Synthesis and biological evaluation of a new sialyl Lewis X mimetic derived from lactose

Article abstract of DOI:10.1021/jo025579t

A sialyl Lewis X (sLe^x) mimetic compound, 2-(trimethylsilyl)ethyl 3-O-carboxymethyl-β-D-galactopyranosyl-(1→4)-[α-L-fucosyl- (1→6)]-β-D-glucopyranoside (2a), has been synthesized in 14 steps from D-lactose. This synthesis features the use of the activated glycosylating donor, lactosyl iodide, in a Koenigs-Knorr sequence, the regioselective derivatization at the C-3 position of the galactose moiety, and the stereoselective construction of a fucose-α(1→6)-lactose linkage. The mimetic was tested for its ability to inhibit human polymorphonuclear leukocyte (hPMNL) adhesion to immobilized recombinant human E-selectin under shear stress conditions.

Full text of DOI:10.1021/jo025579t

Syn th esis a n d Biologica l Eva lu a tion of a New Sia lyl Lew is X
Mim etic Der ived fr om La ctose
Stephanie M. Chervin,† J ohn B. Lowe,§ and Masato Koreeda*,†,‡
Departments of Chemistry and Medicinal Chemistry, University of Michigan,
Ann Arbor, Michigan 48109-1055, and Howard Hughes Medical Institute and Department of Pathology,
University of Michigan Medical School, Ann Arbor, Michigan 48109
koreeda@umich.edu
Received February 2, 2002
A sialyl Lewis X (sLex) mimetic compound, 2-(trimethylsilyl)ethyl 3-O-carboxymethyl-â-D-galacto-
pyranosyl-(1f4)-[R-L-fucosyl-(1f6)]-â-D-glucopyranoside (2a ), has been synthesized in 14 steps from
D-lactose. This synthesis features the use of the activated glycosylating donor, lactosyl iodide, in a
Koenigs-Knorr sequence, the regioselective derivatization at the C-3 position of the galactose
moiety, and the stereoselective construction of a fucose-R(1f6)-lactose linkage. The mimetic was
tested for its ability to inhibit human polymorphonuclear leukocyte (hPMNL) adhesion to
immobilized recombinant human E-selectin under shear stress conditions.
In tr od u ction
through the tissue matrix to the damaged cells, and
releases its arsenal of oxygen radicals, proteases, and
antimicrobial peptides.
The regulated recruitment of leukocytes, the disease-
fighting cells of the immune system, to damaged or
infected tissue has been the subject of extensive inves-
tigation.1 The passage of the leukocytes from the blood
stream to the tissue matrix depends on an initial cell
recognition event between the leukocyte and the vascular
wall. The damaged tissue initiates this important event
by releasing signaling cytokines that stimulate the
endothelial cells of the nearest postcapillary venules to
express the leukocyte adhesion molecules, E- and P-
selectin. The C-type lectin domains of E- and P-selectin
recognizes and weakly binds the blood antigen sialyl
Lewis X (sLex), 1, the tetrasaccharide found on the
terminus of certain leukocyte surface glycoproteins ex-
pressed on the tips of microvilli. The leukocyte surface
is decorated with multiple copies of sLex and the multi-
valent nature of this weak selectin-carbohydrate inter-
action causes the leukocyte to reduce its flow velocity and
roll along the endothelial wall. This initial rolling inter-
action promotes the subsequent firm adhesion mediated
by a protein-integrin interaction of the leukocyte to the
endothelial cell surface. The adhesion of the leukocyte
to the vascular wall achieved, the leukocyte migrates
through the endothelial cell surface of the vessel, travels
While the migration of leukocytes is highly regulated
by the healthy body, the unregulated migration of leu-
kocytes from the vascular structures to healthy tissues
does occur and can have destructive consequences. In
such circumstances, the aggregation of disease-fighting
leukocytes destroys healthy tissues and promotes a
variety of inflammatory conditions such as rheumatoid
arthritis, asthma, lupus, inflammatory bowel disease,
and reperfusion injury. The pharmaceutical industry has
shown great interest in the development of low molecular
weight agents that could inhibit the sLex-mediated cel-
lular adhesion event between the vascular cell wall and
the leukocyte. Such agents may provide effective therapy
for the above conditions that are caused at least in part
by the unregulated migration of leukocytes. The study
of the E-selectin sLex interaction has uncovered the
minimal structural requirements for recognition by E-
selectin and has led to a model of the binding coordina-
tion of sLex to E-selectin.2 On the basis of these data, the
synthetic community has designed a large number of sLex
mimetics and some have demonstrated impressive in-
hibitory effects.3
We proposed the synthesis of sLex mimetic 2. It was
envisioned that our mimetic 2 could be effective alone
as a selectin inhibitor or, if linked to a common antiin-
flammatory drug compound, could serve as a targeting
device for the delivery of the drug (e.g., 2b). It was
† Department of Chemistry, University of Michigan.
§ Howard Hughes Medical Institute and Department of Pathology,
University of Michigan Medical School.
‡ Department of Medicinal Chemistry, University of Michigan.
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10.1021/jo025579t CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/03/2002
5654
J . Org. Chem. 2002, 67, 5654-5662

Evaluation of a New Sialyl Lewis X Mimetic
atoms of Fuc, respectively (results obtained by Macro-
model Calculations using the parameters given in ref 7a,
vide infra).
The fact that the core structure of the Galâ1f4GlcNAc
moiety in sLex is the inexpensive disaccharide D-lactose
prompted us to consider its possible use as a building
block for rapid assembly of sLex mimetics. In keeping
with substantial literature evidence, it was decided to
append the entire L-fucose unit to this lactose core. For
the eventual in vivo studies, the attachment of the fucose
ring needs to be made through a C-glycoside bond to
achieve better in vivo stability against fucosidase diges-
tion. Moreover, the entire NeuAc group was replaced with
the flexible â-methylene carboxylic acid group at C-3 of
the galactose residue.
It was further postulated that a drug molecule with
an accessible hydroxyl group (e.g., salicylic acid or
(3) (a) Roche, D.; Ba¨nteli, R.; Winkler, T.; Casset, F.; Ernst, B.
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envisioned that the carbohydrate portion of a sLex
mimetic-drug conjugate could interact with the selectins
expressed by endothelial cells stimulated by nearby
damaged tissue, thereby delivering the antiinflammatory
drug directly to the site of injury. This mimetic design
replaces the glucosamine residue of sLex with a glucose
residue scaffold and replaces the sialic acid residue with
an ethanoic acid substituent at the C-3 position of the
galactose ring.
Our mimetic design is predicated upon literature data
which implicated that the glucosamine residue of 1 only
served as a rigid spacer between the fucose and galactose
residues and exhibited no coordination to the binding
cavity of E-selectin.2,3w,3x,4 Accordingly, the â-D-lac-
tosamine core in 1 was supplanted with the chemically
more robust â-D-lactose core. The L-fucose residue was
retained, as reports indicated that the hydroxyl groups
at positions C-2 and C-3 of the fucose residue are involved
in coordination to the Ca2+ ion in the binding pocket,5,6
and linked to the lactose scaffold by an R (1f6) glycoside
bond. Literature reports also revealed that only the
carboxylic acid group of the N-acetylneuraminic acid
residue of 1 was involved in coordination to the binding
site.2,3w,3x,4 In 1995, the structure of the E-selectin-bound
sLex was postulated on the basis of transferred nuclear
Overhauser experiments.7 These NMR results suggest
that the tortional angles of the glycosidic bond between
Gal and NeuAc of sLex change dramatically upon binding
with E-selectin to assume the conformer where the COO-
group of NeuAc nearly juxtaposes the 2-OH of Gal. This
provides the direct distances of roughly 8.9 and 9.1 Å
between the NeuAc carboxylate C atom and C-1 and C-2
(4) Rosen, S. V.; Bertozzi, C. R. Curr. Opin. Cell Biol. 1994, 6, 663-
673.
(5) (a) Yuen, C.-T.; Lawson, A. M.; Chai, W.; Larkin, M.; Stoll, M.
S.; Stuart, A. C.; Sullivan, F. X.; Ahern, T. J .; Feizi, T. Biochemistry
1992, 31, 9126-9131. (b) Brandley, B. K.; Kiso, M.; Abbas, S.; Nikrad,
P.; Srivasatava, P.; Foxall, C.; Oda, Y.; Hasegawa, A. Glycobiology
1993, 3, 633-639. (c) DeFrees, S. A.; Gaeta, F. C. A.; Lin, Y.-C.;
Ichikawa, Y.; Wong, C.-H. J . Am. Chem. Soc. 1993, 115, 7549-7550.
(d) Ramphal, J . Y.; Zheng, Z.-L.; Perez, C.; Walker, L. E.; DeFrees, S.
A.; Gaeta, F. C. A. J . Med. Chem. 1994, 37, 3459-3463.
(6) For the proposed coordination of Ca2+ with the 2-OH of Fuc and
the 6-OH of Gal in the E-selectin-bound sLex, see: Siuzdak, G.; Zheng,
Z.-L.; Ramphal, J . Y.; Ichikawa, Y.; Nicolaou, K. C.; Gaeta, F. C. A.;
Chatman, K. S.; Wong, C.-H. Bioorg. Med. Chem. Lett. 1994, 4, 2863-
2866.
