Studies on selection blockers. 5. Design, synthesis, and biological profile of sialyl Lewis x mimetics based on modified serine-glutamic acid dipeptides.

Article abstract of DOI:10.1021/jm970262k

We have rationally designed a sLe(x) mimetic based on molecular modeling, synthesized type II and type II' beta-turn dipeptides (3a,b), and evaluated their biological profiles both in vitro and in vivo. Against E-selectin-sLe(x) binding, the type II beta-turn dipeptide L-Ser-D-Glu 3a (IC50, 13 microM) and the type II' beta-turn dipeptide D-Ser-L-Glu 3b (IC50, 5.5 microM) were 20-100-fold more potent blockers than sLe(x) (1; IC50, 600 microM) and a 3'-sulfated Le(x) analog (2; IC50, 280 microM). On the other hand, other stereoisomers, such as L-Ser-L-Glu 3c and D-Ser-D-Glu 3d, were very weak blockers, with IC50 > 1000 microM for both 3c,d. Against the P- and L-selectins, despite much different stereochemistry of compounds 3a-d, the dipeptides 3a-d were all more potent blockers than either sLe(x) or compound 2. Interestingly, compound 3b provided significant in vivo efficacy against an immunoglobulin E-mediated skin reaction in a mouse model. These findings indicate that sLe(x) mimetics with type II and type II' beta-turn dipeptides could be useful in the design of an active selectin blocker in vitro and/or in vivo.

Full text of DOI:10.1021/jm970262k

3534
J . Med. Chem. 1997, 40, 3534-3541
Stu d ies on Selectin Block er s. 5. Design , Syn th esis, a n d Biologica l P r ofile of
Sia lyl Lew is x Mim etics Ba sed on Mod ified Ser in e-Glu ta m ic Acid Dip ep tid es
Takahiro Tsukida, Yasuyuki Hiramatsu,* Hideki Tsujishita, Takao Kiyoi, Masahiro Yoshida, Kiriko Kurokawa,
Hideki Moriyama, Hiroshi Ohmoto, Yukihisa Wada, Tadayuki Saito, and Hirosato Kondo*
Department of Medicinal Chemistry and Biology, Kanebo, New Drug Discovery Research Laboratories, 1-5-90 Tomobuchi-Cho,
Miyakojima-Ku, Osaka 534, J apan
Received April 21, 1997X
We have rationally designed a sLex mimetic based on molecular modeling, synthesized type II
and type II′ â-turn dipeptides (3a ,b), and evaluated their biological profiles both in vitro and
in vivo. Against E-selectin-sLex binding, the type II â-turn dipeptide L-Ser-D-Glu 3a (IC50, 13
µM) and the type II′ â-turn dipeptide D-Ser-L-Glu 3b (IC50, 5.5 µM) were 20-100-fold more
potent blockers than sLex (1; IC50, 600 µM) and a 3′-sulfated Lex analog (2; IC50, 280 µM). On
the other hand, other stereoisomers, such as L-Ser-L-Glu 3c and D-Ser-D-Glu 3d , were very
weak blockers, with IC50 > 1000 µM for both 3c,d . Against the P- and L-selectins, despite
much different stereochemistry of compounds 3a -d , the dipeptides 3a -d were all more potent
blockers than either sLex or compound 2. Interestingly, compound 3b provided significant in
vivo efficacy against an immunoglobulin E-mediated skin reaction in a mouse model. These
findings indicate that sLex mimetics with type II and type II′ â-turn dipeptides could be useful
in the design of an active selectin blocker in vitro and/or in vivo.
In tr od u ction
Selectins are believed to be involved in the pro-
gression of the clinical manifestations of complicated
diseases, such as chronic inflammation,1 and have
demonstrated a role in the inflammatory response. In
addition, a recent exciting report described that sialyl
Lewis x (1, sLex), which is a ligand for endothelial-
leukocyte adhesion molecules, inhibited angiogenesis
both in vitro and in vivo.2 Such a biologically integral
function makes sLex mimetics an attractive target for
the therapy of these diseases.
We have already reported the design, synthesis, and
biological profile of a 3′-O-sulfated Lex (2) containing a
branched alkyl chain, such as a natural ceramide, a
potent selectin blocker.3 In addition, our previous
studies4 of molecular dynamics simulations of the
compound 2/E-selectin complex suggested the existence
of three essential binding sites of compound 2 for
E-selectin, as follows (Figure 1): (1) The C-2 and C-3
hydroxy groups of fucose coordinate to calcium. (2) The
branched alkyl chain of 2 interacts with two hydropho-
bic regions on the surface of E-selectin. (3) The nega-
tively charged group of the ligand interacts with the
basic residues of E-selectin.5 On the basis of the
information concerning the essential binding sites of
compound 2, we sought to mimic compound 2 by a
simple skeleton. To design simple sLex mimetics, we
focused on compound libraries with simple peptide
backbones, due to the wealth of three-dimensional (3-
D) analysis data of dipeptides.6 In docking models of
compounds/E-selectin, we have screened dipeptide back-
bones conserving the positions of the three essential
functional groups for binding to E-selectin and have
designed a dipeptide backbone, Ser-Glu, as a rational
mimetic of compound 2. To provide support for the
structural hypothesis that led to the design of the
rational mimetics based on molecular modeling, we
F igu r e 1. Binding sites and IC50 of sLex and compound 2
toward E-selectin.
synthesized the type II and type II′ â-turn dipeptides
(3a ,b) (Chart 1) and evaluated their biological profiles
in vitro and in vivo.
In this paper, we describe the derivation strategy for
an active pharmacophore and a conceptual design of
sLex mimetics using MD simulation. We also describe
the synthesis of sLex mimetics and the evaluations in
vitro and in vivo.
X Abstract published in Advance ACS Abstracts, October 1, 1997.
S0022-2623(97)00262-8 CCC: $14.00 © 1997 American Chemical Society

Studies on Selectin Blockers
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3535
Ch a r t 1
Resu lts a n d Discu ssion
In itia l Mod el Con str u ction a n d Molecu la r Dy-
n a m ics Sim u la tion of th e In ter a ction Betw een
Com p ou n d s 3a -d a n d E-Selectin . The initial at-
tempt at docking a peptide backbone containing the
three essential functional groups, the fucose, the car-
boxylic acid, and the hydrophobic branched alkyl chain,
on E-selectin produced almost the same interaction
modes as those observed in our previously reported
complex model of compound 2/E-selectin.4 Next, we
explored the three essential binding sites to E-selectin
by conformational analyses using a molecular dynamics
(MD) technique.7 The crystal structure8 of the lectin
domain of E-selectin was obtained from the Protein Data
Bank (identity code: 1ESL). We undertook the model
building of the complex of compounds 3a -d with
E-selectin.
