Synthesis of fluorescence-labeled galβ1-3Fuc and Galβ1-4Fuc as Probes for the endogenous glyco-epitope recognized by galectins in caenorhabditis elegans

Article abstract of DOI:10.1248/cpb.58.495

To search for the endogenous glyco-epitope in Caenorhabditis elegans, we synthesized labeled Galβ1-3Fuc and Galβ1-4Fuc and examined their binding affinity for C. elegans galectin LEC-6 using frontal affinity chromatography analysis. We developed a new strategy for synthesizing the labeled saccharides, in which the labeling unit, the 2-aminopyridine moiety, is coupled with a spacer unit derived from D-mannitol. Our results indicate that Galβ1-4Fuc is the endogenous glyco-epitope present in C. elegans N-glycans.

Full text of DOI:10.1248/cpb.58.495

April 2010
Chem. Pharm. Bull. 58(4) 495—500 (2010)
495
b
b
Synthesis of Fluorescence-Labeled Gal 1-3Fuc and Gal 1-4Fuc as Probes
for the Endogenous Glyco-Epitope Recognized by Galectins in
Caenorhabditis elegans
Kazusa NISHIYAMA,a Atsushi YAMADA,a Miki TAKAHASHI,a Tomoharu TAKEUCHI,a Ken-ichi KASAI,a
Susumu KOBAYASHI,b Hideaki NATSUGARI,a and Hideyo TAKAHASHI*,a
a School of Pharmaceutical Sciences, Teikyo University; 1091–1 Sagamiko, Sagamihara, Kanagawa 229–0195, Japan: and
b Faculty of Pharmaceutical Sciences, Tokyo University of Science; 2641 Yamazaki, Noda, Chiba 278–8510, Japan.
Received November 14, 2009; accepted January 26, 2010
To search for the endogenous glyco-epitope in Caenorhabditis elegans, we synthesized labeled Galb1-3Fuc
and Galb1-4Fuc and examined their binding affinity for C. elegans galectin LEC-6 using frontal affinity chro-
matography analysis. We developed a new strategy for synthesizing the labeled saccharides, in which the labeling
unit, the 2-aminopyridine moiety, is coupled with a spacer unit derived from D-mannitol. Our results indicate
that Galb1-4Fuc is the endogenous glyco-epitope present in C. elegans N-glycans.
Key words galectin; 2-aminopyridine; frontal affinity chromatography
Galectins are a family of carbohydrate-binding proteins
defined by their affinity for b-galactosides-containing glyco-
conjugates.1—3) Their binding to saccharides results in vari-
ous biological phenomena including development, immunity,
and tumor metastasis.4) It is known that a Galb1-4GlcNAc
(N-acetyllactosamine) unit is the endogenous glyco-epitope
recognized by vertebrate galectins.5—8) Among galectins
from invertebrate species including Caenorhabditis elegans,
specificity for N-acetyllactosamine was reported, although
the existence of the glycan containing N-acetyllactosamine
units has not been confirmed in C. elegans.9—14) This discrep-
ancy raised a question concerning the endogenous glyco-epi-
tope that is recognized by galectins in C. elegans. To search
for it, Takeuchi et al. isolated N-type glycoproteins bound by
C. elegans galectin LEC-6.15) Matrix assisted laser desorp-
tion/ionization-time of flight (MALDI-TOF)/TOF analysis in
Fig. 1. Structures of Fluorescence-Labeled Gal-Fuc Derivatives (1, 2)
conjunction with glycosidase digestion elucidated that the
glycoproteins contain a Gal-Fuc disaccharide unit attached to cence-labeled Gal-Fuc derivatives (1, 2) (Fig. 1).
the innermost GlcNAc (N-acetylglucosamine) residue. Among
several Gal-Fuc units, in which D-galactose and L-fucose are Results and Discussion
b b
Synthesis of Protected Gal 1-3Fuc (14) and Gal 1-
differentially linked at some positions, we assumed that
Galb1-4Fuc should be the endogenous glyco-epitope because 4Fuc (17) As shown in Chart 1, allyl a-L-fucopyranoside
its presence had been confirmed in C. elegans N-glycans.
(3)20) was chosen as the starting material for the synthesis of
In this study, we synthesized Galb1-3Fuc and Galb1-4Fuc Galb1-3Fuc (14) and Galb1-4Fuc (17). Using Kamerling
derivatives and examined their binding affinity for galectin conditions,21) its transformation into cyclic 3,4-O-ortho-
LEC-6 using frontal affinity chromatography (FAC) analy- acetate, followed by 2-O-acetylation and regioselective or-
sis.16) For FAC analysis, saccharides must be labeled with thoester opening by acidic hydrolysis, provided allyl 2,4-O-
a fluorescent agent. Recently, pyridylamination has been diacetyl-a-L-fucopyranoside (6). Similarly, regioselective 4-
widely used as a labeling method for saccharides because of O-acetylation, followed by 2,3-dibenzoylation and deacetyla-
its high sensitivity.17—19) This method, however, has a disad- tion, provided allyl 2,3-O-dibenzoyl-a-L-fucopyranoside (10).
vantage in terms of efficiency: the yield is low in the reduc-
In the presence of trimethylsilyl trifluoromethane sulfonate
tive amination for introducing the pyridylamino group at the (TMSOTf), coupling of the L-fucosyl acceptor 6 with the
reducing end of the saccharide. It is even more disadvanta- 2,3,4,5-tetra-O-acetylated galactosyl donor 11, which was
geous that this reductive amination requires an aldehyde at expected to be an efficient galactosylating agent because of
the anomeric position of the sugar, which causes the loss of neighboring group participation,22) proceeded smoothly to
the characteristic nature of the pyranose ring at the reducing give only the b-linked disaccharide 12 (Chart 2). O-Deallyla-
end. Therefore we investigated another method for obtaining tion of 12 with Pd(PPh3)4, followed by trichloroacetimidation
fluorescence-labeled Gal-Fuc derivatives. In our strategy, the of the hemiacetal formed 13 with trichloroacetonitrile and
fluorescence unit, the 2-aminopyridine moiety, is coupled 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), gave the Galb1-
with Gal-Fuc via a water soluble C6 spacer derived from D- 3Fuc donor 14.23)
mannitol. Here we describe the synthesis of novel fluores-
Similar to the coupling of compounds 11 and 6, the L-
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: hide-tak@pharm.teikyo-u.ac.jp

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Vol. 58, No. 4
Reagents and conditions: (a) TBDMSCl, imidazole, DMF, 0 °C, 79%; (b) Ac2O,
pyridine, r.t., 88%; (c) 1% I2/MeOH, r.t., 42%; (d) DMSO, (COCl)2, TEA, CH2Cl2,
ꢀ78 °C, 97%.
Chart 4. Synthesis of Compound 22
Table 1. Reductive Amination of 22 with 2-Aminopyridine
Reagents and conditions: (a) 1) 2,2-dimethoxypropane, p-TsOH, DMF, r.t.; 2)
Ac2O, pyridine, r.t., 45% (2 steps); (b) 1) TFA, CH2Cl2/H2O, r.t.; 2) (EtO)3CMe, p-
TsOH, DMF, r.t.; (c) AcOH, H2O, r.t., 90% (3 steps); (d) (EtO)3CMe, p-TsOH,
DMF, r.t.; (e) BzCl, pyridine, r.t. 75% (2 steps); (f) AcCl, MeOH/CH2Cl2, r.t.,
57%.
Chart 1. Synthesis of Compounds 6 and 10
Ratio
23 : 24
Entry
Solvent
Concentration (M) Yield (%)
1
2
3
4
5
6
7
8
9
MeOH
EtOH
CH2Cl2
CH3CN
Et2O
THF
THF
DMF
DMF
DMF
0.1
0.1
0.1
0.1
0.1
0.1
0.5
0.1
0.05
0.01
33
28
25
Trace
39
55
25
45
68
51
1 : 0a)
1 : 0a)
1 : 0a)
Reagents and conditions: (a) TMSOTf, CH2Cl2, MS 4 Å, 0 °C, 42%;
(b) Pd(PPh3)4, AcOH, 80 °C, 51%; (c) CCl3CN, DBU, CH2Cl2, 0 °C, 49%.
