The synthesis of compatible solute analogues-solvent effects on selective glycosylation

Article abstract of DOI:10.1016/j.carres.2010.08.007

Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-d-thioglucoside and ethyl 6-O-acetyl-2,3,4-tribenzyl-1-d-thiogalactoside, as a mixture of anomers, were employed in the study of the influence of solvent in the stereoselectivity of the glycosylation reaction with small and reactive acceptors. High α-selectivities were obtained in the glycosylation reactions using NIS/TfOH as activator and ethyl ether as the solvent at -60 °C. Other solvent mixtures such as dichloromethane, THF, THF/ethyl ether and toluene/dioxane were not nearly as selective. The corresponding thiogalactoside underwent similar glycosylations with the same solvents but with low anomer selectivity. These glycosides are key intermediates for the synthesis of new analogues of compatible solutes.

Full text of DOI:10.1016/j.carres.2010.08.007

Carbohydrate Research 346 (2011) 163–168
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
The synthesis of compatible solute analogues—solvent effects
on selective glycosylation
⇑
Eva C. Lourenço, M. Rita Ventura
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2780-901 Oeiras, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2010
Received in revised form 10 August 2010
Accepted 12 August 2010
Available online 24 August 2010
Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-D-thioglucoside and ethyl 6-O-acetyl-2,3,4-tribenzyl-1-D-thiogalacto-
side, as a mixture of anomers, were employed in the study of the influence of solvent in the stereoselec-
tivity of the glycosylation reaction with small and reactive acceptors. High -selectivities were obtained
a
in the glycosylation reactions using NIS/TfOH as activator and ethyl ether as the solvent at ꢀ60 °C. Other
solvent mixtures such as dichloromethane, THF, THF/ethyl ether and toluene/dioxane were not nearly as
selective. The corresponding thiogalactoside underwent similar glycosylations with the same solvents
but with low anomer selectivity. These glycosides are key intermediates for the synthesis of new
analogues of compatible solutes.
Keywords:
a-Glucosides
a-Galactosides
Ó 2010 Elsevier Ltd. All rights reserved.
Compatible solutes
Glycosylation
Thioglycosides
1. Introduction
selectivities for the synthesis of GG (>10:1 cis-1,2-/trans-1,2-isomers)
and GGG (>10:1 cis-1,2-/trans-1,2-isomers) were very good,4 they
In response to stressful conditions such as extreme temperature
and high salt concentration many microorganisms accumulate
were much lower than for the corresponding trans-1,2 selectivity
observed for mannosylation.5 For the formation of glucosyl lactate
(GL) using the same donor and conditions used for GG, a selectivity
of only 4:1 (cis-1,2-/trans-1,2-isomers) was obtained. In order to
optimise the glycosylation process, a study of the effect of solvent
and temperature was carried out. The influence of solvent on the
stereochemical outcome of glycosylations is well documented.6 It
is known that ethyl ether generally greatly favours the formation
of the thermodynamically more stable axial glycoside, by formation
of a diethyl oxonium-ion intermediate having the b-configuration,
due to steric interactions. The 1,2-cis-glycoside is then obtained
by nucleophilic displacement with inversion of configuration.6
compatible solutes.1
A superior thermoprotecting ability was
ascribed to ‘hypersolutes’, which are compatible solutes isolated from
hyperthermophiles, and confirmed by in vitro protein stabilisation
experiments.2,3 In contrast to the solutes more commonly found
in mesophiles, hypersolutes are generally negatively charged, and
most fall into two categories: hexose derivatives such as
nosyl-(1?2)- -glycerate (MG) and -glucosyl-(1?2)-
(GG), and polyol-phosphodiesters like di-myo-inositol phosphate
(DIP). A new trisaccharide (2R)-2-O- -glucopyranosyl-(1?6)-
-glucopyranosyl-2,3-dihydroxypropanoic acid, potassium salt
-glucosyl-(1?6)- -glucosyl-(1?2)- -glycerate (GGG)] iso-
lated from the hyperthermophile Persephonella marina, has recently
a-
D-man-
D
a-
D
D-glycerate
a-D
a-D
[(
a-D
a
-D
D
2. Results and discussions
been synthesised, as well as the related a-D-glucosyl-(1?2)-D-gly-
cerate (GG) (Fig. 1).4 In another study,5 several molecules
chemically related to mannosyl glycerate (MG) were synthesised
(Fig. 1). The effectiveness of the newly synthesised compounds for
the protection of model enzymes against heat-induced denatur-
ation, aggregation and inactivation was studied, revealing that
The main challenge for the synthesis of GG and GGG4 was the
stereoselective formation of the a-glucosidic bonds. Thioglucoside
1, which is readily prepared in just three steps from the correspond-
ing methyl glucoside, was used as the glucosidic donor and in both
glycosylation reactions the a-anomer was the major product (>10:1
a-D-mannosyl-(S)-lactate (ML), a synthetic solute, was superior to
a
/b) using the NIS/TfOH reagent combination. Besides the clear
MG in several applications. This has stimulated the present study
which is aimed towards the production of new synthetic analogues
of GG and GGG. Although the observed cis-1,2 glycosylation
synthetic advantages in obtaining and using thioglucoside 1,4 this
donor has an acetate group at the C-6 position which should favour
the formation of the a
-glucosides (1,2-cis glucosylation)4 as it is a
less reactive donor than the corresponding tetrabenzylated thiog-
lucoside due to the electron-withdrawing substituent at C-6.7 The
effects of remote substituents on glycosylation are obviously less
⇑
Corresponding author. Tel.: +351 214469775; fax: +351 214469789.
