Chemoenzymatic syntheses of linear and branched hemithiomaltodextrins as potential inhibitors for starch-debranching enzymes

Article abstract of DOI:10.1002/1521-3765(20021202)8:23<5447::AID-CHEM5447>3.0.CO;2-H

Oligosaccharides embodying the S-maltosyl-6-thiomaltosyl structure have been readily synthesised by using convergent chemoenzymatic approaches. The key steps for the preparation of these molecules involved: 1) transglycosylation reactions of maltosyl fluo

Full text of DOI:10.1002/1521-3765(20021202)8:23<5447::AID-CHEM5447>3.0.CO;2-H

FULL PAPER
Chemoenzymatic Syntheses of Linear and Branched Hemithiomaltodextrins
as Potential Inhibitors for Starch-Debranching Enzymes
Lionel Greffe,[a] Morten T. Jensen,[b] Florent Chang-Pi-Hin,[c] Sandra Fruchard,[a]
Michael J. O×Donohue,[c] Birte Svensson,[b] and Hugues Driguez*[a]
Abstract: Oligosaccharides embodying
the S-maltosyl-6-thiomaltosyl structure
have been readily synthesised by
using convergent chemoenzymatic ap-
proaches. The key steps for the prepa-
ration of these molecules involved:
1) transglycosylation reactions of malto-
syl fluorides onto suitable acceptors
catalysed by the bacterial transglyco-
sylase, cyclodextrin glycosyltransferase
(CGTase), and 2) the SN2-type displace-
ment of a 6-halide from acetylated
acceptors by activated 1-thioglycoses.
The target molecules, which were ob-
tained in good overall yields, proved to
be useful for investigating substrate
binding in the active sites of several
enzymes that act upon the a-1,6-linkage
of pullulan and/or amylopectin. The
compounds exhibit Ki values in the
2.5 1350 mm range with the different
enzymes, and the highest affinity found
by using these molecules was seen for
the pullulanase from Bacillus acidopul-
lulyticus. Both barley-malt limit dextrin-
ase and pullulanase type II from Ther-
mococcus hydrothermalis only recog-
nised the longest linear thiooligo-
saccharide, while a branched heptasac-
charide was the strongest inhibitor of
pullulanase from Klebsiella planticola.
Keywords: active-site mapping
¥
carbohydrates ¥ enzymatic synthesis
¥ enzymes ¥ oligosaccharides
maltohydrolase [b-amylases; EC 3.2.1.1; GH family 14] and
1,4-a-d-glucan glucohydrolase [glucoamylases; EC 3.2.1.3;
GH family 15]. The a- and b-amylases attack bonds in the
1,4-a-d-glucan-backbone chains of amylopectin to generate
oligosaccharides called a- and b-limit dextrins, respectively.
The a-1,6-bonds in amylopectin and the limit dextrins are
hydrolysed by pullulanases [EC 3.2.1.41, GH family 13], limit
dextrinases [EC 3.2.1.142, GH family 13], and isoamylases
[EC 3.2.1.68, GH family 13]. Moreover, some enzymes such
as amylopullulanase [EC 3.2.1.1/41, GH families 13 or 57] and
glucoamylase [EC 3.2.1.3, GH family 15] can hydrolyse both
the a-1,4 and a-1,6-bonds.[1, 2] As indicated above, glycoside
hydrolases are grouped into different families according to a
classification that is based upon amino acid sequence
similarities. This classification does not coincide perfectly
with the biochemical classification as defined by the Enzyme
Commission based on enzyme specificity. Thus, a given
glycoside hydrolase family can contain several enzymes with
different EC numbers, while enzymes with the same substrate
specificity can act with retention or inversion of the anomeric
configuration and occur with different structural folds, and
hence belong to different structural families.
Introduction
A large proportion of the photosynthetically assimilated
carbon in plants is channelled into the biosynthesis of starch
and sucrose, by far the two most widely used carbohydrate-
based chemicals in food and nonfood industries. Starch
consists of a mixture of two distinct polysaccharide types:
amylose, ideally a linear polymer of 1,4-a-d-glucosyl units,
and amylopectin, a branched polymer containing short
amylose chains linked by a-1,6-branching points. Amylose is
mainly hydrolysed by various amylases, usually classified as
endo-acting 1,4-a-d-glucan glucanohydrolase [a-amylases;
EC 3.2.1.1; GH family 13], or the exo-acting 1,4-a-d-glucan
[a] Dr. H. Driguez, Dr. L. Greffe, S. Fruchard
¬
¬ ¬
Centre de Recherches sur les Macromolecules Vegetales
(CERMAV-CNRS)
¬
Affiliated to the Universite Joseph Fourier
B.P. 53, 38041 Grenoble cedex9 (France)
Fax: ( ‡33)4-76-54-72-03
E-mail: hugues.driguez@cermav.cnrs.fr
[b] Dr. M. T. Jensen, Dr. B. Svensson
Department of Chemistry, Carlsberg Laboratory
Gamle Carlsberg Vej 10, 2500 Copenhagen (Denmark)
Given the structural complexity of amylopectin and the
difficulties associated with the isolation of pure oligomers
from natural sources, there is a high demand for the develop-
ment of strategies for the preparation of compounds that can
be used to determine enzyme specificity. However, few
[c] F. Chang-Pi-Hin, Dr. M. J. O×Donohue
Institut National de la Recherche Agronomique (UMR 614)
8 rue Gabriel Voisin, B.P. 16, 51688 Reims cedex2 (France)
Supporting information for this article is available on the WWW under
http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2002, 8, No. 23
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5447

H. Driguez et al.
FULL PAPER
chemical syntheses of branched oligosaccharides representing
the branch point of amylopectin have been reported.[3, 4]
For many years, we have been involved in the syntheses of
hydrolytically inert substrate analogues, the thiooligosacchar-
ides, that can be used as tools for structural biology and in
biochemical studies of a wide variety of enzymes.[5 7] In such a
molecule, only the scissile bond has to be replaced by a thio-
linkage. The major challenges associated with the synthesis of
potential substrates are concerned with the efficiency and the
accuracy of regiospecific glycosylation. Here we report the
use of chemoenzymatic approaches for the preparation of
potential substrate analogues of a-1,6-degrading enzymes that
enable the distinction of fine differences in enzyme substrate
recognition and catalytic mechanisms.
was therefore also employed in the presence of maltosyl
fluorides for the synthesis of the a-1,4-bonds of the target
compounds. However, in order to prevent self-condensation
of the donors during synthesis, it was necessary to block the
4II-OH of the maltosyl fluorides. Since 4II-O-tetrahydropyr-
anyl-a-cellobiosyl fluoride was previously used successfully
for enzymatic syntheses of b-1,4-oligosaccharides,[10, 11] 4II-O-
tetrahydropyranyl-a-maltosyl fluoride (2) and 6II-bromo-4II-
O-tetrahydropyranyl-a-maltosyl fluoride (3) were used for
the present syntheses. Preferential acetylation of the known
hexaacetyl maltose 4 was readily obtained in excellent
yield by using the 1-acetyloxybenzotriazole procedure
(Scheme 2).[12] The fluorination of the expected compound 5
R2
R
O
R3O
R1O
O
Results and Discussion
HO
AcO
OR1
O
OAc
O
R1O
iii
AcO
O
O
R1O
Although from an industrial point of view the enzymatic
degradation of the branching point of amylopectin is impor-
tant, comparative studies on the substrate specificity of
debranching enzymes of the pullulanase type are lacking
because suitable substrates or substrate analogues have not
been available. Therefore, to facilitate such specificity char-
acterisation, we embarked on the synthesis of molecules
exhibiting the general structure shown in Scheme 1.