1 : 0.3a)
1 : 0.2a)
1 : 0.5a)
1 : 0.3b)
1 : 0.3b)
1 : 0.2b)
Chart 2. Synthesis of Compound 14
10
a) Determined by 1H-NMR, b) after separation by column chromatography.
tion. Therefore we attempted to develop conditions conduc-
tive to the subsequent coupling of the aldehyde 22 and 2-
aminopyridine (Table 1). After confirming that the appropri-
ate acidity (pH 5) was necessary for this reaction, it was also
found that the solvent and concentration were important. When
the alcohols, CH2Cl2, and CH3CN were used as the solvent,
Reagents and conditions: (a) TMSOTf, CH2Cl2, MS 4 Å, 0 °C, 48%;
(b) Pd(PPh3)4, AcOH, 80 °C, 78%; (c) CCl3CN, DBU, CH2Cl2, 0 °C, 97%.
Chart 3. Synthesis of Compound 17
fucosyl acceptor 10 reacted with the galactosyl donor 11 to satisfactory yields were not obtained (Table 1, entries 1—4),
give the b-linked disaccharide 15 (Chart 3). Its transforma- although Et2O, THF, and DMF appeared promising (entries
tion into the glycosyl donor 17 was performed in the same 5—10). In particular, at a rather high dilution in DMF, the
manner as described above for the preparation of 14.
best yield was obtained (entry 9). A detailed examination of
Synthesis of the Spacer Moiety (22) from D-Mannitol the reaction in these solvents allowed us to isolate the minor
Next, we prepared a spacer moiety of the labeled Gal-Fuc product 24, in which the acetyl group migrated to the nucle-
derivatives (Chart 4). We chose commercially available D- ophilic amino group. It is likely that 24 was formed from 23
mannitol as the starting material for this. Primary hydroxyl during the reaction. We therefore evaluated 24 as a useful in-
groups of D-mannitol (18) were protected with t-butyl- termediate for the synthesis of the labeling unit 26. Thus a
dimethylsilyl (TBDMS) to give the tetra-ol 19. Peracetylation mixture of 23 and 24 was acetylated to give the single com-
of 19, followed by selective deprotection of TBDMS, af- pound 25. The TBDMS group of 25 was deprotonated to
forded the alcohol 21.24) To introduce the 2-aminopyridyl form the labeling agent 26 (Chart 5).
group by reductive amination,25—27) 21 was converted into an
aldehyde (22) by oxidation.
Synthesis of Fluorescence-Labeled Gal-Fuc Derivatives
(1, 2) For the synthesis of labeled Galb1-3Fuc and Galb1-
Coupling of the Spacer Moiety with the Aminopyridyl 4Fuc, the labeling agent 26 was reacted with Gal-Fuc 14 in
Group by Reductive Amination After preparing the the presence of TMSOTf to give 27. Fortunately, the glycosy-
spacer unit 22, the next step was introducing the aminopy- lation occurred only at the hydroxyl group without occurring
ridyl group into it. Unfortunately, reductive amination was a at the pyridine-nitrogen, which may be ascribed to a decrease
difficult task in this case. As shown in Table 1, an excess in the nucleophilicity of the nitrogen.28) Finally, deprotection
amount of 2-aminopyridine was necessary owing to the re- of acetyl groups gave the target labeled Galb1-3Fuc 1 (Chart
duced reactivity of the amino group connected to the aro- 6). Labeled Galb1-4Fuc 2 was prepared in a similar manner
matic pyridine ring. This is the essential problem in this reac- (Chart 7).

April 2010
497
unless the otherwise noted. Chemical shifts are given in parts per million
(ppm) downfield from tetramethylsilane as an internal standard and coupling
constants (J) are reported in hertz (Hz). Splitting patterns are abbreviated as
follows: singlet (s), doublet (d), triplet (t), multiplet (m), broad (br). High
resolution mass spectra (HR-MS) were obtained on LCMS-IT-TOF (Shi-
madzu, Kyoto, Japan) for electrospray ionization (ESI). Sodium trifluoroac-
etate (TFA-Na) was used as internal standard for high resolution MS. Opti-
cal 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 FT/IR-410 spectrometer using sodium chloride
plates or potassium bromide pellets. Absorbance frequencies are recorded in
reciprocal centimeters (cmꢀ1). Analytical thin layer chromatography was
carried out using Merck silica gel 60 F254. Column chromatography was
used silica gel Wakogel C-300 (45—60 mm), NH silicagel (Chromatorex®
FujiSilysia Chemical, 100—200 mesh). Reverse phase Column chromatog-
raphy was used ODS-SS10200T.
Reagents and conditions: (a) Ac2O, pyridine, 80 °C, 62%; (b) HF·pyridine, pyri-
dine/THF, r.t., 86%.
Chart 5. Synthesis of Compound 26
Allyl 2-O-Acetyl-3,4-O-isopropylidene-a-L-fucopyranoside (4) To a
solution of 3 (200 mg, 0.98 mmol) in DMF (1 ml) were added 2,2-
dimethoxypropane (0.6 ml, 4.9 mmol) and p-toluenesulfonic acid (p-TsOH)
(30 mg). The mixture was stirred at room temperature for 12 h, yielding a
new product (Rfꢁ0.42) as indicated by TLC (EtOAc/hexane, 1 : 1). Then,
the solution was diluted with EtOAc, washed with saturated aqueous
NaHCO3, dried over Na2SO4 and concentrated. A solution of the residue in
pyridine/acetic anhydride (2 ml, 3 : 1) was stirred for 12 h, when TLC
(EtOAc/hexane, 1 : 1) showed the formation of 4 (Rfꢁ0.72). The mixture
was concentrated using toluene as an azeotropic solvent, and column chro-
matography (EtOAc/hexane, 1 : 3) of the residue afforded 4 (127 mg, 45%).
1H-NMR (400 MHz, CDCl3) d: 1.33 (3H, d, Jꢁ6.8 Hz), 1.32 (3H, s), 1.50
(3H, s), 2.09 (3H, s), 3.96 (1H, m), 4.06 (1H, dd, Jꢁ2.4, 5.2 Hz), 4.13 (2H,
m), 4.30 (1H, dd, Jꢁ5.2, 8.0 Hz), 4.87 (1H, dd, Jꢁ3.6, 8.0 Hz), 4.92 (1H, d,
Jꢁ3.6 Hz), 5.17 (1H, m), 5.26 (1H, m), 5.84 (1H, m). 13C-NMR (100 MHz,
CDCl3) d: 16.3, 21.1, 26.4, 28.1, 63.2, 68.3, 72.0, 73.4, 76.1, 95.1, 109.3,
117.4, 133.5, 170.5. IR (neat) cmꢀ1: 2988, 1741, 1373, 1238, 1130, 1080,
1059, 758. HR-MS (ESI) m/z: 309.1314 (MꢂNa), Calcd for 309.1309,
C14H22O6Na (MꢂNa). [a]D24 ꢀ191.5 (cꢁ0.6, CHCl3).
Reagents and conditions: (a) TMSOTf, CH2Cl2, MS 4 Å, 0 °C, 55%;
(b) NaOMe, MeOH, r.t., 96%.
Chart 6. Synthesis of Compound 1
Allyl 2,4-Di-O-acetyl-a-L-fucopyranoside (6) To a solution of 4
(80 mg, 0.28 mmol) in CH2Cl2 (2 ml) and water (0.2 ml), trifluoroacetic acid
(0.21 ml, 2.8 mmol) was added. The mixture was stirred for 45 min at room
temperature (removal of the isopropylidene group (Rfꢁ0.07) was observed
on TLC). The solution was diluted with CH2Cl2, washed with saturated
aqueous NaHCO3, dried and concentrated. To a solution of the residue in
dry DMF (1 ml) and trimethyl orthoacetate (77 ml, 0.42 mmol), p-TsOH (5.7
mg, 0.03 mmol) was added. After 1 h, 80% acetic acid (2 ml) was added and
stirred for 40 min, the mixture was co-concentrated with toluene and a solu-
tion of the residue in CH2Cl2 was washed with saturated aqueous NaHCO3,
dried, filtered, and concentrated. Then column chromatography (EtOAc/
Reagents and conditions: (a) TMSOTf, CH2Cl2, MS 4 Å, 0 °C, 34%;
(b) NaOMe, MeOH, r.t., 96%.