E-mail address: rventura@itqb.unl.pt (M.R. Ventura).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.08.007

164
E. C. Lourenço, M. R. Ventura / Carbohydrate Research 346 (2011) 163–168
OH
OH
OH
HO
O
O
O
HO
HO
HO
HO
HO
HO
OH
OH
OH
HO
O
O
O
O
HO
HO
GG
CO2 K
MG
CO2 K
OH
OH
O
GGG
CO2 K
OH
HO
OH
OH
HO
OH
HO
O
O
HO
O
O
HO
HO
HO
HO
HO
HO
HO
HO
OH
O
O
O
O
CO2 K
GL
CO2 K
ML
MGlyc
CO2 K
2-MGOH
HO
Figure 1. Compatible solutes and analogues.
important than those at C-2, however a substituent at the C-6 posi-
tion should influence the stereochemical outcome of glycosylation
reactions, by electronically shielding the upper face of the pyranose
ring.6,8
bonds.11 The thioglucosides used were obtained in several steps
and possessed a 4,6-O-cyclohexylidene-protecting group, a donor
similar to the 4,6-O-benzylidene thioglucoside used by Crich to
obtain 1,2-cis glycosides.12 The best solvent systems were found
to be 1:1 mixtures of CHCl3/Et2O and CHCl3/cyclopentylmethyle-
ther (CPME) and the reagents used were 2,6-di-tert-butyl-4-meth-
ylpyridine and a large excess of MeOTf. In most of these studies the
acceptors were other glycosides, mono and higher saccharides, and
no smaller or more reactive alcohols were studied. The acetal-
protecting group is not very stable under acidic glycosylation
conditions and this accounted for the lower yields of the glycosyl-
ation reactions.
Following the same line of research,5 the corresponding
analogues of GG, namely GL,
a-D-glucosyl glycolate (GGlyc) and
a-
D
-glucosyl-(1?6)- -glucosyl lactate (GGL) were required.
a-D
The obvious synthetic route would be to use the thioglucoside 1
as the donor. However, as was earlier reported,4 transglycosylation
of 1 with the less bulky, more reactive, methyl (S)-lactate produced
the glucosyl lactate with an a/b ratio of only 4:1 under the glyco-
sylation reaction conditions used (dichloromethane as solvent at
0 °C—Table 1, entry 1).
This problem prompted us to find a more stable and more easily
Boons and co-workers have reported that the best solvent
prepared donor which we have found in thioglucoside 1.4 During
mixture for obtaining
a
-glucosides from
a
tetrabenzylated
attempts to optimise the a/b ratio for the formation of glucosyl
b-thioglucoside was toluene/1,4-dioxane.9 Ethyl ether was not
lactates, we studied the glycosylation reaction between donor 1
and methyl (S)-lactate using NIS/triflic acid as the promoter and
several solvents or solvent combinations at 0 °C, in the presence
of 4 Å molecular sieves (MS) (Scheme 1, Table 1).
tested alone, but only in mixtures with other solvents. Ethyl ether
with dichloromethane afforded a lower
afforded a considerably higher /b ratio but lower than that
a/b ratio and with toluene
a
obtained with toluene/dioxane mixtures. Although no yields were
presented, it was reported that glycosylations in ether/toluene
were very sluggish and incomplete. Dichloromethane was the
The toluene/dioxane (1:3) solvent combination employed by
Boons and co-workers9 afforded an improved 6:1
a/b ratio, but
the donor was consumed much slower (80 min) and the yield
was only 43% (Table 1, entry 6). The recovered byproduct was
the corresponding reducing monosaccharide. This was formed
particularly in the glycosylation reactions that were slower and
incomplete (see also Table 2) and was probably formed by quench-
ing of the activated donor during the aqueous work-up. Changing
the promoter to NIS/TMSOTf increased the yield to 78% within a
shorter reaction time (20 min), however, the anomeric ratio
worst solvent affording more b anomer (0.7:1
a/b) and the
glycosylation reaction in THF alone was not studied.
More recently,10 Bonnaffé and co-workers have described
optimisation studies for the glycosylation reaction of a 2-azido
trichloroacetimidate disaccharide derived from glucose with
several acceptors, also disaccharides, in order to improve the
a-stereoselectivity. It was shown that dichloromethane was not
the best reaction solvent in terms of both yield and diastereoselec-
tivity and that by changing to ethyl ether, no improvement in the
remained almost the same (
A THF/Et2O (9:1) mixture, that had given good results in the
work of Bonnaffé,10 afforded a 5.8:1
/b mixture but only 60% yield
a/b 6.3:1, Table 1, entry 7).
a/b ratio was observed but the yield was reduced. By performing
a
the glycosylation reaction in THF, the /b ratio was considerably
a
(Table 1, entry 3). Reversing the proportions of THF and Et2O to 1:9
better but the yield was even lower than that obtained in ethyl
ether. After testing several solvent mixtures, they found that the
best reaction solvent was a THF/Et2O (9:1) mixture.
increased the yield considerably to 87%, however the anomeric
ratio was only marginally improved (6:1
Finally, Et2O was used as the sole solvent and pleasingly a higher
anomeric ratio of 7.5:1 /b and 85% yield was obtained (Table 1,
entry 4). However, using neat THF as the solvent, the reaction
a/b, Table 1, entry 2).
In another study, Ito and co-workers observed the effect of
a
solvents during the construction of three sequential
a-glucosidic
OAc
OAc
R = 2: (S)-CH(CH3)CO2Me
3: (R)-CH(CH3)CO2Me
4: (S)-CH(CH)3CO2Bn
5: (R)-CH2CH(OTBDMS)CO2Me
6: -CH2CO2Me
O
O
NIS, TfOH, 4Å MS
BnO
BnO
BnO
BnO
OBn
OBn
OR
SEt
1
Scheme 1. Glycosylation reaction of thioglucoside 1.

E. C. Lourenço, M. R. Ventura / Carbohydrate Research 346 (2011) 163–168
165
was slower and the yield and the selectivity were lower (Table 1,
entry 5). Comparing the results obtained, ethyl ether alone was
clearly the best solvent for this glycosylation reaction.
