Syntheses of Heterobifunctional Candidate Ligands of P-Selectin Containing Both Sulfated Lewis X Trisaccharide and Various Sulfated Peptides

Article abstract of DOI:10.1081/CAR-120026453

Various heterobifunctional glycopeptides containing both 3′-sulfated Lewis x and peptides containing at least one sulfated tyrosine were constructed onto a pentaerythritol backbone following a common strategy that relies on the condensation, through reduc

Full text of DOI:10.1081/CAR-120026453

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Syntheses of Heterobifunctional Candidate Ligands
of P‐Selectin Containing Both Sulfated Lewis X
Trisaccharide and Various Sulfated Peptides
Franck Dagron a & André Lubineau a
a Laboratoire de Chimie Organique Multifonctionnelle, UMR CNRS 8614 , Institut de Chimie
Moléculaire d'Orsay , Université Paris‐Sud , Bât. 420, 91405, Orsay Cedex, France
Published online: 23 Feb 2007.
To cite this article: Franck Dagron & André Lubineau (2003) Syntheses of Heterobifunctional Candidate Ligands of P‐Selectin
Containing Both Sulfated Lewis X Trisaccharide and Various Sulfated Peptides , Journal of Carbohydrate Chemistry, 22:3-4,
481-500, DOI: 10.1081/CAR-120026453
To link to this article: http://dx.doi.org/10.1081/CAR-120026453
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, Nos. 7 & 8, pp. 481–500, 2003
Syntheses of Heterobifunctional Candidate Ligands of
P-Selectin Containing Both Sulfated Lewis X
Trisaccharide and Various Sulfated Peptidesy
*
Franck Dagron and Andre´ Lubineau
Laboratoire de Chimie Organique Multifonctionnelle, Institut de Chimie
Mole´culaire d’Orsay, Universite´ Paris-Sud, Orsay Cedex, France
ABSTRACT
Various heterobifunctional glycopeptides containing both 3’-sulfated Lewis x and
peptides containing at least one sulfated tyrosine were constructed onto a pentaery-
thritol backbone following a common strategy that relies on the condensation, through
reductive amination, of aldehydes 9 or 17 with various peptides having a free terminal
amino group. The corresponding homodimer of 3’-sulfated Lewis x linked to pen-
taerythritol was prepared for comparison of the inhibitory activities.
Key Words: Selectins; Selectin ligands; Sulfated Lewis x; Sulfated tyrosine;
Heterobifunctional ligand.
yThis paper is dedicated to Professor Ge´rard Descotes on the occasion of his 70th birthday.
*
Correspondence: Andre´ Lubineau, Laboratoire de Chimie Organique Multifonctionnelle, UMR
CNRS 8614, Institut de Chimie Mole´culaire d’Orsay, Universite´ Paris-Sud, Baˆt. 420, 91405 Orsay
Cedex, France; Fax: +33-16915 4715; E-mail: lubin@icmo.u-psud.fr.
481
DOI: 10.1081/CAR-120026453
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

482
Dagron and Lubineau
INTRODUCTION
The selectins (E, P and L selectins) are mammalian C-type lectins which mediate
cellular adhesion between blood cells or cancer cells and vascular endothelium. They
participate in a variety of normal and pathological phenomena.[1,2] In particular, P-
selectin which is stored in platelet a-granules and in Weibel–Palade bodies of endo-
thelial cells is redistributed, within minutes, in the plasma membranes upon activation
of the vascular endothelium. Once there, it mediates the initial tethering and rolling of
leucocytes at inflammation sites.[3] The major counter-receptor of P-selectin, PSGL-1
(P-Selectin Glycoprotein Ligand-1), is a dimeric mucin present on leucocytes. It
interacts with P-selectin with high affinity through its N-terminal portion which con-
tains a cluster of three sulfated tyrosines (at positions 5, 7 and 10) and a sialyl Lewis x
(sLex) attached to threonine 16. Indeed, a synthetic glycosulfopeptide corresponding to
the N-terminal part of PSGL-1 was found to bind as efficiently as the dimeric mucin
structure.[4] However, whereas sLex was found essential for tight binding, sulfations of
tyrosine could be modulated. Indeed, only one of the three tyrosines needs to be
sulfated, even if the corresponding polysulfated peptides gave better interactions.[5]
Recently, this was confirmed by analysis of the crystal structure of a construct con-
taining the lectin and EGF domains in complex with the N-terminal domain of human
PSGL-1.[6] Several glycopeptides[7,8] have already been synthesized starting from a
glyco-aminoacid derived from sLex, and extending most of the time the peptide chain
through solid phase peptide synthesis.[9–12] In order to get more insights into the
interaction and to generate more diversity, we decided to develop a strategy compatible
with parallel syntheses, in which the oligosaccharide moiety could be linked, through
reductive amination, to any peptide having a free terminal amino group. Moreover, the
fact that a sulfate, 3-linked to the terminal galactose, can substitute sialic acid ad-
vantageously in several selectin ligands as demonstrated for P-selectin,[13,14] prompted
us to use 3’-sulfated Lewis x as the sugar part. We used the pentaerythritol backbone[15]
as a linker between the oligosaccharide and the peptide chains to get a chance to add, if
needed, a third bioactive part. Finally, to distinguish between the relative contribution
of the two components parts, we synthesized the homobifunctional dimer of 3’-sulfated
Lewis x on the same pentaerythritol backbone.
RESULTS AND DISCUSSION
We first needed a pentaerythritol derivative in which the hydroxyl groups were
differentiated. Starting from the known p-methoxybenzylidene derivative 1,[16]
benzylation under standard conditions followed by reductive opening of the p-me-
thoxybenzylidene (NaBH3CN, TFA, CH2Cl2) gave 3 in 88% yield for the two steps.
Then, condensation of the known trichloroacetimidate 5[17] in the presence of
TMSOTf gave 6 in 86% yield. The N-phthalimido protecting group was then replaced
with N-acetamido by treating 6 with hydrazine acetate and subsequent peracetylation
to give 7 in 89% overall yield. Removal of the p-methoxybenzyl group with cerium
ammonium nitrate gave 8 (84%). Next, Swern oxidation gave the aldehyde 9 (96%)
ready for coupling with the dipeptide H–Gly–Tyr(OH)–OBn (30). Compond 30 was
prepared from commercial Boc–Gly–OH (27) and Tyr(OH)–OBn (28) according to a

Heterobifunctional Candidate Ligands of P-Selectin
483
routine procedure in peptide chemistry in the presence of EDC and HOBt.[18] Thus,
coupling reaction between 27 and 28, gave the dipeptide 29 in 73% yield in which the
Boc protecting group was removed with trifluoroacetic acid to give 30 in 78% yield.
The reductive amination was performed as usual with NaBH3CN in a THF–phosphate
buffer (pH 5) mixture to give 10. Subsequent sulfation of crude 10 with SO3–NMe3 in
DMF gave the N,O-disulfate 11 in 64% yield for the two steps. The benzyl ester was
then selectively cleaved by catalytic hydrogenation (H2, Pd/C) in the presence of
ammonium acetate.[19] The resulting 12 was directly deacetylated with NaOMe in
MeOH to give 13 in 80% overall yield from 11. The 3’-sulfate was then introduced
through the stannylene methodology[20] to give 14 in 49% yield (95% from consumed
13). The reaction was not pushed to completion to avoid concomitant sulfation at C-6.
Finally, complete debenzylation under standard conditions afforded compound 15 in
95% yield bearing an additionnal N-sulfate. Although the later is a priori undesirable,
it deserves nevertheless to be tested as an inhibitor of P-selectin PSGL-1 interaction
and the selective N-desulfation was not attempted. We preferred to focus on an al-
ternative route in which the sulfate groups on both galactose and tyrosine were in-
troduced before the reductive amination. We have therefore prepared the aldehyde 17
already sulfated at O–3’, as a key intermediate to be condensed with sulfated peptides
33 and 41. The aldehyde 17 was prepared from the aldehyde 9 after deacetylation
(NaOMe in MeOH) into 16 (94%) and sulfation using the stannylene methodology
(43%, 81% from consumed 16). Sulfated dipeptide 33 was first prepared from 30
according to standard procedures. The Fmoc protecting group was introduced with N-
(9-fluorenylmethoxycarbonyloxy)-succinimide to give 31 in 80% yield. Then, sulfation
Scheme 1.

484
Dagron and Lubineau
of tyrosine with SO3–DMF complex in DMF afforded 32 (91%) which was hydro-
genolysed quantitatively to give the sulfated peptide 33. Second, we prepared the
oligopeptide 41 containing a cluster of three contiguous sulfated tyrosines linked to 6-
aminohexanoic acid as a spacer. Boc–[Tyr(OH)]3–OBn (36) was prepared step by
step in 64% overall yield, from commercial Boc–Tyr(OH)–OH (34) and H–
Tyr(OH)–OBn (28) as described for peptide 30. The Boc protecting group in 36 was
removed with trifluoroacetic acid to give 37 in 82% yield. Then, conventional cou-
pling with Fmoc–NH(CH2)5–COOH 38[21] gave 39 (82%). Sulfation of the three
tyrosines with SO3–DMF complex gave the intermediate derivative 40 from which the
Fmoc protecting group was removed with morpholine in DMF to afford 41 in 77%
Scheme 2.

