A convenient and efficient synthesis of SLeX analogs

Article abstract of DOI:10.1021/jo960125f

Described is the preparation and use of tetrasaccharide 1, which enables a rapid and preparative scale synthesis of sialyl Lewis X (SLeX) analogs having 1-O- and 2-N-disubstituted glucosamine (GlcN) moieties. Such modifications should bring a dramatic change of the physical and pharmacological properties of the SLeX analogs. Therefore, tetrasaccharide 1 is a convenient intermediate for the synthesis of various SLeX analogs, since it has convertible 2-(trimethylsilyl)ethyl (SE) glycoside and the free amino group on GlcN moiety. The intermediate 1 was constructed from a glucosamine derivative by a highly efficient combined use of enzymatic galactosylation/sialylation and chemical fucosylation. Thus obtained 1 was converted into SLeX analogs by N-substitution followed by transformation of SE glycoside into other glycosides and deprotection. These synthesized analogs were found to inhibit cell adhesion of HL-60 cells to recombinant soluble human E-selectin.

Full text of DOI:10.1021/jo960125f

2938
J . Org. Chem. 1996, 61, 2938-2945
Articles
A Con ven ien t a n d Efficien t Syn th esis of SLeX An a logs
Masaji Hayashi,* Masashi Tanaka, Masanori Itoh, and Hiroshi Miyauchi
Research Center, Sumitomo Pharmaceuticals, Co., Ltd., 1-98, Kasugadenaka 3-chome,
Konohana-ku, Osaka 554, J apan
Received J anuary 19, 1996X
Described is the preparation and use of tetrasaccharide 1, which enables a rapid and preparative
scale synthesis of sialyl Lewis X (SLeX) analogs having 1-O- and 2-N-disubstituted glucosamine
(GlcN) moieties. Such modifications should bring a dramatic change of the physical and
pharmacological properties of the SLeX analogs. Therefore, tetrasaccharide 1 is a convenient
intermediate for the synthesis of various SLeX analogs, since it has convertible 2-(trimethylsilyl)-
ethyl (SE) glycoside and the free amino group on GlcN moiety. The intermediate 1 was constructed
from a glucosamine derivative by a highly efficient combined use of enzymatic galactosylation/
sialylation and chemical fucosylation. Thus obtained 1 was converted into SLeX analogs by
N-substitution followed by transformation of SE glycoside into other glycosides and deprotection.
These synthesized analogs were found to inhibit cell adhesion of HL-60 cells to recombinant soluble
human E-selectin.
The tetrasaccharide sialyl Lewis X antigenic determi-
essential for the binding to selectins. An important
feature of the GlcN moiety is to fix the relative conforma-
tion between the NeuAc-Gal and Fuc residues. Although
it is believed that the GlcN moiety is independent with
respect to binding to selectins, modification of the GlcN
residue could change the physical and pharmacological
properties of the SLeX analogs. Until now, neither
transformation of 2-N-substituents nor combination of
substituents on 1-O- and 2-N-positions of GlcN moiety
has been studied systematically, partly because of their
structural complexity and difficulty in their synthesis.
Generally, oligosaccharides have been synthesized by
enzymatic and/or chemical methods. In the field of
enzymatic synthesis of SLeX and related oligosaccha-
rides, galactosyltransferase (GalT),12 R(2,3)-sialyltrans-
ferase (2,3-ST),13 and cytidine 5′-monophospho-N-acetyl-
neuraminic acid synthetase (CMP-NeuAc synthetase)14
have been available to date, and schemes for their
nant (SLeX, 2) is known to be a ligand for E-selectin1
and P-selectin.2 SLeX exists at the nonreducing termini
of several glycoproteins and glycolipids found on the
surface of neutrophils and plays an important role in
selectin-mediated cell adhesion involved in various as-
pects of immune cell trafficking.3 E-selectin is synthe-
sized and expressed on the lumenal surface of endothelial
cells in areas of tissue injury upon activation by various
cytokines.3,4 Interaction of E-selectin and SLeX on the
surface of leukocytes causes leukocytes to slowly roll
along the surface of endothelial cells. Firm adhesion
occurs as rolling leukocytes interact with integrin Arg-
Gly-Asp (RGD)-containing ligands within intercellular
adhesion molecule (ICAM-1).5 This enables leukocytes
to migrate into surrounding tissue and causes further
damage. Blocking SLeX-E-selectin interactions and
thereby neutrophil emigration should therefore be a novel
therapeutic approach for treating inflammatory diseases.6
Recently, unnatural SLeX analogs with modified Gal,7
Fuc,8 NeuAc,8 and GlcN residues9,10 were prepared as
inhibitors of the SLeX-selectin binding. The biological
properties7-10 and conformational analysis9b,11 of these
analogs indicate that Gal, Fuc, and NeuAc residues are
(6) In preliminary in vivo studies, adminstration of SLeX showed a
marked thrapeutic effect on P-selectin-dependent induced lung injuries
(Mulligan, M. S.; Paulson, J . C.; DeFrees, S. A.; Zeng, Z.-L.; Lowe, J .
B.; Ward, P. A. Nature 1993, 364, 149) and on selectin-dependent
myocardial reperfusion injuries (Buerke, M.; Weyrich, A. S.; Zheng,
Z.; Gaeta, F. C. A.; Forest, M. J .; Lefer, A. M. J . Clin. Invest. 1994, 93,
1140).
X Abstract published in Advance ACS Abstracts, April 1, 1996.
(1) (a) Phillips, M. L.; Nudeman, E.; Gaeta, F. C. A.; Perez, M.;
Singhal, A. K.; Hakomori, S.; Paulson, J . C. Science 1990, 250, 1130.
(b) Walz, G.; Aruffo, A.; Kolanus, W.; Bevilacqua, M.; Seed, B. Science
1990, 250, 1132. (c) Lowe, J . B.; Stoolman, L. M.; Nair, R. P.; Larsen,
R. D.; Berhend, T. L.; Marks, R. M. Cell 1990, 63, 475. (d) Tiemyer,
M.; Swiedler, S. J .; Ishihara, M.; Moreland, M.; Schweingruber, H.;
Hirtzer, P.; Brandley, B. K. Proc. Natl. Acad. Sci. U.S.A. 1991, 88,
1138.
(2) (a) Polley, M. J .; Phillips, M. L.; Wayner, E.; Nudeman, E.;
Singhal, A. K.; Hakomori, S.; Paulson, J . C. Proc. Natl. Acad. Sci.
U.S.A. 1991, 88, 6224. (b) Zhou, Q.; Moore, K. L.; Smith, D. F.; Varki,
A.; McEver, R.; Cummings, R. D. J . Cell. Biol. 1991, 115, 557.
(3) (a) Paulson, J . C. In Adhesion: its role in inflammatory disease;
Harlan, J ., Liu, D., Eds.; W. H. Freeman: New York, 1992; Chapter
2, p 19. (b) Bevilapua, M. P.; Nelson, R. M. J . Clin. Invest. 1993, 91,
379. (c) Springer, T. A. Cell 1994, 76, 301 and reference therein.
(4) (a) Butcher, E. C. Cell 1991, 67, 1033. (b) Shimizu, Y.; Newman,
W.; Tanaka, Y.; Shaw, S. Immunol. Today 1992, 13, 106.
(5) Pytela, R.; Pierschbacher, M. D.; Ginsberg, M. H.; Plow, E. F;
Ruoslahti, E. Science 1986, 231, 1559.
(7) Stahl, W.; Sprengard, U.; Kretzschmar, G.; Kunz, H. Angew.
Chem., Int. Ed. Engl. 1994, 33, 2096.
(8) Brandley, B. K.; Kiso, M.; Abbas, S.; Nikrad, P.; Srivasatava,
O.; Foxall, C.; Oda, Y.; Hasegawa, A. Glycobiology 1993, 3, 633.
(9) (a) Tyrrell, D.; J ames, P.; Rao, N., Foxall, C.; Abbas, S.; Dasgupta,
F.; Hasegawa, A.; Kiso, M.; Asa, D.; Kidd, J .; Brandley, B. Proc. Natl.
Acad. Sci. U.S.A. 1991, 88, 10372. (b) DeFrees, S. A., Gaeta, F. C. A.;
Lin, Y.-C.; Ichikawa, Y.; Wong, C.-H. J . Am. Chem. Soc. 1993, 115,
7549.
(10) Nelson, R. M.; Dolich, S.; Aruffo, A.; Cecconi, O.; Bevilacqua,
M. P. J . Clin. Invest. 1993, 91, 1157.
(11) Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G.-J .; Garcia-
J unceda, E.; Williams, M. A.; Bayer, R.; Ketcham, C.; Walker, L. E.;
Paulson, J . C.; Wong, C.-H. J . Am. Chem. Soc. 1992, 114, 9283.
(12) Purchased from Sigma Chemical Co.
(13) Wen, D. X.; Livinigston, B. D.; Medrihdszky, K. F.; Kelm, S.;
Burlingame, A. L.; Paulson, J . C. J . Biol. Chem. 1992, 267, 21011.
(14) (a) Vann, W. F.; Silver, R. P.; Abeijon, C.; Chang, K.; Aaronson,
W.; Sutton, A.; Finn, C. W.; Lindner, W.; Kotsatos, M. J . Biol. Chem.
1987, 262, 17556. (b) Shames, S. L.; Simon, E. S.; Christpher, C. W.;
Schmid, W.; Whitesides, G. M.; Yang, L.-L. Glycobiology 1991, 1, 187.
S0022-3263(96)00125-9 CCC: $12.00 © 1996 American Chemical Society

Synthesis of SLeX Analogs
Sch em e 1
J . Org. Chem., Vol. 61, No. 9, 1996 2939
Sch em e 2a
utilization in multigram reactions have been developed.11
Although enzymatic glycosylation proceeds regio- and
stereoselectively, there can be major drawbacks with this
enzymatic method which results from strict enzyme-
substrate specificities or insoluble substrates in water.
