Synthesis of a sialic acid dimer derivative, 2′α-O-benzyl Neu5Ac-α-(2→5)Neu5Gc

Article abstract of DOI:10.1021/jo015930v

The preparation of a disaccharide 2, Neu5Ac-α-(2→5)Neu5Gc having a α-benzyl protecting group at the reducing end, by the coupling of the easily accessible building units 4 and 5 is described. Subsequent deprotection of the coupling adduct led to the isolation of the target compound 2 in high yield.

Full text of DOI:10.1021/jo015930v

1376
J . Org. Chem. 2002, 67, 1376-1379
Sch em e 1
Syn th esis of a Sia lic Acid Dim er
Der iva tive, 2′r-O-Ben zyl
Neu 5Ac-r-(2f5)Neu 5Gc
Chien-Tai Ren,† Chien-Sheng Chen,† and
Shih-Hsiung Wu*,†,‡
Institute of Biological Chemistry, Academia Sinica,
Taipei, Taiwan, and Graduate Institute of Biochemical
Science, National Taiwan University, Taipei, Taiwan
shwu@gate.sinica.edu.tw
Received J uly 16, 2001
Abstr a ct: The preparation of a disaccharide 2, Neu5Ac-R-
(2f5)Neu5Gc having a R-benzyl protecting group at the
reducing end, by the coupling of the easily accessible building
units 4 and 5 is described. Subsequent deprotection of the
coupling adduct led to the isolation of the target compound
2 in high yield.
With respect to glycosidic linkages in polysialic acids
(PSA), three kinds of PSA’s have been isolated from
mammalian cells and microorganisms: R(2f8)PSA
(Neu5Ac, Neu5Gc, and KDN), R(2f9)PSA (Neu5Ac), and
R(2f8)/R(2f9) alternating PSA (Neu5Ac).1,2 Recently, a
new PSA chain with Neu5Gc residues ketosidically linked
to the glycolyl group of Neu5Gc ((f5-OglycolylNeu5Gc-
R2f)n, called poly-R-(2f5)-N-glycolylneuraminic acid
(Neu5Gc)), 1, has been isolated from the jelly coat of sea
urchin egg and found to play an important role in the
fertilization of eggs.3-5 In an effort to learn more about
its involvement in the biological processes, an efficient
preparation of this class of compounds would provide easy
access to various structural congeners required for our
study. Here, we report the first synthesis of a sialic acid
dimer derivative, 2′R-O-benzyl Neu5Ac-R-(2f5)Neu5Gc
(2).
Sch em e 2
Our retrosynthetic analysis (Scheme 1) involves the
separate preparation of the substituted glycolic acid 4
and the amine 5 fragments. The amide linkage of the
target molecule would be assembled by the coupling
reaction between compound 4 and 5. This approach was
straightforward and proved to be effective for the con-
struction of protected disaccharides 2′R-O-benzyl Neu5Ac-
R-(2f5)Neu5Gc (2 and 3) in good yield.
The synthesis of compounds 4 and 5 began from a
common precursor, thioglycosides 6 (Scheme 2), prepared
from N-acetylneuraminic acid by the reported procedure.6
Treatment of thioglycosides 6 (R/â ) 1:4) with benzyl
glycolate in the presence of NIS and a catalytic amount
of triflic acid (TfOH)7 at -25 °C gave 7 (62%) and 20% of
the â-anomer 8, and both were separated by flash
chromatography on silica gel as shown in Scheme 2.
Hydrogenolytic removal of the benzyl group of 7 over 10%
Pd on carbon in ethyl acetate cleanly afforded the desired
substituted glycolic acid 4 in quantitative yield.
N,O-deacetylation of the thioglycoside 6â with meth-
anesulfonic acid in methanol8 was followed by selective
protection of the resulting free amino functional group
as N-2,2,2-trichloroethoxycarbonyl carbamate9 to give 9
in 77% yield. (Scheme 3) Peracetylation of 9 by the acetic
anhydride-pyridine protocol afforded 10 in 94% yield.
Reaction of 10 with benzyl alcohol under NIS/TfOH
conditions provided the R-benzyl glycoside 11 (66%) and
the â-benzyl glycoside 12 (14%); both were separated by
flash chromatography on silica gel. Treatment of 11 with
zinc dust in glacial acetic acid at room temperature for 3
h afforded the desired amine 5 in 84% yield.10 It is
interesting to note that amine 5 could be purified by
* To whom correspondence should be addressed, Fax: +886-2-2653-
9142.
† Academia Sinica.
(7) (a) Hasegawa, A.; Nagahama, T.; Ohki, H.; Hotta, K.; Ishida,
H.; Kiso, M. J . Carbohydr. Chem. 1991, 10, 493. (b) Yamamoto, T.;
Teshima, T.; Saitoh, U.; Hoshi, M.; Shiba, T. Tetrahedron Lett. 1994,
35, 2701. (c) Cao, S.; Roy, R. Tetrahedron Lett. 1996, 37, 3421. (d)
Komba, S.; Ishida, H.; Kiso, M.; Hasegawa, A. Carbohydr. Res. 1996,
285, C1. (e) Demchenko, A. V.; Boons, G.-J . Tetrahedron Lett. 1998,
39, 3065. (f) Demchenko, A. V.; Boons, G.-J . Chem. Eur. J . 1999, 5,
1278. (g) Yu, C.-S.; Niikura, K.; Lin, C.-C.; Wong, C.-H. Angew. Chem.,
Int. Ed. 2001, 40, 2900.
‡ National Taiwan University.