Chart 7. Synthesis of Compound 2
The binding affinity of the labeled Galb1-3Fuc 1 and
Galb1-4Fuc 2 for galectin LEC-6 was examined in FAC
analysis. Using an immobilized LEC-6, retardation of the la-
beled Galb1-4Fuc 2 was significantly greater than that of the
1
hexane, 1 : 2 to 1 : 1) of the residue afforded 6 (73 mg, 90%). H-NMR (400
MHz, CDCl3) d: 1.13 (3H, d, Jꢁ6.4 Hz), 2.12 (3H, s), 2.17 (3H, s), 3.99
(1H, m), 4.12 (2H, m), 4.23 (1H, dd, Jꢁ3.6, 10.4 Hz), 4.96 (1H, dd, Jꢁ4.0,
10.4 Hz), 5.03 (1H, d, Jꢁ4.0 Hz), 5.19 (1H, m), 5.23 (1H, m), 5.30 (1H, m),
labeled Galb1-3Fuc 1, the details of which have been re- 5.86 (1H, m). 13C-NMR (100 MHz, CDCl3) d: 16.2, 20.9, 21.1, 64.9, 67.1,
68.6, 71.5, 73.7, 95.4, 117.5, 133.5, 171.1, 171.2. IR (neat) cmꢀ1: 3462,
ported in a separate paper.29) The results support our assump-
tion that Galb1-4Fuc is the endogenous glyco-epitope pres-
ent in C. elegans N-glycans.
2986, 1741, 1433, 1373, 1236, 1132, 1099, 1059, 756. HR-MS (ESI) m/z:
311.1106 (MꢂNa), Calcd for 311.1101, C13H20O7Na (MꢂNa). [a]D24
ꢀ178.9 (cꢁ1.0, CHCl3).
Allyl 4-O-Acetyl-2,3-di-O-benzoyl-a-L-fucopyranoside (9) To
a
stirred solution of allyl fucoside 3 (500 mg, 2.5 mmol) in DMF (7 ml) was
added triethyl orthoacetate (540 ml, 3.0 mmol) and p-TsOH (33 mg, 0.17
mmol). After 30 min (the starting material disappeared by TLC monitoring),
the solution was poured into H2O, and extracted with Et2O. The combined
organic layer was dried over Na2SO4, and concentrated. The resulting
residue was purified by silica gel column chromatography (EtOAc/hexane,
1 : 1) to give 8. To a stirred solution of 8 in pyridine (10 ml) was added BzCl
(0.71 ml, 6.1 mmol). The reaction mixture was stirred at room temperature
for 2.5 h. The reaction mixture was poured into H2O, and extracted with
EtOAc. The combined organic layer was dried over Na2SO4, and concen-
trated. The resulting residue was purified by silica gel column chromatogra-
phy (EtOAc/hexane, 3 : 2) to give 9 (838 mg, 75%, 2 steps).
Conclusion
We synthesized labeled Galb1-3Fuc and Galb1-4Fuc and
examined their binding affinity for galectin LEC-6 using
FAC analysis. To improve the pyridylamination method, we
developed a new strategy, in which the labeling unit, the 2-
aminopyridine moiety, is coupled with a spacer unit derived
from D-mannitol. Our results indicate that Galb1-4Fuc is the
endogenous glyco-epitope present in C. elegans N-glycans.29)
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
methods. The NMR spectra (1H, 13C) were determined on a JEOL 600 MHz
(ECP-600) or 400 MHz (AL-400) spectrometer, using CDCl3 (with TMS for
1H-NMR and chloroform-d for 13C-NMR as the internal reference) solution,
Compound 8: 1H-NMR (600 MHz, CDCl3) d: 1.11 (3H, d, Jꢁ6.6 Hz),
2.14 (3H, s), 3.76 (1H, dd, Jꢁ3.6, 9.6 Hz), 3.95 (1H, dd, Jꢁ3.6, 9.6 Hz),
4.03 (2H, m), 4.18 (1H, m), 4.92 (1H, d, Jꢁ3.6 Hz), 5.18 (1H, dd, Jꢁ1.8,
3.6 Hz), 5.20 (1H, m), 5.28 (1H, m), 5.52 (1H, m). 13C-NMR (150 MHz,
CDCl3) d: 16.1, 20.8, 65.2, 68.8, 69.4, 69.8, 73.1, 97.6, 117.9, 133.5, 171.3.

498
Vol. 58, No. 4
IR (neat) cmꢀ1: 3649, 3422, 2988, 1734, 1421, 1375, 1246, 1132, 1082,
1039, 756. HR-MS (ESI) m/z: 269.0981 (MꢂNa), Calcd for 269.0996,
C11H18O6Na (MꢂNa). [a]D20 ꢀ171.1 (cꢁ0.3, CHCl3).
Jꢁ6.0 Hz), 1.97 (3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.06 (3H, s), 2.14 (3H,
s), 2.15 (3H, s), 3.92 (1H, m), 4.11 (2H, m), 4.26 (1H, m), 4.33 (1H, dd, Jꢁ
3.6, 10.4 Hz), 4.62 (1H, d, Jꢁ7.6 Hz), 4.99 (1H, dd, Jꢁ3.6, 10.8 Hz), 5.10
(1H, dd, Jꢁ7.6, 10.8 Hz), 5.32 (1H, dd, Jꢁ3.6, 10.4 Hz), 5.37 (2H, m), 6.51
(1H, d, Jꢁ3.6 Hz), 8.61 (1H, s). 13C-NMR (100 MHz, CDCl3) d: 16.2, 20.6,
20.6, 20.7, 20.7, 20.8, 20.9, 61.2, 66.9, 67.6, 68.0, 68.8, 70.2, 70.6, 71.1,
73.3, 91.1, 94.4, 95.6, 160.8, 169.3, 169.9, 170.0, 170.0, 170.2, 170.4. IR
(neat) cmꢀ1: 3026, 1747, 1674, 1371, 1234, 1078, 760. HR-MS (ESI) m/z:
744.0836 (MꢂNa), Calcd for 744.0835, C26H34O16Cl3Na (MꢂNa). [a]D20
ꢀ75.0 (cꢁ0.6, CHCl3).
Compound 9: 1H-NMR (600 MHz, CDCl3) d: 1.22 (3H, d, Jꢁ6.6 Hz),
2.17 (3H, s), 4.05 (1H, m), 4.24 (1H, m), 4.34 (1H, m), 5.16 (1H, m), 5.28
(1H, d, Jꢁ3.6 Hz), 5.31 (1H, m), 5.54 (1H, dd, Jꢁ1.2, 3.6 Hz), 5.59 (1H, dd,
Jꢁ3.6, 10.8 Hz), 5.85 (2H, m), 7.37 (4H, m), 7.51 (2H, m), 7.90 (2H, m),
7.99 (2H, m). 13C-NMR (150 MHz, CDCl3) d: 15.9, 20.6, 64.7, 68.7, 68.8,
68.9, 71.4, 95.7, 117.5, 128.3 (ꢃ2), 128.4 (ꢃ2), 129.5 (ꢃ2), 133.1 (ꢃ2),
133.3 (ꢃ2), 133.5, 134.5 (ꢃ2), 165.6, 166.1, 170.4. IR (neat) cmꢀ1: 2986,
2939, 1747, 1726, 1421, 1373, 1280, 1228, 1130, 1072, 1032, 758, 711. HR-
MS (ESI) m/z: 477.1532 (MꢂNa), Calcd for 477.1520, C25H26O8Na (Mꢂ
Na). [a]D20 ꢀ160.8 (cꢁ0.5, CHCl3).
Allyl 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-2,3-di-O-
benzoyl-a-L-fucopyranoside (15) To a stirred solution of 2,3,4,6-tetra-O-
acetyl-a-D-galactopyranosyl-2,2,2-trichloroacetimidate 11 (3 g, 6.3 mmol), 10
(1.7 g, 4.2 mmol) and 4 Å molecular sieves in anhydrous CH2Cl2 (40 ml) was
added TMSOTf (377 ml, 2.1 mmol) at 0 °C under Ar. The mixture was
stirred for 1 h at 0 °C, 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/hexane, 1 : 1) to yield 15 (1.5 g, 48%). 1H-NMR (600 MHz,
CDCl3) d: 1.28 (3H, d, Jꢁ6.6 Hz), 1.91 (3H, s), 1.94 (3H, s), 2.04 (3H, s),
2.09 (3H, s), 3.16 (2H, m), 3.60 (1H, m), 3.98 (1H, m), 4.16 (2H, m), 4.24
(1H, d, Jꢁ3.0 Hz), 4.41 (1H, d, Jꢁ7.8 Hz), 4.91 (1H, dd, Jꢁ3.0, 10.2 Hz),
5.10 (1H, m), 5.14 (1H, d, Jꢁ3.0 Hz), 5.20 (1H, d, Jꢁ3.6 Hz), 5.25 (2H, m),
5.55 (2H, m), 5.49 (1H, m), 7.34 (4H, m), 7.47 (2H, m), 7.96 (2H, m), 8.03
(2H, m). 13C-NMR (150 MHz, CDCl3) d: 15.9, 20.5, 20.5, 20.6, 20.8, 60.4,
65.3, 66.7, 68.6 (ꢃ2), 69.3, 69.9, 70.4, 70.8, 76.5, 95.7, 101.7, 117.2, 128.1
(ꢃ2), 128.3, 129.7 (ꢃ4), 130.0 (ꢃ2), 132.9 (ꢃ2), 133.0, 133.7, 165.8,
166.1, 169.2, 169.9, 170.0, 170.2. IR (neat) cmꢀ1: 3024, 2984, 1751, 1369,
1282, 1222, 1111, 1072, 756, 713. HR-MS (ESI) m/z: 765.2375 (MꢂNa),
Calcd for 765.2365, C37H42O16Na (MꢂNa). [a]D20 ꢀ98.8 (cꢁ1.2, CHCl3).