First, we constructed the initial bound model of 3a
to conserve the interactions of the three functional
groups with E-selectin. The 2- and 3-OH groups of the
fucose were coordinated to the calcium, and the car-
boxylate of the D-Glu was directed to Arg97. Both alkyl
chains (chain A and chain B) were also directed to each
hydrophobic region on the surface of E-selectin; namely,
chain A interacted with one hydrophobic region consist-
ing of Lys111, Lys112, Lys113, Ala9, and Leu114, and
chain B interacted with another hydrophobic region
consisting of Tyr44, Pro46, and Tyr48.
Next, we subjected the initial bound model of 3a to a
200-ps MD simulation with the explicit water solvent
and evaluated the dynamic behavior of the model in
solution. Figure 2 shows the analytical data of the
interactions between the Glu in compound 3a and the
basic residue on E-selectin, as well as the conforma-
tional change of the peptide portion in compound 3a .
As illustrated in Figure 2, the initial interaction be-
tween the carboxylate of 3a and Arg97 in E-selectin was
broken immediately following the MD calculation. In-
stead, the interaction of 3a with Lys111 was observed
at about 130 ps. The hidden surface area of A chain
retained the initial value and fluctuated minimally. On
the other hand, although the hidden surface area of
chain B fluctuated greatly, the hydrophobic interaction
of chain B with proteins would be retained, because the
hidden surface area of chain B retained more than one-
half of the initial value (Figure 3). Surprisingly,
although 3a did not assume the â-turn conformation in
the initial bound model, it was found that the final form
of 3a bound to E-selectin was characterized by the type
II â-turn formation.
F igu r e 2. Evaluation of dynamic behavior of the model in
solution. Bold lines indicate the existence of the â-turn
formation and the ionic interaction between compounds 3a -d
and E-selectin.
characterized by the type II′ â-turn formation. As
shown in Figure 2, the ionic interaction between L-Glu-
Lys 111 and the type II′ â-turn conformation were
conserved during the simulation. The hydrophobic
interaction between this chain and the protein still
existed and did not completely disappear (Figure 3). The
snapshots of the trajectory show the stability of the
interactions involved in the branched alkyl chain (Fig-
ure 4).
We also constructed the initial bound models of 3c,d
and subjected their initial bound models to a 200-ps MD
simulation. However, stable bound forms with E-
selectin were not observed.
Dr u g Design Ba sed on Molecu la r Mod elin g.
Using the MD simulation, we screened some conformers
involving a tight binding form with E-selectin at the
three essential binding sites. The MD calculation was
carried out to investigate the binding mode of 3a -d
with E-selectin, including the explicit solvent water. As
a result, we found that the dipeptides 3a ,b could bind
to E-selectin (Figure 4); namely, the important three
binding sites of 3a ,b, e.g., the fucose, the branched alkyl
chain, and the negatively charged group, tightly inter-
acted with E-selectin. Especially, the negatively charged
groups of 3a ,b, the carboxylic acids of D-Glu and/or
L-Glu, directly formed an ion pair with Lys111 on
E-selectin. This interaction was different from that of
compound 2.5 As illustrated in Figure 4, the most
characteristic structures of compounds 3a ,b involve
â-turn formations: the type II â-turn for 3a and the type
II′ â-turn for 3b. Although the â-turn formation is often
observed at a tetrapeptide unit, as an intramolecular
hydrogen bond between the ith amide oxygen and the
i+3th amide proton, it is also possible that dipeptides
modified at the N- and C-termini with amide bonds
could form a â-turn involving an intramolecular hydro-
gen bond.9 Figure 5 shows the torsion angle values of
3a ,b and the type II and type II′ â-turn tetrapeptides.
Both torsion angle values of 3a ,b were similar to those
of the type II and type II′ â-turn tetrapeptides, respec-
tively. These â-turn structures would play important
roles in conserving the positions of the three essential
functional groups, e.g., the fucose, the branched alkyl
chain, and the negatively charged group, for binding to
The initial bound model of 3b was constructed in a
similar manner to 3a ; however, the carboxylate of L-Glu
was directed to Lys111 like the bound form of 3a . In
the case of 3b, the initial bound conformation was

3536 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Tsukida et al.
F igu r e 3. Change of the ratio of hidden surface area of alkyl chain portions in 3a -d during the simulation in solution: ratio of
the alkyl chain buried in the cavity consisting of Lys111, Lys112, Lys113, Ala9, and Leu114 (A chain) and ratio of the alkyl chain
on the surface of Tyr44, Pro46, and Tyr48 (B chain). Values relative to those in the initial model are shown.
F igu r e 4. Stereorepresentation of the models of E-selectin and 3a or 3b complex models in solution. These models are the
equilibrium structures during MD calculation (200 ps). The structures of 3a ,b are indicated by thick lines. Thin lines indicate the
structure of E-selectin. Only the residues in the ligand-binding site on the protein are shown. Dashed lines indicate interactions
(hydrogen bond, electrostatic interaction, calcium coordination).
E-selectin. On the other hand, the other stereoisomers
(3c,d ) did not bind to E-selectin.
Ch em istr y. For the syntheses of the intermediates
7a -d for dipeptides 3a -d (Scheme 1), enantiomeric Glu
units, an L-Glu unit (L-4) and a D-Glu unit (D-4), which
are commercially available, were treated with 40%
D-5) in quantitative yields. The deprotection of the Boc
groups of the enantiomers (L-5, D-5) with 4 N HCl in
1,4-dioxane at room temperature followed by the cou-
pling with enantiomeric Ser units (L-6, D-6) in the
presence of WSC, 1-ethyl-3-[3-(dimethylamino)propyl]-
carbodiimide hydrochloride, and HOBt, 1-hydroxy-1H-
benzotriazole monohydrate, gave the Ser-Glu dipeptides
7a -d in 80-88% yields.
methylamine/methanol using
a
mixed-anhydride
method10 to afford the corresponding enantiomers (L-5,

Studies on Selectin Blockers
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3537
F igu r e 5. (A) Torsion angles (deg) of two types of â-turns (type II and type II′) with the tetrapeptide. Each residue is denoted
as i, i+1, i+2, i+3. The types of â-turns are defined by the main chain torsion angles of the i+1th and i+2th residues. (B) Torsion
angles (deg) of 3a ,b Ser-Glu main chain of the complex models with E-selectin. 3a structure is classified as type II â-turn; 3b is
type II′.