(7) (a) Scheffler, K.; Ernst, B.; Katopodis, A.; Magnani, J . L.; Wang,
W. T.; Weisemann, R.; Peters, T. Angew. Chem., Int. Ed. Engl. 1995,
34, 1841-1844. (b) Scheffler, K.; Brisson, J .-R.; Weisemann, R.;
Magnani, J . L.; Wong, W. T.; Ernst, B.; Peters, T. J . Biomol. NMR
1997, 9, 423-436.
J . Org. Chem, Vol. 67, No. 16, 2002 5655

Chervin et al.
F IGURE 1. Calculated local minimum energy conformation of carboxylate 2c (R ) CH2CH3).
diflusinal) could be attached to our mimetic by either
acid-promoted glycosylation of 2a 8 or the Ferrier glyco-
sylation of the glycal analogue 3a .9
with I2/CH3COSH11 in good yield (77%). The TMSET
group was introduced by the treatment of the lactosyl
iodide 5 with 2-(trimethylsilyl)ethanol in the presence of
HgCl2 and HgO to give exclusively the â-glycoside, 6a
(58%).10b The removal of the acetate protecting groups
with the use of the Zemple´n conditions12 proceeded
cleanly to give 6b (≈100%). The application of the well-
established, mild, and highly regioselective dialkylstan-
nylene acetal methodology13 was next planned for the
installation of the methylene carboxylic acid group at the
3′-hydroxyl position. Although the selective alkylation
and acylation of the axial 3-OH over the equatorial 4-OH
of the galactose and similar systems14 are well docu-
mented, treatment of the 3′,4′-stanylene intermediate
derivative of the lactose system with less bulky esters of
bromoacetic acid such as methyl and ethyl esters is
reported to result in the formation of the lactone deriva-
tives involving 2′- and 4′-OH groups in addition to the
desired 3-OH derivative.15 Therefore, in an effort to
circumvent this lactone formation problem, the 3,4-
stanylene derivative of 6b was treated with tert-butyl
bromoacetate16 in the presence of catalytic TBAI, result-
The global and local lowest energy conformers of this
new O-fucoside mimetic and a few analogues were sought
by a Macromodel Conformational Search using the Monte
Carlo method (AMBER* Force Field) in a vacuum. The
energy allowed for bond rotation was set as 50 kJ /mol.
Interestingly, the conformational structure virtually
identical with that drawn for 2c was found to be the most
stable conformer of carboxylate 2c. Figure 1 shows the
calculated local minimum energy conformation of car-
boxylate 2c (R ) OCH2CH3) where the carboxylate end
is pointed down as in the case E-selectin-bound sLex. The
results of the calculations revealed that the conformer
matching structure 2c (i.e., with the carboxylate end
pointing up) was estimated to be more stable by only 4.10
kJ /mol. Direct distances between the carboxylate carbon
and C-1 and C-2 in Fuc in the conformer shown in Figure
1B were calculated to be 9.0 and 9.4 Å, respectively,
which are quite close to those values estimated for the
biologically active conformation of sLex.
Resu lts a n d Discu ssion
(10) (a) Lipshutz, B. H.; Pegram, J . J .; Morey, M. C. Tetrahedron
Lett. 1981, 22, 4603-4606. (b) J ansson, K.; Ahlfors, S.; Frejd, T.;
Kihlberg, J .; Magnusson, G. J . Org. Chem. 1988, 53, 5629-5647. (c)
Ekberg, T.; Magnusson, G. Carbohydr. Res. 1993, 246, 119-136. For
reviews on the Koenigs-Knorr glycosidation reaction see: (d) Igarashi,
K. Adv. Carbohydr. Biochem. 1977, 34, 243-283. (e) Schmidt, R. R.
In Comprehensive Organic Synthesis; Trost, B. M., Fleming, I., Eds.;
Pergamon Press: Oxford, UK, 1991; Vol. 6, pp 37-49.
(11) Chervin, S. M.; Abada, P.; Koreeda, M. Org. Lett. 2000, 2, 369-
372.
(12) (a) Zemple´n, G.; Pascu, E. Ber. Dtsch. Chem. Ges. 1929, 62,
1613-1614. See also: (b) Collins, P.; Ferrier, R. Monosaccharides:
Their Chemistry and Their Roles in Natural Products; Wiley: Chich-
ester, UK, 1995; p 361.
(13) Reviews: (a) David, S.; Hanessian, S. Tetrahedron 1985, 41,
643-663. (b) Stanek, J . Top. Curr. Chem. 1990, 154, 209-253. (c)
Davies, A. G. In Organotin Chemistry; VCH: Weinheim, Germany,
1997; pp 173-180. (d) Grindley, T. B. Adv. Carbohydr. Chem. Biochem.
1998, 53, 17-142.
(14) (a) Nashed, M. A.; Anderson, L. Carbohydr. Res. 1977, 56, 419-
422. (b) David, S.; Thieffry, A. J . Chem. Soc., Perkin Trans. 1 1979,
1568-1573. (c) Tsuda, Y.; Haque, Md. E.; Yoshimoto, K. Chem. Pharm.
Bull. 1983, 31, 1612-1624.
Syn th esis of 2a . Our planned synthesis of the sLex
mimetic 2a required access to the D-lactose-derived
requisite intermediate 10 in which only the 6-OH group
is unprotected for the conjugation with the L-fucose unit.
Access to this lactose derivative involved the selective
protection of hydroxyl groups as well as the incorporation
of the ethanoic acid unit onto the 3′-OH group (see
Scheme 1). In an effort first to block the anomeric
hydroxyl group of D-lactose, the inexpensive starting
material D-lactose (4a ) (R and â mixture) was converted
to the per-O-acetyl derivative 4b (≈100%). The installa-
tion of the 2-(trimethylsilyl)ethyl (TMSET) anomeric
blocking group by the two-step Koenig-Knorr method10
required the transformation of the per-O-acetyl lactose
to the 1R-lactosyl halide (e.g., 5) which was accomplished
(15) (a) Huang, H.; Wong, C.-H. J . Org. Chem. 1995, 60, 3100-3106.
(b) Cheng, X.; Khan, N.; Mootoo, D. R. J . Org. Chem. 2000, 65, 2544-
2547. For the clean formation of the lactone formation involving the
4-OH in the galactose system with methyl bromoacetate, see: Hiruma,
K.; Kajimoto, T.; Weitz-Schmidt, G.; Ollman, I.; Wong, C.-H. J . Am.
Chem. Soc. 1996, 118, 9265-9270.
(8) See, e.g.: J ansson, K.; Noori, G.; Magnusson, G. J . Org. Chem.
1990, 55, 3181-3185.
(9) (a) Koreeda, M.; Houston, T. A.; Shull, B. K.; Klemke, E.;
Tuinman, R. J . Synlett 1995, 90-92. (b) Ferrier, R. J .; Prasad, N.;
Sankey, G. H. J . Chem. Soc. C 1969, 587-591.
5656 J . Org. Chem., Vol. 67, No. 16, 2002

Evaluation of a New Sialyl Lewis X Mimetic
SCHEME 1. Ela bor a tion of La ctose Cor e to Key In ter m ed ia te 10a
a
Reagents and conditions: (a) Ac2O, Et3N, DMAP, EtOAc. 100%. (b) I2, CH3COSH, CH2Cl2 77%. (c) 2-(Trimethylsilyl)ethanol, HgO,
HgBr2, CaSO4, CHCl3, 58%. (d) NaOCH3, CH3OH, 100%. (e) Bu2SnO, PhH. (f) BrCH2CO2t-Bu, PhH, 66%. (g) PhCH(OCH3)2, p-TsOH,
CH2Cl2, 90%. (h) t-Bu(Ph)2SiCl, DMAP, CH2Cl2, 79%. (i) Ac2O, DMAP, CH2Cl2, 92%. (j) TBAF, AcOH, THF, 95%.
ing in the highly regioselective alkylation of the 3′-
hydroxy group (7) in 66% yield (or 88% based on the
recovered 6b) together with the recovery of the starting
disaccharide 6b (25%). The 4′- and 6′-hydroxy groups next
were protected smoothly as the diastereochemically pure
benzylidine acetal 8a (83%). The primary 6-hydroxy
group was protected as the tert-butyldiphenylsilyl (TB-
DPS) ether 8b (80%). For ease and uniformity of final
deprotection steps, protection of the remaining hydroxyl
groups as benzyl ethers was deemed most desirable.
However, conversion of 8b to the tri-2,3,2′-O-benzyl ether
could not be realized under the standard basic (NaH/
BnBr/TBAI/DMF) or acidic protocols [Cl3CC(dNH)OCH2-
Ph/TfOH],17 presumably due to unfavorable steric hin-
drance imposed by the juxtaposing (tert-butoxycarbonyl)-
methylene ether at C′-3 and glucose residues at C′-1. In
contrast, acetylation of 8b with Ac2O/DMAP in CH2Cl2
under reflux for 20 min afforded cleanly the 2,3,2′-
triacetate 9 in 92% yield. Finally, the selective removal
of the 6-TBDPS ether group was achieved with refluxing
TBAF/AcOH/THF for 2 h to provide cleanly the desired
alcohol 10 in 95% yield.
