Synthesis of D-trigalacturonic acid methylglycoside and conformational comparison with its sulfur analogue

Article abstract of DOI:10.1271/bbb.80160

D-Trigalacturonic acid methylglycoside (3) was synthesized to evaluate the previously synthesized sulfur analogue 1 by comparison. The NOE experiments revealed that both 3 and 1 took on a similar conformation around their glycosyl linkage.

Full text of DOI:10.1271/bbb.80160

Biosci. Biotechnol. Biochem., 72 (8), 2039–2048, 2008
Synthesis of D-Trigalacturonic Acid Methylglycoside
and Conformational Comparison with Its Sulfur Analogue
Kazunori YAMAMOTO,1 Yu SATO,1 Ayumi ISHIMORI,1 Kazuo MIYAIRI,1
Toshikatsu OKUNO,2 Nobuaki NEMOTO,3 Hiroki SHIMIZU,4
y
1;
Shunichi KIDOKORO,5 and Masaru HASHIMOTO
1Faculty of Agriculture and Life Science, Hirosaki University, 3-Bunkyo-cho, Hirosaki 036-8561, Japan
2University of Akita Nursing and Welfare, 2-3-4 Shimizu, Odate 017-0046, Japan
3JEOL Ltd., 3-1-2 Musasino, Akishima, Tokyo 196-8558, Japan
4Drug-Seeds Discovery Research Laboratory, National Institute
of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan
5Department of Bioengineering, Nagaoka University of Technology,
Kamitomioka-cho, 1603-1 Nagaoka, Niigata 940-2188, Japan
Received March 14, 2008; Accepted April 22, 2008; Online Publication, August 7, 2008
[doi:10.1271/bbb.80160]
D-Trigalacturonic acid methylglycoside (3) was syn-
HO
COOH
O
thesized to evaluate the previously synthesized sulfur
analogue 1 by comparison. The NOE experiments
revealed that both 3 and 1 took on a similar conforma-
tion around their glycosyl linkage.
HO
HO
O
COOH
O
HO
Key words: trigalacturonic acid; sulfur-substituted ana-
logue; endo-polygalacturonase 1; NOE
HO
X
COOH
O
HO
Since glycans play a variety of important roles such
in-cell recognition, accumulation of energy, and stabi-
lization of the cells, organs and frameworks for living
things,1,2) understanding the reaction mechanisms for
glycosidases is very important for their medicinal and
industrial applications.3) Many carbohydrate analogues,
so-called glycomimics, have been developed as molecu-
lar probes for glycosidases in the last decade.4) These
have mainly focused on exo-glycosidases which cleave
the terminal glycosidyl linkages. The mechanistic
studies of endo-glycosidases, which hydrolyze the
internal glycoside bonds, lagged behind in comparison
with the exo-glycosidases, because of the absence of
effective molecular probes.
On the other hand, Miyairi, one of authors in this
report, has isolated endo-polygalacturonase 1 (endo-PG
1) from Stereum purpureum as the substance respon-
sible for the silver-leaf disease on apple.5) We expected
that this enzyme would be ideal for the mechanistic
studies, because endo-PG 1 formed a well-developed
HO
OR
1: X = S, R = CH3
2: X = O, R = H
3: X = O, R = CH3
Fig. 1. Trigalacturonic Acid and Derivatives.
galacturonic acid gave only a complex not with the
trimer, but with two molecules of mono-galacturonic
acid. This mono-galacturonic acid occured during the
experiment due to the function of endo-PG 1 even in the
crystal lattice. In the course of our studies on the
mechanistic characteristics of endo-PG 1, we have
designed and synthesized sulfur-substituted trigalac-
turonic analogue 1 as a stable mimic (Fig. 1).7) In fact,
the complex between 1 and endo-PG 1 was found to be
1000 times more stable than that with the natural
substrate on the basis of a surface plasmon analysis.
However, we met difficulty in a conformational com-
parison with natural trigalacturonic acid (2), because the
reducing end of 2 existed as a mixture of anomers under
˚
single crystal to provide the 3D structure with 0.96 A
resolution.6) However, soaking experiments with tri-
y
To whom correspondence should be addressed. Tel/Fax: +81-172-39-3782; E-mail: hmasaru@cc.hirosaki-u.ac.jp
Abbreviations: TEMPO, 2,2,6,6-tetramethyl-1-piperidinyloxyl, free radical; PhI(OAc)2, iodobenzenediacetate; MPMBr, p-methoxyphenylmethyl
bromide

2040
K. YAMAMOTO et al.
HO
HO
BzO
HO
HO
OH
O
OH
O
COOMe
O
MP
a- c
d
BzO
O
BzO
O
BzO
HO
OMe
OMe
OMe
6
O
5
4
i
MPMO
MPMO
BzO
O
MP
MP
COOMe
O
HO
HO
OH
O
O
O
e, f
g, h
O
O
BzO
SPh
OMe
O
O
10
MPMO
SPh
HO
MPMO
MPMO
MPMO
9
7
OC(=NH)CCl3
8
MP
O
O
HO
HO
COOMe
O
COOH
O
HO
HO
CHO
O
COOMe
O
MPMO
MPMO
n, o
O
HO
p, q
MPMO
MPMO
MPMO
MPMO
O
HO
O
O
MPMO
O
MPMO
COOMe
COOMe
COOH
j, k
m
O
l
O
O
COOMe
O
COOMe
O
O
MPMO
MPMO
HO
BzO
BzO
MPMO
MPMO
HO
O
O
O
BzO
BzO
COOMe
O
COOMe
O
COOH
OMe
OMe
O
12
11
BzO
BzO
HO
BzO
BzO
HO
OMe
OMe
OMe
13
MP= p-methoxyphenylmethylidene
Scheme 1. Synthesis of D-Trigalacturonic Acid Methylglycoside 3.
14
3
Reagents and conditions: (a) TrCl, Py, 100 ꢀC (73%); (b) BzCl, Py, CH2Cl2, 0 ꢀC (95%); (c) AcOH, H2O, 60 ꢀC (75%); (d) TEMPO,
PhI(OAc)2, CH2Cl2, H2O then CH2N2, Et2O (95%, 2 steps); (e) p-MeOPhCH(OMe)2, cat. CSA, DMF, 100 ꢀC (72%); (f) NaH, MPMBr, DMF,
toluene (96%); (g) NBS, acetone, H2O, 0 ꢀC (98%); (h) Cl3CCN, DBU, CH2Cl2, ꢁ15 ꢀC (89%); (i) TESOTf, CH2Cl2, MS4A, ꢁ78 ꢀC (73%);
(j) AcOH, H2O, 50 ꢀC (92%); (k) TEMPO, PhI(OAc)2, CH2Cl2; (l) NaClO2, NaH2PO4, t-BuOH, 2-methyl-2-butene then CH2N2, Et2O (80%,
3 steps); (m) 9, TESOTf, Et2O, MS4A, 0 ꢀC (99%); (n) AcOH, H2O, 50 ꢀC (79%); (o) TEMPO, PhI(OAc)2, CH2Cl2 then NaClO2, NaH2PO4,
t-BuOH, 2-methyl-2-butene, then CH2N2, Et2O (68%, 3 steps); (p) DDQ, CH2Cl2, H2O (64%); (q) 0.3% NaOH aq, THF, H2O (98%).
aqueous conditions. We expected that methyl glycoside
3 would reduce this problem, because the introduction
of a methyl glycoside to the reducing end should fix
the stereochemistry. Our preliminary molecular model-
ing suggested that the methyl glycoside moiety would
not interfere with the complexation with endo-PG 1.
Unfortunately, the methyl glycoside form of trigalac-
turonic acid was not known in the literature, so we
needed to prepare it by ourselves. In this paper, we
describe the synthesis of 3 as well as a preliminary
conformational comparison between S-glycoside 1 and
O-glycoside 3.
the C6 primary alcohol, (ii) benzoylation using two
equivalents of benzoyl chloride at low temperature, and
(iii) acidic hydrolysis of the trityl ether. The benzoyla-
tion reaction proceeded selectively at the sterically less-
hindered C2 and C3 hydroxy groups. It was found that a
8,9)
combination of TEMPO and PhI(OAc)2
took place
the regioselective oxidation of 5 to give the correspond-
ing galacturonic acid derivative which was isolated after
conversion into methyl ester 6 (95% yield in two steps)
by using diazomethane. Glycosyl donor 9 was also
synthesized from phenyl 1-thio-ꢁ-D-galactopyranoside
(7).10) After the C4 and C6 alcohols in 7 had been
simultaneously protected in the form of p-methoxy-
phenylmethylidene acetal,11) the remaining C2 and C3
alcohols were transformed into MPM ethers with
MPMBr/NaH to afford 8 in 69% yield in two steps.
Treatment of 8 with NBS under the aqueous condi-
tion hydrolyzed the phenylthio acetal to provide the
corresponding hemiacetal, which was further converted
into ꢀ-trichloroacetimidate 9 according to Schmidt’s
protocol.12)
Results and Discussion
We initially attempted to prepare 3 from polygalac-
turonic acid by the enzymatic partial degradation of
polygalacturonic acid with endo-PG 1 and subsequent
methyl glycosylation. However, we failed in this, in
spite of many attempts, due to the low yield from
enzymatic degradation and the isolation difficulty in the
final step. We therefore decided to prepare it by
chemical synthesis.
Since our previous studies had revealed that the
carboxylate ester function at the C6 position inhibited
the glycosylation reaction,7) the C6 carboxylic acid
group was introduced after glycosylation. As expected,
9 smoothly reacted with acceptor 6 by using catalytic
This synthesis is outlined in Scheme 1. Commercial
methyl ꢀ-D-galactopyranoside (4) was converted into
4,6-diol 5 by the sequential reactions of (i) tritylation of

Synthesis of D-Trigalacturonic Acid Methylglycoside
2041
TESOTf to stereoselectively give ꢀ-glycoside 10 in
HO
COOH
O
73% yield. The ꢁ-isomer was not found in the 1H-
NMR spectrum. After the p-methoxyphenylmethyli-
dene acetal was selectively removed under the aqueous
acidic condition, C6-OH of the resulting diol was
selectively oxidized under the same conditions to those
described for the oxidation of 5. This oxidation did
not provide the carboxylic acid, but only aldehyde 11,
so it was further oxidized into carboxylic acid with
NaClO2. After treating with CH2N2, dimethyl ester
12 was obtained in 80% yield in three steps. Product
12 carried C4-OH, and this function was further
glycosylated with 9 to result in triglycoside 13 with
ꢀ-stereochemistry.
