Synthesis of new Galβ1→4Fuc segments useful for biological investigations

Article abstract of DOI:10.1248/cpb.59.1307

Useful segments (1, 2) for chemical probes embedded in a Galβ1→4Fuc unit were designed and prepared for characterizing sugar-binding proteins in Caenorhabditis elegans. Segment 1 with an amino group terminus was used as a recognition unit in affinity chro

Full text of DOI:10.1248/cpb.59.1307

October 2011
Note
Chem. Pharm. Bull. 59(10) 1307—1310 (2011)
1307
b →
Synthesis of New Gal 1 4Fuc Segments Useful for Biological
Investigations
Kazusa NISHIYAMA,a Atsushi YAMADA,a Tomoharu TAKEUCHI,a,b Yoichiro ARATA,b Ken-ichi KASAI,a
a
a
,a
*
Tetsuta OSHITARI, Hideaki NATSUGARI, and Hideyo TAKAHASHI
a Teikyo University School of Pharmaceutical Sciences; 1091–1 Sagamiko, Sagamihara, Kanagawa 252–5195, Japan: and
b Faculty of Pharmaceutical Sciences, Josai University; 1–1 Keyakidai, Sakado, Saitama 350–0295, Japan.
Received July 5, 2011; accepted July 25, 2011; published online July 26, 2011
Useful segments (1, 2) for chemical probes embedded in a Galb1→4Fuc unit were designed and prepared
for characterizing sugar-binding proteins in Caenorhabditis elegans. Segment 1 with an amino group terminus
was used as a recognition unit in affinity chromatography. It was revealed that some proteins (annexins and
galectins) in C. elegans have an affinity for Galb1→4Fuc.
Key words galectin; annexin; chemical probe; affinity chromatography
Galectins constitute the carbohydrate-recognition domain derivative containing immobilized Galb1→4Fuc and at-
responsible for b-galactoside binding.1—3) Galectins bind to tempted to identify proteins adsorbed by this adsorbent. We
cell-surface and extracellular-matrix glycans and thereby af- found that various galectins and annexins have an affinity for
fect a variety of cellular processes (e.g., immunity and tumor Galb1→4Fuc.
metastasis).4) It is known that a Galb1→4GlcNAc (N-acetyl-
lactosamine) unit is the endogenous glycoepitope recognized Results and Discussion
by vertebrate galectins.5—8) However, the existence of a gly-
For the synthesis of a Galb1→4Fuc derivative with an
can containing N-acetyllactosamine units has not been con- amino terminus (1), we planned to utilize commercially
firmed in Caenorhabditis elegans.9—14) In the course of in- available D-mannitol as a water-soluble spacer moiety (Chart
vestigation of endogenous counterpart glycans of galectins of 1). Primary hydroxyl groups of D-mannitol were protected as
C. elegans, we found that the motif recognized by LEC-6, TBDMS (tert-butyldimethylsilyl) ethers to give the tetraol
one galectin of C. elegans, is Galb1→4Fuc, instead of 3.17) Peracetylation of 3, followed by selective deprotection
Galb1→4GlcNAc.15—17) Other galectins of C. elegans, LEC- of the TBDMS group, afforded the alcohol 5.17) To introduce
1 and LEC-10, were also found to have an affinity for the amino group, 5 was converted into a tosylate (6), which
Galb1→4Fuc.16,18) These findings indicate that the was subjected to azidation to give 7. The TBDMS group of 7
Galb1→4Fuc unit is a principal disaccharide recognized by was deprotected to form the agent 8. In the presence of TM-
C. elegans galectins. Therefore, it appeared possible that SOTf (trimethylsilyltrifluoromethane sulfonate), coupling of
Galb1→4Fuc would serve as a recognition motif for other the acceptor 8 with the Galb1→4Fuc donor 917) proceeded
sugar-binding proteins in C. elegans. Although the sugar- smoothly to give only the b-linked product (10). Finally, de-
binding abilities of various families of carbohydrate-binding protection of acetyl groups and successive reduction afforded
proteins in C. elegans, such as galectins, C-type lectins, an- the target 1.
nexins etc., have been demonstrated,6,8,19—21) the Galb1→
The synthesis of sulfanylethyl Galb1→4Fuc 2 started
4Fuc-binding ability of most of these proteins has not been from Galb1→4Fuc 917) by glycosylation with 2,2ꢀ-dithiodi-
reported. Synthetic probes containing Galb1→4Fuc are re- ethanol. Compared with the previous synthesis of thioalkyl
garded as good tools for characterizing sugar-binding pro- glycosides,22,23) using the disulfide acceptor 2,2ꢀ-dithiodi-
teins in C. elegans. In the present study, we describe the ethanol provides easier access to the thiol terminus. The re-
preparation of Galb1→4Fuc derivatives with an amino group action proceeded smoothly to provide the disulfide (12) b-se-
(1) or mercapto group (2) via a linker, which could be uti- lectively. Reduction of 12 afforded the thiol (13) in good
lized as recognition units in probes for sugar-binding pro- yield. Deacylation with sodium methoxide in MeOH af-
teins (Fig. 1). Using compound 1, we prepared a Sepharose forded the desired target (2) efficiently (Chart 2).
