Synthesis of potassium (2R)-2-O-α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute

Article abstract of DOI:10.1016/j.carres.2009.06.037

Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-thio-d-glucopyranoside, as a mixture of anomers, was employed for the stereoselective synthesis of the potassium salt of (2R)-2-O-α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-2,3-dihydroxypropanoic acid (α-d-glucosyl-(1→6

Full text of DOI:10.1016/j.carres.2009.06.037

Carbohydrate Research 344 (2009) 2073–2078
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Carbohydrate Research
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Note
Synthesis of potassium (2R)-2-O-a-D-glucopyranosyl-(1?6)-
a-D-glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute
a
Eva C. Lourenço a, Christopher D. Maycock a,b, , M. Rita Ventura
*
a Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2780-901 Oeiras, Portugal
b Faculdade de Ciências da Universidade de Lisboa, Departamento de Química e Bioquímica, 1749-016 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-thio-
D
-glucopyranoside, as a mixture of anomers, was employed for the
-glucopyranosyl-(1?6)- -glucopyran-
-glucosyl-(1?2)- -glyceric acid, GGG),
-anomer was by far the major product of both glycosylation
Received 7 August 2008
Received in revised form 19 June 2009
Accepted 21 June 2009
stereoselective synthesis of the potassium salt of (2R)-2-O-
osyl-2,3-dihydroxypropanoic acid -glucosyl-(1?6)-
a
D
-
D
a-D
(
a
-
D
a
-
D
a
recently isolated compatible solute. The
reactions using NIS/TfOH as activator.
a
Available online 27 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
a-Glucosylation
Hypersolutes
Glucosyl glycerate
Thioglycosides
Glyceric acid
Halotolerant and moderately halophilic microorganisms accu-
mulate compatible solutes to counter fluctuations in the water
activity of their environment.1 Hyperthermophiles (thriving opti-
mally above 80 °C) isolated from marine sources also use the same
general strategy. A superior thermoprotecting ability was soon as-
cribed to these ‘hypersolutes’, and confirmed by in vitro protein
stabilisation experiments.2,3 In contrast to the solutes more com-
monly found in mesophiles, hypersolutes are generally negatively
charged, and most fall into two categories: glyceric acid glycosides
hands, the chemical degradation process was difficult and
unsuitable for large-scale preparation and so we opted for a step-
wise glycosylation strategy. 13C-Labelled GG was prepared photo-
synthetically, in low yield, using a blue-green alga and [13C]
enriched carbon dioxide as a carbon source.6
The main challenge was to form the
a-glucosidic bonds selec-
tively,7 for which there are good procedures but still no general
method. We opted for a thioglycoside donor protocol and the acti-
vation method developed by Crich and co-workers.8,9 The glucosyl
donor, S-phenyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-deoxy-1-
such as a-D-mannosyl-D-glycerate (MG) and a-D-glucosyl-D-glycer-
ate (GG) 1 (Fig. 1), and polyol-phosphodiesters such as di-myo-ino-
thio-b-D-glucopyranoside 3, was activated with 1-benzenesulfinyl
sitol phosphate (DIP). From the hyperthermophile Persephonella
piperidine (BSP) and Tf2O, in the presence of a hindered base
(2,6-di-tert-butyl-4-methylpyridine, DTBMP). The intermediates
(perhaps 1-triflates)9 thus formed were rapidly converted to the
glucoside 4 upon treatment with methyl 3-O-tert-butyldiphenylsi-
marina the new trisaccharide
a-D-glucosyl-(1?6)-a-D-glucosyl-
(1?2)-
D
-glycerate (GGG) 2 has recently been isolated (Fig 1).4
These compounds are available in very small quantities from
their natural sources and chemical synthesis was considered the
best route to obtain GG 1 and GGG 2 in larger amounts for testing
and to confirm the (R) stereochemistry of the glycerate moiety.
