Synthesis of methyl 4′-O-methyl-13C12-β-D- cellobioside from 13C6-D-glucose. Part 1: Reaction optimization and synthesis

Article abstract of DOI:10.1016/j.carres.2005.08.003

A high yielding synthetic route for methyl 4′-O-methyl-β-D- cellobioside starting from D-glucose was established. The reaction conditions optimized with nonlabeled materials were used for the synthesis of methyl 4′-O-methyl-¹³C₁₂-β-D

Full text of DOI:10.1016/j.carres.2005.08.003

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 2428–2435
Note
Synthesis of methyl 40-O-methyl-13C12-b-D-cellobioside from
13C6-D-glucose. Part 1: Reaction optimization and synthesis
a,
a
Yuko Yoneda,a Toshinari Kawada,b,* Thomas Rosenau and Paul Kosma
*
aDepartment of Chemistry and Christian–Doppler Laboratory, University of Natural Resources and
Applied Life Sciences Vienna (BOKU), Muthgasse 18, A-1190 Vienna, Austria
bKyoto Prefectural University, Graduate School of Agriculture, Sakyo-Ku, Shimogamo, Kyoto 606-8522, Japan
Received 20 July 2005; accepted 11 August 2005
Available online 8 September 2005
Abstract—A high yielding synthetic route for methyl 40-O-methyl-b-D-cellobioside starting from D-glucose was established. The
reaction conditions optimized with nonlabeled materials were used for the synthesis of methyl 40-O-methyl-13C12-b-D-cellobioside,
a compound having more than 99% 13C enrichment at each of the twelve pyranose carbon atoms. The labeled compound is required
to study the hydrogen bond network of cellodextrins and cellulose by CPMAS NMR experiments.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Disaccharide; Cellulose model compounds; Isotopic labeling; 13C enrichment
Methyl 40-O-methyl-b-D-cellobioside (12) is the first
cellodextrin model compound for cellulose which was
found to crystallize in two distinct phases.1 Both allo-
morphs were comprehensively characterized including
X-ray crystallography and 13C CPMAS NMR, which
indicated an unexpectedly high effect of the crystal pack-
ing on the chemical shifts.2 The availability of complete
solid-phase structural data of 12 prompted us toward
the synthesis of 13C-perlabeled material, which would
constitute an ideal probe for CPMAS studies of the
hydrogen bond network in cellodextrins and elaboration
of novel suitable CPMAS techniques. Due to the consid-
erable intensity gain in the 13C domain, optimization of
modern two- and multi-dimensional techniques,3 such
as the MAS-J-HMQC,4 and eventually resolution of
the H domain will become possible. With the hydrogen
bond pattern being known from X-ray studies, these
data can be correlated with the CPMAS results. Fur-
thermore, the problem of the assignment of the two sets
of six resonances to the two anhydroglucose units of 12,
which remained an open issue so far, can be solved.
CPMAS on the 13C-perlabeled 12 is expected to provide
novel insights into the dissolution mechanism of higher
cellodextrins in cellulose solvents, such as N-meth-
ylmorpholine-N-oxide or N,N-dimethylacetamide/LiCl,
and of the changes in the hydrogen bond network dur-
ing this process. This will also shed some new light on
the dissolution mechanism of cellulose, which is impor-
tant for a better understanding of technologies requiring
cellulose dissolution, for example, in cellulosic fiber
manufacturing, such as the viscose or Lyocell produc-
tion process.
Although a high-yield synthesis of 12 starting from D-
cellobiose has already been established,5 it could not be
used for the plotted synthesis of the 13C-perlabeled
derivative, as 13C6-D-glucose is the only appropriate,
commercially available starting material. Due to high
costs of this starting material and the requirement of
product yields in the multi-100 mg range the whole syn-
thetic scheme was supposed to be comprehensively opti-
mized with nonlabeled D-glucose, before entering ꢀhot
runsꢁ with perlabeled material. In the present first part
of this study, we wish to communicate an effective syn-
thetic route toward methyl 40-O-methyl-b-D-cellobioside
(12) starting from D-glucose and the synthesis of the
respective labeled compound, while the second will
*
Corresponding authors. Tel./fax: +81 75 703 5647 (T.K.); tel.: +43
1360066071; fax: +43 1360066059 (T.R.); e-mail addresses:
kawada@kpu.ac.jp; trosenau@edv2.boku.ac.at
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.08.003

Y. Yoneda et al. / Carbohydrate Research 340 (2005) 2428–2435
2429
OAc
OH
O
OAc
O
O
AcO
AcO
AcO
AcO
HO
HO
OH
OAc
AcO
OH
D-glucose
OAc
Br
1/1*
2/2*
PhCH(OMe) /
TsOH
OH
O
OAc
2
Ph
O
O
O
O
HO
HO
AcO
AcO
OMe
OMe
OMe
HO
OH
OAc
OH
5/5*
3/3*
4/4*
BnBr / NaH /
Et SiH /
TFA
3
OBn
O
+
-
Bu N
4
I
Ph
O
O
O
HO
BnO
OMe
BnO
OMe
OBn
6/6*
OBn
7/7*
Scheme 1. Synthesis of intermediate 7/7*.
Me-I /
NaH
OBn
OBn
O
O
HO
BnO
MeO
BnO
OMe
OMe
na
OBn
OBn
OBn
8/8*
OBn
O
7/7*
O
BnO
MeO
O
2M TfOH,
AcOH
OMe
OMe
BnO
BF3*Et2O,
-72˚C
OBn
OBn
11/11*
CCl3CN /
DBU
OBn
O
OBn
O
NH
nd
H2 /
Pd/C
MeO
BnO
MeO
BnO
OH
O
CCl3
OH
OH
OBn
OBn
O
HO
MeO
O
9/9*
10/10*
HO
O
OH
12/12*
OH
Scheme 2. Synthesis of the target compound 12/12*.
cover an in-depth solid state structural analysis of the
two allomorphs, placing emphasis on studies of the
hydrogen bond system by CPMAS techniques.
tion as to be expected in the case of acetyl protecting
groups.