After we synthesized the target segments (1, 2), we used
compound 1 as a recognition unit in affinity chromato-
graphy.24) Compound 1 was immobilized onto NHS-activated
Sepharose (GE Healthcare, St. Giles, U.K.), and the immobi-
lized Galb1→4Fuc adsorbent was packed in a disposable
column. The extract of C. elegans was applied to this column
to capture glycoproteins interacting with the Galb1→4Fuc
unit. Three annexins (NEX-1, -2, -3) in addition to galectins
(LEC-1, -2, -4, -6, -9, -10, -12) were found to have affinity
for Galb1→4Fuc. Therefore, the immobilized Galb1→4Fuc
column proved to be useful for the capture of carbohydrate-
binding proteins that interact with this unique sugar struc-
ture.
Fig. 1. Structures of the Galb1→4Fuc Segment with an Amino Terminus
(1) or a Mercapto Terminus (2)
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: hide-tak@pharm.teikyo-u.ac.jp

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Vol. 59, No. 10
Reagents and conditions: (a) TBDMSCl, imidazole, DMF, rt, 79%; (b) Ac2O, pyridine, rt, 88%; (c) 1%
I2-MeOH solution, rt, 42%; (d) p-TsCl, DMAP, pyridine, 60 °C, 72%; (e) NaN3, DMF, 60 °C, 92%; (f) HF-
pyridine, pyridine-THF, rt, 60%; (g) 8, TMSOTf, CH2Cl2, MS4A, 0 °C, 70%; (h) NaOMe, MeOH, rt, 67%;
(i) H2, Pd(OH)2, MeOH, rt, 99%.
Chart 1. Synthesis of Compound 1
Reagents and conditions: (a) 2,2ꢀ-Dithiodiethanol, TMSOTf, CH2Cl2, MS4A, 0 °C, 84%; (b) Zn, HCl, EtOH,
CH2Cl2, rt, 88%; (c) NaOMe, MeOH, rt, 96%.
Chart 2. Synthesis of Compound 2
methods. The NMR spectra (1H and 13C) were determined on a JEOL
600 MHz (ECP-600) or 400 MHz (AL-400) spectrometer, using CDCl3 (with
tetramethylsilane (TMS) for 1H-NMR and chloroform-d for 13C-NMR as the
internal reference) solution, unless the otherwise noted. Chemical shifts are
given in parts per million (ppm) downfield from tetramethylsilane as an in-
ternal standard and coupling constants (J) are reported in hertz (Hz). Split-
ting patterns are abbreviated as follows: singlet (s), doublet (d), triplet (t),
multiplet (m), broad (br). High resolution-mass spectra (HR-MS) were ob-
tained on LCMS-IT-Time-of-Flight (TOF) (Shimadzu, Kyoto, Japan) for
electrospray ionization (ESI). Sodium trifluoroacetate (TFA-Na) was used as
internal standard for high resolution MS. Optical rotations were determined
using a DIP-370 (Shimadzu, Kyoto, Japan) digital polarimeter in 100-mm
cells and the sodium D line (589 nm) at room temperature in the solvent and
concentration indicated. Infrared spectra (IR) were recorded on a JASCO
Fourier Transform (FT)/IR-410 spectrometer using sodium chloride plates
or potassium bromide pellets. Absorbance frequencies are recorded in recip-
rocal centimeters (cmꢁ1). Analytical thin layer chromatography was carried
out using Merck silica gel 60 F254. Column chromatography was carried out
on Wakogel C-300 (45—60 mm), NH silicagel (Chromatorex® FujiSilysia
Because the terminus sulfanyl group of segment 2 is suit-
able for modification with amino acid residues in proteins, it
could be used as a tool for preparing neoglycoconjugates.25)
An in-depth examination of this will be reported by our
group in the future.
Conclusion
We synthesized two types of Galb1→4Fuc-embedded seg-
ments (1, 2) that could be utilized as recognition units in
probes for sugar-binding proteins. We prepared an immobi-
lized Galb1→4Fuc adsorbent from compound 1 and identi-
fied proteins that have an affinity for Galb1→4Fuc.
Experimental
General Procedures All reactions sensitive to air or moisture were con-
ducted under an argon atmosphere. Materials were obtained from commer-
cial suppliers. All anhydrous solvents were purified according to standard

October 2011
1309
Chemical, 100—200 mesh). Reverse phase Column chromatography utilized
ODS-SS10200T.