OAc
AcO
R1O
AcO
F
7: R1 = Ac, R2 = OAc, R3 = H
8: R1 = Ac, R2 = OAc, R3 = THP
2: R1 = H, R2 = OH, R3 = THP
4: R = OH
5: R = OAc
6: R = Br
iv
ii
i
v
9: R1 = Ac, R2 = Br, R3 = H
10: R1 = Ac, R2 = Br, R3 = THP
3: R1 = H, R2 = Br, R3 = THP
iv
v
The 6-thio-isomaltosyl motif is the common disaccharidic
unit of all the compounds prepared in this study. Previous
work has demonstrated that S-a-d-glucopyranosyl-6w-thio-
maltooligosaccharides can be synthesised in high yield[7] by
the nucleophilic displacement of the halide of 6w-iodomal-
tooligosaccharides by using the activated form of fully
acetylated-1-thio-a-d-glucopyranose, which itself is generated
from the corresponding tritylthio derivative 1. This motivated
the present enzymatic approach to the assembly of several
mono- and disaccharide building blocks around the thio unit,
which resulted in the target molecules. Previously, substituted
maltosyl fluorides were used as donor and acceptor molecules
for active-site mapping of cyclodextrin glycosyltransferase
[CGTase; EC 2.4.1.19, GH family 13] and in the enzymatic
synthesis of regularly substituted cyclodextrins.[8, 9] CGTase
Scheme 2. Syntheses of the maltosyl fluorides 2 and 3. i) 1-acetyloxyben-
zotriazole, TEA, CH2Cl2, RT (80%); ii) P(C6H5)3, pyridine, CBr4, 08C to
508C (84%); iii) HF/pyridine, 08C (95% for 7, 90% for 9); iv) dihydro-
pyran, camphorsulfonic acid, CH2Cl2, RT, (98% for 8, 95% for 10);
v) MeONa, MeOH, 08C (98%) for 2 and 3.
was achieved in 95% yield by using a commercially available
pyridine hydrogen fluoride reagent, as described earlier for
fully acetylated maltose and cellobiose derivatives.[13] The 4II-
OH of maltosyl fluoride 7 was tetrahydropyranylated in 98%
yield and 8 was de-O-acetylated with sodium methoxide in
methanol at room temperature to give the corresponding pure
fluoride 2 in almost quantitative yield. Mild and selective
bromination of the free primary hydroxyl group of 4 with
carbon tetrabromide and tri-
phenylphosphine in pyridine,[14]
gave 6 in 84% yield. Fluori-
OH
nation, tetrahydropyranylation
OH
HO
O
OH
and de-O-acetylation, as al-
ready reported for 5, gave 9,
10 and 3 in 90, 95 and 98%
yield, respectively.
OH
OH
O
HO
m
O
O
HO
OH
HO
OH
OH
HO
O
O
HO
O
OH
HO
O
OH
With regard to the prepara-
tion of the donors for the thio-
glycosylation, halide displace-
ment of acetochloro-b-maltose,
generated from peracetyl-b-
maltose (11)[15] and triphenyl-
methyl mercaptan according to
the methodology developed in
the ™gluco∫ and ™galacto∫ ser-
ies[16, 17] gave the acetylated tri-
HO
O
n
O
HO
S
HO
O
O
OH
HO
HO
O
O
HO
OH
HO
O
O
HO
OH
HO
o
Scheme 1. Structure of the target molecules.
5448
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Chem. Eur. J. 2002, 8, No. 23

Thiomaltodextrins as Potential Enzyme Inhibitors
5447 5455
tyl-1-thio-a-maltoside (12) in 31% yield (Scheme 3). This
compound was then transformed into the known donor 13,[18]
by treatment with triethylsilane[19] and trifluoroacetic acid
(TFA) in dichloromethane followed by acetylation, with a
yield of 95%. For the synthesis of the corresponding
trisaccharide 16, instead of optimising the previously de-
scribed procedure for unknown acetochloro-b-maltotriose, we
investigated an alternative enzymatic condensation of the
fluoride 2 and the acceptor 14 derived from 1.[17] The reaction,
catalysed by Bacillus sp. CGTase, afforded the trisaccharide
15 in 86% yield after acetylation. This compound was then
transformed into the desired donor 16 as described for the
preparation of 13 (overall yield 69%).
OAc
O
OAc
O
AcO
AcO
AcO
AcO
OAc
O
OAc
O
i
AcO
AcO
O
O
OAc
AcO
AcO
AcO
AcO
SR
12: R = Tr
13: R = Ac
11
ii
OAc
O
R2O
AcO
OR
OAc
O
AcO
O
RO
RO
O
iv
OAc
O
+
2
AcO
RO
AcO
O
STr
AcO
15: R1 = Tr, R2 = THP
16: R1 = R2 = Ac
1: R = Ac
14: R = H
AcO
SR1
Having confirmed the usefulness of this methodology, we
turned to the exploitation of fluorides 2 and 3 for the
preparation of 6w-halogenomaltooligosaccharides as accep-
tors for the thioglycosylation reactions. The known bromo-
maltose derivative 17[20] may be used for these reactions, but it
can also act as an acceptor for enzymatic transglycosylation
after de-O-acylation. In fact, the condensation of 2 and the
iii
ii
Scheme 3. Syntheses of the 1-S-acetyl-1-thio-glycoses 13 and 16. i) AlCl3,
CHCl3, 08C to RT, then TrSN(Bu)4, toluene, RT (31%); ii) (Et)3SiH, TFA,
CH2Cl2, RT, then Ac2O, pyridine, RT (95% for 13, 69% for 16);
iii) MeONa, MeOH, RT (100%); iv) CGTase, phosphate buffer, 408C,
then Ac2O, pyridine, DMAP (86% overall). Trˆ triphenylmethyl.
known 18[20] gave, after acidic
Br
removal of the ether protecting
group and acetylation, the tet-
rasaccharide 19 in 70% yield as
an anomeric mixture. For the
purposes of spectral analysis, an
aliquot was converted into the
O
BzO
AcO
OAc
O
AcO
O
OAc
AcO
AcO
17
OAc
O
i
AcO
OAc
AcO
pure b-maltotetraosyl deriva-
tive 19b (Scheme 4). To avoid
this additional step, we decided
AcO
O
Br
O
Br
AcO
O
AcO
O
HO
O
OH
O
HO
ii
OAc
O
AcO
HO
AcO
to employ methyl a-d-glucoside
20 as an acceptor for the trans-
glycosylation reaction with 3.
After acidic treatment, methyl
6III-bromo-a-maltotrioside 21
could be either acetylated to
afford 22 in 75% overall yield,
or elongated with the fluoride
2. Following the previously de-
scribed treatment (tetrahydro-
pyranyl (THP) removal and
acetylation), the methyl 6III-
bromomaltopentaoside 24 was
obtained in 52% overall yield
from the fluoride 2.
At this point, having success-
fully prepared all the necessary
precursors, synthesis of the lin-
ear hemithiomaltotetraose 26,
methyl pentaoside 28 and hex-
aoside 30 was attempted. Che-
moselective deprotection and
activation of 1-S-acetylated glu-
cose was achieved by the action
of cysteamine dithioerythritol
in hexamethylphosphoramide
(HMPA), and the resulting thi-
olate was coupled with 6w-iodo-
maltooligosaccharides, result-
2
+
O
O
HO
R2
AcO
OH
HO
AcO
R1
19: R1, R2 = H, OAc
19β: R1= H, R2 = OAc
iii
18
R2
O
R1O
R1O
OR1
OH
R1O
O
OR1
R1O
O
iv
O
HO
HO
3
+
R1O
HO
O
O
OCH3
R1O
R1O
20
21: R1 = H, R2 = Br
22: R1 = Ac, R2 = Br
23: R1 = Ac, R2 = I
OCH3
v
vi
OAc
O
iv
AcO
AcO
OAc
AcO
O
O
Br
O
AcO
vii
AcO
O
21
2
+
OAc
AcO
AcO
O
O
AcO
OAc
O
24
AcO
O
AcO
AcO
OCH3
Scheme 4. Syntheses of the 6-halogeno-6-maltooligosaccharides 19 and 22 24. i) MeONa, MeOH, RT (100%);
ii) a) CGTase, phosphate buffer, 408C; b) HCl (1m), RT; c) Ac2O, pyridine, DMAP (70% overall); iii) HBr/
Ac2O, CH2Cl2, 08C, then AgOAc, AcOH, Ac2O, RT, (52%); iv) a) CGTase, phosphate buffer, 408C; b) HCl (1m),
RT; v) Ac2O, pyridine, DMAP (75%); vi) KI, DMF, 708C, (92%); vii) a) CGTase, phosphate buffer, 408C;
b) HCl (1m), RT; c) Ac2O, pyridine, DMAP (52% overall).
Chem. Eur. J. 2002, 8, No. 23
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H. Driguez et al.
FULL PAPER
OR1
O
ing in the corresponding hemi-
thiomaltodextrins in high yield.[6]
It was also shown that N,N-di-
methylformamide (DMF) was a
good solvent for the effective
substitution of tosylate and bro-
mide at C-6 of mono- and oligo-
saccharides, while the donor was
S-deacetylated and activated in
situ with diethylamine (DEA).[21]
However, for practical reasons,
we preferred the latter procedure
in this work. Condensation of 13
and 17 in DMF in the presence of
DEA gave the expected com-
pound 25 but in only 67% yield
(Scheme 5). To overcome this
drawback with the methyl malto-
trioside 22, an exchange of halo-
gen (Br! I) was achieved by
treatment of the bromo com-
pound with KI in DMF for 2 h
at 708C. Likewise, the iodo de-
rivative 23 was obtained in 92%
yield. The halogen displacement
of 23 under the previous condi-
tions with 13 or 16 gave the
expected penta- (27) or hexasac-
charide (29) with yields of 76%.
De-O-acylation of 25 and de-O-
acetylation of 27 and 29 gave 26,
28 and 30 respectively, in almost
quantitative yields.
Scheme 6 outlines the synthe-
ses of two branched hemithio-
maltodextrins 32 and 34. A one-
pot procedure was adopted for
the transformation of the bromo
derivatives 19 and 24 into the
corresponding iodo analogues,
which were then engaged in cou-
pling reactions with acetylated
1-thio-a-maltose (13). During
thioglycosylation, some de-O-
acetylation occurred, and an
acetylation step was performed.
