially allows access to a wide range of
pure a-anomers which may not be readily accessed by other
methodology.

930
P. M. L. Gonc¸alves et al. / Tetrahedron 60 (2004) 927–932
pressure to afford a crude residue which was purified by
flash chromatography over silica with ethyl acetate–ethanol
(95:5) as eluent, yielding compound 18 (0.45 g, 81%) as a
white solid; mp 80 8C. (Found: C, 51.92; H, 8.06. C12H22O7
requiresC, 51.79;H, 7.97%); [a]2D6 251(c 1.0 inCHCl3);nmax
(CHCl3)/cm21 1730 (CvO); 1H NMR (CDCl3) d 0.96 (3H, t,
J¼8.1 Hz, CH3), 1.26 (3H, t, J¼7.1 Hz, CH3), 1.63–1.71 (2H,
m, CH2CH3), 2.36(2H, t, J¼8.1 Hz, CH2CO), 3.35–3.41(2H,
m), 3.46 (1H, ddd, J¼2.0, 5.1, 9.1 Hz), 3.55–3.65 (2H, m),
3.96 (1H, qd, J¼3.0, 14.2 Hz, CH2) 4.29 (1H, d, J¼7.1 Hz),
4.30 (1H, dd, J¼12.1, 2.0 Hz), 4.49 (1H, dd, J¼12.1, 5.1 Hz);
13C NMR (CDCl3), d 13.9 (CH3), 15.4 (CH3), 18.7 (CH2CH3),
36.4 (CH2CO), 63.4, 66.0, 70.4, 74.0, 74.4, 76.3, 102.8, 174.9
(CO); m/z (CI) [MþNH4]þ 296 (100%).
filtered through Celitew and the buffer evaporated under
reduced pressure. The crude product was chromatographed
over silica with chloroform–methanol (9:1) as eluent
affording 17 as a syrup (1.02 g, 30%). (Found: C, 45.98;
H, 7.88. C8H16O6 required C, 46.15; H, 7.75%); [a]D22
þ147.9 (c 0.57 in MeOH) [lit.15þ150 (c 1.0 in MeOH)]; 1H
NMR (DMSO) d 1.12 (3H, t, J¼7.2 Hz, CH3), 3.01–3.09
(1H, m), 3.14–3.21 (1H, m), 3.32–3.48 (5H, m), 3.56–3.65
(2H, m), 4.63 (1H, d, J¼3.9 Hz); 13C NMR (DMSO) d 15.2
(CH3), 61.1, 62.7, 70.4, 70.5, 72.0, 72.0, 98.4; m/z (CI)
[MþNH4]þ 226 (100%).
3.1.5. Ethyl 6-O-butyryl a-D-glucopyranoside (23). A
solution of ethyl a-D-glucopyranoside 17 (0.22 g,
1.04 mmol) and vinyl butyrate (0.2 cm3, 1.58 mmol) in
THF (10 cm3) was prepared and immersed in a oil bath at
60 8C. After 10 min Novozymew (0.13 g) was added. The
mixture was stirred for 6 h, after which time the enzyme was
filtered off and washed with THF (5 cm3). Excess solvent
was evaporated under reduced pressure and the crude
product was chromatographed over silica with ethyl
acetate–ethanol (95:5) as eluent yielding compound 23 as
a white solid (0.18 g, 60%). (Found: C, 51.80; H, 7.99.
C12H22O7 required C, 51.79; H, 7.97%); mp 70–72 8C;
[a]2D1 þ81.9 (c 1.05 in CHCl3); nmax (CHCl3)/cm21 1730
3.1.3. Ethyl 3,6-O-butyryl b-D-glucopyranoside (19) and
ethyl
2,6-O-butyryl
b-D-glucopyranoside
(20).