2, entry 2), leaving the reaction longer decreased the yield and the
anomeric selectivity (Table 2, entry 3). Thiogalactosides are known
to be more reactive than thioglucosides. This tendency is observed
in glycosylation reactions, for example, p-methylphenyl thiogalac-
toside is 6.4 times more reactive than the corresponding glucoside
in the reaction with MeOH promoted by NIS/TfOH.12 The concept
of an axial polar substituent stabilising a positive charge to a
higher degree than the corresponding equatorial equivalent
explains this observation and supports the concept of very reactive
super-armed thioglycosides.13
The same glycosylation reaction was conducted in dichloro-
methane and ethyl ether at ꢀ60 °C (Table 1). At this temperature,
the anomeric selectivity increased in CH2Cl2 (7:1
a/b, entry 8)
and even though the yield was slightly lower (83%), the reaction
time remained the same (10 min). With Et2O as the solvent, the
donor was consumed more slowly (45 min) but this was largely
compensated by improvement of the
yield (81%) (Table 1, entry 9).
a/b ratio to 9:1 and the good
Changing the solvent to ethyl ether lowered the rate of reaction
Glycosylation with methyl (R)-lactate in CH2Cl2 and in Et2O at
/b ratio
but did not increase the proportion of the a-anomer (a/b 3:1, Table
2, entry 5). This contrasts dramatically with the result obtained for
both 0 and ꢀ60 °C (Table 1, entries 10–13) afforded an
a
very similar to that observed with methyl (S)-lactate as the donor,
indicating that double stereodifferentiation was probably involved
but not very important. With the bulkier benzyl (S)-lactate, the
anomeric selectivity was the highest obtained in Et2O at ꢀ60 °C
the corresponding reaction with thioglucoside 1. At ꢀ60 °C, in both
dichloromethane and ethyl ether, the a/b ratio decreased to 1.5:1
and 2.2:1, respectively (Table 2, entries 4 and 6). Using bulkier
benzyl (S)-lactate as the acceptor, in dichloromethane at 0 °C, no
improvement on the anomeric selectivity was observed (Table 2,
entry 8). However, when the acceptor was benzyl (S)-lactate and
(10:1 a/b Table 1, entry 17), with the additional advantage of the
benzyl ester being removable at the same time as the benzyl
ether-protecting groups, thus avoiding the final basic ester hydro-
lysis to afford the acid salt of GL.4,5 Interestingly, when employing
dichloromethane as the solvent, at 0 °C, the stereoselectivity
obtained with methyl (S)-lactate, methyl (R)-lactate and benzyl
the solvent was ethyl ether at ꢀ60 °C, the
4:1 (Table 2, entry 11). In CH2Cl2, the same was not observed,
the anomeric selectivity at ꢀ60 °C was decreased to 1.7:1 /b
(Table 2, entry 10). The glycosylation reaction of 8 with methyl
glycolate, in dichloromethane at 0 °C, also afforded a similar /b
a/b ratio increased to
a
(S)-lactate was the same (4:1
a
/b, Table 1, entries 1, 10 and 14),
a
indicating that under these conditions the (R),(S)-stereochemistry
of the lactate and the bulkiness of the ester moiety were not
important for the anomeric selectivity.
ratio of 3:1 (Table 2, entry 12) and in the other conditions tested
(Table 2, entries 13–15) the ratio was lower. The different
behaviour of glucose and galactose donors under the same
conditions appears to be related to conformational differences.
The selectivity of the glycosylation reaction is influenced by the
reactivity of its components.6,7 The more reactive galactose donors,
with a non-participating protective group at C-2, afforded lower
Glycosylation with the primary hydroxyl of methyl glycerate4
was also attempted (Table 1, entry 18). This alcohol 7 was obtained
using standard protection reactions. Only the
a-anomer was
observed but the yield of the reaction was low (30%) and thiogluco-
side 1 was recovered. Attempts to carry out the glycosylation
reaction of donor 1 with the small primary alcohol methyl
glycolate in ethyl ether at ꢀ60 °C (Table 1, entry 22) resulted in
anomeric
a
selectivity,7 however, the reactivity of the acceptor also
plays an important role in the selectivity of the reaction. In most
examples cited in the literature,6,9–11 the acceptor is usually
an excellent yield (88%) and the
a/b ratio was 13:1. The low yield
another sugar molecule having a large number of possible
obtained with the acceptor 7 was inherent to its protecting groups.
interactions with the donor, whereas in our case the acceptor is a
relatively small alcohol with few functional groups.
The nature of the promoter has also been reported to have an
influence on the stereoselectivity of the glycosylation reaction,6,9
but studies under the Crich conditions for the glycosylation of
thioglucoside 1 with methyl (S)-lactate did not improve the
selectivity.14
The highest selectivity was obtained when THF was used as
the solvent (a/b 5:1, Table 2, entry 1), however the reaction time
was long, the yield was only 15%, 43% of starting material was
recovered and the hydrolysis product was obtained in 38% yield.
The toluene/dioxane mixture (1:3)9 afforded a low selectivity
In the case of methyl glycolate, the anomeric stereoselectivity
was not improved in CH2Cl2 at ꢀ60 °C (4.2:1
21) compared with the one obtained in the same reaction at 0 °C
(4.2:1 /b, Table 1, entry 19).
a/b, Table 1, entry
a
The rationale behind the design of the analogues synthesised so
far4,5 had been to vary the aglycone part of the molecule. Variation
of the sugar is also of great interest, both for the possible stabilising
properties and also for an understanding of the mechanism of
action of these solutes. To our knowledge, galactose containing
compatible solutes have not been isolated. Thus, the glycosylation
of the thiogalactoside 8, prepared in the same way as the
thioglucoside 1, was attempted. Glycosylation of thiogalactosides
has been less studied than those of thioglucosides but in general
produce lower cis-1,2- selectivities. Once again, the studies that
we found referred to other sugars as the acceptor, not small
unhindered alcohols.6
(a
/b 2.4:1, Table 2, entry 7). The best solvent for the glycosyla-
tion reaction between thiogalactoside 8 and methyl (S)-lactate
was dichloromethane, affording a /b ratio of 3:1 and 87% yield
(Table 2, entry 2). Ethyl ether afforded the same /b ratio of 3:1,
a
a
however the yield was lower. The anomers of galactosides 9, 10
The results of the glycosylation reactions using thiogalactoside
8 as donor and methyl or benzyl (S)-lactate and methyl glycolate
as acceptor are (Scheme 2) summarised in Table 2. The selectivities
of the reactions were generally lower than for the corresponding
glucoside donor. Notable also are the shorter reaction times (Table
and 11 were easier to separate than the corresponding gluco-
sides, and the synthesis of
a- and b-galactosyl-(S)-lactate and
of - and b-galactosylglycolate afforded, after separation, four
a
new solute analogues to be tested for protein stabilisation
properties.