(1) Troy, F. A., II. Glycobiology 1992, 2, 5.
(2) Muhlenhoff, M.; Eckhardt, M.; Gerardy-Schahn, R. Curr. Opin.
Struct. Biol. 1998, 8, 558.
(3) Kitazume, S.; Kitajima, K.; Inoue, S.; Troy, F. A., II; Cho, J .-W.;
Lennarz, W. J .; Inoue, Y. J . Biol. Chem. 1994, 269, 22712.
(4) Kitazume, S.; Kitajima, K.; Inoue, S.; Haslam, S. M.; Morris, H.
R.; Dell, A.; Lennarz, W. J .; Inoue, Y. J . Biol. Chem. 1996, 271, 6694.
(5) Kitazume-Kawaguchi, S.; Inoue, S.; Inoue, Y.; Lennarz, W. J .
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 3650.
(6) Marra, A.; Sinay¨, P. Carbohydr. Res. 1989, 187, 35.
(8) Sugata, T.; Higuchi, R. Tetrahedron Lett. 1996, 37, 2613.
(9) Lapatsanis, L.; Milias, G.; Froussios, K.; Kolovos, M. Synthesis
1983, 671.
10.1021/jo015930v CCC: $22.00 © 2002 American Chemical Society
Published on Web 01/22/2002

Notes
J . Org. Chem., Vol. 67, No. 4, 2002 1377
Sch em e 3
13 by reaction with LiCl in pyridine in 95% yield.13 Basic
hydrolysis of 13 with 0.1 N NaOH cleanly provided the
desired product 2 in 94% yield.
Sch em e 4
In conclusion, the synthetic disaccharide 2′R-O-benzyl
Neu5Ac-R-(2f5)Neu5Gc (2) is more easily traced in
enzymatic reactions as a substrate with a benzyl group
in the reducing end. Application of this synthetic strategy
for the preparation of 2′-R-O-benzyl-R(2f5)-N-glycolyl-
neuraminic acid dimer and oligomer is currently under-
way, and these species will be used as acceptors for the
biosynthetic study of R-(2f5)Neu5Gc polymer.
Exp er im en ta l Section
Gen er a l Con sid er a tion s. NMR spectra were recorded with
Bruker AM-400 (400 MHz) and Bruker Avance DMX-500 (500
MHz) spectrometers. Assignment of 1H NMR spectra was
achieved using 2D methods (COSY, NOESY, HETCOR). Chemi-
cal shifts are expressed in ppm using residual CHCl3 and CHD2-
OD as references. High-resolution FAB-MS was recorded with
a J EOL SX-120 mass spectrometer. Reactions were monitored
by TLC using alumina or glass plates coated with silica gel 60
F254 (Merck) and visualized by using either UV light or by
charring with
a molybdate solution (a 0.02 M solution of
ammonium cerium sulfate dihydrate and ammonium molybdate
tetrahydrate in aqueous 10% H2SO4). Column chromatography
was performed on silica gel 60 (Merck, 70-230 mesh). Methanol
was dried by refluxing with magnesium methoxide and distilled
immediately before use. Dioxane, acetonitrile, and pyridine were
freshly distilled under N2 over CaH2. Molecular sieves (3 Å) were
activated in vacuo at 300 °C for 8 h and stored under N2.
Meth yl [2-(2′-Ben zyloxy-2′-oxoeth yl)-5-a ceta m id o-4,7,8,9-
tetr a -O-a cetyl-3,5-d id eoxy-r- a n d -â-D-glycer o-D-ga la cto-2-
n on u lop yr a n osid ]on a te (7 a n d 8). A mixture of thioglycoside
6 (4.0 g, 6.85 mmol), benzyl glycolate (1.71 g, 10.28 mmol), and
3 Å molecular sieves (6.0 g) in acetonitrile (40 mL) was stirred
for 1 h under N2. After the solution was cooled to -40 °C, NIS
(1.85 g, 8.22 mmol) and TfOH (0.12 mL, 1.37 mmol) were added
successively. The reaction mixture was stirred at -25 °C for 1
h, diluted with DCM (100 mL), and filtered through a pad of
Celite. The Celite was washed with DCM (3 × 20 mL), and the
combined filtrate was washed with saturated aqueous Na2S2O3
(15 mL) and saturated aqueous NaHCO3 (20 mL). The organic
layer was dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash silica gel
chromatography (50-100% gradient EtOAc in hexane) to afford
chromatography and stored at 0 °C for few days without
decomposition.11
The coupling of 4 with 5 (Scheme 4) was conducted in
the presence of EDC and HOBt12 in acetonitrile to give
the fully protected dimer 3 in 81% yield, as shown in
Scheme 4. Finally, transformation of 3 to 2 was per-
formed in two steps. Compound 3 was first converted to
(10) (a) Woodward, R. B.; Heusler, K.; Gosteli, J .; Naegeli, P.;
Oppolzer, W.; Ramage, R.; Ranganathan, S.; Vorbru¨ggen, H. J . Am.
Chem. Soc. 1966, 88, 852. (b) Windholz, T. B.; J ohnston, D. B. R.
Tetrahedron Lett. 1967, 2555. (c) Carson, J . F. Synthesis 1981, 268.
(d) Bergeron, R. J .; McManis, J . S. J . Org. Chem. 1988, 53, 3108. (e)
Zhu, T.; Boons, G.-J . Tetrahedron: Asymmetry 2000, 11, 199.
(11) Sherman, A. A.; Yudina, O. N.; Shashkov, A. S.; Menshov, V.
M.; Nifant’ev, N. E. Carbohydr. Res. 2001, 330, 445.