These limitations make it difficult to synthesize various
kind of oligosaccharide analogs, especially those struc-
tures with bulky or hydrophilic residues.11 Alternatively,
chemical synthesis offers more flexibility in synthetic
strategy and design but requires tedious multiple protec-
tion/deprotection steps.11 In this paper, we describe the
preparation and the application of tetrasaccharide 1
which enables a rapid and preparative scale synthesis
of SLeX analogs having 1-O- and 2-N-disubstituted GlcN
moieties. It was constructed by a highly efficient com-
bined use of enzymatic galactosylation/sialylation and
chemical fucosylation. Tetrasaccharide 1, containing a
2-(trimethylsilyl)ethyl (SE) glycoside15 and a free amino
group on the GlcN moiety, has two attractive advantages
for the synthesis of bioactive SLeX analogs: (a) SE
glycosides can be smoothly transformed into other gly-
cosides via the corresponding glycosyl donors such as
1-chloro sugars15 and (b) desirable functional groups can
be introduced on the amino group by treatment with
desired electrophiles such as acyl halides. Scheme 1
summarizes the synthetic pathway for the preparation
of SLeX analogs from the key intermediate 1.
a
Reagents and conditions: (a) (i) HBr in AcOH, CH2Cl2, -15
°C; (ii) Se-OH, Ag2CO3, MS-4Å, CH2Cl2, -5 °C, 78% overall; (b)
NaOMe, MeOH, 25 °C, 79%; (c) UDP-Glc, UDPGE, BSA, MnCl2,
ClAP, GalT, sodium cacodylate buffer (0.05 mM, pH 7.5), 25 °C,
98%; (d) NeuAc, PEP, MgCl2, MnCl2, CMP, ATP, BSA, HS(CH2)2-
OH, CMP-NeuAc synthetase, MK, PK, PPase, 2,3-ST, HEPES
buffer (0.1 M, pH 7.5); (e) (i) Ac2O, DMAP, pyridine, 25 °C; (ii)
MeOH, 25 °C; (iii) Ac2O, DMAP, pyridine, 25 °C, 88% yield from
7; (f) Pd(PPh3)4, PMHS, THF, 25 °C, 89%; (g) AcOH, MeOH, H2O,
50 °C; (h) Z-Cl, NaHCO3, CH2Cl2, 25 °C, 77% yield from 10; (i)
AgClO4, SnCl2, TMU, Ms-4Å, ClCH2CH2Cl, -20 °C f 25 °C, 85%;
(j) 10% Pd-C, HCOONH4, EtOH, reflux, 95%.
glucosamine derivative 6 was chosen as a starting
material for the synthesis of 1-â-O-protected 2-amino
tetrasaccharide 1. It was prepared from tetraacetate 416
by the Ko¨enigs-Knorr-type glycosylation reaction with
SE alcohol followed by treatment with NaOMe. A first
crucial step in the synthesis would be the enzymatic
galactosylation of monosaccharide 6. On the basis of
previous results,17 however, we expected the glucosamine
derivative 6 to be a suitable substrate for enzymatic
galactosylation under the usual reaction conditions.18,19
The preparative method for the intermediate 1 is
shown in Scheme 2. A 1-â-O- and 2-N-diprotected
(16) Boullanger, P.; Banoub, J .; Descotes, G. Can. J . Chem. 1987,
65, 1343.
(17) DeFrees, S. A. et al. have already found that allyloxycarbonyl
group on the amino group of glucosamine derivatives does not interfere
with enzymatic galactosylation (private communication).
(18) Unverzagt, C.; Kunz, H.; Paulson, J . C. J . Am. Chem. Soc. 1990,
112, 9308.
(15) (a) J ansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J .; Magnusson,
G. J . Org. Chem. 1988, 53, 5629. (b) J ansson, K.; Noori, G.; Magnusson,
G. J . Org. Chem. 1990, 55, 3181.

2940 J . Org. Chem., Vol. 61, No. 9, 1996
Hayashi et al.
Sch em e 3a
Glucosamine derivative 6 was dissolved in 50 mM sodium
cacodylate buffer (pH 7.5), UDP-glucose, bovine serum
albumin (BSA), MnCl2, UDP-galactose 4-epimerase (UD-
PGE), calf intestinal alkaline phosphatase (CIAP), and
GalT were added, and the solution was incubated at 37
°C for 8 days. The reaction mixture was concentrated
and purified by gel filtration to afford lactosamine
derivative 7 in 98% yield. Moreover, it was found that
disaccharide 7 was a well-recognized substrate for enzy-
matic sialylation.20 The sialylation utilized the multi-
enzyme system reported by Ichikawa et al.11 The result-
ing sialylated product 8 was then converted into fully
protected compound 9 in three steps consisting of acety-
lation, methanolysis of the lactone, and successive acety-
lation of a resulting hydroxyl group on the Gal moiety
(88% overall yield from 7).21 The allyloxycarbonyl (Alloc)
group of 9 was removed by reaction of carbamate 8 with
Pd(PPh3)4 in the presence of poly(methylhydrosiloxane)
(PMHS) affording amine 10 in 89% yield. Treatment of
amine 10 in aqueous methanol with a catalytic amount
of acetic acid provided the desired amino alcohol 11 as a
single product.22 The amino group on 11 was reprotected
selectively by benzyloxycarbonyl chloride (Z-Cl) to give
glycosyl acceptor 12 in 77% overall yield from 10.
Introduction of the Fuc group onto the hydroxyl group
of 12 was achieved by using glycosyl fluoride 13,23 in the
presence of AgClO4, SnCl2, and tetramethylurea (TMU)23,24
to provide the tetrasaccharide 14 stereoselectively in 85%
yield. Thus, the desired key intermediate 1 could be
obtained by hydrogenalytic cleavage of the benzyl groups
and Z group of 14 in 95% yield.
a
Reagents and conditions: (a) butyryl chloride, NaHCO3,
CH2Cl2, 25 °C; (b) Ac2O, pyridine, DMAP, 25 °C, 86% yield from
1; (c) 1,1-dichloromethyl methyl ether, ZnCl2, CHCl3, 25 °C; (d)
1-dodecanol, Sn(OTf)2, TMU, MS-4Å, CH2Cl2, 25 °C, 52% yield
from 16a ; (e) EtOH, Sn(OTf)2, TMU, MS4A, CH2Cl2, 25 °C, 41%
yield from 16a ; (f) 3-azido-1-propanol, Sn(OTf)2, TMU, MS-4Å,
CH2Cl2, 25 °C, 38% yield from 16a ; (g) NaOMe, MeOH, H2O, 25
°C, 78% yield; (h) NaOMe, MeOH, H2O, 25 °C, 75% yield.
Compound 1 could be converted into a SLeX analog
3a having lipophilic O-dodecyl/N-butylyl substituents, as
shown in Scheme 3. Utilizing this scheme, the amino
group of 1 was selectively acylated with butyryl chloride
in the presence of NaHCO3 and was then acetylated to
afford butanamide 16a in 86% yield. The SE glycoside
16a was then transformed into dodecyl glycoside 18
utilizing Magnusson’s method15b,25 in 52% yield. The
peracetate 18a was then treated with basic conditions
to give the difunctionalized SLeX analog 3a in 78% yield.
The derivatives 3b-f were synthesized in a similar
manner.
These SLeX analogs were found to inhibit cell adhesion
of human leukocyte (HL-60) to recombinant soluble
F igu r e 1. Inhibition of HL-60 adhesion to recombinant
soluble E-selectin-coated plates: (O) 3a ; (b) 3b; (4) 3c; (2) 3d ;
(0) 3e; (9) 3f.
human E-selectin.26 The IC50 values of analogs 3a , 3b,
3c, 3d , 3e, and 3f are 2.0, 1.0, 1.6, 1.5, 0.1, and 0.1 mM,
respectively, as shown in Figure 1. Herein, IC50 means
the concentration that inhibited cell adhesion of HL-60
to human E-selectin by 50%. Butanamides 3a and 3b
showed almost the same activity as acetamides 3c and
3d as expected from the result reported by Nelson et al.10
In contrast, naphthamides 3e and 3f were indicated to
be five times or more active than acetamides 3c and 3d ,
similar to the result reported by Ramphal et al.22 These
results suggest that modification of GlcN moiety on SLeX
should be of great value for the search of effective
inhibitor of cell adhesion.
(19) For reviews of substrate specificity for GalT, see: (a) Wong,
C.-H.; Ichikawa, Y.; Krach, T.; Narvor, C. G.-L.; Dumas, D. P.; Look,
G. C. J . Am. Chem. Soc. 1991, 113, 8137. (b) Wong, C.-H.; Halcomb,
R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed. Engl. 1995,
34, 521.
(20) For reports on substrate specificities of 2,3-ST, see: (a) Kunz,
H.; Harrews, A. Liebigs Ann. Chem. 1982, 41. (b) Schmidt, R. R.;
Zimmreman, P. Angew. Chem., Int. Ed. Engl. 1986, 25, 725. (c) Sato,
S.; Nunomura, S.; Nakano, T.; Ito, Y.; Ogawa, T. Tetrahedron Lett.
1988, 29, 4097. (d) Ito Y.; Paulson, J . C. J . Am. Chem. Soc. 1993, 115,
1603.
(21) This three-step procedure was developed by Ratcliffe, R. M.
(private communication). See also: DeFrees, S. A.; Gaeta, F. C. A.;
Gaudino, J . J .; Zheng, Z.; Hayashi, M. Int. Pat. 1994, WO 9426760;
Chem. Abstr., 1994.