Sch em e 1a
Sch em e 2a
a
Conditions: (a) 40% CH3NH2/MeOH, Cl-COOEt, Et3N, 86-
90%; (b) 4 N HCl/1,4-dioxane; (c) L-6, D-6, WSC, HOBt, 80-88%
from L-5, D-5.
For the syntheses of compounds 3a -d , the glycosyl-
ation of 7a -d with 2,3,4-tri-O-benzylfucose (8),11 in the
presence of SnCl2/AgOTf as promoters and TMU, tetra-
methylurea, as a weak base, yielded mainly the R-gly-
cosides 9a -d . The deprotection of the Boc groups of
9a -d with TFA in chloroform followed by the coupling
with 1-tetradecylhexadecanoic acid (10) in the presence
of WSC and HOBt for the amide linkage afforded
compounds 11a -d in 65-72% yields. Finally, com-
pounds 11a -d were transformed, by removal of the
protecting benzyl group under general catalytic reduc-
tion conditions, into the desired compounds 3a -d , as
shown in Scheme 2.
a
Conditions: (a) SnCl2, AgOTf, TMU, 4A MS/CHCl3, 60-66%;
(b) TFA/CHCl3; (c) WSC, HOBt, 65-72% from 9a -d ; (d) Pd(OH)2/
MeOH, 72-97%.
Ta ble 1. Blocking Activity of Compounds 2, 3a -d , and sLex
(1)
Biologica l Activities: In h ibition Assa y of E-, P -,
a n d L-Selectin -sLex Bin d in g. The method using
selectin-IgG chimeras reported by Foxall et al. was
followed.12 As shown in Table 1, both dipeptides 3a ,b
were more potent (IC50, 13 µM for 3a , 5.5 µM for 3b)
than compound 2 (IC50, 280 µM) in the ligand-E-
selectin competitive binding assay. In contrast, the
other stereoisomers (3c,d ) showed very weak blocking
activities (IC50, >1000 µM). It was interesting to note
that the biological activities of compounds 3a -d cor-
responded well with the predictions from the molecular
modeling studies, and the remarkable change of the
activities was due to the stereochemistry of the Ser-Glu
dipeptide backbones; namely, the heterochiral amino
acids, 3a (L-Ser-D-Glu) and 3b (D-Ser-L-Glu), were type
IC50, µM
compd
*1
*2
E-selectin
P-selectin
L-selectin
3a
3b
3c
3d
sLex
2
L
D
L
D
D
L
L
D
13
5.5
>1000
>1000
600
0.5
0.7
1.9
6.0
9.4
39
2.1
18
>1000
>1000
280
100
30
II and type II′ â-turn dipeptides, respectively, and the
â-turn formations of compounds 3a ,b could play impor-
tant roles in favorable binding to E-selectin. In addi-

3538 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Tsukida et al.
In conclusion, we succeeded in creating rational
mimetics of a sugar epitope by a simple peptide back-
bone, based on molecular modeling. The sLex mimetics
developed involve the dipeptide backbones, the L-Ser-
D-Glu, and the D-Ser-L-Glu, which are characterized by
type II and type II′ â-turns. Both â-turn dipeptides 3a ,b
showed 50-100-fold more potent inhibitory activity than
sLex and a 3′-sulfated Lex derivative (2) in vitro. In
addition, compound 3b also showed significant in vivo
activity in an IgE-mediated mouse skin reaction model.
These findings indicate that compound 3b could be an
effective antiinflammatory candidate.
Exp er im en ta l Section
Sim u la tion of th e Com p lex in Solu tion Usin g Molec-
u la r Dyn a m ics. The models of complexes were solvated by
the addition of TIP3P water molecules15 which were added
within 25 Å from the center of each compound 3a -d . The
water molecules were then minimized until the root mean
square of the gradients was below 0.05 kcal/(mol‚Å). Following
the minimization, the water molecules were equilibrated by a
5-ps MD calculation at a temperature of 298 K. An integral
time step ∆t ) 2 fs was used. After that, all of the water
molecules, compounds 3a -d , and the binding sites on E-
selectin (Ser6-Met10, Ser43-Tyr94, Trp76-Arg84, Val91-
Val101, Trp104-Ala115) were minimized until the root mean
square of gradients was below 0.05 kcal/(mol‚Å). Part of the
system was then subjected to a 200-ps MD simulation at a
temperature of 298 K. An integral time step ∆t ) 2 fs was
used. The coordinates of the system were recorded at every
0.2 ps during the calculation, for the following analyses of the
trajectory. In the calculations with the explicit water a
dielectric constant, ꢀ ) 1, was used. During MD calculations,
F igu r e 6. Effects of compound 3b and dexamethasone (Dex)
on leukocyte infiltration into skin tissue induced by antigen
in actively sensitized mice. Compound 3b was given intrave-
nously immediately before the antigen challenge. Dexametha-
sone was given orally 2 h before the antigen challenge. Each
column represents the mean of 5 or 6 animals. Vertical bars
indicate SE; **p < 0.01, significantly different from control
group; -, nonsensitized mice.
tion, the inhibitory activities of the other stereoisomers,
3c (L-Ser-L-Glu) and 3d (D-Ser-D-Glu), supported the
importance of the type II and type II′ â-turn formations
for the binding to E-selectin, because homochiral amino
acids, such as L-Ser-L-Glu and/or D-Ser-D-Glu, generally
do not assume type II and type II′ â-turn formations,
owing to the unfavorable steric interaction between the
carbonyl oxygen of the i+1th central amide group and
the side chain of the i+2th residue.13
On the other hand, all of the dipeptides 3a -d (IC50s,
0.5-2.1 µM) were more potent blockers toward P-
selectin than compound 2 (IC50, 100 µM); especially,
dipeptides 3a,b showed 140-200-fold more potent block-
ing activity (IC50, 0.5 µM for 3a , 0.7 µM for 3b) than
compound 2. Furthermore, the blocking activities of
compounds 3c,d toward L-selectin were similar to that
of compound 2 (IC50, 36 µM); however, compounds 3a ,b
still showed 4-6-fold more potent blocking activity (IC50,
6.0 µM for 3a , 9.4 µM for 3b) than compound 2. Toward
the P- and L-selectins, all of the dipeptides 3a -d
showed potent blocking activities, in spite of their
different stereochemical features. These findings indi-
cate that the ligand-binding site to E-selectin would be
different from those to the P- and L-selectins.