Under these experimental con-
ditions, the acetylated hexamer
31 and heptamer 33 were isolated
with yields of 72 and 53%,
respectively. To facilitate NMR
R1O
R1O
OR1
O
i
R1O
13
+
17
O
R1O
R1O
R2O
S
O
OR1
O
R1O
R1O
O
OR1
R1O
25: R1 = Ac, R2 = Bz
26: R1 = R2 = H
ii
R1O
OR
O
R
O
OR
O
RO
RO
O
RO
n
RO
S
O
RO
13
16
+
23
23
OR
RO
RO
i
O
+
O
OR
O
RO
RO
O
27: n = 1, R = Ac
28: n = 1, R = H
RO
ii
RO
OCH3
29: n = 2, R = Ac
30: n = 2, R = H
ii
Scheme 5. Syntheses of the linear target molecules 26, 28, and 30. i) DEA, DMF, RT (36% for 25, 76% for 27
and 29); ii) MeONa, MeOH, RT (100%for 26, 28, and 30).
OR3
OR3
R3O
OR3
OR3
O
OR3
O
R3O
R3O
R3O
O
OR3
O
OR3
O
R3O
O
S
R3O
R3O
O
O
OR3
O
RO
R3O
O
R2
R3O
i
31: R1,R2 = H, OAc, R3 = Ac
32: R1,R2 = H, OH, R3 = H
13
13
+
+
19
R1
R3O
iii
ii
31β: R1= H, R2 = OAc, R3 = Ac
19β
OR
OR
RO
OR
O
OR
OR
O
O
RO
RO
OR
RO
O
OR
RO
O
O
S
RO
RO
O
O
OR
RO
i
analysis
of
this
complex
13
+
24
RO
O
O
structure, 19b was engaged
in the thioglycosylation reac-
tion in place of 19, and both
halogen exchange and acetyla-
tion were omitted. Likewise, 31b
was isolated but in only a 43%
yield.
OR
RO
RO
33: R = Ac
34: R = H
O
O
iii
RO
RO
OCH3
Scheme 6. Syntheses of the branched target molecules 32 and 34. i) a) 19 and KI, DMF, 708C; b) 13, DTE,
DEA, DMF, 08C to RT; c) Ac2O, pyridine, DMAP (72% overall for 31 and 53% overall for 33); ii) DEA, DMF,
RT (43%); iii) MeONa, MeOH, RT (96% for 32 and 99% for 34).
5450
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Chem. Eur. J. 2002, 8, No. 23

Thiomaltodextrins as Potential Enzyme Inhibitors
5447 5455
The structures of the compounds were confirmed by
1H NMR data analysis, the spectra are given as Supporting
Information. Assignments were done on the protected com-
pounds. These spectra were assigned by using COSY, COSY-
relayed and TOCSY 2D experiments employing the Bruker
library pulse sequences.
which sixglucosyl residues are readily accommodated. More-
over, the inefficiency of 28 towards Th activity underlines the
extreme importance of recognition of the additional glucosyl
unit at the nonreducing end of 30.
The structural characterisation is based on comparison of
different types of data obtained on the different compounds.
The general procedure was: identification of a characteristic
signal and assignment of all the protons of the same unit by
using homonuclear-correlation COSY or TOCSY. When units
exhibit the same characteristics, the assignments may be
reversed. The coupling constants were read from the 1D
spectra or from the 2D COSY map when overlaps occurred.
Assignments were verified by using a 1D simulation spectrum
on WIN-DAISY 4.0 software.
13C NMR data were assigned by using model compounds.
For the total attribution given for some derivatives, HMQC
and HMBC experiments were performed by using Bruker
library pulse sequences.
Experimental Section
General procedures: Roman numerals in ascending order are given to the
residues from the reducing end to the terminal unit of the branch. NMR
spectra were recorded at 303 K on a Bruker AC300, Bruker Avance400 or
Varian Unity500. Proton chemical shifts (d) are reported in ppm downfield
from TMS; carbon chemical shifts are reported with reference to internal
solvent.
High- (HRMS) and low-resolution mass spectra were recorded on a
VG ZAB and on a Nermag R-1010C spectrometer, respectively. Optical
rotations were measured with a Perkin Elmer 341 polarimeter. Melting
points were measured on a B¸chi 535 apparatus. Microanalyses were
performed by the ™Laboratoire Central d×Analyses du CNRS∫ (Vernai-
son). Progress of synthesis was monitored by analytical thin-layer
chromatography with silica gel 60 F254 precoated plates (Merck, Darm-
stadt). The enzyme CGTase was from Bacillus sp. and was a gift from
Wacker Industrie S.A. (Lyon, France).
Compounds 26, 28, 30, 32 and 34 were tested as potential
inhibitors during the hydrolysis of pullulan by pullulanases
from Bacillus acidopullulyticus (Ba) and Klebsiella planticola
(Kp), barley limit dextrinase (LD) and pullulanase type II
from Thermococcus hydrothermalis (Th) (Table 1). None of
the compounds was hydrolysed under our experimental
conditions.
All reactions in organic medium were carried out under argon with freshly
distilled solvents. After workup, organic phases were dried over anhydrous
Na2SO4.
Enzyme inhibition assays: The inhibition of pullulanase from Bacillus
acidopullulyticus and Klebsiella planticola (Megazyme) and barley malt
limit dextrinase[22] was determined in a competitive assay with pullulan as
substrate at 408C in sodium acetate (20 mm, pH 5.0). The activity was
calculated from the release of reducing sugar, as measured by the copper
bicinconitate method essentially as described.[22, 23] Km and kcat were
determined in the same assay by using pullulan (0.06 to 10 mgmLÀ1) and
found to be 0.16 mgmLÀ1 and 33 sÀ1 for barley limit dextrinase,
0.24 mgmLÀ1 and 120 sÀ1 for Ba, and 0.09 mgmLÀ1 and 81 sÀ1 for Kp,
respectively. Ki values were calculated by assuming competitive inhibition
from 1/v ˆ (S ‡ Km)/(S ¥ kcat) ‡ Km/(kcat ¥ S ¥ Ki)  [I], in which v is the rate
measured in the absence or presence of inhibitor, S is the substrate
concentration (0.5 mgmLÀ1 for limit dextrinase and 0.25 mgmLÀ1 for Ba
and Kp), and [I] is the concentration of inhibitor (0 2 mm).
Table 1. Inhibitory capacities given as the Ki (mm) of the target compounds
on pullulan hydrolysis by four different enzymes.
LD
Pullulanase
Ba
Kp
Th (type II)
26
28
30
32
34
> 2000
> 2000
ꢀ 500
> 2000
ꢀ 300
ꢀ 200
ꢀ 1350
ꢀ 80
ꢀ 300
2.5
ꢀ 300
ꢀ 625
ꢀ 320
> 2000
> 2000
ꢀ 122
For the inhibition of pullulanase type II from Thermococcus hydrotherma-
lis,[24] measurements were carried out at 808C in the presence of pullulan
(0.1 to 10 mgmLÀ1) as substrate in CaCl2 (5 mm), sodium acetate (50 mm),
pH 5.5. Activity was determined by measuring the release of reducing sugar
by using a previously described modification of the Kidby Davidson
method.[25] The inhibitor concentration was varied over the range 0 to
1 mm, and the data were analysed by using SigmaPlot 2000 software. Ki
values were determined on the basis of a mixed-inhibition model.
> 2000
> 2000
> 2000
ꢀ 545
The smallest hemithiodextrin 26 only affected Ba with a
relatively weak Ki value of about 300 mm. Compound 28,
which is extended in the reducing end by a methyl a-d-
glucosyl unit, increased the affinity of Ba more than 100-fold
to give a Ki of only 2.5 mm. Compounds 28 and 30 affected Kp
with Ki values of about 300 mm and 200 mm, respectively. In
addition, the branched methyl maltoheptaoside 34 was found
to be the best inhibitor for this enzyme with a Ki value of
about 80 mm. Only compound 30 has significant a inhibitory
effect on barley limit dextrinase with a Ki value of about
500 mm. Curiously, the differences of enzyme specificity
revealed by these substrate analogues are not in accordance
with their almost identical affinities measured for the
corresponding natural oligosaccharide substrates (Jensen
and Svensson, unpublished results). This apparent discrep-
ancy remains to be solved.