Heterobifunctional Candidate Ligands of P-Selectin
485
Scheme 3.
yield. The coupling of both sulfated peptides 33 and 41 with aldehyde 17 through
reductive amination, gave 18 (79%) and 20 (77%). Finally, hydrogenolysis led to the
heterobifunctional derivatives 19 and 21 both in 95% yield (Schemes 1–4).
The homobifunctional derivative 26 was prepared through a straightforward se-
quence. Thus, trichloroacetimidate 5 was condensed with the known diol 4[22] in the
presence of TMSOTf to give 22 in 80% yield. Subsequent treatment with hydrazine
acetate, followed by selective N-acetylation gave 23 (66%) which was further sulfated
at O–3’, using the above described methodology, to give the disulfate 24 (40%) along
with the monosulfate 25 (50%). Additional 24 could be obtained by treating 25 with 1
equivalent of dibutyltin oxide and 1 equivalent of SO3–DMF complex to give finally
24 in a total yield of 70%. Lastly, hydrogenolysis of the benzyl group in 24 afforded 26
in 94% yield.
Compounds 15, 19, 21 and 26 are currently being tested as P-selectin ligands.
EXPERIMENTAL
General procedures. All moisture-sensitive reactions were performed under ar-
gon using oven-dried glassware. If necessary, solvents were dried and distilled prior to

486
Dagron and Lubineau
Scheme 4.
use. NMR spectra were recorded with Bruker AM250, AC200, AC250 or DRX 400
1
spectrometers. In CDCl3, H and 13C chemical shifts (d) are reported relative to tet-
ramethylsilane (d 0.00 ppm) and internal chloroform (d 77.0 ppm), respectively. In
D2O, acetone (d 2.22 and 30.5 ppm, respectively) was used as internal reference. Signal
multiplicity is indicated as follow: s for singlet, bs for broad singlet, d for doublet, t for
triplet, q for quadruplet, m for multiplet. In the listing of the NMR data, I, II, III refers
respectively to N-acetylglucosamine, fucose, and galactose. IR spectra were recorded
on a FT-IR Bruker IFS-66 spectrometer. Optical rotations were determined using a
Jasco DIP-370 digital polarimeter. Mass spectra were recorded on a Finnigan MAT 95 S
spectrometer using electrospray ionization. Flash chromatography was performed using
6–35 m silica gel (60) purchased from S.D.S. Company. TLC was run using Merck 60
F254 plates, and visualized first with UV light and second by heating after alcoholic
sulfuric acid treatment. Melting points were measured with a Reichert apparatus and
are uncorrected. Elemental analyses were performed at the Service Central de Micro-
analyses, CNRS, Gif sur Yvette, France.
2,2-Bis-benzyloxymethyl-1,3-p-methoxybenzylidenepropan-1,3-diol (2). NaH
(60% in oil, 2.05 g, 51 mmol) was added to a cooled (0°C) solution of compound 1
(5.0 g, 19.68 mmol) in THF (36 mL). After 5 min, BnBr (5.6 mL, 43 mmol) was added
dropwise. The mixture was then stirred at rt until completion of the reaction. Methanol
was then carefully added, and the mixture was concentrated. Flash chromatography
1
(Petroleum ether/EtOAc, 9/1) of the residue gave 2 (8.26 g, 98%) as colorless oil. H
NMR (250 MHz, CDCl3): d 7.20–7.40 (m, 12 H), 6.85 (d, 2 H, J = 8.8 Hz), 5.35 (s, 1
H), 4.56 (s, 2 H), 4.40 (s, 2 H), 4.14 (d, 2 H, J = 12.0 Hz), 3.87 (d, 2 H), 3.84 (s, 2 H),
3.75 (s, 3 H), 3.31 (s, 2 H). 13C NMR (62.9 MHz, CDCl3): d 159.9, 138.2, 130.8,
128.3, 128.2, 127.5, 127.4, 127.33, 113.5, 101.6, 73.3, 73.2, 70.2, 70.0, 68.7, 55.2, 38.8.
IR(cmÀ1) n 3030, 2972, 2876, 2844, 2787, 1497, 1477, 1455, 1398, 1361, 1199, 1178,
1112, 1098, 1074, 1019, 981, 968, 956, 914, 759, 739, 697.

Heterobifunctional Candidate Ligands of P-Selectin
487
Anal. Calcd for C27H30O5 (434.54): C, 74.63; H, 6.96; O, 18.41. Found: C, 74.81;
H, 7.04; O, 18.24.
2,2-Bis-benzyloxymethyl-3-p-methoxybenzyloxymethylpropan-1-ol (3). Tri-
fluoroacetic acid (1.5 mL, 19.7 mmol) was added to a cooled (0°C) solution of
compound 2 (1 g, 2.3 mmol) and NaBH3CN (2.2 g, 3.5 mmol) in THF (14 mL). The
mixture was stirred for 20 min, then diluted with CH2Cl2, neutralized with NEt3 and
finally washed with satd aq NaHCO3 and water. The aq layers were extracted with
CH2Cl2. The organic phases were pooled, filtered off silicone treated paper and con-
centrated. Flash chromatography of the residue (EtOAc/petroleum ether, 2/8) gave 3
1
(0.9 g, 90%) as a colorless oil. H NMR (250 MHz, CDCl3): d 7.23–7.38 (m, 10 H),
7.19 (d, 2 H, J = 8.8 Hz), 6.83 (d, 2 H), 4.47 (s, 4 H), 4.40 (s, 2 H), 3.77 (s, 3 H), 3.76
(d, 2 H, J = 6 Hz), 3.55 (s, 4 H), 3.53 (s, 2 H), 2.89 (t, 1 H, CH2OH). 13C NMR (62.9
MHz, CDCl3): d 159.1, 138.3, 130.3, 129.0, 128.3, 127.5, 127.3, 113.7, 73.4, 73.1, 70.8,
70.5, 66.1, 55.2, 44.9. IR (cmÀ1): n 3497, 3063, 3030, 3004, 2863, 1717, 1612, 1586,
1513, 1496, 1453, 1422, 1362, 1302, 1248, 1207, 1173, 1091, 1030, 820, 737, 698.
Anal. Calcd for C27H32O5 (436.55): C, 74.29; H, 7.39; O, 18.32. Found: C, 74.26;
H, 7.42; O, 18.59.
2,2-Bis-benzyloxymethyl-1-O-{2,3,4,6-tetra-O-acetyl- -D-galactopyranosyl)-
!
!
(1
phthalimido- -D-glucopyranosyloxy}-3-p-methoxybenzyloxypropane (6). Molecular
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl-(1
3)]-6-O-benzyl-2-deoxy-2-
˚
sieves (4 A, 100 mg) were added to a stirred solution of the trichloroacetimidate 5
(219 mg, 0.17 mmol) and compound 3 (364 mg, 0.85 mmol) in CH2Cl2 (1.2 mL). After
5 min, the mixture was cooled to À30°C, and a solution of trimethylsilyl triflate in
CH2Cl2 (0.1 M, 170 mL) was added. The stirred mixture was then allowed to warm up
to 0°C over a period of 3 h. NEt3 (50 mL) was then added and the mixture was
concentrated. Flash chromatography of the residue (petroleum ether/EtOAc, 7/3) gave 6
28
1
(228 mg, 86%) as a colorless foam. [a]D À24 (c 1.07, CH2Cl2). H NMR (250 MHz,
CDCl3): d 7.60 (m, 4 H), 7.42–6.98 (m, 32 H), 6.77 (d, 2 H, J = 8.5 Hz), 5.23 (d, 1 H,
III III
,4
III III
,2
III III
J3
= 3.5 Hz, H-4III), 5.00 (dd, 1 H, J1
= 8.0, J2
,3
= 10.0 Hz, H-2III), 4.99
I
I
II II
,2
(d, 1 H, J1 ,2 = 8.5 Hz, H-1I), 4.83 (d, 1 H, J1
Jgem = 12.0 Hz, OCH2Ph), 4.78 (d, 1 H, Jgem = 12.0 Hz, OCH2Ph), 4.77 (m, 1 H, H-3I),
= 3.5 Hz, H-1II), 4.81 (d, 1 H,
III III
,4
4.76 (dd, 1 H, J3
= 3.5 Hz, H-3III), 4.72 (d, 1 H, H-1III), 4.68 (s, 2 H, OCH2Ph),
4.65 (m, 1 H, H-5II), 4.59 (d, 1 H, J = 12.0 Hz, OCH2Ph), 4.44 (dd, 1 H, J2 ,3 = 10.5
Hz, H-2I), 4.42 (d, 2 H, J = 12.0 Hz, 2 OCH2Ph), 4.27 (d, 2 H, J = 12.0 Hz, OCH2Ph),
4.22–4.05 (m, 8 H, H-4I, H-6III, 2 OCH2Ph, OCH2Ph–OMe), 3.97 (d, 1 H, J = 9.0 Hz),
I
I
3.96 (dd, 1 H, J5
= 5.5 Hz, J6
,
= 10.5 Hz, H-6’III), 3.95–3.72 (m, 7 H, H-2II,
III III
,6’
III III
6’
H-3II, H-6I, H-6’I, CH3–O), 3.63 (bs, 1 H, H-4II), 3.56 (m, 2 H, H-5I, H-5III), 3.42 (d, 1
H, J = 9.0 Hz), 3.36–3.21 (m, 6 H), 2.01 (s, 6 H, 2 OAc), 1.94, 1.81 (2 s, 6 H, 2 OAc),
II II
,6
1.20 (d, 3 H, J5
= 6.5 Hz, H-6II). 13C NMR (62.9 MHz, CDCl3): d 170.0, 169.8,
168.7, 138.8, 138.7, 138.1, 137.7, 130.7, 134.0, 128.7, 128.6, 128.2, 128.1, 127.9,
127.8, 127.4, 127.1, 127.0, 126.9, 123.4, 113.5, 99.4, 98.7, 97.2, 79.7, 76.9, 75.1, 74.5,
71.9, 70.9, 70.2, 68.8, 66.7, 66.7, 74.0, 73.4, 72.9, 72.7, 72.3, 69.1, 68.7, 68.5, 67.3,
60.1, 56.5, 55.2, 45.12, 20.7, 20.6, 20.5. IR (cmÀ1): n 3451, 3063, 3030, 2934, 2871,
1755, 1716, 1613, 1514, 1497, 1454, 1386, 1368, 1302, 1249, 1220, 1171, 1135, 1047,
955, 912, 821, 738, 723, 698.

