Ligand Recognition by E- and P-Selectin
J . Org. Chem., Vol. 63, No. 15, 1998 5141
1H NMR (400 MHz, D2O) δ 4.53 (d, 1H, J ) 7.9 Hz), 4.49 (d,
1H, J ) 7.5 Hz), 4.09 (dd, 1H, J ) 3.1, 9.9 Hz), 3.97 (dd, 1H,
J ) 1.9, 12.0 Hz), 3.93 (d, 1H, J ) 3.1 Hz), 3.91-3.80 (m, 5H),
3.74-3.52 (m, 13H), 3.67 (s, 3H), 2.73 (dd, 1H, J ) 4.6, 12.6
Hz), 2.37 (t, 2H, J ) 7.4 Hz), 2.01 (s, 3H), 2.00 (s, 3H), 1.78 (t,
1H, J ) 12.6 Hz), 1.60-1.50 (m, 4H), 1.35-1.26 (m, 2H); 13C
NMR (100 MHz, CD3OD) δ 177.6, 175.1, 174.4, 174.0, 102.6,
101.2, 99.9, 78.3, 75.5, 75.2, 74.8, 72.9, 72.5, 71.8, 70.3, 69.5,
68.4, 68.1, 67.5, 62.6, 61.1, 60.1, 55.1, 52.1, 51.7, 39.7, 33.7,
28.3, 24.7, 24.0, 23.6, 22.2; HRMS (FAB+, NBA/CsI) calcd for
C32H53N2O21Cs (M - H + 2Cs+) m/e 1067.1249, found 1067.1261.
5-(Meth oxyca r bon yl)p en tyl (5-Aceta m id o-3,5-d id eoxy-
D-glycer o-r-D-ga la cto-n on -2-u lopyr an osylon ic acid)-(2f3)-
â-D-ga la ctop yr a n osyl-(1f4)-[r-L-fu cop yr a n osyl-(1f3)]-2-
a ceta m id o-2-d eoxy-â-D-glu cop yr a n osid e (3). To a mixture
of compound 2 (76 mg, 94.7 µmol), monoammonium GDP-Fuc
(85 mg, 140 µmol), MnCl2‚4H2O (1 M in H2O) (280 µL, 280
µmol), MES buffer (100 mM, pH 6.0) (7 mL), and water (6.4
mL) were added R-1,3-fucosyltransferase V (324 µL, 700 mU)
and alkaline phosphatase (20 µL, 100 U). The mixture was
gently shaken at 37 °C for 5 days, filtered, and evaporated.
Purification by size-exclusion chromatography (Bio-Gel P-2,
2.5 × 70 cm, 50 mM NH4HCO3) gave 3 (81 mg, 90%) as a white
solid after lyophilization: Rf 0.25 (i-PrOH/1 M NH4OAc, 4:1);
1H NMR (500 MHz, D2O) δ 5.06 (d, 1H, J ) 4.0 Hz), 4.48 (d,
1H, J ) 7.8 Hz), 4.47 (d, 1H, J ) 8.3 Hz), 4.05 (dd, 1H, J )
3.2, 9.8 Hz), 3.97 (dd, 1H, J ) 2.3, 12.3 Hz), 3.92-3.79 (m,
10H), 3.74 (d, 1H, J ) 3.5 Hz), 3.67-3.51 (m, 10H), 3.65 (s,
3H), 3.50 (dd, 1H, J ) 7.8, 9.8 Hz), 2.72 (dd, 1H, J ) 4.6, 12.4
Hz), 2.35 (t, 2H, J ) 7.5 Hz), 1.99 (s, 3H), 1.98 (s, 3H), 1.76 (t,
1H, J ) 12.2 Hz), 1.60-1.49 (m, 4H), 1.32-1.25 (m, 2H), 1.13
(d, 3H, J ) 6.7 Hz); 13C NMR (125 MHz, D2O) δ 178.3, 175.8,
174.9, 174.3, 102.4, 101.7, 100.2, 99.4, 76.4, 76.0, 75.64, 75.59,
74.1, 73.7, 72.7, 72.5, 71.1, 70.0, 69.9, 68.94, 68.87, 68.5, 68.0,
67.4, 63.4, 62.2, 60.4, 56.6, 52.8, 52.4, 40.4, 34.4, 29.0, 25.4,
24.7, 23.0, 22.8, 16.0; HRMS (FAB+, NBA/CsI) calcd for
ethylene glycols of varying chain length (20-24). The
products were assayed for inhibitory potency on binding
of a sLea-polymer to immobilized E- and P-selectin. In
the E-selectin assay all dimers had lower IC50 values than
the sLex monomer. Interestingly, the highest binding
enhancements obtained (5.6 and 4.9 times, respectively)
are similar to previous studies in which two sLex moieties
were connected by significantly shorter and more rigid
linkers resulting in 5-6 times more active dimers.17a,b,19
Obviously it is possible to obtain the same binding
enhancement with completely different spacers. Four of
the five sLex carboxylic acids which were obtained as side
products during the cross-linking reaction displayed
significantly improved inhibitory potency in the P-selectin
assay. The lowest IC50 value was observed for the
compound with the shortest side chain (26, n ) 2) thus
being ca. 20-40 times39 more potent than unmodified
sLex. It is noted that the affinity may depend on the
assay system. These findings, however, should be of
importance for the design of new multivalent forms of
sLex as well as sLex mimetics as high-affinity selectin
ligands.