(22) Ramphal, J . Y.; Hiroshige, M.; Lou, B.; Guadino, J .; Hayashi,
M.; Chen, S. M.; Chiang, L. C.; Gaeta, F. C. A.; DeFrees, S. A. J . Med.
Chem., in press.
(23) Nicolaou, K. C.; Hummel, C. W.; Bockovich, N. J .; Wong, C.-H.
J . Chem. Soc., Chem. Commun. 1991, 15, 872.
(24) Mukaiyama, T.; Murai, Y.; Shoda, S. Chem. Lett. 1981, 431.
(25) Lubineau, A., Gallic, J . L.; Malleron, A. Tetrahedron Lett. 1987,
28, 5041.
(26) For the evaluation method of inhibition of HL-60 adhesion to
recombinant soluble human E-selectin, see: DeFrees, S. A.; Kosch, W.;
Way, W.; Paulson, J . C.; Sabesan, S.; Halcomb, R. L.; Huang, D.-H.;
Ichikawa, Y.; Wong, C.-H. J . Am. Chem. Soc. 1995, 117, 66.

Synthesis of SLeX Analogs
J . Org. Chem., Vol. 61, No. 9, 1996 2941
amount of dichloromethane, and the solution was added
dropwise to heptane (500 g) at 0 °C. The resulting precipitate
was collected by filtration to afford 6 (28.6 g, 79% yield) as
colorless crystals: Rf ) 0.70 (developed with a 10:2:1 mixture
of ethyl acetate, ethanol, and water); mp 134-138 °C; 1H NMR
(CDCl3) δ 5.90 (1H, m), 5.75 (1H, d, J ) 8.3 Hz), 5.30 (1H, dd,
J ) 1.3 and 17.2 Hz), 5.18 (1H, dd, J ) 1.3 and 10.2 Hz), 5.05
(1H, s), 4.72 (1H, s), 4.55 (1H, d, J ) 5.6 Hz), 4.48 (1H, m),
3.96 (1H, m), 3.85 (2H, m), 3.80-3.23 (6H, m), 2.41 (1H, s),
To further demonstrate utility of this synthetic ap-
proach, the above methodology was used to prepare the
SLeX derivative 19. Thus obtained compound 19 has
been used as a precursor to allow coupling of the
oligosaccharide to lipids.27 Moreover, compound 19 can
be used for coupling to proteins such as bovine serum
albumin and provide neoglycoproteins which are useful
as antigens.28
In conclusion, our synthetic approach to intermediate
1 provides ready access to SLeX analogs with modified
GlcN residues. We continue to study the synthesis and
structure-activity relationships of SLeX both in vitro and
in vivo and will report the results in due course.
0.99-0.85 (2H, m), and 0.00 (9H, s). Anal. Calcd for C15H29
-
NO7Si: C, 49.57; H, 8.04; N, 3.85. Found: C, 49.40; H, 7.92;
N, 4.03.
2-(Tr im eth ylsilyl)eth yl â-D-Ga la ctop yr a n osyl-(1f4)-O-
(2-N-(a llyloxyca r bon yl)-2-a m in o-2-d eoxy-â-D-glu cop yr a -
n osid e) (7). Galactosyltransferase (GalT, EC 2.4.1.22, 30 U)
and uridine 5′-diphosphogalactose 4′-epimerase (UDPGE, EC
5.1.3.2, 240 U) were added to a solution containing 50 mM
aqueous sodium cacodylate (pH 7.5, 120 mL), 5% aqueous
bovine serum albumin (BSA, 2.46 mL), 0.35 M aqueous
manganese(II) chloride (0.78 mL), 0.50 M aqueous sodium
azide (1.68 mL), calf intestinal alkaline phosphatase (CIAP,
EC 3.1.3.1, 1320 U), uridine 5′-diphosphoglucose disodium salt
(UDP-Glc‚2Na, 4.56 g, 8.05 mmol), and monosaccharide 6 (2.18
g, 6.00 mmol). The reaction mixture was inverted several
times and allowed to stand at 37 °C for 4 days, and then
additional GalT (10 U) was added to the mixture. After
standing at 37 °C for an additional 4 days, the mixture was
filtered through a pad of Celite. The filtrate was concentrated
under reduced pressure, and the residue was purified by gel
filtration chromatography. Disaccharide 7 was afforded as a
white solid after lyophilization (3.01 g, 98% yield): Rf ) 0.38
(developed with a 10:2:1 mixture of ethyl acetate, ethanol, and
Exp er im en ta l Section
1H-NMR (270 MHz) and 13C-NMR (125 MHz) spectra were
recorded in the indicated solvent. Melting points (mp) were
uncorrected. All reactions involving nonaqueous solutions
were carried out under a nitrogen atomosphere. All reactions
were monitored by thin-layer chromatography carried out on
silica gel glass-backed plates (0.25 mm). Silica gel column
chromatography was performed on silica gel 60 (230-400 or
70-230 mesh) using the indicated solvents. Gel filtration
chromatography was performed on Bio-Gel P-2 gel (65 ( 20
µm). Dichloromethane and 1,2-dichloroethane were dried over
4 Å molecular sieves (MS-4Å). Tetrahydrofuran (THF) was
dried over MS-5Å.
2-(Tr im eth ylsilyl)eth yl 2-N-(Allyloxycar bon yl)-2-am in o-
2-d eoxy-3,4,6-tr i-O-a cetyl-â-D-glu cop yr a n osid e (5). Hy-
drogen bromide in acetic acid (25% assay, 388.4 g, 1.20 mmol)
was added dropwise to a stirred solution of 2-N-(allyloxycar-
bonyl)-2-amino-2-deoxy-1,3,4,6-tetra-O-acetyl-â-D-glucopyra-
nose (4) (172.56 g, 0.40 mmol) in dichloromethane (1035 mL)
at -15 °C over a 1 h period, and the mixture was stirred at
-15 °C for 2 h. Then, the mixture was washed with water,
5% aqueous sodium hydrogen carbonate, and water in that
order. The organic layer was dried over MS-4Å and then
filtered. The filtrate was added dropwise to the mixture of
2-(trimethylsilyl)ethanol (94.6 g, 0.80 mmol), silver carbonate
(331 g, 1.20 mmol), and powdered MS-4Å (429 g) in dichlo-
romethane (1035 mL) at -5 °C over a 90 min period. After
being stirred for 1 h at -5 °C, the reaction mixture was filtered
through a pad of Celite and the filtrate was washed with 5%
aqueous sodium hydrogen carbonate and water in that order.
The organic layer was concentrated under reduced pressure,
and the resulting residue was dissolved in toluene (245 g). The
solution was added dropwise into stirred hexane (817 g), and
the resulting crystals were collected by filtration to afford 5
(153.0 g, 78% yield) as colorless crystals: Rf ) 0.57 (developed
with a 1:1 mixture of hexane and ethyl acetate); mp 70-72
1
water); H NMR (D2O) δ 5.96 (1H, m), 5.33 (1H, d, J ) 17.1
Hz), 5.25 (1H, d, J ) 10.5 Hz), 4.74-4.50 (3H, m), 4.47 (1H, d,
J ) 7.6 Hz), 4.06-3.40 (14H, m), 1.08-0.93 (2H, m), and 0.00
(9H, s).
2-(Tr im eth ylsilyl)eth yl (5-Aceta m id o-3,5-d id eoxy-r-D-
glycer o-D-ga la cto-2-n on u lop yla n osylon ic a cid )-(2f3)-O-
(â-D-ga la ctop yr a n osyl)-(1f4)-O-[2-N-(a llyloxyca r bon yl)-
2-a m in o-2-d eoxy-â-D-glu cop yr a n osid e] (8). (-)-N-Acetyl-
neuraminic acid (6.30 g, 20.3 mmol), phospho(enol)pyruvate
trisodium salt monohydrate (PEP‚3Na‚H2O, 11.58 g, 49.5
mmol), 1.0 M aqueous magnesium chloride (20.3 mL), 1.0 M
aqueous manganese(II) chloride (5.38 mL), 1.0 M aqueous
potassium chloride (20.3 mL), cytidine 5′-monophosphate
(CMP, 656 mg, 2.03 mmol), adenosine 5′-triphosphate (ATP,
112 mg, 0.203 mmol), 5% aqueous BSA (16.3 mL), and
2-mercaptethanol (64 µL) were added to a solution of 7 (8.30
g, 16.8 mmol) in HEPES buffer (pH 7.5, 820 mL). The pH of
the solution was adjusted to 7.5 with 1.0 M aqueous sodium
hydroxide, and the enzymes, inorganic pyrophosphatase (PPase,
EC 3.6.1.1, 2444 U), myokinase (MK, EC 2.7.4.1, 32587 U),
pyrvate kinase (PK, EC 2.7.1.40, 52956 U), cytidine 5′-
monophospho-N-acetylneuraminic acid synthetase (CMP-
NeuAc synthetase, EC 2.7.7.43, 62 U), and R(2,3)-sialyl
transferase (2,3-ST, EC 2.4.99.4, 162 U) were added to the
solution. After standing at room temperature for 5 days, the
mixture was diluted with excess methanol and concentrated
under reduced pressure to give 8. The resulting amorphous
solid was subjected to the next reaction without further
purification: Rf ) 0.49 (developed with a 4:2:1 mixture of ethyl
1
°C; H NMR (CDCl3) δ 5.81 (1H, m), 5.27-5.08 (4H, m), 4.96
(1H, t, J ) 9.6 Hz), 4.65-4.45 (2H, m), 4.47 (1H, d, J ) 4.3
Hz), 4.20 (1H, dd, J ) 4.6 and 11.9 Hz), 4.04 (1H, dd, J ) 2.3
and 11.9 Hz), 3.89 (1H, m), 3.65 (1H, m), 3.63-3.45 (2H, m),
1.99 (3H, s), 1.94 (3H, s), 1.93 (3H, s), 0.94-0.80 (2H, m), and
-0.08 (9H, s). Anal. Calcd for C21H35NO10Si‚H2O: C, 49.69;
H, 7.35; N, 2.76. Found: C, 49.82; H, 6.98; N, 2.81.