488
Dagron and Lubineau
Anal. Calcd for C89H97NO24 (1564.76): C, 68.32; H, 6.25; N, 0.90; O, 24.54.
Found: C, 68.23; H, 6.38; N, 0.92; O, 24.51.
2,2-Bis-benzyloxymethyl-1-{O-(2,3,4,6-tetra-O-acetyl- -D-galactopyranosyl)-
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl-(1
!
!
(1
3)]-2-acetamido-6-O-ben-
zyl-2-deoxy- -D-glucopyranosyloxy}-3-p-methoxybenzyloxypropane (7). A solution
of compound 6 (256 mg, 0.16 mmol) in a mixture of hydrazine hydrate and ethanol (1/
9, 3 mL) was refluxed for 20 h. The mixture was then cooled to rt then concentrated.
Pyridine (3 mL) and acetic anhydride (2 mL) were then added to the residue. After 18
h at rt, the reaction mixture was concentrated. Flash chromatography of the residue
24
(petroleum ether/EtOAc, 6/4) gave 7 (216 mg, 89%) as a colorless powder. [a]D À50
1
(c 1.06, CH2Cl2). H NMR (200 MHz, CDCl3): d 7.42–7.20 (m, 30 H, Ph), 7.18 (d, 2
H, J = 8.5 Hz), 6.81 (d, 2 H), 5.63 (d, 1 H, JNH,2 = 8.5 Hz, NH), 5.28 (d, 1 H,
I
III III
,4
III III
,2
II II
,2
J3
J1
= 3.5 Hz, H-4III), 5.10 (d, 1 H, J1
= 3.5 Hz, H-1II), 5.03 (dd, 1 H,
= 8.0, J2 III = 10.5 Hz, H-2III), 4.95 (d, 1 H, J = 12.0 Hz, OCHPh), 4.84 (dd, 1
III
,3
H, H-3III), 4.83 (d, 1 H, J = 12.0 Hz, OCHPh), 4.81–4.64 (m, 4 H, 4 OCHPh), 4.61 (d,
I
I
1 H, J1 ,2 = 6.5 Hz, H-1I), 4.58 (d, 1 H, J = 12.0 Hz, OCHPh), 4.54 (d, 1 H, H-1III),
4.48–4.29 (m, 8 H, H-5II, 3 OCH2Ph, OCHPh), 4.18–3.90 (m, 4 H, H-2II, H-6III, H-6’III,
H-4I), 3.88 (dd, 1 H, J2 II = 10.5 Hz, J3
= 3.5 Hz, H-3II), 3.80–3.66 (m, 6 H, H-2I,
II
II II
,4
,3
H-6I, H-3I, CH3O), 3.63 (bs, 1 H, H-4II), 3.60–3.40 (m, 9 H, H-5I, H-5III), 2.01, 1.98,
II II
1.96 and 1.92 (4 s, 12 H, 4 OAc), 1.68 (s, 3 H, NHAc), 1.15 (d, 3 H, J5
= 6.5 Hz, H-
,6
6II). 13C NMR (50 MHz, CDCl3): d 169.9, 169.7, 169.4 (C O), 158.8, 138.8, 138.5,
137.9, 130.8, 128.8, 128.4, 128.4, 128.3, 127.8, 127.7, 127.6, 127.2, 127.0, 113.5, 100.7,
99.3, 97,3 (C-1I, C-1II, C-1III), 79.6, 77.0, 76.3, 73.9, 70.5, 70.3, 68.8, 66.7, 66.4, 74.3,
74.2, 73.3, 72.8, 72.6, 69.2, 68.9, 68.7, 68.2, 60.3, 55.5, 55.1, 45.4, 23.1, 20.7, 20.5,
16.6. IR(cmÀ1): n 3410, 3088, 3063, 3030, 2931, 2871, 1754, 1672, 1612, 1586, 1514,
1497, 1454, 1396, 1302, 1249, 1220, 1171, 1099, 1056, 954, 911, 819, 738, 698.
Anal. Calcd for C83H97NO23 (1476.69): C, 67.51; H, 6.62; N, 0.95; O, 24.92.
Found: C, 67.49; H, 6.73; N, 1.11; O, 24.63.
2,2-Bis-benzyloxymethyl-3-{O-(2,3,4,6-tetra-O-acetyl- -D-galactopyranosyl)-
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl)-(1
!
!
(1
3)]-2-acetamido-6-O-ben-
zyl-2-deoxy-b-D-glucopyranosyloxy}-propan-1-ol (8). Ammonium cerium nitrate
(349 mg, 0.64 mmol) was added to a vigorously stirred solution of compound 7 (119 mg,
0.08 mmol) in a 9/1 mixture of acetonitrile and water (1 mL). After 5 min, CH2Cl2 was
added, and the mixture washed with satd aq NaHCO3, then with water. The aq layers
were washed with CH2Cl2. Then, the organic phases were pooled, filtered using silicone
treated paper and concentrated. Flash chromatography of the residue (EtOAc/petroleum
26
ether, 65/35) gave 8 (92 mg, 84%) as a colorless powder. [a]D À53 (c 1.13, CH2Cl2).
I
1H NMR (250 MHz, CDCl3): d 7.55–7.10 (m, 30 H, Ph), 5.86 (d, 1 H, JNH,2 = 8.5 Hz,
III III
,4
II II
,2
NH), 5.28 (d, 1 H, J3
= 3.5 Hz, H-4III), 5.27 (d, 1 H J1
= 3.0 Hz, H-1II), 2.00,
II II
1.98, 1.96, 1.93 (4 s, 12 H, 4 OAc), 1.72 (s, 3 H, NHAc), 1.14 (d, 3 H, J5
= 6.5 Hz,
,6
H-6II). 13C NMR (62.9 MHz, CDCl3): d 170.1, 169.9, 169.2 (C O), 138.7, 138.6,
138.5, 138.3, 137.7, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5,
127.4, 127.3, 127.1, 100.6, 99.3, 97.8 (C-1I, C-1II, C-1III), 79.7, 76.9, 76.4, 74.4, 74.3,
73.8, 70.5, 68.8, 73.5, 73.3, 72.5, 70.4, 69.6, 68.2, 66.6, 66.4, 64.6, 60.3, 55.5, 45.0, 23.1,

Heterobifunctional Candidate Ligands of P-Selectin
489
20.7, 20.5, 16.6. IR (cmÀ1): n 3418, 3088, 3063, 3031, 2934, 2873, 1754, 1672, 1524,
1497, 1454, 1369, 1221, 1168, 1134, 1100, 1054, 954, 910, 814, 739, 698.
Anal. Calcd for C75H89NO22 (1356.54): C, 66.41; H, 6.61; N, 1.03; O, 25.95.
Found: C, 66.54; H, 6.44; N, 1.18; O, 25.76.
2,2-Bis-benzyloxymethyl-3-{O-(2,3,4,6-tetra-O-acetyl- -D-galactopyranosyl)-
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl)-(1
!
!
(1
3)]-2-acetamido-6-O-ben-
zyl-2-deoxy- -D-glucopyranosyloxy}-propanal (9). DMSO (81.5 mL, 1.15 mmol)
was added dropwise to a cooled (À78°C) solution of oxalyl chloride (20 mL) in CH2Cl2
(500 mL). After 15 min, a solution of compound 8 (97 mg, 77 mmol) in CH2Cl2 (0.5
mL) was added and the resulting mixture was stirred at À78°C for 30 min. Then, NEt3
(160 mL, 1.15 mmol) was added and the reaction mixture was slowly warmed up to
0°C over 3 h. The reaction was then diluted with CH2Cl2, washed with a 5% aq
solution of KH2PO4 then with water. The aq layers were extracted with CH2Cl2. Then,
the organic phases were pooled, filtered using silicone treated paper, and concentrated.
Flash chromatography of the residue (EtOAc/petroleum ether, 1/1) gave 9 (94 mg,
24
1
96%) as a colorless syrup. [a]D À53 (c 1.05, CH2Cl2). H NMR (250 MHz, CDCl3): d
I
9.65 (s, 1 H, CHO), 7.45–7.15 (m, 30 H, Ph), 5.72 (d, 1 H, JNH,2 = 8.5 Hz, NH), 5.27
III III
,4
II II
,2
(d, 1 H, J3
III III
= 3.5 Hz, H-4III), 5.04 (d, 1 H, J1
= 3.5 Hz, H-1II), 5.02 (dd, 1 H,
= 11.0 Hz, H-2III), 4.95 (d, 1 H, J = 12 Hz, CHPh), 4.85 (d, 1
III III
,3
J1
= 8.0 Hz, J2
,2
H, J = 11.5 Hz, CHPh), 4.82 (d, 1 H, H-3III), 4.76 (d, 1 H, CHPh), 4.70 (d, 1 H, J = 12
Hz, CHPh), 4.69 (d, 1 H, J1 ,2 = 7.0 Hz, H-1I), 4.68 (d, 1 H, CHPh), 4.60 (d, 1 H,
I
I
CHPh), 4.52 (d, 1 H, H-1III), 4.44 (s, 2 H, OCH2Ph), 4.39 (s, 2 H, OCH2Ph), 4.38 (d, 1
III III
,6
III III
,6’
H, CHPh), 4.32 (m, 1 H, H-5II), 4.12 (dd, 1 H, J5
= 8.0 Hz, J6
= 10.5 Hz, H-2II), 4.05 (t, 1 H, J2 ,3
= 11.0 Hz, H-
I I
II II
,3
6III), 4.09 (d, 1H, J = 9.5 Hz), 4.09 (dd, 1 H, J2
I
I
III III
,6’
= 7.0 Hz, J3 ,4 = 7.0 Hz, H-3I), 3.97 (dd, 1 H, J5
= 6.0 Hz, H-6’III), 3.93 (t, 1 H,
J4 ,5 = 7.0 Hz, H-4I), 3.87 (dd, 1 H, J3
= 2.5 Hz, H-3II), 3.80–3.53 (m, 10 H), 3.48
I
I
II II
,4
(m, 1 H, H-5I), 2.00, 1.99, 1.96, 1.93 (4 s, 4 OAc), 1.72 (s, 1 H, NHAc), 1.13 (d, 1 H,
II II
,6
J5
= 6.5 Hz, H-6II). 13C NMR (62.9 MHz, CDCl3): d 203.7 (CHO), 170.1, 170.0,
169.4 (C O), 138.8, 138.7, 138.5, 138.0, 137.8, 128.5, 128.4, 128.3, 128.2, 128.0,
127.9, 127.8, 127.6, 127.4, 127.4, 127.1 (arom), 100.5, 99.4, 97.9 (C-1I, C-1II, C-1III),
79.8, 76.9, 76.4, 74.2, 74.1, 73.8, 70.5, 70.4, 68.8, 66.7, 66.5, 74.4, 73.1, 73.4, 72.6,
68.3, 67.8, 67.6, 67.5, 60.4, 56.1, 55.1, 23.1, 20.7, 20.6, 20.5, 16.7. IR (cmÀ1): n 3411,
3063, 3030, 2872, 1754, 1676, 1523, 1497, 1454, 1369, 1220, 1168, 1134, 1100, 1057,
955, 910, 815, 739, 698.
Anal. Calcd for C75H87NO22 (1354.52): C, 66.51; H, 6.47; N, 1.03; O, 25.99.
Found: C, 65.67; H, 6.53; N, 1.12; O, 25.75.
Sodium salt of N-Sulfo-N-{2,2-bis-benzyloxymethyl-3-(O-(2,3,4,6-tetra-O-acetyl-
!
-D-galactopyranosyl)-(1
4)-O-[(2,3,6)-tri-O-benzyl- -L-fucopyranosyl)-
!
(1
3)]-2-acetamido-6-O-benzyl-2-deoxy-b-D-glucopyranosyloxy)propyl}-Gly-
Tyr(SO3À)-O-benzyl (11). Dipeptide 30 (25 mg, 0.057mmol) was added to a solu-
tion of aldehyde 9 (70 mg, 0.052 mmol) in THF (0.9 mL). Then, were added suc-
cessively, phosphate buffer (1 M, pH 5, 0.1 mL) and sodium cyanoborohydride (4 mg,
0.062 mmol). After 3 h, the mixture was diluted with CH2Cl2 and washed with water.
The organic phase was then concentrated and dried in vacuo. A solution of SO3–NMe3