(2,3,6-Tri-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-1,2,3,6-tetra-O-acetyl-b-
d-glucopyranose (5): A solution of compound 4 (6.08 g, 10.2 mmol),
1-(acetyloxy)benzotriazole (2.0 g, 1.2 equiv) and triethylamine (2.1 mL,) in
dichloromethane (60 mL) was stirred for 20 h at room temperature. The
resulting solution was evaporated, and crystallisation in diethyl ether gave
the monohydroxy compound 5 (5.2 g, 80%). M.p. 1768C, (lit: 175
1778C);[12] [a]D25 ˆ ‡46 (c ˆ 0.88 in CHCl3), (lit: ‡46.5);[12] 1H NMR
(300 MHz, CDCl3) see Supporting Information; 13C NMR (75 MHz,
CDCl3) d ˆ 171.1 168.7 (7  OCOCH3), 95.75 (C-1II), 91.09 (C-1I), 74.94,
72.88, 72.31, 71.46, 70.84, 70.77, 70.03, 68.47 (C-2I,II, C-3I,II, C-4I,II, C-5I,II),
62.37 (C-6I,II), 20.53 20.26 (OCOCH3); elemental analysis calcd (%) for
C26H36O18: C 49.306, H 5.70; found C 48.97, H 5.85.
(2,3-Di-O-acetyl-6-bromo-6-deoxy-a-d-glucopyranosyl)-(1 ! 4)-1,2,3,6-
tetra-O-acetyl-b-d-glucopyranose (6): Triphenylphosphine (4 g, 2 equiv)
and tetrabromomethane (2.57 g, 1.02 equiv) were added to a solution of
compound 4 (5.0 g, 7.86 mmol) in pyridine (54 mL) at 08C. After the
solution had been stirred for 15 min at 08C and then for 3 h at 508C,
methanol (5 mL) was added, and the reaction mixture was concentrated
and co-evaporated with toluene. Purification by flash chromatography
(EtOAc/petroleum ether 1:1, v/v) gave 6 (4.2 g, 84%). [a]2D5 ˆ ‡55.3 (c ˆ
Like LD, Th was not affected by 26 or 28. However, 30 and
34 did inhibit Th, giving Ki values of 122 mm and 545 mm,
respectively (Table 1). Clearly, compared with Ba, which was
strongly inhibited by 28, Th must possess a larger active site in
1
0.77 in CHCl3); H NMR (300 MHz, CDCl3) see Supporting Information;
Chem. Eur. J. 2002, 8, No. 23
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0823-5451 $ 20.00+.50/0
5451

H. Driguez et al.
FULL PAPER
13C NMR (75 MHz, CDCl3) d ˆ 171.1 168.8 (6  OCOCH3), 95.67 (C-1I),
and evaporation of solvent and co-evaporation with toluene (3 Â 20 mL)
gave crude acetochloro-b-maltose, which was used in the next step without
further purification and characterisation. A solution of this compound in
toluene (23 mL) was added to a slurry of tetrabutylammonium triphenyl-
methanethiolate, prepared as already described[16] with triphenylmethyl-
thiol (4 g, 14.4 mmol). The mixture was stirred at room temperature for 3 h,
then evaporated. The residue, after purification by flash chromatography
(1: CH2Cl2, 2: EtOAc/petroleum ether 1:1, v/v), gave compound 12 (3.99 g,
91.18 (C-1II), 74.96, 73.12, 72.44, 71.73, 71.28, 70.82, 70.72, 70.14 (C-2I,II
,
C-3I,II, C-4I,II, C-5I,II), 62.61 (C-6I), 32.66 (C-6II), 20.75 20.38 (OCOCH3);
elemental analysis calcd (%) for C24H33BrO16: C 43.85, H 5.06, Br 12.15;
found C 43.75, H 5.29, Br 11.76.
(2,3,6-Tri-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-a-d-glu-
copyranosyl fluoride (7): In a plastic vessel, a solution of compound 5 (5.0 g,
7.9 mmol) in hydrogen fluoride/pyridine (30 mL, 7:3) was stirred at 08C for
30 min, then diluted with dichloromethane (20 mL) and poured in a plastic
beaker containing an ice-cooled solution of ammonia (25 mL, 3m). The
organic layer was washed with saturated aq. sodium hydrogen carbonate
until neutralisation, dried (Na2SO4) and concentrated under reduced
pressure. Flash column chromatography (petroleum ether/EtOAc 1:2, v/v)
of the residue with triethylamine neutralised silica gave the a-fluoride 7
(4.45 g, 95%).[a]2D5 ˆ ‡93 (c ˆ 0.8 in CHCl3); 1H NMR (300 MHz, CDCl3)
see Supporting Information; 13C NMR (75 MHz, CDCl3) d ˆ 171.3 169.7
(6  OCOCH3), 103.56 (d, 1JCF ˆ 229.3 Hz, C-1I), 95.84 (C-1II), 71.78/71.66/
71.58/71.21/70.72/70.30/69.99/68.69 (C-2I,II, C-3I,II, C-4I,II, C-5I,II), 62.36/62.12
(C-6I, C-6II), 20.71/20.41 (OCOCH3); elemental analysis calcd (%) for
C24H33FO16: C 48.32, H 5.58, F 3,18%; found C 48.34, H 5.82, F 2.98.
1
31%). [a]2D5 ˆ ‡147 (c ˆ 0.56 in CHCl3); H NMR (400 MHz, CDCl3) see
Supporting Information; 13C NMR (100 MHz, CDCl3) d ˆ 170.62 169.39
(7 Â OCOCH3), 144.32 (CC5H5), 129.82/127.88/127.06 (CC5H5), 95.70
(C-1II), 81.84 (C-1I), 73.42 (C-4I), 72.96 (C-3I), 70.26 (C2I), 69.95 (C-2II),
69.79 (C-5I), 69.32 (C-3II, CC5H5), 68.33 (C-5II), 67.95 (C-4II), 62.77 (C-6I),
‡
61.31 (C-6II), 20.95 20.56 (OCOCH3); ES HRMS: calcd for C45H50O17S
‡
[M ‡Na]: 917.2666, found: 917.2667.
(2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-1-S-
acetyl-1-thio-a-d-glucopyranose (13): Triethylsilane (645 mL, 4.0 mmol)
and trifluoroacetic acid (18 mL) were added to a stirred solution of
derivative 12 (1.2 g, 1.7 mmol) in CH2Cl2 (28 mL) at room temperature.
The solution was stirred for 45 min, then evaporated, and the residue was
acetylated in a mixture of acetic anhydride and pyridine (1:1, v/v, 18 mL).
After 12 h at room temperature, the reaction mixture was cooled to 08C,
and methanol was added (10 mL). Evaporation of the solution, usual
workup and flash chromatography (EtOAc/petroleum ether 1:1, v/v) gave
13 (1.08 g, 95%). [a]D25 ˆ ‡68 (c ˆ 0.72 in CHCl3) (lit: ‡68);[18] 13C NMR
(75 MHz, CDCl3) d ˆ 191.55 (SCOCH3), 170.28 169.22 (7  OCOCH3),
(2,3,6-Tri-O-acetyl-4-O-tetrahydropyranyl-a-d-glucopyranosyl)-(1 ! 4)-
2,3,6-tri-O-acetyl-a-d-glucopyranosyl fluoride (8): Freshly distilled dihy-
dropyran (2.3 mL) and camphorsulfonic acid (86 mg) were added to a
solution of compound 7 (3.0 g, 5.0 mmol) in CH2Cl2 (120 mL). After 3 h at
room temperature, the solution was diluted with CH2Cl2 (120 mL) and
successively washed with H2O and saturated aqueous NaHCO3. The
organic layer was dried, concentrated and purified by flash chromatog-
raphy (EtOAc/petroleum ether 1:1, v/v) to give compound 8 in 98% yield.
13C NMR (75 MHz, CDCl3) d ˆ 170.54 169.31 (6  OCOCH3), 103.56
(d,1JCF ˆ 229.3 Hz, C-1I), 102.05/101.62 (CH THP group), 95.72 (C-1II),
75.28/73.59/71.75/71.68/71.48/70.77/70.43/70.38/70.26/69.99/69.84/69.74 (C-
95.77 (C-1II), 79.74 (C-1I), 72.88/72.76/71.80/69.84/69.33/69.45/67.88 (C-2I,II
,
C-3I,II
, ,
C-4I,II C-5I,II), 62.61/61.34 (C-6I,II), 31.24 (SCOCH3), 20.41
‡
(OCOCH3); DCIMS: m/z ˆ 712 [M ‡NH4].
Triphenylmethyl-1-thio-a-d-glucopyranoside (14): This compound was
¡
obtained in quantitative yield by Zemplen de-O-acetylation of known
2I,II, C-3I,II, C-4I,II, C-5I,II), 64.03/63.34/62.61/62.14/62.1/62.02 (C-6I, C-6II
R
compound 1 (200 mg, 0.33 mmol) with sodium methoxide (1m 1% v/v) in
1
MeOH (20 mL). [a]2D5 ˆ ‡216.4 (c ˆ 0.83 in MeOH); H NMR (300 MHz,
and S, CH2 THP group), 20.76 20.41 (OCOCH3), 20.09/19.67 (CH2 THP
CDCl3) see Supporting Information; 13C NMR (100 MHz, CD3OD) d ˆ
146.25 (CC5H5), 131.32/128.69/127.84 (CC5H5), 87.42 (C-1), 76.06 (C-3),
‡
group); DCIMS: m/z: 698 [M ‡NH4]; elemental analysis calcd (%) for
C29H41FO17: C 51.18, H 6.07, F 2.79; found: C 50.98, H 6.21, F 2.73.