Glucosyl glycerate (GG)-terminated oligosaccharides have been
studied previously5 and a synthesis by chemical degradation of
leucrose has been reported with the characterisation and confir-
mation of the structure and stereochemistry a salient feature of
this work. In fact, the oligosaccharides studied appear to have a
glucosyl-(1?6)-glucosyl-(1?2)-glycerate terminal. This motif ap-
pears to be widely dispersed in nature. We found that, in our
lylglycerate 9, obtained from D
-serine10 in good yield (72%), and no
b-anomer was detected by NMR (Scheme 1). Even though this was
a good method for our purposes, we experienced some difficulties
with the lability of the benzylidene-protecting group and for the
GGG synthesis an important aspect was the need to have the
6-OH group free for the second glycosylation reaction. Removal
of the 4,6-benzylidene-protecting group at this stage would release
two hydroxyl groups and opened up the possibility of side reac-
tions during subsequent glycosylation processes, reducing the
overall efficiency. Thus, we looked for another suitable glucosyl do-
nor and for our purposes a simple glucose derivative having a dif-
ferentially protected 6-OH group was needed. 6-Hydroxysugars,
that is sugars having all other positions protected, have been used
* Corresponding author. Tel.: +351 21 4469775; fax: +351 21 4469789.
E-mail address: maycock@itqb.unl.pt (C.D. Maycock).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.06.037

2074
E. C. Lourenço et al. / Carbohydrate Research 344 (2009) 2073–2078
tate, hydrolytic removal of the acetates, followed by perbenzyla-
OH
tion of the remaining hydroxyls and finally acidolysis of the trityl
group and acetylation of the 6-OH group. Treatment of methyl tet-
ra-O-benzylglucopyranoside with a mixture of sulfuric acid, acetic
acid and acetic anhydride affords directly the diacetate 7 in 91%
yield.16 This could then be converted to thioglycoside 8, as a mix-
ture of anomers, by acid catalysed thiolysis. Roy14 and more re-
O
HO
HO
OH
OH
O
HO
HO
O
OH
O
HO
HO
O
OH
1
cently Iadonisi and co-workers17 have reported highly
a selective
OH
CO2K
O
OH
glycosylation reactions of acetate 8. Although the 6-O-acetyl group
could disarm the system slightly, this group could also participate
in the stabilisation of the intermediate carbocation and favour for-
2
CO2K
a
-glycoside.18 Uronic acid ester glycosylation has
Figure 1. Two natural glycerate solutes.
mation of the
shown that anomer selectivity is considerably reduced19 indicating
that the carbonyl group of similar compounds does not participate.
The use of large glucosyl acceptors may have the effect of increas-
for a variety of applications. The connection to resins11 for solid-
supported reactions and directed glycosidations through tethers
and templates12 at the 6-OH have also been reported. They are,
obviously, also useful for the preparation of uronic acids and
6-deoxysugars.13 The methods available for the preparation of these
useful molecules have normally involved an extended sequence of
protection and deprotection reactions.14 Thus 6-acetoxy perbenzy-
lated glucose 8 (b-anomer) has previously been prepared by
ing
Applying the Crich protocol for the glycosylation of donor
8 (2.7:1,
:b),17 with glycerate acceptor 9 afforded 10 in 76%
yield; however, the simpler NIS–TfOH21 mixture afforded pre-
dominantly (>10:1) the -anomer in 96% yield and the product
a
-selectivity.20
a
a
mixture was easily separated. The major isomer showed a dou-
blet at 5.15 ppm (J = 3.2 Hz) in the 1H NMR corresponding to the
the selective tritylation of the 6-OH group of 1-ethylthio-b-D-
glucopyranoside, obtained by the thiolysis15 of glucosepentaace-
a-anomer.
OH
TBDPSO
CO2Me
Ph
Ph
9
O
O
O
O
O
O
SPh
a
BnO
BnO
OBn
BnO
OTBDPS
CO2Me
O
3
4
Scheme 1. Reagents and conditions: (a) PhSONC5H10, DTBMP, Tf2O, MS, CH2Cl2, 72%.