490
Dagron and Lubineau
(143 mg, 1.03 mmol) in DMF (1 mL) was added, and the mixture was stirred at 50°C
for 10 h. After cooling to rt, excess of sulfur trioxide was destroyed with MeOH (0.25
mL). After 1 h, the mixture was diluted with ethyl acetate (10 mL) and washed with
satd aq NaHCO3, then with water. Aqueous phases were extracted with ethyl acetate
and the combined organic phases were dried (Na2SO4) then concentrated. Flash
chromatography of the residue (EtOAc/MeOH/NEt3, from 9/1/0.01 to 8/2/0.01) gave 11
(62 mg, 64%) as a white solid. [a]D À28 (c 1.22, MeOH). 1H NMR [250 MHz,
29
CDCl3–CD3OD (8/2)]: d 7.45–7.15 (m, 37 H), 7.02 (d, 2 H, J = 8.5 Hz), 2.03, 1.99,
II II
,6
1.96, 1.92, 1.87 (5 s, 15 H, 4 OAc and NHAc), 1.21 (d, 3 H, J5
= 6.5 Hz, H-6II).
13C NMR [62.9 MHz, CDCl3–CD3OD (8/2)]: d 172.4, 172.0, 170.8, 170.6, 170.4,
169.8 (C O), 151.8, 139.0, 138.8, 138.6, 138.0, 137.8, 135.3, 132.7, 130.5 128.9,
128.8, 128.6, 128.3, 128.1, 128.0, 127.9, 127.6, 127.1, 122.1, 102.3, 98.8 and 97.1 (C-
1I, C-1II, C-1III), 79.6, 77.8, 77.4, 75.3, 75.1, 74.7, 74.4, 74.0, 73.7, 73.6, 73.5, 73.1,
72.8, 72.6, 71.1, 70.6, 69.6, 69.4, 69.1, 68.3, 67.8, 67.7, 67.6, 67.0, 66.6, 60.5, 55.9,
54.6, 53.5, 51.1, 45.5, 37.1, 23.3, 20.8, 20.7, 20.6, 16.8. IR (cmÀ1): n 3450, 3063, 3032,
2928, 1755, 1669, 1541, 1507, 1455,1371, 1218, 1173, 1100, 1049, 955, 914, 864, 840,
819, 741, 698, 643. MS (electrospray, negative ion mode) m/z calcd for
C93H105N3O31S2Na2: [M–Na]À 1846.6, [M–2 Na]2À 911.8. Found: 1846.7, 911.8.
Sodium salt of N-Sulfo-N-{2,2-bis-benzyloxymethyl-3-(O-( -D-galactopyranos-
!
!
yl)-(1
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl)-(1
3)]-2-acetamido-6-O-
benzyl-2-deoxy-b-D-glucopyranosyloxy)propyl}–Gly–Tyr(SO3À)–OH (13). Ammo-
nium acetate (4 mg) and Pd/C (5%, 50 mg) were added to a solution of compound 11
(50 mg, 0.028 mmol) in MeOH (3 mL). The mixture was stirred under H2 atmosphere
(1 atm) for 30 min, then filtered through a pad of celite and concentrated. Sodium
methylate in MeOH (0.5 M, 1 mL) was added to the residue and the solution was left
at rt for 2 h. The mixture was then neutralized with acetic acid and concentrated.
Reverse phase chromatography of the residue (C18 silica gel, water/MeOH from 7/3 to
29
1
4/6) gave 13 (34 mg, 80%) as a colorless foam. [a]D À41 (c 2.1, MeOH). H NMR
(200 MHz, CDCl3): d 7.50–7.10 (m, 34 H), 5.34 (d, 1 H, J1
= 3.5 Hz, H-1II), 1.98
II II
,2
= 6.5 Hz, H-6II). 13C NMR (62.9 MHz, CDCl3): d
II II
,6
(s, 3 H, NHAc), 1,18 (d, 3 H, J5
178.1, 173.5, 173.2 (C O), 152.4, 140.4, 140.2, 140.1, 140.0, 139.6, 139.3, 136.2,
131.2, 129.6, 129.4, 129.3, 128.8, 128.7, 128.5, 128.4, 122.4, 103.6, 102.9, 97.8 (C-1I,
C-1II, C-1III), 80.0, 79.7, 77.1, 76.4, 76.3, 75.2, 74.8, 74.7, 74.6, 74.2, 74.1, 73.6, 73.2,
72.6, 71.0, 70.9, 69.9, 69.1, 68.4, 67.8, 63.2, 57.1, 56.8, 55.2, 52.2, 46.3, 39.1, 23.9,
16.9. IR (cmÀ1): n 3423, 1637, 1558, 1541, 1506, 1456, 1418, 1209, 553, 520, 507.
HRMS (electrospray, negative ion mode) m/z calcd for C78H90N3O27S2Na3: [M–3
Na + H]2À 1565.52814. Found: 1565.52818.
Sodium salt of N-Sulfo-N-{2,2-bis-benzyloxymethyl-3-(O-(3-sulfo- -D-galacto-
!
!
pyranosyl)-(1
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl)-(1
3)]-2-acet-
amido-6-O-benzyl-2-deoxy- -D-glucopyranosyloxy)propyl}–Gly–Tyr(SO3À)–OH
(14). A suspension of dibutyltin oxide (4 mg, 15 mmol) and compound 13 (19 mg,
12 mmol) in toluene (5 mL) was heated under reflux for 12 h with continual removal
of water using a Dean–Stark apparatus and concentrated. A solution of SO3–NMe3 in
DMF (0.1 M, 125 mL) was added to the residue, and the resulting mixture was stirred
for 5 h at rt. The mixture was then diluted with EtOAc and washed with a satd aq

Heterobifunctional Candidate Ligands of P-Selectin
491
NaHCO3 solution. The aq phase was extracted with EtOAc, and the combined organic
layers were dried (Na2SO4) then concentrated. Reverse phase chromatography (silica
gel C18, water/MeOH, from 1/0 to 6/4) gave 14 along with the starting material 13 (9
mg). Fractions containing compound 14 were passed through a cation exchange resin
column (Biorad AG50WX-8, sodium form) which was eluted with MeOH to give 14
29
as the pure sodium salt (10 mg, 49%, 95% from consumed 13). [a]D À25 (c 1.92,
MeOH). 1H NMR (200 MHz, CD3OD): d 7.45–7.12 (m, 34 H), 5.34 (d, 1 H,
II II
,2
II II
,6
J1
= 3.5 Hz, H-1II), 1.98 (s, 3 H, NHAc), 1.15 (d, 3 H, J5
= 6.5 Hz, H-6II). 13C
NMR (50 MHz, CD3OD): d 177.6, 173.5, 173.2 (C O), 152.4, 140.6, 140.2, 140.1,
139.7, 139.5, 136.2, 131.4, 129.6, 129.5, 129.3, 129.2, 129.0, 128.8, 128.6, 128.4,
128.3, 122.4, 103.2, 103.1, 97.8 (C-1I, C-1II and C-1III), 81.8, 79.9, 77.1, 76.9, 76.4,
75.3, 74.3, 74.2, 73.6, 71.1, 71.0, 70.2, 69.1, 68.4, 67.7, 63.4, 57.5, 57.1, 57.05, 52.6,
46.3, 39.2, 24.0, 16.8. IR (cmÀ1): n 3435, 2928, 2089, 1644, 1506, 1454, 1402, 1235,
1164, 1098, 1049, 867, 736, 697, 577, 560, 552, 519, 503, 499. HRMS (electrospray,
negative ion mode) m/z calcd for C78H89N3O30S3Na4: [M–4 Na + H]3À 1644.47713.
Found: 1644.47631.
Sodium salt of N-Sulfo-N-{2,2-bis-hydroxymethyl-3-(O-(3-sulfo- -D-galactopyr-
!
!
anosyl)-(1
4)-O-[ -L-fucopyranosyl)-(1
3)]-2-acetamido-2-deoxy-b-D-gluco-
pyranosyloxy)propyl}–Gly–Tyr(SO3À)–OH (15). A solution of compound 14 (6 mg,
3.4mmol)andPd(OH)2/C(20%, 10mg)inamixtureofmethanolandwater(1/1, 0.5mL)was
stirredunderH2 atmosphere(1atm)for24h.Thereactionmixturewasthenfilteredthrougha
padofceliteandelutedwithwaterfromacationexchangeresincolumn(BioradAG50WX-8,
sodium form). Freeze-drying of the resulting aq solution gave 15 (4 mg, 95%) as a white
29
powder.[a]D À25(c1.02,H2O).1HNMR(250MHz,D2O):d7.33(d,2H,J = 8.0Hz),7.25
(d,2H,J = 8.0Hz),5.12(d,1H,J1 II = 3.5Hz,H-1II),4.58(d,1H,J1 III = 8.0Hz,H-1III),
II
III
,2
,2
4.48 (dd, 1H, J = 5.5, 6.5Hz), 4.32(dd, 1H, J2 III = 9.0Hz, J3
= 2.5Hz, H-3III), 4.32
III
III III
,4
,3
(d, 1 H, J1 ,2 = 8.0 Hz, H-1I), 4.27 (d, 1 H, H-4III), 2.04 (s, 3 H, NHAc), 1.17 (d, 3 H,
I
I
II II
,6
J5
= 6.5 Hz, H-6II). 13C NMR (62.9 MHz, D2O): d 174.5–172.3 (C O), 150.3, 135.5,
130.9, 121.8, 101.7, 101.3, 98.8 (C-1I, C-1II, C-1III), 80.4, 75.2, 74.9, 74.8, 73.5, 72.2, 69.5,
69.4, 68.0, 66.9, 68.3, 61.6, 60.9, 59.8, 56.2, 56.1, 50.9, 45.6, 37.4, 22.6, 15.5. HRMS
(electrospray, negative ion mode) m/z calcd for C36H53N3O30S3Na4: [M–4 Na + H]3À
1104.19543. Found: 1104.19545.
!
2,2-Bis-benzyloxymethyl-3-{O-(b-D-galactopyranosyl)-(1
4)-O-[2,3,6-tri-O-
3)]-2-acetamido-6-O-benzyl-2-deoxy- -D-gluco-
!
benzyl- -L-fucopyranosyl)-(1
pyranosyloxy}propanal (16). A solution of NaOMe in MeOH (0.2M, 0.65 mL) was
added to the aldehyde 43 (88 mg, 0.065 mmol). The mixture was stirred for 1 h, then
neutralized with AcOH (7 mL) and concentrated. Flash chromatography of the residue
28
(EtOAc/ MeOH from 1/0 to 95/5) gave 16 (73 mg, 94 %) as a white solid. [a]D À66
1
(c 2.75, MeOH). H NMR (200 MHz, CDCl3): d 9.65 (s, 1 H, CHO), 7.5–7.05 (m, 30
H, Ph), 5.23 (bs, 1 H, H-1II), 4.96 (m, 1 H, H-5II), 1.79 (s, 3 H, NHAc), 1.12 (d, 3 H,
II II
,6
J5
= 6.5 Hz, H-6II). 13C NMR [62.9 MHz, CDCl3–CD3OD (8/2)]: d 204.2 (CHO),
171.2 (CONH), 139.0, 138.8, 138.2, 138.1, 138.0, 128.7, 127.4, 101.8, 101.6, 97.0 (C-
1I, C-1II, C-1III), 79.2, 78.1, 76.2, 75.7, 74.7, 74.0, 73.6, 72.5, 71.8, 69.2, 66.9, 75.3,
73.6, 73.4, 73.3, 68.5, 68.0, 67.8, 67.7, 62.5, 56.3, 23.2, 16.4. IR (cmÀ1): n 3420 ; 3063 ;
3030 ; 2925 ; 2872 ; 1727 ; 1656 ; 1540 ; 1497 ; 1454 ; 1366 ; 1312 ; 1208 ; 1098 ; 1066 ;