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-2,3-di-O-benzoyl-
L-fucopyranose (16) To a stirred solution of 15 (1.1 g, 1.5 mmol) in AcOH
(10 ml) was added Pd(PPh3)4 (504 mg, 0.44 mmol). The reaction mixture
was stirred at 80 °C for 1 h and cooled to room temperature, to which was
added toluene and concentrated. The resulting residue was purified by silica
gel column chromatography (EtOAc/hexane, 2 : 3) to give 16 (802 mg, 78%,
a:bꢁ1 : 1).
Allyl 2,3-Di-O-benzoyl-a-L-fucopyranoside (10) To a stirred solution
of 9 (118 mg, 0.3 mmol) in CH2Cl2 (0.5 ml) were added AcCl (0.3 ml) and
MeOH (2.2 ml). The reaction mixture was stirred at room temperature for
12 h. The reaction mixture was poured into H2O, and extracted with CH2Cl2.
The combined organic layer was dried over Na2SO4, and concentrated. The
resulting residue was purified by silica gel column chromatography
(EtOAc/hexane, 1 : 4) to give 10 (61 mg, 57%) and recovery of 9 (20 mg,
17%). 1H-NMR (600 MHz, CDCl3) d: 1.35 (3H, d, Jꢁ6.6 Hz), 4.05 (1H, m),
4.16 (1H, br), 4.22 (1H, m), 4.26 (1H, m), 5.15 (1H, m), 5.26 (1H, d, Jꢁ3.6
Hz), 5.30 (1H, m), 5.64 (1H, dd, Jꢁ3.6, 10.8 Hz), 5.73 (1H, dd, Jꢁ3.0, 10.8
Hz), 5.85 (1H, m), 7.38 (4H, m), 7.51 (2H, m), 7.99 (4H, m). 13C-NMR
(150 MHz, CDCl3) d: 16.0, 65.5, 68.6, 68.7, 70.8, 71.7, 95.7, 117.3, 128.4
(ꢃ2), 128.5 (ꢃ2), 129.7 (ꢃ2), 129.8 (ꢃ2), 133.2, 133.3 (ꢃ2), 133.6 (ꢃ2),
165.8, 166.1. IR (neat) cmꢀ1: 3649, 3067, 2984, 1718, 1280, 1165, 1111,
1070, 1030, 758, 711. HR-MS (ESI) m/z: 435.1427 (MꢂNa), Calcd for
435.1414, C23H24O7Na (MꢂNa). [a]D20 ꢀ152.6 (cꢁ1.0, CHCl3).
Allyl 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→3)-2,4-di-O-
acetyl-a-L-fucopyranoside (12) To a stirred solution of 2,3,4,6-tetra-O-
acetyl-a-D-galactopyranosyl-2,2,2-trichloroacetimidate 11 (1.82 g, 3.7 mmol),
6 (710 mg, 2.5 mmol) and 4 Å molecular sieves in anhydrous CH2Cl2 (24 ml)
was added TMSOTf (223 ml, 1.23 mmol) at 0 °C under Ar. The mixture was
stirred for 1.5 h at 0 °C, and then neutralized with saturated aqueous
NaHCO3. The mixture was extracted with CH2Cl2, and the organic phase
was dried, filtered, and concentrated. The residue was purified by silica gel
1
Compound 16 (a : bꢁ1 : 1 mixture): 1H-NMR (600 MHz, CDCl3) d: 1.24
(3H, d, Jꢁ6.6 Hz), 1.31 (3H, d, Jꢁ6.6 Hz), 1.85 (3H, s), 1.86 (3H, s), 1.90
(3H, s), 1.91 (3H, s), 2.00 (3H, s), 2.02 (3H, s), 2.04 (3H, s), 2.06 (3H, s),
3.12 (4H, m), 3.56 (2H, m), 3.79 (1H, m), 4.14 (1H, d, Jꢁ3.0 Hz), 4.21 (2H,
m), 4.37 (3H, m), 4.70 (1H, m), 4.88 (2H, m), 5.12 (3H, m), 5.21 (2H, dd,
Jꢁ7.8, 10.2 Hz), 5.43 (1H, m), 5.52 (2H, m), 7.28-7.98 (20H, m). 13C-NMR
(150 MHz, CDCl3) d: 16.0, 16.2, 20.5, 20.5, 20.5, 20.6, 60.3, 60.4, 65.3,
66.7, 66.8, 68.9, 69.3, 69.4, 69.5, 70.2, 70.4, 70.4, 70.5, 70.8, 71.8, 72.6,
75.5, 76.5, 90.8, 96.0, 101.7, 128.1, 128.1, 128.3, 129.3, 129.5, 129.6,
129.7, 129.8, 129.9, 130.0, 133.1, 133.2, 133.3, 133.4, 165.8, 166.0, 166.1,
166.7, 169.2, 169.9, 170.0, 170.0, 170.1, 170.1, 170.2. IR (neat) cmꢀ1: 3447,
3022, 1749, 1369, 1219, 1176, 1111, 1072, 758, 711. HR-MS (ESI) m/z:
725.2041 (MꢂNa), Calcd for 725.2052, C34H38O16Na (MꢂNa). [a]D20
ꢀ141.6 (cꢁ0.9, CHCl3).
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→4)-2,3-di-O-benzyl-
a-L-fucopyranosyl Trichloroacetimidate (17) To a stirred solution of 16
(429 mg, 0.61 mmol) in anhydrous CH2Cl2 (6 ml) were added trichloroace-
tonitrile (490 ml, 4.9 mmol) and DBU (18 ml, 0.12 mmol) at 0 °C. After the
mixture was stirred for 30 min at 0 °C, the solvent was removed in vacuo.
The residue was purified by silica gel column chromatography (EtOAc/
hexane, 1 : 2 to 1 : 1) to give 17 (504 mg, 97%). 1H-NMR (600 MHz, CDCl3)
d: 1.34 (3H, d, Jꢁ6.6 Hz), 1.92 (3H, s), 1.95 (3H, s), 2.05 (3H, s), 2.09 (3H,
s), 3.11 (1H, dd, Jꢁ6.6, 11.4 Hz), 3.19 (1H, dd, Jꢁ7.2, 11.4 Hz), 3.62 (1H,
m), 4.36 (2H, m), 4.44 (1H, d, Jꢁ8.4 Hz), 4.93 (1H, dd, Jꢁ3.6, 10.8 Hz),
5.16 (1H, m), 5.28 (1H, dd, Jꢁ8.4, 10.8 Hz), 5.57 (1H, dd, Jꢁ3.6, 10.8 Hz),
5.83 (1H, dd, Jꢁ3.6, 10.8 Hz), 6.65 (1H, d, Jꢁ3.6 Hz), 7.32 (2H, m), 7.37
(2H, m), 7.48 (2H, m), 7.94 (2H, m), 8.03 (2H, m), 8.53 (1H, s). 13C-NMR
(150 MHz, CDCl3) d: 16.0, 20.5, 20.5, 20.6, 20.8, 60.4, 66.7, 67.2, 68.3,
69.2, 69.8, 70.6, 70.7, 75.7, 91.0, 94.3, 101.7, 128.1, 128.3 (ꢃ2), 129.2,
129.7 (ꢃ3), 129.8, 130.0 (ꢃ2), 133.2, 133.3, 160.8, 165.4, 166.1, 169.1
(ꢃ2), 170.0, 170.2. IR (neat) cmꢀ1: 3024, 1751, 1371, 1221, 1118, 1070,
758, 711. HR-MS (ESI) m/z: 868.1144 (MꢂNa), Calcd for 868.1148,
C36H38O16Na (MꢂNa). [a]D20 ꢀ117.4 (cꢁ0.8, CHCl3).