Scheme 1 shows the synthetic route to intermediate 7,
starting from D-glucose. Methyl b-D-glucoside (4) was
prepared via glucose pentaacetate (1), acetobromoglu-
cose (2) and methyl 2,3,4,6-tetra-O-acetyl-b-D-glucoside
(3). Protection of all hydroxyl groups in 4 except 4-OH
was accomplished by a reaction sequence comprising the
introduction of a 4,6-O-benzylidene group giving 5, 2,3-
di-O-benzylation to afford compound 6, and regioselec-
tive opening of the benzylidene ring with triethylsilane
(TES)/trifluoroacetic acid (TFA) to obtain compound
7 in 27.2% overall yield relative to D-glucose.
Intermediate 7 can be directly used as the glycosyl
acceptor. Preparation of the glycosyl donor—starting
from 7 as well—required three additional steps, as shown
in Scheme 2. Methylation of 4-OH gave compound 8,
and hydrolysis of the 1-OMe group was achieved by acid
treatment. In the case of trichloroacetic acid as the
catalyst, no reaction was observed. Trifluoroacetic acid
Dictated by the requirement of optimum yields of 12/
12*ꢂ—not necessarily implying to take to shortest syn-
thetic route—the path shown in Schemes 1 and 2 was
chosen. From retrosynthetic analysis, methyl 40-O-
methyl-b-D-cellobioside (12) was to be synthesized
through a glycosidation reaction between a methyl
2,3,6-tri-O-protected b-D-glucoside and 4-O-methyl-
2,3,6-tri-O-protected b-D-glucosyl donor. Methyl 2,3,6-
tri-O-benzyl-b-D-glucoside (7) was selected as the
common intermediate, which could be used directly as
a glycosyl acceptor and as precursor of glucosyl imidate
10. Benzyl group protection was chosen to avoid migra-
ꢂ Here and in the following the nonlabeled compounds are given in
bold numbers, while isotopic labeling is indicated with an asterisk
following the compound number.

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Y. Yoneda et al. / Carbohydrate Research 340 (2005) 2428–2435
(TFA)6 and/or trifluoromethanesulfonic acid (TfOH)7
gave much better results, although a minor side reaction
was noted, possibly a hydrolytic cleavage of the 6-O-ben-
zyl group. Finally, 2 M triflic acid was chosen as the
catalyst. The main product of the hydrolysis, 2,3,6-
tri-O-benzyl-4-O-methyl-a/b-D-glucose (9), underwent
trichloroacetimidoylation to a mixture of a- and b-imi-
dates 10 in an approximate ratio of 5:1 as estimated by
TLC and NMR spectroscopy.
For the glycosidation of benzyl protected trichloro-
acetimidate 10 no neighbour group participation would
be expected, as in the case of acyl protection. Thus, only
the a-imidate should be used to construct a b-glycosidic
linkage, assuming that the reaction proceeds according
to a SN2-type mechanism. However, preliminary exper-
iments with neat b-imidate indicated also the b-glycoside
was produced to some part. We, thus, decided to use the
mixture of both imidates in the glycosidation reaction.
In addition, imidate mixture 10 proved to be too reac-
tive to withstand purification by flash chromatography
on silica gel without significant yield penalties. There-
fore, work-up of the imidoylation reaction was done
by rapid filtration through a short alumina column to
remove the diazabicyclo[5.4.0]undec-7-ene (DBU) cata-
lyst. Evaporation of the solvents afforded a mixture of
the anomeric imidates in 92% yield, which was directly
used in the glycosylation step. Purification and separa-
tion of the a/b-glycosides was performed afterwards,
to afford 63% of the desired b-isomer 11.
The overall yield of imidate mixture 10 from the inter-
mediate 7 was 64%. As the common intermediate 7 is
split into glycosyl acceptor and glycosyl donor, 61% of
7 are to be used for the preparation of imidate 10, while
the remaining 39% of 7 are taken directly as the glycosyl
acceptor, provided 7 and 10 are to be applied in an equi-
molar ratio (see Table 1).
The glycosylation reaction was performed at ꢀ72 ꢁC
in CH2Cl2 with BF3 etherate as the catalyst. Aceto-
nitrile, which possibly would have had a beneficial nitri-
lium effect on the yield, was not tested as the solvent.
The initial molar ratio of imidate 10 and glycosyl accep-
tor 7 (nd/na) affected the overall yield of cellobiose deriv-
ative 11 relative to the common intermediate 7 in a
rather complex manner (Table 1). The first trial was con-
ducted with a molar ratio (nd/na) of 1:1. The yield of 11
was 40.4% after purification and separation from the
corresponding maltose derivative, while 25% of acceptor
7 remained as nonreacted starting material. The overall
yield of cellobiose derivative 11 relative to compound 7
was then 40.4% · 0.39 = 15.8%. In order to consume
acceptor 7 completely, nd/na was set to 1.33:1. As a
result, the yield of 11 in the glycosylation increased to
63.2%, and the overall yield to 20.9%, superior to that
of the first trial. However, even in the case of excess
donor, still 9% of the starting compound 7 remained
unused. The third run was conducted with an nd/na of
1.33/(1–0.09). This gave a better glycosylation yield
(66.1%) than that of the second trial, but a less optimal
overall yield (19.8%) due to the smaller na (0.30). In con-
clusion, an initial molar ratio nd/na of 1.33:1 proved to
be the best variant among those examined and was used
further. The fully benzyl-protected cellobiose derivative
11 was finally hydrogenated with 10% Pd/C as the cata-
lyst to afford the target compound methyl 40-O-methyl-
b-D-cellobioside (12) in 90.9% yield.