NOE
COOH
1"
HO
H
HO
H
4'
O
HO
O
HO
1'
X
H
COOH
O
4
H
NOE
HO
HO
OMe
1: X = S
3: X = O
Fig. 2. NOE Correlations Observed around the Glycoside Bond in
1 and 3.
The carboxylic acid function at the non-reducing
end was provided by a similar sequence: (i) acidic
removal of the p-methoxyphenylmethylidene acetal, (ii)
TEMPO/PhI(OAc)2 oxidation giving the corresponding
C6 aldehyde, (iii) oxidation with NaClO2, and (iv)
esterification with CH2N2. The last process, methyl ester
formation, was required for effective silica gel column
chromatographic purification. The MPM groups were
then removed by DDQ oxidation, giving the corre-
sponding pentaol in 64% yield. Finally, a basic treat-
ment under aqueous conditions removed all benzoate
and methyl esters to achieve the synthesis of 3 after
passing the crude mixture through an ion-exchange
column (Dowex 50W, Hþ form). Our synthesis afforded
a sufficient amount of 3 (25 mg in total) for NMR and
subsequent detailed calorimetric experiments.
Fig. 3. Stable Conformations of Models
X (O-glycoside) and
Y (S-glycoside) Obtained by Theoretical Calculations.
The additional lines are those penetrating through pyranose
oxygens and corresponding C3s.
A preliminary conformational analysis of 1 and 3 was
carried out by NMR experiments. NOESY is a powerful
tool to investigate distances between two proton
atoms.13) In the carbohydrate research field, this tech-
nique has enabled the geometric-relationships of pyr-
anose (or furanose) rings to be deduced in an oligosac-
charide.14–16) The 600-MHz NMR instrument provided
provided by a conformational search19) with semiempir-
ical AM1.20)
As shown in Fig. 3, it was found that the pyranose
rings in the stable conformers of models X (O-glycoside)
and Y (S-glycoside) took on similar orientations. The
distances between C4H and C10H for both models X and
Y were small, showing good accordance with exper-
1
the H-NMR spectra for 1 and 3, both with satisfying
signal separation, but it gave very poor NOESY spectra.
Generally, when the product of correlation time ꢂ and
Larmor frequency ! is small, the maximum NOE
intensity is positive, while it becomes negative when !ꢂc
is large. The maximum NOE intensity closes to zero and
the number of cross peaks observed in NOESY spectra
drastically decreases when !ꢂc is around 1.13.17) For-
tunately, in our case, a 920-MHz NMR spectrometer
turned these invisible NOEs by the 600-MHz spectrom-
eter into observable as negative NOEs. There was no
remarkable difference in the NOE pattern between 1 and
3. Both compounds 1 and 3 afforded NOE correlations at
100$40 and 10$4 with similar intensity, suggesting that
the glycoside bonds in each compound took on similar
conformations (Fig. 2).
˚
imental NOEs. However, that for model Y (2.92 A)
˚
was larger compared to that for model X (2.13 A),
and the geometry of the pyranose rings was slightly
different between models X and Y. Thus, we concluded
previously synthesized 1 mimicked 3 with slightly higher
steric energy at the glycosyl bond being cleaved by
endo-PG 1.
As described, we synthesized methyl glycoside 3. The
NOE and modeling experiments suggested that previ-
ously synthesized 1 would mimic natural 2, but with
slightly higher steric energy around the glycoside moiety
to be cleaved. Since it is known that the enzymes would
prefer the substrates taking on a strained conformation,
this difference might be an advantage in complexation
with an enzyme. Thus, further investigation by calori-
metric experiments is underway in our laboratories.
This was also supported by molecular modeling
calculations. The stable conformers for models X (O-
glycoside) and Y (S-glycoside) were estimated by
18)
structural optimization with Hartree-Fock 6-31Gꢂ of
the tentatively obtained stable conformers which were

2042
K. YAMAMOTO et al.
24
Experimental
123 ꢀC; ½ꢀꢄD +63.3 (c 0.94, CHCl3); IR (KBr) cmꢁ1
:
3400, 2930, 1490, 1445, 1150, 1075, 1045, 765;
1H-NMR (CDCl3) ꢃ: 2.05 (d, 1H, J ¼ 9:5 Hz, C2OH),
2.46 (d, 1H, J ¼ 2:7 Hz, C4OH), 2.58 (d, 1H, J ¼
5:3 Hz, C3OH), 3.38 (dd, 1H, J ¼ 5:9, 9.4 Hz, C6HH),
3.43 (s, 3H, OCH3), 3.43 (dd, 1H, J ¼ 5:9, 9.4 Hz,
C6HH), 3.71 (ddd, 1H, J ¼ 3:7, 5.3, 9.5 Hz, C3H), 3.80
(dt, 1H, J ¼ 3:7, 9.5 Hz, C2H), 3.82 (brt, 1H, J ¼
5:9 Hz, C5H), 4.03 (brdd, 1H, J ¼ 2:7, 3.7 Hz, C4H),
4.82 (d, 1H, J ¼ 3:7 Hz, C1H), 7.23 (m, 3H, aromatic
protons), 7.30 (m, 6H, aromatic protons), 7.46 (m, 6H,
aromatic protons); 13C-NMR (CDCl3) ꢃ: 55.44 (OCH3),
63.17 (C6), 69.00 (C5), 69.60 (C4), 69.88 (C2), 71.35
(C3), 87.05 (OCPh3), 99.36 (C1), 127.14, 127.92,
128.61, 143.69 (aromatic carbons); negative-FABMS
(%, rel. int.) m=z: 436 (12, [M]ꢁ), 435 (41, ½M ꢁ Hꢄꢁ),
259 (19, [Ph3CO]ꢁ), 243 (16, [Ph3C]ꢁ), 193 (61,
[M-Ph3C]ꢁ), 148 (100, [M-Ph3COCH2-OCH3]ꢁ); neg-
ative-FAB-HRMS: calcd. for C26H27O6 ½M ꢁ Hꢄꢁ,
435.1808; found, m=z 435.1811.
General methods. Melting point (mp) data were
determined with Yanako MP-J3 micro-melting point
apparatus and are uncorrected. Optical rotation values
were measured by a HORIBA SEPA300 high-sensitivity
1
polarimeter. H- and 13C-NMR spectra were measured
by a JEOL ALPHA 400 spectrometer for structural
determinations, and a JEOL ECA 920 spectrometer was
used for quantitative NOE experiments. In the 1H-NMR
spectra, the chemical shifts are expressed in ppm
downfield from the signal for trimethylsilane used as
an internal standard in the case of CDCl3. When another
solvent was employed, the remaining proton signals in
deuterosolvent C6HD5 (7.15 ppm), CHD2OD (3.30
ppm), or HDO (4.63 ppm) were used as the internal
standards. Splitting patterns are designated as s (singlet),
d (doublet), t (triplet), m (multiplet), and br (broad). In
the 13C-NMR spectra, the 13C chemical shifts of the
solvents were used as the internal standard (13CDCl3,
77.0 ppm; 13C6D6, 128.0 ppm; or 13CD3OD, 49.5 ppm).
For the 13C-NMR spectra measured in D2O, the default
offset was employed and we did not perform corrections.
Assignments of the signals are according to the
numbering based on IUPAC nomenclature. IR spectra
were obtained with a Horiba FT-720 Fourier transform
infrared spectrometer in a KBr cell. Measurements of
field desorption and fast atom bombardment mass
spectra (FD-MS and FAB-MS, respectively) were
performed with JEOL JMS AX500 or JEOL JMS
SX102A spectrometers. Whenever MS spectra were
measured in the negative mode, ‘‘negative’’ is men-
tioned. MS analyses for unstable compounds such as
glycosyl imidates were not performed. Analytical and
preparative thin-layer chromatography was carried out
by using pre-coated Merck silica gel 60F254 (Art.
1.05715) plates. The silica gel used for column
chromatography was Merck silica gel 60 (Art.
1.07734). All reactions were carried out in an N2 or
Ar atmosphere by using dried solvents, except for the
aqueous conditions. Dichloromethane and tetrahydro-
furan were freshly distilled from diphosphorus pent-
oxide and benzophenone-ketyl, respectively. Molecular
sieves 4A were finely powdered and activated (200 ꢀC
in vacuo for 1 h) before use.
A solution of the 6-O-triphenylmethyl ether (1.59 g,
3.64 mmol) in CH2Cl2 (100 ml) was stirred with benzoyl
chloride (1.02 g, 7.26 mmol) and pyridine (576 mg,
7.28 mmol) at 0 ꢀC. After stirring for 5 min, the cooling
bath was removed, and the mixture was stirred for
30 min more at room temperature. The mixture was
poured into H2O (100 ml) and extracted with AcOEt
(80 ml ꢃ 3). The organic layers were washed with brine
(80 ml), combined, dried over MgSO4, and then con-
centrated in vacuo. Silica gel column chromatography of
the residue (AcOEt:hexane = 10:90) gave methyl 2,3-
di-O-benzoyl-6-O-triphenylmethyl-ꢀ-D-galactopyranoside
24
(2.24 g, 95%) as a viscous oil, ½ꢀꢄD +93.4 (c 0.93,
CHCl3); IR (film) cmꢁ1: 3500, 3060, 2935, 1725, 1450,
1280, 1105; 1H-NMR (C6D6) ꢃ: 3.11 (s, 3H, OCH3), 3.38
(dd, 1H, J ¼ 4:1, 9.9 Hz, C6HH), 3.56 (dd, 1H, J ¼ 5:9,
9.9 Hz, C6HH), 3.78 (brdd, 1H, J ¼ 4:1, 5.9 Hz, C5H),
3.96 (brd, 1H, J ¼ 3:0 Hz, C4H), 5.29 (d, 1H,
J ¼ 3:6 Hz, C1H), 6.01 (dd, 1H, J ¼ 3:0, 10.5 Hz,
C3H), 6.12 (dd, 1H, J ¼ 3:6, 10.5 Hz, C2H), 6.83–7.18
(m, 15H, aromatic protons), 7.58 (m, 6H, aromatic
protons), 8.10 (brdd, 2H, J ¼ 1:5, 8.1 Hz, aromatic
protons), 8.14 (brdd, 2H, J ¼ 1:5, 8.1 Hz, aromatic
protons); 13C-NMR (C6D6) ꢃ: 54.89 (OCH3), 64.56
(C6), 69.29 (C5), 69.67 (C4), 69.79 (C2), 71.43 (C3),
87.49 (OCPh3), 97.97 (C1), 127.35, 128.53, 129.11,
130.05, 130.10, 133.02, 133.04, 144.40 (aromatic
carbons), 165.85, 166.24 (each þArC=O); FABMS (%,
rel. int.) m=z: 667 (28, ½M þ Naꢄ ), 243 (100, [CPh3]þ);
FAB-HRMS: calcd. for C26H27O6Na ½M þ Naꢄþ,
667.2308; found, m=z 667.2280.