chromatography (EtOAc/toluene, 1 : 2) to yield 12 (645 mg, 42%). H-NMR
(600 MHz, CDCl3) d: 1.12 (3H, d, Jꢁ6.6 Hz), 1.97 (3H, s), 2.05 (3H, s),
2.06 (3H, s), 2.09 (3H, s), 2.12 (3H, s), 2.14 (3H, s), 3.91 (1H, m), 4.01 (1H,
m), 4.07 (2H, m), 4.15 (1H, m), 4.23 (2H, m), 4.56 (1H, d, Jꢁ7.8 Hz), 4.98
(1H, dd, Jꢁ3.6, 10.2 Hz), 5.04 (1H, d, Jꢁ3.6 Hz), 5.08 (1H, dd, Jꢁ7.8, 10.2
Hz), 5.12 (1H, dd, Jꢁ3.6, 10.2 Hz), 5.21 (1H, m), 5.25 (1H, m), 5.30 (1H,
m), 5.36 (1H, dd, Jꢁ1.2, 3.6 Hz), 5.87 (1H, m). 13C-NMR (150 MHz,
CDCl3) d: 15.9, 20.4, 20.5, 20.5, 20.7, 20.7, 60.9, 64.8, 66.7, 68.4, 68.5,
68.8, 70.3, 70.9, 71.1, 74.0, 95.6, 99.9, 117.5, 133.5, 169.3, 169.9 (ꢃ2),
170.0, 170.3, 170.5. IR (neat) cmꢀ1: 3024, 2986, 2941, 1747, 1433, 1371,
1232, 1167, 1132, 1079, 756. HR-MS (ESI) m/z: 641.2065 (MꢂNa), Calcd
for 641.2052, C27H38O16Na (MꢂNa). [a]D24 ꢀ70.4 (cꢁ0.7, CHCl3).
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→3)-2,4-di-O-acetyl-L-
fucopyranose (13) To a stirred solution of 12 (645 mg, 1.0 mmol) in
HOAc (7.0 ml) was added Pd(PPh3)4 (361 mg, 0.31 mmol). The reaction
mixture was stirred at 80 °C for 1.5 h and cooled to room temperature, to
which was added toluene and concentrated. The resulting residue was puri-
fied by silica gel column chromatography (EtOAc/hexane, 2 : 3) to give 13
(305 mg, 51%, a : bꢁ3 : 1).
Compound 13a: 1H-NMR (600 MHz, CDCl3) d: 1.13 (3H, d, Jꢁ10.2 Hz),
1.97 (3H, s), 2.06 (3H, s), 2.06 (3H, s), 2.12 (3H, s), 2.12 (3H, s), 2.14 (3H,
s), 3.91 (1H, m), 4.09 (1H, m), 4.21 (1H, m), 4.27 (1H, dd, Jꢁ4.8, 15.6 Hz),
4.30 (1H, m), 4.57 (1H, d, Jꢁ11.4 Hz), 4.98 (1H, dd, Jꢁ5.4, 15.6 Hz), 5.09
(1H, dd, Jꢁ11.4, 15.6 Hz), 5.15 (1H, dd, Jꢁ5.4, 15.6 Hz), 5.27 (1H, m),
5.37 (1H, d, Jꢁ5.4 Hz), 5.43 (1H, t, Jꢁ5.4 Hz). 13C-NMR (150 MHz,
CDCl3) d: 15.9, 20.5, 20.6, 20.6, 20.7, 20.8, 20.9, 60.9 65.0, 66.8, 68.9,
68.9, 70.4, 70.9, 71.2, 73.6, 91.0, 100.1, 169.4, 170.1, 170.1, 170.3, 170.4,
170.6. IR (neat) cmꢀ1: 3462, 2988, 1749, 1373, 1236, 1078, 760. HR-MS
(ESI) m/z: 601.1736 (MꢂNa), Calcd for 601.1739, C24H34O16Na (MꢂNa).
[a]D24 ꢀ47.2 (cꢁ0.6, CHCl3).
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→3)-2,4-di-O-acetyl-a-
L-fucopyranosyl Trichloroacetimidate (14) To a stirred solution of 13
(80 mg, 0.14 mmol) in anhydrous CH2Cl2 (1.4 ml) was added trichloroace-
tonitrile (111 ml, 1.1 mmol) and DBU (4.5 ml, 0.03 mmol) at 0 °C. The mix-
ture was stirred for 1 h at 0 °C. The solvent was removed in vacuo. The
residue was purified by silica gel column chromatography (EtOAc/hexane,
1,6-Di-O-(t-butyldimethylsilyl)-D-mannitol (19) To a stirred solution
of D-mannitol (18) (45 g, 0.25 mol) in DMF (165 ml) were added TBDMSCl
(74 g, 0.54 mol) and imidazole (55 g, 0.54 mol) at 0 °C. The mixture was
stirred for 1 h at 0 °C. Then H2O was added to the mixture and the suspen-
1
1 : 1) to give 14 (49 mg, 49%). H-NMR (400 MHz, CDCl3) d: 1.17 (3H, d,

April 2010
499
tion was extracted with EtOAc and the combined organic layers were dried
over Na2SO4 and concentrated. The resulting residue was purified by silica
gel column chromatography (EtOAc/hexane, 1 : 3) to give 19 (80 g, 79%).
1H-NMR (400 MHz, CDCl3) d: 0.00 (6Hꢃ2, s), 0.81 (9Hꢃ2, s), 3.12 (1Hꢃ
2, d, Jꢁ4.8 Hz), 3.52 (1Hꢃ2, d, Jꢁ6.0 Hz), 3.70 (4Hꢃ2, m). 13C-NMR
(100 MHz, CDCl3) d: ꢀ5.4 (ꢃ4), 18.3 (ꢃ2), 25.9 (ꢃ6), 64.3 (ꢃ2), 70.9 (ꢃ
2), 72.1 (ꢃ2). IR (neat) cmꢀ1: 3422, 2955, 2932, 2858, 1471, 1257, 1217,
1070, 839, 760. HR-MS (ESI) m/z: 411.2602 (MꢂH), Calcd for 411.2593,
C18H43O6Si2 (MꢂH). [a]D20 ꢀ1.26 (cꢁ0.5, CHCl3).
2,3,4,5-Tetra-O-acetyl-1,6-di-O-(t-butyldimethylsilyl)-D-mannitol (20)
To a stirred solution of 19 (6 g, 0.015 mol) in pyridine (15 ml) was added
Ac2O (7 ml, 0.073 mol) at 0 °C. The mixture was stirred for 12 h at room
temperature. Then H2O was added to the mixture and the suspention was ex-
tracted with EtOAc and the combined organic layers were dried over Na2SO4
and concentrated. The resulting residue was purified by silica gel column
chromatography (EtOAc/hexane, 1 : 5) to give 20 (7.7 g, 88%). 1H-NMR
(400 MHz, CDCl3) d: ꢀ0.01 (3Hꢃ2, s), 0.00 (3Hꢃ2, s), 0.85 (9Hꢃ2, s),
2.02 (3Hꢃ2, s), 2.05 (3Hꢃ2, s), 3.59 (1Hꢃ2, dd, Jꢁ7.8, 16.8 Hz), 3.71 (1H
ꢃ2, dd, Jꢁ5.4, 16.8 Hz), 4.94 (1Hꢃ2, m), 5.45 (1Hꢃ2, d, Jꢁ12.0 Hz). 13C-
NMR (100 MHz, CDCl3) d: ꢀ5.54 (ꢃ4), 18.1 (ꢃ2), 20.9 (ꢃ2), 21.0 (ꢃ2),
25.6 (ꢃ6), 61.4 (ꢃ2), 67.8 (ꢃ2), 70.8 (ꢃ2), 169.5 (ꢃ2), 169.7 (ꢃ2).
IR (neat) cmꢀ1: 2955, 2932, 2858, 1749, 1371, 1253, 1222, 1118, 1064,
1035, 758. HR-MS (ESI) m/z: 579.3015 (MꢂH), Calcd for 579.3015,
C26H51O10Si2 (MꢂH). [a]D20 ꢂ18.9 (cꢁ0.5, CHCl3).
21.0, 25.7 (ꢃ3), 40.6, 61.7, 67.9, 68.7, 69.4, 70.6, 107.7, 113.0, 137.3,
147.6, 158.3, 169.4, 170.0, 170.4, 170.7. IR (neat) cmꢀ1: 3020, 2957, 2932,
2853, 1743, 1371, 1217, 1057, 839, 761. HR-MS (ESI) m/z: 541.2558 (Mꢂ
Na), Calcd for 541.2576, C25H40O9N2Si2Na (MꢂNa). [a]D20 ꢂ75.9 (cꢁ0.1,
CHCl3).