2-(Tr im eth ylsilyl)eth yl 2-N-(Allyloxycar bon yl)-2-am in o-
2-d eoxy-â-D-glu cop yr a n osid e (6). Sodium methoxide in
methanol (28% assay, 6.0 g, 0.02 mmol) was added to a stirred
solution of 5 (49.0 g, 0.10 mmol) in methanol (150 mL) at room
temperature. After 1 h, the mixture was concentrated under
reduced pressure and diluted with dichloromethane (720 g).
The solution was added to stirred water (480 g), and the
mixture was neutrilized with 1 N hydrochloric acid. The
organic layer was then separated and concentrated under
reduced pressure. The residue was dissolved in a small
1
acetate, ethanol, and water); H NMR (D2O) δ 5.96 (1H, m),
5.34 (1H, dd, J ) 1.3 and 17.1 Hz), 5.25 (1H, dd, J ) 1.3 and
10.5 Hz), 4.60-4.51 (4H, m), 4.14-3.37 (21H, m), 2.75 (1H,
dd, J ) 4.6 and 12.5 Hz), 2.03 (3H, s), 1.80 (1H, t, J ) 12.2
Hz), 1.05-0.82 (2H, m), and 0.00 (9H, s).
On e-P ot P r ep a r a tion of 2-(Tr im eth ylsilyl)eth yl [Meth -
yl (5-a cet a m id o-3,5-d id eoxy-4,7,8,9-t et r a -O-a cet yl-r-D-
glycer o-D-ga la ct o-2-n on u lop yr a n osylon a t e )]-(2f3)-O-
(2,4,6-t r i-O-a cet yl-â-D-ga la ct op yr a n osyl)-(1f4)-O-[2-N-
(a llyloxyca r bon yl)-2-a m in o-2-d eoxy-3,6-d i-O-a cetyl-â-D-
glu cop yr a n osid e] (9). Acetic anhydride (227 mL) and
4-(dimethylamino)pyridine (0.50 g) were added to a stirred
solution of crude 8 (obtained above) in pyridine (377 mL) at 0
°C. After stirring for 12 h, methanol (580 mL) was added
dropwise to the mixture at 0 °C. The mixture was warmed to
(27) DeFrees, S. A. Private communication. See also: DeFrees, S.
A.; Phillips, L.; Zalipsky, S.; Guo, L. Submitted.
(28) Rice, K. G. In Neoglycoconjugates: Preparation and Applica-
tions; Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego, 1994;
Chapter 9, pp 285-321.

2942 J . Org. Chem., Vol. 61, No. 9, 1996
Hayashi et al.
room temperature, stirred for 1 day, and concentrated under
reduced pressure. The residue was treated with pyridine (240
mL) and acetic anhydride (150 mL) at 0 °C, and the mixture
was stirred at room temperature for an additional 3 h. Then,
methanol (500 mL) was added dropwise to the mixture at 0
°C. After stirring at 0 °C for 30 min, the mixture was
concentrated under reduced pressure. The residue was dis-
solved in ethyl acetate and washed with saturated copper(II)
sulfate, saturated sodium hydrogen carbonate, and brine in
that order. The organic layer was dried over anhydrous
sodium sulfate, filtered, and concentrated under reduced
pressure. The resulting residue was purified by silica gel
column chromatography eluting with hexane, ethyl acetate,
and ethanol (10/10/2) to afford 9 (16.77 g, 88% yield from 6)
as a pale yellow crystalline powder: Rf ) 0.40 (developed with
a 10:10:3 mixture of hexane, ethyl acetate, and ethanol); mp
>200 °C dec; IR (KBr) 2958, 1741, 1670, 1542, 1372, 1230,
10/2) to afford 9 (156 mg, 92% yield) as a colorless crystalline
powder. This sample showed identical physical and spectral
properties with those recorded on 9 obtained above.
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
lactopyr an osyl)-(1f4)-O-(2-am in o-2-deoxy-3,6-di-O-acetyl-
â-D-glu cop yr a n osid e) (10). Tetrakis(triphenylphosphine)-
palladium(0) [Pd(PPh3)4] (3.30 g) and poly(methylhydrosilox-
ane) (PMHS, 1.6 mL) were added to a stirred solution of 9 (16.5
g, 13.7 mmol) in THF (165 mL) at room temperature. After
stirring for 2.5 h, Pd(PPh3)4 (3.30 g) and PMHS (1.6 mL) were
added again. After an additional 12 h, the mixture was diluted
with dichloromethane and washed with water. The organic
layer was dried over anhydrous magnesium sulfate, filtered,
and concentrated under reduced pressure. The resulting
residue was purified by silica gel column chromatography
eluting with hexane, ethyl acetate, and methanol (10/10/1) to
afford 10 (13.61 g, 89% yield) as a pale yellow amorphous
solid: Rf ) 0.19 (developed with a 10:10:3 mixture of hexane,
1
1047 cm-1; H NMR (CDCl3) δ 5.86 (1H, m), 5.55-3.50 (29H,
m), 3.85 (3H, s), 2.59 (1H, dd, J ) 4.3 and 12.5 Hz), 2.24 (3H,
s), 2.15 (3H, s), 2.10 (3H, s), 2.08 (3H, s), 2.07 (3H, s), 2.06
(3H, s), 2.05 (3H, s), 2.04 (3H, s), 2.00 (3H, s), 1.85 (3H, s),
1.68 (1H, t, J ) 12.5 Hz), 0.98-0.91 (2H, m), and -0.01 (9H,
s). Anal. Calcd for C51H76N2O29Si: C, 50.66; H, 6.33; N, 2.32.
Found: C, 50.30; H, 6.32; N, 2.29.
1
ethyl acetate, and ethanol); H NMR (CDCl3) δ 5.50 (1H, m),
5.38 (1H, m), 5.11-4.85 (4H, m), 4.65-3.45 (19H, m), 3.83 (3H,
s), 2.76 (1H, t, J ) 8.9 Hz), 2.60 (1H, dd, J ) 4.6 and 12.9 Hz),
2.26 (3H, s), 2.15 (3H, s), 2.10 (3H, s), 2.08 (3H, s), 2.07 (3H,
s), 2.06 (3H, s), 2.05 (3H, s), 2.04 (3H, s), 1.99 (3H, s), 1.84
(3H, s), 1.70 (1H, m), 1.05-0.93 (2H, m), and 0.01 (9H, s). Anal.
Stepwise P r epar ation of 2-(Tr im eth ylsilyl)eth yl [Meth -
yl (5-a cet a m id o-3,5-d id eoxy-4,7,8,9-t et r a -O-a cet yl-r-D-
glycer o-D-ga la ct o-2-n on u lop yr a n osylon a t e )]-(2f3)-O-
(2,4,6-t r i-O-a cet yl-â-D-ga la ct op yr a n osyl)-(1f4)-O-[2-N-
(a llyloxyca r bon yl)-2-a m in o-2-d eoxy-3,6-d i-O-a cetyl-â-D-
glu cop yr a n osid e] (9). Acetic anhydride (3.5 mL) and 4-(di-
methylamino)pyridine (10 mg) were added to a stirred solution
of 8 (554 mg, 0.678 mmol) in pyridine (9.0 mL) at 0 °C. After
stirring for 1 day, the mixture was concentrated under reduced
pressure. The residue was dissolved in ethyl acetate and
washed with saturated sodium hydrogen carbonate and brine
in that order. The organic layer was dried over anhydrous
sodium sulfate, filtered, and concentrated under reduced
pressure. The resulting residue was purified by silica gel
column chromatography eluting with hexane, ethyl acetate,
and acetone (1/1/0.5) to afford lactone 8a (704 mg, 92% yield)
as a colorless crystalline powder: Rf ) 0.45 (developed with a
1:1:1 mixture of hexane, ethyl acetate, and acetone); mp 116-
120 °C; IR (KBr) 2958, 1748, 1655, 1542, 1372, 1236, 1036
Calcd for C47
Found: C, 48.85; H, 6.49; N, 2.45.
H72N2O27Si‚2H2O: C, 48.62; H, 6.60; N, 2.41.
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
la ctop yr a n osyl)-(1f4)-O-(6-O-a cetyl-2-a m in o-2-d eoxy-â-
D-glu cop yr a n osid e) (11). Acetic acid (0.72 mL) was added
to a solution of 10 (13.61 g, 12.1 mmol) in methanol (1089 mL)
and water (272 mL). The mixture was warmed to 50 °C and
stirred at the temperature for 1 day. Concentration under
reduced pressure afforded crude 11 as a pale yellow amorphous
solid which was subjected to the sequential reaction without
further purification: Rf ) 0.76 (developed with a 9:1 mixture
of chloroform and methanol); 1H NMR (CDCl3) δ 5.50 (1H, m),
5.40 (1H, m), 5.12-4.85 (4H, m), 4.65-3.48 (20H, m), 3.83 (3H,
s), 2.76 (1H, t, J ) 8.7 Hz), 2.56 (1H, dd, J ) 4.3 and 12.5 Hz),
2.27 (3H, s), 2.15 (3H, s), 2.10 (3H, s), 2.08 (3H, s), 2.07 (3H,
s), 2.06 (3H, s), 2.05 (3H, s), 2.00 (3H, s), 1.85 (3H, s), 1.67
(1H, t, J ) 12.5 Hz), 1.02-0.92 (2H, m), and 0.01 (9H, s). Anal.
Calcd for C45H70N2O26Si‚H2O: C, 49.08; H, 6.59; N, 2.54.
Found: C, 48.75; H, 6.56; N, 2.60.