Methyl 2,3-di-O-benzoyl-ꢀ-D-galactopyranoside (5).
Commercial methyl ꢀ-D-galactopyranoside (8.48 g,
43.7 mmol) was stirred with chlorotriphenylmethane
(14.0 g, 50.2 mmol) in pyridine (50 ml) at 100 ꢀC for
30 min. The mixture was poured into H2O (200 ml) and
extracted with AcOEt (150 ml ꢃ 3). The organic layers
were washed with brine (100 ml), combined, dried over
MgSO4, and then concentrated in vacuo. Silica gel
column chromatography of the residue (AcOEt 100%)
gave the corresponding 6-O-triphenylmethyl ether
(14.0 g, 73%) as a white solid. Recrystallization from
AcOEt:hexane (50:50) gave colorless needles, mp 122–
A solution of methyl 2,3-di-O-benzoyl-6-O-triphenyl-
methyl-ꢀ-D-galactopyranoside (2.24 g, 3.48 mmol) was
stirred in 60% aqueous acetic acid solution (8.0 ml) at
60 ꢀC for 30 min. After cooling, the mixture was
concentrated in vacuo. Silica gel column chromatogra-
phy of the residue (AcOEt:hexane = 60:40) gave 5
23
(1.05 g, 75%) as an oil, ½ꢀꢄD +157 (c 1.02, CHCl3); IR

Synthesis of D-Trigalacturonic Acid Methylglycoside
2043
(film) cmꢁ1: 3440, 2935, 1720, 1280, 1105, 1030, 710;
1H-NMR (CDCl3) ꢃ: 2.45 (br, 1H, C6OH), 3.07 (br, 1H,
C4OH), 3.44 (s, 3H, OCH3), 4.00 (m, 3H, C6H2, C5H),
4.47 (brs, 1H, C4H), 5.22 (d, 1H, J ¼ 1:9 Hz, C1H),
5.70 (m, 2H, C2H, C3H), 7.36 (m, 4H, aromatic
protons), 7.50 (m, 2H, aromatic protons), 7.98 (m, 4H,
aromatic protons); 13C-NMR (CDCl3) ꢃ: 55.44 (OCH3),
62.73 (C6), 68.98 (C2), 69.09 (C5), 69.32 (C4), 71.02
(C3), 97.55 (C1), 128.30, 128.35, 129.23, 129.31,
129.69, 129.76, 133.22, 133.25 (aromatic carbons),
165.91, 166.21 (each ArC=O); negative-FABMS (%,
rel. int.) m=z: 402 (3.7, [M]ꢁ), 401 (6.0, ½M ꢁ Hꢄꢁ), 297
(9.3, [M-PhCO]ꢁ), 121 (100, [PhCOO]ꢁ); negative-
FAB-HRMS: calcd. for C21H21O8 ½M ꢁ Hꢄꢁ, 401.1236;
found, m=z 401.1251.
give a crude solid. Recrystallization from AcOEt:hexane
(30:70) gave phenyl 4,6-O-(4-methoxyphenylmethyli-
dene)-1-thio-ꢁ-D-galactopyranoside (2.74 g, 72%) as
colorless needles, mp 151–154 ꢀC; ½ꢀꢄD
23
ꢁ7:5 (c
1.50, CHCl3); IR (KBr) cmꢁ1: 3410, 2910, 1615,
1515, 1250, 1165, 825; H-NMR (CDCl3) ꢃ: 2.48 (brd,
1
1H, J ¼ 9:0 Hz, C2OH), 2.51 (d, 1H, J ¼ 1:2 Hz,
C3OH), 3.55 (brdd, 1H, J ¼ 1:4, 1.7 Hz, C5H), 3.69
(m, 2H, C2H, C3H), 3.82 (s, 3H, OCH3), 4.02 (dd, 1H,
J ¼ 1:7, 12.4 Hz, C6HH), 4.20 (brd, 1H, J ¼ 1:9, C4H),
4.37 (dd, 1H, J ¼ 1:4, 12.4 Hz, C6HH), 4.51 (m, 1H,
C1H), 5.47 (s, 1H, ArCH), 6.86 (brd, 2H, J ¼ 8:7 Hz,
aromatic protons), 7.33 (m, 5H, aromatic protons),
7.69 (brdd, 2H, J ¼ 2:0, 8.2 Hz, aromatic protons);
13C-NMR (CDCl3) ꢃ: 55.33 (OCH3), 68.86 (C2), 69.25
(C6), 70.06 (C5), 73.82 (C3), 75.30 (C4), 87.00 (C1),
101.29 (ArC(OR)2), 113.57, 127.82, 128.21, 128.93,
130.14, 130.71, 133.76, 160.33 (aromatic carbons);
negative-FABMS (%, rel. int.) m=z: 389 (2.1,
½M ꢁ Hꢄꢁ), 375 (1.3, [M-CH3]ꢁ), 148 (100), 109 (91,
[PhS]ꢁ); negative-FAB-HRMS: calcd. for C20H21O8S
½M ꢁ Hꢄꢁ, 389.1059; found, m=z 389.1057.
Methyl (methyl 2,3-di-O-benzoyl-ꢀ-D-galactopyrano-
sid)uronate (6). A suspension of 5 (1.05 g, 2.61 mmol)
in a mixture of CH2Cl2 (10 ml) and H2O (5.0 ml) was
stirred with PhI(OAc)2 (4.33 g, 13.4 mmol) and TEMPO
(80.0 mg, 512.0 mmol) at room temperature for 10 min.
The mixture was poured into H2O (70 ml) and extracted
with AcOEt (40 ml ꢃ 3). The extracts were washed with
brine (50 ml), dried over MgSO4, combined, and then
concentrated in vacuo. After the residue had been
diluted with THF (8.0 ml), ethereal diazomethane was
added until the yellow color did not disappear. After
concentration in vacuo, silica gel column chromatog-
raphy (AcOEt:hexane = 30:70) of the residue gave 6
Sodium hydride (washed with hexane, 3.21 g,
8.22 mmol) was slowly added to a DMF solution
(20 ml) of the foregoing product (3.21 g, 8.22 mmol) at
room temperature. Upon the addition of the substrate,
H2 gas was bubbled. After stirring for 30 min, 50%
toluene solution of MPMBr (13.2 g, 32.8 mmol, freshly
prepared from anisic alcohol and PBr3) was added at
0 ꢀC. After 10 min, the cooling bath was removed, and
the mixture was stirred at room temperature for 30 min.
Methanol (5.0 ml) and triethylamine (5.0 ml) were
added to decompose the excess reagent. After stirring
for an additional 30 min, the mixture was poured into
H2O (100 ml) and extracted with AcOEt (70 ml ꢃ 3).
The organic layers were successively washed with H2O
(50 ml), and brine (50 ml), combined, and dried over
MgSO4. After concentration, the residue was purified
by silica gel column chromatography (AcOEt:hexane =
23
(1.07 g, 95%) as an oil, ½ꢀꢄD +107 (c 0.95, CHCl3); IR
(film) cmꢁ1: 3940, 2955, 1725, 1450, 1280, 1100, 1025,
915, 710; 1H-NMR (CDCl3) ꢃ: 3.47 (s, 3H, OCH3), 3.80
(s, 3H, C6OCH3), 4.65 (brs, 1H, C5H), 4.73 (brs, 1H,
C4H), 5.31 (d, 1H, J ¼ 2:7 Hz, C1H), 5.71 (dd, 1H,
J ¼ 2:7, 10.7 Hz, C2H), 5.75 (dd, 1H, J ¼ 1:9, 10.7 Hz,
C3H), 7.34 (m, 4H, aromatic protons), 7.48 (m, 2H,
aromatic protons), 7.98 (m, 4H, aromatic protons);
13C-NMR (CDCl3) ꢃ: 52.50 (C6OCH3), 56.06 (OCH3),
68.23 (C3), 68.88 (C4), 69.81 (C5), 70.16 (C2), 97.76
(C1), 128.16, 128.24, 128.27, 129.06, 129.64, 129.66,
133.15, 133.23 (aromatic carbons), 165.61, 165.83
(each ArC=O), 1þ68.59 (C6); FDMS (%, rel. int.) m=z:
431 (64, ½M þ Hꢄ ), 398 (100, [M-CH3OH]þ), 341 (26,
[MH-(COOCH3)-CH3O]þ), 308 (42, [M-PhCOOH]þ);
FD-HRMS: calcd. for C22H23O9 ½M þ Hꢄþ, 431.1342;
found, m=z 431.1335.