1
Compound (24): H-NMR (600 MHz, CDCl3) d: 0.00 (3H, s), 0.01 (3H,
s), 0.85 (9H, s), 2.01 (3H, s), 2.06 (3H, s), 2.08 (3H, s), 2.16 (3H, s), 3.36
(1H, m), 3.61 (1H, dd, Jꢁ5.4, 11.4 Hz), 3.71 (2H, m), 5.02 (2H, m), 5.45
(2H, m), 6.67 (2H, m), 7.67 (1H, m), 8.13 (1H, dd, Jꢁ1.2, 6.0 Hz). 13C-
NMR (150 MHz, CDCl3) d: ꢀ5.6 (ꢃ2), 18.1, 20.6, 20.7, 20.8, 20.9, 25.7
(ꢃ3), 42.5, 61.6, 67.7, 68.0, 68.8, 70.3, 108.1, 112.6, 141.4, 145.7, 155.2,
169.5, 170.2, 170.6, 171.0.
2,3,4,5-Tetra-O-acetyl-6-O-(t-butyldimethylsilyl)-1-deoxy-1-(N-acetyl-
N-2-pyridinylamino)-D-mannitol (25) To a stirred solution of 23 (286
mg, 0.53 mol) in pyridine (1 ml) was added Ac2O (0.5 ml) at room tempera-
ture. The mixture was stirred for 12 h at 80 °C. Then H2O was added to the
mixture and the suspension was extracted with EtOAc and the combined or-
ganic layers were dried over Na2SO4 and concentrated. The resulting residue
was purified by silica gel column chromatography (EtOAc/toluene, 1 : 1) to
give 25 (191 mg, 62%) and recovery of 23 (42 mg, 15%). 1H-NMR (400
MHz, CDCl3) d: 0.03 (6H, d, Jꢁ0.3 Hz), 0.85 (9H, s), 1.78 (3H, s), 1.98
(3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.11 (3H, s), 3.57 (1H, dd, Jꢁ5.6, 11.6
Hz), 3.69 (1H, dd, Jꢁ4.0, 11.6 Hz), 4.07 (1H, dd, Jꢁ8.8, 14.4 Hz), 4.18 (1H,
d, Jꢁ13.6 Hz), 4.97 (1H, m), 5.23 (1H, m), 5.33 (1H, dd, Jꢁ2.4, 8.0 Hz),
5.40 (1H, dd, Jꢁ2.4, 8.4 Hz), 7.20 (2H, dd, Jꢁ4.8, 7.2 Hz), 7.74 (1H, m),
8.49 (1H, m). 13C-NMR (100 MHz, CDCl3) d: ꢀ5.5 (ꢃ2), 18.2, 20.8, 20.8,
20.9, 21.0, 22.9, 25.7 (ꢃ3), 47.9, 61.5, 67.9, 69.2, 69.3, 70.8, 121.5, 127.3,
130.4, 138.4, 149.0, 169.5, 169.7, 169.8, 170.1, 170.4. IR (neat) cmꢀ1: 2957,
2932, 2858, 1749, 1471, 1437, 1371, 1221, 1122, 1062, 754. HR-MS (ESI)
m/z: 583.2687 (MꢂH), Calcd for 583.2681, C27H43O10N2Si (MꢂH). [a]D20
ꢂ53.3 (cꢁ0.5, CHCl3).
2,3,4,5-Tetra-O-acetyl-1-deoxy-1-(N-acetyl-N-2-pyridinylamino)-D-
mannitol (26) To the solution of 25 (60 mg, 0.10 mmol) in pyridine (200
ml), THF (100 ml), and HF-pyridine (343 ml) were added and the mixture
was stirred for 30 min at room temperature. The reaction mixture was
poured into saturated aqueous NaHCO3 solution, and extracted with CH2Cl2.
The combined organic layer was dried over Na2SO4, and concentrated. The
resulting residue was purified by silica gel column chromatography
(EtOAc/toluene, 4 : 1) to give 26 (42 mg, 86%). 1H-NMR (400 MHz, CDCl3)
d: 1.70 (3H, s), 1.90 (3H, s), 2.01 (3H, s), 2.05 (3H, s), 2.07 (3H, s), 3.43
(1H, m), 3.64 (1H, m), 3.93 (1H, m), 4.15 (1H, d, Jꢁ14.4 Hz), 4.74 (1H, m),
5.26 (3H, m), 7.10 (1H, m), 7.15 (1H, m), 7.69 (1H, m), 8.43 (1H, m). 13C-
NMR (100 MHz, CDCl3) d: 20.7, 20.7, 20.8, 21.0, 22.9, 48.2, 60.4, 67.7,
68.9, 69.2, 70.1, 121.4, 128.2, 129.0, 138.2, 149.2, 169.9, 170.1, 170.2,
170.6, 171.9. IR (neat) cmꢀ1: 3464, 3016, 1747, 1664, 1437, 1371, 1224,
1047, 756. HR-MS (ESI) m/z: 491.1658 (MꢂNa), Calcd for 491.1636,
C21H28O10N2Na (MꢂNa). [a]D25 ꢂ26.8 (cꢁ0.2, CHCl3).
2,3,4,5-Tetra-O-acetyl-6-O-(t-butyldimethylsilyl)-D-mannitol (21)
The compound 20 (6.4 g, 0.011 mol) was added to the 1% I2/methanol solu-
tion (40 ml) and stirred for 18 h at room temperature. Then 3% Na2S2O3 was
added to the mixture and the suspension was extracted with CH2Cl2 and the
combined organic layers were dried over Na2SO4 and concentrated. The
resulting residue was purified by silica gel column chromatography
(EtOAc/hexane, 2 : 3) to give 21 (2.1 g, 42%) and recovery of 20 (3.2 g,
1
52%). H-NMR (400 MHz, CDCl3) d: 0.00 (3H, s), 0.06 (3H, s), 0.86 (9H,
s), 2.03 (3H, s), 2.06 (3H, s), 2.08 (3H, s), 2.13 (3H, s), 2.55 (1H, m), 3.50
(1H, m), 3.59 (1H, dd, Jꢁ4.8, 11.4 Hz), 3.66 (1H, dd, Jꢁ3.0, 11.4 Hz), 3.72
(1H, m), 4.77 (1H, m), 5.05 (1H, m), 5.35 (1H, dd, Jꢁ1.8, 10.2 Hz), 5.48
(1H, dd, Jꢁ1.8, 9.0 Hz). 13C-NMR (150 MHz, CDCl3) d: ꢀ5.5 (ꢃ2), 18.1,
20.6, 20.8, 20.9, 21.0, 25.6 (ꢃ3), 60.5, 61.8, 67.6, 67.8, 70.0, 70.4, 169.4,
169.7, 170.3, 171.9. IR (neat) cmꢀ1: 3504, 2932, 2858, 1749, 1471, 1371,
1226, 1035, 839, 777. HR-MS (ESI) m/z: 487.1975 (MꢂNa), Calcd for
487.1970, C20H36O10SiNa (MꢂNa). [a]D20 ꢂ14.4 (cꢁ0.5, CHCl3).
2,3,4,5-Tetra-O-acetyl-6-O-(t-butyldimethylsilyl)-D-mannose (22) To
a cold solution of DMSO (141 ml, 1.99 mmol) in dry CH2Cl2 (10 ml) at
ꢀ78 °C, (COCl)2 (84 ml, 0.99 mmol) was added slowly and the mixture was
stirred for 15 min. Then the mixture was added to the solution of 21
(308 mg, 0.66 mmol) in dry CH2Cl2 (10 ml). After being stirred for 2 h at
ꢀ78 °C, the mixture was warmed to r.t., treated with triethylamine (370 ml,
2.65 mmol) and then washed with saturated aqueous NH4Cl solution. The re-
action products were extracted with CH2Cl2, dried over Na2SO4 and concen-
trated. The resulting residue was purified by silica gel column chromatogra-
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1→3)-2,4-di-O-acetyl-b-
L-fucopyranosyl-(1→6)-2,3,4,5-tetra-O-acetyl-1-deoxy-1-(N-acetyl-N-2-
pyridinylamino)-D-mannitol (27) To the solution of 14 (49 mg, 0.07
mmol) and 26 (48 mg, 0.10 mmol) in dry CH2Cl2 (1 ml), containing activated
molecular sieves (4 Å, 25 mg), was stirred at 0 °C. TMSOTf (12 ml, 0.07
mmol) was added, and the mixture was stirred for 30 min, when TLC
(CH2Cl2/MeOH, 10 : 1) showed the formation of a new spot. The mixture
was neutralized with pyridine, then filtered and washed with aqueous
NaHCO3 solution, dried, filtered, and concentrated. Column chromatography
(NH silicagel, EtOAc/toluene, 10 : 1) of the residue afford 27 (36 mg, 55%).