In Vivo Activities: Effects on IgE-Med ia ted Sk in
Rea ction in Mou se Ea r s. Next, we estimated the in
vivo activity of compound 3b, which was the most potent
in vitro. The in vivo efficacy of compound 3b was
assessed according to a previous paper.3 Compound 3b
was administered intravenously at 5 h after an oval-
bumin (OA) challenge. Measurements of neutrophil
infiltration in mouse ears were made at 24 h, as
assessed by myeloperoxidase (MPO). As indicated in
Figure 6, the IgE-mediated skin reactions in the mouse
ears were significantly inhibited by dexamethasone at
3 mg/kg, po. In addition, compound 3b (10 mg/kg, iv)
also showed a reduced MPO content in the mouse ears.
The IgE-mediated skin reactions have been studied in
an experiment using mouse E-, P-, and L-selectin mAb,
and the requisite role of selectins in this model has been
confirmed.14 Therefore, the in vivo efficacy of compound
3b in the IgE-mediated skin reaction seems to be due
to the selectin blocking activities.
all of the bond lengths were fixed by SHAKE algorithm.
A
nonbonded cutoff of 9 Å was used in all of the above calcula-
tions. The atoms coordinated to the calcium ion were fixed
by harmonic position constraints with a force constant of 50
kcal/(mol‚Å). All calculations were performed using AMBER
4.0.1 with the force field published by Cornell et al.16 The
force-field parameters for the carbohydrate protion of 3a -d
were derived from GLYCAM-93, a general force field for
carbohydrates that is suitable to AMBER.
An a lyses of th e Tr a jector y. Various analyses of the
trajectory from MD calculations in solution were performed
with several in-house programs. For the interactions observed
in the initial model complex, we calculated the distances of
each interaction from the sets of coordinates in the trajectory.
For the analysis of the behavior of the branched alkyl chains,
we calculated the “ratio of hidden surface area” as a measure
of hydrophobic interactions with the protein. This indicator
was calculated in the following way: for each set of coordinates
in the trajectory, we calculated the solvent accessible surface
area of the alkyl chain portions. The area calculations were
performed in two ways: one was the area of free alkyl chains
(Afree), which was calculated after the deletion of the protein
coordinates, and the other was the area of bound alkyl chains
(Acomplex), calculated in the presence of the protein. Then, the
ratio of hidden surface areas was calculated by the following
equation:
ratio of hidden surface area (%) )
(Afree - Acomplex)/Afree × 100
As indicated by the above equation, the ratio of hidden
surface area is the ratio of the solvent accessible surface area
of the alkyl chains that was buried in the protein. A larger
percentage of hidden surface area reflects wider alkyl chain
contacts with the protein surface. The calculation of solvent
accessible surface area was done by the methods of Richmond17
and Wesson and Eisenberg.18 Hydrogen atoms were not
included in the calculation.
In h ibition Assa y of E-, P -, a n d L-Selectin -sLex Bin d -
in g. The construction of the selectin-immunoglobulin was

Studies on Selectin Blockers
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3539
carried out according to the previous paper. A solution of sLex-
pentaceramide, in a 1:1 mixture of methanol and distilled
water, was pipetted into microtiter plate wells (96 wells, Falcon
PRO-BIND) at 100 pmol/50 µL/well and was adsorbed by
evaporating the solvent. The wells were washed twice with
distilled water, blocked with 5% BSA (bovine serum albumin)-
PBS (phosphate-buffered saline) for 1 h at room temperature,
and washed three times with PBS.
Separately, a 1:1 volumetric mixture of a 1:500 dilution in
1% BSA-PBS of biotin-anti-human IgG (Fc) (BioSource In-
ternational Inc., lot 1201)/streptavidin-alkaline phosphatase
(Zymed Lab Inc., lot 50424702) and a E-selectin-immuno-
globulin fusion protein (E-selectin-Ig) was incubated at room
temperature for 30 min to form a complex. The test com-
pounds were dissolved in distilled water at 10 mM and finally
diluted to final concentrations at 100, 25, 6.25, and 1.56 µM,
respectively. Reactant solutions were prepared by incubating
30 µL of this solution at each concentration with 30 µL of the
above complex solution for 30 min at room temperature. This
reactant solution was then added to the above microtiter wells
at 50 µL/well and incubated at 37 °C for 45 min. The wells
were washed three times with PBS and distilled water,
respectively, followed by addition of p-nitrophenylphosphate
(1 mg/mL) and 0.01% MgCl2 in 1 M diethanolamine (pH 9.8)
at 50 mL/well. The reactant mixture was developed for 120
min at 23 °C, and absorbance at 405 nm was measured.
Percent binding was calculated by the following equation:
N-(ter t-Bu t oxyca r b on yl)-D-glu t a m ic a cid 1-m et h yl-
a m id e 5-ben zyl ester (D-5): yield 86.7%; [R]D +6° (c ) 0.1,
1
CHCl3); mp 120-123 °C; H NMR (DMSO-d6) δ 1.37 (s, 9H),
1.65-1.82 (m, 1H), 1.83-2.00 (m, 1H), 2.36 (t, 2H, J ) 7.7
Hz), 2.57 (d, 3H, J ) 4.5 Hz), 3.82-3.98 (m, 1H), 5.08 (s, 2H),
6.90 (d, 1H, J ) 8.2 Hz), 7.25-7.45 (m, 5H), 7.76 (d, 1H, J )
4.5 Hz). Anal. (C18H26N2O5) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Dip ep tid es
7a -d : N-(ter t-Bu toxyca r bon yl)-D-ser yl-L-glu ta m ic Acid
1-Meth yla m id e 5-Ben zyl Ester (7b). To the L-5 (10.0 g, 28.5
mM) was added 4 N HCl/1,4-dioxane (30 mL), and the solution
was stirred for 0.5 h at room temperature. The mixture was
concentrated, and the residue was dissolved in DMF (200 mL),
added to Et3N (5.4 mL), and then cooled to 0 °C. WSC (6.6 g,
34.2 mM) and HOBt (5.2 g, 34.2 mM) were added to the
mixture, and this was stirred for 18 h at room temperature.