Novozymew (0.21 g) was added to a solution of ethyl
6-O-butyryl b-D-glucopyranoside 18 (0.44, 1.59 mmol) and
vinyl butyrate (0.4 cm3, 3.16 mmol) in THF (10 cm3). The
reaction mixture was heated to 60 8C and stirred for 7 days,
after which time the enzyme was filtered off and washed
with THF (10 cm3). The solvent was evaporated under
reduced pressure to afford a crude residue which was
chromatographed over silica with ethyl acetate–ethanol
(95:5) as eluent and re-chromatographed over silica gel with
ethyl acetate–petroleum ether (2:1) as eluent affording
compound 19 (0.28 g, 50%) and compound 20 (0.17 g,
30%). Compound 19. (Found: C, 54.97; H, 8.17. C16H28O8
requires C, 55.16; H, 8.10%); [a]2D7 217.6 (c 1.1 in CHCl3);
mp 36–38 8C; nmax (CHCl3)/cm21 1730 (CvO); 1H NMR
(CDCl3) d 0.94–0.99 (6H, m, CH3), 1.26 (3H, t, J¼7.1 Hz,
1
(CvO); H NMR (CDCl3) d 0.96 (3H, t, J¼7.1 Hz, CH3),
1.25 (3H, t, J¼7.1 Hz, CH3), 1.61–1.73 (2H, m, CH2CH3),
2.36 (2H, t, J¼8.1 Hz, CH2CO), 3.45 (1H, t, J¼9.1 Hz),
3.47–3.60 (2H, m), 3.67 (1H, br s, OH), 3.72–3.85 (3H, m),
4.26 (1H, dd, J¼12.1, 2.0 Hz), 4.47 (1H, dd, J¼12.1,
4.0 Hz), 4.89 (1H, d, J¼4.0 Hz); 13C NMR (CDCl3) d 13.6
(CH3), 15.0 (CH3), 18.4 (CH2CH3), 36.0 (CH2CO), 63.1
(CH2), 63.9, 69.8, 70.0, 72.0, 74.4, 98.0, 174.4 (CvO); m/z
(CI) [MþNH4]þ 296 (100%).
CH3), 1.62–1.74 (4H, m, 2£CH2CH3), 2.35 (2H, t,
0
J¼8.1 Hz, CH2CO), 2.40 (2H, t, J¼7.1 Hz, CH2CO),
3.46–3.55 (3H, m), 3.63 (1H, qd, J¼2.0, 16.2 Hz), 3.96
(1H, qd, J¼3.0, 14.2 Hz, CH2), 4.34 (1H, d, J¼8.1 Hz),
4.35–4.44 (2H, m), 4.93 (1H, t, J¼9.1 Hz); 13C NMR
(CDCl3) d 13.5 (CH3), 13.6 (CH3), 15.1 (CH3), 18.4
(CH2CH3), 18.4 (CH2CH3), 36.0 (CH2CO), 36.2 (CH2CO),
63.0 (CH2), 65.7 (CH2), 69.4, 72.13, 74.4, 77.6, 102.6, 174.0
(CvO), 175.1 (CvO); m/z (CI) [MþNH4]þ 366 (88%).
Compound 20. (Found: C, 54.96; H, 8.15. C16H28O8
requires C, 55.16; H, 8.10%); [a]2D6 248.8 (c 1.1 in
3.2. Separation of anomers of ethyl D-glucopyranoside
Novozyme 435w (0.53 g) was added to a solution of ethyl
D-glucopyranoside (0.88 g, 4.24 mmol) and vinyl butyrate
(1.1 cm3, 8.66 mmol) in THF (20 cm3). The reaction
mixture was immersed in a oil bath at 60 8C and stirred
for 4 days, after which time the enzyme was filtered off and
washed with THF (10 cm3). The excess solvent was
evaporated under reduced pressure and the crude product
was chromatographed over silica gel using a gradient of
ethyl acetate–petroleum ether (2:1) to ethyl acetate (100%)
as eluent, affording compound 19 (0.27 g, 18%), compound
20 (0.19 g, 13%), and compound 23 (0.57 g, 48%).