23
25:75) to give 8 (4.98 g, 96%) as an oil, ½ꢀꢄD +1.7
(c 1.25, CHCl3); IR (film) cmꢁ1: 2860, 1610, 1515,
1
1250, 1170, 1100, 1035, 820; H-NMR (CDCl3) ꢃ: 3.37
(brdd, 1H, J ¼ 1:3, 1.4 Hz, C5H), 3.57 (dd, 1H,
J ¼ 3:3, 9.2 Hz, C3H), 3.79, 3.80, 3.83 (each s, 3H,
OCH3), 3.86 (t, 1H, J ¼ 9:2 Hz, C2H), 3.95 (dd, 1H,
J ¼ 1:4, 12.4 Hz, C6HH), 4.10 (brd, 1H, J ¼ 3:3 Hz,
C4H), 4.33 (dd, 1H, J ¼ 1:3, 12.4 Hz, C6HH), 4.58
(d, 1H, J ¼ 9:2 Hz, C1H), 4.62 (s, 2H, ArCH2O), 4.63,
4.66 (each d, 1H, J ¼ 12:3 Hz, ArCHHO), 5.43 (s, 1H,
ArCH), 6.82 (brd, 2H, J ¼ 8:7 Hz, aromatic protons),
6.87 (brd, 2H, J ¼ 8:6 Hz, aromatic protons), 6.91
(brd, 2H, J ¼ 8:8 Hz, aromatic protons), 7.16–7.27
(m, 5H, aromatic protons), 7.33 (brd, 2H, J ¼ 8:7 Hz,
aromatic protons), 7.44 (brd, 2H, J ¼ 8:6 Hz, aromatic
protons), 7.70 (brdd, 2H, J ¼ 2:1, 7.8 Hz, aromatic
protons); 13C-NMR (CDCl3) ꢃ: 55.24, 55.27, 55.34
(each OCH3), 69.37 (C6), 69.84 (C5), 71.46 (ArCH2O),
73.79 (C4), 75.05 (ArCH2O), 75.18 (C2), 80.95 (C3),
Phenyl 2,3-bis-O-(4-methoxyphenylmethyl)-4,6-O-(4-
methoxyphenylmethylidene)-1-thio-ꢁ-D-galactopyranoside
(8). A solution of phenyl-1-thio-ꢁ-D-galactopyranoside
(7); (2.62 g, 9.62 mmol) in DMF (20 ml) was stirred with
4-methoxybenzaldehyde dimethylacetal (3.50 g, 19.2
mmol) and camphorsulfonic acid (22.3 mg, 96.0 mmol)
at 100 ꢀC for 10 min. After cooling, the mixture was
poured into 5% aqueous NaHCO3 solution (100 ml) and
extracted with AcOEt (70 ml ꢃ 3). The extracts were
washed with H2O (50 ml) and brine (50 ml), combined,
dried over MgSO4, and then concentrated in vacuo to

2044
K. YAMAMOTO et al.
86.59 (C1), 101.24 (ArC(OR)2), 113.49, 113.73,
113.76, 127.36, 127.91, 128.82, 129.37, 129.81,
130.23, 130.57, 130.76, 132.67, 132.88, 159.25,
159.27, 160.14 (aromatic carbons); FDMS (%, rel.
int.) m=z: 631 (38, ½M þ Hꢄþ), 630 (100, ½Mꢄþ);
FD-HRMS: calcd. for C36H38O8S [M]þ, 630.2287;
found, m=z 630.2276.
A solution of the product (489 mg, 908 mmol) in
CH2Cl2 (8.0 ml) was stirred with CCl3CN (656 mg,
4.54 mmol) in the presence of DBU (45.6 mg, 6.94
mmol) at ꢁ15 ꢀC for 30 min. After concentrating in
vacuo, the residue was purified by silica gel column
chromatography (AcOEt:hexane = 30:70) to give 9
(556 mg, 89%) as an oil. 1H-NMR (CDCl3) ꢃ: 3.80,
3.80 (each s, 3H, OCH3), 3.80 (m, 1H, C5H), 3.81
(s, 3H, OCH3), 3.97 (dd, 1H, J ¼ 1:3, 12.6 Hz, C6HH),
4.02 (dd, 1H, J ¼ 3:3, 10.1 Hz, C3H), 4.19 (brd, 1H,
J ¼ 3:3 Hz, C4H), 4.24 (dd, 1H, J ¼ 3:4, 10.1 Hz, C2H),
4.24 (dd, 1H, J ¼ 1:1, 12.6 Hz, C6HH), 4.66, 4.70 (each
d, 1H, J ¼ 11:5 Hz, ArCHHO), 4.70, 4.75 (each d, 1H,
J ¼ 11:8 Hz, ArCHHO), 5.45 (s, 1H, ArCH), 6.59
(d, 1H, J ¼ 3:4 Hz, C1H), 6.82–6.90 (m, 6H, aromatic
protons), 7.25 (brd, 2H, J ¼ 8:6 Hz, aromatic protons),
7.29 (brd, 2H, J ¼ 8:6 Hz, aromatic protons), 7.44
(brd, 2H, J ¼ 8:7 Hz, aromatic protons), 8.55 (s, 1H,
C(=NH)CCl3). This sample gradually decomposed, so it
was immediately used for the next step.
2,3-Bis-O-(4-methoxyphenylmethyl)-4,6-O-(4-methoxy-
phenyl-methylidene)-ꢀ-D-galactopyranosyl trichloroacet-
imidate (9). A solution of 8 (515 mg, 817.0 mmol) in a
mixture of acetone (10.0 ml) and H2O (1.0 ml) was
stirred with NBS (356 mg 2.0 mmol) at 0 ꢀC. After
5 min, 10% Na2S2O3 (4.0 ml) was added to the mixture.
After concentrating in vacuo, the residue was diluted
with AcOEt (150 ml) and then washed with H2O
(60 ml). The aqueous solution was extracted with AcOEt
(50 ml ꢃ 2). Each organic layer was washed with brine
(50 ml), combined, dried over MgSO4, and then con-
centrated in vacuo to give a white solid. Recrystalliza-
tion (from AcOEt:hexane = 50:50) gave 2,3-bis-O-(4-
methoxyphenylmethyl)-4,6-O-(4-methoxyphenylmethy-
lidene)-D-galactopyranose as needles (431 mg, 98%), mp
Methyl {methyl 2,3-di-O-benzoyl-4-O-[20,30-bis-O-(4-
methoxyphenylmethyl)-40,60-O-(methoxyphenlylmethyli-
dene)-ꢀ-D-galactopyranosyl]-ꢀ-D-galactopyranosid}ur-
onate (10). Triethylsilyl trifluoromethanesulfonate (1.8
mg, 6.8 mmol) was added to a suspension of 6 (30.1 mg,
69.9 mmol), 9 (138.7 mg, 0.2 mmol), and powdered
4A molecular sieves (43 mg) in CH2Cl2 (0.5 ml) at
ꢁ78 ꢀC. After stirring for 5 min, triethylamine (50 ml)
was adding, and the mixture was allowed to warm
to room temperature. After filtering through a cotton
pad, the filtrate was concentrated in vacuo. Purification
of the residue by silica gel column chromatography
(AcOEt:hexane = 25:75) gave 10 (48.8 mg, 73%) as an
22
124–130 ꢀC; ½ꢀꢄD +39.8 (c 0.75, CHCl3); IR (film)
cmꢁ1: 3435, 2930, 1610, 1515, 1250, 1195, 1035, 825.
The 1H-NMR spectrum indicated that the sample
consisted of a mixture of anomers (ꢀ:ꢁ = 67:33 in
CDCl3); 1H-NMR (CDCl3) ꢃ: 2.86 (d, 1H ꢃ 0.67,
J ¼ 1:2 Hz, C1OH (ꢀ-anomer)), 2.94 (d, 1H ꢃ 0.33,
J ¼ 7:4 Hz, C1OH (ꢁ-anomer)), 3.37 (brdd, 1H ꢃ 0.33,
J ¼ 1:2, 1.6 Hz, C5H (ꢁ-anomer)), 3.54 (dd, 1H ꢃ 0.33,
J ¼ 3:6, 9.5 Hz, C3H (ꢁ-anomer)), 3.73 (dd, 1H ꢃ 0.33,
J ¼ 7:7, 9.5 Hz, C2H (ꢁ-anomer)), 3.80, 3.80, 3.80
(each s, 3H ꢃ 0.33, OCH3 (ꢁ-anomer)), 3.81, 3.81, 3.81
(each s, 3H ꢃ 0.67, OCH3 (ꢀ-anomer)), 3.81 (1H, m,
C5H (ꢀ-anomer)), 3.91 (dd, 1H ꢃ 0.67, J ¼ 3:6, 9.7 Hz,
C3H (ꢀ-anomer)), 3.97–4.03 (m, 1H ꢃ 0.67, 1H ꢃ
0.33, 1H ꢃ 0.67, C6HH (ꢀ-anomer), C6HH (ꢁ-anomer),
C2H (ꢀ-anomer)), 4.08 (brd, 1H ꢃ 0.33, J ¼ 3:6 Hz,
C4H (ꢁ-anomer)), 4.15 (brd, 1H ꢃ 0.67, J ¼ 3:6 Hz,
C4H (ꢀ-anomer)), 4.20 (dd, 1H ꢃ 0.67, J ¼ 1:6,
12.3 Hz, C6HH (ꢀ-anomer)), 4.28 (dd, 1H ꢃ 0.33, J ¼
1:6, 12.5 Hz, C6HH (ꢁ-anomer)), 4.62 (d, 1H ꢃ 0.33,
J ¼ 11:2 Hz, ArCHHO (ꢁ-anomer)), 4.65 (d, 1H ꢃ
0.33, J ¼ 7:7 Hz, C1H (ꢁ-anomer)), 4.68 (d, 1H ꢃ 0.67,
J ¼ 12:2 Hz, ArCHHO (ꢀ-anomer)), 4.69 (s, 2H ꢃ
0.67, ArCH2O (ꢀ-anomer)), 4.72 (d, 1H ꢃ 0.67, J ¼
12:2 Hz, ArCHHO (ꢀ-anomer)), 4.78 (d, 1H ꢃ 0.33,
J ¼ 10:7 Hz, ArCHHO (ꢁ-anomer)), 4.81 (d, 1H ꢃ
0.33, J ¼ 11:2 Hz, ArCHHO (ꢁ-anomer)), 4.82 (d,
1H ꢃ 0.33, J ¼ 10:7 Hz, ArCHHO (ꢁ-anomer)), 5.31
(dd, 1H ꢃ 0.67, J ¼ 1:2, 3.5 Hz, C1H (ꢀ-anomer)), 5.44
(s, 1H ꢃ 0.33, ArCH (ꢁ-anomer)), 5.45 (s, 1H ꢃ 0.67
ArCH (ꢀ-anomer)), 6.84–7.48 (m, 12H, aromatic pro-
tons); FABMS (%, rel. int.) m=z: 561 (46, ½M þ Naꢄþ),
417 (61, [M-CH3OPhCH2]þ), 121 (100, [PhCOO]þ);
FAB-HRMS: calcd. for C30H34O9Na ½M þ Naꢄþ,
561.2101; found, m=z 561.2108.