The N-iodosuccinimide-promoted coupling of thiophen-
yl per-O-benzyl-â-L-fucoside (11)18 with 10 proceeded
smoothly with high R-selectivity to give trisaccharide 12
(80%, R:â ) 10:1) (Scheme 2). Interestingly, the use of
the R-anomer of 11, thiophenyl per-O-benzyl-R-L-fucoside,
resulted in predominant formation of a â-fucoside linkage
(R:â ) 1:3) in a similar yield. The removal of the three
benzyl and benzylidine protecting groups in trisaccharide
12 required a long reaction period (2 d, H2, Pd/C, ethanol).
Hydrogenolysis for shorter reaction periods (<10 h)
resulted in the selective cleavage of the three benzyl ether
groups leading, upon subsequent acetylation, to ben-
zylidene 13a . To facilitate in isolation and identification
of the deprotected trisaccharide, the crude trisaccharide
(16) (a) Prodger, J . C.; Bamford, M. J .; Gore, P. M.; Holmes, D. S.;
Saez, V.; Ward, P. Tetrahedron Lett. 1995, 36, 2339-2342. (b) Prodger,
J . C.; Bamford, M. J .; Bird, M. I.; Gore, P. M.; Holmes, D. S.; Priest,
R.; Saez, V. Bioorg. Med. Chem. 1996, 4, 793-801.
(17) Wessel, H.-P.; Iverson, T.; Bundle, D. R. J . Chem. Soc., Perkin
Trans. 1 1985, 2247-2249.
(18) (a) Komba, S.; Ishida, H.; Kiso, M.; Hasegawa, A. Bioorg. Med.
Chem. 1996, 4, 1833-1847. (b) Paquette, L. A.; Barriault, L.; Pissar-
nitski, D.; J ohnson, J . N. J . Am. Chem. Soc. 2000, 122, 619-631.
J . Org. Chem, Vol. 67, No. 16, 2002 5657

Chervin et al.
SCHEME 2. Syn th esis of O-Glycosid e Mim etic
hydrolyze after long reaction periods (14 h, 20 °C).
Interestingly, the use of nonanhydrous ethyl ether made
the transformation extremely sluggish and no hydrolysis
product formation was visible by TLC after 3 days of
stirring. Upon completion of exhaustive hydrolysis, the
crude potassium salt was taken up into water and
carefully protonated to pH 5 with cationic acid exchange
resin. Purification of the compound by Sephadex G-10
chromatography gave pure trisaccharide 2a in quantita-
tive yield. The structure of the trisaccharide was con-
firmed by 1H and 13C NMR spectroscopy, and NMR peak
Com p ou n d 2a a
1
assignments were validated by H-1H COSY and 1H-
13C HSQC experiments.
Biologica l Eva lu a tion . Compound 2a was tested for
its ability to inhibit human polymorphonuclear leukocyte
(hPMNL) adhesion to immobilized recombinant human
E-selectin20 under shear stress conditions.21 The assay
involved pumping a suspension of hPMNL in buffer
containing a concentration of the small molecule inhibitor
over a parallel plate flow chamber coated with hE-
selectin-hIgG chimera. The flowing and rolling of the
hPMNL cells were recorded by video over 2 min, and the
total number of rolling cells at three time intervals were
quantified with the aid of an analysis program. The
inhibitory activity of compound 2a was tested against the
negative control compound 3′-N-acetylneuraminyl-N-
acetyllatosamine (3′-SLN)22 and the native ligand sLex
(1) (both purchased from Calbiochem, San Diego). The
native ligand, sLex, showed a 68% reduction of activity
at 1 mM vs the negative control compound 14, respec-
tively. However, compound 2a showed no inhibition at a
concentration of 1 mM, and likewise no significant
inhibition at concentrations of up to 4 mM.
a
Reagents and conditions: (a) BF3‚Et2, PhSH, THF, 95%.18 (b)
NaOCH3, CH3OH, 100%.18 (c) NaH, BnBr, THF, DMF, 90%. (d)
NIS, p-TsOH, CH2Cl2, 80%, R:â ) 10:1. (e) H2, Pd/C. CH3OH. (f)
Ac2O, Et3N, DMAP, CH2Cl2, 71%. (g) KOt-Bu, Et2O, H2O, 100%.
was converted immediately to the per-O-acetyl derivative
13b (71%, purified yield for 2 steps). The complete
structure of this trisaccharide product 13b was ascer-
Con clu sion s
A new sLex mimetic compound, 2-(trimethylsilyl)ethyl
3-O-carboxymethyl-â-D-galactopyranosyl-(1f4)-[R-L-fuco-
syl-(1f6)]-â-D-glucopyranoside (2a ), was designed and
has been synthesized from D-lactose in 14 steps (longest
linear sequence). Our route features the use of an
activated glycosylating agent, per-O-acetylated lactosyl
iodide, prepared with I2/CH3C(dO)SH in the initial
Koenigs-Knorr sequence, the regioselective derivatiza-
tion at C-3′, and the stereoselective construction of the
fucose-R(1f6)-lactose linkage. However, biological testing
revealed that this sLex mimetic was inactive up to 4 mM
in an E-selectin binding assay. In light of the unremark-
able E-selectin inhibitory property of compound 2a , it
appears unlikely that the trisaccharide moiety would
perform well as the targeting device for the delivery of
an antiinflammatory drug. The results of a Macromodel
Conformational Search (Monte Carlo/AMBER* Force
Fields/vacuum) of sLex indicated the distance between
the sialic acid carboxylate carbon and the fucose anomeric
carbon of sLex in its bound form to be 9.1 Å. This value
is similar to literature estimates of the distance between
the two carbon centers.7 A similar Macromodel Confor-
1
tained by H and 13C NMR and all NMR signal assign-
1
ments were verified by H-1H gCOSY and HSQC (1H-
13C correlation) experiments on a 500 MHz NMR
instrument.
The hydrolysis of the eight acetate esters and the tert-
butyl ester in trisaccharide 16b remained. In view of the
inherent acid sensitivity of the trisaccharide, the use of
the typical Lewis acid-mediated methods for deprotection
of the tert-butyl ester (e.g., CF3CO2H; AcOH; TMSOTf/
TEA; TiCl4) was suspected to result in extensive decom-
position of the trisaccharide. Indeed, exposure of the
trisaccharide to these acidic conditions resulted in mas-
sive decomposition of the sugar. In search for mild,
nonacidic conditions for the hydrolysis of a tert-butyl
ester, we adopted the method of Gassman19 that used the
highly nucleophilic anhydrous hydroxide ion generated
from potassium tert-butoxide and stoichiometric water
in anhydrous diethyl ether at room temperature for the
deprotection of simple hindered esters. Accordingly,
octaacetate tert-butyl ester 13b was subjected to 36 mol
equiv each of KOt-Bu and H2O in Et2O at room temper-
ature, which resulted in the formation of the fully
deprotected trisaccharide acid 2a in quantitative yield.
TLC analysis of the crude reaction mixture indicated that
the tert-butyl group was sluggish to hydrolyze but did
(20) Watsin, S. R.; Imai, Y.; Fennie, C.; Geoffroy, J . S.; Rosen, S. D.
J . Cell Biol. 1990, 110, 2221-2229.
(21) (a) Lawrence, M. B.; Mcintire, L. V.; Eskin, S. G. Blood 1987,
70, 1284-1290. (b) Lawrence, M. B.; Springer, T. A. J . Immunol. 1993,
151, 6338-6346.
(19) Gassman, P. G.; Schenk, W. N. J . Org. Chem. 1977, 42, 918-
920.
(22) Ichikawa, Y.; Shen, G.-J .; Wong, C.-H. J . Am. Chem. Soc. 1991,
113, 4698-4700.
5658 J . Org. Chem., Vol. 67, No. 16, 2002

Evaluation of a New Sialyl Lewis X Mimetic
Stark trap was removed. Tetrabutylammonium iodide (1.84
g, 0.50 equiv) and tert-butyl bromoacetate (5.8 mL, 4.0 equiv)
were added. The reaction mixture was heated at reflux for 16
h and then cooled to room temperature. The solid residue
obtained upon removal of the organic solvent was purified by
silica gel column chromatography (CH2Cl2 to CH2Cl2/MeOH
95:5) to give the tert-butyl ester 7 as a white powder (3.68 g,
66%), together with the recovered starting disaccharide 6b
(1.10 g). Data for 7: 1H NMR [400 MHz, acetone-d6 (1.5 mL)/
D2O (2 drops)] 4.42 (d, 1H, H-1′, J 1′,2′ ) 7.7 Hz), 4.31 (d, 1H,
H-1, J 1,2 ) 7.7 Hz), 4.26 and 4.22 (ABq, 2H, -CH2CO2t-Bu,
J AB )17.2 Hz), 4.06 (dd, 1H, H-4′, J 4′,3′ ) 3.3 Hz, J 4′,5′ ) 1.1
Hz), 3.96 (ddd, 1H, -OCHHCH2TMS, J ) 10.6, 9.5, 5.9 Hz),
3.84 and 3.82 (ABq, 2H, H-6, H-7, J AB ) 12.5 Hz, the 3.84 and
3.82 peaks further split by 3.5 Hz), 3.79 and 3.75 (ABq, 2H,
H-6′, H-7′, J AB ) 11.0 Hz, the 3.79 and 3.74 peaks further split
by 7.0 and 5.1 Hz, respectively), 3.72 (dd, 1H, H-2′, J 2′,3′ ) 9.5
mational Search carried out on the projected Ferrier
reaction product 2c resulted in the crucial distance of 9.0
Å between the carboxylate carbon and the fucose ano-
meric carbon. On these grounds, analogue 2a had been
anticipated to be comparable to sLex in its E-selectin
inhibitory activity. It is interesting to note that more
recent NMR studies23 through the use of 13C-enriched
sLex have shown that the orientation of the NeuAc-Gal
linkage in the E-selectin-bound sLex differs from that
postulated earlier by Sheffler et al.7 Although our flexible
mimetic could adopt an equivalent conformation even
more easily, the considerably flexible nature of our
mimetic in terms of its entire conformation may in turn
be the drawback of this mimetic. This line of analysis
seems quite relevant in light of recent successes by
Thoma and co-workers in the design of E-selectin an-
tagonists guided by the principle of preorganization of
the bioactive conformation of sLex.3u,v Additionally, the
X-ray-based structure of E-selectin bound to sLex recently
achieved by Camphausen24 should provide a further
guide for our design of sLex mimetic compounds.