1
CHCl3); mp 48 8C; nmax CHCl3/cm21 1760 (CvO); H
NMR (CDCl3) d 0.94–0.99 (6H, m, 2£CH3), 1.19 (3H, t,
J¼7.1 Hz, CH3), 1.63–1.73 (4H, m, 2£CH2CH3), 2.36 (4H,
t, J¼7.1 Hz, 2£CH2CO), 3.41–3.48 (2H, m), 3.51–3.62
(2H, m), 3.89 (1H, qd, J¼2.0, 16.2 Hz), 4.32 (1H dd, J¼2.0,
12.1 Hz), 4.43 (1H, d, J¼8.1 Hz), 4.44 (1H dd, J¼2.0,
12.1 Hz), 4.76 (1H, dd, J¼9.1, 8.1 Hz); 13C NMR (CDCl3) d
13.8 (CH3), 13.9 (CH3), 15.4 (CH3), 18.7 (CH2CH3), 18.8
(CH2CH3), 36.4 (CH2CO), 36.6 (CH2CO), 63.3 (CH2), 65.7
(CH2), 71.1, 74.1, 74.1, 75.7, 101.0, 174.0 and 174.8
(CvO); m/z (CI) [MþNH4]þ 366 (37%).
3.2.1. Benzyl b-D-glucopyranoside (14). b-Glucosidase
from almonds (120 mg) was added to a solution of
D-glucose (0.81 g, 4.48 mmol) in distilled water (2 cm3)
and benzyl alcohol (18 cm3). The solution was stirred for
30 h at 50 8C, after which time the enzyme was filtered off
and washed with distilled water (5 cm3). The excess of
benzyl alcohol was removed under reduced pressure at
90 8C and 1 mbar. Flash chromatography of the residue over
silica with chloroform–methanol (9:1) as the eluent
afforded compound 14 (0.52 g, 43%) as a white solid.
(Found: C, 57.73; H, 6.77. C13H18O6 requires C, 57.77; H,
6.71%); mp 104 8C (lit.16 120–121 8C); [a]2D7 252 (c 0.5 in
MeOH) [lit.16 255.1 (c 1.0 in MeOH)]; nmax (CHCl3)/cm21
3.1.4. Ethyl a-D-glucopyranoside (17). A mixture of
Amberlite IR-120 (Hþ) (3.5 g) and anhydrous D-glucose
(3.02 g, 16.76 mmol) in ethanol (25 cm3) was refluxed for
20 h. After cooling and filtration of the resin, excess ethanol
was evaporated under reduced pressure affording a yellow
syrup which was dissolved in pH 5.0 buffer (25 cm3) and
heated to 35 8C. b-Glucosidase from almonds (0.047 g) was
added to the reaction mixture. After 3 days the solution was
1
1140 (CvO); H NMR (DMSO) d 3.02–3.19 (3H, m),

P. M. L. Gonc¸alves et al. / Tetrahedron 60 (2004) 927–932
931
3.44–3.52 (2H, m), 3.71 (1H, dd, J¼11.6, 4.0 Hz), 4.24
(1H, d, J¼7.8 Hz), 4.49 (1H, t, J¼6.1 Hz), 4.59 (1H, d,
J¼12.4 Hz, CH2), 4.83 (1H, d, J¼12.4 Hz, 0CH2), 4.88 (2H,
dd, J¼11.6, 4.8 Hz), 7.26–7.41 (5H, m, Ar); 13C NMR
(DMSO) d 48.9, 61.4, 69.7, 70.4, 73.8, 77.0, 77.3, 102.4,
127.6, 127.9, 128.4, 138.4; m/z (CI) [MþNH4]þ 288
(100%).