(12) (a) Sheehan, J . C.; Preston, J .; Cruickshank, P. A. J . Am. Chem.
Soc. 1965, 87, 2492. (b) Nozaki, S.; Muramatsu, I. Bull. Chem. Soc.
J pn. 1982, 55, 2165. (c) Ho, G.-J .; Emerson, K. M.; Mathre, D. J .;
Shuman, R. F.; Grabowski, E. J . J . Org. Chem. 1995, 60, 3569. (d)
Nozaki, S. Chem. Lett. 1997, 1.
(13) (a) Elsinger, F.; Schreiber, J .; Eschenmoser, A. Helv. Chim. Acta
1960, 43, 113. (b) Fujita, S.; Numata, M.; Sugimoto, M.; Tomita, K.;
Ogawa, T. Carbohydr. Res. 1994, 263, 181. (c) Huang, B.-G.; Locke, R.
D.; J ain, R. K.; Matta, K. L. Bioorg. Med. Chem. Lett. 1997, 7, 1157.

1378 J . Org. Chem., Vol. 67, No. 4, 2002
Notes
the R-anomer 7 (2.72 g, 62%) and the â-anomer 8 (0.88 g, 20%)
as a white foam. 7: 1H NMR (400 MHz, CDCl3) δ 7.33-7.28 (m,
5H), 5.33 (ddd, J ) 8.3, 5.5, 2.7 Hz, 1H), 5.28 (d, J ) 9.9 Hz,
1H), 5.27 (dd, J ) 8.3, 2.2 Hz, 1H), 5.14 (dd, J ) 25.2, 12.3 Hz,
2H), 4.90 (ddd, J ) 12.0, 10.2, 4.8 Hz, 1H), 4.29 (dd, J ) 32.1,
16.4 Hz, 2H), 4.24 (dd, J ) 12.5, 3.0, 1H), 4.06-4.04 (m, 1H),
4.01 (t, J ) 10.2 Hz, 1H), 3.91 (dd, J ) 10.2, 2.2 Hz, 1H), 3.70
(s, 3H), 2.82 (dd, 1H, J ) 12.0, 4.8 Hz, 1H), 2.09 (s, 3H), 2.08(s,
3H), 2.00 (s, 3H), 1.99-1.97 (m, 4H), 1.84 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 170.93 (C), 170.57 (C), 170.23 (C), 170.06 (C),
169.98 (C), 169.12 (C), 167.50 (C), 135.39 (C), 128.48 (CH), 128.30
(CH), 98.15 (C), 72.51 (CH), 68.93 (CH), 68.38 (CH), 67.14 (CH),
66.58 (CH2), 62.28 (CH2), 61.81 (CH2), 52.90 (CH3), 49.30 (CH),
37.53 (CH2), 23.11 (CH3), 21.04 (CH3), 20.79 (CH3), 20.74 (CH3),
20.66 (CH3); LRMS (FAB; m/z (%)) 663 (M + Na+, 12), 640 (MH+,
12), 580 (25), 474 (17), 414 (100), 196 (10), 154 (16), 91 (91), 58
(36); HRMS (FAB, MH+) calcd for C29H38O15N 640.2241, found
640.2223. 8: 1H NMR (400 MHz, CDCl3) δ 7.36-7.30 (m, 5H),
5.35 (d, J ) 10.4 Hz, 1H), 5.27 (dd, J ) 3.9, 2.1 Hz, 1H), 5.23
(ddd, J ) 10.9, 6.5, 4.9 Hz, 1H), 5.16 (dd, J ) 20.3, 12.2 Hz,
2H), 5.15-5.11 (m, 1H), 4.73 (dd, J ) 12.3, 2.5, 1H), 4.26 (d, J
) 1.02 Hz, 2H), 4.16 (dd, J ) 10.4, 2.1 Hz, 1H), 4.09 (q, J ) 10.4
Hz, 1H), 3.95 (dd, J ) 12.3, 8.0 Hz, 1H), 3.70 (s, 3H), 2.46 (dd,
J ) 13.1, 4.9 Hz, 1H), 2.09 (s, 3H), 2.00 (m, 4H), 1.97 (s, 3H),
1.93 (s, 3H), 1.82 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.76
(C), 170.54 (C), 170.48 (C), 170.21 (C), 169.99 (C), 169.22 (C),
166.69 (C), 135.22 (C), 128.60 (CH), 128.52 (CH), 128.48 (CH),
98.56 (C), 72.17 (CH), 71.71 (CH), 68.62 (CH), 68.38 (CH), 66.95
(CH2), 62.51 (CH2), 61.52 (CH2), 52.84 (CH3), 48.92 (CH), 36.86
(CH2), 23.03 (CH3), 20.87 (CH3), 20.79 (CH3), 20.68 (CH3), 20.65
(CH3); LRMS (FAB; m/z (%)) 663 (M + Na+, 42), 640 (17), 580
(25), 474 (18), 414 (100), 154 (23), 91 (74), 58 (68); HRMS (FAB,
MH+) calcd for C29H38O15N 640.2241, found 640.2243.