1H-NMR (600 MHz, CDCl3) d: 1.17 (3H, d, Jꢁ6.6 Hz), 1.79 (3H, s), 1.96
(3H, s), 2.01 (3H, s), 2.03 (3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.06 (3H, s),
2.06 (3H, s), 2.11 (3H, s), 2.12 (3H, s), 2.14 (3H, s), 3.68 (1H, m), 3.71 (1H,
dd, Jꢁ3.6, 12.0 Hz), 3.80 (1H, dd, Jꢁ6.6, 12.0 Hz), 3.87 (1H, m), 3.91 (1H,
dd, Jꢁ3.6, 10.2 Hz), 4.06 (1H, m), 4.07 (1H, dd, Jꢁ7.2, 10.8 Hz), 4.16 (1H,
dd, Jꢁ6.0, 10.8 Hz), 4.19 (1H, m), 4.45 (1H, d, Jꢁ8.4 Hz), 4.53 (1H, d, Jꢁ
7.8 Hz), 4.96 (1H, dd, Jꢁ3.6, 10.8 Hz), 4.99 (1H, m), 5.04 (1H, dd, Jꢁ7.8,
10.8 Hz), 5.12 (1H, m), 5.18 (1H, d, Jꢁ3.6 Hz), 5.25 (1H, m), 5.30 (1H, m),
5.34 (1H, m), 5.35 (1H, dd, Jꢁ1.8, 3.6 Hz), 7.22 (2H, m), 7.76 (1H, m), 8.49
(1H, dd, Jꢁ1.8, 5.4 Hz). 13C-NMR (150 MHz, CDCl3) d: 16.2, 20.5, 20.6,
20.7, 20.7, 20.7, 20.7, 20.8, 20.8, 20.8, 20.9, 22.9, 47.8, 60.9, 66.1, 66.7,
68.2, 68.8, 68.8, 69.2, 69.3, 69.3, 69.4, 69.5, 69.8, 70.5, 71.1, 99.5, 100.1,
121.4, 122.0, 129.1, 138.1, 149.1, 163.3, 169.3, 169.4, 169.6, 169.9, 170.0,
170.1, 170.2, 170.2, 170.3, 170.6. IR (neat) cmꢀ1: 3020, 1749, 1371, 1217,
1132, 1076, 756, 669. HR-MS (ESI) m/z: 1029.3557 (MꢂH), Calcd for
1029.3558, C45H61O25N2 (MꢂH). [a]D25 ꢂ7.16 (cꢁ0.3, CHCl3).
1
phy (EtOAc/hexane, 2 : 1) to give 22 (300 mg, 97%). H-NMR (400 MHz,
CDCl3) d: 0.00 (3H, s), 0.01 (3H, s), 0.85 (9H, s), 2.03 (3H, s), 2.06 (3H, s),
2.09 (3H, s), 2.16 (3H, s), 3.62 (1H, dd, Jꢁ6.6, 16.8 Hz), 3.71 (1H, dd, Jꢁ
4.8, 16.8 Hz), 5.01 (2H, m), 5.51 (2H, m), 9.42 (1H, s). 13C-NMR (100
MHz, CDCl3) d: ꢀ5.6 (ꢃ2), 18.2, 20.5, 20.6, 20.7, 21.0, 25.8 (ꢃ3), 61.8,
67.9, 68.2, 70.7, 74.8, 170.4, 170.7, 170.9, 170.9, 196.6. HR-MS (ESI) m/z:
485.1831 (MꢂH), Calcd for 485.1813, C20H35O10Si (MꢂH).
2,3,4,5-Tetra-O-acetyl-6-O-(t-butyldimethylsilyl)-1-deoxy-1-(2-
pyridinylamino)-D-mannitol (23) and 3,4,5-Tri-O-acetyl-6-O-(t-butyl-
dimethylsilyl)-1-deoxy-1-(N-acetyl-N-2-pyridinylamino)-D-mannitol (24)
To a solution of aldehyde 22 (50 mg, 0.11 mmol) and 4 Å molecular sieves in
anhydrous DMF (2 ml) were added 2-aminopyridine (102 mg, 1.1 mmol) and
AcOH (840 ml), and the mixture was stirred for 23 h at room temperature.
Then NaBH3CN in THF solution (0.22 mmol, 216 ml) was added to the mix-
ture. After 24 h, the mixture was neutralized with saturated aqueous
NaHCO3. The mixture was extracted with CH2Cl2, and the organic phase
was dried, filtered, and concentrated. The residue was purified by silica gel
chromatography (EtOAc/toluene, 3 : 2) to yield 23 (30 mg, 50%) and 24 (10
mg, 18%).
1
Compound (23): H-NMR (600 MHz, CDCl3) d: 0.00 (3H, s), 0.06 (3H,
s), 0.85 (9H, s), 1.94 (3H, s), 2.04 (3H, s), 2.07 (3H, s), 2.12 (3H, s), 3.20
(1H, m), 3.59 (1H, dd, Jꢁ6.0, 11.4 Hz), 3.69 (1H, dd, Jꢁ3.6, 11.4 Hz), 3.97
(1H, m), 4.96 (1H, m), 5.03 (1H, m), 5.42 (1H, dd, Jꢁ2.4, 9.0 Hz), 5.49 (1H,
dd, Jꢁ2.4, 9.0 Hz), 6.38 (1H, d, Jꢁ8.4 Hz), 6.53 (1H, m), 7.36 (1H, m), 8.02
(1H, m). 13C-NMR (150 MHz, CDCl3) d: ꢀ5.6 (ꢃ2), 18.1, 20.7, 20.8, 20.9,
O-b-D-Galactopyranosyl-(1→3)-O-b-L-fucopyranosyl-(1→6)-1-deoxy-

500
Vol. 58, No. 4
1-(2-pyridinylamino)-D-mannitol (1) To the solution of 27 (17 mg, 0.017
mmol) and NaOMe (5.4 mg, 0.099 mmol) in MeOH (1 ml) was stirred at
room temperature for 5 h. Then the mixture was concentrated, and the
residue was purified by reverse phase silica gel chromatography (H2O/
3) Vasta G. R., Ahmed H., Odom E. W., Curr. Opin. Struct. Biol., 14,
617—630 (2004).
4) Rabinovich G. A., Toscano M. A., Jackson S. S., Vasta G. R., Curr.
Opin. Struct. Biol., 17, 513—520 (2007).
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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., 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).
1
MeOH, 1 : 1 to 1 : 4) to yield 1 (9 mg, 96%). H-NMR (600 MHz, CD3OD)
d: 1.29 (3H, d, Jꢁ6.6 Hz), 3.50 (1H, dd, Jꢁ3.6, 10.2 Hz), 3.55 (2H, m), 3.60
(2H, m), 3.64 (1H, m), 3.68 (3H, m), 3.77 (3H, m), 3.81 (4H, m), 3.90 (1H,
d, Jꢁ9.0 Hz), 4.07 (1H, dd, Jꢁ4.8, 10.2 Hz), 4.31 (1H, d, Jꢁ7.8 Hz), 4.40
(1H, d, Jꢁ7.8 Hz), 6.55 (1H, m), 6.65 (1H, d, Jꢁ9.0 Hz), 7.44 (1H, m), 7.88
(1H, dd, Jꢁ1.2, 4.8 Hz). 13C-NMR (150 MHz, CD3OD) d: 16.7, 46.5, 62.6,
70.3, 70.3, 70.5, 70.7, 71.2, 71.4, 71.7, 72.0, 72.3, 72.6, 74.7, 77.2, 81.6,
102.7, 104.6, 110.5, 113.4, 139.0, 147.1, 160.7. HR-MS (ESI) m/z: 567.2396
(MꢂH), Calcd for C23H38O14N2, 567.2396. [a]D25 ꢀ22.1 (cꢁ0.2, EtOH).