(3H, t, J¼7.1 Hz, CH3), 1.62–1.71 (2H, m, CH2CH3), 2.35
(2H, t, J¼7.1 Hz, CH2CO), 3.33 (1H, t, J¼9.1 Hz), 3.5 (1H,
dd, J¼3.0, 9.1 Hz), 3.74–3.78 (2H, m), 4.19 (1H, dd, J¼2.0,
12.1 Hz), 4.42 (1H, dd, J¼4.0, 12.1 Hz), 4.53 (1H, d,
J¼11.1 Hz), 4.72 (1H, d, J¼11.1 Hz), 4.95 (1H, d,
J¼4.0 Hz), 7.28–7.37 (5H, m); 13C NMR (CDCl3) d 14.0
(CH3), 14.5 (CH2CH3), 18.7 (CH2CO), 36.4, 60.7, 63.3,
70.3, 70.4, 70.5, 72.5, 74.7, 98.0, 128.5, 128.9, 137.2, 174.7
(CvO). (Found: [MþNH4]þ, 358.186 C17H28O7N requires
[MþNH4]þ, 358.187); m/z (CI) [MþNH4]þ 358 (100%).
3.2.2. Benzyl 6-O-butyryl b-D-glucopyranoside (21).
Novozyme 435w (0.10 g) was added to a solution of
b-benzyl D-glucopyranoside 14 (0.31 g, 1.14 mmol) and
vinyl butyrate (0.3 cm3, 2.36 mmol) in dry THF (10 cm3)
and immersed in a oil bath at 60 8C with stirring. After 3 h
the reaction was complete by TLC and was cooled to room
temperature; the excess solvent was evaporated under
reduced pressure. Flash chromatography of the residue
over silica with EtOAc–EtOH (95:5) as eluent gave
compound 21 as a white solid (0.38 g, 98%). (Found: C,
59.77; H, 7.17. C17H24O7 requires C, 59.99; H, 7.11%); mp
65 8C; [a]2D3 255.7 (c 1.1 in CHCl3); nmax (CHCl3)/cm21
3.2.5. Benzyl 2,6-O-butyryl a-D-glucopyranoside (24).
Lipase PS-C II (0.054 g) was added to a slurry of benzyl
6-O-butyryl a-D-glucopyranoside 22 (0.03 g, 0.09 mmol) in
vinyl butyrate (5 cm3) and immersed in a oil bath at 30 8C
and stirred for 3 days. The enzyme was filtered off and
washed with THF (5 cm3), before the solvent was
evaporated under reduced pressure. Flash chromatography
of the crude residue over silica with ethyl acetate–
petroleum ether (2:1) as eluent afforded compound 24 as a
yellow syrup (0.032 g, 86%); [a]2D4 þ46 (c 0.5 in CHCl3);
n
max (CHCl3)/cm21 1720 (CvO); 1H NMR (CDCl3) d 0.94
(3H, t, J¼8.1 Hz, CH3), 0.97 (3H, t, J¼7.1 Hz, CH3), 1.59–
1.73 (4H, m, 2£CH2CH3), 2.39–2.39 (4H, m, 2£CH2CO),
3.41 (1H, t, J¼10.1 Hz), 3.82 (1H, ddd, J¼10.1, 4.0,
2.0 Hz), 4.04 (1H, t, J¼10.1 Hz), 4.17 (1H, dd, J¼12.1,
2.0 Hz), 4.48–4.53 (2H, m), 4.67–4.71 (2H, m), 5.11 (1H,
d, J¼4.0 Hz), 7.27–7.36 (5H, m, Ar); 13C NMR (CDCl3) d
13.9, 14.0, 18.5, 18.7, 36.3, 36.4, 63.1, 70.2, 70.2, 70.9,
71.7, 73.3, 95.9, 128.2, 128.3, 128.8, 137.4, 173.8, 174.9.
(Found: [M]þ411.203 C21H31O8 requires [M]þ, 411.202);
m/z (CI) [M]þ 411 (6%).