‡
75.11 (C-5), 73.38 (C-2), 71.28 (C-4), 69.99 (CC5H5), 62.10 (C-6); ES
(2,3,-Di-O-acetyl-6-bromo-6-deoxy-a-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-
O-acetyl-a-d-glucopyranosyl fluoride (9): In a plastic vessel, a solution of
compound 6 (2.04 g, 3.11 mmol) in hydrogen fluoride/pyridine (20 mL, 7:3)
stirred at 08C for 30 min, was worked up and purified as already described
for the preparation of 7. The a-fluoride 9 was obtained (1.71 g, 90%).
[a]2D5 ˆ ‡95.7 (c ˆ 0.67 in CHCl3); 1H NMR (300 MHz, CDCl3) see
Supporting Information; 13C NMR (75 MHz, CDCl3) d ˆ 171.33/169.76
‡
HRMS: calcd for C25H26O5S [M ‡Na]: 461.1399, found: 461.1402; calcd for
‡
C25H26O5S [M ‡K]: 477.1138, found: 477.1155.
Triphenylmethyl (2,3,6-Tri-O-acetyl-4-O-tetrahydropyranyl-a-d-glucopyr-
anosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-
O-acetyl-1-thio-a-d-glucopyranoside (15): CGTase (207 mL) was added to
a solution of THP-fluoride 2 (50 mg, 0.12 mmol) and compound 14
(1.2 equiv) in a sodium phosphate buffer (3 mL, 0.1m, pH 7.0). The reaction
mixture was gently shaken in an oven at 408C for 2 h, freeze-dried and
acetylated (acetic anhydride/pyridine 1:1, v/v, 10 mL) in the presence of a
trace of dimethylaminopyridine. After 12 h at 708C, the reaction mixture
was cooled to 08C, quenched by adding MeOH (5 mL) and concentrated in
vacuo. The residue was dissolved in CH2Cl2 and washed with water and
saturated aq. NaHCO3. The organic layers were concentrated, coevapo-
rated with toluene, and purified by flash chromatography (EtOAc/
petroleum ether 1:1, v/v) to generate the title compound 15 (127 mg,
86%); 13C NMR (75 MHz, CDCl3) d ˆ 170.69 169.20 (OCOCH3), 144.35
(CC5H5), 129.79/127.80/126.94 (CH CPh3), 101.14/100.67 (CH THP group),
95.77/95.65 (C-1II,III), 81.66 (C-1I), 76.58/75.28/73.93/73.57/72.59/72.42/71.78/
1
(5  OCOCH3), 103.54 (d, JCF ˆ 227.8 Hz, C-1I), 95.72 (C-1II), 71.80/71.31/
70.7/70.35/70.06 (C-2I,II, C-3I,II, C-4I,II, C-5I,II), 62.17 (C-6I), 32.71 (C-6II),
‡
‡
20.9 20.36 (OCOCH3); ES HRMS: calcd for C22H30BrFO14 [M ‡Na]:
639.0701, found: 639.0705.
(2,3,6-Tri-O-acetyl-6-bromo-6-deoxy-4-O-tetrahydropyranyl-a-d-gluco-
pyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-a-d-glucopyranosyl fluoride (10):
Compound 9 (1.72 g, 2.79 mmol) was treated as described for its analogue
14. The expected fluoride 10 was obtained as a mixture of diastereoisomers
‡
‡
(1.8 g, 95%). ES HRMS: calcd for C27H38BrFO15 [M ‡Na]: 723.1276,
found: 723.1281.
(4-O-Tetrahydropyranyl-a-d-glucopyranosyl)-(1 ! 4)-a-d-glucopyranosyl
fluoride (2): Fluoride 8 (1.55 g, 2.28 mmol) in methanol (50 mL) was
treated with sodium methoxide (1m, 2.5 mL) for 30 min at room temper-
ature. The mixture was then cooled to 08C and neutralised with Amberlite
71.56/70.36/70.26/69.79/69.60/69.25/68.76 (C-2I,II,III
,
C-3I,II,III
,
C-4I,II,III
,
C-5I,II,III, CC5H5), 63.91/63.20/63.00/62.54/62.24/62.00 (C-6I,II, C-6III, CH2
THP group), 20.85 20.46 (OCOCH3), 20.16/19.53 (CH2 THP
‡
IRN 120 (H ) resin, the resin was removed by filtration, and the filtrate was
‡
‡
group); ES HRMS: calcd for C60H72O25S [M ‡Na]: 1247.3981, found:
concentrated. The free fluoride 2 was dissolved in water and freeze dried
(956 mg, 98%). In this form, compound 2 was stable for several weeks at
1247.3989.
(2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-a-
d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-1-S-acetyl-1-thio-a-d-gluco-
pyranose (16): Triethylsilane (35.8 mL, 0.22 mmol) and trifluoroacetic acid
(1.2 mL) were added to a stirred solution of the THP derivative 15 (81 mg,
0.66 mmol) in CH2Cl2 (2.0 mL) at room temperature. The solution was
stirred for 45 min, then evaporated, and the residue was acetylated in a
mixture of acetic anhydride/pyridine (1.5:2, v/v, 3.5 mL). After 12 h at room
temperature and workup as already described for 13, flash chromatography
(EtOAc/petroleum ether 1.5:1, v/v) gave 16 (45 mg, 69%). [a]2D5 ˆ ‡183
(c ˆ 0.27 in CHCl3); 1H NMR (400 MHz, CDCl3) see Supporting Informa-
tion; 13C NMR (100 MHz, CDCl3) d ˆ 191.51 (SCOCH3), 170.3 169.2
(OCOCH3), 96.02 (C-1II), 95.65 (C-1III), 79.75 (C-1I), 73.84 (C-5III), 72.71
‡
‡
À188C. ES HRMS: calcd for C17H29FO11 [M ‡Na]: 451.1592, found:
451.1596.
(6-Bromo-6-deoxy-4-O-tetrahydropyranyl-a-d-glucopyranosyl)-(1 ! 4)-a-
d-glucopyranosyl fluoride (3). The fluoride 10, de-O-acetylated and treated
‡
as described for 8, afforded 3 in 98% yield (683 mg). ES HRMS: calcd for
‡
C17H28BrFO10 [M ‡Na]: 513.0748, found: 513.0747.
Triphenylmethyl
(2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-
2,3,6-tri-O-acetylthio-a-d-glucopyranoside (12): AlCl3 (2 g) was added to
a solution of octaacetyl-b-maltose 11 (10 g, 14.7 mmol) in anhydrous
chloroform (42 mL) at 08C. After being stirred for 15 min, the solution was
allowed to warm up to room temperature. After 30 min, Celite filtration
5452
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0823-5452 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 23

Thiomaltodextrins as Potential Enzyme Inhibitors
5447 5455
(C-3I), 72.47 (C-5II), 71.76 (C-3II, C-4I), 70.34 (C-2II), 70.04 (C-2III), 69.41
(C-2I), 69.35 (C-3III), 68.94 (C-5I), 68.47 (C-4II), 67.86 (C-4III), 62.88 (C-6I),
Methyl (2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-
acetyl-a-d-glucopyranosyl)-(1 ! 4)-(2,3,di-O-acetyl-6-bromo-6-deoxy-a-
d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-a-d-glucopyranosyl)-(1 !
4)-2,3,6-tri-O-acetyl-a-d-glucopyranoside (24): 6II-Bromomaltosyl fluoride
‡
62.23 (C-6II), 61.32 (C-6II), 31.24 (SCOCH3), 20.41 (OCOCH3); ES
‡
HRMS: calcd for C40H54O26S [M ‡Na]: 1005.2522, found: 1005.2519.