OAc
OH
OBn
b
O
O
O
a
BnO
BnO
HO
HO
BnO
BnO
OBn
OH
OBn
OAc
OMe
OAc
OMe
5
6
7
OAc
d
c
O
O
BnO
BnO
BnO
BnO
OH
OBn
BnO
SEt
O
TBDPSO
OTBDPS
CO2Me
8
CO2Me
10
OH
9
OH
e
f
O
O
BnO
BnO
BnO
BnO
BnO
BnO
O
O
OH
CO2Me
OTBDPS
CO2Me
12
11
OH
O
g
h
HO
HO
1
OH
O
OH
CO2Me
13
Scheme 2. Reagents and conditions: (a) BnBr, NaH, DMF, 0 °C/rt, 88%; (b) Ac2O, AcOH, H2SO4, 0 °C, 91%; (c) EtSH, BF3ꢀOEt2, CH2Cl2, 0 °C, 80%; (d) NIS, TfOH, CH2Cl2, 96%; (e) Na,
MeOH, 0 °C, 94%; (f) TBAF, THF, 90%; (g) H2, Pd, AcOEt/EtOH, 35 psi, 99%; (h) KOH 2 M, H2O, rt.

E. C. Lourenço et al. / Carbohydrate Research 344 (2009) 2073–2078
2075
OAc
OAc
O
O
a
BnO
BnO
b
BnO
BnO
+
8
11
BnO
BnO
O
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
O
O
OH
CO2Me
OTBDPS
CO2Me
15
14
OH
OH
e
d
O
c
O
BnO
BnO
2
HO
HO
BnO
OH
O
O
O
O
BnO
BnO
HO
HO
BnO
OH
O
O
OH
CO2Me
OH
CO2Me
16
17
Scheme 3. Reagents and conditions: (a) NIS–TfOH, CH2Cl2, 0 °C, 89%; (b) TBAF, THF, 88%; (c) Na, MeOH, 0 °C, 95%; (d) H2, Pd, 35 psi, 99%; (e) KOH 2 M, H2O rt.
1. Experimental
Table 1
Comparison of 13C NMR chemical shifts for the potassium salt of GG 1 with data from
the literature
1.1. General
GG
Glucosyl moiety
Glyceryl moiety
1H NMR spectra were obtained at 400 MHz in CDCl3 or D2O with
chemical shift values (d) in ppm downfield from tetramethylsilane
in the case of CDCl3, and 13C NMR spectra were obtained at
100.61 MHz in CDCl3 or D2O. Assignments are supported by 2D
correlation NMR studies. Medium pressure preparative column
chromatography: Silica Gel Merck 60 H. Analytical TLC: Alumin-
13C, d ppm
C-1
C-2
C-3
C-4
C-5
C-6
C-1
C-2
C-3
NH4 salt5
99.0
99.0
73.7
73.8
74.9
74.8
71.0
71.1
73.2
73.2
62.1
62.1
178.5
178.6
80.6
80.7
64.7
64.7
þ
K+ salt
ium-backed Silica Gel Merck 60 F254. Specific rotations (½a D20
ꢁ
) were
Methanolysis of the 6-O-acetyl group of fully protected GG 10
furnished alcohol 11 (Scheme 2) which was used for the second
glycosylation reaction (Scheme 3). Removal of the silyl ether, fol-
lowed by hydrogenolysis of the benzyl groups and finally hydroly-
sis of the methyl ester afforded the potassium salt of GG 1, which
was purified by ion exchange chromatography. The NMR data (Ta-
ble 1) of the synthetic product and those of an authentic sample of
the natural product were identical and corresponded well to the
data for the salts.5
measured using an automatic polarimeter. Reagents and solvents
were purified and dried according to Ref. 22. All the reactions were
carried out in an inert atmosphere (argon), except when the sol-
vents were undried. Compounds 1 and 2 were purified by anion ex-
change chromatography using a QAE-Sephadex A25 column eluted
with sodium bicarbonate buffer (pH 9.8) gradient (5 mM to 1 M).
The combined relevant fractions were lyophilised and passed
through a H+ Dowex 50W-X8 column eluting with water. Again
the relevant fractions were combined and the pH adjusted to
4.5–5.0 with ultra-pure potassium hydroxide solution and concen-
trated to dryness.