The solvent was removed under reduced pressure, and the oily
residue was dissolved in AcOEt (300 mL), washed with 0.1 N
HCl, saturated sodium hydrogen carbonate, and brine, dried
(MgSO4), and concentrated. The precipitate was filtered, and
recrystallization from AcOEt-n-hexane gave 7b (10.3 g,
82.5%) as a white crystal: [R]D -5° (c ) 0.1, CHCl3); mp 127-
128 °C; 1H NMR (DMSO-d6) δ 1.38 (s, 9H), 1.65-1.85 (m, 1H),
1.90-2.15 (m, 1H), 2.30-2.40 (m, 2H), 2.58 (d, 3H, J ) 4.6
Hz), 3.54 (t, 2H, J ) 5.6 Hz), 3.85-4.00 (m, 1H), 4.13-4.29
(m, 1H), 4.88 (t, 1H, J ) 5.6 Hz), 5.07 (s, 2H), 6.82 (d, 1H, J
) 6.9 Hz), 7.30-7.45 (m, 5H), 7.77 (d, 1H, J ) 4.6 Hz), 8.03
(d, 1H, J ) 8.3 Hz). Anal. (C21H31N3O7) C, H, N.
% binding ) (X - C)/(A - C) × 100
N-(ter t-Bu toxycar bon yl)-L-ser yl-D-glu tam ic acid 1-m eth -
yla m id e 5-ben zyl ester (7a ): yield 80.6%; [R]D +5° (c ) 0.1,
wherein X is the absorbance of wells containing the test
compounds at each concentration, C is the absorbance of wells
not containing the selectin-Ig and test compounds, and A is
the absorbance of control wells not containing the test com-
pounds. Inhibition of P- or L-selectin-sLex binding was
repeated except the P-selectin-Ig or L-selectin-Ig was replaced
for E-selectin-Ig. The results of inhibitory activities are
presented in Table 1 as IC50 values. The number of replicates
is two.
1
CHCl3); mp 124-127 °C; H NMR (DMSO-d6) δ 1.38 (s, 9H),
1.66-1.88 (m, 1H), 1.92-2.12 (m, 1H), 2.30-2.42 (m, 2H), 2.57
(d, 3H, J ) 4.6 Hz), 3.54 (t, 2H, J ) 5.6 Hz), 3.86-4.00 (m,
1H), 4.13-4.30 (m, 1H), 4.88 (t, 1H, J ) 5.7 Hz), 5.07 (s, 2H),
6.82 (d, 1H, J ) 6.9 Hz), 7.30-7.45 (m, 5H), 7.77 (d, 1H, J )
4.5 Hz), 8.03 (d, 1H, J ) 8.1 Hz). Anal. (C21H31N3O7) C, H,
N.
N-(ter t-Bu toxycar bon yl)-L-ser yl-L-glu tam ic acid 1-m eth -
yla m id e 5-ben zyl ester (7c): yield 85.2%; [R]D -22° (c ) 0.1,
P r otective Effects on IgE-Med ia ted Sk in Rea ction in
Mice Ea r s. Eight week-old female BALB/c mice were sensi-
tized by intraperitoneal injection with 3 µg of ovalbumin (OA)
and 4 mg of alum. Reactions to OA were elicited by intra-
cutaneous injection of 10 µg of OA to each ear of mice 2 weeks
after the sensitization. After 24 h, the mice were killed, and
6-mm diameter biopsies of the ears were taken and weighed
as an index of tissue swelling. As an index of neutrophil
infiltration into tissue, myeloperoxidase activity in biopsy
homogenates (in 50 mM potassium phosphate, pH 6.0, with
0.5% hexadecyltrimethylammonium bromide) was measured
as the degradation of H2O2 in the presence of o-dianisidine.
Compound 3b dissolved in 0.1 N NaOH-water solution was
given intravenously immediately before the antigen challenge.
Dexamethasone suspended in 0.5% carboxymethyl cellulose
was given orally 2 h before the OA challenge. Results are
expressed as mean ( SE. A one-way analysis of variance with
Dunnett’s test was used to determine statistical significance.19
Gen er a l P r oced u r e for th e P r ep a r a tion s of En a n tio-
m er ic Glu Un its (L-5, D-5): N-(ter t-Bu toxyca r bon yl)-L-
glu ta m ic Acid 1-Meth yla m id e 5-Ben zyl Ester (L-5). To a
solution of N-(tert-butoxycarbonyl)-L-glutamic acid 5-benzyl
ester (L-4, 10.0 g, 29.6 mM) in THF (100 mL) was added Et3N
(3.9 g, 38.5 mM), and then the mixture cooled to 0 °C. Ethyl
chlorocarbonate (4.2 g, 38.7 mM) was added to the mixture,
this was stirred for 2 min, and then 40% methylamine/
methanol (6.9 g, 74.0 mM) was added to the mixture. After
the mixture stirred for 2 h gradually returning to room
temperature, AcOEt (200 mL) was added to the mixture, and
the mixture was washed with 1 N HCl, saturated sodium
hydrogen carbonate, and brine, dried (MgSO4), and concen-
trated. The precipitate was filtered and washed with Et2O to
give L-5 (9.3 g, 90.3%) as a white crystal: [R]D -6° (c ) 0.1,
1
CHCl3); mp 110-113 °C; H NMR (DMSO-d6) δ 1.37 (s, 9H),
1.68-1.86 (m, 1H), 1.90-2.11 (m, 1H), 2.30-2.45 (m, 2H), 2.56
(d, 3H, J ) 4.6 Hz), 3.55 (t, 2H, J ) 5.4 Hz), 3.90-4.15 (m,
1H), 4.20-4.35 (m, 1H), 4.98 (t, 1H, J ) 5.4 Hz), 5.10 (s, 2H),
6.79 (d, 1H, J ) 7.5 Hz), 7.25-7.45 (m, 5H), 7.78 (d, 1H, J )
4.6 Hz), 7.96 (d, 1H, J ) 8.5 Hz). Anal. (C21H31N3O7) C, H,
N.