1
1720 (CvO); H NMR (CDCl3) d 0.96 (3H, t, J¼8.1 Hz,
CH3), 1.62–1.71 (2H, m, CH2CH3), 2.36 (2H, t, J¼7.1 Hz,
CH2CO), 3.35–3.43 (2H, m), 3.51 (1H, t, J¼8.1 Hz), 4.30–
4.32 (1H, m), 4.33 (1H, d, J¼7.1 Hz), 4.40 (1H, d,
J¼4.0 Hz), 4.43 (1H, d, J¼4.0 Hz), 4.59 (1H, d,
J¼12.1 Hz, CH2Ph) 4.89 (1H, d, J¼12.1 Hz, CH2Ph),
7.27–7.36 (5H, m, Ar); 13C NMR (CDCl3) d 13.6 (CH3),
18.4 (CH2CH3), 36.0 (CH2CO), 63.1, 70.0, 71.0, 73.5, 73.9,
75.9, 101.4, 128.1, 128.2, 128.5, 136.8, 174.4 (CvO); m/z
(CI) [MþNH4]þ 358 (100%).
3.2.3. Benzyl a-D-glucopyranoside (16). b-Glucosidase
from almonds (0.06 g) was added to a solution of benzyl-D-
glucopyranoside (0.42 g, 1.56 mmol) in a citric acid buffer
pH 5.0 (20 cm3). The reaction mixture was stirred for 3 days
at 50 8C. The enzyme was filtered off and the buffer was
evaporated under reduced pressure. The crude product was
chromatographed over silica with chloroform–methanol
(9:1) as eluent yielding compound 16 (0.28 g, 67%).
(Found: C, 57.49; H, 6.77. C13H18O6 required C, 57.77;
H, 6.71%); mp 102 8C (lit.17 122 8C); [a]2D2 þ134.7 (c 1.01
in MeOH) [lit.17þ133.5 (c 2.5 in H2O)]; 1H NMR (DMSO)
d 3.04–3.10 (1H, m), 3.20–3.25 (1H, m), 3.41–3.50 (3H,
m), 3.63 (1H, dd, J¼6.1, 9.1 Hz), 4.41–4.48 (2H, m), 4.66
(1H, s, OH), 4.70 (2H, d, J¼6.1 Hz), 4.73 (1H, d,
J¼3.0 Hz), 4.83 (1H, d, J¼5.1 Hz), 7.25–7.38 (5H, m);
13C NMR (DMSO) d 61.3, 68.2, 70.7, 72.3, 73.4, 73.7, 98.2,
127.6, 127.8, 128.5, 138.5; m/z (CI) [MþNH4]þ, 288
(100%).
3.2.6. 2,6-O-Butyryl-D-glucopyranoside (25). Benzyl 2,6-
butyryl a-D-glucopyranoside 24 (0.35 g, 0.86 mmol) was
dissolved in ethyl acetate, followed by the addition of Pd/C
(10%) (0.11 g). The reaction mixture was purged with
hydrogen and then placed under hydrogen pressure
(0.2 bar), with stirring, for 7 days at room temperature.
After completion of the reaction, the catalyst was filtered off
and washed with ethyl acetate. The solvent was evaporated
under reduced pressure and the crude product was
chromatographed over silica using ethyl acetate–petroleum
ether (2:1) as eluent affording compound 25 (0.22 g, 75%),
as a mixture of a- and b-anomers. (Found: C, 52.63; H,
7.62. C14H24O8 requires C, 52.49; H, 7.55%); [a]2D2 þ49.6
1
(c 0.6 in CHCl3); nmax (CHCl3)/cm21 1740 (CvO); H
NMR (CDCl3) d 0.93–0.99 (12H, m), 1.62–1.72 (8H, m)
2.33–2.41 (8H, m), 3.23 (1H, bs), 3.43 (2H, t, J¼9.2 Hz),
3.52 (1H, ddd, J¼9.9, 4.8, 2.2 Hz), 3.63–3.71 (3H, m), 3.78
(1H, bs) 4.00–4.05 (3H, m), 4.33 (2H, m), 4.43–4.49 (2H,
m), 4.64 (1H, t, J¼7.3 Hz), 4.67–4.72 (2H, m), 5.41 (1H,
bs); 13C NMR (CDCl3) d 13.9, 13.9 and 14.0, 18.7, 18.7,
36.3, 36.3, 60.8, 63.3, 63.4, 69.9, 70.9, 71.2 73.5, 74.5, 74.7,
75.8, 90.9, 95.9, 175.2 and 175.2 (CvO); m/z (CI)
[MþNH4]þ 338 (64%).