Having optimized the procedure, the reaction se-
quence was repeated step-by-step in parallel with nonla-
beled and labeled materials. The yields for both runs
were generally equal or close to equal. The total yield
of 12 (12*) starting from D-glucose (13C6-D-glucose)
was 4.84%, with 5 g of 13C6-D-glucose affording
13
250 mg of C-perlabeled methyl 40-O-methyl-13C12-b-
D-cellobioside: sufficient amounts for the planned ana-
lytical studies.
The signal pattern in the 13C soln NMR spectrum of
12* is rather complex due to primary signal splitting
1
by homonuclear 13C–13C J-couplings, and additional
splitting by geminal and vicinal 13C–13C couplings. Fig-
1
ure 1 displays the H-decoupled spectrum which shows
four distinct regions of resonances: the anomeric car-
bons, the C-4 carbons, the C-6 carbons, and the complex
region of C-2, C-3, and C-5.
For comparison, the solid-state NMR spectrum of
12* is given in Figure 2, the trace shown being the result
of a single scan—which illustrates the intensity gain
achieved by the isotopic enrichment. The spectrum
displayed is that of a microcrystalline mixture of both
allomorphs, and is thus slightly different from the
Table 1. Optimization of educt ratios and yields for the glycosylation reaction
Yield for glycosylationb (%)
Nonreacted 7
Yield of 11 from 7
a
Molar ratio nd/na
na
ac
bd
a + b
1/1 = 1
1.33/1 = 1.33
1.33/0.91 = 1.46
0.39*n0
0.33*n0
0.30*n0
11.6
19.9
24.0
40.4
63.2
66.1
52.0
83.1
90.1
25
9
Trace
15.8
20.9
19.8
a n0 starting yield (D-glucose).
b Calculated based on na.
c Maltose derivative.
d Cellobiose derivative 11.

Y. Yoneda et al. / Carbohydrate Research 340 (2005) 2428–2435
2431
and the corresponding experimental procedures are
not listed in the following. All intermediates and the
product showed satisfying purity data (elemental analy-
sis), which in some cases were sustained by HRMS data.
1.1. Methyl 4,6-O-benzylidene-b-D-glucopyranoside (5)
To a soln of methyl b-D-glucopyranoside (4, 3.57 g,
18.4 mmol) in 1,4-dioxane (80 mL) were added benzal-
dehyde dimethyl acetal (3.35 mL, 22.1 mmol) and p-tol-
uenesulfonic acid monohydrate (35.0 mg, 0.184 mmol)
at rt. The soln was stirred under diminished pressure
(4 kPa) for 1 h at 30 ꢁC. A second portion of benzalde-
hyde dimethyl acetal (2.80 mL, 18.4 mmol) was added,
and stirring was continued for 2 h. The reaction mixture
was neutralized with solid NaHCO3. Solids were filtered
off and the filtrate was evaporated to dryness. The fil-
trate was purified by flash column chromatography
(1:19 MeOH–CH2Cl2) to give a colorless syrup which
spontaneously crystallized to afford 5 (4.12 g, 79.3%)
as colorless crystals: Rf 0.74 (1:9 MeOH–CH2Cl2); mp
171–173 ꢁC [lit. 174–175 ꢁC];8 [a]D ꢀ61.4 (c 0.8, CHCl3)
[lit. [a]D ꢀ53.3 (c 0.5, CHCl3)];8 1H NMR: d 2.70 (d,
J2,2-OH 2.3 Hz, 1H, 2-OH), 2.85 (d, J3,3-OH 2.5 Hz, 1H,
3-OH), 3.49 (dt, J5,4 = J5,6a = 9.2 Hz, J5,6b 5.1 Hz, 1H,
H-5), 3.51 (ddd, J1,2 7.6 Hz, J2,3 9.2 Hz, J2,2-OH 2.3 Hz,
1H, H-2), 3.56 (t, J3,4 = J4,5 = 9.2 Hz, 1H, H-4), 3.59
(s, 3H, 1-OMe), 3.80 (t, J5,6a = J6a,6b = 9.2 Hz, 1H, H-
6a), 3.83 (dt, J2,3 = J3,4 = 9.2 Hz, J3,3-OH 2.3 Hz, 1H,
H-3), 4.34 (d, J1,2 7.6 Hz, 1H, H-1), 4.37 (dd, J5,6b
5.1 Hz, J6a,6b 9.2 Hz, 1H, H-6b), 5.55 (s, 1H, CHPh),
7.36–7.40 (m, 3H, Ph), 7.48–7.51 (m, 2H, Ph); 13C
NMR: d 57.54 (1-OMe), 66.39 (C-5), 68.66 (C-6),
73.18 (C-3), 74.57 (C-2), 80.58 (C-4), 101.93 (CHPh),
104.13 (C-1), 126.25, 128.35, 129.30, 136.90 (Ph). Anal.
Calcd for C14H18O6: C, 59.57; H, 6.43. Found: C, 59.62;
H, 6.55. The 1H and 13C NMR data agree well with one
literature report,8 but disagree with another.9
Figure 1. Proton-decoupled 13C NMR solution spectrum of 12*,
having >99% 13C enrichment at the 12 cellobiose carbon atoms.
