Chemoenzymatic synthesis and fluorescent visualization of cell-surface selectin-bound sialyl Lewis X derivatives

Article abstract of DOI:10.1002/(SICI)1521-3765(20000103)6:1<162::AID-CHEM162>3.0.CO;2-9

Sialyl Lewis x (sLe^x) derivatives conjugated to readily visualized molecular labels are useful chemical probes to study selectin-carbohydrate interactions. Localization of the selectins on the surface of leukocytes and activated endothelial cel

Full text of DOI:10.1002/(SICI)1521-3765(20000103)6:1<162::AID-CHEM162>3.0.CO;2-9

FULL PAPER
Chemoenzymatic Synthesis and Fluorescent Visualization of Cell-Surface
Selectin-Bound Sialyl Lewis X Derivatives
Valentin Wittmann, Arun K. Datta, Kathryn M. Koeller, and Chi-Huey Wong*[a]
Abstract: Sialyl Lewis x (sLex) derivatives conjugated to readily visualized
molecular labels are useful chemical probes to study selectin ± carbohydrate
interactions. Localization of the selectins on the surface of leukocytes and activated
Keywords: chemoenzymatic synthe-
endothelial cells can be detected through fluorescence of bound selectin ligands.
sis ´ oligosaccharides ´ regioselec-
Herein we present a short chemoenzymatic synthesis of a fluorescently labeled
tive glycosylation ´ selectin ´ sialyl
bivalent sLex conjugate. The use of an amino-substituted monovalent sLex to obtain
Lewis x
fluorescent- and biotin-labeled sLex derivatives is also described. The cell-staining
utility of the fluorescent sLex conjugates is demonstrated for a HUVEC cell line
expressing E-selectin and for CHO-K1 cells expressing either L- or E-selectin.
HO
OH
Introduction
H3C
OH
O
O
Leukocyte adhesion to the vascular endothelium is a defining
event in the inflammatory response. In the initial stages of this
multistep process, leukocytes transiently tether and roll on the
endothelial layer through adhesive interactions between the
selectins and their carbohydrate ligands.[1] The tetrasacchar-
ides sialyl Lewis x (sLex),[2] sialyl Lewis a (sLea),[3] and sulfated
derivatives thereof[4] have been identified as minimal carbo-
hydrate epitopes recognized by selectins. Studies involving
bi-,[5±8, 18] tri-,[7, 9, 10] tetra-,[11] and polyvalent[12±17] sLex deriva-
tives have suggested that the selectin ± ligand interaction may
be multivalent in nature.
Bivalent sLex derivative 1, previously reported by this
laboratory, inhibits binding of HL-60 cells to immobilized
E-selectin five times more efficiently than sLex itself.[6a, b]
Fluorescent derivatives of 1 therefore are of interest as cell-
staining reagents[19] and as tools in the development of a
fluorescence-based[20] E-selectin binding assay.[21]
HO
CO2H
O
OH
OH
O
O
AcHN
O
NHAc
O
HO
OH
OH
HO
HO
OH
O
HO
CO2H
O
R
O
OH
OH
O
AcHN
NHAc
O
HO
OH
OH
HO
OH
H3C
OH
HO
1: R = OEt
HN
H
N
2: R =
N
B
F
O
O
O
N
F
NH-Cbz
20: R = HN
As such, N-glycoconjugate 2 was selected as a primary
synthetic target in this study. The b-alanine spacer at the
carbohydrate reducing terminus facilitated the incorporation
of molecular probes at a position unlikely to interfere with the
selectin ± ligand interaction. The strategy for the synthesis of 2
consisted of three stages: 1) chemical synthesis of trisacchar-
ide-b-alanine conjugate 17, 2) tandem enzymatic introduction
of six peripheral carbohydrates, and 3) attachment of the
fluorescent label through amide coupling.
Chemoenzymatic synthesis of monomeric sLex derivative
22 has been previously reported by this laboratory.[18] Mono-
meric labeled sLex derivatives were readily obtained through
conjugation of 22 to molecular probes containing activated
esters.
The utility of labeled sLex-derivatives as cell-staining
reagents was demonstrated for human umbilical vein endo-
thelial cells (HUVEC) expressing E-selectin or chinese
hamster ovary (CHO) cells expressing either L- or E-selectin.
[a] Prof. Dr. C.-H. Wong, Dr. V. Wittmann,
Dr. A. K. Datta, K. M. Koeller
Department of Chemistry and The Skaggs Institute
for Chemical Biology
The Scripps Research Institute, 10550 North Torrey Pines Road
La Jolla, CA 92037 (USA)
Fax : (‡ 1)858-784-2409
E-mail: wong@scripps.edu
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Chem. Eur. J. 2000, 6, No. 1