oil, ½ꢀꢄD +58.4 (c 0.60, CHCl3); IR (film) cmꢁ1: 2935,
23
1730, 1515, 1250, 1100, 1030, 830, 710; 1H-NMR
(CDCl3) ꢃ: 3.36, 3.41 (each brd, 1H, J ¼ 12:7 Hz,
C60HH), 3.49, 3.60, 3.76, 3.78, 3.78 (each s, 3H, OCH3),
3.85 (brs, 1H, C50H), 3.96 (dd, 1H, J ¼ 3:2, 10.2 Hz,
C20H), 4.04 (brd, 1H, J ¼ 3:1 Hz, C40H), 4.08 (dd, 1H,
J ¼ 3:1, 10.2 Hz, C30H), 4.64 (d, 1H, J ¼ 11:4 Hz,
ArCHHO), 4.66 (brs, 1H, C5H), 4.66 (s, 2H, ArCH2O),
4.71 (d, 1H, J ¼ 11:4 Hz, ArCHHO), 4.86 (brd, 1H,
J ¼ 2:5 Hz, C4H), 4.96 (d, 1H, J ¼ 3:2 Hz, C10H), 5.24
(s, 1H, ArCH), 5.32 (d, 1H, J ¼ 3:4 Hz, C1H), 5.66 (dd,
1H, J ¼ 2:5, 11.0 Hz, C3H), 5.72 (dd, 1H, J ¼ 3:4,
11.0 Hz, C2H), 6.80–6.88 (m, 6H, aromatic protons),
7.22–7.40 (m, 10H, aromatic protons), 7.48 (m, 2H,
aromatic protons), 7.89 (brd, 2H, J ¼ 7:3 Hz, aromatic
protons), 7.99 (brd, 2H, J ¼ 7:3 Hz, aromatic protons);
13C-NMR (CDCl3) ꢃ: 52.39, 55.17, 55.20, 55.25, 56.16,
(each OCH3), 63.47 (C50), 68.26 (C2), 68.94 (C60),
69.73 (C5), 70.39 (C3), 71.73, 73.19 (each ArCH2O),
74.46 (C20), 74.68 (C40), 75.41 (C30), 76.49 (C4), 97.88
(C1), 100.40 (C10), 100.60 (ArC(OR)2), 113.35, 113.58,
113.63, 127.61, 127.61, 128.21, 128.45, 128.60, 129.02,
129.23, 129.26, 129.46, 129.73, 129.75, 129.80, 130.60,
130.79, 130.97, 133.38, 133.53, 159.05, 159.05, 159.91

Synthesis of D-Trigalacturonic Acid Methylglycoside
2045
(aromatic carbons), 165.82, 166.02 (each ArC=O),
167.90 (C6); FABMS (%, rel. int.) m=z: 973 (1.9,
½M þ Naꢄþ), 829 (1.2, [M-CH3OPhCH]þ), 121 (100,
[PhCOO]þ); FAB-HRMS: calcd. for C52H54O17Na
½M þ Naꢄþ, 973.3259; found, m=z 973.3230.
temperature. The mixture was stirred for 5 min at room
temperature, poured into H2O (40 ml), and extracted
with AcOEt (30 ml ꢃ 3). The organic layers were
washed with brine (30 ml), combined, dried over
MgSO4, and then concentrated in vacuo. After diluting
with THF (2.0 ml), an ethereal solution of diazomethane
was added until the yellow color did not disappear. After
concentrating in vacuo, silica gel column chromatog-
raphy (AcOEt:hexane = 40:60) of the residue gave 12
Methyl {methyl 2,3-di-O-benzoyl-4-O-[20,30-bis-O-(4-
methoxyphenylmethyl)-60-methyl-ꢀ-D-galactopyranuro-
nosyl]-ꢀ-D-galactopyranosid}uronate (12). A solution
of 10 (348 mg, 366 mmol) in 60% aqueous acetic acid
solution (8.0 ml) was stirred at 50 ꢀC for 20 min. After
cooling, the mixture was concentrated in vacuo. The
residue was purified by silica gel column chromatog-
raphy (AcOEt:hexane = 50:50) to give the correspond-
23
(58.2 mg, 80%) as an oil, ½ꢀꢄD +92.7 (c 0.74, CHCl3);
IR (film) cmꢁ1: 3455, 2935, 1730, 1510, 1250, 1095,
1030, 710; 1H-NMR (CDCl3) ꢃ: 3.41, 3.48, 3.64 (each s,
3H, OCH3), 3.75 (dd, 1H, J ¼ 3:2, 9.9 Hz, C20H), 3.76,
3.80 (each s, 3H, OCH3), 4.04 (dd, 1H, J ¼ 3:3, 9.9 Hz,
C30H), 4.33 (ddd, 1H, J ¼ 1:1, 1.6, 3.3 Hz, C40H), 4.58,
4.62 (each d, 1H, J ¼ 12:0 Hz, ArCH2O), 4.62, 4.68
(each d, 1H, J ¼ 10:9 Hz, ArCH2O), 4.68 (brs, 1H,
C5H), 4.73 (dd, 1H, J ¼ 1:2, 1.6 Hz, C50H), 4.82 (brd,
1H, J ¼ 2:7 Hz, C4H), 4.94 (d, 1H, J ¼ 3:2 Hz, C10H),
5.33 (d, 1H, J ¼ 3:5 Hz, C1H), 5.61 (dd, 1H, J ¼ 3:5,
10.9 Hz, C2H), 5.71 (dd, 1H, J ¼ 2:7, 10.9 Hz, C3H),
6.80 (brd, 2H, J ¼ 8:7 Hz, aromatic protons), 6.89 (brd,
2H, J ¼ 8:6 Hz, aromatic protons), 7.22–7.28 (m, 4H,
aromatic protons), 7.32–7.37 (m, 4H, aromatic protons),
7.49 (m, 2H, aromatic protons), 7.93–7.97 (m, 4H,
aromatic protons); 13C-NMR (CDCl3) ꢃ: 49.66, 52.00,
52.44, 55.21, 56.19 (each OCH3), 68.44 (C2), 68.60
(C40), 69.72 (C5), 69.95 (C3), 70.82 (C50), 72.49, 73.18
(each ArCH2O), 74.20 (C20), 76.40 (C30), 77.21 (C4),
97.85 (C1), 99.69 (C10), 113.74, 113.89, 128.38, 128.48,
128.96, 129.20, 129.61, 129.69, 129.76, 129.98, 129.98,
130.25, 133.25, 133.31, 159.24, 159.39 (aromatic
carbons), 165.88, 166.01 (each ArC=O), 167.92,
168.63 (each C=O); FABMS (%, rel. int.) m=z: 883
(44, ½M þ Naꢄþ), 121 (100, [CH3OPhCH2]þ), 105
(96, [PhCO]þ); FAB-HRMS: calcd. for C45H48O17Na
½M þ Naꢄþ, 883.2789; found, m=z 883.2816.
22
ing diol (280 mg, 92%) as a viscous oil, ½ꢀꢄD +93.7
(c 0.52, CHCl3); IR (film) cmꢁ1: 3450, 2935, 1730,
1
1510, 1250, 1095, 710; H-NMR (CDCl3) ꢃ: 3.49, 3.62
(each s, 3H, OCH3), 3.63 (dd, 1H, J ¼ 3:4, 12.0 Hz,
C60HH), 3.69, (dd, 1H, J ¼ 3:2, 10.0 Hz, C20H), 3.71
(dd, 1H, J ¼ 4:5, 12.0 Hz, C60HH), 3.74, 3.80 (each s,
3H, OCH3), 3.92 (dd, 1H, J ¼ 3:2, 10.0 Hz, C30H), 4.02
(dd, 1H, J ¼ 1:1, 3.2 Hz, C40H), 4.16 (ddd, 1H, J ¼ 1:1,
3.4, 4.5, Hz, C50H), 4.51, 4.57 (each d, 1H, J ¼ 11:7 Hz,
ArCH2O), 4.61 (d, 1H, J ¼ 11:1 Hz, ArCHHO), 4.69
(d, 1H, J ¼ 1:8 Hz, C5H), 4.70 (d, 1H, J ¼ 11:1 Hz,
ArCHHO), 4.71 (brd, 1H, J ¼ 1:8 Hz, C4H), 4.79
(d, 1H, J ¼ 3:2 Hz, C10H), 5.25 (brs, 1H, C1H), 5.75
(m, 2H, C2H, C3H), 6.78 (brd, 2H, J ¼ 8:7 Hz, aromatic
protons), 6.90 (brd, 2H, J ¼ 8:7 Hz, aromatic protons),
7.22–7.36 (m, 8H, aromatic protons), 7.48 (m, 2H,
aromatic protons), 7.93–7.96 (m, 4H, aromatic pro-
tons); 13C-NMR (CDCl3) ꢃ: 52.43, 55.14, 55.23, 56.19
(each OCH3), 62.95 (C60), 68.20 (C2 or 3), 69.16 (C40),
69.98 (C5), 70.11 (C2 or 3), 70.96 (C50), 72.66, 73.17
(each ArCH2O), 75.72 (C20), 76.64 (C30), 76.88 (C4),
98.17 (C1), 99.98 (C10), 113.73, 113.88, 128.34, 128.49,
128.94, 129.08, 129.62, 129.77, 129.83, 129.87, 130.25,
130.39, 133.34, 133.42, 159.24, 159.36 (aromatic
carbons), 165.90, 166.98 (each ArC=O), 168.01 (C6);
FABMS (%, rel. int.) m=z: 855 (61, ½M þ Naꢄþ), 121
(100, [CH3OPhCH2]þ), 105 (96, [PhCO]þ); FAB-
HRMS: calcd. for C44H48O16Na ½M þ Naꢄþ, 855.2840;
found, m=z 855.2802.