Hz, J 2′,1′ ) 7.7 Hz), 3.64 (ddd, 1H, H-5′, J 5′,6′ ) 7.0 Hz, J 5′,7′′
)
5.1 Hz, J 5′,4′ ) 1.1 Hz), 3.59 (ddd, 1H, -OCHHCH2TMS, J )
10.3, 9.5, 5.9 Hz), 3.51-3.47 (m, 2H, H-3, H-4), 3.45 (dd, 1H,
H-3′, J 3′,2′ ) 9.5 Hz, J 3′,4′ ) 3.3 Hz), 3.41-3.37 (m, 1H, H-5),
3.21 (dd, 1H, H-2, J 2,3 ) 9.2 Hz, J 2,1 ) 7.7 Hz), 1.48 (s, 9H,
-C(CH3)3), 0.89-1.02 (m, 2H, -OCH2CH2TMS), 0.03 (s, 9H,
-Si(CH3)3); 13C NMR [100.6 MHz, acetone-d6 (1.5 mL)/D2O (2
drops)] 172.64, 105.09 (C-1′), 103.49 (C-1), 84.40 (C-3′), 82.85
(-C(CH3)3), 82.71 (C-4), 76.31 (C-5′), 76.25 (C-3), 75.75 (C-5),
74.54 (C-2), 70.86 (C-2′), 68.18 (-CH2CO2t-Bu), 67.21 (-OCH2-
CH2TMS), 66.84 (C-4′), 62.62 (C-6), 62.08 (C-6′), 28.21
Exp er im en ta l Section
Gen er a l. All reactions were performed under an atmo-
sphere of N2 unless otherwise indicated. The reaction solvents
diethyl ether (over benzophenone/Na), tetrahydrofuran (over
benzophenone/Na), and dichloromethane (over CaCl2) were
distilled freshly from laboratory stills. Other reaction solvents
(benzene, toluene, methanol, ethanol, N,N-dimethylformamide,
dimethyl sulfoxide) were distilled as needed and stored in
sealed bottles over molecular sieves. All glassware was dried
in a laboratory oven (100 °C) prior to use. Solutions were
concentrated at room temperature by rotary evaporation. Flash
column chromatography was performed with E. Merck 200-
300 silica gel and N2 gas pressure. Analytical thin-layer
chromatography was performed with EM Science Silica Gel
60 F254 0.2-mm precoated silica gel aluminum sheets with UV
indicator. Compounds were visualized by using carbazole/
sulfuric acid stain.
(-C(CH3)3), 18.76 (-CH2CH2TMS), -1.23 (-Si(CH3)3). [R]25
D
-6.2 (CHCl3, c 0.75). Anal. Calcd for C23H44O13Si: C, 49.63;
H, 7.97. Found: C, 49.35; H, 7.81. 1H-1H gCOSY, 1H-13C
HSQC.
2-(Tr im eth ylsilyl)eth yl 4,6-O-Ben zylid in e-[3-O-[(ter t-
bu toxyca r bon yl)m eth yl]-â-D-ga la ctop yr a n osyl]-(1f4)-â-
D-glu cop yr a n osid e (8a ). To a 250-mL, round-bottomed flask
equipped with a condenser, a stir bar, rubber septum, and a
N2 inlet were added 7 (2.37 g, 4.25 mmol), CH2Cl2 (150 mL),
benzaldehyde dimethylacetal (1.3 mL, 2.0 equiv), and p-TsOH‚
H2O (0.10 g). The reaction mixture was heated at reflux for
20 min. The reaction mixture was cooled and was washed with
aq satd NaHCO3, water, and brine, dried (Na2SO4), and
concentrated by rotary evaporation. The residue was purified
by silica gel column chromatography (CH2Cl2/MeOH, 95:5) to
give the product as a colorless oil (2.27 g, 83%): 1H NMR (400
MHz, CDCl3) 7.50-7.47 (m, 2H, Ar), 7.38-7.32 (m, 3H, Ar),
5.54 (s, 1H, -CHPh), 4.90 (s, 1H, -OH), 4.50 (d, 1H, H-1′, J 1′,2′
) 7.7 Hz), 4.34 (d, 1H, H-1, J 1,2 ) 7.7 Hz), 4.32 and 4.03 (ABq,
2H, -CH2CO2t-Bu, J AB ) 17.2 Hz), 4.30-4.27 (m, 3H, H-4′,
H-6′, -OH), 3.89 (ddd, 1H, H-6, J 6,7 ) 12.5 Hz, J 6,-OH ) 5.8
Hz, J 6,5 ) 4.0 Hz), 4.09-3.97 (m, 4H, H-2′, H-7, H-7′,
-OCHHCH2TMS), 3.72 (dd, 1H, H-3, J 3,2 ) 8.8 Hz, J 3,4 ) 8.8
Hz), 3.66 (dd, 1H, H-4, J 4,5 ) 9.1 Hz, J 4,3 ) 8.8 Hz), 3.60 (ddd,
1H, -OCHHCH2TMS, J ) 15.4, 9.5, 5.8 Hz), 3.53 (br, 1H,
Nuclear magnetic spectra were recorded on either a Varian
Inova 400 MHz or a Varian Inova 500 MHz spectrometer. The
chemical shifts are reported from low field to high field with
respect to tetramethylsilane as the internal standard (δ )
1
0.00). The data for H spectra are recorded as follows: chemical
shift (peak shape, integration, H-assignment if unambiguous,
and J values if resolvable). Peak shapes are described as
follows: s for singlet, d for doublet, dd for doublet of doublets,
ddd for doublet of doublet of doublets, q for quartet, m for
multiplet, dq for doublet of quartets, ABq for AB quartet, and
br for broadened. Coupling constants are given as J values in
hertz. The data for 13C spectra are reported from low field to
high field with respect to tetramethylsilane as the internal
standard (δ ) 0.00) as follows: chemical shift (C-assignment).
1H-1H (gCOSY) and 1H-13C (HSQC) 2D experiments were
performed on many compounds to aid in peak assignments.
2-(Tr im et h ylsilyl)et h yl [3-O-[(ter t-Bu t oxyca r b on yl)-
m eth yl]-â-D-galactopyr an osyl]-(1f4)-â-D-glu copyr an oside
(7). To a 500-mL, round-bottomed flask equipped with a
Dean-Stark trap, a condenser, a stir bar, a rubber septum,
and a N2 inlet were added lactoside 6b (4.41 g, 9.96 mmol),
dibutyltin oxide (2.9 g, 1.2 equiv), and benzene (325 mL). The
reaction mixture was heated at reflux with removal of water
for 5.5 h. The reaction mixture was cooled, and the Dean-
H-5′), 3.47 (ddd, 1H, H-5, J 5,4 ) 9.1 Hz, J 5,6 ) 4.0 Hz, J 5,7
)
3.7 Hz), 3.42 (dd, 1H, H-3′, J 3′,2′ ) 9.9 Hz, J 3′,4′ ) 3.6 Hz), 3.41
(dd, 1H, H-2, J 2,3 ) 8.8 Hz, J 2,1 ) 7.7 Hz), 3.20 (dd, 1H,
-CH2OH, J OH,7 ) 8.8 Hz, J OH,6 ) 5.8 Hz), 2.49 (s, 1H, -OH),
1.47 (s, 9H, -C(CH3)3), 1.10-0.95 (m, 2H, -OCH2CH2TMS),
0.26 (s, 9H, -Si(CH3)3); 13C NMR (100.6 MHz, CDCl3) 170.96
(R2CO), 137.22 (Ar), 128.98 (Ar), 128.11 (Ar), 126.28 (Ar),
104.19 (C-1′), 102.04 (C-1), 101.24 (-CHPh), 82.90 (-C(CH3)3),
81.39, 81.38, 74.68 (C-3), 74.52 (C-5), 73.56 (C-2), 73.29 (C-
4′), 69.01 (C-6′), 68.90 (C-2′), 66.85 (C-5′), 67.54 (-CH2CO2t-
Bu), 67.49 (-OCH2CH2TMS), 62.30 (C-6), 28.13 (-C(CH3)3),
18.32 (-OCH2CH2TMS), -1.33 (-Si(CH3)3). [R]22D +8.3 (CHCl3,
c 0.58,). Anal. Calcd for C30H48O13Si: C, 55.88; H, 7.50.