N-(ter t-Bu toxycar bon yl)-D-ser yl-D-glu tam ic acid 1-m eth -
yla m id e 5-ben zyl ester (7d ): yield 82.9%; [R]D +18° (c ) 0.1,
1
CHCl3); mp 110-112 °C; H NMR (DMSO-d6) δ 1.37 (s, 9H),
1.70-1.85 (m, 1H), 1.90-2.10 (m, 1H), 2.30-2.45 (m, 2H), 2.56
(d, 3H, J ) 4.5 Hz), 3.55 (t, 2H, J ) 5.6 Hz), 3.90-4.05 (m,
1H), 4.15-4.30 (m, 1H), 4.99 (t, 1H, J ) 5.5 Hz), 5.07 (s, 2H),
6.80 (d, 1H, J ) 7.5 Hz), 7.25-7.45 (m, 5H), 7.78 (d, 1H, J )
4.6 Hz), 7.97 (d, 1H, J ) 8.1 Hz). Anal. (C21H31N3O7) C, H,
N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 9a -d by
Glycosyla tion of 7a -d w ith 2,3,4-Tr i-O-ben zylfu cose
Don or 8: N-(ter t-Bu toxyca r bon yl)-O-(2,3,4-tr i-O-ben zyl-
r-L-fu cop yr a n osyl)-D-ser yl-L-glu t a m ic Acid 1-Met h yl-
a m id e 5-Ben zyl Ester (9b). A mixture of 4A molecular
sieves (2.0 g), AgOTf (0.59 g, 2.28 mM), and SnCl2 (0.43 g, 2.28
mM) in CHCl3 (15 mL) was stirred for 5 h under nitrogen
atmosphere at room temperature and cooled to 40 - 50 °C.
TMU (0.66 g, 5.70 mM), 2,3,4-tri-O-benzyl-L-fucosyl fluoride
(8) (1.00 g, 2.28 mM) in CHCl3 (1 mL), and compound 7b (0.50
g, 1.14 mM) in CHCl3 (3 mL) were added to the mixture
successively, and the mixture stirred for 1 h at the same
temperature and then for 20 h gradually returning to room
temperature. The precipitate was filtered off, and the filtrate
was concentrated. The residue was purified by thin-layer
chromatography developing with 1:1 AcOEt/cyclohexane to
give 9b (0.60 g, 62.0%) as a white crystal: [R]D -59° (c ) 0.1,
CHCl3); mp 77-81 °C; 1H NMR (CDCl3) δ 1.13 (d, 3H, J ) 6.5
Hz), 1.43 (s, 9H), 1.7-2.1 (m, 1H), 2.1-2.3 (m, 1H), 2.3-2.6
(m, 2H), 2.67 (d, 3H, J ) 4.8 Hz), 3.5-3.7 (m, 2H), 3.8-4.0
(m, 3H), 4.0-4.25 (m, 1H), 4.25-4.45 (m, 1H), 4.5-5.0 (m, 7H),
1
CHCl3); mp 124-125 °C; H NMR (DMSO-d6) δ 1.37 (s, 9H),
1.66-1.82 (m, 1H), 1.83-2.00 (m, 1H), 2.35 (t, 2H, J ) 7.6
Hz), 2.57 (d, 3H, J ) 4.5 Hz), 3.83-3.95 (m, 1H), 5.08 (s, 2H),
6.90 (d, 1H, J ) 8.2 Hz), 7.25-7.45 (m, 5H), 7.76 (d, 1H, J )
4.6 Hz). Anal. (C18H26N2O5) C, H, N.

3540 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Tsukida et al.
5.0-5.2 (m, 2H), 5.5 (bs, 1H), 6.07 (bs, 1H), 7.1-7.45 (m, 20H).
Anal. (C48H59N3O11) C, H, N.
5.06 (s, 2H), 6.2-6.3 (m, 1H), 6.82 (d, 1H, J ) 6.7 Hz), 7.06 (d,
1H, J ) 8 Hz), 7.15-7.4 (m, 20H). Anal. (C73H109N3O10) C, H,
N.
N-(ter t-Bu t oxyca r b on yl)-O-(2,3,4-t r i-O-b en zyl-r-L-fu -
cop yr a n osyl)-L-ser yl-D-glu t a m ic a cid 1-m et h yla m id e
5-ben zyl ester (9a ): yield 66.0% as a syrup; [R]D -43° (c )
0.1, CHCl3); 1H NMR (CDCl3) δ 1.11 (d, 3H, J ) 6 Hz), 1.45 (s,
9H), 1.8-2.0 (m, 1H), 2.0-2.25 (m, 1H), 2.3-2.6 (m, 2H), 2.71
(d, 3H, J ) 5 Hz), 3.47 (dd, 1H, J ) 5, 10 Hz), 3.65 (d, 1H, J
) 2 Hz), 3.81 (q, 1H, J ) 7 Hz), 3.92 (dd, 1H, J ) 3, 10 Hz),
4.03 (dd, 1H, J ) 4, 10 Hz), 7.0-7.4 (m, 20H). Anal.
(C48H59N3O11) C, H, N.
N-(2-Tetr a d ecylh exa d eca n oyl)-O-(2,3,4-tr i-O-ben zyl-r-
L-fu cop yr a n osyl)-D-ser yl-D-glu ta m ic a cid 1-m eth yla m id e
5-ben zyl ester (11d ): yield 54.5%; [R]D -19° (c ) 0.1, CHCl3);
mp 130-132 °C; 1H NMR (CDCl3) δ 0.8-0.95 (m, 6H), 1.13
(d, 3H, J ) 6.5 Hz), 1.1-1.7 (m, 53H), 1.9-2.4 (m, 4H), 2.69
(d, 3H, J ) 4.8 Hz), 3.56 (dd, 1H, J ) 9.1, 10.8 Hz), 3.69 (d,
1H, J ) 1.8 Hz), 3.75-4.0 (m, 3H), 4.1 (dd, 1H, J ) 3.5, 6.5
Hz), 4.2-4.35 (m, 1H), 4.45-4.55 (m, 1H), 4.63 (d, 1H, J )
11.6 Hz), 4.68-4.9 (m, 4H), 4.95 (d, 1H, J ) 11.6 Hz), 5.03 (d,
1H, J ) 3.7 Hz), 5.07 (s, 2H), 6.34 (q, 1H, J ) 4.8 Hz), 6.43 (d,
1H, J ) 6.1 Hz), 7.1-7.5 (m, 20H). Anal. (C73H109N3O10) C,
H, N.