For the GGG synthesis, thioglycoside 8 was again the glycosyl
donor and the acceptor was alcohol 11. This glycosylation reaction
failed under Crich conditions, but was successfully carried out
using NIS–TfOH, affording predominantly the
a-anomeric dissa-
charide 14 (>10:1 :b, 89% yield). Successive deprotection of the si-
a
1.2. 1,6-Di-O-acetyl-2,3,4-tri-O-benzyl-a/b-D-glucopyranoside 7
lyl ether, of the acetate at 6-OH, the benzyl groups and hydrolysis
of the methyl ester afforded GGG 2 as the potassium salt (Scheme
3). The NMR data of the synthesised product were identical to
Concentrated sulfuric acid (1 mL) was added dropwise to a solu-
tion of 6 (5.4 g, 9.8 mmol) in acetic acid/acetic anhydride (1:1,
50 mL) at 0 °C. After complete conversion of the starting material
(TLC) saturated aqueous NaHCO3 (50 mL) was added and the mix-
ture was extracted with AcOEt (3 ꢂ 80 mL), the combined organic
phases were dried (MgSO4), filtered and the solvent removed.
The crude product was purified by medium pressure column
chromatography (20/80 AcOEt/hexane) to afford the product 7
those of a sample of natural GGG4 (Table 2), confirming the
cerate stereochemical assignment for this solute.
D-gly-
In conclusion, the efficient syntheses of two natural solutes, GG
and GGG, have been achieved. A readily available simple glucosyl
donor was applied to afford
a-glucosides with high selectivity,
and offers a very easy method for preparing 1,6-linked di and high-
er saccharides.
Table 2
Comparison of 13C NMR chemical shifts for the synthetic Potassium salt of GGG 2 with data from the natural product
GGG
a
(1?6)glucosyl moiety
a
(1?2)glucosyl moiety
Glyceryl moiety
C-2
13C, d ppm
C-1
C-2
C-3
C-4
C-5
C-6
C-1
C-2
C-3
C-4
C-5
C-6
C-1
C-3
Natural
Synthesised
98.3
98.4
72.0
72.0
73.5
73.7
70.0
70.1
72.3
72.4
60.8
61.0
98.0
98.0
71.9
72.0
73.9
74.0
69.9
70.0
71.1
71.3
66.0
66.9
177.3
177.5
79.7
79.5
63.6
63.7

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E. C. Lourenço et al. / Carbohydrate Research 344 (2009) 2073–2078
(mixture of anomers) as a viscous colourless foam (4.71 g, 91%).
mmax (film): 1744 (C@O) cmꢃ1 1H NMR (CDCl3): d 7.36–7.24 (m),
(CDCl3): d 7.38–7.37 (5H, m), 7.37–7.22 (25H, m), 5.16 (1H, d,
J = 3.6 Hz, H-1), 5.02 (1H, d, J = 10.6 Hz), 4.89 (2H, dd, J = 11.9 Hz,
J = 3.1 Hz), 4.76 (1H, d, J = 10.5 Hz), 4.70 (1H, d, J = 11.8 Hz), 4.54
(1H, d, J = 11.2 Hz), 4.49 (1H, dd, J = 6.3 Hz; J = 4.3 Hz), 4.15–3.94
(6H, m), 3.73 (3H, s), 3.60 (1H, dd, J = 9.7 Hz, J = 3.5 Hz), 3.48 (1H,
t, J = 9.5 Hz), 1.99 (3H, s), 1.03 (9H, s). 13C NMR (CDCl3): d 170.6
(C@O), 170.2 (C@O), 138.7, 138.1, 138.0, 135.6, 135.5, 133.0,
132.8, 129.8, 129.8, 128.4, 128.3, 128.2, 128.2, 128.1, 127.8,
127.7, 127.6, 94.8 (anomeric-C), 81.6, 75.8, 74.8, 72.0, 71.9, 69.0,
65.8, 64.6, 62.9, 51.9, 26.7, 20.8, 19.1. HR-MS: calcd for C49H56O10Si
[M]+: 559.2130; found: 559.2125.
.
6.32 (d, J = 3.5 Hz), 5.62 (d, J = 8.2 Hz), 5.01–4.55 (m), 4.31–4.21
(m), 4.01–3.92 (m), 3.67 (dd, J = 9.7 Hz, J = 3.5 Hz), 3.60–3.53 (m),
2.15 (s), 2.05 (s), 2.04 (s), 2.02 (s). 13C NMR (CDCl3): d 170.6,
169.3, 138.4, 137.5, 137.4, 128.5, 128.4, 128.2, 128.1, 128.0,
127.8, 127.7, 89.6, 81.6, 78.8, 76.6, 75.7, 75.2, 75.0, 73.2, 71.0,
62.6, 21.0, 20.8. Anal. Calcd for C31H34O8: C, 69.65; H, 6.41. Found:
C, 69.80; H, 6.42.