Exp er im en ta l Section
Gen er a l Meth od s. LacNAc derivative 131 and monoam-
monium GDP-Fuc40 were prepared according to published
procedures. Trisodium phosphoenolpyruvate (PEP‚3Na), CTP,
ATP, nucleoside monophosphate kinase (NMK), pyruvate
kinase (PK), pyrophosphatase (PPase), and alkaline phos-
phatase (type VII-N, from bovine intestinal mucosa, P-2276)
were purchased from Sigma (St. Louis, MO). Anhydrous DMF
was purchased from Aldrich (Milwaukee, WI). CMP-NeuAc
synthetase, R-2,3-sialyltransferase, and R-1,3-fucosyltrans-
ferase V were a kind donation from Cytel (San Diego, CA).
Dichloromethane and triethylamine were distilled from cal-
cium hydride. Flash chromatography (FC) was performed on
Mallinckrodt silica gel 60 (230-400 mesh). Analytical thin-
layer chromatography was performed using silica gel 60 F254
precoated glass plates from Merck (Darmstadt, Germany);
compound spots were visualized by quenching of fluorescence
and/or by charring after treatment with cerium molybdophos-
hate. Size-exclusion chromatography was performed on Bio-
Gel P-2 gel (fine) and Bio-Gel P-4 gel (fine) (Bio-Rad Labora-
tories, Hercules, CA). NMR spectra were recorded on Bruker
AM-250, AMX-400, or AMX-500 spectrometers. 1H NMR
chemical shifts are referenced to residual protic solvent (CDCl3,
δH ) 7.26; D2O, δH ) 4.80) or the internal standard TMS (δH
) 0.00). 13C NMR chemical shifts are referenced to the solvent
signal (CDCl3, δC ) 77.0) or to DMSO-d6 (δC ) 39.5) as external
standard. High-resolution mass spectra (HRMS) were re-
corded using fast atom bombardment (FAB) method in a
m-nitrobenzyl alcohol matrix doped with NaI or CsI.
5-(Meth oxyca r bon yl)p en tyl (5-Aceta m id o-3,5-d id eoxy-
D-glycer o-r-D-ga la cto-n on -2-u lopyr an osylon ic acid)-(2f3)-
â-D-ga la ctop yr a n osyl-(1f4)-2-a ceta m id o-2-d eoxy-â-D-glu -
cop yr a n osid e (2). A solution of the lactosamine derivative
131 (395 mg, 0.77 mmol), NeuAc (255 mg, 0.83 mmol), PEP‚
3Na (452 mg, 1.92 mmol), MgCl2‚6H2O (32 mg, 0.16 mmol),
MnCl2‚4H2O (5 mg, 0.04 mmol), KCl (12 mg, 0.16 mmol), CTP
(40 mg, 0.08 mmol), ATP (4.3 mg, 0.008 mmol), and mercap-
toethanol (54 µL) in HEPES buffer (200 mM, pH 7.5) (5.6 mL)
was adjusted with 1 N NaOH to pH 7.5, and the enzymes NMK
(5 U), PK (200 U), PPase (20 U), CMP-NeuAc synthetase (0.9
U), and R-2,3-sialyltransferase (0.16 U) were added to the
solution. The mixture was gently stirred under an argon
atmosphere at room temperature for 5 days. The mixture was
concentrated, and the residue was chromatographed on silica
gel (EtOAc/MeOH/0.02% aqueous CaCl2, 5:2:1) to give recov-
ered 1 (140 mg, 35%) and the title compound (393 mg, 47%):
C
38H64N2O25Cs (M + Cs+) m/e 1081.2853, found 1081.2889.