110
100
90
80
70
60
50
Figure 2. Proton-decoupled 13C NMR CPMAS solid-state spectrum
of 12*, having >99% 13C enrichment at the 12 cellobiose carbon atoms.
spectra obtained for the two allomorphs of 12. For the
CPMAS and solid state structural studies, which will
be reported in part 2 of this work, this sample as well
as both neat, crystalline phases of 12* will be employed.
1.2. Methyl 4,6-O-benzylidene-13C6-b-D-glucopyranoside
(5*)
Yield: 3.238 g (11.23 mmol, 63%) from 4* (3.563 g,
17.70 mmol); mp 172–177 ꢁC; [a]D ꢀ62.4 (c 0.5, CHCl3).
Anal. Calcd for C813C6H18O6 (288.25): C, 58.35; H,
6.30. Found: C, 58.30; H, 6.38.
1. Experimental
Melting points (mp) are uncorrected. TLC was per-
formed on silica gel plates (Kieselgel 60 F254, E. Merck),
flash column chromatography on silica gel (Wakogel
FC-40). 1H and 13C NMR spectra were recorded in
CDCl3 if not otherwise stated, on a JNM-500 FT-
1.3. Methyl 2,3-di-O-benzyl-4,6-O-benzylidene-b-D-
glucopyranoside (6)
1
NMR (Jeol) at 500 MHz for H and 125 MHz for 13C,
NaH (2.27 g, 56.6 mmol, as 60% dispersion in mineral
oil), tetra-n-butylammonium iodide (262 mg, 0.708
mmol) and benzyl bromide (4.04 mL, 34.0 mmol) were
added to a soln of 5 (4.00 g, 14.2 mmol) in anhyd THF
(150 mL) at 0 ꢁC. The soln was refluxed overnight. After
the reaction mixture was cooled to rt, excess NaH was
with tetramethylsilane (TMS) as the internal standard.
Chemical shifts are given in ppm, coupling constants
(J) in hertz. The signals were assigned by homo- and het-
eronuclear two-dimensional techniques. Compounds 1–
4 were synthesized according to standard techniques,

2432
Y. Yoneda et al. / Carbohydrate Research 340 (2005) 2428–2435
quenched with MeOH. The reaction mixture was diluted
with EtOAc (approx. 200 mL), washed neutral with
water, washed with brine, dried over Na2SO4, and evap-
orated to dryness. The residue was purified by flash
column chromatography (n-hexane, then 1:49 EtOAc–
toluene) to give a colorless syrup. The syrup was crystal-
lized from EtOH/n-hexane to afford 6 (4.21 g, 64.1%) as
colorless crystals: Rf 0.45 (1:4 EtOAc–n-hexane); mp
119–120 ꢁC [lit. 119–120 ꢁC,10 mp 119–119 ꢁC11]; [a]D
ꢀ35.0 (c 1.0, CHCl3) [lit. [a]D ꢀ35.8 (c 3, CHCl3),10
to give a colorless syrup, which was crystallized from
EtOH/n-hexane to afford 7 (3.32 g, 80.7%) as colorless
crystals: Rf 0.49 (1:2 EtOAc–n-hexane); mp 71–73 ꢁC
[lit. 64–65 ꢁC];14 [a]D ꢀ12.6 (c 1.0, CHCl3) [lit. [a]D
1
ꢀ17 (c 1.0, CHCl3),14 [a]D ꢀ9 (c 1.05, CHCl3)15]; H
NMR: d 2.61 (d, J4,4-OH 2.3 Hz, 1H, 4-OH), 3.40 (dd,
J1,2 7.6 Hz, J2,3 8.9 Hz, 1H, H-2), 3.44 (ddd, J4,5
8.9 Hz, J5,6a 5.3 Hz, J5,6b 3.9 Hz, 1H, H-5), 3,45 (t,
J2,3 = J3,4 = 8.9 Hz, 1H, H-3), 3.57 (s, 3H, 1-O Me),
3.58 (dt, J3,4 = J4,5 = 8.9 Hz, J4,4-OH 2.3 Hz, 1H, H-4),
3.70 (dd, J5,6a 5.3 Hz, J6a,6b 10.6 Hz, 1H, H-6a), 3.77
(dd, J5,6b 3.9 Hz, J6a,6b 10.6 Hz, 1H, H-6b), 4.32 (d,
J1,2 = 7.6 Hz, 1H, H-1), 4.56, 4.60, 4.70, 4.72, 4.92,
4.93 (six d, 6H, CH2Ph), 7.26–7.37 (m, 15H, Ph); 13C
NMR: d 57.07 (1-OMe), 70.14 (C-6), 71.40 (C-4),
73.59 (CH2Ph), 73.98 (C-5), 74.60, 75.20 (2 · CH2Ph),
81.69 (C-2), 83.89 (C-3), 104.67 (C-1), 127.64–128.47,
137.84, 138.38, 138.51 (Ph). Anal. Calcd for C28H32O6:
C, 72.39; H, 6.94. Found: C, 72.36; H, 7.06. NMR data
agreed with the literature references, which give either
only 13C data,16 or only 13C data without assignment,14
or a differing carbon signal assignment.15
1
[a]D ꢀ37 (c 1.4, CHCl3)11]; H NMR: d 3.41 (br dt,
J4,5 9.7 Hz, J5,6a 10.1 Hz, J5,6b 5.0 Hz, 1H, H-5), 3.45
(br t, J1,2 7.6 Hz, J2,3 9.2 Hz, 1H, H-2), 3.58 (s, 3H, 1-
OMe), 3.68 (br t, J3,4 9.2 Hz, J4,5 9.7 Hz, 1H, H-4),
3.75 (t, J2,3 = J3,4 = 9.2 Hz, 1H, H-3), 3,79 (br t, J5,6a
10.1 Hz, J6a,6b 10.3 Hz, 1H, H-6a), 4.37 (dd, J5,6b
5.0 Hz, J6a,6b 10.3 Hz, 1H, H-6b), 4.42 (d, J1,2 7.6 Hz,
1H, H-1), 4.76, 4.80, 4.87, 4.91 (four d, 4H, CH2Ph),
5.57 (s, 1H, CHPh), 7.23–7.50 (m, 15H, Ph); 13C
NMR: d 57.44 (1-OMe), 65.90 (C-5), 68.74 (C-6),
75.07, 75.23 (CH2Ph), 80.72 (C-3), 81.43 (C-4), 82.11
(C-2), 101.06 (CHPh), 105.16 (C-1), 125.95–128.90,
137.25, 138.32, 138.41 (Ph). Anal. Calcd for C28H30O6:
1
C, 72.81; H, 6.54. Found: C, 72.69; H, 6.65. H NMR
1.6. Methyl 2,3,6-tri-O-benzyl-13C6-b-D-glucopyranoside
(7*)
data agree with the literature, which either presented
only data for H-4, H-5, and H-6,12 or gave no signal
assignment.13
Yield: 1.882 g (4.00 mmol, 87%) from 6* (2.192 g,
4.62 mmol); mp 70–71 ꢁC; [a]D ꢀ19.5 (c 0.2, CHCl3).