A suspension of the diol thus obtained (70.6 mg,
84.8 mmol) in CH2Cl2 (2.0 ml) was stirred with
PhI(OAc)2 (141 mg, 437.8 mmol) and TEMPO (19.9
mg, 127.4 mmol) at room temperature for 10 min. The
mixture was poured into H2O (40 ml) and extracted with
AcOEt (30 ml ꢃ 3). The organic layers were washed
with brine (30 ml), combined, dried over MgSO4, and
then concentrated in vacuo to give corresponding
C6-aldehyde 11 (70.6 mg, 99%). Since this sample
gradually decomposed, it was immediately used for the
next step. After crude 11 had been dissolved in a mixture
of 2-methyl-2-propanol (10 ml) and 2-methyl-2-butene
(23.8 mg, 339.4 mmol), sodium dihydrogenphosphate
dehydrate (79.4 mg, 508.9 mmol) and sodium chlorite
(30.7 mg, 339.5 mmol) were successively added at room
Methyl {methyl 2,3-di-O-benzoyl-4-O-{20,30-bis-O-(4-
methoxyphenylmethyl)-40-O-[200,300-bis-O-(4-methoxyphen-
ylmethyl)-400,600-O-(methoxyphenlylmethylidene)-ꢀ-D-ga-
lactopyranosyl]-60-methyl-ꢀ-D-galactopyranuronosyl}-ꢀ-
D-galactopyranosid}uronate (13). A 0.16 M solution of
triethylsilyl trifluoromethanesulfonate in Et2O (10 ml)
was added at 0 ꢀC to a suspension of a mixture of 12
(14.0 mg, 16.3 mmol), 7 (33.3 mg, 48.8 mmol), and pow-
dered 4A molecular sieves (20 mg) in Et2O (1.0 ml).
After stirring for 10 min, triethylamine (10 ml) was
added to quench the reaction. The mixture was filtered
through a cotton pad, and the filtrate was concentrated in
vacuo. Purification of the residue by silica gel column
chromatography (benzene:AcOEt = 80:20) gave 13
22
(22.0 mg, 99%) as an oil, ½ꢀꢄD +69.7 (c 1.15, CHCl3);
IR (film) cmꢁ1: 2935, 1730, 1610, 1515, 1250, 1095,
1030, 825, 720; 1H-NMR (CDCl3) ꢃ: 2.93 (s, 3H,
OCH3), 3.40 (dd, 1H, J ¼ 1:5, 12.6 Hz, C600HH), 3.49
(s, 3H, OCH3), 3.57 (dd, 1H, J ¼ 1:7, 12.6 Hz, C600HH),
3.63 (brdd, 1H, J ¼ 1:5, 1.7 Hz, C500H), 3.70 (dd, 1H,

2046
K. YAMAMOTO et al.
J ¼ 3:3, 10.2 Hz, C300H), 3.74, 3.75, 3.76, 3.76, 3.76,
3.79 (each s, 3H, OCH3), 3.83 (dd, 1H, J ¼ 3:3, 10.3 Hz,
C20H), 3.86 (dd, 1H, J ¼ 3:2, 10.2 Hz, C200H), 3.89 (brd,
1H, J ¼ 3:3 Hz, C400H), 4.01 (dd, 1H, J ¼ 2:3, 10.3 Hz,
C30H), 4.37 (brd, 1H, J ¼ 2:3 Hz, C40H), 4.50 (d, 1H,
J ¼ 12:0 Hz, ArCHHO), 4.51 (d, 1H, J ¼ 12:4 Hz,
ArCHHO), 4.52 (s, 2H, ArCH2O), 4.56 (d, 1H, J ¼
12:0 Hz, ArCHHO), 4.61 (brs, 1H, C50H), 4.63, 4.67
(each d, 1H, J ¼ 11:4 Hz, ArCH2O), 4.69 (brs, 1H,
C5H), 4.75 (d, 1H, J ¼ 12:4 Hz, ArCHHO), 4.90 (d, 1H,
J ¼ 3:2 Hz, C100H), 4.94 (brd, 1H, J ¼ 2:4 Hz, C4H),
5.13 (d, 1H, J ¼ 3:3 Hz, C10H), 5.24 (s, 1H, ArCH), 5.34
(d, 1H, J ¼ 2:9 Hz, C1H), 5.67 (dd, 1H, J ¼ 2:9,
11.0 Hz, C2H), 5.71 (dd, 1H, J ¼ 2:4, 11.0 Hz, C3H),
6.76–6.88 (m, 10H, aromatic protons), 7.18 (brd, 2H,
J ¼ 8:7 Hz, aromatic protons), 7.25–7.37 (m, 12H,
aromatic protons), 7.48 (m, 2H, aromatic protons),
7.88 (brdd, 2H, J ¼ 1:3, 8.3 Hz, aromatic protons), 7.94
(brdd, 2H, J ¼ 1:4, 8.6 Hz, aromatic protons); 13C-
NMR (CDCl3) ꢃ: 51.52, 52.52, 55.13, 55.18, 55.18,
55.21, 55.23, 56.16 (each OCH3), 62.85 (C500), 68.35
(C3), 69.12 (C600), 69.57 (C5), 70.16 (C2), 71.23, 71.33,
72.30 (each ArCH2O), 72.63 (C50), 72.78 (ArCH2O),
73.00 (C200), 74.11 (C20), 74.48 (C400), 75.74 (C300),
76.14 (C4), 76.20 (C30), 76.33 (C40), 97.80 (C1), 98.82
(C10), 99.78 (C100), 100.59 (ArC(OR)2), 113.30, 113.45,
113.51, 113.53, 113.72, 127.61, 128.37, 128.49, 128.96,
129.00, 129.16, 129.16, 129.61, 129.72, 129.80, 129.90,
130.39, 130.40, 130.70, 130.83, 131.05, 133.24, 133.30,
158.86, 159.95, 159.07, 159.09, 159.84 (aromatic
carbons), 165.87, 165.87 (each ArC=O), 167.88,
168.06 (each C=O); FABMS (%, rel. int.) m=z: 1403
(4.2, ½M þ Naꢄþ), 121 (100, [CH3OPhCH2]þ), 105
(82, [PhCO]þ); FAB-HRMS: calcd. for C75H80O25Na
½M þ Naꢄþ, 1403.4886; found, m=z 1403.4907.
(d, 1H, J ¼ 12:1 Hz, ArCHHO), 4.65 (d, 1H, J ¼
0:9 Hz, C50H), 4.65, 4.68 (each d, 1H, J ¼ 12:1 Hz,
ArCH2O), 4.68 (brs, 1H, C5H), 4.70 (d, 1H, J ¼ 12:1
Hz, ArCHHO), 4.81 (brs, 1H, C100H), 4.91 (brd, 1H,
J ¼ 2:1 Hz, C4H), 5.06 (d, 1H, J ¼ 3:3 Hz, C10H),
5.35 (d, 1H, J ¼ 2:8 Hz, C1H), 5.66 (dd, 1H, J ¼ 2:8,
10.9 Hz, C2H), 5.70 (dd, 1H, J ¼ 2:1, 10.9 Hz, C3H),
6.78–6.81 (m, 4H, aromatic protons), 6.81–6.89 (m, 4H,
aromatic protons), 7.18–7.21 (m, 4H, aromatic protons),
7.26–7.36 (m, 8H, aromatic protons), 7.48 (m, 2H,
aromatic protons), 7.88 (brdd, 2H, J ¼ 1:3, 8.3 Hz,
aromatic protons), 7.95 (brdd, 2H, J ¼ 1:3, 8.3 Hz,
aromatic protons); 13C-NMR (CDCl3) ꢃ: 51.68, 52.54,
55.12, 55.21, 55.21, 55.25, 56.19 (each OCH3),
62.96 (C600), 68.39 (C3), 69.09 (C400), 69.61 (C5),
69.70 (C500), 70.23 (C2), 71.37 (C50), 72.20, 72.57,
72.63, 73.01 (each ArCH2O) 73.79 (C20), 74.82 (C200 or
300), 76.18 (C30), 76.53 (C4), 77.20 (C200 or 300), 77.81
(C40), 97.82 (C1), 99.21 (C100), 99.25 (C10), 113.61,
113.61, 113.79, 113.82, 128.38, 128.46, 129.00, 129.20,
129.36, 129.56, 129.69, 129.69, 129.74, 129.88, 130.13,
130.28, 130.56, 130.59, 133.22, 133.31, 159.07, 159.09,
159.25, 159.28 (aromatic carbons), 165.86, 165.94
(each ArC=O), 167.99, 168.02 (each C=O); FABMS
(%, rel. int.) m=z: 1285 (0.6, ½M þ Naꢄþ), 121 (100,
[CH3OPhCH2]þ); FAB-HRMS: calcd. for C67H74O24Na
½M þ Naꢄþ, 1285.4468; found, m=z 1285.4490.
A suspension of the product (20.0 mg, 15.8 mmol) in
CH2Cl2 (1.0 ml) was stirred with PhI(OAc)2 (26.2 mg,
81.3 mmol) and TEMPO (1.2 mg, 7.7 mmol) at room
temperature for 30 min. The mixture was poured into
H2O (20 ml) and extracted with AcOEt (15 ml ꢃ 3).