OAc
OAc
BnO
BnO
BnO
BnO
O
O
NIS, TfOH, 4Å MS
ROH, solvent
R = 9: (S)-CH(CH3)CO2Me
10: (S)-CH(CH)3CO2Bn
11: -CH2CO2Me
OBn
OBn
SEt
OR
8
Scheme 2. Glycosylation reactions of thiogalactoside 8.

166
E. C. Lourenço, M. R. Ventura / Carbohydrate Research 346 (2011) 163–168
Table 1
3. Conclusions
The effect of solvent and temperature on the stereoselectivity of the glycosylation of 1
Ethyl ether was the solvent of choice for the glycosylation
reaction of thioglucoside 1 with small, reactive lactate and glyco-
late esters using NIS/TfOH as the promoter. This result was
expected, ethyl ether is considered a participating solvent and
the diethyl oxonium-ion formed in situ preferentially adopts
equatorial orientation, leading towards the axial glycosidic bond
formation.6 Generally, with dichloromethane as the solvent, per-
forming the reaction at ꢀ60 °C improved the anomeric selectivity
compared to the same reaction at 0 °C. Similar glycosylations with
the thiogalactoside 8 did not afford such high stereoselectivity, and
changing the solvent or lowering the reaction temperature did not
improve the results. Comparing these results with those reported
in the literature shows once more that the stereoselectivity of
the glycosylation reaction depends on many factors and that it is
difficult to find a general method to obtain 1,2-cis glycosides even
when using donors and acceptors of similar structure. Most of the
reported studies6 describing solvent effects in glycosylation use
bulky and much less reactive glycosides as acceptors and the
results cannot be applied to simpler molecules.
Entry ROH
T
(°C)
Solvent
t
Yield
a/b
(min) (%)
1
2
3
4
5
6
Methyl (S)-lactate
0
0
0
0
0
0
CH2Cl2
10
10
10
10
45
80
91
87
60
4:1
6:1
5.8:1
7.5:1
6.5:1
6:1
Methyl (S)-lactate
Methyl (S)-lactate
Methyl (S)-lactate
Methyl (S)-lactate
Methyl (S)-lactate
THF/Et2O (1:9)
THF/Et2O (9:1)
Et2O
85
THF
72
Toluene/
dioxane (1:3)
Toluene/
43a
7
Methyl (S)-lactate
0
20
78
6.3:1
b
dioxane (1:3)
8
9
Methyl (S)-lactate
Methyl (S)-lactate
Methyl (R)-lactate
Methyl (R)-lactate
Methyl (R)-lactate
Methyl (R)-lactate
Benzyl (S)-lactate
Benzyl (S)-lactate
Benzyl (S)-lactate
Benzyl (S)-lactate
ꢀ60 CH2Cl2
10
45
8
15
10
45
10
15
10
45
83
81
85
76
82
83
93
82
97
81
7:1
9:1
4:1
7.9:1
6.5:1
8.3:1
4:1
8.3:1
6.1:1
10:1
ꢀ60 Et2O
10
11
12
13
14
15
16
17
0
0
CH2Cl2
Et2O
ꢀ60 CH2Cl2
ꢀ60 Et2O
0
0
CH2Cl2
Et2O
ꢀ60 CH2Cl2
ꢀ60 Et2O
OTBDMS
18
ꢀ60 Et2O
45
30c
1:0
HO
The glucosides2, 3, 4 and 6 and galactosides 9, 10and 11 were key
CO2Me
7
intermediates in the synthesis of
(R)-lactate, -glucosylglycolate,
- and -galactosylglycolate for testing as protein stabilisers.
a
-glucosyl-(S)-lactate,
a-glucosyl-
19
20
21
22
Methyl glycolate
Methyl glycolate
Methyl glycolate
Methyl glycolate
0
0
CH2Cl2
Et2O
10
45
10
45
93
85
96
88
4.2:1
4.4:1
4.2:1
13:1
a
a- and b-galactosyl-(S)-lactate and
a
a
ꢀ60 CH2Cl2
ꢀ60 Et2O
4. Experimental
4.1. General
a
b
c
Hydrolysis product also recovered.
TMSOTf was used instead of TfOH.
Starting material was recovered.
1H NMR spectra were obtained at 400 MHz in CDCl3 with
chemical shift values (d) in ppm downfield from tetramethylsilane,
and 13C NMR spectra were obtained at 100.61 MHz in CDCl3. Assign-
ments are supported by 2D correlation NMR studies. Medium pres-
sure preparative column chromatography: Silica Gel Merck 60 H.
Analytical TLC: Aluminium-backed Silica Gel Merck 60 F254. Re-
agents and solvents were purified and dried according to Ref. 15.
All the reactions were carried out under an inert atmosphere
(argon). Thioglycosides 1 and 7 were prepared as described in Ref. 4.