5-[[(2-Am in oeth yl)am in o]car bon yl]pen tyl (5-Acetam ido-
3,5-d id eoxy-D-glycer o-r-D-ga la cto-n on -2-u lop yr a n osyl-
on ic a cid )-(2f3)-â-D-ga la ctop yr a n osyl-(1f4)-[r-L-fu cop y-
r a n osyl-(1f3)]-2-a cet a m id o-2-d eoxy -â-D -glu cop yr a n o-
sid e (4). A mixture of compound 3 (52 mg, 54.8 µmol) and
ethylenediamine (3 mL) was stirred under argon at 70 °C for
24 h. The mixture was evaporated, and ethanol (2 × 3 mL)
and toluene (2 × 3 mL) were evaporated from the remainder.
Size-exclusion chromatography (Bio-Gel P-2, 2.5 × 70 cm, 50
mM NH4HCO3) gave 4 (49.5 mg, 92%) as a white solid after
1
lyophilization: Rf 0.37 (i-PrOH/1 M NH4OAc, 2:1); H NMR
(400 MHz, D2O) δ 5.08 (d, 1H, J ) 3.9 Hz), 4.50 (d, 1H, J )
7.8 Hz), 4.49 (d, 1H, J ) 8.1 Hz), 4.06 (dd, 1H, J ) 2.9, 9.8
Hz), 4.01-3.79 (m, 11H), 3.76 (d, 1H, J ) 3.1 Hz), 3.71-3.49
(m, 11H), 3.45 (t, 2H, J ) 6.1 Hz), 3.08 (t, 2H, J ) 6.0 Hz),
2.75 (dd, 1H, J ) 4.6, 12.5 Hz), 2.26 (t, 2H, J ) 7.4 Hz), 2.01
(s, 3H), 2.00 (s, 3H), 1.77 (t, 1H, J ) 12.1 Hz), 1.60-1.50 (m,
4H), 1.34-1.26 (m, 2H), 1.15 (d, 3H, J ) 6.6 Hz); 13C NMR
(100 MHz, D2O) δ 180.3, 177.5, 176.6, 176.3, 104.1, 103.5,
102.1, 101.1, 78.1, 77.7, 77.4, 77.3, 75.8, 75.4, 74.3, 72.9, 71.7,
71.6, 70.8, 70.5, 70.1, 69.8, 69.1, 65.0, 64.0, 62.1, 58.3, 54.1,
42.2, 41.6, 39.7, 38.0, 30.8, 27.2, 24.7, 24.5, 17.7; ESIMS (H2O,
neg) calcd for C39H67N4O24 (M - H+) m/e 975, found 975.
Gen er a l P r oced u r es for th e Alk yla tion of Glycols.
Meth od A. The glycol (5 mmol) was added to a stirred
suspension of NaH (95%) (278 mg, 11 mmol) in anhydrous
DMF (25 mL). After 2 h at room temperature, the mixture
was slowly transferred via syringe to a cooled (0 °C) flask
containing tert-butyl bromoacetate (2.95 mL, 20 mmol) using
an additional 10 mL of DMF to achieve a complete transfer
and stirred for 30 min at 0 °C and for 2 h at room temperature.
After addition of a few milliliters of saturated NH4Cl solution,
the mixture was evaporated and distributed between ethyl
acetate and saturated NH4Cl solution. The organic layer was
washed with saturated NH4Cl solution, saturated NaHCO3
solution, and brine, dried (Na2SO4), and evaporated.
Meth od B. A mixture of 50% aqueous NaOH (15 mL),
toluene (15 mL), and the glycol (5 mmol) was cooled to 0 °C,
(40) Wittmann, V.; Wong, C.-H. J . Org. Chem. 1997, 62, 2144-2147.