Anal. Calcd for C2213C6H32O6 (470.51): C, 71.51; H,
6.86. Found: C, 71.52; H, 6.87. HRMS: Calcd for
[C2213C6H32O6 + Na+]: 493.2298. Found: 493.2512
(DM = 0.0214 Da).
1.4. Methyl 2,3-di-O-benzyl-4,6-O-benzylidene-13C6-b-D-
glucopyranoside (6*)
Yield: 2.247 g (4.80 mmol, 44%) from 5* (3.179 g,
11.03 mmol); mp 121–122 ꢁC; [a]D ꢀ33.1 (c 0.7, CHCl3).
Anal. Calcd for C2213C6H30O6 (468.49 g molꢀ1): C,
71.82; H, 6.46. Found: C, 71.77; H, 6.56. HRMS: Calcd
for [C2213C6H30O6 + Na+]: 491.2141 g molꢀ1. Found:
491.2291 g molꢀ1 (DM = 0.0150 Da).
1.7. Methyl 2,3,6-tri-O-benzyl-4-O-methyl-b-D-gluco-
pyranoside (8)
NaH (380 mg, 9.47 mmol, as 60% dispersion in mineral
oil) was added to a soln of 7 (2.20 g, 4.74 mmol) in
anhyd THF (30 mL) at rt, and the soln was stirred for
30 min. Methyl iodide (590 lL, 9.47 mmol) was added
to this soln, which was further stirred at rt for 2 h. After
quenching excess NaH with MeOH, the reaction mix-
ture was diluted with EtOAc (approx. 100 mL), neutral-
ized with water, washed with brine, dried over Na2SO4,
and evaporated to dryness. The residue was purified by
flash column chromatography (1:19 EtOAc–toluene) to
give 8 (2.24 g, 98.9%) as a colorless syrup: Rf 0.56 (1:4
EtOAc–n-hexane); [a]D +14.0 (c 1.0, CHCl3); 1H
NMR: d 3.29 (dd, J3,4 9.2 Hz, J4,5 9.6 Hz, 1H, H-4),
3.37 (ddd, J4,5 9.6 Hz, J5,6a 4.9 Hz, J5,6b 1.9 Hz, 1H,
H-5), 3.39 (dd, J1,2 7.8 Hz, J2,3 9.2 Hz, 1H, H-2), 3.48
(s, 3H, 4-OMe), 3.53 (t, J2,3 = J3,4 = 9.2 Hz, 1H, H-3),
3.57 (s, 3H, 1-OMe), 3.69 (dd, J5,6a 4.9 Hz, J6a,6b
10.8 Hz, 1H, H-6a), 3.76 (dd, J5,6b 1.9 Hz, J6a,6b 10.8
Hz, 1H, H-6b), 4.28 (d, J1.2 7.8 Hz, 1H, H-1), 4.57,
1.5. Methyl 2,3,6-tri-O-benzyl-b-D-glucopyranoside (7)
Triethylsilane (1.69 mL, 10.6 mM) and trifluoroacetic
acid (0.82 mL, 10.6 mmol) were added to a soln of 6
(4.10 g, 8.86 mmol) in anhyd CH2Cl2 (100 mL) at 0 ꢁC.
The soln was stirred at rt for 2 h. Another charge of tri-
ethylsilane (1.42 mL, 8.86 mmol) and trifluoroacetic
acid (0.68 mL, 8.86 mmol) was added at 0 ꢁC, and the
stirring at rt was continued for 2 h. A third portion of
triethylsilane (1.42 mL, 8.86 mmol) and trifluoroacetic
acid (0.68 mL, 8.86 mmol) was added again at 0 ꢁC,
and the reaction mixture stirred at rt for 2 h. The reac-
tion mixture was diluted with EtOAc (approx.
200 mL), neutralized with saturated aq NaHCO3,
washed with 0.05 M aq HCl, neutralized with saturated
aq NaHCO3, washed with brine, dried over Na2SO4,
and evaporated to dryness. The residue was purified
by flash column chromatography (1:19 EtOAc–toluene)

Y. Yoneda et al. / Carbohydrate Research 340 (2005) 2428–2435
2433
4.65, 4.69, 4.77, 4.88, 4.90 (6 d, 6H, 3CH2Ph), 7.24–7.36
(m, 15H, 3Ph); 13C NMR: d 57.05 (1-OMe), 60.65 (4-
OMe), 69.03 (C-6), 73.47, 74.70, 75.53 (CH2Ph), 74.84
(C-5), 79.78 (C-4), 82.10 (C-2), 84.52 (C-3), 104.63 (C-
1), 127.56–128.29, 138.22, 138.54, 138.62 (Ph). Anal.