492
Dagron and Lubineau
912 ; 812 ; 737 ; 697. MS (electrospray, positive ion mode) m/z calcd for C67H79NO18:
[M + Na]+ 1208.5. Found: 1208.4.
Anal. Calcd for C67H79NO18, 0.5 H2O: C, 67.32; H, 6.75; N, 1.17; O, 24.27.
Found: C, 67.37; H, 6.53; N, 1.06; O, 25.15.
Sodium salt of 2,2-bis-benzyloxymethyl-3-{O-(3-O-sulfo- -D-galactopyranosyl)-
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl)-(1
!
!
(1
3)]-2-acetamido-6-O-ben-
zyl-2-deoxy- -D-glucopyranosyloxy}propanal (17). A suspension of dibutyltin oxide
(31 mg, 126 mmol) and aldehyde 15 (75 mg, 63 mmol) in toluene (15 mL) was heated
under reflux for 18 h with continual removal of water using a Dean–Stark apparatus
and concentrated. A solution of SO3–NMe3 (10 mg) in DMF (0.6 mL) was added to
the residue, and the resulting mixture was stirred for 5 h at rt. The mixture was diluted
with ethyl acetate and washed with a satd aq NaHCO3 solution. The aq phase was
extracted with ethyl acetate, and the combined organic layers were dried (Na2SO4) and
concentrated. Flash chromatography of the residue (EtOAc/MeOH, 9/1) gave 17 along
with the starting material 15 (17 mg). Fractions containing 17 were passed through a
cation exchange resin column (Biorad AG50WX-8, sodium form) which was further
eluted with MeOH to give 17 as the pure sodium salt (51 mg, 63%, 81% from con-
28
1
sumed 15). [a]D À49 (c 1.21, CH2Cl2). H NMR [250 MHz, CDCl3–CD3OD (8/2)]: d
= 3.5 Hz, H-1II),
II II
,2
9.64 (s, 1 H, CHO), 7.50–7.10 (m, 30 H, Ph), 5.18 (d, 1 H, J1
1.83 (s, 3 H, NHAc), 1.15 (d, 3 H, J5
= 7.5 Hz, H-6II). 13C NMR [62.9 MHz,
II II
,6
CDCl3–CD3OD (8/2)]: d 204.2 (CHO), 171.2 (NHAc), 139.0, 138.8, 138.2, 138.1,
138.0, 128.7, 127.4, 101.8, 101.6, 97.0 (C-1I, C-1II, C-1III), 79.2, 78.1, 76.2, 75.7, 74.7,
74.0, 73.6, 72.5, 71.8, 69.2, 66.9, 75.3, 73.6, 73.4, 73.3, 68.5, 68.0, 67.8, 67.7, 62.5,
56.3, 23.2, 16.4. IR (cmÀ1): n 3426, 3088, 3063, 3031, 2925, 2858, 1956, 1881, 1813,
1728, 1658, 1545, 1497, 1454, 1367, 1258, 1213, 1157, 1100, 1058, 1027, 875, 810,
737, 697. MS (electrospray, negative ion mode) m/z calcd for C67H78NO21SNa: [M–
Na]À 1264.5. Found: 1264.2.
Anal. Calcd for C67H78NO21SNa, H2O: C, 61.60; H, 6.17; N, 1.09; S, 2.45; Found:
C, 61.56; H, 6.51; N, O.99; S, 2.27.
Sodium salt of N-{2,2-bis-benzyloxymethyl-3-(O-(3-sulfo- -D-galactopyranosyl)-
!
!
(1
4)-O-[2,3,4,tri-O-benzyl- -L-fucopyranosyl)-(1
3)]-2-acetamido-6-O-ben-
zyl-2-deoxy- -D-glucopyranosyloxy}propyl]–Gly–Tyr(SO3À)–OH (18). A solution
of aldehyde 17 (13 mg, 10 mmol) and dipeptide 33 (17 mg, 49 mmoL) in a 1/1/1
mixture of THF, MeOH and pH5 phosphate buffer (0.5 mL) was heated to 50°C. After
5 min, NaBH3CN (2 mg) was added and the mixture was kept at 50°C for 15 h. The
mixture was then cooled to rt, diluted with CH2Cl2 and washed with water. The aq
phase was extracted with CH2Cl2 and the combined organic layers were dried (Na2SO4)
and concentrated. Reverse phase chromatography (C18 silica gel, water/MeOH, from
90/10 to 75/25) followed by cation exchange resin column (Biorad AG50WX-8, so-
26
dium form) eluted with MeOH gave 18 as pure sodium salt (13 mg, 79%). [a]D À47
1
(c 1.00, MeOH). H NMR [250 MHz, CDCl3–CD3OD (1/1)]: d 7.45–7.10 (m, 34 H),
= 3.5 Hz, H-1II), 1.91 (s, 3 H, NHAc), 1.14 (d, 3 H, J5
= 6.5
II II
,2
II II
,6
5.31 (d, 1 H, J1
Hz, H-6II). 13C NMR [62.9 MHz, CDCl3–CD3OD (1/1)]: d 175.0, 173.1 (C O),
152.2, 139.8, 139.5, 138.7, 138.5, 138.3, 134.5, 130.7, 129.1, 128.9, 128.5, 128.0,

Heterobifunctional Candidate Ligands of P-Selectin
493
122.1, 102.5, 97.3 (C-1I, C-1II, C-1III), 62.9, 56.6, 55.5, 55.4, 52.2, 43.4 et 38.0, 30.3,
23.6, 16.6. IR (cmÀ1): n 3440, 3088, 3066, 3032, 2925, 2855, 1733, 1683, 1676, 1654,
1636, 1558, 1541, 1507, 1498, 1455, 1384, 1261, 1213, 1159, 1094, 1049, 1028, 738,
698. HRMS (electrospray, negative ion mode) m/z calcd for C78H91N3O27S2Na2: [M–
Na]2À, 1565.52814. Found: 1565.52798.
Sodium salt of N-{2,2-bis-hydroxymethyl-3-(O-(3-sulfo- -D-galactopyranosyl)-
!
!
(1
4)-O-[ -L-fucopyranosyl)-(1
3)]-2-acetamido-2-deoxy- -D-glucopyr-
anosyloxy)propyl}–Gly–Tyr(SO3À)–OH (19). A suspension of compound 18 (8 mg,
5 mmol) and Pd(OH)2/C (20%, 10 mg) in a 1:1 mixture of MeOH and water was stirred
under H2 atmosphere (1 atm) for 24 h. The reaction mixture was then filtered through a
pad of celite and eluted with water from a cation exchange resin column (Biorad
AG50WX-8, sodium form). Freeze-drying of the resulting aq solution gave 19 (5 mg,
29
1
95%) as a white powder. [a]D À19 (c 1.00, H2O). H NMR (250 MHz, D2O): d
7.48–7.15 (m, 4 H), 5.12 (bs, 1 H, H-1II), 4.57 (d, 1 H, J1
III III
(dd, 1 H, Ja,b = 5.5 Hz, Ja,b’ = 6.5 Hz), 4.32 (dd, 1 H, J2
= 7.5 Hz, H-1III), 4.48
III III
,2
III III
,4
= 9.0 Hz, J3
= 2.5
,3
I
I
Hz, H-3III), 4.32 (d, 1 H, J1 ,2 = 8.0 Hz, H-1I), 4.27 (d, 1 H, H-4III), 2.04 (s, 3 H,
II II
,6
NHAc), 1.18 (d, 3 H, J5
= 6.5 Hz, H-6II). 13C NMR (50 MHz, D2O): d 174.5,
150.6, 135.1, 131.3, 121.6, 101.7, 98.8 (C-1I, C-1II, C-1III), 80.4, 75.4, 75.1, 74.9,
73.7, 72.2, 69.5, 68.0, 63.1, 61.6, 59.8, 56.1, 55.1, 46.3, 36.9, 22.6, 15.5. IR (cmÀ1): n
3437, 2922, 2860, 1620, 1383, 1253, 1053, 868, 820, 778. HRMS (electrospray,
negative ion mode) m/z calcd for C36H55N3O27S2Na2: (M–2 Na+)2À, 1057.21847;
found: 1057.21802.
Tetrabutylammonium salt of N-{2,2-bis-benzyloxymethyl-3-(O-(3-sulfo- -D-ga-
lactopyranosyl)-(1
!
!
4)-O-[2,3,4,tri-O-benzyl- -L-fucopyranosyl)-(1
3)]-2-
acetamido-6-O-benzyl-2-deoxy-b-D-glucopyranosyloxy)propyl}-6-aminohexanoyl-
[Tyr(SO3À)]3-O-benzyl (20). A solution of aldehyde 17 (40 mg, 30 mmol) and peptide
41 (55 mg, 33 mmol) in a water–THF mixture (1/1, 0.2 mL) was heated to 40°C. After 10
min, NaBH3CN (4 mg) was added and the resulting mixture was kept at 40°C for 18 h.
After cooling to rt, the mixture was diluted with EtOAc and washed with water. Water
was extracted with EtOAc and the organic layers were combined, dried (Na2SO4) then
concentrated. Column chromatography (LH20, CH2Cl2/MeOH, 1/1) followed by elution
with MeOH from a cation exchange resin column (Biorad AG50WX-8, tetra-
29
butylammonium form) gave 20 as pure tetrabutylammonium salt (68 mg, 77%). [a]D
1
À31 (c 1.52, CH2Cl2). H NMR [250 MHz, CDCl3–CD3OD (8/2)]: d 7.45–7.00 (m, 47
= 3.0 Hz, H-1II), 5.12 (s, 2 H, OCH2Ph), 2.08–1.85 (m, 2 H),
II II
H), 5.33 (d, 1 H, J1
1.90 (s, 3 H, NHAc), 1.13 (d, 3 H, J5
,2
= 6.5 Hz, H-6II). 13C NMR [62.9 MHz, CDCl3–
II II
,6
CD3OD (8/2)]: d 174.8, 172.2, 172.1, 172.0, 171.6, 171.3 (C O), 152.2, 151.9, 151.8,
139.4, 139.1, 138.3, 138.1, 137.9, 137.8, 135.4, 133.5, 133.0, 132.5, 132.4, 121.5, 121.4
et 121.1, 102.0, 101.7, 96.7 (C1I, C1II, C1III), 81.2, 79.2, 78.5, 77.8, 76.1, 75.9, 75.5,
75.1, 73.7, 73.2, 73.0, 72.6, 70.9, 70.3, 70.2, 69.8, 68.0, 67.9, 67.4, 66.8, 62.9, 58.8
(NBu4 ), 56.5 (C-2I), 54.3, 54.2, 54.1, 52.4, 50.9, 42.9, 37.0, 36.1, 35.9, 25.5, 25.3, 25.0,
+
23.9 (NBu4 ), 23.4 (NHAc), 19.8 (NBu4 ), 16.4 (C-6II), 13.7 (NBu4 ). IR (cmÀ1): n
3471, 3063, 3030, 2937, 2875, 1756, 1716, 1497, 1454, 1427, 1387, 1368, 1316, 1218,
1170, 1134, 1100, 1048, 955, 911, 874.0, 740, 722, 698, 643. HRMS (electrospray,
+
+
+