12.3 Hz, 2H), 4.70 (d, J ) 12.3 Hz, 1H), 4.59 (d, J ) 10.3 Hz,
1H), 4.25 (ddd, J ) 15.8, 11.8, 4.7 Hz, 1H), 3.88-3.55 (m, 10H),
3.45 (s, 3H), 2.69 (dd, J ) 13.7, 4.7 Hz, 1H), 2.00 (dd, J ) 13.7,
12.0 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 169.51 (C), 156.91
(C), 137.33 (C), 130.27 (C), 129.79 (CH), 97.23 (C), 91.37 (C),
75.26 (CH2), 73.54 (CH), 71.08 (CH), 70.96 (CH), 70.84 (CH),
69.55 (C), 67.41 (CH), 65.09 (CH2), 55.98 (CH), 52.74 (CH3), 42.63
(CH2). LRMS (FAB; m/z (%)): 576 ((M + Na + 6)+, 2), 574 ((M
+ Na + 4)+, 8), 572 ((M + Na + 2)+, 22), 570 ((M + Na)+, 20),
554 ((MH + 6)+, 1), 552 ((MH + 4)+, 3), 550 ((MH + 2)+, 8), 548
(MH+, 7), 442 (5), 440 (18), 438 (19), 307 (14), 154 (100), 136
(77), 107 (25), 89 (27), 77 (26). HRMS (FAB, MH+): calcd for
C
22H32O15N 548.0316, found 548.0326.
Meth yl [P h en yl 5-(2,2,2-tr ich lor oeth oxycar bon ylam in o)-
4,7,8,9-t et r a -O-a cet yl-3,5-d id eoxy-2-t h io-â-D-glycer o-D-ga -
la cto-2-n on u lop yr a n osid ]on a te (10). A solution of compound
9 (4.23 g, 7.71 mmol) in pyridine (6.0 mL), and acetic anhydride
(6.0 mL) was stirred at room temperature for 24 h. After the
mixture was cooled to 0 °C, methanol (3.0 mL) was added over
10 min, and the mixture was stirred at room temperature for
30 min. The volatile material was removed under reduced
pressure, and the residue was purified by flash silica gel
chromatography (20-50% gradient EtOAc in hexane) to give 10
as a white foam (5.2 g, 94%). 1H NMR (400 MHz, CDCl3): δ
7.46-7.44 (m, 2H), 7.37-7.27 (m, 3H), 5.51 (t, J ) 2.4 Hz, 1H),
5.46 (ddd, J ) 10.7, 10.7, 4.9 Hz, 1H), 5.25 (br, 1H), 5.01 (ddd,
J ) 2.3, 2.4, 8.0 Hz, 1H), 4.91 (d, J ) 12.1 Hz, 1H), 4.68 (dd, J
) 10.7, 2.4 Hz, 1H), 4.50 (d, J ) 12.3, 2.3 Hz, 1H), 4.47 (dd, J )
12.3, 2.3 Hz, 1H), 4.04 (dd, J ) 12.3, 8.0 Hz, 1H), 3.77 (q, J )
10.7 Hz, 1H), 3.60 (s, 3H), 2.74 (dd, J ) 13.8, 4.9 Hz, 1H), 2.12
(s, 3H), 2.11-2.03 (m, 4H), 1.98 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 171.53 (C), 171.02 (C), 170.94 (C), 170.70 (C), 168.79
(C), 154.94 (C), 136.78 (CH), 130.37 (CH), 129.75 (CH), 129.49
(C), 96.02 (C), 89.45 (C), 75.20 (CH2), 73.47 (CH), 73.30 (CH),
69.45 (CH), 69.30 (CH), 63.13 (CH2), 53.23 (CH3), 52.44 (CH),
38.19 (CH2), 21.64 (CH3), 21.46 (CH3), 21.39 (CH3), 21.34 (CH3).
LRMS (FAB; m/z (%)): 742 ((M + Na + 4)+, 1), 740 ((M + Na +
2)+, 2), 738 ((M + Na)+, 2), 718 ((MH + 2)+, 1), 716 (MH+, 1),
658 (4), 656 (4), 609 (4), 607 (5), 550 (30), 548 (100), 546 (99),
488 (15), 486 (15), 368 (31), 366 (31), 330 (35), 328 (36), 154 (43),
136 (45), 78 (20). HRMS (FAB, MH+): calcd for C28H35O14NCl3
716.0738, found 716.0729.
Meth yl [2-(2′-Oxido-2′-oxoeth yl)-5-acetam ido-4,7,8,9-tetr a-
O-a cetyl-3,5-d id eoxy-r-D-glycer o-D-ga la cto-2-n on u lop yr a -
n osid ]on a te (4). To a solution of compound 7 (1.60 g, 2.50
mmol) in EtOAc (50 mL) was added 10% Pd on carbon (262 mg).
The mixture was stirred under hydrogen at 1 atm for 20 h until
TLC analysis indicated that the reaction had gone to completion.