(3H, m, H6, H6ꢀ, Fuc H5), 3.61 (1H, m, Gal H5), 3.41 (1H, dd, Jꢂ3.6, 13.8,
H1), 3.22 (1H, dd, Jꢂ6.0, 13.8, H1ꢀ), 3.15 (2H, m, Gal H6, Gal H6ꢀ), 2.12
(3H, s, –COCH3), 2.07 (3Hꢃ2, s, –COCH3), 2.06 (3H, s, –COCH3), 2.04
(3H, s, –COCH3), 1.97 (3H, s, –COCH3), 1.93 (3H, s, –COCH3), 1.82 (3H,
s, –COCH3), 1.37 (1H, d, Jꢂ6.0, Fuc Me); 13C-NMR (150 MHz, CDCl3) d:
170.2, 170.1, 170.0ꢃ2, 169.8, 169.7, 169.3, 168.9, 166.0, 164.9, 133.2ꢃ2,
133.0ꢃ2, 130.1ꢃ2, 129.8ꢃ2, 128.3ꢃ2, 128.2ꢃ2, 101.8 (Fuc C1), 100.4
(Gal C1), 75.4 (Fuc C4), 73.1 (Fuc C3), 70.8 (Gal C3), 70.5 (Gal C5), 70.2
(C6), 69.2 (Gal C2), 68.8 (C5), 68.6 (C3), 68.6 (Fuc C4), 68.5 (C2), 67.9
(C4), 66.7 (Gal C4), 65.4 (Fuc C5), 60.3 (Gal C6), 50.5 (C1), 20.8, 20.7,
20.6, 20.6, 20.6, 20.5, 20.5, 20.5, 16.0 (Fuc C6); IR (neat) 3025, 2984, 2939,
2106, 1748, 1371, 1282, 1219, 1111, 1071, 757, 714 cmꢁ1; HR-MS (ESI)
Calcd for C48H57O24N3 [MꢄNa]ꢄ: 1082.3224 Found 1082.3219.
b-D-Galactopyranosyl-(1→4)-b-L-fucopyranosyl-(1→6)-1-deoxy-1-
azido-D-mannitol (11) To a solution of 10 (89 mg, 0.084 mmol) and
NaOMe (68 mg, 1.26 mmol) in MeOH (0.28 ml) was stirred at room temper-
ature for 12 h. Then the mixture was concentrated, and the residue was puri-
fied by column chromatography (CH2Cl2/MeOHꢂ1 : 1) to give 11 (29 mg,
0.06 mmol, 67%) as a clear oil. [a]D23 ꢁ1.2 (cꢂ0.3 in MeOH); 1H-NMR
(600 MHz, D2O) d: 4.32 (2H, m, Gal H1, Fuc H1), 3.92 (1H, m, H3), 3.88
(1H, d, Jꢂ3.0, Fuc H4), 3.74 (5H, m, H2, H4, H5, Gal H4, Fuc H5), 3.56
(7H, m, Gal H6, Gal H6ꢀ, Gal H5, Gal H3, Fuc H2, Fuc H3, H1), 3.40 (2H,
m, GalH2, H6), 3.36 (1H, dd, Jꢂ6.6, 13.2, H1ꢀ), 3.29 (1H, dd, Jꢂ6.6, 12.6,
H6ꢀ), 1.21 (3H, d, Jꢂ6.6, Fuc Me); 13C-NMR (150 MHz, D2O) d: 103.4,
104.0, 79.8, 75.8, 73.0, 72.7, 72.0, 71.9, 71.5, 70.3, 70.2, 69.9, 69.3, 69.2,
67.2, 61.8, 54.6, 16.0; HR-MS (ESI) Calcd for C18H33O14N3 [MꢁH]ꢁ:
514.1890 Found 514.1883.
2,3,4,5-Tetra-O-acetyl-6-O-(t-butyldimethylsilyl)-1-O-(p-toluenesul-
fonyl)-D-mannitol (6) To a solution of 5 (1.2 g, 2.58 mmol) in pyridine
(26 ml) were added p-TsCl (2.5 g, 12.9 mmol) and DMAP (158 mg,
1.29 mmol) at room temperature. The mixture was stirred at 60 °C for 12 h
and water was added. After extraction with EtOAc, the organic phase was
dried over Na2SO4, and the solvent was removed. The residue was purified
by column chromatography (eluent hexane : EtOAcꢂ4 : 1) to give 6 (1.2 g,
1.88 mmol, 72%) as a clear yellow oil. [a]D24 ꢁ19.9 (cꢂ1.0 in CHCl3); H-
1
NMR (600 MHz, CDCl3) d: 7.76 (2H, d, Jꢂ8.4), 7.33 (2H, d, Jꢂ8.4), 5.42
(2H, m, H3, H4), 4.98 (1H, m, H5), 4.90 (1H, m, H2), 4.16 (1H, dd, Jꢂ3.0,
10.8, H1), 4.02 (1H, dd, Jꢂ4.2, 10.8, H1ꢀ), 3.69 (1H, dd, Jꢂ3.6, 11.4, H6),
3.57 (1H, dd, Jꢂ4.8, 11.4, H6ꢀ), 2.44 (3H, s, –PhCH3), 2.04 (3H, s,
–COCH3), 2.03 (3H, s, –COCH3), 2.02 (3H, s, –COCH3), 1.99 (3H, s,
–COCH3), 0.85 (9H, s, –Si(CH3)2C(CH3)3), 0.00 (3H, s, –Si(CH3)2C(CH3)3),
ꢁ0.01 (3H, s, –Si(CH3)2C(CH3)3); 13C-NMR (150 MHz, CDCl3) d: 170.0,
169.7, 169.4 (ꢃ2), 145.0, 132.6, 129.8, 128.1, 70.6 (C5), 67.6 (C2), 67.5
(C3), 67.4 (C4), 66.9 (C1), 61.3 (C6), 25.6 (ꢃ3, –Si(CH3)2C(CH3)3), 21.0,
20.7, 20.6, 20.6, 18.1 (–Si(CH3)2C(CH3)3), ꢁ5.6 (ꢃ2, –Si(CH3)2C(CH3)3);
IR (neat) 2955, 2930, 2857, 1752, 1370, 1217, 1030, 837 cmꢁ1; HR-MS
(ESI) Calcd for C27H42O12SSi [MꢄNa]ꢄ: 641.2058 Found 641.2043.
2,3,4,5-Tetra-O-acetyl-1-azide-6-O-(t-butyldimethylsilyl)-1-deoxy-D-
mannitol (7) To a solution of 6 (793 mg, 1.28 mmol) in DMF (12.8 ml)
was added NaN3 (125 mg, 1.92 mmol) at room temperature. The suspension
was stirred for 12 h at 60 °C. The mixture was diluted with H2O and was ex-
tracted with EtOAc. The combined organic layers were dried (MgSO4) and
evaporated in vacuo. The crude product was purified by column chromatog-
raphy (hexane : EtOAcꢂ1 : 3) to give 7 (574 mg, 1.17 mmol, 92%) as a clear
oil. [a]D24 ꢁ23.7 (cꢂ1.0 in CHCl3); 1H-NMR (600 MHz, CDCl3) d: 5.43
(2H, m, H3, H4), 5.01 (1H, m, H2), 4.95 (1H, m, H5), 3.69 (1H, dd, Jꢂ3.6,
11.4, H6), 3.58 (1H, dd, Jꢂ5.4, 11.4, H6ꢀ), 3.47 (1H, dd, Jꢂ3.6, 13.2, H1),
3.26 (1H, dd, Jꢂ5.6, 13.2, H1ꢀ), 2.08 (3H, s, –COCH3), 2.07 (3H, s,
–COCH3), 2.05 (3H, s, –COCH3), 2.04 (3H, s, –COCH3), 0.86 (9H, s,
–Si(CH3)2C(CH3)3), 0.01 (3H, s, –Si(CH3)2C(CH3)3), 0.00 (3H, s,
–Si(CH3)2C(CH3)3); 13C-NMR (150 MHz, CDCl3) d: 170.7, 169.9, 169.8,
169.5, 70.7 (C5), 68.7 (C2), 68.3 (C3), 67.7 (C4), 61.5 (C6), 50.7 (C1), 25.7
(ꢃ3, –Si(CH3)2C(CH3)3), 21.0, 20.8, 20.7, 20.7, 18.1 (–Si(CH3)2C(CH3)3),
ꢁ5.6 (ꢃ2, –Si(CH3)2C(CH3)3); IR (neat) 2955, 2931, 2858, 2106, 1753,
b-D-Galactopyranosyl-(1→4)-b-L-fucopyranosyl-(1→6)-1-deoxy-1-
amino-D-mannitol (1) To a solution of 11 (51 mg, 0.10 mmol) in MeOH
(0.3 ml) was added Pd(OH)2 (20.0 mg). The reaction was stirred for 2 h
under a H2 atmosphere and then filtered through cotton. Then the mixture
was concentrated, and the residue was purified by column chromatography
(EtOAc/2-propanol/H2Oꢂ1 : 1 : 1) to give 1 (47 mg, 0.10 mmol, 99%) as a
clear oil. [a]D23 0.9 (cꢂ1.0 in H2O); H-NMR (600 MHz, D2O) d: 8.39 (2H,
1
s, NH2), 4.40 (2H, m, Gal H1, Fuc H1), 4.01 (1H, m, H3), 3.96 (1H, br, Fuc
H4), 3.86 (1H, br, Gal H4), 3.81 (4H, m, H2, H4, H5, Fuc H5), 3.65 (6H, m,
Gal H6, Gal H6ꢀ Gal H5, Gal H3, Fuc H2, Fuc H3), 3.47 (2H, m, Gal H2,
H6), 3.36 (2H, m, H6ꢀ, H1), 3.00 (1H, dd, Jꢂ9.0, 12.6, H1ꢀ), 1.28 (3H, d,
Jꢂ6.6, Fuc Me); 13C-NMR (150 MHz, D2O) d: 103.4, 104.0, 79.8, 75.8,
73.1, 72.7, 72.0, 71.8, 71.6, 71.5, 69.8, 69.3, 69.2, 67.8, 67.3, 61.8, 43.2,
16.0; HR-MS (ESI) Calcd for C18H35O14N1 [MꢁH]ꢁ: 488.1985 Found
488.1975.