N-(ter t-Bu t oxyca r b on yl)-O-(2,3,4-t r i-O-b en zyl-r-L-fu -
cop yr a n osyl)-L-ser yl-L-glu t a m ic a cid 1-m et h yla m id e
5-ben zyl ester (9c): yield 64.2%; [R]D -53° (c ) 0.17, CHCl3);
1
mp 141-142 °C; H NMR (CDCl3) δ 1.09 (d, 3H, J ) 6.5 Hz),
1.45 (s, 9H), 1.65-1.9 (m, 1H), 2.05-2.55 (m, 3H), 2.74 (d, 3H,
J ) 4.8 Hz), 3.4-3.5 (m, 1H), 3.63 (d, 1H, J ) 1.6 Hz), 3.75-
3.9 (m, 2H), 4.02 (dd, 1H, J ) 3.6, 10.1 Hz), 4.12-4.28 (m,
2H), 4.35-4.5 (m, 1H), 4.5-4.95 (m, 7H), 5.06 (s, 2H), 6.04
(bs, 1H), 6.33 (bs, 1H), 7.05 (d, 1H, J ) 8.3 Hz), 7.2-7.4 (m,
20H). Anal. (C48H59N3O11‚0.5H2O) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 3a -d : N-(2-
Tetr a d ecylh exa d eca n oyl)-O-(r-L-fu cop yr a n osyl)-D-ser yl-
L-glu ta m ic Acid 1-Meth yla m id e (3b). To a solution of 11b
(80 mg, 0.067 mM) in ethanol (50 mL) was added 20%
Pd(OH)2/C (80 mg), and the mixture was stirred for 4 h under
3-4 atmospheric pressure of hydrogen. The precipitate was
filtered off, and the filtrate was concentrated in vacuo. The
residue was crystallyzed with water to give 3b (46 mg, 82.5%)
as a white crystal: [R]D -62° (c ) 0.1, MeOH); mp 179-183
N-(ter t-Bu t oxyca r b on yl)-O-(2,3,4-t r i-O-b en zyl-r-L-fu -
cop yr a n osyl)-D-ser yl-D-glu t a m ic a cid 1-m et h yla m id e
5-ben zyl ester (9d ): yield 61.8%; [R]D -42° (c ) 0.1, CHCl3);
1
1
mp 109-116 °C; H NMR (CDCl3) δ 1.13 (d, 3H, J ) 6.5 Hz),
°C; H NMR (DMSO-d6) δ 0.75-0.9 (m, 6H), 1.06 (d, 3H, J )
1.44 (s, 9H), 1.6-1.75 (m, 1H), 2.0-2.15 (m, 1H), 2.15-2.4 (m,
2H), 2.71 (d, 3H, J ) 4.8 Hz), 3.55-3.7 (m, 2H), 3.75-4.0 (m,
3H), 4.07 (dd, 1H, J ) 3.7, 10.2 Hz), 4.1-4.25 (m, 1H), 4.25-
4.4 (m, 1H), 4.6-5.0 (m, 7H), 5.07 (s, 2H), 5.52 (bs, 1H), 6.4
(bs, 1H), 7.1-7.5 (m, 20H). Anal. (C48H59N3O11) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 11a -d :
N-(2-Tetr a d ecylh exa d eca n oyl)-O-(2,3,4-tr i-O-ben zyl-r-L-
fu cop yr a n osyl)-D-ser yl-L-glu ta m ic Acid 1-Meth yla m id e
5-Ben zyl Ester (11b). To a solution of 9b (0.5 g, 0.59 mM)
in CH2Cl2 (10 mL) was added TFA (10 mL) at 0 °C, and the
mixture was stirred for 2 h at room temperature and then
concentrated. The residue was dissolved in AcOEt (100 mL),
washed with saturated sodium carbonate, and dried (MgSO4),
and the solvent was removed in vacuo. The residue was
dissolved in DMF (50 mL), 2-tetradecylhexadecanoic acid (294
mg, 0.65 mM) was added to the solution, and the mixture was
dissolved by heating and then cooled to room temperature.
WSC (170 mg, 0.89 mM) and HOBt (136 mg, 0.89 mM) were
added to the solution. After the mixture stirred for 20 h,
AcOEt (100 mL) was added to the solution, and the mixture
was washed with 1 N HCl, saturated sodium hydrogen
carbonate, and brine successively, dried (MgSO4), and concen-
trated. The residue was purified by thin-layer chromatogra-
phy developing with 25:1 CHCl3/methanol to give 11b (456 mg,
65.0%) as a white crystal: [R]D -44° (c ) 0.1, CHCl3); mp 118-
120 °C; 1H NMR (CDCl3) δ 0.8-0.95 (m, 6H), 1.12 (d, 3H, J )
6.5 Hz), 1.15-1.7 (m, 52H), 1.75-1.92 (m, 1H), 1.92-2.1 (m,
1H), 2.1-2.3 (m, 2H), 2.3-2.6 (m, 2H), 2.66 (d, 3H, J ) 4.8
Hz), 3.45-3.65 (m, 2H), 3.78-3.95 (m, 3H), 4.08 (dd, 1H, J )
4.2, 10.2 Hz), 4.15-4.25 (m, 2H), 4.25-4.4 (m, 1H), 4.6-5.0
(m, 7H), 5.05 (d, 1H, J ) 11.9 Hz), 5.1 (d, 1H, J ) 12.3 Hz),
6.0-6.1 (m, 1H), 6.47 (d, 1H, J ) 6.7 Hz), 7.15-7.4 (m, 20H).
Anal. (C73H109N3O10) C, H, N.
6.4 Hz), 1.1-1.55 (m, 52H), 1.6-1.8 (m, 1H), 1.8-2.1 (m, 1H),
2.1-2.3 (m, 3H), 2.58 (d, 3H, J ) 4.5 Hz), 3.4-3.6 (m, 3H),
3.6-3.85 (m, 2H), 4.1-4.3 (m, 1H), 4.34 (d, 1H, J ) 4.5 Hz),
4.4-4.55 (m, 1H), 7.45 (d, 1H, J ) 4.4 Hz), 7.97 (d, 1H, J )
7.0 Hz), 8.05 (d, 1H, J ) 8.0 Hz). Anal. (C45H85N3O10) C, H,
N.
N-(2-Tetr a d ecylh exa d eca n oyl)-O-(r-L-fu cop yr a n osyl)-
L-ser yl-D-glu ta m ic a cid 1-m eth yla m id e (3a ): yield 85.0%;
[R]D -40° (c ) 0.1, MeOH); mp 183-187 °C; 1H NMR (DMSO-
d6) δ 0.75-0.95 (m, 6H), 1.05 (d, 3H, J ) 6.5 Hz), 1.1-1.55
(m, 52H), 1.55-1.75 (m, 1H), 1.75-2.0 (m, 1H), 2.05-2.3 (m,
3H), 2.57 (d, 3H, J ) 4.5 Hz), 3.3-3.55 (m, 4H), 3.67 (q, 1H, J
) 6.8 Hz), 3.85 (dd, 1H, J ) 4.3, 9.2 Hz), 4.1-4.35 (m, 3H),
4.45-4.55 (m, 2H), 7.68 (d, 1H, J ) 8 Hz), 7.86 (q, 1H, J ) 4.5
Hz), 8.13 (d, 1H, J ) 7.9 Hz), 11.9 (bs, 1H). Anal. (C45H85N3O10‚
1H2O) C, H, N.