Table 2
Effect of solvent and temperature on the stereoselectivity of the glycosylation of 8
Entry ROH
T
Solvent
t
Yield
a/b
(°C)
(min) (%)
1
2
Methyl (S)-
lactate
Methyl (S)-
lactate
Methyl (S)-
lactate
Methyl (S)-
lactate
Methyl (S)-
lactate
Methyl (S)-
lactate
Methyl (S)-
lactate
Benzyl (S)-
lactate
Benzyl (S)-
lactate
Benzyl (S)-
lactate
Benzyl (S)-
lactate
Methyl
glycolate
Methyl
glycolate
Methyl
glycolate
Methyl
0
0
0
THF
60
5
15a
87
68
83
70
86
54b
92
84
82
86
87
92
96
95
5:1
CH2Cl2
CH2Cl2
3:1
3
30
5
2.6:1
1.5:1
3:1
4
ꢀ60 CH2Cl2
Et2O
4.2. General glycosylation procedure
5
0
40
40
10
5
A suspension of thioglycoside donor (0.15 mmol), acceptor
(0.15 mmol) and 4 Å MS in the solvent/mixture of solvents
indicated in Tables 1 and 2 (1 mL) was stirred for 1 h at room
temperature then cooled to 0 °C. N-Iodosuccinimide (0.19 mmol)
6
ꢀ60 Et2O
2.2:1
2.5:1
2.8:1
2.7:1
1.7:1
4:1
7
0
Toluene/dioxane
(1:3)
CH2Cl2
and TfOH (0.9 lL) were added at 0 °C and when the reaction was
8
0
complete (TLC), 10% Na2S2O3 aqueous solution (2 mL) and satd
aq NaHCO3 (1 mL) were added and the mixture was extracted with
CH2Cl2 (3 ꢁ 5 mL); the combined organic phases were dried
(MgSO4), filtered and the solvent was removed under vacuum.
The crude product was purified by PLC (3:7 EtOAc/hexane). The
9
0
Et2O
40
10
40
5
10
11
12
13
14
15
ꢀ60
ꢀ60 Et2O
a/b ratio of the isolated product was measured by comparison of
0
0
CH2Cl2
Et2O
3:1
the integral of the methyl group of the lactate moiety signals in
1H NMR (400 MHz, CDCl3) spectra. Yields and
described in Tables 1 and 2.
a/b ratio values are
15
5
2.2:1
1.2:1
1.4:1
ꢀ60 CH2Cl2
ꢀ60 Et2O
4.2.1. Methyl (2S)-2-(6-O-acetyl-2,3,4-tri-O-benzyl-a/b-D-
15
glucopyranosyl)propanoate 2
max (film): 1743 cmꢀ1 (C@O). 1H NMR (CDCl3): d 7.42–7.24 (m,
glycolate
m
a
15H, Bn aromatic), 5.01 (d, J = 10.7 Hz, 1H, Bn), 4.89–4.76 (m, 3H,
43% of starting material and 38% the hydrolysis product were recovered.
18.2% of the hydrolysis product was recovered.
b
Bn), 4.75 (d, J = 3.0 Hz, 1H, H-1 ( )), 4.63 (d, J = 12.0 Hz, 1H, Bn),
a
4.57 (d, J = 10.9 Hz, 1H, Bn), 4.50 (d, J = 6.2 Hz, H-1 (b)), 4.27 (dd,
J = 12.2 Hz, J = 3.9 Hz, 1H, H-6), 4.15–4.02 (m, 4H, H-3, H-5, H-6,
CH(CH3)CO2Me), 3.71 (s, –OMe (b)), 3.70 (s, 3H, –OMe (
3.48 (m, 2H, H-2, H-4), 2.03 (s, –OAc (b)), 2.00 (s, 3H, –OAc (
a
)), 3.55–
1.50 (d, J = 5.5 Hz, CH(CH3)CO2Me (b)), 1.44 (d, J = 5.4 Hz, 3H,
CH(CH3)CO2Me (
)). 13C NMR (CDCl3): d 172.9, 172.7, 138.6,
a)),
a

E. C. Lourenço, M. R. Ventura / Carbohydrate Research 346 (2011) 163–168
167
138.5, 138.0, 135.6, 135.5, 132.8, 129.9, 129.8, 128.5–127.4, 102.4
(C-1(b)), 97.8 (C-1( )), 84.3, 82.0, 81.6, 80.1, 75.7, 75.6, 75.1,
75.0, 74.6, 74.4, 73.4, 72.8, 71.5, 62.0, 61.8, 52.1, 52.0, 19.1, 18.0.
OAc), 0.91 (s, 9H, tert-Butyl), 0.13 (s, 6H, SiMe2). 13C NMR (CDCl3):
d 172.2, 170.7, 138.6, 138.3, 138.1, 128.6–127.6, 97.1 (C-1), 81.7,
79.2, 77.1, 75.7, 74.8, 72.6, 72.0, 68.8, 65.0, 63.1, 52.0, 25.7, 20.8,
18.3, ꢀ4.9.
a
4.2.2. Methyl (2S)-2-(6-O-acetyl-2,3,4-tri-O-benzyl-a/b-D-
glucopyranosyl)propanoate 2—Crich conditions
4.2.6. Methyl 2-(6-O-acetyl-2,3,4-tri-O-benzyl-a/b-D-
To a solution of 1 (0.060 g, 0.11 mmol), 1-benzenesulfinyl piper-
idine (0.023 g, 0.11 mmol), 2,6-di-tert-butyl-4-methylpyridine
(0.045 g, 0.22 mmol) and 4 Å powdered molecular sieves in
dichloromethane (1.5 mL) at ꢀ60 °C was added Tf2O (0.021 mL,
glucopyranosyl)acetate 6
mmax (film): 1742 cmꢀ1 (C@O). 1H NMR (CDCl3): 7.32–7.25 (m,
15H, Bn aromatic), 5.05 (d, J = 4.0 Hz, 1H, H-1 (a)), 5.03 (d,
J = 11.2 Hz, 1H, Bn), 4.88 (d, J = 10.8 Hz, 1H, Bn), 4.86 (d, J =
11.9 Hz, 1H, Bn), 4.81 (d, J = 10.8 Hz, 1H, Bn), 4.77 (d, J = 11.9 Hz,
1H, Bn), 4.55 (d, J = 10.9 Hz, 1H, Bn), 4.51 (d, J = 7.7 Hz, H-1 (b)),
4.27 (d, J = 16.6 Hz, 1H, OCH2CO2Me), 4.25–4.24 (m, 2H, H-6), 4.15
(d, J = 16.6 Hz, 1H, OCH2CO2Me), 4.06 (t, J = 9.2 Hz, 1H, H-3), 3.91
0.12 mmol). After 5 min,
a solution of methyl (S)-lactate
(0.016 mL, 0.16 mmol) in dichloromethane (0.5 mL) was added.