The organic layers were washed with brine (15 ml),
combined, dried over MgSO4, and then concentrated
in vacuo. After diluting with a mixture of 2-methyl-2-
propanol (0.5 ml) and 2-methyl-2-butene (4.4 mg, 63.0
mmol), sodium dihydrogenphosphate dehydrate (14.8
mg, 94.9 mmol) and sodium chlorite (5.7 mg, 63.0 mmol)
were successively added at room temperature. After
stirring for 30 min, the mixture was poured into H2O
(20 ml) and extracted with AcOEt (15 ml ꢃ 3). The
organic layers were washed with brine (15 ml), com-
bined, dried over MgSO4, and then concentrated in
vacuo. After diluting with THF (1.0 ml), an ethereal
solution of diazomethane was added until the yellow
color did not disappear. After concentrating in vacuo,
silica gel column chromatography (AcOEt:hexane =
40:60) of the residue gave 14 (14.0 mg, 68%) as an oil,
Methyl {methyl 2,3-di-O-benzoyl-4-O-{20,30-bis-O-
(4-methoxyphenylmethyl)-40-O-[200,300-bis-O-(4-methoxy-
phenylmethyl)-600-methyl-ꢀ-D-galactopyranuronosyl]-60-
methyl-ꢀ-D-galactopyranuronosyl}-ꢀ-D-galactopyrano-
sid}uronate (14). A solution of 13 (21.1 mg, 15.4 mmol)
in 90% aqueous acetic acid solution (1.0 ml) was stirred
at 50 ꢀC for 20 min. After cooling, the mixture was
concentrated in vacuo. Silica gel column chromatogra-
phy of the residue (AcOEt:hexane = 70:30) gave the
23
corresponding diol (15.4 mg, 79%) as an oil, ½ꢀꢄD
+75.0 (c 0.84, CHCl3); IR (film) cmꢁ1: 3450, 2935,
1730, 1510, 1250, 1095, 1030, 710; 1H-NMR (CDCl3) ꢃ:
2.33 (brd, 1H, J ¼ 8:9 Hz, C600OH), 2.51 (brs, 1H,
C400OH), 3.04 (s, 3H, OCH3), 3.48 (m, 1H, C600HH),
3.49 (s, 3H, OCH3), 3.57–3.65 (m, 3H, C600HH, C200H,
C300H), 3.70, 3.73, 3.75, 3.76, 3.79 (each s, 3H, OCH3),
3.83 (dd, 1H, J ¼ 3:3, 10.3 Hz, C20H), 3.87–3.90
(m, 2H, C400H, C500H), 4.03 (dd, 1H, J ¼ 2:5, 10.3 Hz,
C30H), 4.27 (dd, 1H, J ¼ 0:9, 2.5 Hz, C40H), 4.43
(d, 1H, J ¼ 12:1 Hz, ArCHHO), 4.45 (d, 1H,
J ¼ 10:6 Hz, ArCHHO), 4.51 (d, 1H, J ¼ 12:1 Hz,
ArCHHO), 4.53 (d, 1H, J ¼ 10:6 Hz, ArCHHO), 4.59
23
½ꢀꢄD +67.2 (c 0.75, CHCl3); IR (film) cmꢁ1: 3450,
2935, 1730, 1510, 1250, 1100, 1030, 820, 715; 1H-NMR
(CDCl3) ꢃ: 2.92, 3.49 (each s, 3H, OCH3), 3.54 (dd, 1H,
J ¼ 3:2, 10.0 Hz, C300H), 3.59 (s, 3H, OCH3), 3.62
(d, 1H, J ¼ 3:0, 10.0 Hz, C200H), 3.70, 3.74, 3.74, 3.79,
3.80 (each s, 3H, OCH3), 3.28 (dd, 1H, J ¼ 3:3, 10.5 Hz,
C20H), 4.03 (dd, 1H, J ¼ 2:2, 10.5 Hz, C30H), 4.23 (brd,
1H, J ¼ 3:2 Hz, C400H), 4.25 (brd, 1H, J ¼ 2:2 Hz,
C40H), 4.39 (d, 1H, J ¼ 12:3 Hz, ArCH2O), 4.43 (s,
2H, ArCH2O), 4.52 (d, 1H, J ¼ 12:3 Hz, ArCH2O), 4.55

Synthesis of D-Trigalacturonic Acid Methylglycoside
2047
(brs, 1H, C50H), 4.57 (d, 1H, J ¼ 11:8 Hz, ArCH2O),
4.62 (d, 1H, J ¼ 12:7 Hz, ArCH2O), 4.68 (brs, 1H, C5H),
4.73 (d, 1H, J ¼ 11:8 Hz, ArCH2O), 4.80 (d, 1H,
J ¼ 12:7 Hz, ArCH2O), 4.85 (brs, 1H, C500H), 4.88 (d,
1H, J ¼ 3:3 Hz, C100H), 4.91 (brd, 1H, J ¼ 2:3 Hz, C4H),
5.05 (d, 1H, J ¼ 3:3 Hz, C10H), 5.35 (d, 1H, J ¼ 3:0 Hz,
C1H), 5.67 (dd, 1H, J ¼ 3:0, 11.0 Hz, C2H), 5.71 (dd,
1H, J ¼ 2:3, 11.0 Hz, C3H), 6.72 (brd, 2H, J ¼ 8:4 Hz,
aromatic protons), 6.83–6.90 (m, 6H, aromatic protons),
7.11 (brd, 2H, J ¼ 8:5 Hz, aromatic protons), 7.19 (brd,
2H, J ¼ 8:5 Hz, aromatic protons), 7.24–7.38 (m, 8H,
aromatic protons), 7.48 (m, 2H, aromatic protons), 7.81
(brd, 2H, J ¼ 7:5 Hz, aromatic protons), 7.94 (brd, 2H,
J ¼ 7:5 Hz, aromatic protons); 13C-NMR (CDCl3) ꢃ:
51.49, 52.08, 52.59, 55.07, 55.17, 55.20, 55.24, 56.19
(each OCH3), 68.27 (C400), 68.36 (C3), 69.47 (C5), 70.08
(C2), 70.42 (C500), 71.08 (C50), 71.82 (C20), 71.94, 72.16,
72.61, 72.63 (each ArCH2O), 72.96 (C200), 75.74 (C4),
75.87 (C30), 77.21 (C300), 77.21 (C40), 97.79 (C1), 98.64
(C10), 99.20 (C100), 113.58, 113.68, 113.70, 113.79,
128.39, 128.52, 128.95, 129.18, 129.35, 129.47, 129.54,
129.69, 129.72, 129.87, 130.03, 130.14, 130.26, 130.34,
133.27, 133.31, 159.01, 159.01, 159.15, 159.29 (aro-
matic carbons), 165.75, 165.82 (each ArC=O), 167.76,
168.09, 168.47 (each C=O); FABMS (%, rel. int.) m=z:
1313 (8.4, ½M þ Naꢄþ), 121 (100, [CH3OPhCH2]þ), 105
(76, [PhCO]þ); FAB-HRMS: calcd. for C68H74O25Na
½M þ Naꢄþ, 1313.4417; found, m=z 1313.4409.
129.63, 129.81, 130.35, 130.48, 130.65, 130.79, 134.65,
134.71 (aromatic carbons), 167.32, 167.40 (each
ArC=O), 169.84, 170.02, 171.67 (each C=O); FABMS
(%, rel. int.) m=z: 833 (50, ½M þ Naꢄþ), 121 (86,
[PhCOO]þ), 105 (100, [PhCO]þ); FAB-HRMS: calcd.
for C36H42O21Na ½M þ Naꢄþ, 833.2038; found, m=z
833.2101. The product (10.3 mg, 12.7 mmol) was stirred
in a mixture of THF (1.0 ml) and a 0.3% NaOH aqueous
solution (1.5 ml) at room temperature for 30 min. The
mixture was passed through an ion-exchange column
(DOWEX 50W, Hþ form). Lyophilization of the eluent
24
gave 3 (7.0 mg, 98%) as an amorphous powder, ½ꢀꢄD
1
+107.7 (c 1.25, H2O); H-NMR (D2O) ꢃ: 3.25 (s, 3H,
OCH3), 3.56 (dd, 1H, J ¼ 3:9, 10.3 Hz, C200H), 3.60
(dd, 1H, J ¼ 3:9, 10.7 Hz, C20H), 3.67 (dd, 1H, J ¼ 3:8,
10.6 Hz, C2H), 3.76 (dd, 1H, J ¼ 3:4, 10.3 Hz, C300H),
3.82 (dd, 1H, J ¼ 3:1, 10.6 Hz, C3H) 3.87 (dd, 1H,
J ¼ 3:0, 10.7 Hz, C30H), 4.16 (dd, 1H, J ¼ 1:4, 3.4 Hz,
C400H), 4.29 (brd, 1H, J ¼ 3:0 Hz, C40H), 4.30 (dd, 1H,
J ¼ 0:7, 3.1 Hz, C4H), 4.47 (d, 1H, J ¼ 0:7 Hz, C5H),
4.77 (d, 1H, J ¼ 3:8 Hz, C1H), 4.88 (d, 1H, J ¼ 3:9 Hz,
C100H), 4.91 (d, 1H, J ¼ 1:4 Hz, C500H), 4.93 (brs, 1H,
C50H), 4.94 (d, 1H, J ¼ 3:9 Hz, C10H); 13C-NMR (D2O)
ꢃ: 58.22 (OCH3), 70.15 (C2), 70.35 (C200), 70.37 (C20),
70.57 (C30), 70.63 (C3), 71.24 (C300), 72.03 (C5), 72.48
(C400), 72.72 (C50), 73.45 (C500), 80.69 (C4), 80.96 (C40),
102.12 (C1), 102.46 (C100), 102.59 (C10), 174.51,
174.72, 175.35 (each C=O);ꢁnegative-FABMS (%, rel.
int.) m=z: 599 (4.5, ½M ꢁ Hꢄ ), 148 (100, [C5H8O5]ꢁ);
negative-FAB-HRMS: calcd. for C19H27O19 ½M ꢁ Hꢄꢁ,
559.1147; found, m=z 559.1169.