162±171
The synthetic sLex conjugates were shown to bind specifically
to each selectin, in a manner similar to that of anti-selectin
monoclonal antibodies (mAb).
yields based on azide 5). In nitromethane, however, the
regioselectivity was acceptable, yielding 62% of 7, 7% of 8,
and 9% of 9.[28] The position of the newly formed glycosidic
bond in 7 was unambiguously deduced from the coupling
pattern in the 1H NMR spectrum of 7.[29] Acetylation of 7 and
removal of the isopropylidene group gave diol 11 (Scheme 2).
In contrast to acceptor 5, glycosylation of the 2-O-acetylated
Results and Discussion
Synthetic route to divalent 2: The structure GlcNAcb1,3(Glc-
NAcb1,6)GalbOR represents the branch point of the I blood
group antigen and the core structure of bivalent sLex
derivative 2. The preparation of N-glycosides of this core
structure has not been previously reported. The application of
glycosyl azides in the synthesis of N-glycoconjugates is well
established,[22] and this strategy was therefore chosen here.
Specifically, Kunz et al. have utilized sLex glycosyl azides in
the formation of multivalent sLex conjugates.[9, 10] As follows,
the main task in the synthesis of fluorescent conjugate 2 was to
develop an efficient route to 3,6-diglycosylated galactosyl
azides.
OAc
O
AcO
AcO
O
PhtN
O
b)
a)
7
O
O
N3
OAc
10
OAc
O
OAc
O
AcO
AcO
AcO
AcO
O
O
PhtN
c)
PhtN
HO
HO
AcO
AcO
AcO
O
O
O
O
N3
HO
N3
NPht
OAc
OAc
11
12
Scheme 2. a) Ac2O, pyridine, 97%; b) 80% HOAc, 74%; c) 6 (1.5 equiv),
AgOTf, collidine, CH2Cl2, 208C, 84%.
Synthesis of trisaccharide azide 12: In order to circumvent
extensive protecting group manipulations, the goal was to
synthesize 12 following the concept of minimal protection and
regioselective glycosylation.[23] The initial approach[24] was
based on 3,4-O-isopropylidene derivative 5 as a substrate for
preferred glycosylation at the primary 6-OH group. Thus,
trisaccharide 12 was available in eight steps from penta-O-
acetyl-b-d-galactopyranose (3) (Schemes 1 and 2). Treatment
acceptor 11 proceeded with remarkable regioselectivity at the
equatorial 3-position and furnished 12 in 84% yield.[30] In this
reaction, dichloromethane was the solvent of choice, since the
use of nitromethane under otherwise identical conditions
resulted in incomplete reaction. However, in both cases,
glycosylation at the 4-position of the 3,4-diol could not be
detected. Since it is also possible to selectively glycosylate 4,6-
diols in galactopyranosides at the 6-position,[6b, 23c,e,f] it was
expected that the 2-O-acetylated 3,4,6-triol 14 would be a
promising glycosyl acceptor for a simultaneous introduction
of two glucosamine residues in the 3- and 6-positions of the
galactose ring.[31]
OAc
O
AcO
AcO
Br
O
AcO
AcO
HO
HO
OH
O
OAc
O
OH
O
NPht
a)-c)
d)
6
e)
O
N3
OAc
N3
OH
OAc
OH
5
3
4
OAc
O
In an alternative synthetic strategy, 14 was efficiently
obtained by making use of a 1,2-orthoester (Scheme 3).
AcO
AcO
O
OAc
O
O
OH
O
PhtN
AcO
AcO
O
O
O
N3
PhtN
AcO
AcO
AcO
+
+
O
O
O
O
O
N3
O
HO
HO
HO
HO
AcO
AcO
AcO
OH
O
OH
O
O
O
OH
e)
O
f), g)
d)
a)-c)
NPht
O
N3
12
N3
3
NPht
O
O
OAc
8
9
7
14
OEt
13
OAc
11 %
7 %
32 %
9 %
37 %
62 %
CH2Cl2:
OAc
O
O
AcO
AcO
AcO
AcO
CH3NO2:
O
AcHN
AcHN
h), i)
Scheme 1. a) HBr, HOAc, b) NaN3, Bu4NHSO4, EtOAc/NaHCO3 soln;
AcO
AcO
OAc
O
OAc
O
c) NaOMe, MeOH, 92% (three steps); d) dimethoxypropane, p-TsOH,
H
O
O
AcO
AcO
AcO
AcO
NH-Cbz
N
‡
O
O
N3
DMF; then Et3HN TsO , MeOH, H2O, reflux 3 h, 86%; e) 6 (1.2 equiv),
NHAc
OAc
O
NHAc
OAc
AgOTf, collidine, 208C.
16
15
OH
O
HO
HO
of known galactosyl azide 4[25] with dimethoxypropane gave
3,4-O-isopropylidene derivative 5 in 86% yield after cleav-
age[26] of the mixed acetal at the 6-hydroxyl. Small amounts of
the 4,6-isomer (4%) and the 2,3:4,6-di-O-isopropylidene
compound (2%) were also isolated from the reaction mixture.
Diol acceptor 5 was then glycosylated with donor 6.[27]
Unexpectedly, when dichloromethane was employed as the
solvent, only 37% of the desired (1,6)-linked disaccharide 7
was formed. The remainder of the reaction products were
undesired regioisomer 8 (11%) and trisaccharide 9 (32%,
O
j)
AcHN
OH
HO
H
O
O
HO
HO
NH-Cbz
N
O
NHAc
OH
O
17
Scheme 3. a) HBr, HOAc; b) Bu4NBr, EtOH, collidine; c) NaOMe,
MeOH, 87% (three steps); d) TMS-N3 (10 equiv), THF, rt to reflux, then
80% HOAc, 90%; e) 6 (2.5 equiv), AgOTf, collidine, CH2Cl2, 308C,
70%; f) ethylene diamine; g) Ac2O, pyridine, 90% (two steps); h) H2/Pd-C;
i) Cbz-b-Ala-OH, HBTU, HOBt, iPr2NEt, 67% (two steps); j) NaOMe,
MeOH, 86%.
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163