1371, 1220, 1062, 1033, 839, 779 cmꢁ1
; HR-MS (ESI) Calcd for
C20H35O9N3Si [MꢄNa]ꢄ: 512.2035 Found 512.2044.
2,3,4,5-Tetra-O-acetyl-1-azido-1-deoxy-D-mannitol (8) 7 (270 mg,
0.55 mmol) was dissolved in pyridine (3.7 ml) and tetrahydrofuran (THF)
(1.8 ml), then 30% HF-pyridine solution (1.84 ml) was added. After being
stirred for 10 min, the mixture was poured into saturated NaHCO3, and the
desired compound was extracted with EtOAc, dried over Na2SO4 and evapo-
rated in vacuo. The crude product was purified by column chromatography
(Et2O : CH2Cl2ꢂ1 : 1) to give 8 (124 mg, 0.33 mmol, 60%) as a clear oil.
Bis (2-Ethyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-2,3-di-
O-benzoyl-b-L-fucopyranoside) Disulfide (12) To the solution of
9
(822 mg, 0.97 mmol) and 2,2ꢀ-dithiodiethanol (50 mg, 0.33 mmol) in dry
CH2Cl2 (3.5 ml), containing activated molecular sieves 4A (40 mg), was
stirred at 0 °C. TMSOTf (0.04 ml, 0.16 mmol) was added, and the mixture
was stirred for 1 h, and then neutralized with saturated aqueous NaHCO3.
The mixture was extracted with CH2Cl2, and the organic phase was dried, fil-
tered, and concentrated. The residue was purified by silica gel chromatogra-
phy (EtOAc/tolueneꢂ3 : 2) to yield 12 (414 mg, 0.81 mmol, 84%). [a]D23
ꢁ110.9 (cꢂ0.25 in CHCl3); 1H-NMR (600 MHz, CDCl3) d: 7.97 (2Hꢃ2,
m), 7.91 (2Hꢃ2, m), 7.44 (2Hꢃ2, m), 7.31 (4Hꢃ2, m), 5.64 (1H, dd,
Jꢂ8.4, 10.8, Fuc H2), 5.21 (1Hꢃ2, dd, Jꢂ7.8, 10.2, Gal H2), 5.53 (1Hꢃ2,
dd, Jꢂ1.2, 3.6, Gal H4), 5.07 (1Hꢃ2, dd, Jꢂ3.6, 10.8, Fuc H3), 4.87
(1Hꢃ2, dd, Jꢂ3.6, 10.2, Gal H3), 4.63 (1Hꢃ2, d, Jꢂ8.4, Fuc H1), 4.36
(1Hꢃ2, d, Jꢂ7.8, Gal H1), 4.14 (1Hꢃ2, d, Jꢂ3.6, Fuc H4), 4.02 (1Hꢃ2, m,
–CH2CH2S), 3.76 (2Hꢃ2, m, –CH2CH2S, Fuc H5), 3.55 (1Hꢃ2, m, Gal
H5), 3.11 (2Hꢃ2, m, Gal H6, Gal H6ꢀ), 2.79 (1Hꢃ2, m, –CH2CH2S), 2.76
(1Hꢃ2, m, –CH2CH2S), 2.05 (3Hꢃ2, s, OAc), 2.02 (3Hꢃ2, s, OAc), 1.90
(3Hꢃ2, s, OAc), 1.86 (3Hꢃ2, s, OAc), 1.31 (3Hꢃ2, d, Jꢂ6.6, Fuc Me);
13C-NMR (150 MHz, CDCl3) d: 170.2 (ꢃ2), 170.1 (ꢃ2), 170.0 (ꢃ2), 169.2
(ꢃ2), 166.1 (ꢃ2), 165.3 (ꢃ2), 133.2 (ꢃ2), 133.1 (ꢃ2), 130.1 (ꢃ2), 129.7
(ꢃ2), 128.3 (ꢃ2), 128.2 (ꢃ2), 101.8 (ꢃ2, Gal C1), 100.8 (ꢃ2, Fuc C1),
75.5 (ꢃ2, Fuc C4), 73.1 (ꢃ2, Fuc C3), 70.7 (ꢃ2, Gal C3), 70.5 (ꢃ2, Fuc
C5), 70.2 (ꢃ2, Gal C5), 69.3 (ꢃ2, Gal C2), 68.5 (ꢃ2, Fuc C2), 66.7 (ꢃ2,
Gal C4), 66.2 (ꢃ2, –CH2CH2S), 60.3 (ꢃ2, Gal C6), 38.8 (ꢃ2, –CH2CH2S),
20.8 (ꢃ2), 20.6 (ꢃ2), 20.6 (ꢃ2), 20.5 (ꢃ2), 16.1 (ꢃ2, Fuc C6); IR (neat)
2875, 1750, 1603, 1451, 1369, 1283, 1251, 1222, 1113, 1071, 1030, 757,
713 cmꢁ1; HR-MS (ESI) Calcd for C72H82O32S2 [MꢄNa]ꢄ: 1545.4123
Found 1545.4123.