After 5 min at ꢀ60 °C, satd aq NaHCO3 was added (5 mL) and the
mixture extracted with dichloromethane (3 ꢁ 5 mL), dried with
anhyd MgSO4 and concentrated. Purification by preparative layer
(dt, J = 10.1 Hz, J = 3.1 Hz, 1H, H-5), 3.76 (s, 3H, CO2Me (
(s, CO2Me (b)), 3.59 (dd, J = 9.6 Hz, J = 3.6 Hz, 1H, H-2 (
3.50 (m, 1H, H-2 (b)), 3.48 (dd, J = 9.7 Hz, J = 9.2 Hz, 1H, H-4), 2.03
(s, OAc (b)), 2.02 (s, 3H, OAc (
)). 13C NMR (CDCl3): d 170.7, 170.0,
138.6, 137.9, 137.8, 128.5, 128.47, 128.45, 128.3, 128.1, 128.0,
127.95, 127.92, 127.7, 103.2 (C-1(b)), 96.3 (C-1( )), 81.6, 79.3,
77.0, 75.8, 75.0, 72.7, 69.3, 63.3, 62.9, 51.9, 20.8. Anal. Calcd for
32H36O9: C, 68.07; H, 6.43. Found: C, 68.60, H, 6.54.
a
)), 3.75
chromatography (30:70 EtOAc/hexane) afforded glycoside
(0.051 g, 77%, /b 4:1) as a very viscous oil.
2
a)), 3.54–
a
a
4.2.3. Methyl (2R)-2-(6-O-acetyl-2,3,4-tri-O-benzyl-a/b-D-
glucopyranosyl)propanoate 3
a
m
max (film): 1742 cmꢀ1 (C@O). 1H NMR (CDCl3): d 7.43–7.25 (m,
15H, Bn aromatic), 5.10 (d, J = 3.4 Hz, 1H, H-1 (
a)), 5.06 (d, J =
C
10.8 Hz, 1H, Bn), 4.89 (d, J = 11.9 Hz, 1H, Bn), 4.88 (d, J = 10.7 Hz,
1H, Bn), 4.81 (d, J = 10.8 Hz, 1H, Bn), 4.72 (d, J = 11.9 Hz, 1H, Bn),
4.55 (d, J = 10.7 Hz, 1H, Bn), 4.48 (d, J = 7.7 Hz, H-1 (b)), 4.37 (q,
J = 7.0 Hz, 1H, CH(CH3)CO2Me), 4.26 (d, J = 3.4 Hz, 2H, H-6), 4.08 (t,
J = 9.2 Hz, 1H, H-3), 3.83 (ddd, J = 10.0 Hz, J = 3.2 Hz, J = 3.2 Hz, 1H,
4.2.7. Ethyl 6-O-acetyl-2,3,4-tri-O-benzyl-1-thio-
galactopyranoside 8
a
/bꢀ
D-
Alpha anomer (56% yield): ½a D20
ꢂ
+113.8 (c 2.32, CH2Cl2). mmax
(film): 1743 cmꢀ1 (C@O). 1H NMR (CDCl3): d 7.40–7.27 (m, 15H,
Bn aromatic), 5.49 (d, 1H, J = 3.5 Hz, H-1), 4.95 (d, 1H, J = 7.0 Hz,
Bn), 4.86 (d, 1H, J = 7.5 Hz, Bn), 4.76–4.66 (m, 3H, Bn), 4.61 (d, 1H,
J = 7.0 Hz, Bn), 4.31–4.25 (m, 2H, H-2, H-5), 4.18 (dd, 1H,
J = 4.5 Hz, J = 2.8 Hz, H-6), 4.05 (dd, 1H, J = 3.3 Hz, J = 3.8 Hz, H-6),
3.85–3.84 (m, 1H, H-4), 3.79 (dd, 1H, J = 1.8 Hz, J = 4.5 Hz, H-3),
2.62–2.44 (m, 2H, SCH2CH3), 1.97 (s, 3H, OAc), 1.27 (t, 3H,
J = 4.5 Hz, SCH2CH3). 13C NMR (CDCl3): d 138.7, 138.2, 138.1,
128.5–127.5, 83.2 (C-1), 79.4, 76.4, 74.7, 74.5, 73.7, 72.5, 68.9,
H-5), 3.76 (s, 3H, OMe (
J = 3.6 Hz, 1H, H-2), 3.48 (t, J = 9.7 Hz, 1H, H-4), 2.04 (s, OAc (b)),
2.02 (s, 3H, OAc ( )), 1.50 (d, J = 7.0 Hz, 3H, CH(CH3)CO2Me ( )),
1.46 (d, J = 6.8 Hz, CH(CH3)CO2Me (b)). 13C NMR (CDCl3): d 172.9
(b), 172.5 ( ), 170.7 (b), 170.6 ( ), 138.6 ( ), 138.4 (b), 138.2 (b),
137.9 ( ), 137.6 ( ), 137.5 (b), 128.5–127.6, 103.4 (C-1(b)), 95.3
(C-1( )), 84.5 (b), 81.8 (b), 81.5 ( ), 78.9 ( ), 77.1 ( ), 75.7 ( ),
75.6 (b), 75.5 (b), 75.2 ( ), 75.0 (b), 74.9 (b), 72.9 (b), 72.3 ( ), 69.7
), 69.3 ( ), 62.9 ( ), 62.8 (b), 51.9 ( ), 20.7 ( ), 18.4 ( ), 18.2 (b).