494
Dagron and Lubineau
negative ion mode) m/z calcd for C107H122N5O37S4: [M–3 N+Bu4]3À 2196.67015.
Found: 2196.66564.
Sodium salt of N-{2,2-bis-hydroxymethyl-3-(O-(3-sulfo- -D-galactopyranosyl)-
!
!
(1
4)-O-[ -L-fucopyranosyl)-(1
3)]-2-acetamido-2-deoxy-b-D-glucopyr-
anosyloxy)propyl}-6-aminohexanoyl-[Tyr(SO3À)]3–OH (21). A suspension of com-
pound 20 (18 mg, 6.2 mmol) and Pd(OH)2/C (20%, 20 mg) in a mixture of MeOH and
water (1/1, 1 mL) was stirred under H2 atmosphere (1 atm) for 24 h. The reaction
mixture was then filtered through a pad of celite and eluted with water from a cation
exchange resin column (Biorad AG50WX-8, sodium form). Freeze-drying of the
29
resulting aq solution gave 21 (10 mg, 95%) as a white powder. [a]D À32 (c 0.5, H2O).
1H NMR (400 MHz, D2O): d 7.35–7.10 (m, 12 H), 5.13 (d, 1 H, J1
= 3.5 Hz, H-1II),
II II
,2
4.88–4.76 (m, 1 H, H-5II), 4.70–4.53 (m, 3 H), 4.50 (d, 1 H, J1 ,2 = 8.0 Hz, H-1I), 4.32
I
I
= 9.5 Hz, H-3III), 4.26 (d, 1 H, H-4III), 2.16–2.02 (m, 2 H), 2.04 (s, 3
III III
,3
(dd, 1 H, J2
II II
,6
H, NHAc), 1.64–1.48 (m, 2 H), 1.42–1.24 (m, 2 H), 1.17 (d, 3 H, J5
= 6.5 Hz, H-
6II), 1.01–0.80 (m, 2 H). 13C NMR (62.9 MHz, D2O): d 176.3, 174.6, 172.8, 171.8
(C O), 150.2, 135.7, 134.8, 134.4, 130.8, 130.7, 121.8, 121.6, 101.7 (C-1III), 101.5 (C-
1I), 98.8 (C-1II), 80.4 (C-3III), 75.4, 74.9, 74.0, 73.6, 72.2, 69.4, 68.0, 67.0, 62.0, 61.6,
61.4, 59.9, 56.0 (C-2I), 54.5, 54.4, 54.2, 50.3, 49.2, 43.2, 37.4, 36.7, 36.3, 35.4, 25.1,
25.0, 24.9, 22.6 (NHAc), 15.5. IR (cmÀ1): n 3423, 2927, 2852, 1734, 1653, 1558, 1540,
1507, 1384, 1254, 1165, 1051, 1018, 871, 834, 643, 624. HRMS (electrospray, negative
ion mode) m/z calcd for C58H79N5O37S4Na4: [M–4 Na–SO3+ H]3À 1486.38469. Found:
1486.38414.
2,2-Bis-benzyloxymethyl-1,3-bis-{O-(2,3,4,6-tetra-O-acetyl- -D-galactopyr-
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl)-(1
!
!
anosyl)-(1
3)]-6-O-benzyl-2-
deoxy-2-phthalimido-b-D-glucopyranosyloxy}propane (22). Compound 4 (59 mg,
0.19 mol) was added to a solution of trichloroacetimidate 5 (630 mg, 0.49 mmol) in
CH2Cl2 (2.5 mL). The stirred solution was cooled to 0°C, and a solution of TMSOTf in
CH2Cl2 (0.1M, 38 mL) was added dropwise. The mixture was kept at 0°C for 1 h,
quenched with NEt3 (50 mL) and concentrated. Flash chromatography of the residue
27
(toluene/EtOAc, 3.5/1) gave 22 (392 mg, 80%) as a white foam. [a]D À35 (c 1.38,
1
CH2Cl2). H NMR (250 MHz, CDCl3): d 7.58 (m, 8 H, 2 NPhth), 7.42–6.94 (m, 50 H,
III III
,4
III III
,2
Ph), 5.21 (d, 2 H, J3
III III
= 3.5 Hz, 2 H-4III), 4.99 (dd, 2 H, J1
= 8.5,
= 10.5 Hz, 2 H-2III), 4.81 (d, 2 H, J1 ,2 = 8.5 Hz, 2 H-1I), 4.80 (d, 2 H,
I I
J2
,3
Jgem = 12.0 Hz, 2 OCHPh), 4.77–4.69 (m, 6 H, 2 H-1II, 2 H-3III, 2 OCHPh), 4.68–4.59
(m, 10 H, 2 H-1III, 2 H-3I, 2 H-5II, 2 OCH2Ph), 4.58 (d, 2 H, J = 12.0 Hz, 2 OCHPh),
4.41 (d, 2 H, J = 12.0 Hz, 2 OCHPh), 4.37 (d, 2 H, J = 12.0 Hz, 2 OCHPh), 4.33 (dd, 2
H,J2 ,3 = 10.5 Hz, 2 H-2I), 4.23 (d, 2 H, J = 12.0 Hz, 2 OCHPh), 4.11 (t, 2 H,
I
I
I
I
III III
,6’
J3 ,4 = 10.5 Hz, 2 H-4I), 4.11 (dd, 2 H, J6
= 10.5, 2 H-6III), 3.93 (dd, 2 H,
III III
,6’
J5
= 5.5 Hz, 2 H-6’III), 3.91–3.68 (m, 14 H, 2 H-2II, 2 H-3III, 2 H-6I, 2 H-6’I, 2
III III
,5
OCH–C8, 4 OCHPh), 3.61 (bs, 2 H, 2 H-4II), 3.47 (ddd, 2 H, J4
= 1.0,
= 8.5 Hz, 2 H-5III), 3.40 (d, 2 H, J5 ,6 = 10.0 Hz, 2 H-5I), 3.19 (d, 2 H, J = 9.0
III III
,6
I I
J5
II II
,6
Hz, 2 OCH–C8), 2.00, 1.99, 1.93, 1.79 (4 s, 24 H, 8 OAc), 1.20 (d, 6 H, J5
= 6.5
Hz, 6 H-6II). 13C NMR (50 MHz, CDCl3): d 170.0, 169.8, 168.8 (C O), 138.9, 138.6,
138.1, 137.8, 131.5, 134.1, 128.6, 128.3, 128.1, 127.9, 127.8, 127.4, 127.1, 126.9,
126.7, 123.4, 99.4, 98.6. 97.2 (C-1I, C-1II, C-1III), 79.7, 77.2, 76.9, 75.0, 74.4, 71.8,

Heterobifunctional Candidate Ligands of P-Selectin
495
70.9, 70.2, 68.8, 66.6, 66.2, 74.1, 73.4, 72.7, 72.3, 68.6, 67.9, 67.4, 60.1, 56.4, 44.9,
20.5, 16.7. IR (cmÀ1): n 3471, 3063, 3030, 2937, 2875, 1756, 1716, 1497, 1454, 1427,
1387, 1368, 1316, 1218, 1170, 1134, 1100, 1048, 955, 911, 874.0, 740, 722, 698, 643.
Anal. Calcd for C143H154N2O42 (2572.81): C, 66.76; H, 6.03; N, 1.09; O, 26.12.
Found: C, 66.37; H, 6.37; N, 1.28; O, 26.38.
!
2,2-Bis-benzyloxymethyl-1,3-bis-{O-( -D-galactopyranosyl)-(1
tri-O-benzyl- -L-fucopyranosyl)-(1
4)-O-[2,3,6-
3)]-2-acetamido-6-O-benzyl-2-deoxy-b-D-
!
glucopyranosyloxy}propane (23). A solution of 22 (563 mg, 0.219 mmol) in a 1/2
mixture of ethylenediamine and ethanol (2 mL) was heated under reflux for 18 h, then
concentrated. A 1/1 mixture of pyridine and acetic anhydride (2 mL) was then added to
the residue, and the resulting mixture was stirred at 40°C for 12 h. Solvents were
coevaporated with toluene and the residue diluted with CH2Cl2. The solution was
washed with satd aq NaHCO3 and water, then concentrated. Flash chromatography of
the residue (EtOAc/petroleum ether, 52/48) gave the peracetylated intermediate (353
mg) which was added to a solution of sodium methanolate in MeOH (0.1 M, 1.5 mL).
After 1 h at rt, acetic acid (8 mL) was added and the mixture concentrated. Flash
chromatography of the residue (EtOAc/CH2Cl2 /MeOH, 50/42/8) gave 23 (296 mg,
25
1
66%) as a colorless powder. [a]D À68 (c 1.12, CH2Cl2). H NMR [400 MHz, CDCl3–
= 3.0 Hz, 2 H-1II), 1.90–
II II
,2
CD3OD (8/2)]: d 7.40–7.50 (m, 50 H), 5.19 (d, 2 H, J1
= 5.0 Hz, 6 H-6II). 13C NMR [50 MHz,
II II
1.60 (m, 6 H, 2 NHAc), 1.09 (d, 6 H, J5
,6
CDCl3–CD3OD (8/2)]: d 175.8, 170.8 (NHAc), 138.9, 138.8, 138.7, 138.5, 138.4,
138.2, 137.9, 137.8, 137.7, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.5, 127.4,
127.3, 127.1, 101.9, 101.3, 100.8, 100.0, 97.2, 97.0 (C-1I, C-1’I, C-1II, C-1’II, C-1III, C-
1’III), 59.5 (C-2I), 45.3, 23.3, 21.3 (NHAc), 16.6, 16.5 (C-6II, C-6’II). IR (cmÀ1): n
3418, 3088, 3063, 3030, 3005, 2930, 2874, 1720.4, 1660, 1585, 1540, 1497, 1454,
1368, 1313, 1280, 1209, 1157, 1094, 1054, 1028, 913, 805, 737, 697. HRMS (elec-
trospray, positive ion mode) m/z calcd for C115H138N2O32: [M + 2Na]2 +, 2104.90281.
Found: 2104.90162.
Anal. Calcd for C115H138N2O32, 2 H2O: C, 65.89; H, 6.83; N, 1.34; O, 25.95.
Found: C, 65.89; H, 6.79; N, 1.21; O, 25.57.
Sodium salt of 2,2-bis-benzyloxymethyl-1,3-bis-(O-{3-O-sulfo- -D-galactopyr-
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl)-(1
!
!
anosyl)-(1
3)]-2-acetamido-
6-O-benzyl-2-deoxy-b-D-glucopyranosyloxy}propane (24). A suspension of 23 (103
mg, 0.05 mmol) and dibutyltin oxide (27 mg, 0.11mmol) in toluene (10 mL) was
heated under reflux for 12 h with continual removal of water using a Dean–Stark
apparatus and concentrated. A solution of SO3–NMe3 (15 mg, 0.11 mmol) in THF (0.5
mL) was added to the residue, and the mixture was kept at rt for 14 h. The reaction
mixture was diluted with EtOAc and washed with satd aq NaHCO3 and water. The aq
phases were extracted with EtOAc and the combined organic layers were dried
(Na2SO4) and concentrated. Flash chromatography of the residue (EtOAc/MeOH, from
95/5 to 85/15) gave 24 along with the monosulfated compound 25. Separately, fractions
containing 24 and fractions containing 25 were passed through a cation exchange resin
column (Biorad AG50WX-8, sodium form) eluted with MeOH to give the bis sulfate
24 (45 mg, 40%) and the mono sulfate 25 (54 mg, 50%) as pure sodium salts. Following
the same procedure, but using 1 equiv of dibutyltin oxide and 1 equiv of SO3–NMe3,