1
[a]D24 19.9 (cꢂ1.0 in CHCl3); H-NMR (600 MHz, CDCl3) d: 5.45 (1H, dd,
Jꢂ1.8, 9.6, H3), 5.38 (1H, dd, Jꢂ1.8, 9.6, H4), 5.15 (1H, m, H2), 4.83 (1H,
m, H5), 3.73 (1H, br, H6), 3.52 (1H, br, H6ꢀ), 3.45 (1H, dd, Jꢂ3.6, 13.8,
H1), 3.28 (1H, dd, Jꢂ6.0, 13.8, H1ꢀ), 2.48 (1H, br, OH), 2.15 (3H, s,
–COCH3), 2.11 (6H, s, –COCH3), 2.10 (3H, s, –COCH3); 13C-NMR
(150 MHz, CDCl3) d: 171.6, 170.2, 169.8, 169.7, 69.9 (C5), 68.4 (C2), 68.2
(C3), 67.5 (C4), 60.5 (C6), 51.0 (C1), 21.0, 20.7, 20.7, 20.6; IR (neat) 2977,
2941, 2107, 1748, 1372, 1216, 1053 cmꢁ1; HR-MS (ESI) Calcd for
C14H21O9N3 [MꢄNa]ꢄ: 398.1170 found 398.1171.
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-2,3-di-O-benzoyl-
b-L-fucopyranosyl-(1→6)-2,3,4,5-tetra-O-acetyl-1-deoxy-1-azido-D-man-
nitol (10) A solution of 8 (88 mg, 0.23 mmol) and 9 (297 mg, 0.35 mmol)
in dry CH2Cl2 (3.5 ml) was stirred with activated molecular sieves 4 A
(180 mg) at room temperature for 1 h. Then the reaction mixture was cooled
to 0 °C, and TMSOTf (32 ml, 0.18 mmol) was added. After 60 min, the reac-
tion mixture was neutralized with saturated aq NaHCO3 and was extracted
with CH2Cl2. The combined organic layers were dried (Na2SO4) and evapo-
rated in vacuo. The crude product was purified by column chromatography
(toluene/EtOAcꢂ2 : 1) to give 10 (173 mg, 0.16 mmol, 70%) as a clear oil.
[a]D24 ꢁ64.6 (cꢂ0.3 in CHCl3); 1H-NMR (600 MHz, CDCl3) d: 8.03 (2H,
dd, Jꢂ1.2, 8.4), 7.97 (2H, dd, Jꢂ1.2, 8.4), 7.51 (2H, m), 7.38 (4H, m), 5.61
(1H, dd, Jꢂ8.4, 10.8, Fuc H2), 5.39 (1H, dd, Jꢂ2.4, 7.8, H4), 5.32 (1H, dd,
Jꢂ2.4, 8.4, H3), 5.26 (1H, dd, Jꢂ7.8, 10.8, Gal H2), 5.16 (1H, dd, Jꢂ0.6,
3.6, Gal H4), 5.10 (1H, dd, Jꢂ3.6, 10.8, Fuc H3), 5.02 (1H, m, H5), 4.98
(1H, m, H2), 4.93 (1H, dd, Jꢂ3.6, 10.8, Gal H3), 4.65 (1H, d, Jꢂ8.4, Fuc
H1), 4.42 (1H, d, Jꢂ7.8, Gal H1), 4.19 (1H, d, Jꢂ3.6, Fuc H4), 3.77—3.84
2-Sulfanylethyl 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-
2,3-di-O-benzoyl-b-L-fucopyranoside (13) To 4.4 ml of ethanol/acetoni-
trile, 1 : 1 by volume, was added 12 (200 mg, 0.13 mmol). This was treated

1310
Vol. 59, No. 10
with zinc dust (188 mg, 2.89 mmol) and 0.05 ml of concentrated HCl, and
the reaction mixture was stirred for 1 h at room temperature. The reaction
mixture was filtered to remove zinc and zinc salts, and the solvents were re-
moved by rotary evaporation. The residue was dissolved in CH2Cl2, the re-
sulting solution was washed once each with water, and the organic layer was