1
1
Found: C, 55.80; H, 7.75. H-1H gCOSY, H-13C HSQC.
2-(Tr im eth ylsilyl)eth yl 4,6-O-Ben zylid in e-[3-O-[(ter t-
bu toxyca r bon yl)m eth yl]-â-D-ga la ctop yr a n osyl]-(1f4)-6-
O-ter t-bu tyld ip h en ylsilyl-â-D-glu cop yr a n osid e (8b). To a
250-mL round-bottomed flask equipped with a condenser, a
stir bar, rubber septum, and a N2 inlet were added 8a (2.27 g,
(23) Scheffller, K.; Ernst, B.; Katopodis, A.; Magnani, J . L.; Wang,
W.-T.; Weisemann, R.; Peters, T. Angew. Chem., Int. Ed. Engl. 1995,
34, 1841-1844.
(24) Somers, W. S.; Tang, J .; Shaw, G. D.; Camphausen, R. T. Cell
2000, 103, 467-479.
J . Org. Chem, Vol. 67, No. 16, 2002 5659

Chervin et al.
3.52 mmol), THF (180 mL), tert-butylchlorodiphenylsilane (3.7
mL, 4.0 equiv), and DMAP (0.86 g, 2.0 equiv). The reaction
mixture was heated at reflux for 21 h and then cooled to room
temperature and quenched with water. The organic layer was
washed with satd aq NaHCO3, water, and brine and dried
(MgSO4). The solvent was evaporated and the residue was
purified by silica gel column chromatography (CH2Cl2/acetone
5:1) to obtain the TBDPS ether as a colorless oil (2.46 g, 79%):
1H NMR (400 MHz, CDCl3) 7.80-7.76 (m, 4H, Ar), 7.51-7.48
(m, 2H, Ar), 7.43-7.31 (m, 9H, Ar), 5.53 (s, 1H, -CHPh), 4.51
and 3.93 (ABq, 2H, -CH2CO2t-Bu, J AB ) 17.2 Hz), 4.49 (d,
1H, H-1′, J 1′,2′ ) 7.7 Hz), 4.32 (d, 1H, H-1, J 1,2 ) 7.3 Hz), 4.28-
4.26 (m, 2H, H-4′, H-6′), 4.16 (dd, 1H, H-6, J 6,7 ) 11.5 Hz, J 6,5
) 4.0 Hz), 4.06 and 3.98 (m, 3H, H-7, H-7′, -OCHHCH2TMS),
3.96 (ddd, 1H, H-2′, J 2′,3′ ) 9.7 Hz, J 2′,1′ ) 7.7 Hz, J 2′,OH ) 1.7
Hz), 3.74 (dd, 1H, H-2, J 3,2 ) 9.0 Hz, J 3,4 ) 9.0 Hz), 3.69 (dd,
1H, H-2, J 4,3 ) 9.0 Hz, J 4,5 ) 9.0 Hz), 3.60-3.58 (m, 1H,
-OCHHCH2TMS), 3.49-3.44 (m, 3H, H-2, H-5, H-5′), 3.36 (dd,
1H, H-3′, J 3′,2′ ) 9.7 Hz, J 3′,4′ ) 3.5 Hz), 1.48 (s, 9H, -C(CH3)3),
1.09-1.02 (m, 2H, -OCH2CH2TMS), 1.04 (s, 9H, -Si(Ph)2C-
(CH3)3), 0.03 (s, 9H, -Si(CH3)3); 13C NMR (100.6 MHz, CDCl3)
170.99 (R2CO), 137.50 (Ar), 135.89 (Ar), 135.70 (Ar), 133.73
(Ar), 133.16 (Ar), 129.60 (Ar), 129.53 (Ar), 129.12 (Ar), 128.23
(Ar), 127.67 (Ar), 127.55 (Ar), 126.49 (Ar), 104.18 (C-1′), 101.78
(C-1), 101.34 (-CHPh), 82.69 (-C(CH3)3), 81.57 (C-3′), 80.67
(C-3), 75.08, 74.51 (C-4), 73.86, 73.43 (C-4′), 69.59 (C-2′), 68.89
(C-6′), 67.54 (-CH2CO2t-Bu), 66.97 (-OCH2CH2TMS), 66.81
(C-2), 62.66 (C-6), 28.10 (-C(CH3)3), 26.78 (-SiC(CH3)3), 19.34
(-SiC(CH3)3), 18.35 (-OCH2CH2TMS), -1.37 (-Si(CH3)3).
Found: C, 61.43; H, 7.40. HRMS Calcd for C52H72O16NaSi2:
m/z 1031.4257. Found: m/z 1031.4222. 1H-1H gCOSY, 1H-
13C HSQC.
2-(Tr im eth ylsilyl)eth yl [2-O-Acetyl-4,6-O-ben zylid in e-
3-O-[(ter t-bu toxycar bon yl)m eth yl]-â-D-galactopyr an osyl]-
(1f4)-2,3-d i-O-a cetyl-â-D-glu cop yr a n osid e (10). To a 250-
mL, round-bottomed flask equipped with a stir bar, a condenser,
a rubber septum, and a N2 inlet were placed TBDPS ether 9
(1.75 g, 1.73 mmol) and anhydrous THF (100 mL). Acetic acid
(0.20 mL, 2.0 equiv) and tetrabutylammonium fluoride (3.46
mL of 1 M solution in THF, 2.0 equiv) were added, and the
reaction mixture was heated at reflux for 2 h. The reaction
mixture was cooled to room temperature and quenched with
water. The organic layer was first washed with satd aq
NaHCO3 and then with water. The organic phase was con-
centrated, and the residue thus obtained was purified by silica
gel column chromatography (hexanes/ethyl acetate 2:1) to
afford alcohol 10 as a white foam (1.21 g, 91%): 1H NMR (400
MHz, CDCl3) 7.47-7.45 (m, 2H, Ar), 7.37-7.32 (m, 3H, Ar),
5.53 (s, 1H, -CHPh), 5.25 (dd, 1H, H-2′, J 2′,3′ ) 10.2 Hz, J 2′,1′
) 8.0 Hz), 5.20 (dd, 1H, H-3, J 3,2 ) 9.9 Hz, J 3,4 ) 9.5 Hz), 4.88
(dd, 1H, H-2, J 2,3 ) 9.9 Hz, J 2,1 ) 8.1 Hz), 4.55 (d, 1H, H-1,
J 1,2 ) 8.1 Hz), 4.54 (d, 1H, H-1′, J 1′,2′ ) 8.0 Hz), 4.48 (d br.,
1H, H-4′, J 4′,3′ ) 3.3 Hz, J 4′,5′ < 1.0 Hz), 4.29 and 4.05 (ABq,
2H, H-6′, H-7′, J AB ) 12.4 Hz), 4.17 and 4.01 (ABq, 2H, -CH2-
CO2t-Bu, J AB ) 16.8 Hz), 3.91 (dd, 1H, H-4, J 4,3 ) 9.5 Hz, J 4,5
) 9.5 Hz), 3.91 and 3.82 (ABq, 2H, H-6, H-7, J AB ) 12.1 Hz),
3.93 (ddd, 1H, -OCHHCH2TMS, J ) 9.9, 9.9, 6.6 Hz), 3.69
(dd, 1H, H-3′, J 3′,2′ ) 10.2 Hz, J 3′,4′ ) 3.3 Hz), 3.54 (ddd, 1H,
-OCHHCH2TMS, J ) 9.9, 9.9, 6.6 Hz), 3.41 (m br., 2H, H-5,
H-5′), 2.02 (s, 3H, Ac), 2.10 (s, 3H, Ac), 1.42 (s, 9H, -C(CH3)3),
0.96-0.88 (m, 2H, -OCH2CH2TMS), 0.00 (s, -Si(CH3)3); 13C
NMR (100.6 MHz, CDCl3) 170.59 (R2CO), 169.95 (R2CO),
169.86 (R2CO), 169.16 (R2CO), 137.81 (Ar), 129.30 (Ar), 128.40
(Ar), 126.86 (Ar), 101.70 (-CHPh), 101.16 (C-1′), 100.63 (C-
1), 81.89 (-C(CH3)3), 78.35 (C-3′), 75.22 (C-5), 74.94 (C-4′),
74.68 (C-4), 72.79 (C-3), 70.95(C-2′), 70.90 (C-2), 68.90 (C-6′),
67.94 (-OCH2CH2TMS), 67.54 (-CH2CO2t-Bu), 66.75 (C-5′),
60.61 (C-6), 28.29 (-C(CH3)3), 21.18 (-OCH3), 20.99 (-OCH3),
20.93 (-OCH3), 18.22 (-OCH2CH2TMS), 1.23 (-Si(CH3)3).
[R]22 -4.6 (CHCl3, c 0.63). Anal. Calcd for C46H66O13Si2: C,
D
62.56; H, 7.53. Found: C, 62.49; H, 7.64. 1H-1H gCOSY, 1H-
13C HSQC.