3.2.4. Benzyl 6-O-butyryl a-D-glucopyranoside (22).
Vinyl butyrate (0.2 cm3, 1.58 mmol) was added to a solution
of benzyl a-D-glucopyranoside 16 (0.25 g, 0.91 mmol) in
THF (20 cm3) and stirred for 5 min at room temperature.
Novozyme 435w (0.10 g) was added and the reaction
mixture was heated to 60 8C and stirred for 1 h. The enzyme
was filtered and washed with THF (10 cm3). The solvent
was evaporated under reduced pressure and the crude
product chromatographed over silica with ethyl acetate–
ethanol (95:5) as eluent yielding compound 22 (0.30 g,
97%). (Found: C, 60.20; H, 7.15. C17H24O7 requires C,
59.99; H, 7.11%); mp 72 8C; [a]2D1 þ77.8 (c 1.0 in CHCl3);
3.3. Separation of anomers from benzyl D-glucopyrano-
side
Novozyme 435w (0.054 g) and lipase PS-C II (0.22 g) was
added to benzyl D-glucopyranoside (0.31 g, 1.15 mmol) in
vinyl butyrate (5 cm3). The reaction was stirred for 4 days at
n
max (CHCl3)/cm21 1729 (CvO); 1H NMR (CDCl3) d 0.95

932
P. M. L. Gonc¸alves et al. / Tetrahedron 60 (2004) 927–932
40 8C, after which time the enzymes were filtered off and
washed with THF (10 cm3). Excess solvent was evaporated
under reduced pressure and the crude product chromato-
graphed over silica with ethyl acetate–petroleum ether (2:1)
as eluent affording compound 21 (0.141 g, 36%) and
compound 24 (0.13 g, 28%).
[a]2D4 þ96.1 (c 1.55 in MeOH); 1H NMR (DMSO) d 3.41–
3.56 (2H, m), 3.59–3.69 (3H, m) 3.73 (1H, br s, OH), 4.31–
4.34 (1H, br s, OH), 4.23 (1H, d, J¼12.1 Hz), 4.49–4.55
(3H, m), 4.68 (1H, d, J¼12.1 Hz), 4.76 (1H, d, J¼3.2 Hz),
7.26–7.69 (5H, m, Ar); 13C NMR (DMSO) d 61.0, 68.2,
68.7, 69.2, 70.0, 71.8, 98.6, 127.6, 127.8, 128.4, 138.5.
(Found: [MþNH4]þ, 288.145 C13H18O6N requires
[MþNH4]þ, 288.145); m/z (CI) [MþNH4]þ 288 (100%).
3.3.1. Benzyl 6-O-butyryl a-D-galactopyranoside (28)
and benzyl 2,6-O-butyryl b-D-galactopyranoside (29).
Novozyme 435w (0.12 g) was added to a solution of benzyl
D-galactopyranoside (0.12 g, 0.43 mmol) and vinyl butyrate
(0.1 cm3, 0.79 mmol) in THF (10 cm3). The reaction
mixture was heated to 60 8C and stirred for 7.5 h; the
enzyme was filtered off and washed with THF (10 cm3).
Excess solvent was evaporated under reduced pressure, and
the crude product was chromatographed over silica with
ethyl acetate–ethanol (95:5) as eluent yielding compound
28 (0.067 g, 46%) and compound 29 (0.041 g, 23%).