a)), 3.72 (s, OMe (b)), 3.58 (dd, J = 9.6 Hz,
a
a
a
a
a
a
a
a
a
a
a
a
a
a
(a
a
a
a
a
a
63.4, 23.5, 20.8, 14.7. HR-MS: calcd for C
31H36O6SH+ [M]+:
537.23054; found: 537.23155. Beta anomer (23% yield): ½a D20
ꢂ
4.2.4. Benzyl (2S)-2-(6-O-acetyl-2,3,4-tri-O-benzyl-
a/b-D
-
+17.1 (c 1.18, CH2Cl2). mmax (film): 1742 cmꢀ1 (C@O). 1H NMR
(CDCl3): d 7.39–7.26 (m, 15H, Bn aromatic), 4.98 (d, 1H, J = 7.3 Hz,
Bn), 4.89 (d, 1H, J = 6.3 Hz, Bn), 4.83–4.74 (m, 3H, Bn), 4.65 (d, 1H,
J = 7.5 Hz, Bn), 4.41 (d, 1H, J = 6.0 Hz, H-1), 4.22 (dd, 1H, J = 4.3 Hz,
J = 2.8 Hz, H-6), 4.05 (dd, 1H, J = 3.8 Hz, J = 3.3 Hz, H-6), 3.87–3.82
(m, 2H, H-2, H-4), 3.58–3.53 (m, 2H, H-3, H-5), 2.80–2.64 (m, 2H,
SCH2CH3), 1.96 (s, 3H, OAc), 1.29 (t, 3H, J = 4.5 Hz, SCH2CH3). 13C
NMR (CDCl3): d 170.5, 138.28, 138.25, 138.22, 128.5–127.6, 85.4
(C-1), 84.1, 78.4, 75.9, 75.8, 74.3, 73.3, 73.1, 63.3, 24.9, 20.8, 15.1.
HR-MS: calcd for C31H36O6SH+ [M]+: 537.23054; found: 537.23002.
glucopyranosyl)propanoate 4
m
max (film): 1739 cmꢀ1 (C@O). 1H NMR (CDCl3): d 7.34–7.24 (m,
20H, Bn aromatic), 5.18–5.09 (m, 2H, Bn ester), 4.99 (d, J = 10.7 Hz,
1H, Bn), 4.86 (d, J = 10.9 Hz, 1H, Bn), 4.82 (d, J = 11.0 Hz, 1H, Bn),
4.79 (d, J = 12.1 Hz, 1H, Bn), 4.77 (1H, d, J = 3.1 Hz, H-1 (a)), 4.63
(d, J = 12.0 Hz, 1H, Bn), 4.54 (d, J = 11.0 Hz, 1H, Bn), 4.51 (1H, d,
J = 7.8 Hz, H-1 (b)), 4.20–4.09 (m, 3H, H-5, H-6, CH(CH3)CO2Bn),
4.06–3.99 (m, 2H, H-3, H-6), 3.54–3.47 (m, 2H, H-2, H-4), 2.01 (s,
OAc (b)), 1.97 (s, 3H, OAc (
(b)), 1.45 (d, J = 6.8 Hz, 3H, CH(CH3)CO2Bn (
d 172.1 (b), 171.9 ( ), 170.7 (b), 170.6 ( ), 138.6 (
138.4 (b), 138.1 ( ), 138.0 ( ), 137.7 (b), 135.4 ( ), 128.6–127.6,
102.4 (C-1(b)), 97.3 (C-1( )), 84.4 (b), 81.8 ( ), 79.7 ( ), 77.1 (b),
77.0 ( ), 75.7 ( ), 75.0 (b), 74.8 (
72.9 (b), 72.8 (b), 69.1 ( ), 66.7 (
19.1 (b), 17.8 ( ).
a
)), 1.51 (d, J = 6.8 Hz, CH(CH3)CO2Bn
)). 13C NMR (CDCl3):
), 138.5 (b),
a
a
a
a
4.2.8. Methyl (2S)-2-(6-O-acetyl-2,3,4-tri-O-benzyl-a/b-D-
a
a
a
galactopyranosyl)propanoate 9
a
a
a
mmax (film): 1743 cmꢀ1 (C@O). 1H NMR (CDCl3): 7.45–7.25 (m,
a
a
a
), 74.5 (b), 73.8 (
a
), 73.3 (
a
),
),
Bn aromatic,
Bn, and b), 4.85 (s, 1H, H-1
b), 4.45 (d, J = 7.6 Hz, H-1 b), 4.21–3.95 (m, H-6
CH(CH3)CO2Me , H-2 , H-6 , H-6 b, H-3 ), 3.93 (sl, 1H, H-4
), 3.86 (dd, J = 7.6 Hz, J = 9.6 Hz, 1H, H-2 b), 3.75 (d, J = 2.2 Hz,
H-4 b), 3.69 (s, 3H, –OMe b), 3.67 (s, 3H, –OMe ), 3.58–3.44 (m,
2H, H-3 b, H-5 b), 1.96 (s, 3H, –OAc b), 1.95 (s, 3H, –OAc ), 1.49
(d, J = 6.9 Hz, 3H, CH(CH3)CO2Me b), 1.43 (d, J = 6.8 Hz, 3H,
CH(CH3)CO2Me
). 13C NMR (CDCl3): 173.0, 172.9, 170.5, 138.8,
a
and b), 5.12 (d, J = 10.8 Hz, 1H, Bn), 4.98–4.58 (m,
), 4.51–4.44 (m, 1H, CH(CH3)CO2Me
, H-6 b, H-5
a
a
), 63.0 (b), 62.7 (
a
), 20.8 (
a
a
a
a
a
a,
a
a
a
a
4.2.5. Methyl (2R)-tert-butyldimethylsilyl-3-(6-O-acetyl-2,3,4-
tri-O-benzyl- -glucopyranosyl)propanoate 5
a
a
-D
a
mmax (film): 1744 cmꢀ1 (C@O). 1H NMR (CDCl3): 7.38–7.25 (m,
15H, Bn aromatic), 4.95 (d, J = 10.8 Hz, 1H, Bn), 4.88 (d,
J = 11.1 Hz, 1H, Bn), 4.87 (d, J = 3.5 Hz, 1H, H-1), 4.77 (d,
J = 10.8 Hz, 1H, Bn), 4.72–4.66 (m, 2H, Bn), 4.56 (d, J = 11.1 Hz,
1H, Bn), 4.45 (dd, J = 4.4 Hz, J = 6.2 Hz, 1H, CH2CH(OTBDMS)-
CO2Me), 4.24–4.22 (m, 2H, H-6), 4.00 (t, J = 9.2 Hz, 1H, H-3), 3.88
(dt, J = 10.0 Hz, J = 3.0 Hz, 1H, H-5), 3.81–3.75 (m, 1H,
CH2CH(OTBDMS)CO2Me), 3.72–3.67 (m, 1H, CH2CH(OTBDMS)-
CO2Me), 3.70 (s, OMe), 3.54–3.44 (m, 2H, H-2, H-4), 2.01 (s, 3H,
a
a
138.7, 138.5, 138.3, 138.1, 128.6, 128.4, 128.37, 128.3, 128.2,
128.1, 127.9, 127.8, 127.7, 127.6, 127.57, 127.53, 127.45, 127.43,
102.6 (C-1
a
), 98.0 (C-1 b), 81.9 (C-3 b), 79.1 (C-2 b), 78.9
), 74.9, 74.57, 74.53, 74.2, 73.7, 73.4, 73.2,
(C-3 ), 76.3 (C-2
a
a
72.8, 72.5, 72.1 (C-5 b), 68.9 (C-5
a), 63.1 (C-6 a), 62.9 (C-6 b),
51.98, 51.94, 20.7, 19.1, 17.8.