2-(Tr im eth ylsilyl)eth yl [2-O-Acetyl-4,6-O-ben zylid in e-
[3-O-(ter t-bu toxycar bon yl)m eth yl]-â-D-galactopyr an osyl]-
(1f4)-2,3-d i-O-a cetyl-6-O-ter t-bu tyld ip h en ylsilyl-â-D-glu -
copyr an oside (9). To a 250-mL, round-bottomed flask equipped
with a stir bar, a condenser, rubber septum, and a N2 inlet
were added triol 8b (4.46 g, 5.0 mmol), CH2Cl2 (150 mL),
DMAP (3.7 g, 6.0 equiv), and acetic anhydride (2.85 mL, 6.0
equiv). The reaction mixture was stirred and heated at reflux
for 20 min. The reaction mixture then was quenched with
water and the organic layer washed first with satd aq NaHCO3
and then with water, dried (NaSO4), and concentrated. Puri-
fication of the residue by silica gel column chromatography
(hexanes/ethyl acetate 10:1 to 1:1) afforded the triacetate 9
as a colorless oil (4.6 g, 92%): 1H NMR (400 MHz, CDCl3)
7.79-7.74 (m, 3H, Ar), 7.47-7.32 (m, 12H, Ar), 5.51 (s, 1H,
-CHPh), 5.25 (dd, 1H, H-2′, J 2′,3′ ) 10.3 Hz, J 2′1′ ) 8.0 Hz),
5.19 (dd, 1H, H-3, J 3,4 ) 9.8 Hz, J 3,2 ) 9.5 Hz), 4.98 (dd, 1H,
H-2, J 2,3 ) 9.5 Hz, J 2,1 ) 8.0 Hz), 4.76 (d, 1H, H-1′, J 1′,2′ ) 8.0
Hz), 4.47 (d, 1H, H-1, J 1,2 ) 8.0 Hz), 4.43 (d br., 1H, H-4′, J 4′,3′
) 3.3 Hz, J 4′,5′ < 1.0 Hz), 4.32 (dd, 1H, H-6′, J 6′,7′ ) 12.4 Hz,
[R]21 -5.5 (CHCl3, c 0.87). Anal. Calcd for C36H54O16Si: C,
D
56.09; H, 7.06. Found: C, 55.85; H, 7.24. 1H-1H gCOSY, 1H-
13C HSQC.
P h en yl 2,3,4-Tr i-O-ben zyl-1-th io-â-L-fu cop yr a n ose (11).
To a 10-mL, round-bottomed flask equipped with a stir bar,
rubber septum, and N2 inlet was added phenyl 1-thio-â-L-
fucopyranoside (42 mg, 0.16 mmol) and DMF (3 mL). Sodium
hydride (38 mg of 60% suspension in mineral oil, 6.0 mol equiv)
was added to the flask and the suspension stirred for 10 min
at room temperature. Benzyl bromide (0.11 mL, 6.0 mol equiv)
and tetra(n-butyl)ammonium iodide (cat.) were added, and the
mixture was stirred for 2 h at room temperature. The reaction
mixture was then quenched with water (4 mL) and diluted
with ether (10 mL). The layers were separated and the aqueous
layer was back extracted with ether (10 mL). The combined
organic layers were washed with satd aq NH4Cl and concen-
trated. The resulting oil was purified by silica gel column
chromatography (hexanes f hexanes/ethyl acetate 3:1) to give
benzyl ether 1118a as a white solid (74 mg, 90%).
2-(Tr im eth ylsilyl)eth yl [2-O-Acetyl-4,6-O-ben zylid in e-
3-O-[(ter t-bu toxycar bon yl)m eth yl]-â-D-galactopyr an osyl]-
(1f4)-[2,3,4-tr i-O-ben zyl-r-L-fu cosyl-(1f6)]-2,3-di-O-acetyl-
â-D-glu cop yr a n osid e (12). Fucoside 11 (38.0 mg, 0.0700
mmol) and lactoside 10 (64.6 mg, 1.2 equiv) were dissolved in
CH2Cl2 (5 mL) in a 10-mL, round-bottomed flask containing
molecular sieves and equipped with a rubber septum and N2
inlet. To the flask were added N-iodosuccinimide (23 mg, 2.0
equiv) and p-TsOH‚H2O (cat.). The mixture became purple
instantly, indicating the generation of molecular iodine. After
the solution was stirred for 15 min, the reaction mixture was
decanted by pipet into 3 mL of aq satd Na2S2O3. The organic
layer was concentrated by rotary evaporation. The residue was
J 6′,5′ ) 1.5 Hz), 4.29 and 3.90 (ABq, 2H, -CH2CO2t-Bu, J AB
)
16.8 Hz), 4.12 (dd, 1H, H-4, J 4,3 ) 9.8 Hz, J 4,5 ) 9.8 Hz), 4.06-
3.94 (m, 4H, H-6, H-7, H-7′, -OCHHCH2TMS), 3.58-3.52 (m,
1H, -OCHHCH2TMS), 3.55 (dd, 1H, H-3′, J 3′,2′ ) 10.3 Hz, J 3′,4′
) 3.3 Hz), 3.35 (d br, 1H, H-5, J 5,4 ) 9.8 Hz), 3.25 (br, 1H,
H-5′), 2.05 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.86 (s, 3H, Ac), 1.41
(s, 9H, -C(CH3)3), 1.07 (s, 9H, -Si(Ph)2C(CH3)3), 1.02-0.94
(m, 2H, -OCH2CH2TMS), 0.02 (s, 9H, -Si(CH3)3). 13C NMR
(100.6 MHz, CDCl3) 170.69 (R2CO), 169.80 (R2CO), 169.76
(R2CO), 168.71 (R2CO), 137.62 (Ar), 136.03 (Ar), 135.50 (Ar),
133.76 (Ar), 132.25 (Ar), 129.86 (Ar), 129.82 (Ar), 129.11 (Ar),
128.21 (Ar), 127.83 (Ar), 127.66 (Ar), 126.66 (Ar), 101.52
(-CHPh), 100.54 (C-1′), 100.21 (C-1), 81.61 (-C(CH3)3), 78.47
(C-3′), 75.24 (C-5), 74.93 (C-4′), 74.32 (C-4), 72.53 (C-3), 71.76
(C-2), 70.38 (C-2′), 68.75 (C-6′), 67.32 (-CH2CO2t-Bu), 66.68
(-OCH2CH2TMS), 66.52 (C-5′), 61.23 (C-6), 28.07 (-C(CH3)3),
26.82 (-Si(Ph)2C(CH3)3), 19.40 (-Si(Ph)2C(CH3)3), 20.85, 20.80,
20.78, 17.97 (-OCH2CH2TMS), -1.32 (-Si(CH3)3). [R]23D +17.6
(CHCl3, c 0.86). Anal. Calcd for C52H72O16Si2: C, 61.88; H, 7.19.
5660 J . Org. Chem., Vol. 67, No. 16, 2002

Evaluation of a New Sialyl Lewis X Mimetic
purified by silica gel column chromatography to give the
trisaccharide as a foam (46.9 mg, 56%): 1H NMR (500 MHz,
CDCl3) 7.48-7.30 (m, 20H, Ar), 5.31 (s, 1H, -CHPh), 5.15 (dd,
1H, H-3, J 3,2 ) 10.0 Hz, J 3,4 ) 9.5 Hz), 5.09 (dd, 1H, H-2′, J 2′,3′
) 10.0 Hz, J 2′,1′ ) 8.1 Hz), 4.99 and 4.69 (ABq, 2H, -CH2Ph,
J AB ) 11.5 Hz), 4.94 (d, 1H, H-1′′, J 1′′,2′′ ) 3.4 Hz), 4.92 (dd,
1H, H-2, J 2,3 ) 10.0 Hz, J 2,1 ) 7.8 Hz), 4.88 (s br, 2H, -CH2-
Ph), 4.87 and 4.67 (ABq, 2H, -CH2Ph, J AB ) 11.7 Hz), 4.62
(d, 1H, H-1′, J 1′,2′ ) 8.1 Hz), 4.44 (d, 1H, H-1, J 1,2 ) 7.8 Hz),
4.09-3.93 (m, 9H, H-4, H-7, H-5′, H-6′, H-7′, H-2′′, H-5′′, -CH2-
CO2t-Bu), 3.91-3.87 (m, 1H, -OCHHCH2TMS), 3.85 (dd, 1H,
(-OCH2CH2TMS), 15.72 (C-6′′), -1.38 (-Si(CH3)3). HRMS.
Calcd for C48H70O23NaSi: m/z 1065.3975. Found: m/z 1065.4015.
1
1H-1H gCOSY, H-13C HSQC.