1.3. Ethyl 6-O-acetyl-2,3,4-tri-O-benzyl-1-thio-a/b-D-
glucopyranoside 8
1.6. Methyl 3-O-tert-butyldiphenylsilyl-(2R)-2-O-(2,3,4-tri-O-
To a solution of diacetate 7 (4.3 g, 8.0 mmol) in CH2Cl2 (20 mL)
at 0 °C was added ethanethiol (2.7 mL, 24.2 mmol), followed by
BF3ꢀOEt2 (1.54 mL, 12.1 mmol). After 3 h at 0 °C, the reaction was
quenched with saturated aqueous NaHCO3 (20 mL), extracted with
CH2Cl2 (3 ꢂ 20 mL), the combined organic phases were dried
(MgSO4) and concentrated. Purification by medium pressure col-
umn chromatography (10/90 AcOEt/hexane) afforded the thiogly-
coside 8 (mixture of anomers) as a viscous colourless foam
benzyl-a-D-glucopyranosyl)-2,3-dihydroxypropanoate 11
A solution (7.6 mL) of Na (0.046 g) in MeOH (10 mL) was added
to a stirred solution of the acetate 10 (2.0 g, 2.4 mmol) in MeOH
(7.5 mL) at 0 °C. After 30 min saturated aqueous NH4Cl (15 mL)
was added. The aqueous phase was extracted with AcOEt
(3 ꢂ 20 mL) and the combined organic extracts were dried
(MgSO4), filtered and the solvent was removed. Purification by
medium pressure column chromatography (10/90 to 20/80
(3.43 g, 80%). mmax (film): 1742 (C@O) cmꢃ1 1H NMR (CDCl3): d
.
7.33–7.26 (m), 5.37 (d, J = 5.3 Hz), 4.99–4.54 (m), 4.47 (d,
J = 9.8 Hz), 4.34–4.20 (m), 3.91–3.79 (m), 3.56–3.44 (m), 2.80–
2.66 (m), 2.62–2.44 (m), 2.03 (s), 2.01 (s), 1.34–1.26 (m). 13C
NMR (CDCl3): d 170.7, 170.6, 138.5, 138.3, 137.9, 137.8, 137.7,
137.6, 128.5–127, 86.6, 85.2, 83.0, 82.4, 81.7, 79.5, 77.6, 77.1,
76.9, 75.8, 75.7, 75.5, 75.1, 75.0, 72.3, 68.9, 63.4, 63.1, 25.2, 23.7,
20.9, 20.8, 15.1, 14.8. Anal. Calcd for C31H36O6S: C, 69.38; H, 6.76;
S, 5.97. Found: C, 69.13; H, 6.44; S, 5.97.
AcOEt/hexane) afforded the glyceryl
a
-glycoside 11 as a viscous
colourless foam (1.79 g, 94%). ½a D20
ꢁ
+17.2 (c 1.49, CH2Cl2). FT-IR
(film) 1753 cmꢃ1 (C@O) 1H NMR (CDCl3): d 7.70–7.67 (5H, m),
7.44–7.24 (25H, m), 5.14 (1H, d, J = 3.6 Hz, H-1), 4.99 (1H, d,
J = 10.8 Hz), 4.90 (1H, d, J = 11.5 Hz), 4.88 (1H, d, J = 11.2 Hz), 4.78
(1H, d, J = 10.1 Hz), 4.71 (1H, d, J = 11.7 Hz), 4.62 (1H, d,
J = 11.3 Hz), 4.46 (1H, dd, J = 6.1 Hz, J = 4.3 Hz), 4.13–3.95 (3H, m),
3.35–3.79 (1H, m), 3.74 (3H, s), 3.70–3.45 (5H, m), 1.03 (9H, s).
13C NMR (CDCl3): d 170.3 (C@O), 138.8, 138.4, 138.2, 135.6,
135.5, 133.0, 132.8, 129.7, 128.3, 128.1, 128.0, 127.7, 127.6,
127.5, 95.0 (anomeric-C), 81.5, 79.3, 76.9, 75.7, 74.8, 74.7, 72.1,
71.2, 64.6, 61.6, 51.9, 26.7, 19.1. HR-MS: calcd for C47H54O9Si
[M]+: 790.3531; found: 790.3500.