496
Dagron and Lubineau
additional 24 (33 mg) could be obtained from the monosulfate 25 in 60% yield giving a
30
70% total yield of 24 from 23. [a]D À52 (c 1.37, CH2Cl2). 1H NMR [250 MHz, CDCl3–
= 2.5 Hz, 2 H-1II), 1.86
II II
,2
CD3OD (8/2)]: d 7.18–7.42 (m, 50 H), 5.22 (d, 2 H, J1
= 6.5 Hz, H-6III). 13C NMR [50 MHz, CDCl3–
II II
(s, 6 H, NHAc), 1.14 (d, 6 H, J5
,6
CD3OD (8/2)]: d 171.9 (C O), 138.9, 138.6, 138.5, 138.1, 137.9, 137.7, 128.7,
128.5, 128.0, 127.8, 127.7, 127.6, 127.4, 102.6, 101.7, 97.0 (C-1I, C-1II, C-1III), 62.3
(C-6III), 55.8 (C-2I), 45.5, 23.3 (NHAc), 16.4 (C-6II). IR (cmÀ1): n 3422, 3088.5,
3064, 3032, 2925, 2874, 2856, 1959, 1882, 1720.2, 1665, 1552, 1497, 1454, 1370,
1261, 1212, 1157, 1099, 1059, 1027, 805, 737, 697. MS (electrospray, negative ion
mode) m/z calcd for C115H136N2O38S2Na2: [M–2 Na]+, 1108.4. Found: 1108.9.
Anal. Calcd for C115H136N2O38S2Na2, 4 H2O: C, 59.12; H, 6.21; N, 1.20; S, 2.74;
Found: C, 59.16; H, 6.81; N, 1.09; S, 2.51.
Sodium salt of 2,2-bis-benzyloxymethyl-1,3-bis-{O-(3-O-sulfo- -D-galactopyr-
4)-O-[2,3,6-tri-O-benzyl- -L-fucopyranosyl)-(1
!
!
anosyl)-(1
3)]-2-acetamido-
6-O-benzyl-2-deoxy- -D-glucopyranosyloxy}propane (26). A suspension of com-
pound 24 (39 mg, 17 mmol) and Pd(OH)2/C (20%, 40 mg) in a mixture of MeOH and
water (1/1, 1 mL) was stirred under H2 atmosphere (1 atm) for 36 h. The reaction
mixture was then filtered through a pad of celite and eluted with water from a cation
exchange resin column (Biorad AG50WX-8, sodium form). Freeze-drying of the
27
resulting aq solution gave 26 (22 mg, 94%) as a white powder. [a]D À51 (c 0.375,
1
II II
,2
H2O). H NMR (400 MHz, D2O): d 5.13 (d, 2 H, J1
= 3.5 Hz, 2 H-1II), 4.84–4.80
(m, 2 H, 2 H-5II), 4.57 (d, 2 H, J1
= 8.0 Hz, 2 H-1III), 4.45 (d, 2 H, J1 ,2 = 8.0 Hz,
III III
,2
I I
III III
,3
III III
,4
2 H-1I), 4.32 (dd, 2 H, J2
= 10.0 Hz, J3
= 3.0 Hz, 2 H-3III), 4.26 (d, 2 H, 2
H-4III), 3.91 (dd, 2 H, 2 H-2I), 3.71–3.60 (m, 4 H, 2 H-3I, 2 H-2III), 2.04 (s, 6 H, 2
II II
,6
NHAc), 1.17 (d, 6 H, J5
= 6.5 Hz, H-6II). 13C NMR (50MHz, D2O): d 174.4
(NHAc), 102.0 (C-1I), 101.7 (C-1III), 98.8 (C-1II), 80.4 (C-3III), 75.4, 74.8, 73.6, 72.2,
69.4, 68.0, 66.9, 61.6, 60.9, 59.9, 56.2 (C-2I), 22.5 (NHAc), 15.5. IR (cmÀ1): n 3429,
2934, 1652.1, 1559, 1507, 1388, 1248, 1163, 1072, 1035, 871, 810, 771. HRMS
(electrospray, negative ion mode) m/z calcd for C45H76N2O38S2Na2: (M–2 Na+)2À
,
1316.35175. Found: 1316.35216.
Boc–Gly–Tyr(OH)–OBn (29). Diisopropyl ethylamine (2 mL, 11.42 mmol)
was added to a solution of commercial Boc–Gly–OH (27) (1g, 5.71 mmol) and H–
Tyr(OH)–OBn (28) (2.3 g, 5.19 mol) in CH2Cl2 (18 mL). HOBt (841 mg, 6.23 mmol)
and EDC (1.19 g, 6.23 mmol) were then added. After 18 h, the reaction mixture was
diluted with CH2Cl2 and washed with satd aq NaHCO3. Aqueous solution was
extracted with CH2Cl2 and the combined organic layers were dried (Na2SO4) and
concentrated. Flash chromatography of the residue (toluene/acetone, 77/23) gave 29
29
(1.6 g, 73%) as a colorless gum. [a]D + 7 (c 1, CH2Cl2). 1H NMR (200 MHz,
CDCl3): d 7.42–7.25 (m, 5 H, Ph), 6.79 (d, 2 H, J = 8.0 Hz), 6.63 (d, 2 H, J = 8.0 Hz),
5.17 (d, 1 H, J = 12.0 Hz, OCHPh), 5.09 (d, 1 H, OCHPh), 4.86 (m, 1 H), 3.74 (m, 2
H), 3.12 (m, 4 H), 1.43 (s, 9 H, 3 CH3). 13C NMR (50 MHz, CDCl3): d 171.4, 169.5
(C O), 155.5, 134.9, 130.3, 128.6, 126.4, 115.6, 80.4, 67.3, 53.2, 44.0, 36.9, 28.2. IR
(cmÀ1): n 3370, 3067, 3044, 2979, 2931, 1742, 1672, 1615, 1595, 1516, 1455, 1392,
1367, 1251, 1213, 1166, 1116, 1050, 1029, 944, 914, 858, 827.
Anal. Calcd for C23H28N2O6, O.5 H2O: C, 63.14; H, 6.68; N, 6.40; O, 23.77.
Found: C, 63.20; H, 6.77; N, 6.49; O, 23.18.

Heterobifunctional Candidate Ligands of P-Selectin
497
H–Gly–Tyr(OH)–OBn (30). Trifluoroacetic acid (1.5 mL) was added to a
cooled (0°C) solution of compound 29 (948 mg, 2.21 mmol) in CH2Cl2 (3 mL) and the
reaction mixture was allowed to warm up at rt. After 1 h, solvents were coevaporated
three times with CH2Cl2. Flash chromatography of the residue (CH2Cl2/MeOH, from
28
1
88/12 to 85/15) gave 30 as trifluoroacetate (2.545 g, 78%). [a]D + 3 (c 1, MeOH). H
NMR [250 MHz, CDCl3–CD3OD (8/2)]: d 7.41–7.24 (m, 5 H, Ph), 6.92 (d, 2 H,
J = 8.5 Hz), 6.70 (d, 2 H, J = 8.5 Hz), 5.23 (s, 2 H, O–CH2–Ph), 4.75 (dd, 1 H,
J = 6.0, 8.0 Hz), 3.61 (bs, 2 H), 3.07 (dd, 1 H, J = 14.5, 6.0 Hz), 2.91 (dd, 1 H,
J = 14.5, 8 Hz). 13C NMR [62.9 MHz, CDCl3–CD3OD (8/2)]: d 171.6, 166.1 (C O),
156.1, 135.2, 130.4, 128.8, 128.6, 126.8, 115.6, 67.6 (OCH2Ph), 54.5, 40.4, 36.9. IR
(cmÀ1): n 3318, 3093, 2974, 1736, 1724, 1664, 1612, 1596, 1549, 1514, 1458, 1441,
1378, 1350, 1287, 1190, 1135, 1021, 958, 914, 841, 799, 752, 724, 698.
Anal. Calcd for C20H21N2O6F3, 1.5 H2O: C, 51.17; H, 5.15; N, 5.97. Found: C,
50.78; H, 5.01; N, 5.35.
H–Gly–Tyr(OSO3Na)–OH (33). N-(9-Fluorenylmethoxycarbonyloxy)succinimide
(534 mg, 1.58 mmol) and NaHCO3 (133 mg, 1.6 mmol) were added to a cooled (0°C)
and stirred solution of dipeptide 30 (350 mg, 0.79 mmol) in a mixture of acetone/water
(4/1, 3 mL). After 15 h, the reaction mixture was diluted with CH2Cl2 and washed with
water. The aq phases were extracted with CH2Cl2 and the combined organic layers
were dried (Na2SO4) and concentrated. Flash chromatography of the residue (petroleum
ether/EtOAc, 6/4 to 4/6) gave 31 (350 mg, 80%). A solution of of SO3–DMF complex
(486 mg, 3.178 mmol) in DMF (2.5 mL) was then added, and the mixture was stirred at
70°C for 12 h. After cooling, satd aq NaHCO3 was added and the resulting mixture was
extracted with EtOAc. The combined organic layers were dried (Na2SO4) and con-
centrated. Flash chromatography of the residue (EtOAc/MeOH, 1/0 to 9/1) followed by
cation exchange chromatography (Biorad AG50WX-8, sodium form, MeOH) gave 32
(376 mg, 90 % from 31) as a colorless foam. The residue was then dissolved in MeOH
(25 mL) and Pd(OH)2/C (20%, 350 mg) was added. The resulting mixture was stirred
under an H2 atmosphere (1 atm) for 16 h, then filtered through a pad of celite washed
thoroughly with MeOH. Solvents were evaporated and the residue was dissolved in a
mixture of ether and water. The aq phase was extracted several time with ether, then
freeze-dried to give 33 (196 mg, 100% from 32, 72% from 30) as a white powder.
29
1
[a]D + 15 (c 1.40, H2O). H NMR (250 MHz, D2O): d 7.3 (d, 2 H, J = 9.0 Hz),
7.24 (d, 2 H, J = 9.0 Hz), 4.53 (dd, 1 H, J = 4.5, 9.0 Hz), 3.78 (d, 1 H, J = 16.0 Hz),
3.65 (d, 1 H, J = 16 Hz), 3.26 (dd, 1 H, J = 14.0 Hz), 2.94 (dd, 1 H). 13C NMR (62.9
MHz, D2O): d 177.7 (NHCO), 166.4 (COOH), 150.2, 135.8, 130.6, 121.8, 56.5,
40.5, 37.2. IR (cmÀ1): n 3469, 3299, 3090, 2959, 2632, 1912, 1685, 1608, 1506, 1442,
1400, 1262, 1234, 1164, 1106, 1049, 1017, 916, 867, 780, 746, 700, 644, 623. HRMS
(electrospray, negative ion mode) m/z calcd for C11H13N2O7SNa: [M–Na]À,
317.044348. Found: 317.044260.
Boc–Gly–[Tyr(OH)]3–OBn (36). Diisopropylethylamine (1.57 mL, 9 mmol)
was added to a solution of Boc–Tyr(OH)–OH (34) (761 mg, 2.71 mmol) and
Tyr(OH)–OBn (28) (1 g, 2.26 mmol) in CH2Cl2 (7.5 mL). After 5 min, HOBt (366 mg,
2.71 mmol) and EDC (519 mg, 2.71 mmol) were added. After stirring for 18 h, the mixture
was diluted with CH2Cl2 then washed with satd aq NaHCO3 and water. The aq phases
were extracted with CH2Cl2, and the combined organic layers were dried (Na2SO4) then