Calcd for C29H34O6: C, 72.78; H, 7.16. Found: C,
72.61; H, 7.13. The NMR data agree with those avail-
able for the 2,3-p-methoxybenzylated derivative.17
91.22 (C-1a), 97.36 (C-1b), 127.83–128.34, 134.77–
138.66 (Ph). Anal. Calcd for C28H32O6: C, 72.39; H,
6.94. Found: C, 72.39; H, 7.08. The 1H NMR data agree
with those of an anomeric mixture (a/b = 1:0.6).18
1.10. 2,3,6-Tri-O-benzyl-4-O-methyl-13C6-a,b-D-gluco-
pyranose (9*)
Yield: 0.920 g (1.96 mmol, 78%) from 8* (1.217 g,
2.51 mmol), mp 94–97 ꢁC, [a]D +36.4 (c 0.5, CHCl3).
Anal. Calcd for C2213C6H32O6 (470.51): C, 71.51; H,
6.86. Found: C, 71.44; H, 6.96. HRMS: calcd for
[C2213C6H32O6 + Na+]: 493.2298 g. Found: 493.2133
g molꢀ1 (DM = ꢀ0.0165 Da).
1.8. Methyl 2,3,6-tri-O-benzyl-4-O-methyl-13C6-b-D-
glucopyranoside (8*)
Yield: 1.245 g (2.57 mmol, 98%) from 7* (1.237 g,
2.63 mmol); [a]D +14.0 (c 1.0, CHCl3). Elemental analy-
sis not available. HRMS: Calcd for [C2313C6H34O6 +
Na+]: 507.2454 gmolꢀ1
.
Found: 507.2343 g molꢀ1
1.11. 2,3,6-Tri-O-benzyl-4-O-methyl-a-D-glucopyranosyl
trichloroacetimidate (10a/10a*) and 2,3,6-tri-O-benzyl-4-
O-methyl-b-D-glucopyranosyl trichloroacetimidate (10b/
10b*)
(DM = ꢀ0.0111 Da).
1.9. 2,3,6-Tri-O-benzyl-4-O-methyl-a,b-D-glucopyranose
(9)
To a soln of 9 (1.55 g, 3.34 mmol) in CH2Cl2 (20 mL)
DBU (200 lL, 1.33 mmol) was added under stirring
for 30 min at rt. Then, trichloroacetonitrile (1.34 mL,
13.3 mmol) was added, and the soln was stirred at ambi-
ent temperature for 2 h. The reaction mixture was di-
luted with n-hexane (5 mL), and the residue was
filtered using a column of alumina (B grade, activity 1,
by ICN Biomedicals GmbH, approx. 30 g) to remove
DBU. The filtrate was evaporated to give the mixture
of 10a and 10b (1.63 g, 80.2% recovery) as a pale yellow
syrup, which was directly used for the next reaction. For
analysis, the residue was purified by flash column chro-
matography (1:19 EtOAc–toluene containing 1% (v/v)
of triethylamine) to separate 10a and 10b(a/b = 10/1,
w/w).
To a soln of 8 (2.15 g, 4.49 mmol) in acetic acid (45 mL)
was added a 2 M soln of TfOH in water (9.0 mL). The
soln was heated to 80 ꢁC and then stirred for 3 h. The
reaction mixture was diluted with CH2Cl2 (approx.
200 mL), neutralized with saturated aq NaHCO3,
washed with brine, dried over Na2SO4, and evaporated
to dryness. The residue was purified by flash column
chromatography (1:6 EtOAc–toluene) to give a colorless
syrup, which was crystallized from EtOH/n-hexane to
afford 9 (1.68 g, 80.6%) as colorless crystals: Rf 0.33
and 0.38 (1:2 EtOAc/n-hexane); mp 96–98 ꢁC; [a]D
1
+38.1 (c 1.0, CHCl3); H NMR: d 1.75 (br s, 0.65H,
1a-OH), 3.25 (t, J3b,4b = J4b,5b = 9.4 Hz, 0.35H, H-4b),
3.31 (t, J3a,4a = J4a,5a = 9.3 Hz, 0.65H, H-4a), 3.35 (t,
J
1b,2b = J2b,3b = 8.0 Hz, 0.35H, H-2b), 3.36 (br s,
2,3,6-Tri-O-benzyl-4-O-methyl-a-D-glucopyranosyl tri-
chloroacetimidate (10a); Rf 0.44 (1:4 EtOAc–n-hexane);