1.4. Methyl (2R)-3-O-tert-butyldiphenylsilyl-2,3-
dihydroxypropanoate 9
To a solution of methyl (2R)-2,3-dihydroxypropanoate8 (3.8 g,
28.3 mmol) in pyridine (19 mL) at rt was added TBDPSCl (8.0 mL,
30.5 mmol), followed by a catalytic amount (about 5 mg) of DMAP.
After 16 h at rt, the reaction was quenched with H2O (20 mL), ex-
tracted with CH2Cl2 (3 ꢂ 20 mL) and the combined organic phases
were dried (MgSO4) and concentrated. Purification by medium
pressure column chromatography (5/95 AcOEt/hexane) afforded
1.7. Methyl (2R)-2-O-(2,3,4-tri-O-benzyl-a-D-glucopyranosyl)-
2,3-dihydroxypropanoate 12
To a solution of 11 (3.00 g, 3.8 mmol) in THF (15 mL) at rt was
added Bu4NF (1.20 g, 4.6 mmol). The reaction mixture was stirred
for 3 h and then water was added. The mixture was extracted
with AcOEt (3 ꢂ 20 mL), dried (MgSO4) and concentrated to furnish
the product 9 as viscous colourless oil (9.10 g, 79%). ½a D20
ꢃ22.2 (c
ꢁ
1.0, CHCl3). FT-IR (film): 3510, 1745 (C@O). 1H NMR (CDCl3): d
7.67–7.61 (4H, m), 7.46–7.36 (6H, m), 4.24 (1H, t, J = 2.9 Hz), 3.98
(1H, dd, J = 10.6 Hz, J = 2.9 Hz), 3.91 (1H, dd, J = 10.6 Hz,
J = 2.9 Hz), 3.79 (3H, s), 1.04 (9H, s). 13C NMR (CDCl3): d 173.3,
135.6, 135.5, 133.0, 132.9, 129.8, 127.8, 127.7, 71.9, 65.8, 52.4,
26.7, 19.3. Anal. Calcd for C20H26O4Si: C, 67.00; H, 7.31. Found: C,
67.12; H, 7.45.
a
yellow viscous residue. Purification by medium pressure
column chromatography (50/50 to 100% AcOEt) afforded
the product 12 as a very viscous colourless foam (1.89 g, 90%).
½
a 2D0 +49.5 (c 1.16, CH2Cl2). 1H NMR (CDCl3): d 7.43–7.26 (18H,
ꢁ
m), 5.20 (1H, d, J = 3.6 Hz, H-1), 5.00 (1H, d, J = 10.8 Hz), 4.92–
4.80 (3H, m), 4.70 (1H, d, J = 11.4 Hz), 4.62 (1H, d, J = 10.8 Hz),
4.36 (1H, t, J = 4.3 Hz), 4.03 (1H, t, J = 9.3 Hz), 3.93 (2H, d,
J = 4.5 Hz), 3.76 (3H, s), 3.75–3.52 (4H, m), 2.09 (2H, br s). 13C
NMR (CDCl3): d 170.1, 138.6, 137.9, 137.7, 128.5, 128.4, 128.3,
127.9, 94.9 (anomeric-C), 81.3, 79.4, 77.1, 75.7, 75.1, 74.6, 72.4,
71.6, 63.3, 61.7, 52.1. HR-MS: calcd for C31H36O9 [M]+: 552.2354;
found: 552.2333.