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-(N-acetyl-N-
2-pyridinylamino)-D-mannitol (28) To the solution of 17 (88 mg, 0.10
9) Guérardel Y., Balanzino L., Maes E., Leroy Y., Coddeville B., Oriol
R., Strecker G., Biochem. J., 357, 167—182 (2001).
mmol) and 26 (58 mg, 0.12 mmol) in dry CH2Cl2 (2 ml), containing activated 10) Schachter H., Curr. Opin. Struct. Biol., 14, 607—616 (2004).
molecular sieves (4 Å, 40 mg), was stirred at 0 °C. TMSOTf (10 ml, 0.05
mmol) was added, and the mixture was stirred for 1 h, when TLC (CH2Cl2/
MeOH, 10 : 1) showed the formation of a new spot. The mixture was neu-
tralized with pyridine, then filtered and washed with aqueous NaHCO3 solu-
tion, dried, filtered, and concentrated. Column chromatography (NH sil-
icagel, EtOAc/toluene, 10 : 1) of the residue afforded 28 (41 mg, 34%). H-
NMR (600 MHz, CDCl3) d: 1.30 (3H, d, Jꢁ6.6 Hz), 1.66 (3H, s), 1.86 (3H,
s), 1.86 (3H, s), 1.90 (3H, s), 1.96 (3H, s), 1.99 (3H, s), 2.00 (3H, s), 2.04 14) Paschinger K., Gutternigg M., Rendic D., Wilson I. B., Carbohydr.
11) Cipollo J. F., Awad A. M., Costello C. E., Hirschberg C. B., J. Biol.
Chem., 280, 26063—26072 (2005).
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Morris H., Cremer P. S., Dell A., Adang M. J., Aroian R. V., Science,
307, 922—925 (2005).
1
13) Hanneman A. J., Rosa J. C., Ashline D., Reinhold V. N., Glycobiology,
16, 874—890 (2006).
(3H, s), 2.06 (3H, s), 3.09 (1H, m), 3.14 (1H, m), 3.54 (1H, m), 3.67 (1H,
Res., 343, 2041—2049 (2008).
dd, Jꢁ6.0, 12.0 Hz), 3.75 (2H, m), 3.95 (1H, m), 4.08 (1H, m), 4.35 (1H, d, 15) Takeuchi T., Hayama K., Hirabayashi J., Kasai K., Glycobiology, 18,
Jꢁ8.4 Hz), 4.58 (1H, d, Jꢁ7.8 Hz), 4.86 (1H, dd, Jꢁ3.6, 10.8 Hz), 5.03 (2H,
m), 5.08 (1H, m), 5.21 (3H, m), 5.31 (1H, m), 5.53 (2H, m), 7.13 (2H, m),
7.29 (4H, m), 7.43 (2H, m), 7.66 (1H, m), 7.89 (2H, m), 7.96 (2H, m), 8.41
882—890 (2008).
16) Kasai K., Oda Y., Nishikata M., Ishii S., J. Chromatogr., 376, 33—47
(1986).
(1H, m). 13C-NMR (150 MHz, CDCl3) d: 16.0, 20.4, 20.5, 20.6, 20.7, 20.7, 17) Hase S., Ibuki T., Ikenaka T., J. Biochem., 95, 197—203 (1984).
20.7, 20.8, 20.9, 22.9, 47.8, 60.3, 65.3, 65.7, 66.7, 68.4, 68.5, 68.6, 69.2,
69.2, 69.4, 70.1, 70.5, 70.8, 73.1, 100.2, 101.7, 121.4, 128.1, 128.1, 128.2,
128.2, 128.3, 129.0, 129.6, 129.7, 129.9, 129.9, 130.0, 130.1, 133.0, 133.1,
138.1, 149.0. 163.3, 165.0, 166.0, 169.6, 169.9, 170.0, 170.1, 170.1, 170.1,
170.2, 170.2. IR (neat) cmꢀ1: 2986, 1747, 1371, 1226, 1178, 1111, 1070,
756, 713. HR-MS (ESI) m/z: 1153.3853 (MꢂH), Calcd for 1153.3871,
C55H65O25N2 (MꢂH). [a]D25 ꢀ71.8 (cꢁ0.5, CHCl3).
O-b-D-Galactopyranosyl-(1→4)-O-b-L-fucopyranosyl-(1→6)-1-deoxy-
1-(2-pyrydinylamino)-D-mannitol (2) To the solution of 28 (20 mg,
0.017 mmol) and NaOMe (6 mg, 0.10 mmol) in MeOH (1 ml) was stirred
at room temperature for 12 h. Then the mixture was concentrated, and
18) Hase S., Ikenaka T., Matsushima Y., Biochem. Biophys. Res.
Commun., 85, 257—263 (1978).
19) Natsuka S., Hase S., Ikenaka T., Anal. Biochem., 167, 154—159
(1987).
20) Garegg P. J., Norberg T., Carbohydr. Res., 52, 235—240 (1976).
21) Souza A. C., Halkes K. M., Meeldijk J. D., Verkleij A. J., Vliegenthart
J. F. G., Kamerling J. P., Eur. J. Org. Chem., 2004, 4323—4339 (2004).
22) Cheng H., Cao X., Xian M., Fang L., Cai T. B., Ji J. J., Tunac. J. B.,
Sun D., Wang P. G., J. Med. Chem., 48, 645—652 (2005).
23) Ustyuzhanina N., Krylov V., Grachev A., Gerbst A., Nifantiev N., Syn-
thesis, 23, 4017—4031 (2006).
the residue was purified by reverse phase silica gel chromatography 24) Itoh K., Huang Z., Liu H., Org. Lett., 9, 879—882 (2007).
(H2O/MeOH, 1 : 1) to yield 2 (9 mg, 96%). 1H-NMR (600 MHz, CD3OD) d: 25) Kajihara Y., Kamiyama D., Yamamoto N., Sakakibara T., Izumi M.,
1.36 (3H, d, Jꢁ6.6 Hz), 3.48 (3H, m), 3.55 (2H, m), 3.61 (2H, m), 3.68 (1H,
m), 3.74 (4H, m), 3.82 (4H, m), 3.87 (1H, m), 4.05 (1H, dd, Jꢁ4.8,
10.2 Hz), 4.28 (1H, d, Jꢁ7.8 Hz), 4.30 (1H, d, Jꢁ7.8 Hz), 6.56 (1H, m), 6.63
(1H, d, Jꢁ9.0 Hz), 7.45 (1H, m), 7.88 (1H, dd, Jꢁ1.2, 5.4 Hz). 13C-NMR
(150 MHz, CD3OD) d: 16.6, 62.5, 70.3, 70.5, 71.2, 71.3, 72.0, 72.2, 72.8,
72.9, 73.0, 73.3, 74.0, 74.5, 77.1, 81.0, 105.0, 105.8, 110.7, 113.4, 139.1,
147.9, 160.6. HR-MS (ESI) m/z: 567.2373 (MꢂH), Calcd for 567.2396,
C23H39O14N2 (MꢂH). [a]D25 ꢀ40.6 (cꢁ0.3, EtOH).
Hashimoto H., Carbohydr. Res., 339, 1545—1550 (2004).
26) Zhan W., Liang Z., Zhu A., Kurtkaya S., Shim H., Snyder J. P., Liotta
D. C., J. Med. Chem., 50, 5655—5664 (2007).
27) Boys M. L., Schretzman L. A., Chandrakumar N. S., Tollefson M. B.,
Mohler S. B., Downs V. L., Penning T. D., Russell M. A., Wendt J. A.,
Chen B. B., Stenmark H. G., Wu H., Spangler D. P., Clare M., Desai B.
N., Khanna I. K., Nguyen M. N., Duffin T., Engleman V. W., Finn M.
B., Freeman S. K., Hanneke M. L., Keene J. L., Klover J. A., Nickols
G. A., Nickols M. A., Steininger C. N., Westlin M., Westlin W., Yu Y.
X., Wang Y., Dalton C. R., Norring S. A., Bioorg. Med. Chem. Lett.,
16, 839—844 (2006).
Acknowledgements We wish to thank Ms. J. Shimode, and Ms. A.
Kawaji for spectroscopic measurements. This work was partially supported
by a Grant-in-Aid for Scienctific Research from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
28) Unfortunately, glycosyl donors 14 and 17 are hydrolyzable during the
reaction, which decreased the yield of the products.
29) Takeuchi T., Nishiyama K., Sugiura K., Takahashi M., Yamada A.,
Kobayashi S., Takahashi H., Natsugari H., Kasai K., Glycobiology, 19,
1503—1510 (2009).
References and Notes
1) Kasai K., Hirabayashi J., J. Biochem., 119, 1—8 (1996).
2) Cooper D. N., Biochim. Biophys. Acta, 1572, 209—231 (2002).