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
la c t o p y r a n o s y l)-(1f4)-O -[6-O -a c e t y l-2-a m i n o -2-N -
(ben zyloxyca r bon yl)-2-d eoxy-â-D-glu cop yr a n osid e] (12).
Benzyl chloroformate (Z-Cl, 2.6 mL, 18.2 mmol) was added
dropwise to a mixture of 11 and powdered sodium hydrogen
carbonate (3.05 g, 36.3 mmol) in dichloromethane (262 mL)
at room temperature. The mixture was stirred at room
temperature for 12 h and then diluted with ethyl acetate. The
solution was washed with water, dried over anhydrous mag-
nesium sulfate, filtered, and concentrated under reduced
pressure. The resulting residue was purified by silica gel
column chromatography eluting with hexane, ethyl acetate,
and methanol (10/10/1) to give 12 (11.30 g, 77% yield from 10)
1
cm-1; H NMR (CDCl3) δ 6.13 (1H, d, J ) 10.2 Hz), 5.82 (1H,
m), 5.38-5.02 (8H, m), 4.70-4.32 (7H, m), 4.25-3.79 (8H, m),
3.67-3.43 (4H, m), 2.47 (1H, br.), 2.37 (1H, dd, J ) 5.3 and
13.2 Hz), 2.14 (3H, s), 2.04 (3H, s), 2.03 (3H, s), 2.02 (3H, s),
2.01 (3H, s), 2.00 (3H, s), 1.99 (3H, s), 1.95 (3H, s), 1.82 (3H,
s), 1.77 (1H, t, J ) 13.2 Hz), 0.87 (2H, m), and -0.06 (9H, s).
Anal. Calcd for C48H70N2O27Si‚H2O: C, 49.99; H, 6.29; N, 2.43.
Found: C, 49.90; H, 6.23; N, 2.47.
4-(Dimethylamino)pyridine (5.2 mg, 0.043 mmol) was added
to a solution of lactone 8a (244 mg, 0.215 mmol) in methanol
(3.0 mL), and the solution was stirred at room temperature
for 18 h. The solution was then concentrated under reduced
pressure, and the resulting residue was purified by silica gel
column chromatography eluting with hexane, ethyl acetate,
and acetone (1/1/0.5) to give ester 8b (176 mg, 70% yield) as a
colorless crystalline powder: Rf ) 0.37 (developed with a 1:1:1
mixture of hexane, ethyl acetate, and acetone); mp 102-105
°C; 1H NMR (CDCl3) δ 5.87 (1H, m), 5.45-4.25 (17H, m), 4.10-
3.40 (12H, m), 3.86 (3H, s), 2.83 (1H, br), 2.67 (1H, dd, J ) 4.6
and 12.5 Hz), 2.15 (3H, s), 2.14 (3H, s), 2.06 (15H, s), 2.02 (3H,
s), 1.89 (3H, s), 1.84 (1H, t, J ) 12.5 Hz), 0.93 (2H, m), and
-0.01 (9H, s). Anal. Calcd for C49H74N2O28Si‚2 H2O: C, 48.91;
H, 6.53; N, 2.33. Found: C, 49.10; H, 6.33; N, 2.57.
Acetic anhydride (1.0 mL) and 4-(dimethylamino)pyridine
(5.0 mg) were added to a stirred solution of ester 8b (164 mg,
0.141 mmol) in pyridine (3.0 mL) at 0 °C. After stirring for 1
day, the mixture was concentrated under reduced pressure.
The residue was dissolved in ethyl acetate and washed with
saturated sodium hydrogen carbonate and brine in that order.
The organic layer was dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure. The
resulting residue was purified by silica gel column chroma-
tography eluting with hexane, ethyl acetate, and ethanol (10/
as a colorless crystalline powder: Rf
) 0.71 (developed with a
9:1 mixture of chloroform and methanol); mp 115-120 °C; 1H
NMR (CDCl3) δ 7.34-7.27 (5H, m, Ph-H), 5.51 (1H, m), 5.41
(1H, m), 5.15-3.25 (26H, m), 3.82 (3H, s), 2.58 (1H, dd, J )
4.6 and 12.5 Hz), 2.28 (3H, s), 2.17 (3H, s), 2.11 (3H, s), 2.10
(3H, s), 2.09 (3H, s), 2.07 (3H, s), 2.02 (3H, s), 2.01 (3H, s),
1.86 (3H, s), 1.66 (1H, t, J ) 12.5 Hz), 0.95-0.82 (2H, m), and
-0.01 (9H, s). Anal. Calcd for C53H76N2O28Si‚H2O: C, 51.53;
H, 6.36; N, 2.27. Found: C, 51.56; H, 6.38; N, 2.26.
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
la ct op yr a n osyl)-(1f4)-O-[2,3,4-t r i-O-b en zyl-r-L-fu cop y-
r a n osyl-(1f3)-O]-[6-O-a cet yl-2-a m in o-2-N-(b en zyloxy-

Synthesis of SLeX Analogs
J . Org. Chem., Vol. 61, No. 9, 1996 2943
ca r bon yl)-2-d eoxy-â-D-glu cop yr a n osid e] (14). Powdered
MS-4Å (2.6 g), 1,1,3,3-tetramethylurea (TMU, 3.30 mL, 27.6
mmol), and 2,3,4-tri-O-benzyl-L-fucosyl fluoride (13) (12.0 g,
27.5 mmol) were added to a stirred solution of 12 (5.60 g, 4.60
mmol) in 1,2-dichloroethane (25 mL). After stirring for 90 min,
the mixture was shielded from light, cooled to -20 °C, and
treated with tin(II) chloride (3.49 g, 18.4 mmol) and silver
perchlorate (3.85 g, 18.4 mmol). The reaction mixture was
allowed to warm to room temperature over a 90 min period
and stirred for 24 h at room temperature. The suspension was
filtered through a pad of Celite, and the filtrate was washed
with water. The organic layer was separated, dried over
anhydrous sodium sulfate, filtered, and concentrated under
reduced pressure. The resulting residue was purified by silica
gel column chromatography eluting with hexane, ethyl acetate,
and methanol (10/10/1) to give 14 (6.37 g, 85% yield) as a
colorless crystalline powder: Rf ) 0.41 (developed with a 10:
10:3 mixture of hexane, ethyl acetate, and methanol); mp 102-
108 °C; 1H NMR (CDCl3) δ 7.46-7.24 (20H, m), 5.43 (1H, d, J
) 9.5 Hz), 5.46 (1H, d, J ) 9.5 Hz), 5.20-3.50 (36H, m), 3.94
(3H, s), 2.60 (1H, dd, J ) 4.6 and 12.5 Hz), 2.24 (3H, s), 2.18
(3H, s), 2.10 (3H, s), 2.08 (3H, s), 2.07 (3H, s), 2.04 (6H, s),
1.88 (3H, s), 1.84 (3H, s), 1.70 (1H, t, J ) 12.5 Hz), 1.26 (3H,
d, J ) 6.3 Hz), 0.94-0.84 (2H, m), and 0.00 (9H, s). Anal.
Calcd for C80H104N2O32Si‚3H2O: C, 56.93; H, 6.57; N, 1.66.
Found: C, 56.72; H, 6.22; N, 1.71.
fu copyr an osyl-(1f3)-O]-[6-O-acetyl-2-(acylam in o)-2-deoxy-
â-D-glu cop yr a n osid e] (16). Triol 15 was treated with acetic
anhydride (20 mL) and 4-(dimethylamino)pyridine (80 mg) in
pyridine (40 mL) at room temperature. After the mixture was
stirred for 2 h, methanol (25 mL) was added dropwise to the
mixture at 0 °C. The mixture was stirred at room temperature
for 0.5 h and then concentrated in vacuo. The resulting
residue was diluted with ethyl acetate and washed with
saturated copper(II) sulfate, saturated sodium hydrogen car-
bonate and brine in that order. The organic layer was dried
over anhydrous sodium sulfate, filtered, and concentrated
under reduced pressure. The residue was purified by silica
gel column chromatography to afford 16.
Gen er a l P r oced u r e for Con ver sion of th e 1-O-SE
Gr ou p to Oth er Glycosid es. Syn th esis of Alk yl [Meth yl
(5-a ceta m id o-3,5-d id eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glyc-
er o-D-ga la cto-2-n on u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-
t r i-O-a cet yl-â-D-ga la ct op yr a n osyl)-(1f4)-O-[2,3,4-t r i-O-
a cet yl-r-L-fu cop yr a n osyl-(1f3)-O]-[6-O-a cet yl-2-(a cyl-
a m in o)-2-d eoxy-â-D-glu cop yr a n osid e] (18). 1,1-Dichloro-
methyl methyl ether (32.0 µL, 0.355 mmol) and zinc chloride
(2.4 mg, 17.6 µmol) were added to a stirred solution of 16 (0.071
mmol) in chloroform (5.0 mL) at room temperature. After
stirring for 6 h, the mixture was concentrated under reduced
pressure to give crude 17.
A solution of alcohol (0.178 mmol) and TMU (17.0 µL, 0.142
mmol) in dichrolomethane (1.0 mL) was added dropwise to the
stirred suspension of 17 obtained above, tin(II) trifluo-
romethanesulfonate [Sn(OTf)2] (59.0 mg, 0.142 mmol), and
powdered MS-4Å (95.0 mg) in dichloromethane (6.0 mL) at
room temperature. After the mixture was stirred for 8 h,
additional Sn(OTf)2 (59.0 mg, 0.142 mmol), alcohol (0.178
mmol) and TMU (17.0 µL, 0.142 mmol) were added to the
mixture. The mixture was stirred for 16 h at room tempera-
ture and then filtered through a pad of Celite. The filtrate
was washed with saturated sodium hydrogen carbonate and
brine in that order, dried over anhydrous magnesium sulfate,
and concentrated under reduced pressure. The resulting
residue was purified by preparative thin layer chromatography
(silica gel 60, 0.5 mm, 20 cm × 20 cm, E. Merck) developed
with a mixture of chloroform and methanol to give 18.