168
E. C. Lourenço, M. R. Ventura / Carbohydrate Research 346 (2011) 163–168
4.2.9. Benzyl (2S)-2-(6-O-acetyl-2,3,4-tri-O-benzyl-
a
/b-
D
-
2006. E.L. acknowledges FCT for a Ph.D. grant SFRH/47702/2008.
We thank CERMAX for the use of the NMR spectrometers, which
were purchased within the framework of the National Programme
for Scientific Re-equipment, contract REDE/1517/RMN/2005, with
funds from POCI 2010 (FEDER) and Fundação para a Ciência e a
Tecnologia (FCT). This work was motivated by collaboration with
Professor Helena Santos.
galactopyranosyl)propanoate 10
mmax (film): 1744 cmꢀ1 (C@O). 1H NMR (CDCl3): 7.37–7.23 (m,
20H, Bn aromatic), 5.15–5.07 (m, 2H, Bn ester), 4.97–4.89 (m, 3H,
Bn), 4.86–4.84 (m, 1H, H-1(a)), 4.82–4.49 (m, 3H, Bn), 4.46 (d,
J = 7.6 Hz, H-1 (b)), 4.19–4.12 (m, 2H, H-5, CH(CH3)CO2Bn),
4.10–3.96 (m, 4H, H-2, H-3, H-6), 3.87 (br s, 1H, H-4), 1.93 (s,
OAc (b)), 1.92 (s, 3H, OAc (
(b)), 1.44 (d, J = 6.8 Hz, 3H, CH(CH3)CO2Bn (
172.2, 170.4, 138.7, 138.5, 138.2, 128.2–127.4, 102.6 (C-1(b)),
98.0 (C-1( )), 81.9 (b), 79.1 (b), 78.9 ( ), 76.3 ( ), 74.9 (b), 74.6
), 74.5 ( ), 74.2 (b), 73.7 (b), 73.5 ( ), 73.2, 72.9 (b), 72.4 (b),
72.2 (b), 68.9, 66.6 ( ), 66.5 (b), 63.0 ( ), 62.9 (b), 20.8, 17.8.
a
)), 1.50 (d, J = 6.9 Hz, CH(CH3)CO2Bn
a
)). 13C NMR (CDCl3):
References
1. da Costa, M. S.; Santos, H.; Galinski, E. A. Adv. Biochem. Eng. Biotechnol. 1998, 61,
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a
a
a
a
(a
a
a
a
4.2.10. Methyl 2-(6-O-acetyl-2,3,4-tri-O-benzyl-a/b-D-
galactopyranosyl)acetate 11
mmax (film): 1743 cmꢀ1 (C@O). 1H NMR (CDCl3): 7.43–7.26 (m,
Bn aromatic,
H-1( )) 4.99–4.91 (m, Bn), 4.85–4.72 (m, Bn), 4.67–4.60 (m, Bn),
4.48 (d, J = 7.6 Hz, 1H, H-1(b)), 4.39–4.26 (m, CH2CO2Me),
4.22–3.98 (m, H-6 , H-6 b, CH2CO2Me, H-2 , H-6 b, H-6 , H-5
, H-3 ), 3.93–3.88 (m, H-2 b, H-4 ), 3.77 (br s, H-4 b, CO2Me
b), 3.75 (br s, CO2Me ), 3.56–3.48 (m, 2H, H-3 b, Y-5 b), 1.98 (s,
3H, –OAc
), 1.97 (s, 3H, –OAc b). 13C NMR (CDCl3): 170.5, 170.2,
138.7, 138.3, 138.1, 138.0, 128.5–128.1, 103.3 (C-1(b)), 97.3
(C-1( )), 81.8 (b), 79.0 (b), 78.6 ( ), 76.0 ( ), 75.0 (b), 74.7 ( ),
74.6 ( ), 74.3 (b), 73.7 ( ), 73.6 (b), 73.1 ( ), 72.9 (b), 72.2 (b),
69.1 ( ), 65.5 (b), 63.6 ( ), 63.4 ( ), 63.0 (b), 51.9, 20.8.
a and b), 5.08 (d, J = 10.0 Hz, Bn), 5.05 (d, J = 3.6 Hz,
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
13. Pedersen, C. M.; Nordstrom, L. U.; Bols, M. J. Am. Chem. Soc. 2007, 129, 9222–
9235.
14. Crich, D.; Cai, W. J. Org. Chem. 1999, 64, 4926–4930.
15. Armarego, W. L. F.; Chai, C. L. L. Purification of Laboratory Chemicals, 5th ed.;
Elsevier, 2003.
Acknowledgements
We acknowledge the generous financial support provided by
Fundação para a Ciência e Tecnologia (FCT) PTDC/BIO/70806/