1H NMR: d 3.49 (s, 3H, 4-OMe), 3.50 (t, J3,4 9.4 Hz, J4,5
9.6 Hz, 1H, H-4), 3.66 (dd, J5,6a 1.9 Hz, J6a,6b 10.9 Hz,
1H, H-6a), 3.70 (dd, J5,6b 3.5 Hz, J6a,6b 10.9 Hz, 1H,
H-6b), 3.73 (dd, J1,2 3.3 Hz, J2,3 9.4 Hz, 1H, H-2), 3.89
(ddd, J5,6a 1.9 Hz, J5,6b 3.5 Hz, J4,5 10.9 Hz, 1H, H-5),
3.95 (t, J2,3 = J3,4 = 9.4 Hz, 1H, H-3), 4.49, 4.60, 4.66,
4.72, 4.81, 4.92 (6d, 6H, 3 CH2Ph), 6.48 (d, J1,2
3.3 Hz, 1H, H-1), 8.57 (s, 1H, NH).
2,3,6-Tri-O-benzyl-4-O-methyl-b-D-glucopyranosyl tri-
chloroacetimidate (10b); Rf 0.31 (1:4 EtOAc–n-hexane);
1H NMR: d 3.45 (dd, J3,4 8.8 Hz, J4,5 9.67 Hz, 1H, H-4),
3.50 (s, 3H, 4-OMe), 3.54 (ddd, J5,6a 2.1 Hz, J5,6b 3.9 Hz,
J4,5 9.7 Hz, 1H, H-5), 3.95 (t, J2,3 = J3,4 = 8.8 Hz, 1H,
H-3), 3.73 (dd, J1,2 7.8 Hz, J2,3 8.8 Hz, 1H, H-2), 3.72
(dd, J5,6a 3.9 Hz, J6a,6b 11.2 Hz, 1H, H-6a), 3.76 (dd,
J5,6b 2.1 Hz, J6a,6b 11.2 Hz, 1H, H-6b), 4.56, 4.65, 4.74,
4.81, 4.87, 4.92 (6d, 6H, 3CH2Ph), 5.72 (d, J1,2 7.8 Hz,
1H, H-1), 8.69 (s, 1H, NH).
0.35H, 1b-OH), 3.42 (ddd, J5b,6bb 0.7 Hz, J5b,6ab
5.3 Hz, J4b,5b 9.4 Hz, 0.35H, H-5b), 3.46 (s, 1.05H,
4-OMeb), 3.47 (s, 1.95H, 4-OMea), 3.52 (dd, J1a,2a
3.2 Hz, J2a,3a 9.3 Hz, 0.65H, H-2a), 3.53 (dd, J2b,3b
8.0 Hz, J3b,4b 9.4 Hz, 0.35H, H-3b), 3.62 (dd, J5b,6ab
5.3 Hz, J6ab,6bb 10.6 Hz, 0.35H, H-6ab), 3.64 (dd,
J5a,6aa 0.7 Hz, J6aa,6ba 10.5 Hz, 0.65H, H-6aa), 3.67
(dd, J5a,6ba 3.8 Hz, J6aa,6ba 10.5 Hz, 0.65H, H-6ba),
3.71 (dd, J5b,6bb 0.7 Hz, J6ab,6bb 10.6 Hz, 0.35H, H-
6bb), 3.87 (t, J2a,3a = J3a,4a = 9.3 Hz, 0.65H, H-3a),
3.94 (ddd, J5a,6aa 0.7 Hz, J5a,6ba 3.8 Hz, J4a,5a 9.3 Hz,
0.65H, H-5a), 4.50, 4.55, 4.611, 4.614, 4.67, 4.74, 4.75,
4.78, 4.82, 4.88, 4.91, 4.93 (12d, 12H, CH2Ph), 4.60 (d,
J1b,2b 8.0 Hz, 0.35H, H-1b), 5.19 (d, J1a,2a 3.2 Hz,
0.65H, H-1a), 7.24–7.38 (m, 15H, Ph); 13C NMR: d
58.77 (4-OMeb), 60.64 (4-OMea), 68.64 (C-6a), 69.03
(C-6b), 70.19 (C-5a), 73.19, 73.43, 73.50, 74.70, 75.58
(CH2Ph), 74.63 (C-5b), 79.50 (C-4a), 79.69 (C-2a),
79.80 (C-4b), 81.61 (C-3a), 82.87 (C-2b), 84.39 (C-3b),

2434
Y. Yoneda et al. / Carbohydrate Research 340 (2005) 2428–2435
1.12. Methyl 2,3,6-tri-O-benzyl-4-O-methyl-b-D-gluco-
pyranosyl-(1!4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside
(11)
MeOH–CH2Cl2); mp 204–210 ꢁC [lit. 196–198 ꢁC5];
[a]D ꢀ16.4 (c 1.0, water) [lit. [a]D ꢀ11 (c 0.4, water)5];
1H NMR (D2O): d 3.39 (t, J3 ;4 ¼ J4 ;5 ¼ 9:4 Hz, 1H,
H-40), 3.47 (broad dd, J1,2 8.0 Hz, J2,3 8.9 Hz, 1H, H-
0
0
0
0
2), 3.49 (dd, J1 ;2 8:0 Hz; J2 ;3 9:4 Hz, 1H, H-20), 3.65
0
0
0
0
To a soln of 7 (935 mg, 2.01 mmol) and 10 (1.63 g,
2.68 mmol) in anhyd CH2Cl2 (50 mL) was added BF3ÆE-
t2O (34 lL, 0.27 mmol) at ꢀ72 ꢁC. After the soln was
stirred at ꢀ72 ꢁC for 1 h, the reaction mixture was
neutralized with triethylamine. The residue was purified
by flash column chromatography (1:9 EtOAc–toluene)
to give 11 (1.16 g, 63.1%) as a colorless syrup: Rf
0.32 (1:9 EtOAc–toluene); [a]D +24.5 (c 1.0, CHCl3)
[lit. [a]D +23 (c 0.5, CHCl3)5]; 1H NMR: d 3.18
0
0
0
0
0
0
(ddd, J5 ;6 b 2:3 Hz; J5 ;6 a 5:5 Hz; J4 ;5 9:4 Hz, 1H,
H-50), 3.72 (s, 3H, 40-OMe), 3.74 (s, 3H, 1-OMe),
3.74–3.80 (m, 4H, H-30, H-4, H-3, H-5), 3.91 (dd,
J5 ;6 a 5:5 Hz; J6 a;6 b 12:4 Hz, 1H, H-60a), 3.97 (dd,
0
0
0
0
0
0
J5,6a 4.8 Hz, J6a,6b 12.5 Hz, 1H, H-6a), 4.07 (dd, J5 ;6 b
2:3 Hz; J6 a;6 b 12:4 Hz, 1H, H-60b), 4.16 (dd, J5,6b
2.0 Hz, J6a,6b 12.5 Hz, 1H, H-6b), 4.56 (d, J1,2 8.0 Hz,
0
0
1H, H-1), 4.64 (d, J1 ;2 8:0 Hz, 1H, H-10), 4.80 (br s,
6H, OH); 13C NMR (D2O): d 57.54 (1-OMe), 60.30
(40-OMe), 60.45 (C-6), 60.73 (C-60), 73.22 (C-2), 73.55
(C-20), 74.69 (C-3), 75.10 (C-50), 75.35 (C-5), 75.51 (C-
30), 79.08 (C-4), 79.49 (C-40), 102.80 (C-10), 103.42 (C-
1). Anal. Calcd for C14H26O11: C, 45.40; H, 7.08.