2-(Tr im eth ylsilyl)eth yl [2,4,6-Tr i-O-a cetyl-3-O-(ter t-bu -
toxyca r bon yl)m eth yl-â-D-ga la ctop yr a n osyl]-(1f4)-[2,3,4-
tr i-O-acetyl-r-L-fu cosyl-(1f6)]-2,3-di-O-acetyl-â-D-glu copy-
r a n osid e (13b). Triacetate 12 (46.9 mg, 0.0487 mmol) was
placed in a 10-mL, round-bottomed flask equipped with a stir
bar. To the flask were added methanol (distilled, 6 mL) and
10% Pd/C (53 mg, 1.0 mol equiv). The flask was equipped with
a gas inlet adapter and H2-filled balloon. The suspension was
stirred for 2 d with replenishment of the H2 balloon when
necessary. The reaction mixture was then passed through a
short column of Celite to remove the particulates, and the
solvent was evaporated. The resulting residue was dissolved
in CH2Cl2 (3 mL) and stirred with triethylamine (69 µL, 10
mol equiv), acetic anhydride (46 µL, 10 mol equiv), and DMAP
(cat.). The mixture was stirred at room temperature under an
atmosphere of nitrogen for 2 h until TLC analysis indicated
that no starting material remained. The reaction mixture was
quenched with the addition of water, and the organic layer
was concentrated. Purification of the crude material by silica
gel column chromatography (CH2Cl2) gave the octaacetate 13b
as a colorless film (37 mg, 71%): 1H NMR (500 MHz, CDCl3)
5.39 (dd, 1H, H-4′, J 4′,3′ ) 3.4 Hz, J 4′,5′ < 1.0 Hz), 5.35 (dd, 1H,
H-3′′, J 3′′,2′′ ) 10.5 Hz, J 3′′,4′′ ) 3.1 Hz), 5.32 (dd, 1H, H-4′′, J 4′′,3′′
) 3.4 Hz, J 4′′,5′′ < 1.0 Hz), 5.18 (dd, 1H, H-3, J 3,2 ) 9.7 Hz, J 3,4
) 9.2 Hz), 5.16 (d, 1H, H-1′′, J 1′′,2′′ ) 3.6 Hz), 5.12 (dd, 1H,
H-2′′, J 2′′,3′′ ) 10.5 Hz, J 2′′,1′′ ) 3.6 Hz), 4.99 (dd, 1H, H-2′, J 2′,3′
) 10.0 Hz, J 2′,1′ ) 8.1 Hz), 4.86 (dd, 1H, H-2, J 2,3 ) 9.7 Hz, J 2,1
) 8.1 Hz), 4.54 (d, 1H, H-1′, J 1′,2′ ) 8.1 Hz), 4.45 (d, 1H, H-1,
J 1,2 ) 8.1 Hz), 4.26 (dq, 1H, H-5′′, J 5′′,6′′ ) 6.6 Hz, J 5′′,4′′ < 1.0
Hz), 4.14-4.11 (m, 2H, H-6′, H-7′), 3.98-3.90 (m, 2H, H-6,
-OCHHCH2TMS), 3.89 and 3.29 (ABq, 2H, -CH2CO2t-Bu, J AB
) 17.1 Hz), 3.78-3.74 (m, 2H, H-4, H-7), 3.81 (ddd br, 1H,
H-5′, J 5′,6′ ) 7.3 Hz, J 5′,7′ ) 6.8 Hz, J 5′,4′ < 1.0 Hz), 3.61 (dd,
1H, H-3′, J 3′,2′ ) 10.0 Hz, J 3′,4′ ) 3.4 Hz), 3.54-3.49 (m, 2H,
H-5, -OCHHCH2TMS), 2.17 (s, 3H, Ac), 2.14 (s, 6H, Ac), 2.08
(s, 6H, Ac), 2.04 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.00 (s, 3H, Ac),
1.44 (s, 9H, -OC(CH3)3), 1.15 (d, 3H, -CH3, J 6′′,5′′ ) 6.6 Hz),
1.00-0.83 (m, 2H, -CH2CH2TMS), 0.00 (s, -Si(CH3)3); 13C
NMR (100.6 MHz, CDCl3) 175.75 (R2CO), 175.24 (R2CO),
173.16 (R2CO), 170.70 (R2CO), 170.32 (R2CO), 170.20 (R2CO),
170.68 (R2CO), 169.82 (R2CO), 169.80 (R2CO), 100.77 (C-1′),
100.08 (C-1), 96.50 (C-1′′), 81.53 (-C(CH3)3), 78.26 (C-3′), 75.92
(C-4), 74.21 (C-5), 73.17 (C-3), 72.06 (C-2), 71.27 (C-4′′), 70.92
(C-2′), 70.67 (C-5′), 68.63 (C-2′′), 67.85 (C-3′′), 67.25 (-OCH2-
CH2TMS), 66.43 (-CH2CO2t-Bu), 66.22 (C-6), 65.42 (C-4′),
64.81 (C-5′′), 61.49 (C-6′), 28.12 (-C(CH3)3), 21.01, 20.90, 20.84,
20.79, 20.78, 20.68, 20.67, 20.64, 17.91 (-OCH2CH2TMS),
H-4′, J 4′,3′ ) 3.2 Hz, J 4′,5′ < 1.0 Hz), 3.75 (dd, 1H, H-4′′, J 4′′,3′′
)
1.7 Hz, J 4′′,5′′ < 1.0 Hz), 3.71 (dd, 1H, H-6, J 6,7 ) 10.5 Hz, J 6,5
) 2.2 Hz), 3.58-3.50 (m, 1H, -OCHHCH2TMS), 3.43 (dd, 1H,
H-5, J 5,4 ) 9.7 Hz, J 5,6 < 1.0 Hz), 3.35 (dd, 1H, H-3′, J 3′,2′
10.0 Hz, J 3′,4′ ) 3.2 Hz), 2.12 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.01
(s, 3H, Ac), 1.39 (s, 9H, -C(CH3)3), 1.10 (d, 3H, H-6′′, J 6′′,5′′
)
)
6.3 Hz), 0.92-0.86 (m, 2H, -OCH2CH2TMS), -0.02 (s, 9H,
-Si(CH3)3); 13C NMR (100.6 MHz, CDCl3) 171.91 (R2CO),
170.99 (R2CO), 170.32 (R2CO), 168.20 (R2CO), 140.44 (Ar),
139.90 (Ar), 139.19 (Ar), 130.36 (Ar), 129.91 (Ar), 129.85 (Ar),
129.84 (Ar), 129.73 (Ar), 129.62 (Ar), 129.52 (Ar), 129.22 (Ar),
129.16 (Ar), 129.13 (Ar), 128.99 (Ar), 128.94 (Ar), 127.97 (Ar),
101.24 (-CHPh), 100.26 (C-1), 99.93 (C-1′), 96.68 (C-1′′), 81.29
(-C(CH3)3), 79.31, 77.89 (C-3′), 77.30, 74.93 (-CH2Ph), 74.27
(C-4′), 73.94, 73.86, 73.81 (C-5), 72.68, 72.47 (C-3), 71.68 (C-
2), 70.65 (C-2′), 68.63 (C-3′′), 68.63 (overlap), 66.86, 66.52,
66.16, 65.65, 63.11 (C-6), 27.99 (-C(CH3)3), 21.11 (Ac), 20.74
(Ac), 20.65 (Ac), 17.90 (-OCH2CH2TMS), 16.48 (C-6′′), -1.44
(-Si(CH3)3). [R]20 -20.4 (CHCl3, c 0.99). HRMS Calcd for
D
1
C
63H82O20NaSi: m/z 1209.5066. Found: m/z 1209.5073. H-
13C HSQC.
2-(Tr im eth ylsilyl)eth yl [2-O-Acetyl-4,6-O-ben zylid in e-
3-O-[(ter t-bu toxycar bon yl)m eth yl]-â-D-galactopyr an osyl]-
(1f4)-[2,3,4-tr i-O-acetyl-r-L-fu cosyl-(1f6)]-2,3-di-O-acetyl-
â-D-glu cop yr a n osid e (13a ). To a 5-mL, round-bottomed flask
equipped with a stir bar and balloon was added 12 (0.047 g,
4.0 × 10-2mmol), ethanol (4 mL, absolute, distilled), and 10%
Pd/C (8.5 mg, 20 mol %); the balloon was filled with H2 four
times over a 23-h period. The reaction mixture was filtered
through Celite, and the solvent was evaporated. The residue
was dissolved in CH2Cl2 (4 mL). To the solution was added
triethylamine (excess), acetic anhydride (excess), and DMAP
(cat.). The reaction mixture was allowed to stir for 1.25 h at
room temperature. Column chromatography of the reaction
mixture gave hexaacetate 13a as a colorless film (24 mg, 57%,
2 steps): 1H NMR (500 MHz, CDCl3) 7.48-7.46 (m, 2H, Ar),
7.36-7.34 (m, 3H, Ar), 5.53 (s, 1H, -CHPh), 5.37 (dd, 1H,
H-3′′, J 3′′,2′′ ) 10.7 Hz, J 3′′,4′′ ) 3.4 Hz), 5.31 (dd, 1H, H-4′′, J 4′′,3′′
) 3.4 Hz, J 4′′,5′′ ) 1.0 Hz), 5.23 (dd, 1H, H-2′, J 2′,3′ ) 10.3 Hz,
J 2′,1′ ) 8.0 Hz), 5.19 (dd, 1H, H-3, J 3,2 ) 9.7 Hz, J 3,4 ) 9.5 Hz),
15.79 (C-6′′), -1.39 (-Si(CH3)3). [R]20 -6.1 (CHCl3, c 0.87).