The catalyst was removed by filtration through a pad of Celite,
and the cake was washed with EtOAc (3 × 20 mL). The filtrate
was concentrated under reduced pressure to give 4 as a white
foam (1.37 g, 100%). 1H NMR (400 MHz, CDCl3): δ 5.81-5.56
(br, 1H), 5.49 (d, J ) 9.1 Hz, 1H), 5.37 (ddd, J ) 8.2, 5.8, 2.5 Hz,
1H), 5.30-5.28 (m, 1H), 4.96 (ddd, J ) 12.3, 9.8, 4.5 Hz, 1H),
4.30 (dd, J ) 44.3, 16.7 Hz, 2H), 4.29 (dd, J ) 12.5, 2.5 Hz, 1H),
4.07 (dd, J ) 12.5, 5.8 Hz, 1H), 4.06-4.00 (m, 2H), 3.81 (s, 3H),
2.70 (dd, J ) 12.9, 4.5 Hz, 1H), 2.13 (s, 3H), 2.04-1.97 (m, 7H),
1.89 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 172.31 (C), 171.69
(C), 171.49 (C), 170.99 (C), 170.85 (C), 168.26 (C), 98.91 (C), 73.18
(CH), 69.59 (CH), 69.21 (CH), 67.87 (CH), 63.06 (CH2), 62.29
(CH2), 53.72 (CH3), 50.07 (CH), 38.01 (CH2), 23.68 (CH3), 21.71
(CH3), 21.46 (CH3), 21.36 (CH3). LRMS (FAB; m/z (%)): 572 (M
+ Na+, 25), 550 (MH+, 12), 490 (43), 414 (100), 372 (13), 154
Meth yl [Ben zyl 5-(2,2,2-tr ich lor oeth oxyca r bon yla m in o)-
4,7,8,9-tetr a -O-a cetyl-3,5-d id eoxy-r- a n d â-D-glycer o-D-ga -
la cto-2-n on u lop yr a n osid ]on a te (11 a n d 12). A mixture of
compound 10 (5.2 g, 7.25 mmol), benzyl alcohol (1.10 mL, 10.87
mmol), and 3 Å molecular sieves (4.8 g) in acetonitrile (40 mL)
was stirred for 1 h under N2. After the solution was cooled to
-40 °C, NIS (1.96 g, 8.70 mmol) and TfOH (0.25 mL, 2.90 mmol)
were added successively. The reaction mixture was stirred at
-25 °C for 1 h, diluted with DCM (100 mL), and filtered through
a pad of Celite. The Celite was washed with DCM (3 × 20 mL),
and the combined filtrate was washed with saturated aqueous
Na2S2O3 (15 mL) and saturated aqueous NaHCO3 (20 mL). The
organic layer was dried over MgSO4, filtered, and concentrated
under reduced pressure. The residue was purified by flash silica
gel chromatography (10-30% gradient EtOAc in hexane) to
afford the R-anomer 11 (3.4 g, 66%) and the â-anomer 12 (0.7 g,
(57), 136 (52), 55 (42). HRMS (FAB, MH+): calcd for C22H32O15
550.1722, found 550.1766.
N
Meth yl [P h en yl 5-(2,2,2-tr ich lor oeth oxycar bon ylam in o)-
3,5-dideoxy-2-th io-â-D-glycer o-D-galacto-2-n on u lopyr an osid]-
on a te (9). To a solution of thioglycoside 6â (15.0 g, 25.70 mmol)
in methanol (100 mL) was added methanesulfonic acid (2.0 mL,
30.83 mmol), and the resulting mixture was stirred at 65 °C for
24 h. After it was cooled to room temperature, the solution was
neutralized with Dowex 1×8-50 ion-exchange resin. The resin
was removed by filtration and washed with methanol (2 × 40
mL). The filtrate was concentrated to afford a pale brown foam.
Without purification, the crude material was dissolved in 1 M
sodium hydrogen carbonate aqueous solution (27 mL). A solution
of succinimidyl 2,2,2-trichloroethyl carbonate (8.2 g, 28.28 mmol)
in dioxane (30 mL) was added in one portion, and the resulting
mixture was stirred vigorously for 1 h. Dioxane was removed
under reduced pressure, and the remaining alkaline suspension
was extracted with EtOAc (3 × 150 mL). The combined organic
layer was washed with water (2 × 20 mL), dried over Na2SO4,
filtered, and concentrated. The residue was purified by flash
silica gel chromatography (0-10% gradient methanol in EtOAc)
to provide 9 as a white foam (10.86 g, 77%). 1H NMR (400 MHz,
CDCl3): δ 7.65-7.63 (m, 2H), 7.37-7.30 (m, 3H), 4.96 (d, J )
1
14%) as a white foam. 11: H NMR (400 MHz, CDCl3) δ 7.37-
7.27 (m, 5H), 5.47 (ddd, J ) 8.9, 4.9, 4.7 Hz, 1H), 5.41 (dd, J )
8.9, 1.8 Hz, 1H), 5.00 (ddd, J ) 12.1, 9.9, 4.4 Hz, 1H), 4.93-
4.90 (dm, J ) 12.1 Hz, 2H), 4.82 (d, J ) 12.0 Hz, 1H), 4.49 (d,