Found: C, 45.35; H, 7.11. The data are consistent with
those previously published.5
0
0
0
0
0
0
0
0
(dd, J5 ;6 b 1:6 Hz; J5 ;6 a 4:6 Hz; J4 ;5 9:3 Hz, 1H, H-
50), 3.320 (dd, J1 ;2 7:6 Hz; J2 ;3 9:3 Hz, 1H, H-20),
0
0
0
0
3.324 (t, J3 ;4 ¼ J4 ;5 ¼ 9:3 Hz, 1H, H-40), 3.35 (ddd,
J5,6a 1.3 Hz, J5,6b 4.1 Hz, J4,5 9.6 Hz, 1H, H-5), 3.39
(dd, J1,2 7.7 Hz, J2,3 9.0 Hz, 1H, H-2), 3.41 (t,
0
0
0
0
J2 ;3 ¼ J3 ;4 ¼ 9:3 Hz, 1H, H-30), 3.47 (s, 3H, 40-OMe),
0
0
0
0
3.52 (dd, J5 ;6 a 4:6 Hz; J6 a;6 b 11:0 Hz, H-60a), 3.55 (s,
3H, 1-OMe), 3.57 (dd, J2,3 9.0 Hz, J3,4 9.2 Hz, 1H, H-
0
0
0
0
3), 3.67 (dd, J5 ;6 b 1:6 Hz; J6 a;6 b 11:0 Hz, 1H, H-60b),
3.71 (dd, J5,6a 1.3 Hz, J6a,6b 11.0 Hz, 1H, H-6a), 3.82
(dd, J5,6b 4.1 Hz, J6a,6b 11.0 Hz, 1H, H-6b), 4.00 (dd,
J3,4 9.2 Hz, J4,5 9.6 Hz, 1H, H-4), 4.28 (d, J1,2 7.7 Hz,
1H, H-1), 4.40, 4.42, 4.43, 4.58, 4.68, 4.72, 4.73, 4.77,
4.78, 4.83, 4.83, 5.05 (12d, 12H, 6CH2Ph), 4.46 (d,
0
0
0
0
1.15. Methyl 4-O-methyl-13C6-b-D-glucopyranosyl-
(1!4)-13C6-b-D-glucopyranoside (12*)
Yield: 0.250 g (0.645 mmol, 64%) from 11* (0.779 g,
0.844 mmol) 77%, mp 197–203 ꢁC, [a]D ꢀ15.5 (c 0.66,
13
water). Anal. Calcd for C2 C12H26O11 (382.26): C,
J1 ;2 7:6 Hz, 1H, H-10), 7.17–7.35 (m, 30H, 6Ph); 13C
NMR: d 57.00 (1-OMe), 60.54 (40-OMe), 68.10 (C-6),
68.88 (C-60), 73.21 (CHPh), 74.86, 74.95, 74.96, 75.50
(C-5, C-50, CHPh), 76.56 (C-4), 79.86 (C-40), 81.66 (C-
2), 82.49 (C-20), 82.72 (C-3), 84.77 (C-30), 102.59 (C-
10), 104.59 (C-1), 127.43–128.31, 138.13, 138.45,
138.47, 138.58, 138.59, 139.24 (Ph). Elemental analysis
not performed. 1H NMR data agreed with the literature.5
0
0
44.00; H, 6.86. Found: C 44.10; H 6.74. HRMS: Calcd
13
for [C2 C12H26O11 + Na+]: 405.1775. Found: 405.1625
(DM = ꢀ0.0150 Da).
Acknowledgements
The authors thank Dr. Andreas Hofinger and Dr. Dan-
iel Kolarich, both of the Department of Chemistry at
the University of Natural Resources and Applied Life
Sciences (BOKU), for recording the NMR spectra and
high-resolution MS spectra, respectively. The financial
support of the Fonds zur Fo¨rderung der wissenschaftli-
chen Forschung (FWF), project P-17426-N11 is grate-
fully acknowledged.
1.13. Methyl 2,3,6-tri-O-benzyl-4-O-methyl-13C6-b-D-
glucopyranosyl-(1!4)-2,3,6-tri-O-benzyl-13C6-b-D-gluco-
pyranoside (11*)
Yield: 0.781 g (0.846 mmol, 64%) from 7* (0.623 g,
1.324 mmol) and 10* (1.186 g, 1.88 mmol). Elemental
analysis not performed. HRMS: Calcd for
13
[C44 C12H62O11 + Na+]: 945.4592 g molꢀ1
.
Found:
945.4471 (DM = ꢀ0.0121 Da).
References
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