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
la ctop yr a n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O-[6-
O-a cetyl-2-a m in o-2-d eoxy-â-D-glu cop yr a n osid e] (1).
A
mixture of 14 (6.25 g, 3.83 mmol), ammonium formate (10 g),
and 10% palladium on activated carbon (Pd-C, wet, 10 g) in
ethanol (125 mL) was refluxed with stirring for 8 h. Additional
ammonium formate (10 g) and 10% Pd-C (wet, 10 g) were
added to the mixture, and the mixture was refluxed for an
additional 8 h. The mixture was filtered through a pad of
Celite, and the filtrate was concentrated under reduced
pressure to give amine 1 (4.45 g, 95% yield) as a colorless
crystalline powder: Rf ) 0.44 (developed with a 10:2:1 mixture
of ethyl acetate, ethanol, and water); mp 149-152 °C; IR (KBr)
3420, 2960, 1745, 1655, 1373, 1232, 1042 cm-1 1H NMR
;
Gen er a l P r oced u r e for Dep r otection . Syn th esis of
Alk yl (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 )-(2f3)-O-(â-D-ga la ctop yr a n o-
syl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-[2-(a cyla m in o)-
2-d eoxy-â-D-glu cop yr a n osid e] (3). To a stirred solution of
18 (36.8 µmol) in methanol (5.0 mL) was added sodium
methoxide in methanol (28% assay, 50 µL) at room tempera-
ture. After stirring for 7 h, water (2.5 mL) was added to the
mixture and the mixture was stirred for 2 days. The mixture
was then concentrated under reduced pressure, purified by gel
filtration chromatography, and lyophilized to afford 3.
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
la ctop yr a n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-(6-
O -a c e t y l -2 -a m i n o -2 -N -b u t y r y l -2 -d e o x y -â-D -g l u c o -
p yr a n osid e) (15a ). The title compound was obtained as a
crude residue from 1, which was then used in the next reaction
without further purification: Rf ) 0.63 (developed with a 10:
2:1 mixture of ethyl acetate, ethanol, and water).
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
la ct op yr a n osyl)-(1f4)-O-[2,3,4-t r i-O-a ce t yl-r-L -fu co-
p yr a n osyl-(1f3)-O]-(6-O-a cet yl-2-a m in o-2-N-b u t yr yl-2-
d eoxy-â-D-glu cop yr a n osid e) (16a ). The title compound was
obtained as colorless crystals in 86% yield from 1: Rf ) 0.56
(developed with a 9:1 mixture of chloroform and methanol);
mp 152-155 °C; 1H NMR (CDCl3) δ 5.50-3.45 (30H, m), 3.85
(3H, s), 2.59 (1H, dd, J ) 4.3 and 12.5 Hz), 2.20 (3H, s), 2.15
(3H, s), 2.13 (3H, s), 2.12 (3H, s), 2.09 (3H, s), 2.08 (3H, s),
2.07 (3H, s), 2.06 (3H, s), 2.04 (3H, s), 2.00 (3H, s), 2.00 (2H,
t, J ) 5.2 Hz), 1.94 (3H, s), 1.85 (3H, s), 1.70-1.56 (3H, m),
1.18 (3H, d, J ) 6.6 Hz), 0.97-0.85 (2H, m), 0.88 (3H, t, J )
(CDCl3) δ 5.48-5.40 (2H, m), 5.34 (1H, d, J ) 3.3 Hz), 5.13
(1H, d, J ) 10.6 Hz), 4.96-4.84 (3H, m), 4.68-4.53 (4H, m),
4.35-3.50 (22H, m), 3.83 (3H, s), 2.75 (1H, t, J ) 9.4Hz), 2.57
(1H, dd, J ) 4.6 and 12.5 Hz), 2.20 (3H, s), 2.15 (3H, s), 2.12
(3H, s), 2.09 (3H, s), 2.06 (9H, s), 1.99 (3H, s), 1.84 (3H, s),
1.67 (1H, t, J ) 12.5 Hz), 1.33 (3H, d, J ) 6.6 Hz), 0.99-0.91
(2H, m), and 0.01 (9H, s); 13C NMR (CDCl3) δ 170.9, 170.8,
170.6, 170.5, 170.4, 170.1, 169.4, 169.3, 167.9, 103.1, 100.0,
99.6, 96.8, 79.0, 75.2, 73.3, 72.3, 72.0, 71.3, 71.0, 70.9, 69.7,
69.5, 69.4, 68.0, 67.7, 67.4, 66.6, 66.0, 62.3, 61.7, 61.6, 58.9,
53.2, 49.1, 37.4, 23.1, 21.5, 20.9, 20.8, 20.8, 20.7, 20.7, 20.6,
18.1, 16.3, and -1.4; MS m/ z (FAB+) calcd for C51H80O30N2Si
1228, found 1229 (base peak; M + H+). Anal. Calcd for
C51H80N2O30Si‚3H2O: C, 47.73; H, 6.75; N, 2.18. Found: C,
47.92; H, 6.66; N, 2.41.
Gen er a l P r oced u r e for N-Acyla tion . Syn th esis of
2-(Tr im eth ylsilyl)eth yl [Meth yl (5-acetam ido-3,5-dideoxy-
4,7,8,9-t e t r a -O-a ce t yl-r-D-glycer o-D-ga la ct o-2-n on u lo-
p yr a n osylon a te)]-(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la cto-
p yr a n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-[6-O-
a cetyl-2-(a cyla m in o)-2-d eoxy-â-D-glu cop yr a n osid e] (15).
Powdered sodium hydrogen carbonate (171 mg, 2.04 mmol)
and acyl chloride (1.02 mmol) were added to a solution of
amine 1 (500 mg, 0.406 mmol) in dichloromethane (40 mL) at
room temperature. After the mixture was stirred for 20 h,
methanol (10 mL) and pyridine (10 mL) were added to the
reaction mixture. After 15 min, the mixture was concentrated
under reduced pressure to afford amide 15, which was then
used in the next reaction without further purification.
Gen er a l P r oced u r e for F u cose Acetyla tion . Syn th esis
of 2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-
d id eoxy-4,7,8,9-t et r a -O-a cet yl-r-D-glycer o-D-ga la cto-2-
n on u lop yr a n osylon a te)]-(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-
g a la c t o p y r a n o s y l)-(1f4)-O -[2,3,4-t r i -O -a c e t y l-r-L -

2944 J . Org. Chem., Vol. 61, No. 9, 1996
Hayashi et al.
7.6 Hz), and 0.00 (9H, s). Anal. Calcd for C61H92N2O34Si‚
2H2O: C, 50.13; H, 6.62; N, 1.92. Found: C, 50.27; H, 6.57;
N, 1.99.
silica gel column chromatography eluting with hexane, ethyl
acetate, and acetone (1/1/1) to afford 16c (489 mg, 86% yield)
as pale yellow crystals: Rf ) 0.58 (developed with a 1:1:2
mixture of hexane, ethyl acetate, and acetone); IR (KBr) 2967,
Dodecyl [Meth yl (5-acetam ido-3,5-dideoxy-4,7,8,9-tetr a-
O-acetyl-r-D-glycer o-D-ga la cto-2-n on u lopyr an osylon ate)]-
(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-(1f4)-
O-[2,3,4-t r i-O-a cet yl-r-L-fu cop yr a n osyl-(1f3)-O]-(6-O-
a c e t y l -2 -a m i n o -2 -N -b u t y r y l -2 -d e o x y -â-D -g l u c o -
p yr a n osid e) (18a ). The title compound was obtained as a
pale yellow amorphous solid in 52% yield from 16a : Rf ) 0.47
(developed with a 9:1 mixture of chloroform and methanol);
1H NMR (CDCl3) δ 5.50-3.35 (30H, m), 3.84 (3H, s), 2.56 (1H,
dd, J ) 4.3 and 12.5 Hz), 2.19 (3H, s), 2.14 (3H, s), 2.12 (3H,
s), 2.11 (3H, s), 2.08 (3H, s), 2.07 (3H, s), 2.06 (3H, s), 2.05
(6H, s), 1.98 (3H, s), 1.93 (3H, s), 2.10-1.90 (2H, m),1.83 (3H,
s), 1.75-1.45 (5H, m), 1.35-1.05 (18H, m), 1.17 (3H, d, J )
6.3 Hz), 0.93 (3H, t, J ) 7.2 Hz), and 0.86 (3H, t, J ) 7.2 Hz).
Dodecyl (5-Acetam ide-3,5-dideoxy-r-D-glycer o-D-ga la cto-
2-n on u lop yr a n osylon ic a cid )-(2f3)-O-(â-D-ga la ctop yr a -
n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-(2-a m in o-2-
N-bu tyr yl-2-d eoxy-â-D-glu cop yr a n osid e) (3a ). The title
compound was obtained as a colorless lyophilized solid in 78%
yield from 18a : Rf ) 0.48 (developed with a 12:10:3 mixture
of chloroform, methanol, and 15 mM aqueous calcium chloride);
1H NMR (CD3OD) δ 4.92 (1H, d, J ) 4.0 Hz) 4.39 (1H, d, J )
7.6 Hz), 4.30 (1H, d, J ) 7.6 Hz), 3.95-3.30 (25H, m), 2.75
(1H, dd, J ) 4.3 and 12.5 Hz), 2.06 (2H, m), 1.89 (3H, s), 1.65-
1.35 (5H, m), 1.17 (18H, br.s), 1.03 (3H, d, J ) 6.6 Hz), 0.85
(3H, t, J ) 7.4 Hz), and 0.78 (3H, t, J ) 6.6 Hz); MS m/ z
(FAB+) calcd for C45H79N2O23Na 1038, found 1039 (M + H+).