dried over anhydrous Na2SO4 and evaporated in vacuo. The crude product
was purified by column chromatography (toluene/EtOAcꢂ1 : 2) to give 13
(176 mg, 0.23 mmol, 88%). [a]D24 ꢁ103.9 (cꢂ0.25 in CHCl3); 1H-NMR
(600 MHz, CDCl3) d: 8.04 (2H, dd, Jꢂ1.2, 8.4), 7.98 (2H, dd, Jꢂ1.2, 8.4),
7.51 (2H, m), 7.38 (4H, m), 5.7 (1H, dd, Jꢂ7.8, 10.2, Fuc H2), 5.28 (1H, dd,
Jꢂ7.8, 10.2, Gal H2), 5.17 (1H, d, Jꢂ3.0, Gal H4), 5.13 (1H, dd, Jꢂ3.0,
10.2, Fuc H3), 4.94 (1H, dd, Jꢂ3.0, 10.2, Gal H3), 4.69 (1H, d, Jꢂ7.8, Fuc
H1), 4.43 (1H, d, Jꢂ7.8, Gal H1), 4.2 (1H, d, Jꢂ3.0, Fuc H4), 3.98 (1H, m,
–CH2CH2SH), 3.83 (1H, m, Fuc H5), 3.67 (1H, m, –CH2CH2SH), 3.62 (1H,
4) Rabinovich G. A., Toscano M. A., Jackson S. S., Vasta G. R., Curr.
Opin. Struct. Biol., 17, 513—520 (2007).
5) Lobsanov Y. D., Gitt M. A., Leffler H., Barondes S. H., Rini J. M., J.
Biol. Chem., 268, 27034—27038 (1993).
6) Hirabayashi J., Hashidate T., Arata Y., Nishi N., Nakamura T., Hi-
rashima M., Urashima T., Oka T., Futai M., Muller W. E. G., Yagi F.,
Kasai K., Biochim. Biophys. Acta, 1572, 232—254 (2002).
7) Pace K. E., Lebestky T., Hummel T., Arnoux P., Kwan K., Baum L. G.,
J. Biol. Chem., 277, 13091—13098 (2002).
8) Nemoto-Sasaki Y., Hayama K., Ohya H., Arata Y., Kaneko M. K.,
Saitou N., Hirabayashi J., Kasai K., Biochim. Biophys. Acta, 1780,
1131—1142 (2008).
9) Guérardel Y., Balanzino L., Maes E., Leroy Y., Coddeville B., Oriol
R., Strecker G., Biochem. J., 357, 167—182 (2001).
m, Gal H5), 3.19 (2H, m, Gal H6, Gal H6ꢀ), 2.69 (1H, m, –CH2CH2SH), 10) Schachter H., Curr. Opin. Struct. Biol., 14, 607—616 (2004).
2.61 (1H, m, –CH2CH2SH), 2.11 (3H, s), 2.09 (3H, s), 1.97 (3H, s), 1.93 11) Cipollo J. F., Awad A. M., Costello C. E., Hirschberg C. B., J. Biol.
(3H, s), 1.52 (1H, m, -SH), 1.38 (3H, d, Jꢂ6.6, Fuc Me); 13C-NMR
Chem., 280, 26063—26072 (2005).
(150 MHz, CDCl3) d: 170.2, 170.1, 170.0, 169.0, 166.1, 165.1, 133.2, 133.0, 12) Griffitts J. S., Haslam S. M., Yang T., Garczynski S. F., Mulloy B.,
130.1, 129.7, 128.3, 128.2, 101.8 (Gal C1), 100.9 (Fuc C1), 75.6 (Fuc C4),
73.2 (Fuc C3), 70.8 (Gal C3), 70.5 (Gal C5), 70.2 (Fuc C5), 69.9
(–CH2CH2SH), 69.3 (Gal C2), 68.6 (Fuc C2), 66.7 (Gal C4), 60.3 (Gal C6),
24.5 (–CH2CH2SH), 20.8, 20.6, 20.6, 20.5, 16.1 (Fuc C6); IR (neat) 2984,
2940, 2876, 2575, 1750, 1603, 1451, 1369, 1282, 1251, 1222, 1112, 1071,
1030, 756, 713 cmꢁ1; HR-MS (ESI) Calcd for C36H42O16S [MꢄNa]ꢄ:
785.2086 Found 785.2089.
Morris H., Cremer P. S., Dell A., Adang M. J., Aroian R. V., Science,
307, 922—925 (2005).