1.5. Methyl 3-O-tert-butyldiphenylsilyl-(2R)-2-O-(6-O-acetyl-
2,3,4-tri-O-benzyl-a-D-glucopyranosyl)-2,3-
dihydroxypropanoate 10
A suspension of thioglycoside 8 (2.38 g, 4.4 mmol), glycerate
9 (1.56 g, 4.4 mmol) and 4 Å MS in CH2Cl2 (15 mL) was stirred
at rt for 1 h. N-Iodosuccinimide (1.25 g, 5.6 mmol) and TfOH
(0.026 mL) were added, at 0 °C. After 30 min, 10% Na2S2O3 aqueous
solution (20 mL) and saturated NaHCO3 aqueous solution (10 mL)
were added and the mixture was extracted with CH2Cl2
(3 ꢂ 20 mL), the combined organic phases were dried (MgSO4), fil-
tered and the solvent removed under vacuum. The crude product
was purified by medium pressure column chromatography (3/7
AcOEt/hexane) to afford 3.23 g of glycoside 10 (90%) as a colourless
1.8. Potassium (2R)-2-O-(a-D-glucopyranosyl)-2,3-
dihydroxypropanoate 1
Benzyl ether 12 (1.89 g, 3.4 mmol) in AcOEt/EtOH (20 mL/
10 mL) was hydrogenated at 35 psi in the presence of a catalytic
amount of Pd/C 10% (0.05 equiv). After 3 h, the reaction mixture
was filtered, the solvent was evaporated and the residue dried un-
der vacuum to afford 13 as a water soluble viscous colourless foam
(0.95 g, 99%). This material was sufficiently pure to proceed to the
next step.
viscous foam (12:1 mixture of
a
- and b-anomers).
a
-Anomer ½a 2D0
ꢁ
+45.2 (c 1.45, CH2Cl2). FT-IR (film) 1744 cmꢃ1 (C@O). 1H NMR

E. C. Lourenço et al. / Carbohydrate Research 344 (2009) 2073–2078
2077
To a solution of 13 (0.95 g, 3.4 mmol) in H2O (10 mL) was added
a 2 M KOH aqueous solution (1.65 mL). The reaction was stirred
overnight at rt. The solution was neutralised with HCl 10% and
the solvent evaporated to afford a very viscous colourless foam.
This was purified by ion exchange chromatography as described
in the general procedures to form the potassium salt 1 (0.51 g,
49%). Its NMR data were identical to those of the natural sample4
and comparable to those found by Walton et al.5 for ammonium
product 16 as a viscous colourless foam (1.47 g, 95%). ½a D20
ꢁ
+77.0 (c
1.13, CH2Cl2). 1H NMR (CDCl3): d7.37–7.25 (30H, m), 5.10 (1H, br s),
5.02–4.44 (14H, m), 4.11–3.93 (6H, m), 3.74 (3H, s), 3.66–3.50 (8H,
m). 13C NMR (CDCl3): d 170.2 (C@O), 138.7, 138.6, 138.1, 138. 0,
137.9, 137.8, 128.4–127.6, 97.4 (anomeric-C), 95.0 (anomeric-C),
81.9, 81.8, 79.9, 79.5, 77.8, 77.3, 75.7, 75.6, 75.2, 75.1, 75.0, 73.0,
72.4, 70.9, 70.6, 66.5, 63.4, 61.8, 52.1. HR-MS: calcd for C58H64O14
[M]+: 984.4290; found: 984.4275.
salt (Table 1). ½a D20
ꢁ
+97.6 (c 2.08, H2O). FT-IR 1605 cmꢃ1 (C@O).
1H NMR (D2O): d 5.00 (1H, d, J = 3.7 Hz, H-1), 4.39 (1H, t,
J = 3.5 Hz), 3.89–3.87 (2H, m), 3.78–3.62 (4H, m), 3.49 (1H, dd,
J = 9.8 Hz, J = 3.9 Hz), 3.34 (1H, t, J = 9.1 Hz). 13C NMR (D2O): d
178.6 (C@O), 99.0 (C-1), 80.7 (C-2 glycerate), 74.8 (C-3), 73.8 (C-
2), 73.2 (C-5), 71.1 (C-4), 64.7 (C-3 glycerate), 62.1 (C-6).
1.12. Potassium (2R)-2-O-
a-D-glucopyranosyl-(1?6)-a-D-
glucopyranosyl-2,3-dihydroxypropanoate 2
Benzyl ether 16 (1.47 g, 1.5 mmol) in AcOEt/EtOH (20/10 mL)
was hydrogenated at 50 psi in the presence of Pd/C 10%
(0.25 equiv). After 3 h, the reaction mixture was filtered and the
solvent was evaporated to afford ester 17 as a very viscous colour-
less foam (0.640 g, 99%).