J ) 12.1 Hz, 1H), 4.43 (d, J ) 12.0 Hz, 1H), 4.32 (dd, J ) 12.5,
2.7 Hz, 1H), 4.24 (dd, J ) 10.7, 1.8 Hz, 1H), 4.15 (dd, J ) 12.5,
4.9 Hz, 1H), 3.74-3.66 (m, 4H), 2.73 (dd, J ) 12.1, 4.4 Hz, 1H),
2.17 (s, 3H), 2.16 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 1.97-1.91
(m, 1H); 13C NMR (100 MHz, CDCl3) δ 171.32 (C), 171.05 (C),
170.77 (C), 170.62 (C), 168.90 (C), 154.74 (C), 137.78 (C), 128.88
(CH), 128.47 (CH), 128.35 (CH), 99.05 (C), 96.05 (C), 75.17 (CH2),
72.61 (CH), 69.17 (CH), 68.85 (CH), 68.06 (CH), 67.53 (CH2),
62.85 (CH2), 53.30 (CH3), 52.36 (CH), 38.91 (CH2), 21.74 (CH3),
21.46 (CH3), 21.40(CH3); LRMS (FAB; m/z (%)) 738 ((M + Na +
2)+, 1), 736 ((M + Na)+, 1), 714 (MH+, 1), 656 (5), 654 (5), 609
(5), 607 (5), 550 (12), 548 (38), 546 (40), 488 (8), 486 (8), 370 (5),
368 (13), 366 (15), 332 (17), 330 (17), 328 (5); HRMS (FAB, MH+)
calcd for C28H35O14NCl3 714.1123, found 714.1113. 12: 1H NMR
(400 MHz, CDCl3) δ 7.29-7.19 (m, 5H), 5.37 (dd, J ) 5.0, 1.7

Notes
J . Org. Chem., Vol. 67, No. 4, 2002 1379
Hz, 1H), 5.35 (ddd, J ) 11.0, 11.0, 5.0 Hz, 1H), 5.23 (ddd, J )
7.0, 5.0, 2.4 Hz, 1H), 5.10 (d, J ) 9.9, 1H), 4.80 (d, J ) 12.1 Hz,
1H), 4.64 (dd, J ) 12.5, 2.4 Hz, 1H), 4.42 (quin, J ) 11.8 Hz,
3H), 4.06 (dd, J ) 12.5, 7.0 Hz, 1H), 4.01 (dd, J ) 10.7, 1.7 Hz,
1H), 3.70-3.58 (m, 4H), 2.55 (dd, J ) 12.9, 5.0 Hz, 1H), 2.08 (s,
3H), 1.95 (s, 3H), 1.91 (s, 3H), 1.83-1.76 (m, 4H); 13C NMR (100
MHz, CDCl3) δ 170.63 (C), 170.45 (C), 170.08 (C), 167.29 (C),
154.21 (C), 136.53 (C), 128.52 (CH), 127.91 (CH), 127.28 (CH),
98.36 (C), 95.41 (C), 74.48 (CH2), 71.17 (CH), 68.45 (CH), 68.30
(CH), 68.67 (CH2), 62.15 (CH2), 52.67 (CH3), 51.71 (CH), 37.48
(CH2), 20.87 (CH3), 20.78 (CH3), 20.73 (CH3); LRMS (FAB; m/z
(%)) 738 ((M + Na + 2)+, 1), 736 ((M + Na)+, 1), 716 ((MH +
2)+, 1), 714 (MH+, 1), 674 (2), 672 (2), 656 (23), 654 (24), 552 (1),
550 (8), 548 (22), 546 (23), 506 (4), 504 (4), 488 (4), 486 (4), 386
(3), 384 (3), 368 (7), 366 (6), 330 (7), 328 (7); HRMS (FAB, MH+)
calcd for C28H35O14NCl3 714.1123, found 714.1114.
Meth yl [Ben zyl 5-am in o-4,7,8,9-tetr a-O-acetyl-3,5-dideoxy-
r-D-glycer o-D-ga la cto-2-n on u lop yr a n osid ]on a te (5). To a
solution of compound 11 (0.235 g, 0.329 mmol) in glacial acetic
acid (5.0 mL) was added freshly activated zinc dust (1.5 g). The
mixture was stirred at room temperature for 3 h, diluted with
DCM (80 mL), and filtered. The filtrate was washed with
saturated aqueous NaHCO3 solution, dried over Na2SO4, filtered,
and concentrated. The residue was purified by flash silica gel
chromatography (50-100% gradient EtOAc in hexane) to afford
5 (0.151 g, 84%) as a white foam. 1H NMR (400 MHz, CDCl3):
δ 7.33-7.25 (m, 6H), 5.50 (dd, J ) 3.5, 2.0 Hz, 1H), 5.46 (dd, J
) 10.0, 2.0 Hz, 1H), 4.80-4.77 (m, 1H), 4.79 (d, J ) 8.0 Hz, 1H),
4.44 (d, J ) 8.8 Hz, 1H), 4.34 (dd, J ) 12.6, 2.0 Hz, 1H), 4.29
(dd, J ) 12.6, 3.5 Hz, 1H), 3.97 (d, J ) 10.0 Hz, 1H), 3.70 (s,
3H), 2.77 (dd, J ) 12.5, 4.6 Hz, 1H), 2.64 (t, J ) 10.0 Hz, 1H),
2.19 (s, 3H), 2.18 (s, 3H), 2.11 (s, 3H), 2.07 (s, 3H), 1.78 (t, J )
12.5 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 171.09 (C), 170.68
(C), 170.42 (C), 170.08 (C), 168.05 (C), 137.34 (C), 128.17 (CH),
127.75 (CH), 127.59 (CH), 98.35 (C), 74.12 (CH), 71.21 (CH),
67.99 (CH), 66.83 (CH), 66.60 (CH2), 61.98 (CH2), 52.54 (CH3),
51.17 (CH), 37.74 (CH2), 21.11 (CH3), 20.97 (CH3), 20.93 (CH3),
20.77 (CH3), 20.71 (CH3). LRMS (FAB; m/z (%)): 580 ((M + 41)+,
33), 540 (MH+, 35), 480 (8), 432 (5), 372 (5), 192 (8), 126 (8), 91
(100). HRMS (FAB, MH+): calcd for C25H34O12N2 540.2081,
found 540.2090.