Anal. Calcd for C45H79N2O23Na‚5H2O: C, 47.86; H, 7.94; N,
2.48. Found: C, 47.75; H, 7.82; N, 2.46.
Eth yl [Meth yl (5-a ceta m id o-3,5-d id eoxy-4,7,8,9-tetr a -
O-acetyl-r-D-glycer o-D-ga la cto-2-n on u lopyr an osylon ate)]-
(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-(1f4)-
O-[2,3,4-t r i-O-a cet yl-r-L-fu cop yr a n osyl-(1f3)-O]-(6-O-
a ce t yl-2-a m in o-2-N-b u t yr yl-2-d e oxy-â-D-glu cop yr a n o-
sid e) (18b). The title compound was obtained as a pale yellow
amorphous solid in 41% yield from 16a : Rf ) 0.43 (developed
with a 9:1 mixture of chloroform and methanol); 1H NMR
(CDCl3) δ 5.50-3.40 (30H, m), 3.85 (3H, s), 2.59 (1H, dd, J )
4.3 and 12.5 Hz), 2.19 (3H, s), 2.17 (3H, s), 2.15 (3H, s), 2.13
(3H, s), 2.11 (3H, s), 2.09 (3H, s), 2.07 (3H, s), 2.06 (3H, s),
2.05 (3H, s), 1.99 (3H, s), 1.94 (3H, s), 2.10-1.90 (2H, m), 1.84
(3H, s), 1.68-1.55 (3H, m), 1.17 (3H, d, J ) 6.3 Hz), 1.15 (3H,
t, J ) 6.9 Hz), and 0.93 (3H, t, J ) 7.4 Hz).
1745, 1670, 1542, 1438, 1373, 1240, 1048 cm-1 1H NMR
;
(CDCl3) δ 5.55-3.45 (30H, m), 3.86 (3H, s), 2.59 (1H, dd, J )
4.3 and 12.6 Hz), 2.21 (3H, s), 2.16 (3H, s), 2.14 (3H, s), 2.12
(3H, s), 2.10 (3H, s), 2.08 (3H, s), 2.07 (3H, s), 2.06 (3H, s),
2.04 (3H, s), 2.00 (3H, s), 1.96 (3H, s), 1.95 (3H, s), 1.85 (3H,
s), 1.68 (1H, t, J ) 12.6 Hz), 1.19 (3H, d, J ) 6.6 Hz), 0.94-
0.84 (2H, m), and -0.01 (9H, s). Anal. Calcd for C59H88N2-
O34Si‚3H2O: C, 48.82; H, 6.53; N, 1.93. Found: C, 48.71; H,
6.33; N, 2.00.
Dodecyl [Meth yl (5-acetam ido-3,5-dideoxy-4,7,8,9-tetr a-
O-acetyl-r-D-glycer o-D-ga la cto-2-n on u lopyr an osylon ate)]-
(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-(1f4)-
O-[2,3,4-t r i-O-a cet yl-r-L-fu cop yr a n osyl-(1f3)-O]-(6-O-
a cetyl-2-a ceta m id e-2-d eoxy-â-D-glu cop yr a n osid e) (18c).
The title compound was obtained as a pale yellow amorphous
solid in 46% yield from 16c: 1H NMR (CDCl3) δ 5.69 (1H, d,
J ) 7.5 Hz), 5.55-3.30 (29H, m), 3.83 (3H, s), 2.56 (1H, dd, J
) 4.3 and 12.5 Hz), 2.19 (3H, s), 2.14 (3H, s), 2.12 (3H, s),
2.11 (3H, s), 2.09 (3H, s), 2.06 (6H, s), 2.04 (6H, s), 1.98 (3H,
s), 1.93 (6H, s), 1.84 (3H, s), 1.67 (1H, t, J ) 12.5 Hz), 1.51
(2H, m), 1.35-1.00 (18H, m), 1.17 (3H, d, J ) 6.3 Hz), and
0.86 (3H, t, J ) 6.4 Hz).
Dod ecyl (5-Acet a 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 )-(2f3)-O-(â-D-ga la cto-
p yr a n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-(2-a ce-
ta m id o-2-d eoxy-â-D-glu cop yr a n osid e) (3c). The title com-
pound was obtained as a colorless lyophilized solid in 43% yield
from 18c: Rf ) 0.57 (developed with a 12:10:3 mixture of
chloroform, methanol, and 15 mM aqueous calcium chloride);
1H NMR (D2O) δ 5.01 (1H, d, J ) 4.0 Hz), 4.50-4.40 (2H, m),
4.00-3.30 (25H, m), 2.68 (1H, dd, J ) 4.3 and 12.5 Hz), 1.95
(6H, s), 1.71 (1H, t, J ) 12.5 Hz), 1.45 (2H, m), 1.25-1.10 (18H,
m), 1.08 (3H, d, J ) 6.3 Hz), and 0.77 (3H, t, J ) 6.6 Hz); MS
m/ z (FAB+) calcd for C43H76N2O23Si 988, found 989 (M + H+),
1011 (M + Na+).
Eth yl [Meth yl (5-a ceta m id o-3,5-d id eoxy-4,7,8,9-tetr a -
O-acetyl-r-D-glycer o-D-ga la cto-2-n on u lopyr an osylon ate)]-
(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-(1f4)-
O-[2,3,4-t r i-O-a cet yl-r-L-fu cop yr a n osyl-(1f3)-O]-(6-O-
a cetyl-2-a ceta m id o-2-d eoxy-â-D-glu cop yr a n osid e) (18d ).
The title compound was obtained as a pale yellow amorphous
solid in 72% yield from 16c: 1H NMR (CD3OD) δ 5.55-3.40
(30H, m), 3.87 (3H, s), 2.58 (1H, d, J ) 4.3 and 12.5 Hz), 2.18
(3H, s), 2.14 (3H, s), 2.11 (3H, s), 2.10 (3H, s), 2.08 (3H, s),
2.06 (3H, s), 2.05 (3H, s), 2.04 (3H, s), 2.03 (3H, s), 1.97 (3H,
s), 1.94 (3H, s), 1.91 (3H, s), 1.81 (3H, s), 1.51 (1H, t, J ) 12.5
Hz), 1.14 (3H, d, J ) 6.9 Hz), and 1.13 (3H, t, J ) 7.2 Hz).
Eth yl (5-Aceta m id e-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 )-(2f3)-O-(â-D-ga la ctop yr a -
n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-(2-a cetoa m -
ido-2-deoxy-â-D-glu copyr an oside) (3d). The title compound
was obtained as a colorless lyophilized solid in 87% yield from
18d : 1H NMR (D2O) δ 5.00 (1H, d, J ) 4.2 Hz), 4.48-4.40
(2H, m), 4.00-3.40 (25H, m), 2.72 (1H, dd, J ) 4.3 and 12.5
Hz), 1.94 (6H, s), 1.71 (1H, t, J ) 12.5 Hz), 1.07 (3H, d, J )
6.6 Hz), and 1.06 (3H, t, J ) 6.9 Hz); MS m/ z (FAB+) calcd
for C33H56N2O23Si 848, found 871 (M + Na+).
Eth yl (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 )-(2f3)-O-(â-D-ga la ctop yr a -
n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-(2-a m in o-2-
N-b u t yr yl-2-d eoxy-â-D-glu cop yr a n osid e) (3b ). The title
compound was obtained as a colorless lyophilized solid in 75%
yield from 18b: Rf ) 0.48 (developed with a 12:10:3 mixture
of chloroform, methanol, and 15 mM aqueous calcium chloride);
1H NMR (D2O) δ 5.00 (1H, d, J ) 4.0 Hz), 4.48-4.40 (2H, m),
4.00-3.39 (25H, m), 2.66 (1H, dd, J ) 4.3 and 12.2 Hz), 2.15
(2H, t, J ) 5.6 Hz), 1.92 (3H, s), 1.69 (1H, t, J ) 12.2 Hz), 1.51
(2H, m), 1.06 (3H, d, J ) 6.3 Hz), 1.05 (3H, t, J ) 6.9 Hz), and
0.83 (3H, t, J ) 7.4 Hz); MS m/ z (FAB+) calcd for C35H59N2O23-
Na 898, found 899 (base peak; M + H+). Anal. Calcd for
C35H59N2O23Na‚5H2O: C, 42.51; H, 7.03; N, 2.83. Found: C,
42.26; H, 6.67; N, 2.79.
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
la ctop yr a n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-[6-
O-a cet yl-2-a m in o-2-d eoxy-2-N-(2-n a p h t h oyl)-â-D-glu co-
p yr a n osid e] (15e). The title compound was obtained as a
crude residue from 1, which was then used in the next reaction
without further purification: Rf ) 0.70 (developed with a 10:
2:1 mixture of ethyl acetate, ethanol, and water).
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
la ctop yr a n osyl)-(1f4)-O-[2,3,4-tr i-O-a cetyl-r-L-fu cop yr -
a n osyl-(1f3)-O]-[6-O-a ce t yl-2-a m in o-2-d e oxy-2-N -(2-
n a p h th oyl)-â-D-glu cop yr a n osid e] (16e). The title com-
2-(Tr im et h ylsilyl)et h yl [Met h yl (5-a cet a m id o-3,5-d i-
d eoxy-4,7,8,9-tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on -
u lop yr a n osylon a t e)]-(2f3)-O-(2,4,6-t r i-O-a cet yl-â-D-ga -
la ctop yr a n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-(6-
O-acetyl-2-acetam ido-2-deoxy-â-D-glu copyr an oside) (16c).