3
(100 mg, 0.20 mmol) and methyl a-d-glucopyranoside 20 (43.5 mg,
(2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-a-
d-glucopyranosyl)-(1 ! 4)-(2,3,di-O-acetyl-6-bromo-6-deoxy-a-d-gluco-
pyranosyl)-(1 ! 4)-1,2,3,6-tetra-O-acetyl-d-glucopyranose (19): Maltosyl
fluoride 2 (250 mg, 0.58 mmol)and the known 6II-bromomaltose[20] 18
(235 mg, 1 equiv) in phosphate buffer (0.1m, pH 7.0, 25 mL) were incubated
with CGTase (750 mL)at 408C for 1 h, then the mixture was boiled for 5 min
and filtered through a cotton plug. The filtrate was acidified down to pH 2.0
with 1ùm HCl and stirred at room temperature for 20 min. The solution
was then neutralised with aqueous ammonia, lyophilised and acetylated
under standard conditions. Usual workup and flash chromatography
(EtOAc/petroleum ether 2:1.5, v/v) gave the acetylated derivative 19
1.1 equiv) in phosphate buffer (0.1m, pH 7.0, 3.7 mL) were incubated with
CGTase (130 mL)at 408C for 1 h, then the mixture was boiled for 5 min and
filtered through a cotton plug. The filtrate was acidified (pH 2.0) with HCl
(1ùm) and stirred at room temperature for 20 min, then neutralised with
aqueous ammonia. Fluoride 2 (112 mg, 1.3 equiv) and CGTase (130 mL) in
phosphate buffer (0.1m, pH 7.0, 1 mL) were added to this solution of crude
21. After 2 h of incubation, the boiling and the neutralisation steps were
repeated, and were followed by acetylation and workup of the mixture as
described for the preparation of 19. Flash chromatography (1: Et2O, 2 :
Et2O/acetone 10:1, v/v) gave 19 (92 mg, 36%) and the expected compound
1
24 (161 mg, 52%). [a]2D5 ˆ ‡105 (c ˆ 0.25 in CHCl3); H NMR (400 MHz,
1
3
(521 mg, 70%). H NMR (300 MHz, CDCl3) d ˆ 6.62 (d, J ˆ 3.7 Hz, 1H;
H1a-1), 5.69 (d, 1H, 3J ˆ 8.0 Hz H1b-1; 13C NMR (75 MHz, CDCl3) d ˆ
170.56 169.88 (OCOCH3), 96.01 95.64 (C-1II,III,IV), 91.21 (C-1Ib), 88.81
(C-1Ia), 75.14/74.56/73.63/73.51/72.89/72.40/72.15/71.73/70.99/70.40/70.15/
CDCl3) see Supporting Information; 13C NMR (100 MHz, CDCl3) d ˆ
170.45 169.56 (OCOCH3), 96.58/95.87/95.65/95.53 (C-1I,II,III,IV,V), 76.79/
74.18/73.67/72.64/72.34/71.73/71.26/70.36/69.97/69.40/69.11/68.94/68.77/
68.45/67.98/67.54 (C-2I,II,III,IV,V, C-3I,II,III,IV,V, C-4I,II,III,IV,V, C-5I,II,III,IV,V), 63.00/
62.71/62.56/61.36 (C-6I,II,IV,V), 55.33 (OCH3), 33.45 (C-6III), 20.83 20.51
69.97/69.71/69.39/69.18/68.96/68.47/68.01 (C-2I,II,III,IV, C-3I,II,III,IV, C-4I,II,III,IV
,
C-5I,II,III,IV), 62.72/61.38 (C-6I,III,IV), 33.38 (C-6II), 20.77 20.48 (OCOCH3);
elemental analysis calcd (%) for C24H33BrO16: C 47.07, H 5.29, Br 6.26;
found: C 47.25, H 5.52, Br 6.34.
‡
‡
(OCOCH3); ES HRMS: calcd for C61H83BrO40 [M ‡Na]: 1557.3542,
found: 1557.3542.
(2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-S-(2,3,6-tri-O-acetyl-
a-d-glucopyranosyl)-(1 ! 6)-(2,3-di-O-acetyl-4-O-benzoyl-6-thio-a-d-glu-
copyranosyl)-(1 ! 4)-1,2,3,6-tetra-O-acetyl-b-d-glucopyranose (25): Com-
pound 13 (251 mg, 0.361 mmol) was added to a solution of bromide 17
(260 mg, 0.34 mmol) in DMF (2 mL) with diethylamine (594 mL,
5.7 mmol). The reaction mixture was stirred under argon for 12 h at room
temperature, then evaporated and coevaporated (Â3) with toluene. The
residue was purified by flash chromatography (EtOAc/petroleum ether 2:1,
v/v), and 25 was obtained (303 mg, 67%). [a]2D5 ˆ ‡121 (c ˆ 0.48 in CHCl3);
1H NMR (400 MHz, CDCl3) see Supporting Information; 13C NMR
(100 MHz, CDCl3) d ˆ 170.72 168.84 (OCOCH3), 165.39 (COC6H5),
133.79 (COCC5H5), 130.02/128.65/128.52 (COCC5H5), 95.64/95.53 (C-1II,IV),
91.30 (C-1I), 82.29 (C-1III), 75.29/73.02/73.01/71.12/71.00/70.67/70.28/70.14/
69.95/69.40/68.46/68.45/67.99 (C-2I,II,III,IV, C-3I,II,III,IV, C-4I,II,III,IV, C-5I,II,III,IV),
(2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-a-
d-glucopyranosyl)-(1 ! 4)-(2,3,di-O-acetyl-6-bromo-6-deoxy-a-d-gluco-
pyranosyl)-(1 ! 4)-1,2,3,6-tetra-O-acetyl-b-d-glucopyranose (19b): An ali-
quot of the anomeric mixture 19 (520 mg, 0.4 mmol) was dissolved in dry
CH2Cl2 (20 mL) and cooled to 08C. HBr (30% w/v in AcOH, 10 mL) was
added. After being stirred for 1.5 h, the mixture was diluted in CH2Cl2 and
washed with ice-cold H2O and ice-cold saturated aq. NaHCO3. The
resulting crude bromide (500 mg) and AgOAc (707 mg, equiv.) in Ac2O/
AcOH (10 mL, 1:1 v/v) were stirred in the dark overnight at room
temperature. The mixture was diluted with CH2Cl2 and filtered trough a
Celite bed, and the filtrate was washed with saturated aqueous NaHCO3,
dried and concentrated. Flash chromatography (EtOAc/petroleum ether
1.5:1, v/v) gave 19b (271 mg, 52%). a]2D5 ˆ ‡86 (c ˆ 0.25 in CHCl3);
1H NMR (400 MHz, CDCl3) see Supporting Information, 13C NMR
(75 MHz, CDCl3) d ˆ 170.42 168.7 (OCOCH3), 96.01/95.55 (C-1II,III,IV),
91.21 (C-1Ib), 75.14/74.45/73.62/72.91/72.32/71.73/71.07/70.97/70.38/70.31/
‡
62.76/61.43 (C-6I,III,IV), 30.03 (C-6II), 20.88 20.58 (OCOCH3); ES HRMS:
‡
calcd for C57H72O34S [M ‡Na]: 1355.3523, found: 1355.3516.
(a-d-Glucopyranosyl)-(1 ! 4)-S-(a-d-glucopyranosyl)-(1 ! 6)-(6-thio-a-
d-glucopyranosyl)-(1 ! 4)-d-glucopyranose (26): Acylated 25 (200 mg,
0.15 mmol) was de-O-acylated by treatment with sodium methoxide (1m,
5 mL) in methanol (50 mL) for 1 h at room temperature. The mixture was
69.97/69.40/69.16/68.91/68.44/67.98 (C-2I,II,III,IV
,
C-3I,II,III,IV
,
C-4I,II,III,IV
,
C-5I,II,III,IV), 62.72/61.38 (C-6I,III,IV), 33.35 (C-6II), 20.77 20.51 (OCOCH3);
‡
FABMS: m/z ˆ 1299 [M ‡Na].
‡
Methyl (2,3,6-Tri-O-acetyl-6-bromo-6-deoxy-a-d-glucopyranosyl)-(1 ! 4)-
(2,3,6-tri-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-a-d-glu-
copyranoside (22): CGTase (207 mL) was added to a solution of fluoride 3
(80 mg, 0.163 mmol) and methyl a-d-glucopyranoside 20 (95 mg, 3 equiv)
in sodium phosphate buffer (7 mL, 0.1m, pH 7.0). The solution was
incubated for 14 h at 408C, and workup was performed as described for
compound 19. Compound 22 (118 mg, 75%) was isolated after flash
chromatography (Et2O). [a]2D5 ˆ ‡112.3 (c ˆ 0.44 in CHCl3); 1H NMR
(300 MHz, CDCl3) see Supporting Information; 13C NMR (75 MHz,
CDCl3) d ˆ 171.2 168.7 (OCOCH3), 96.58 (C-1I), 95.57/95.42 (C-1II,III),
73.72/72.86 (C-4I,II), 72.56 (C-3I), 71.58 (C-3II), 71.23 (C-2I), 70.53 (C-4III),
70.34/70.12 (C-2II,III), 69.09/69.04/68.98/67.52 (C-3III, C-5I,II,III), 62.96/62.53
neutralised with Amberlite IR 12(H ) resin, the resin was removed by
filtration, and the filtrate was concentrated. The residue was dissolved in
water and extracted with Et2O. Freeze-dried compound 26 was obtained
(97.3 mg, 95%). 13C NMR (75 MHz, D2O) d ˆ 100.13 (C-1II,IV), 96.11
(C-1Ib), 92.19 (C-1Ia), 85.29 (C-1III), 77.55/76.57/75.01/74.29/73.58/73.21/
73.05/72.97/72.45/72.10/71.81/71.62/71.19/70.39/69.71 (C-2I,II,III,IV, C-3I,II,III,IV
,
‡
C-4I,II,III,IV, C-5I,II,III,IV), 61.51/60.87 (C-6I,III,IV), 30.98 (C-6II); ES HRMS:
calcd for C24H42O20S [M ‡Na]: 705.1888, found: 705.1874.