Meth yl {2-Ben zyl-5-glycolyla m id o-4,7,8,9-tetr a -O-a cetyl-
3,5-d id eoxy-5-Oglycolyl-[m eth yl (5-a ceta m id o-4,7,8,9-tetr a -O-
a cetyl-3,5-d id eoxy-r-D-glycer o-D-ga la cto-2-n on u lop yr a n o-
syl)on a t e ]-r-D-glyc e r o-D-ga la c t o-2-n on u lop yr a n osid }-
on a te (3). To a solution of the compound 4 (192 mg, 0.349 mmol)
and compound 5 (200 mg, 0.371 mmol) in acetonitrile (4.0 mL)
was added sequentially NaHCO3 (84 mg, 1.0 mmol), HOBt (10
mg, 0.084 mmol), and EDC (100 mg, 0.525 mmol). The resulting
mixture was stirred at room temperature for 18 h. The solvent
was removed under reduced pressure, and the residue was
purified by flash silica gel chromatography (0-2% gradient
MeOH in EtOAc) to give compound 3 (0.302 g, 81%) as a white
foam. 1H NMR (400 MHz, d6-benzene): δ 7.43 (d, J ) 7.4 Hz,
2H), 7.15-7.12 (m, 2H), 7.04 (t, J ) 7.4 Hz, 1H), 6.46 (d, J )
10.1 Hz, 1H), 5.94 (ddd, J ) 10.0, 7.4, 2.7 Hz, 1H), 5.72 (dd, J )
7.4, 2.2 Hz, 1H), 5.68 (ddd, J ) 7.9, 6.0, 2.5 Hz, 1H), 5.49 (dd, J
) 7.9, 2.2 Hz, 1H), 5.30 (ddd, J ) 12.3, 10.3, 4.7 Hz, 1H), 5.13
(d, J ) 12.2 Hz, 1H), 4.82 (ddd, J ) 12.3, 10.7, 4.7 Hz, 1H), 4.78-
4.63 (m, 4H), 4.50 (dd, J ) 10.7, 2.2 Hz, 1H), 4.41-4.32 (m, 3H),
4.11 (dd, J ) 10.7, 2.4 Hz, 1H, 3.47 (s, 3H), 3.18 (s, 3H), 2.94
(dd, J ) 12.3, 4.5 Hz, 1H), 2.64 (dd, J ) 12.3, 4.5 Hz, 1H), 2.30
(t, J ) 12.3 Hz, 1H), 2.18 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H), 2.07
(s, 3H), 1.98 (t, J ) 12.3 Hz, 1H), 1.94 (s, 3H), 1.85 (s, 3H), 1.84
(s, 3H), 1.72 (s, 3H), 1.68 (s, 3H). 13C NMR (100 MHz, d6-
benzene): δ 170.81 (C), 170.69 (C), 170.46 (C), 170.41 (C), 169.73
(C), 169.17 (C), 169.05 (C), 168.76 (C), 138.35 (C), 128.81(CH),
128.68 (CH), 128.56 (CH), 128.14 (C), 99.68 (C), 99.36 (C), 74.01
(CH), 70.26 (CH), 69.41 (CH), 69.36 (CH), 68.66 (CH), 67.83 (CH),
67.61 (CH2), 64.88 (CH2), 63.56 (CH2), 63.26 (CH2), 53.16 (CH3),
52.45 (CH3), 50.07 (CH), 49.29 (CH), 39.29 (CH2), 37.73 (CH2),
23.31 (CH3), 21.51 (CH3), 31.37 (CH3), 31.14 (CH3), 21.09 (CH3),
20.86 (CH3), 20.77 (CH3), 20.67 (CH3). LRMS (FAB; m/z (%)):
1093 ((M + Na)+, 3), 1071 (MH+, 4), 1011 (12), 903 (61), 474
(24), 430 (25), 414 (97), 252 (18), 196 (26), 136 (29), 91 (100).
HRMS (FAB, MH+): calcd for C47H63O26N2 1071.3669, found
1071.3677.
Lith iu m {2-Ben zyl-5-glycolylam ido-4,7,8,9-tetr a-O-acetyl-
3,5-d id eoxy-5-Oglycolyl-[lith iu m (5-a ceta m id o-4,7,8,9-tetr a -
O-a cetyl-3,5-d id eoxy-r-D-glycer o-D-ga la cto-2-n on u lop yr a -
n osyl)on a t e]-r-D-glycer ol-D-ga la ct o-2-n on u lop yr a n osid }-
on a te (13). To a solution of 3 (180 mg, 0.168 mmol) in pyridine
(4.0 mL) was added LiCl (61 mg, 1.44 mmol). The solution was
heated at 120 °C for 16 h, and the solvent was removed under
reduced pressure. The dark brown residue was purified by flash
silica gel chromatography (5-20% gradient MeOH in DCM) to
afford the dilithium salt 13 as a white foam (168.5 mg, 95%).
1H NMR (400 MHz, d6-DMSO): δ 8.20 (d, J ) 5.3 Hz, 1H), 7.74
(d, J ) 9.8 Hz, 1H), 7.34-7.22 (m, 5H), 5.36 (ddd, J ) 7.1, 7.1,
3.0 Hz, 1H), 5.20 (ddd, J ) 7.0, 7.0, 2.8 Hz, 1H), 5.11-5.07 (m,
2H), 4.97 (ddd, J ) 11.4, 11.4, 4.7 Hz, 1H), 4.87 (ddd, J ) 11.4,
11.4, 4.7 Hz, 1H), 4.75 (d, J ) 11.3 Hz, 1H), 4.65 (d, J ) 12.0
Hz, 1H), 4.47 (d, J ) 12.0 Hz, 1H), 4.43-4.37 (m, 2H), 4.19 (dd,
J ) 12.0, 2.8 Hz, 1H), 4.03 (dd, J ) 12.0, 7.0 Hz, 1H), 3.82 (q, J
) 11.4 Hz, 1H), 3.78-3.60 (m, 3H), 2.70 (dd, J ) 11.4, 4.7 Hz,
1H), 2.44 (dd, J ) 11.4, 4.7 Hz, 1H), 2.05 (s, 3H), 2.03 (s, 3H),
2.00 (s, 3H), 1.96 (s, 3H), 1.95 (s, 6H), 1.91 (s, 3H), 1.88 (s, 3H),
1.63 (s, 3H), 1.43 (t, J ) 11.4 Hz, 1H), 1.39 (t, J ) 11.4 Hz, 1H).