Acetic anhydride (3.5 mL) and 4-(dimethylamino)pyridine (20
mg) were added to a solution of 1 (500 mg, 0.406 mmol) in
pyridine (6.0 mL) at room temperature. After stirring for 18
h, methanol (6.0 mL) was added dropwise to the mixture at 0
°C. The mixture was stirred at room temperature for 1 h and
then concentrated in vacuo. The resulting residue was diluted
with ethyl acetate and washed with saturated sodium hydro-
gen carbonate and brine in that order. The organic layer was
dried over anhydrous sodium sulfate, filtered, and concen-
trated under reduced pressure. The residue was purified by

Synthesis of SLeX Analogs
J . Org. Chem., Vol. 61, No. 9, 1996 2945
pound was obtained as colorless crystals in 86% yield from 1:
Rf ) 0.38 (developed with a 1:1:1 mixture of hexane, ethyl
acetate, and acetone); mp 156-158 °C; 1H NMR (CDCl3) δ 8.28
(1H, s), 7.92-7.82 (4H, m), 7.58-7.53 (2H, m), 6.37 (1H, d, J
) 9.6 Hz), 5.60-4.65 (143H, m), 4.56 (1H, dd, J ) 4.0 and 9.9
Hz), 4.45-3.80 (11H, m), 3.86 (3H, s), 3.70-3.45 (3H, m), 2.59
(1H, dd, J ) 4.6 and 12.5 Hz), 2.23 (3H, s), 2.17 (3H, s), 2.11
(3H, s), 2.08 (9H, s), 2.07 (9H, s), 2.00 (3H, s), 1.93 (3H, s),
1.85 (3H, s), 1.70 (1H, t, J ) 12.5 Hz), 1.18 (3H, d, J ) 6.6
Hz), 0.90-0.82 (2H, m), and -0.08 (9H, s). Anal. Calcd for
C68H92N2O34Si‚2H2O: C, 52.84; H, 6.26; N, 1.81. Found: C,
52.86; H, 6.28; N, 1.84.
Dodecyl [Meth yl (5-acetam ido-3,5-dideoxy-4,7,8,9-tetr a-
O-acetyl-r-D-glycer o-D-ga la cto-2-n on u lopyr an osylon ate)]-
(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-(1f4)-
O-[2,3,4-t r i-O-a cet yl-r-L-fu cop yr a n osyl-(1f3)-O]-[6-O-
a cetyl-2-a m in o-2-d eoxy-2-N-(2-n a p h th oyl)-â-D-glu cop yr a -
n osid e] (18e). The title compound was obtained as a pale
yellow amorphous solid in 74% yield from 16e: Rf ) 0.54
(developed with a 19:1 mixture of chloroform and methanol);
1H NMR (CDCl3) δ 8.27 (1H, s), 7.92-7.81 (4H, m), 7.58-7.53
(2H, m), 6.28 (1H, d, J ) 8.9 Hz), 5.56-3.75 (26H, m), 3.86
(3H, s), 3.63 (2H, m), 3.40 (1H, m), 2.59 (1H, dd, J ) 4.5 and
12.5 Hz), 2.22 (3H, s), 2.17 (3H, s), 2.11 (3H, s), 2.08 (6H, s),
2.07 (9H, s), 2.04 (3H, s), 2.00 (3H, s), 1.93 (3H, s), 1.85 (3H,
s), 1.75-1.55 (3H, m), 1.35-1.00 (18H, m), 1.19 (3H, d, J )
6.3 Hz), 0.87 (3H, t, J ) 7.2 Hz).
Dod ecyl (5-Acet a 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 )-(2f3)-O-(â-D-ga la cto-
pyr an osyl)-(1f4)-O-[r-L-fu copyr an osyl-(1f3)-O]-[2-am in o-
2-d eoxy-2-N-(2-n a p h th oyl)-â-D-glu cop yr a n osid e] (3e). The
title compound was obtained as a colorless lyophilized solid
in 89% yield from 18e: Rf ) 0.56 (developed with a 12:10:3
mixture of chloroform, methanol, and 15 mM aqueous calcium
chloride); 1H NMR (CD3OD) δ 8.28 (1H, s), 7.87-7.80 (4H, m),
7.50-7.45 (2H, m), 5.04 (1H, d, J ) 4.0 Hz), 4.72 (1H, m), 4.46
(1H, d, J ) 7.6 Hz), 4.10-2.90 (25H, m), 2.78 (1H, dd, J ) 4.5
and 12.5 Hz), 1.91 (3H, s), 1.63 (1H, t, J ) 12.5 Hz), 1.38 (2H,
m), 1.20-0.90 (18H, m), 1.06 (3H, d, J ) 6.6 Hz), 0.79 (3H, t,
J ) 7.2 Hz); MS m/ z (FAB+) calcd for C52H79N2O23Na 1122,
found 1123 (M + H+). Anal. Calcd for C52H79N2O23Na‚
5.5H2O: C, 51.10; H, 7.42; N, 2.29. Found: C, 51.07; H, 7.54;
N, 2.50.
Eth yl [Meth yl (5-a ceta m id o-3,5-d id eoxy-4,7,8,9-tetr a -
O-acetyl-r-D-glycer o-D-ga la cto-2-n on u lopyr an osylon ate)]-
(2f3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-(1f4)-
O-[2,3,4-t r i-O-a cet yl-r-L-fu cop yr a n osyl-(1f3)-O]-[6-O-
a cetyl-2-a m in o-2-d eoxy-2-N-(2-n a p h th oyl)-â-D-glu cop yr a -
n osid e] (18f). The title compound was obtained as a pale
yellow amorphous solid in 77% yield from 16e: Rf ) 0.30
(developed with a 1:1:1 mixture of hexane, ethyl acetate, and
acetone); H NMR (CDCl3) δ 8.29 (1H, s), 7.99-7.81 (4H, m),
1
7.59-7.52 (2H, m), 6.37 (1H, d, J ) 8.9 Hz), 5.60-3.50 (26H,
m), 3.86 (3H, s), 3.67-3.45 (3H, m), 2.59 (1H, dd, J ) 4.3 and
12.5 Hz), 2.23 (3H, s), 2.17 (3H, s), 2.12 (3H, s), 2.11 (3H, s),
2.09 (6H, s), 2.08 (6H, s), 2.05 (3H, s), 2.01 (3H, s), 1.94 (3H,
s), 1.86 (3H, s), 1.71 (1H, t, J ) 12.5 Hz), 1.19 (3H, d, J ) 6.6
Hz), 1.12 (3H, t, J ) 7.2 Hz).
Eth yl (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 )-(2f3)-O-(â-D-ga la ctop yr a -
n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-(1f3)-O]-[2-a m in o-2-
d eoxy-2-N-(2-n a p h th oyl)-â-D-glu cop yr a n osid e] (3f). The
title compound was obtained as a colorless lyophilized solid
in 98% yield from 18f: Rf ) 0.51 (developed with a 12:10:3
mixture of chloroform, methanol, and 15 mM aqueous calcium
chloride); 1H NMR (D2O) δ 8.23 (1H, s), 7.97-7.87 (3H, m),
7.72 (1H, dd, J ) 2.0 and 8.9 Hz), 7.60-7.52 (2H, m), 5.09
(1H, d, J ) 4.0 Hz), 4.64 (1H, m), 4.46 (1H, d, J ) 7.9 Hz),
4.10-3.35 (25H, m), 2.66 (1H, dd, J ) 4.6 and 12.5 Hz), 1.92
(3H, s), 1.70 (1H, t, J ) 12.5 Hz), 1.06 (3H, d, J ) 6.3 Hz),
0.99 (3H, t, J ) 7.2 Hz); MS m/ z (FAB+) calcd for C42H59N2O23-
Na 982, found 983 (base peak; M + H+). Anal. Calcd for
C42H59N2O23Na‚8H2O: C, 44.76; H, 6.71; N, 2.49. Found: C,
44.45; H, 6.51; N, 2.54.
3-Azidopr opyl [Meth yl (5-acetam ido-3,5-dideoxy-4,7,8,9-
tetr a -O-a cetyl-r-D-glycer o-D-ga la cto-2-n on u lop yr a n osy-
lon ate)]-(2f3)-O-(2,4,6-tr i-O-acetyl-â-D-galactopyr an osyl)-
(1f4)-O-[2,3,4-t r i-O-a cet yl-r-L-fu cop yr a n osyl-(1f3)-O]-
[6-O -a c e t y l-2-a m in o -2-N -b u t y r y l-2-d e o x y -â-D -g lu c o -
p yr a n osid e] (19). The title compound was obtained as a pale
yellow amorphous solid in 39% yield from 16a : Rf ) 0.47
(developed with a 9:1 mixture of chloroform and methanol);
IR (KBr) 2958, 2102, 1746, 1656, 1546, 1373, 1232, 1047 cm-1
;
1H NMR (CDCl3) δ 5.50-3.50 (28H, m), 3.85 (3H, s), 3.62 (2H,
t, J ) 5.4 Hz), 3.37 (2H, m), 2.58 (1H, dd, J ) 4.3 and 12.5
Hz), 2.19 (3H, s), 2.15 (3H, s), 2.13 (3H, s), 2.12 (3H, s), 2.09
(3H, s), 2.08 (3H, s), 2.06 (9H, s), 2.00 (3H, s), 1.94 (3H, s),
1.84 (3H, s), 1.75-1.60 (5H, m), 1.17 (3H, d, J ) 6.6 Hz), and
0.94 (3H, t, J ) 7.3 Hz).
Ack n ow led gm en t. We are grateful to Drs. J . C.
Paulson and R. Bayer in Cytel Corporation (La J olla,
CA) for providing us with enzymes (2,3-ST and CMP-
NeuAc synthetase). We appreciate Drs. R. M. Ratcliffe
and S. A. DeFrees in Cytel Corporation for their helpful
discussions. We also thank Miss N. Kawamura for
evaluation of the biological activity.
J O960125F