Compound 28. (Found: C, 59.94; H, 7.12. C17H24O7
requires C, 59.99; H, 7.11%); [a]2D1 þ92.5 (c 0.56 in
CHCl3); mp 106–108 8C; nmax (CHCl3)/cm21 1735
Acknowledgements
ˆ
The generosity of BAT and FCT (Fundac¸a˜o para a Ciencia e
Tecnologia) is acknowledged (studentship to P.G.).
References and notes
1. For some of the early work see: Seino, H.; Uchibori, T.;
Nishitani, T.; Inamasu, S. J. Am. Oil Chem. Soc. 1984, 61,
1761.
1
(CvO); H NMR (CDCl3) d 0.96 (3H, t, J¼7.6 Hz, CH3),
1.62–1.72 (2H, m, CH2CH3), 2.34 (2H, t, J¼7.6 Hz,
CH2CO), 3.79–3.87 (2H, m), 3.97 (1H, d, J¼2.0 Hz),
4.01 (1H, t, J¼6.6 Hz), 4.23 (1H, dd, J¼11.6, 6.5 Hz), 4.41
(1H, dd, J¼11.6, 6.1 Hz), 4.54 (1H, d, J¼11.6 Hz, CH2Ph),
4.76 (1H, d, J¼11.6 Hz), 5.05 (1H, d, J¼3.5 Hz), 7.31–7.40
(5H, m, Ar); 13C NMR (CDCl3) d 13.7 (CH3), 18.3 (CH2),
36.0 (CH2), 63.0, 68.3, 68.8, 69.4, 69.8, 70.8, 97.6, 128.1,
128.6, 136.7, 173.7(CvO; m/z (CI) 358 [MþNH4]þ.
Compound 29. (Found: C, 61.33; H, 7.37. C21H30O8 require
C, 61.45; H, 7.37%); [a]2D2 224.2 (c 0.8 in CHCl3); nmax
(CHCl3)/cm21 1738 (CvO); 1H NMR (CDCl3) d 0.94 (3H,
t, J¼7.6 Hz, CH3), 0.97 (3H, t, J¼7.6 Hz, 0CH3), 1.60–1.72
(4H, m, CH2CH3), 2.29–2.36 (4H, m, CH2CO), 3.60–3.67
(2H, m), 3.87 (1H, d, J¼2.9 Hz), 4.32 (1H, dd, J¼11.4,
6.7 Hz), 4.43 (1H, dd, J¼11.4, 6.4 Hz), 4.45 (1H, d,
J¼7.9 Hz), 4.62 (1H, d, J¼12.1 Hz), 4.88 (1H, d,
J¼12.1 Hz), 5.01 (1H, dd, J¼7.9, 9.5 Hz), 7.26–7.36 (5H,
m, Ar); 13C NMR (CDCl3) d 13.9, 14.0, 18.7, 18.7, 36.4,
36.5, 62.7, 69.1, 70.7, 72.5, 73.1, 73.6, 99.8, 128.2, 128.2,
128.7, 137.3, 174.0 and 174.6 (CvO); m/z (CI) 428
[MþNH4]þ.
2. Akoh, C. C.; Mutua, L. N. Enzyme Microb. Technol. 1994, 16,
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351. Fernandez-Mayoralas, A. Top. Curr. Chem. 1997, 186.
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¨
12. Adelhurst, K.; Bjorkling, F.; Godtfredsen, S. E.; Kirk, O.
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3.3.2. Benzyl a-D-galactopyranoside (27). Benzyl 6-O-
butyryl a-D-galactopyranoside 28 (0.065 g, 0.19 mmol) was
added to a solution of sodium methoxide in methanol
(0.08 M 10 cm3). The reaction mixture was stirred for 3 min
after which time the solvent was evaporated under reduced
pressure affording a crude product, which was chromato-
graphed over silica with chloroform–methanol (9:1) as
eluent, yielding compound 27 as a syrup (0.047 g, 91%);
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