‡
Methyl (2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-S-(2,3,6-tri-O-
acetyl-a-d-glucopyranosyl)-(1 ! 6)-(2,3,4-tri-O-acetyl-6-thio-a-d-gluco-
pyranosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-2,3,6-
tri-O-acetyl-a-d-glucopyranoside (27): Compound 13 (83.3 mg, 0.12 mmol)
was added to a solution of iodide 23 (101 mg, 0.10 mmol) in DMF (2 mL)
with diethylamine (16 mL, 0.15 mmol). The mixture was treated as already
described for the preparation of 25. Flash chromatography (Et2O/acetone
10:1, v/v) afforded the expected compound 27 (116 mg, 76%). [a]2D5 ˆ ‡156
(c ˆ 0.42 in CHCl3); 1H NMR (400 MHz, CDCl3) see Supporting Informa-
tion; 13C NMR (75 MHz, CDCl3) d ˆ 170.61 169.41 (OCOCH3), 95.64/
96.54/95.62/95.32 (C-1I,II,III,V), 82.3 (C-1IV), 73.92/73.03/72.49/72.34/72.24/
71.90/71.21/70.92/70.43/70.13/69.95/69.88/69.82/69.34/68.94/68.72/68.41/
67.99/67.49 (C-2I,II,III,IV,V, C-3I,II,III,IV,V, C-4I,II,III,IV,V, C-5I,II,III,IV,V), 63.07/62.71/
62.69/61.41 (C-6I,II,IV,V), 55.30 (OCH3), 29.93 (C-6III), 20.78 20.48
‡
(C-6I,II), 55.27 (OCH3), 31.15 (C-6III), 21.00 20.38(OCOCH3); ES HRMS:
‡
calcd for C37H51BrO24 [M ‡Na]: 981.1851, found: 981.1843.
Methyl (2,3,6-Tri-O-acetyl-6-iodo-6-deoxy-a-d-glucopyranosyl)-(1 ! 4)-
(2,3,6-tri-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-a-d-glu-
copyranoside (23): A mixture of bromo derivative 22 (200 mg, 0.21 mmol)
and KI (41.54 mg, 1.2 equiv) in DMF (2 mL) was stirred at 708C for 2 h.
After being cooled to room temperature, the solution was diluted with
water and extracted with EtOAc, dried and concentrated under reduced
pressure. Flash chromatography (EtOAc/petroleum ether 1:1, v/v) gave the
expected compound 23 (193 mg, 92%). [a]2D5 ˆ ‡52 (c ˆ 0.6 in CHCl3);
1H NMR (400 MHz, CDCl3) see Supporting Information; 13C NMR
(100 MHz, CDCl3) d ˆ 170.55 169.75 (OCOCH3), 96.57/95.57/95.39
(C-1I,II,III), 73.65/73.02/72.59/72.22/71.48/71.26/70.33/69.02/68.81/67.50 (C-
2I,II,III, C-3I,II,III, C-4I,II,III, C-5I,II,III), 62.99/62.67 (C-6I,II), 55.36 (OCH3),
‡
‡
(OCOCH3); ES HRMS: calcd for C63H86O41S [M ‡Na]: 1553.4263,
‡
found: 1553.4260; calcd for C63H86O41S [M ‡K]: 1569.4002, found:
1569.3976.
Methyl (a-d-Glucopyranosyl)-(1 ! 4)-S-(a-d-glucopyranosyl)-(1 ! 6)-(6-
thio-a-d-glucopyranosyl)-(1 ! 4)-(a-d-glucopyranosyl)-(1 ! 4)-a-d-gluco-
pyranoside (28): De-O-acetylation of 27 (54.5 mg, 35.6 mmol) by treatment
‡
20.90 20.58 (OCOCH3), 4.06 (C-6III); ES HRMS: calcd for C37H51IO24
‡
[M ‡Na]: 1029.1713, found: 1029.1694.
Chem. Eur. J. 2002, 8, No. 23
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0823-5453 $ 20.00+.50/0
5453

H. Driguez et al.
FULL PAPER
with sodium methoxide (1m, 500 mL) in methanol (20 mL), then neutral-
isation with resin (H ) and lyophilisation gave 28 in quantitative yield
precipitated by the addition of ice-cold water. The precipitate was filtered
on Celite, dissolved in CH2Cl2 and dried. Flash chromatography as
described for 31 gave 31b (28 mg, 43%). [a]2D5 ˆ ‡374 (c ˆ 0.75 in CHCl3);
1H NMR (400 MHz, CDCl3) see Supporting Information; 13C NMR
(75 MHz, CDCl3) d ˆ 170.63 168.72 (OCOCH3), 95.98/95.70/95.64
(C-1II,III,IV,VI), 91.22 (C-1Ib), 83.16 (C-1V), 75.32/74.90/72.98/72.87/72.65/
‡
(31 mg). [a]2D5 ˆ ‡206 (c ˆ 0.37 in water); 13C NMR (125 MHz, D2O)
d ˆ 99.99/99.66/99.30 (C-1I,II,III,V), 85.12 (C-1IV), 77.35/77.25/76.69/
74.14/73.53/73.06/72.91/72.81/72.28/71.95/71.70/71.46/71.23/71.05/70.28/69.52
(C-2I,II,III,IV,V, C-3I,II,III,IV,V, C-4I,II,III,IV,V, C-5I,II,III,IV,V), 61.09/60.71 (C-6I,II,IV,V),
‡
‡
55.30 (OCH3), 30.78 (C-6III); ES HRMS: calcd for C31H54O25S [M ‡Na]:
72.10/71.80/71.02/70.30/69.99/69.35/69.17/68.64/68.45/67.97 (C-2I,II,III,IV,V,VI
,
‡
881.2773, found: 881.2576; calcd for C31H54O25S ([M À H‡2Na]: 903.2392,
C-3I,II,III,IV,V,VI, C-4I,II,III,IV,V,VI, C-5I,II,III,IV,V,VI), 62.69/61.36 (C-6I,III,IV,V,VI), 30.26
‡
found: 903.2393.
(C-6IIa,IIb), 20.83 20.51 (OCOCH3); FABMS: m/z ˆ 1870 [M ‡Na].
(a-d-Glucopyranosyl)-(1 ! 4)-(a-d-glucopyranosyl)-(1 ! 4)-S-[(a-d-glu-
copyranosyl)-(1 ! 4)-(a-d-glucopyranosyl)-(1 ! 6)]-(6-thio-a-d-glucopyr-
anosyl)-(1 ! 4)-d-glucopyranose (32): An aliquot of sodium methoxide
(1m, 1 mL) was added to a solution of 31 (300 mg, 0.16 mmol) in methanol
(30 mL). The mixture was stirred for 2 h at room temperature, neutralised
Methyl (2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-
acetyl-a-d-glucopyranosyl)(1 ! 4)-S-(2,3,6-tri-O-acetyl-a-d-glucopyrano-
syl)-(1 ! 6)-(2,3,4-tri-O-acetyl-6-thio-a-d-glucopyranosyl)-(1 ! 4)-(2,3,6-
tri-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-a-d-glucopyra-
noside (29): A solution of 23 (40 mg, 39.7 mmol) was treated with 16
(46.2 mg, 1.2 equiv) as described for the preparation of 25, but in the
presence of 1,4-dithioerytritol (250 mg). After being stirred for 12 h at
room temperature under argon, the reaction mixture was acetylated
(pyridine/Ac2O, 2 mL, 1:1, v/v). Workup as described for 13 and flash
chromatography (EtOAc/petroleum ether 3:1, v/v) gave the expected
compound 29 (52 mg, 76%). [a]2D5 ˆ ‡158 (c ˆ 0.44 in CHCl3); 1H NMR
(400 MHz, CDCl3) see Supporting Information; 13C NMR (100 MHz,
CDCl3) d ˆ 170.66 169.54 (OCOCH3), 96.57/95.78/95.65/95.61/95.32
(C-1I,II,III,V,VI), 82.26 (C-1IV), 73.94/73.86/72.54/72.43/72.21/72.17/71.97/
71.74/71.25/70.92/70.46/70.36/70.13/70.02/69.92/69.88/69.37/68.97/68.89/
‡
with Amberlite IRN120 (H ), concentrated, diluted with water and freeze
dried. The residue, diluted with water, was filtered through a C18 cartridge
Sep-pak Plus (Waters) and pure 32 was obtained in 96% yield (157 mg).