Methyl O-(ꢀ-D-galactopyranuronosyl)-(10!4)-O-(ꢀ-
D-galactopyranuronosyl)-(100!40)-ꢀ-D-galactopyranosi-
duronic acid (3). A suspension of 14 (26.4 mg, 20.4
mmol) in a mixture of CH2Cl2 (1.0 ml) and H2O (0.1 ml)
was vigorously stirred with DDQ (23.2 mg, 102.2 mmol)
at room temperature for 6 h. After concentrating, silica
gel column chromatography (acetone:CH2Cl2 = 80:20)
920-MHz NMR measurements. Sample solutions for
NMR measurements were prepared by dissolving in
99.9% D2O, the sample pH not being adjusted. Shigemi
NMR sample tubes matched with D2O were used. 920-
MHz NMR spectra were measured by a JEOL spec-
trometer at Institute for Molecular Science, Okazaki,
Japan. The sample was not spun, and the spectra were
recorded at a temperature of 298 K. The water signal
was suppressed by DANTE (Delay Alternating with
Nutation for Tailored Exitation) method.21) One-dimen-
sional 1H-NMR experiments were performed with a
spectral width of 11,510.12891 Hz, 64 K data points and
8 scans. Both the two-dimensional ROESY14,15) and
NOESY16) spectra were recorded in the phase-sensitive
mode, with a mixing time of 300 msec. and with 2048 ꢃ
512 data points, and were zero-filled to yield 2048 ꢃ
2048 data matrices. The two-dimensional {13C}-1H
HSQC22) spectrum was recorded without 13C decoupling
during the acquisition period and with 2048 ꢃ 128 data
points. The number of scans for all spectra was 8. Time
domain data in both dimensions were multiplied by a
sine bell squared function. All 2D NMR spectra were
processed by NMRPipe software,23) and the signals were
assigned by Sparky 3 (Goddard, T., and Kneller, D. G.,
SPARKY 3, University of California, San Francisco, CA,
USA) run under Windows XP.
of the residue gave the MPM deprotected pentaol
23
(14.9 mg, 90%) as an oil, ½ꢀꢄD
+32.4 (c 0.59,
CH3OH); IR (film) cmꢁ1: 3420, 2925, 1730, 1580,
1450, 1275, 1105, 1020, 715; 1H-NMR (CD3OD) ꢃ:
3.12, 3.50 (each s, 3H, OCH3), 3.59 (dd, 1H, J ¼ 3:8,
10.3 Hz, C200H), 3.66 (dd, 1H, J ¼ 3:8, 10.7 Hz, C20H),
3.68 (dd, 1H, J ¼ 3:4, 10.3 Hz, C300H), 3.73, 3.84 (each
s, 3H, OCH3), 4.02 (dd, 1H, J ¼ 2:9, 10.7 Hz, C30H),
4.15 (dd, 1H, J ¼ 1:4, 3.4 Hz, C400H), 4.32 (brd, 1H,
J ¼ 2:9 Hz, C40H), 4.74 (d, 1H, J ¼ 3:8 Hz, C100H), 4.76
(brs, 1H, C50H), 4.84 (brs, 1H, C5H), 4.91 (brd, 1H,
J ¼ 3:0 Hz, C4H), 5.02 (d, 1H, J ¼ 3:8, C10H), 5.05 (d,
1H, J ¼ 1:4 Hz, C500H), 5.27 (d, 1H, J ¼ 3:5 Hz, C1H),
5.58 (dd, 1H, J ¼ 3:0, 11.0 Hz, C3H), 5.68 (dd, 1H,
J ¼ 3:5, 11.0 Hz, C2H), 7.34 (m, 4H, aromatic protons),
7.52 (m, 2H, aromatic protons), 7.84 (brd, 2H, J ¼
7:4 Hz, aromatic protons), 7.90 (brd, 2H, J ¼ 7:1 Hz,
aromatic protons); 13C-NMR (CD3OD) ꢃ: 52.47, 52.59,
53.19, 56.55 (each OCH3), 69.33 (C20), 69.40 (C2),
69.40 (C30), 69.62 (C200), 70.70 (C300), 70.97 (C5), 71.81
(C400), 71.91 (C3), 72.48 (C50), 72.81 (C500), 79.00 (C4),
80.50 (C40), 99.20 (C1), 102.18 (C100), 102.92 (C10),

2048
K. YAMAMOTO et al.
der Marel, G. A., Thioglycuronides: synthesis and
application in the assembly of acidic oligosaccharide.
Org. Lett., 13, 2165–2168 (2004).
Acknowledgments
Part of this work was supported by Grants-in-Aid for
Scientific Research (no. 19380064) from Japan Society
for Promotion of Science and the ‘‘Nanotechnology
Network Project’’ of the Ministry of Education, Culture,
Sports, Science and Technology (MEXT), and the Joint
Studies Program of the Institute for Molecular Science
(IMS), Japan. The authors thank Professor Yasuhiro
Uozumi and Ms. Michiko Nakano at Institute for
Molecular Science (Okazaki, Japan) for supporting
measurement of 920-MHz NMR data.
10) Khiar, N., and Martin-Lomas, M., A highly convergent
synthesis of the tetragalactose moiety of the glycosyl
phosphatidyl inositol anchor of the variant surface
glycoprotein of Trypanosoma brucei. J. Org. Chem.,
60, 7017–7021 (1995).
11) Wong, C.-H., Moris-Varas, F., Hung, S.-C., Marron,
T. G., Lin, C.-C., Gong, K. W., and Weitz-Schmidt, G.,
Small molecules as structural and functional mimics of
sialyl Lewis X tetrasaccharide in selectin inhibition:
a remarkable enhancement of inhibition by additional
negative charge and/or hydrophobic group. J. Am.
Chem. Soc., 119, 8152–8158 (1997).
12) Schmidt, R. R., and Jung, K.-H., Oligosaccharide
synthesis with trichloroacetoimidates. In ‘‘Preparative
Carbohydrate Chemistry,’’ ed. Hanessian, S., Marcel
Dekker Inc., New York, pp. 283–312 (1997).
13) Claridge, T. D. W., High-resolution NMR techniques in
organic chemistry. In ‘‘Tetrahedron Organic Chemistry
Series,’’ eds. Baldwin, J. E., and Williams, R. M.,
Elsevier, Oxford (1999).
References
1) ‘‘Comprehensive Glycoscience from Chemistry to
Systems Biology’’ Vol. 2, eds. Kamerling, J. P., Boons,
G.-J., Lee, Y. C., Suzuki, A., Taniguchi, N., and
Voragen, A. G. J., Elsevier, Oxford (2007).
2) ‘‘Comprehensive Glycoscience from Chemistry to
Systems Biology’’ Vol. 3, eds. Kamerling, J. P., Boons,
G.-J., Lee, Y. C., Suzuki, A., Taniguchi, N., and
Voragen, A. G. J., Elsevier, Oxford (2007).
3) ‘‘Comprehensive Glycoscience from Chemistry to
Systems Biology’’ Vol. 4, eds. Kamerling, J. P., Boons,
G.-J., Lee, Y. C., Suzuki, A., Taniguchi, N., and
Voragen, A. G. J., Elsevier, Oxford (2007).
14) Bothner-By, A. A., Stephens, R. L., Lee, J.-M., Warren,
C. D., and Jeanloz, R. W., Structure determination of a
tetrasaccharide: transient nuclear Overhauser effects in
the rotating frame. J. Am. Chem. Soc., 106, 811–813
(1984).
´
´
4) Bols, M., Lopez, O., and Ortega-Caballero, F., Glyco-
sidase inhibitors; structure, activity, synthesis, and
medical relevance. In ‘‘Comprehensive Glycoscience
from Chemistry to Systems Bbiology’’ Vol. 1, eds.
Kamerling, J. P., Boons, G.-J., Lee, Y. C., Suzuki, A.,
Taniguchi, N., and Voragen, A. G. J., Elsevier, Oxford,
pp. 815–884 (2007).
15) Bax, A., and Davis, D. G., Practical aspects of two-
dimensional transverse NOE spectroscopy. J. Magn.
Reson., 63, 207–213 (1985).
16) Jeener, J., Meier, B. N., Bachmann, P., and Ernst, R. R.,
Investigation of exchange processes by two-dimensional
NMR spectroscopy. J. Chem. Phys., 71, 4546–4553
(1979).
5) Miyairi, K., Okuno, T., and Sawai, K., Purification and
properties of endopolygalacturonase I from Stereum
purpureum, a factor inducing silver-leaf symptoms
on apple trees. Agric. Biol. Chem., 49, 1111–1118
(1985).
6) Shimizu, T., Nakatsu, T., Miyairi, K., Okuno, T., and
Kato, H., Crystallization and preliminary X-ray study
of endopolygalacturonase from the pathogenic fungus
Stereum purpureum. Acta Crystallogr. D, 57, 1171–1173
(2001).
7) Yamamoto, K., Watanabe, N., Matsuda, H., Oohara, K.,
Araya, T., Hashimoto, M., Miyairi, K., Okazaki, I.,
Saito, M., Shimizu, T., Kato, H., and Okuno, T., Design,
synthesis, and enzymatic property of a sulfur-substituted
analogue of trigalacturonic acid. Bioorg. Med. Chem.
Lett., 15, 4932–4935 (2005).
17) Neuhaus, D., and Williamson, M., ‘‘The Nuclear
Overhauser Effect in Structural and Conformational
Analysis,’’ VCH Publishers, New York (1989).
18) Leach, A. R., ‘‘Molecular Modelling: Principles and
Applications’’ second edition, Person Education Limited,
Harlow, pp. 26–105 (2001).
19) Leach, A. R., ‘‘Molecular Modelling: Principles and
Applications’’ second edition, Person Education Limited,
Harlow, pp. 457–506 (2001).
20) Bingham, R. C., Dewar, M. J. S., and Lo, D. H., Ground
states of molecules. XXV. MINDO/3. Improved version
of the MINDO semiempirical SCF-MO method. J. Am.
Chem. Soc., 97, 1285–1293 (1975).
21) Morris, G. A., and Freeman, R., Selective exitation in
Fourier transform nuclear magnetic resonance. J. Magn.
Reson., 29, 433–462 (1978).
8) De Mico, A., Margarita, R., Parlanti, L., Vescovi, A.,
and Piancatelli, G., A versatile and highly selective
hypervalent iodine (III)/2,2,6,6-tetramethyl-1-piperidi-
nyloxyl-mediated oxidation of alcohols to carbonyl
compound. J. Org. Chem., 62, 6974–6977 (1997).
22) Bodenhausen, G., and Ruben, D. J., Natural abundance
nitrogen-15 NMR by enhanced heteronuclear spectros-
copy. Chem. Phys. Lett., 69, 185–189 (1980).
23) Delaglio, F., Grzesiek, S., Vuister, G., Zhu, G., Pfeifer,
J., and Bax, A., NMRPipe: a multidimensional spectral
processing system based on UNIX pipes. J. Biol. NMR,
6, 277–293 (1995).
´
9) van den Bos, L. J., Codee, J. D. C., van der Toorn, J. C.,
Boltje, T. J., van Boom, J. H., Overkleeft, H. S., and van