13C NMR (100 MHz, d6-DMSO): δ 170.60 (C), 170.09 (C), 169.68
(C), 169.50 (C), 169.41 (C), 169.15 (C), 139.06 (C), 127.97 (CH),
127.57 (CH), 126.96 (CH), 100.459 (C), 71.14 (CH), 70.78 (CH),
70.25 (CH), 68.79 (CH), 68.32 (CH), 67.98 (CH), 65.44 (CH2),
62.59 (CH2), 62.42 (CH2), 48.97 (CH), 48.66 (CH), 39.13 (CH2),
38.46 (CH2), 22.46 (CH3), 21.10 (CH3), 20.92 (CH3), 20.81 (CH3),
20.67 (CH3), 20.53 (CH3). LRMS (FAB; m/z (%)): 1087 ((M+2Na)+,
0.5), 1061 ((M + 18)+, 2.3), 1055 ((M + 2Li)+, 1), 313 (21), 160
(78), 154 (70), 136 (62), 91 (100). HRMS (FAB, MH+): calcd for
C45H61O27N2 1061.3462, found 1061.3485.
Oglycolyl-(5-Aceta m id o-3,5-d id eoxy-r-D-glycer o-D-ga la cto-
2-n on u lop yr a n osylon ic a cid )-(2f5)-2-ben zyl-5-glycolyla -
m ido-3,5-dideoxy-r-D-glycer o-D-galacto-2-n on u lopyr an oson -
ic a cid (2). Compound 13 (168.5 mg, 0.160 mmol) was treated
with 0.1 M aqueous NaOH (15 mL) at room temperature for 3
h. The reaction mixture was cooled to 0 °C, neutralized with
Amberlite IR-120 (H+) ion-exchange resin, and filtered, and the
resin was washed with water (2 × 5 mL). The filtrate was
lyophilized to give the crude product as a light brown solid. The
crude product was purified by flash silica gel chromatography
(20-60% gradient MeOH in DCM) to yield compound 2 as a
colorless powder (104 mg, 94%). 1H NMR (500 MHz, D2O): δ
7.48-7.43 (m, 5H), 4.82 (d, J ) 10.9 Hz, 1H), 4.58 (d, J ) 10.9
Hz, 1H), 4.36 (d, J ) 15.4 Hz, 1H), 4.17 (d, J ) 15.4 Hz, 1H),
2.84 (dd, J ) 12.4, 4.9 Hz, 1H), 2.80 (dd, J ) 12.4, 4.9 Hz, 1H),
2.08 (s, 3H), 1.85 (t, J ) 12.4 Hz, 1H), 1.74 (t, J ) 12.4 Hz, 1H).
13C NMR (100 MHz, D2O): δ 175.46 (C), 174.05 (C), 173.62 (C),
173.30 (C), 137.46 (C), 129.19 (CH), 129.12 (CH), 128.74 (CH),
101.38 (C), 100.79 (C), 73.17 (CH), 72.75 (CH), 72.29 (CH), 71.94
(CH), 68.64 (CH), 68.54 (CH), 68.50 (CH), 68.35 (CH), 67.52
(CH2), 63.49 (CH2), 63.02 (CH2), 62.98 (CH2), 52.23 (CH), 52.16
(CH), 40.92 (CH2), 39.88 (CH2), 22.46 (CH3). 1H NMR (500 MHz,
d6-DMSO): δ 8.50 (d, J ) 8.4 Hz, 1H), 8.01 (s, 1H), 7.36-7.27
(m, 5H), 4.74 (d, J ) 11.6 Hz, 1H), 4.45 (d, J ) 15.8 Hz, 1H),
4.17 (d, J ) 15.8 Hz, 1H), 3.98 (d, J ) 15.8 Hz, 1H), 2.71 (d, J
) 10.8 Hz, 1H), 2.58 (dd, J ) 12.3, 4.3 Hz, 1H), 1.89 (s, 3H),
1.62 (t, J ) 12.3 Hz, 1H), 1.47 (t, J ) 10.8 Hz, 1H). 13C NMR
(100 MHz, d6-DMSO): δ 172.80 (C), 170.90 (C), 170.85 (C),
169.39 (C), 138.16 (C), 128.22 (CH), 127.61 (CH), 127.51 (CH),
99.38 (C), 98.80 (C), 73.93 (CH), 72.79 (CH), 71.81 (CH), 70.98
(CH), 69.61 (CH), 68.46 (CH), 66.38 (CH), 66.05 (CH), 65.57
(CH2), 63.95 (CH2), 63.67 (CH2), 60.83 (CH2), 53.30 (CH), 52.86
(CH), 40.77 (CH2), 39.18 (CH2), 22.52 (CH3). LRMS (FAB; m/z
(%)): 729 ((M + Na)+, 0.6), 707 (MH+, 0.5), 523 (1.5), 505 (1.2),
154 (100), 136 (70). HRMS (FAB, MH+): calcd for C29H43O18N2
707.2511, found 707.2515.
Ack n ow led gm en t. We thank the National Science
Council, Taiwan, for financial support.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectral data for selective compounds 2, 3, 7, 8, 11, 12, and
13. This material is available free of charge via the Internet
at http://pubs.acs.org.
J O015930V