13) Hanneman A. J., Rosa J. C., Ashline D., Reinhold V. N., Glycobiology,
16, 874—890 (2006).
14) Paschinger K., Gutternigg M., Rendic D., Wilson I. B. H., Carbohydr.
Res., 343, 2041—2049 (2008).
15) Takeuchi T., Hayama K., Hirabayashi J., Kasai K., Glycobiology, 18,
882—890 (2008).
16) Takeuchi T., Nishiyama K., Sugiura K., Takahashi M., Yamada A.,
Kobayashi S., Takahashi H., Natsugari H., Kasai K., Glycobiology, 19,
1503—1510 (2009).
17) Nishiyama K., Yamada A., Takahashi M., Takeuchi T., Kasai K.,
Kobayashi S., Natsugari H., Takahashi H., Chem. Pharm. Bull., 58,
495—500 (2010).
2-Sulfanylethyl b-D-Galactopyranosyl-(1→4)-b-L-fucopyranoside (2)
To a solution of 13 (86 mg, 0.11 mmol) and NaOMe (43 mg, 0.79 mmol) in
MeOH (2 ml) was stirred at room temperature for 12 h. Then the mixture
was concentrated, and the residue was purified by reverse phase silica gel
chromatography (H2O/MeOHꢂ1 : 1) to give 2 (42 mg, 0.11 mmol, 96%).
1
[a]D25 ꢁ3.7 (cꢂ0.2 in CH3OH); H-NMR (600 MHz, CD3OD) d: 4.27 (1H,
d, Jꢂ7.8, Fuc H1), 4.23 (1H, d, Jꢂ7.8, Gal H1), 4.03 (1H, m, –CH2CH2SH),
3.82 (2H, m, –CH2CH2SH, Fuc H4), 3.76 (1H, d, Jꢂ3.0, Gal H4), 3.72 (2H,
m, Fuc H5, Gal H6), 3.64 (1H, dd, Jꢂ4.2, 11.4, Gal H6ꢀ), 3.58 (1H, dd,
18)
Maduzia L. L., Yu E., Zhang Y., J. Biol. Chem., 286, 4371—4381
(2011).
Jꢂ7.8, 10.2, Gal H2), 3.51 (1H, m, Gal H5), 3.39 (2H, m, Gal H3, Fuc H3), 19) Arata Y., Hirabayashi J., Kasai K., J. Biol. Chem., 276, 3068—3077
3.39 (1H, dd, Jꢂ7.8, 10.2, Fuc H2), 2.94 (2H, t, Jꢂ6.6, –CH2CH2SH), 1.32
(3H, d, Jꢂ6.6, Fuc Me); 13C-NMR (150 MHz, CD3OD) d: 105.7 (Gal C1),
104.9 (Fuc C1), 80.9 (Fuc C4), 77.0 (Gal C5), 74.4 (Fuc C3), 74.1 (Gal C3),
73.1 (Fuc C2), 72.9 (Gal C2), 72.1 (Fuc C5), 70.3 (Gal C4), 69.5
(–CH2CH2SH), 62.5 (Gal C6), 39.5 (–CH2CH2SH), 16.7 (Fuc C6); HR-MS
(ESI) Calcd for C14H26O10S [MꢄNa]ꢄ: 409.1153 Found 409.1139.
(2001).
20) Satoh A., Miwa H. E., Kojima K., Hirabayashi J., Matsumoto I., J.
Biochem., 128, 377—381 (2001).
21) Nishioka S., Aikawa J., Ida M., Matsumoto I., Street M., Tsujimoto
M., Kojima-Aikawa K., J. Biochem., 141, 47—55 (2007).
22) Murakami T., Yoshioka K., Sato Y., Tanaka M., Niwa O., Yabuki S.,
Bioorg. Med. Chem. Lett., 21, 1265—1269 (2011).
Acknowledgements We wish to thank Ms. J. Shimode, and Ms. M.
Takahashi for spectroscopic measurements. This work was supported in part
by a Grant-in-Aid for Scientific Research (C) (21590021) from the Japan
Society for the Promotion of Sciences.
23) Oka H., Onaga T., Koyama T., Guo C.-T., Suzuki Y., Esumi Y., Hatano
K., Terunuma D., Matsuoka K., Bioorg. Med. Chem., 17, 5465—5475
(2009).
24) Details regarding the biochemical experiences have been reported in
our precedent paper: Takeuchi T., Nishiyama K., Yamada A., Tamura
M., Takahashi H., Natsugari H., Aikawa J., Kojima-Aikawa K., Arata
Y., Kasai K., Carbohydr. Res., 346, 1837—1841 (2011).
25) Lee R. T., Lee Y. C., “Glycosciences,” ed. by Gabius H.-J., Gabius S.,
Chapman & Hall, Weinheim, 1997, pp. 55—77.
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