To a solution of ester 17 (0.50 g, 1.12 mmol) in H2O (5 mL) was
added 2 M KOH (1.05 mL). After all the starting material had been
consumed (3 h), the pH was adjusted to 7 with HCl 10% and the
solvent evaporated to afford crude 2 as a viscous colourless foam
which was purified by ion exchange chromatography as indicated
in the general procedures and afforded 2 (0.33 g, 63%) which was
detected homogeneous by NMR. Its NMR data were identical to
1.9. Methyl 3-O-tert-butyldiphenylsilyl-(2R)-2-O-[6-O-acetyl-
2,3,4-tri-O-benzyl-a-D-glucopyranosyl-(1?6)-2,3,4-tri-O-
benzyl-1-O- -glucopyranosyl]-2,3-dihydroxypropanoate 14
a
-D
A suspension of 8 (1.74 g, 3.2 mmol), 11 (2.38 g, 3.0 mmol) and
4 Å MS in CH2Cl2 (35 mL) was stirred at rt for 1 h. A solution of N-
iodosuccinimide (0.96 g, 4.2 mmol) and TfOH (0.020 lL) in 10 mL
of CH2Cl2 was added at 0 °C. After 30 min, 10% Na2S2O3 aqueous
solution (20 mL) and saturated NaHCO3 aqueous solution (20 mL)
were added and the mixture was extracted with CH2Cl2
(3 ꢂ 30 mL), the combined organic phases were dried (MgSO4), fil-
tered and the solvent was removed under vacuum. The crude prod-
uct was purified by medium pressure column chromatography (10/
90 AcOEt/hexane) to afford 3.47 g of 14 (84%) as a colourless vis-
those of a natural sample4 (Table 2). ½a D20
ꢁ
+105.3 (c 0.83, H2O).
FT-IR: 1604 cmꢃ1 (C@O). 1H NMR (D2O): d 5.04 (1H, d, J = 3.5 Hz,
H-1), 4.96 (1H, d, J = 3.3 Hz), 4.20 (1H, dd, J = 5.0 Hz, J = 3.2 Hz),
3.99–3.71 (10H, m), 3.60–3.40 (4H, m). 13C NMR (D2O): d 177.5
(C@O), 98.4 (anomeric-C), 98.0 (anomeric-C), 79.5, 74.0, 73.7,
72.4, 72.0, 71.3, 70.1, 70.0, 66.9, 63.7, 61.0. HR-MS: calcd for
C15H25O14 [M]ꢃ: 429.12498; found: 429.13007.
cous foam. ½a 2D0
ꢁ
+21.1 (c 1.39, CH2Cl2). 1H NMR (CDCl3): d 7.68
(5H, br s), 7.35–7.20 (35H, m), 5.15 (1H, s), 5.00–4.74 (8H, m),
4.65–4.51 (6H, m), 4.15–3.89 (7H, m), 3.79–3.71 (2H, m), 3.71
(3H, s) 3.61–3.43 (5H, m), 1.95 (3H, s), 1.02 (9H, s). 13C NMR
(CDCl3): d 170.7 (C@O), 170.3 (C@O), 138.9, 138.6, 138.3, 138. 0,
135.6, 135.5, 133.1, 132.9, 129.7, 128.3, 128.2, 128.1, 127.8,
127.5, 127.4, 97.1 (anomeric-C), 94.8 (anomeric-C), 81.6, 79.9,
79.5, 75.6, 74.9, 74.6, 72.1, 71.0, 68.7, 64.7, 63.0, 51.9, 26.7, 20.8,
19.2. HR-MS: calcd for C76H84O15Si [M]+: 1264.5574; found:
1264.5556.
Acknowledgements
We wish to acknowledge the analysis department at the ITQB
for providing elemental analyses. We also acknowledge the gener-
ous financial support provided by FCT POCI/BIA-PRO/57263/2004
and CRAFT Hotsolutes Project (FP6-2002-SME-1). We also thank
the Mass spectrometry unit at the Faculty of Sciences at the Uni-
versity of Lisbon for HRMS.
1.10. Methyl (2R)-2-O-[6-O-acetyl-2,3,4-tri-O-benzyl-a-D-
glucopyranosyl-(1?6)-2,3,4-tri-O-benzyl-1-O-a-D-
glucopyranosyl]-2,3-dihydroxypropanoate 15
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a