N-(2-Tetr a d ecylh exa d eca n oyl)-O-(r-L-fu cop yr a n osyl)-
L-ser yl-L-glu ta m ic a cid 1-m eth yla m id e (3c): yield 72.8%;
[R]D -45° (c ) 0.08, MeOH); mp 171-173 °C; 1H NMR (DMSO-
d6) δ 0.75-0.95 (m, 6H), 1.06 (d, 3H, J ) 6.5 Hz), 1.1-1.6 (m,
52H), 1.6-2.0 (m, 2H), 2.1-2.3 (m, 3H), 2.58 (d, 3H, J ) 4.5
Hz), 3.4-3.6 (m, 3H), 3.74 (q, 1H, J ) 6.9 Hz), 3.86 (dd, 1H, J
) 5.7, 9.3 Hz), 4.15-4.4 (m, 3H), 4.45-4.6 (m, 2H), 7.63 (d,
1H, J ) 8.2 Hz), 7.82 (q, 1H, J ) 4.4 Hz), 8.09 (d, 1H, J ) 8.7
Hz), 11.9 (bs, 1H). Anal. (C45H85N3O10) C, H, N.
N-(2-Tetr a d ecylh exa d eca n oyl)-O-(r-L-fu cop yr a n osyl)-
D-ser yl-D-glu ta m ic a cid 1-m eth yla m id e (3d ): Yield 96.7%;
[R]D -32° (c ) 0.1, MeOH); mp 155-157 °C; 1H NMR (DMSO-
d6) δ 0.75-0.9 (m, 6H), 1.05 (d, 3H, J ) 6.5 Hz), 1.1-1.55 (m,
52H), 1.6-2.05 (m, 2H), 2.1-2.3 (m, 3H), 2.57 (d, 3H, J ) 4.5
Hz), 3.55-3.8 (m, 2H), 4.1-4.25 (m, 1H), 4.4-4.6 (m, 1H), 4.6-
4.7 (m, 1H), 7.66 (q, 1H, J ) 4.4 Hz), 7.83 (d, 1H, J ) 8.4 Hz),
7.92 (d, 1H, J ) 8.3 Hz). Anal. (C45H85N3O10‚1.5H2O) C, H,
N.
N-(2-Tetr a d ecylh exa d eca n oyl)-O-(2,3,4-tr i-O-ben zyl-r-
L-fu cop yr a n osyl)-L-ser yl-D-glu ta m ic a cid 1-m eth yla m id e
5-ben zyl ester (11a ): yield 61.1%; [R]D -29° (c ) 0.1, CHCl3);
Refer en ces
1
mp 119-120 °C; H NMR (CDCl3) δ 0.75-0.95 (m, 6H), 1.13
(1) (a) Berg, E. L.; Yoshino, T.; Rott, L. S.; Robinson, M. K.; Warnock,
R. A.; Kishimoto, T. K.; Picker, L. J .; Butcher, E. C. The
Cutaneous Lymphocyte Antigen is a Skin Lymphocyte Homing
Receptor for the Vascular Lectin Endothelial Cell-Leukocyte
Adhesion Molecule 1. J . Exp. Med. 1991, 174, 1461-1466. (b)
Picker, L. J .; Kishimoto, T. K.; Smith, C. W.; Warnock, R. A.;
Butcher, E. C. ELAM-1 is an Adhesion Molecule for Skin-
Homing T Cells. Nature 1991, 349, 796-799. (c) Lorant, D. E.;
Topham, M. K.; Whatley, R. E.; McEver, R. P.; Mclntyre, T. M.;
Prescott, S. M.; Zimmerman, G. A. Inflammatory Roles of
P-selectin. J . Clin. Invest. 1993, 92, 559-570. (d) Symon, F. A.;
Walsh, G. M.; Watson, S. R.; Wardlaw, A. J . Eosinophil Adhesion
to Nasal Polyp Endothelium is P-Selectin-Dependent. J . Exp.
Med. 1994, 80, 371-376. (e) Akbar, A. N.; Salmon, M.; J anossy,
G. The Synergy Between Naive and Memory T Cells During
Activation. Immunol. Today 1991, 12, 184-188. (f) Duijvestijn,
A. M.; Horst, E.; Pals, S. T.; Rouse, B. N.; Steere, A. C.; Picker,
(d, 3H, J ) 6 Hz), 1.1-1.7 (m, 52H), 1.75-2.0 (m, 2H), 2.0-
2.2 (m, 1H), 2.25-2.6 (m, 2H), 2.73 (d, 3H, J ) 5 Hz), 3.37
(dd, 1H, J ) 5, 9 Hz), 3.67 (s, 1H), 3.85 (q, 1H, J ) 6 Hz), 3.97
(dd, 1H, J ) 2, 10 Hz), 4.06 (dd, 1H, J ) 3, 10 Hz), 7.0-7.4
(m, 20H). Anal. (C73H109N3O10) C, H, N.
N-(2-Tetr a d ecylh exa d eca n oyl)-O-(2,3,4-tr i-O-ben zyl-r-
L-fu cop yr a n osyl)-L-ser yl-L-glu ta m ic a cid 1-m eth yla m id e
5-ben zyl ester (11c): yield 71.8%; [R]D -44° (c ) 0.24, CHCl3);
mp 133-135 °C; 1H NMR (CDCl3) δ 0.8-0.95 (m, 6H), 1.13
(d, 3H, J ) 6.5 Hz), 1.1-1.6 (m, 52H), 1.7-2.0 (m, 2H), 2.0-
2.25 (m, 1H), 2.25-2.55 (m, 2H), 2.71 (d, 3H, J ) 4.8 Hz), 3.37
(dd, 1H, J ) 5.9, 9.4 Hz), 3.67 (d, 1H, J ) 1.7 Hz), 3.85-3.95
(m, 2H), 4.06 (dd, 1H, J ) 3.6, 10.1 Hz), 4.21 (dd, 1H, J ) 2.8,
9.5 Hz), 4.3-4.45 (m, 1H), 4.5-4.6 (m, 1H), 4.55-4.95 (m, 7H),

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