13C NMR (100 MHz, D2O) d ˆ 99.24/99.22/99.18/98.87/98.71 (C-1II,III,IV,VI),
95.22 (C-1Ib), 91.31(C-1Ia), 84.76 (C-1V), 79.57/76.77/76.47/76.45/76.15/
75.66/74.09/73.44/73.37/72.74/72.67/72.46/71.20/71.18/71.06/70.96/70.81/
70.72/70.34/70.26/69.56/69.46/68.79/68.72 (C-2I,II,III,IV,V,VI
,
C-3I,II,III,IV,V,VI
,
C-4I,II,III,IV,V,VI, C-5I,II,III,IV,V,VI), 60.52/60.38/59.94 (C-6I,III,IV,V,VI), 30.13 (C-6II);
‡
‡
ES HRMS: calcd for C36H62O30S [M ‡Na]: 1029.2944, found: 1029.2946;
‡
calcd for C36H62O30S [M ‡K]: 1045.2684, found: 1045.2773.
, , ,
68.73/68.43/68.40/67.91/67.50 (C-2I,II,III,IV,V,VI C-3I,II,III,IV,V,VI C-4I,II,III,IV,V,VI
Methyl (2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-
acetyl-a-d-glucopyranosyl)-(1 ! 4)-S-[(2,3,4,6-tetra-O-acetyl-a-d-gluco-
pyranosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-a-d-glucopyranosyl)-(1 ! 6)]-(2,3
di-O-acetyl-6-thio-a-d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-d-glu-
copyranosyl)-(1 ! 4)-2,3,6-tri-O-acetyl-a-d-glucopyranoside (33): Methyl
6-bromo-maltopentaoside 24 (50 mg, 32.5 mmol) and then thio maltose 13
(34 mg, 48.7 mmol) were treated as already described for 19 and 13 during
the preparation of 31. The expected compound 33 was obtained (36 mg,
C-5I,II,III,IV,V,VI), 63.05/62.88/62.72/62.23 (C-6I,II,IV,V,VI), 55.33 (OCH3), 29.89
‡
(C-6III), 20.83 20.36 (OCOCH3); ES HRMS: calcd for C75H102O49S
‡
[M ‡Na]: 1841.5108, found: 1841.5106
Methyl (a-d-Glucopyranosyl)-(1 ! 4)-(a-d-glucopyranosyl)(1 ! 4)-S-(a-
d-glucopyranosyl)-(1 ! 6)-(6-thio-a-d-glucopyranosyl)-(1 ! 4)-(a-d-glu-
copyranosyl)-(1 ! 4)-a-d-glucopyranoside (30): De-O-acetylation of 29
(40.0 mg, 21.2 mmol) by treatment with sodium methoxide (1m, 200 mL) in
methanol (20 mL), and treatment as described for the preparation of 28
gave 30 in quantitative yield (22 mg). [a]2D5 ˆ ‡186 (c ˆ 0.41 in water);
13C NMR (100 MHz, D2O) d ˆ 100.18/100.14/100.00/99.84/99.48 (C-1I,II,III,V,-
VI), 85.31 (C-1IV), 77.64/77.61/77.44/77.12/74.28/73.88/73.71/73.24/73.09/
72.99/72.44/72.19/72.12/71.94/71.879/71.65/71.60/71.40/71.23/71.16/70.46/
69.69 (C-2I,II,III,IV,V,VI, C-3I,II,III,IV,V,VI, C-4I,II,III,IV,V,VI, C-5I,II,III,IV,V,VI), 61.28/60.85
53%). [a]2D5 ˆ ‡188 (c ˆ 0.32 in CHCl3); H NMR (400 MHz, CDCl3) see
1
Supporting Information; 13C NMR (100 MHz, CDCl3) d ˆ 170.66 169.50
(OCOCH3), 96.58/95.92/95.68/95.60/95.40 (C-1I,II,III,IV,V,VII), 83.47 (C-1VI),
74.49/73.72/72.99/72.82/72.64/72.07/71.95/71.82/71.31/71.21/70.88/70.43/
70.32/70.23/70.03/69.87/69.35/69.15/68.75/68.66/68.43/67.92/67.53
2I,II,III,IV,V,VI,VII C-3I,II,III,IV,V,VI,VII C-4I,II,III,IV,V,VI,VII C-5I,II,III,IV,V,VI,VII), 62.99/
62.83/62.71/62.61/61.35 (C-6I,II,IV,V,VI,VII), 55.37 (OCH3), 29.36 (C-6III),
(C-
,
,
,
‡
(C-6I,II,IV,V,VI), 55.45 (OCH3), 30.97 (C-6III); ES HRMS: calcd for
‡
‡
‡
20.90 20.59 (OCOCH3); ES HRMS: calcd for C87H118O57S [M ‡Na]:
C37H64O30S [M ‡Na]: 1043.3101, found: 1043.3001
2129.5953, found: 2129.5977.
(2,3,4,6-Tetra-O-acetyl-a-d-glucopyranosyl)-(1 ! 4)-(2,3,6-tri-O-acetyl-a-
d-glucopyranosyl)-(1 ! 4)-S-[(2,3,4,6-tetra-O-acetyl-a-d-glucopyranosyl)-
(1 ! 4)-(2,3,6-tri-O-acetyl-a-d-glucopyranosyl)-(1 ! 6)]-(2,3,di-O-acetyl-
6-thio-a-d-glucopyranosyl)-(1 ! 4)-1,2,3,6-tetra-O-acetyl-d-glucopyranose
(31): A mixture of bromo derivative 19 (472 mg, 0.37 mmol) and KI (95 mg,
1.5 equiv) in DMF (2.5 mL) was stirred at 708C for 2 h under argon. After
the mixture had been cooled to 08C, acetylated 1-thio-a-maltose 13
(550 mg, 7.9 mmol), 1,4-dithioerytritol (62 mg) and diethylamine (125 mL,
1.2 mmol) were added. After it had been stirred at room temperature for
12 h, the solution was concentrated, and the residue was acetylated
(pyridine/Ac2O, 17 mL, 10:7, v/v) in the presence of a catalytic amount of
DMAP at 708C for 2 h . Following incubation, the reaction mixture was
cooled (08C), and MeOH (7 mL) was added. The solution was concen-
trated, diluted with CH2Cl2 and washed with water, saturated aq. sodium
hydrogen carbonate and aq. KHSO4 (10%). The organic phase was dried
and concentrated under reduced pressure. Flash chromatography (1: Et2O,
2: Et2O/acetone 10:1, v/v) gave the expected compound 31 (490 mg, 72%).
1H NMR (400 MHz, CDCl3) see Supporting Information; 13C NMR
(100 MHz, CDCl3) d ˆ 170.67 168.48 (OCOCH3), 95.94/95.64 (C-1II,III,IV,-
VI), 91.22 (C-1Ib), 88.87 (C-1Ia), 83.29/83.20 (C-1Va,Vb), 75.34/74.76/72.85/
72.62/72.32/72.10/71.80/71.19/71.02/70.31/70.01/69.87/69.72/69.34/69.17/
68.63/68.42/67.93 (C-2I,II,III,IV,V,VI, C-3I,II,III,IV,V,VI, C-4I,II,III,IV,V,VI, C-5I,II,III,IV,V,VI),
62.68/61.35 (C-6I,III,IV,V,VI), 30.25/30.22 (C-6IIa,IIb), 20.86 20.54 (OCOCH3);
Methyl (a-d-glucopyranosyl)-(1 ! 4)-(a-d-glucopyranosyl)-(1 ! 4)-S-[a-
d-glucopyranosyl)-(1 ! 4)-(a-d-glucopyranosyl)-(1 ! 6)]-(6-thio-a-d-glu-
copyranosyl)-(1 ! 4)-(d-glucopyranosyl)-(1 ! 4)-a-d-glucopyranoside
(34): Compound 33 (20 mg, 0.94 mmol) was de-O-acetylated as already
described for the preparation of 32. The expected compound 34 was
obtained (11 mg, 99%). [a]2D5 ˆ ‡174 (c ˆ 0.15 in water); 13C NMR
(100 MHz, CDCl3) d ˆ 100.12/99.77/99.45 (C-1I,II,III,IV,V,VII), 85.65 (C-1VI),
80.4/77.37/77.22/77.01/74.27/73.86/73.65/73.37/73.21/73.06/72.08 -71.86/71.70/
71.57/71.39/71.25/71.15/70.51/70.40/69.65 (C-2I,II,III,IV,V,VI,VII, C-3I,II,III,IV,V,VI,VII
,
C-4I,II,III,IV,V,VI,VII
,
C-5I,II,III,IV,V,VI,VII), 61.16 60.81 (C-6I,II,IV,V,VI,VII), 55.42
(OCH3), 30.00 (C-6III); ES HRMS: calcd for C43H74O35S [M ‡Na]:
‡
‡
‡
1205.3629, found: 1205.3635; calcd for C43H74O35S [M ‡K]: 1227.3449,
found: 1227.3460.
Acknowledgements
This work was supported by the CNRS and the European Union 4th
Framework Program on Biotechnology (BIO4-CT98 0022).
‡
‡
ES HRMS: calcd for C76H102O50S [M ‡Na]: 1869.5057, found: 1869.5057.
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¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0823-5454 $ 20.00+.50/0
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Received: April 24, 2002 [F4040]
Chem. Eur. J. 2002, 8, No. 23
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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