498
Dagron and Lubineau
concentrated. Flash chromatography of the residue (toluene/acetone, 77/23) gave 35 (1.06
g, 88%) as a gum. The residue was dissolved in CH2Cl2 (5mL) then cooled to 0°C.
Trifluoroacetic acid (1 mL) was then added and the mixture was stirred overnight at rt.
Solvents were coevaporated three time with CH2Cl2, and the residue dried in vacuo. The
resulting trifluoroacetate salt was dissolved in CH2Cl2 (10 mL) and Boc–Tyr(OH)–OH
(0.67 g, 2.38 mmol), then diisopropylethylamine (1 mL, 5.74 mmol) were added, followed
after 5 min by HOBt (322 mg, 2.38 mmol) and EDC (456 mg, 2.38 mmol). After 15 h, the
mixture was diluted with CH2Cl2, then washed with satd aq NaHCO3 and with water. The
aq phases were extracted with CH2Cl2, then the combined organic layers were concen-
trated. Flash chromatography of the residue (Toluene/acetone, 68/32) gave 36 (1.00 g,
27
1
72%) as a colorless gum. [a]D À13 (c 1.03, acetone). H NMR (250 MHz, CDCl3): d
8.30 (bs, 3 H, OH), 7.59 (d, 1 H, J = 7.5 Hz, NH), 7.45–7.25 (m, 5 H), 7.10–6.90 (m, 6
H), 6.78–6.62 (m, 6 H), 6.07 (d, 1 H, J = 7.5 Hz, NH), 5.11 (s, 2 H, OBn), 4.78–4.63 (m,
2 H), 4.35–4.22 (m, 1 H), 3.22–2.70 (m, 6 H), 1.28 (bs, 9 H, CH3). 13C NMR (62.9 MHz,
CD3COCD3): d 172.3, 171.8 and 171.5 (3 C O), 157.1, 156.8, 156.2, 136.8, 131.3,
131.1, 129.2, 129.0, 128.9, 128.5, 127.9, 116.0, 115.8, 79.4 (OC(CH3)3), 67.1, 57.1, 55.0,
37.9, 37.7, 37.5, 28.4 (CH3). IR (cmÀ1): n 3319, 3028, 2977, 2928, 2363, 1654, 1614,
1516, 1446, 1368, 1232, 1171, 1104, 1049, 1018, 892, 827, 752, 698.
Anal. Calcd for C39H43N3O9, H2O: C, 65.44; H, 6.34; N, 5.87. Found: C, 65.09; H,
6.65; N, 5.51.
Fmoc–NH(CH2)5CO[Tyr(OH)]3–OBn (39). Trifluoroacetic acid (3 mL) was
added to a cooled (0°C) solution of the tripeptide 36 (411 mg, 0.59 mmol) in CH2Cl2
(3 mL). After 3 h the reaction mixture was concentrated. Flash chromatography of the
residue (EtOAc/MeOH, from 95/5 to 85/15) gave 37 (0.343 g, 82%) as a colorless gum,
a part of which was directly used in the coupling reaction. Thus, NEt3 (0.42 mmol, 74
mL) was added to a solution of 37 (151 mg, 0.21 mmol) and acid 38 (75 mg, 0.21
mmol) in CH2Cl2 (800 mL). After 5 min, EDC (45 mg, 0.23 mmol) and HOBt (31 mg,
0.23 mmol) were added. The mixture was stirred for 18 h, diluted with EtOAc and
washed successively with satd aq NaHCO3, 10% aq KH2PO4 and water. The organic
phase was dried (Na2SO4) then concentrated. Flash chromatography of the residue
27
(toluene/acetone, 6/4) gave 39 (161 mg, 82%) as a white powder. [a]D À13 (c 1.5,
1
MeOH). H NMR (250 MHz, CD3OD): d 7.78 (d, 2 H, J = 7.0 Hz), 7.62 (d, 2 H,
J = 7.0 Hz), 7.42–7.20 (m, 9 H), 7.03–6.88 (m, 6 H), 6.69–6.61 (m, 6 H), 5.05 (s, 2
H, OCH2Ph), 4.63 (t, 1 H, J = 6.5 Hz), 4.59–4.50 (m, 2 H), 4.34 (d, 2 H, J = 7.0 Hz),
4.20 (t, 1 H), 3.12–2.62 (m, 8 H), 2.10 (d, 2 H, J = 7.5 Hz), 1.60–1.00 (m, 6 H). 13C
NMR (62.9 MHz, CD3OD): d 176.0, 173.5, 173.1, 172.5 (4 C O), 158.4 (O–CONH),
157.4, 157.3, 157.2, 145.3, 142.6, 136.9, 131.3, 131.2, 131.1, 129.5, 129.7, 128.3,
128.1, 126.2, 68.0, 67.5, 56.0, 55.7, 48.5, 41.6, 38.3, 37.8, 36.7, 30.5, 27.1, 26.5. IR
(cmÀ1): n 3284, 3067, 2924, 2856, 1735, 1687, 1638, 1614, 1539, 1515, 1450, 1384,
1231, 1172, 1104, 1002, 825, 758, 740, 712, 695. MS (electrospray, positive ion mode)
m/z calcd for C55H56N4O10: [M + Na]+ 955.4. Found: 955.9.
Anal. Calcd for C55H56N4O10, H2O: C, 69.46; H, 6.15; N, 5.89. Found: C, 69.42;
H, 6.24; N, 5.65.
H2N–(CH2)5CO–[Tyr(OSO3NBu4)3–OBn (41). Compound 39 (200 mg, 0.21
mmol) was added to a solution of SO3–DMF complex (503 mg, 3.2 mmol) in DMF

Heterobifunctional Candidate Ligands of P-Selectin
499
(2 mL). The mixture was maintained at 50°C for 20 h then cooled to 0°C. A solution
of NBu4HCO3 (prepared by passing CO2 through an 0.75 M aq solution of NBu4OH)
was then added until neutrality. The reaction mixture was extracted with CH2Cl2 and the
combined organic layers were filtered using silicone treated paper then concentrated.
Column chromatography of the residue (LH20, CH2Cl2/MeOH, 1/1) gave the interme-
diate 40 (450 mg) slightly contaminated with tetrabutyl ammonium salts. Part of the
residue (50 mg, 26 mmol) was then dissolved in a mixture of morpholine and DMF (1/4,
200 mL). After 20 h, the reaction mixture was directly subjected to a column chroma-
tography (LH20, CH2Cl2/MeOH, 1/1). Elution with the same solvent mixture gave 41 (34
29
1
mg, 77%). [a]D À13 (c 1.03, MeOH). H NMR [250 MHz, CDCl3–CD3OD (1/1)]: d
7.40–7.05 (m, 17 H), 5.15 (d, 2 H, OCH2Ph), 4.75 (t, 1 H, J = 7.0 Hz), 4.62 (dd, 1 H,
J = 9.0, 6.0 Hz), 4.54 (dd, 1 H, J = 3.5, 12.0 Hz), 3.29–2.97 (m, 29 H), 2.85 (dd, 1 H,
J = 9.0, 14.5 Hz), 2.68 (t, 2 H, J = 7.0 Hz), 2.07 (t, 2 H, J = 7.0 Hz), 1.70–1.54 (m, 24
H), 1.52–1.25 (m, 30 H), 1.02 (t, 36 H, J = 7.0 Hz, (CH3–C3H6)4N+). 13C NMR (62.9
MHz, CDCl3): d 175.4, 172.7, 172.2, 171.7 (4 C O), 152.3, 152.1, 151.9, 135.9, 134.4,
133.5, 133.0, 130.5, 130.4, 129.1, 129.0, 121.8, 121.7, 121.6, 67.7, 59.0, 55.2, 54.8, 54.4,
39.9, 37.4, 37.2, 36.7, 35.7, 26.8, 25.4, 25.0, 24.2, 20.1, 13.8. IR (cmÀ1): n 3422, 3307,
3060, 3034, 2963, 2936, 2876, 1746, 1667, 1611, 1507, 1488, 1464, 1383, 1268, 1238,
1218, 1171, 1044, 1018, 863, 741, 697, 642, 618. HRMS (electrospray, negative ion
mode) m/z calcd for C56H79N5O17S3: [M–2 NBu4]2À 1189.46331. Found: 1189.46410.
ACKNOWLEDGMENTS
University of Paris-Sud and CNRS are acknowledged for their financial support
and the Ministe`re de l’Education Nationale de la Recherche et de la Technologie is
acknowledged for a fellowship to FD.
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Received January 16, 2003
Accepted June 18, 2003