D
HRMS. Calcd for C45H70O25NaSi: m/z 1061.3873. Found: m/z
1061.3844. ESIHRMS. Calcd for C45H70O25NaSi: m/z 1061.3873.
5.16 (d, 1H, H-1′′, J 1′′,2′′ ) 3.6 Hz), 5.12 (dd, 1H, H-2′′, J 2′′,3′′
)
10.7 Hz, J 2′′,1′′ ) 3.6 Hz), 4.88 (dd, 1H, H-2, J 2,3 ) 9.7 Hz, J 2,1
) 8.0 Hz), 4.57 (d, 1H, H-1′, J 1′,2′ ) 8.0 Hz), 4.47 (dd, 1H, H-4′,
J 4′,3′ ) 3.4 Hz, J 4′,5′ < 1.0 Hz), 4.46 (d, 1H, H-1, J 1,2 ) 8.0 Hz),
4.35-4.32 (m, 2H, H-5′′, H-7′), 4.18 and 3.98 (ABq, 2H, -CH2-
CO2t-Bu, J AB ) 16.8 Hz), 4.08 (d br, 1H, H-6′, J 6′,7′ ) 10.9 Hz),
3.97-3.91 (m, 1H, -OCHHCH2TMS), 3.93-3.84 (m, 2H, H-6,
H-7), 3.81 (dd, 1H, H-4, J 4,5 ) 9.7 Hz, J 4,3 ) 9.5 Hz), 3.69 (dd,
1H, H-3′, J 3′,2′ ) 10.3 Hz, J 3′,4′ ) 3.4 Hz), 3.54-3.48 (m, 3H,
H-5, H-5′, -OCHHCH2TMS), 2.18, 2.10, 2.07, 2.03, 2.02, 2.01,
1.39 (s, 9H, -OC(CH3)3), 1.12 (d, 3H, -CH3, J 6′′,5′′ ) 6.3 Hz),
0.98-0.83 (m, 2H, -CH2CH2TMS), 0.00 (s, -Si(CH3)3); 13C
NMR (100.6 MHz, CDCl3) 170.50 (R2CO), 170.49 (R2CO),
170.27 (R2CO), 169.82 (R2CO), 169.81 (R2CO), 169.61 (R2CO),
168.97 (R2CO), 137.67 (Ar), 129.08 (Ar), 128.20 (Ar), 126.61
(Ar), 101.41 (-CHPh), 100.59 (C-1′), 100.29 (C-1), 96.46 (C-
1′′), 81.61 (-C(CH3)3), 77.95 (C-3′), 75.07 (C-4), 74.70 (C-4′),
74.31, 72.56 (C-3), 71.93 (C-2), 71.36 (C-4′′), 70.74 (C-2′), 68.87
(C-6′), 68.87 (C-2′′), 67.70 (C-3′′), 67.21 (-OCH2CH2TMS),
66.80 (-CH2CO2t-Bu), 66.55, 66.27 (C-6), 64.90 (C-5′′), 28.07
(-C(CH3)3), 21.01, 21.01, 20.73, 20.73, 20.73, 20.64, 17.93
1
1
Found: m/z 1061.3868. H-1H gCOSY, H-13C HSQC.
2-(Tr im eth ylsilyl)eth yl 3-O-Ca r boxym eth yl-â-D-ga la c-
top yr a n osyl-(1f4)-[r-L-fu cosyl-(1f6)]-â-D-glu cop yr a n o-
sid e (2a ). Octaacetate tert-butyl ester 13b (16 mg, 1.6 × 10-2
mmol) was dissolved in dry diethyl ether (4 mL). Water (10.1
µL, 36 mol equiv) and potassium tert-butoxide (63 mg, 36 mol
equiv) were added. The resulting slurry was stirred for 14 h
at room temperature until TLC analysis indicated that the
starting material had been consumed. The reaction mixture
was quenched by the addition of water (2 mL). The aqueous
phase was acidified to pH 5 with cationic acid-exchange resin.
The resin was removed by filtration, and the filtrate was
concentrated. The syrup thus obtained was purified by gravity
chromatography with water as the eluent (Sephadex G-10,
1-cm-diameter column, 3-in. high). The water was removed
under high vacuum to give the trisaccharide 2a as a white
powder (19 mg): 1H NMR (500 MHz, D2O) 4.77 (d,1H, H-1′′,
J 1′′,2′′ ) 3.6 Hz), 4.41 (d, 1H, H-1′, J 1′,2′ ) 7.8 Hz), 4.35 (d, 1H,
H-1, J 1,2 ) 8.1 Hz), 3.97 (dq, 1H, H-5′′, J 5′′,6′′ ) 6.6 Hz, J 5′′,4′′
J . Org. Chem, Vol. 67, No. 16, 2002 5661

Chervin et al.
<1.0 Hz), 3.94-3.92 (m, 3H, H-4′, -CH2), 3.89-3.82 (m, 2H,
-CHH, -CHHCH2TMS), 3.77 (dd, 1H, H-3′′, J 3′′,2′′ ) 10.2 Hz,
J 3′′,4′′ ) 2.9 Hz), 3.72-3.50 (m, 9H, H-4, H-5, H-5′, H-2“, H-4”,
-CHH, -CH2, -OCHHCH2TMS), 3.49-3.32 (m, 2H, H-3,
H-2′), 3.34 (dd, 1H, H-3′, J 3′,2′ ) 10.0 Hz, J 3′,4′ ) 2.7 Hz), 3.15
(dd, 1H, H-2, J 2,3 ) 8.3 Hz, J 2,1 ) 8.1 Hz), 1.08-0.95 (m, 1H,
-OCH2CHHTMS), 0.85-0.78 (m, 1H, -OCH2CHHTMS), -0.16
(s, 9H, -Si(CH3)3); 13C NMR (100.6 MHz, D2O) 86.16 (R2CO),
103.02 (C-1′), 102.03 (C-1), 99.58 (C-1′′), 82.31 (C-3′), 78.12,
75.46, 74.82, 74.01, 73.35 (C-2), 72.30, 70.22, 69.90, 69.88 (C-
3′′), 68.74 (-CH2), 68.56 (-OCH2CH2TMS), 67.04 (C-5′′), 66.72
(-CH2), 65.76 (C-4′), 61.60 (-CH2), 18.08 (-OCH2CH2TMS),
of IgG-hE-sel solution (which was determined to give the
optimum number of binding events by prior analysis) was
mounted on the flow chamber and clamped to the stage of the
inverted-stage microscope. Solutions of the test compounds in
water (10 mM) and a test solution containing no inhibitor were
prepared. Each test solution (100 µL) was combined with
rolling media (400 µL) and neutrophil suspension (500 µL) and
agitated immediately before analysis. One-third of the result-
ing mixture was transferred to the assay inlet. The mixture
was perfused through the chamber at a shear rate of 0.38
dynes/cm2 for 2 min and the rolling action recorded on video.
Each experiment was performed in triplicate and the chamber
was washed with rolling media between each run.
15.86 (C-6′′), -2.43 (-Si(CH3)3). [R]20 -10.7 (H2O, c 0.46).
D
ESIHRMS. Calcd for C25H46O17SiNa: m/z 669.2402. Found:
The video was analyzed at snapshot intervals of 60, 90, and
120 s for each run. Interacting cells appeared as spherical,
well-defined objects whereas noninteracting flowing cells ap-
peared as long horizontal segmented streaks. The number of
interacting cells was tabulated for each shot snapshot image
and the average of three runs for each test suspension was
calculated.
1
1
m/z 669.2445. H-1H gCOSY, H-13C HSQC.
Deter m in a tion of E-Selectin /Leu k ocyte In h ibition .
One week prior to performing the rolling assay, the tissue
culture plates to be used with the parallel plate flow assembly
were coated with recombinant human E-selectin. Each plate
(35 mm, polystyrene) was spotted with 40 µL of a solution of
Protein A (Sigma) in Tris-HCl buffer (20 µg/mL, pH 9.0) and
incubated for 18 h at 4 °C. The Protein A solution was removed
(aspiration) and each plate was washed with TBS buffer (3 ×
40 µL). Each plate was spotted with 80 µL of BSA/TBS/NaN3
blocking buffer (pH 7.5) and incubated for 3 h at room
temperature. The blocking buffer was removed (aspiration) and
each plate was washed with TBS buffer (5 × 80 µL). The plates
were spotted with varying concentrations of chimera IgG-
human E-selectin (IgG-hE-sel) in BSA/TBS/NaN3 buffer (3
plates each at dilutions of 1:10, 1:20, and 1:30). The plates
were incubated for 2 h at 4 °C, washed with TBS buffer (3 ×
40 µL), and stored over BSA/TBS/NaN3 buffer at 4 °C until
use. Human polymorphonuclear leukocytes were collected from
fresh blood (20 mL) and suspended in rolling media (0.2% BSA/
HEPES/2 mM CaCl2) at a concentration of 1 × 106 cells/mL
and used within 4 h of isolation.
Ack n ow led gm en t. We thank Ms. Bronia Petryniak
of Howard Hughes Medical Institute, University of
Michigan Medical School, for her assistance in the
biological testings. The work presented was supported
in part by a grant from Glycosyn Pharmceuticals (to
M.K.). J .B.L. is an Investigator of the Howard Hughes
medical Institute, and was supported in part by a grant
from the NIH (CA 71932).
Su p p or tin g In for m a tion Ava ila ble: NMR spectral data
(1H, 13C, 1H-1H gCOSY, and HSQC) for new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
The experimental apparatus was assembled as described
by Lawrence.21 A culture plate coated with the 1:10 dilution
J O025579T
5662 J . Org. Chem., Vol. 67, No. 16, 2002