C.-H. Wong et al.
FULL PAPER
Bromination[32] of pentaacetate 3, followed by cyclization[33]
and deprotection gave orthoester 13[34] as a mixture of epimers
(endo/exo 83:17). The orthoester functionality served as a
means to both distinguish the 2-OH group from the remaining
hydroxyls and activate the anomeric carbon. Thus, treatment
of triol 13 with trimethylsilyl azide[35] provided triol 14 in a
single step in 90% yield. In this case, acidic work-up was
necessary to remove TMS ethers generated in situ. Silver
triflate promoted diglycosylation of 14 with donor 6 in
dichloromethane resulted in the formation of trisaccharide
12 in a yield of 70%. Small amounts of intermediate 11
formed in the reaction were easily removed by flash
chromatography. With this approach, trisaccharide 12 was
accessible from pentaacetate 3 in only five steps and high
overall yield.
prevent product inhibition[39] by UDP, CMP, and GDP,
respectively, and to facilitate product isolation from these
nucleotides by size-exclusion chromatography. Notably, 17,
18, and 19 were accepted as substrates by the transferases
despite the presence of the unnatural Cbz-b-alanine group at
the reducing terminus. Finally, Cbz-protected glycoconjugate
20 was deprotected hydrogenolytically and reacted with
BODIPY-succinimidyl ester 21, leading to fluorescently
labeled nonasaccharide-b-alanine conjugate
2 in 89%
yield.
In summary, following the protecting group strategy
presented in Schemes 3 and 4, the synthesis of 2 was
accomplished in only 15 steps from commercially available 3.
Synthesis of fluorescent- and biotin-labeled derivatives 23 and
25: Recently, the synthesis of amino-substituted sLex deriv-
ative 22 was described. Compound 22 was employed in the
preparation of sLex dimers with oligoethylene glycol based
spacers of varying chain length.[18] As shown in Scheme 5, 22
was also coupled to the succinimidyl esters 21 and 24 to
produce fluorescent BODIPY-labeled sLex derivative 23 and
biotinylated sLex derivative 25 in 83% and 65% yields,
respectively. Thus, labeled monovalent sLex derivatives were
also easily accessible by short chemoenzymatic routes.
Synthesis of trisaccharide-b-alanine conjugate 17: Deprotec-
tion of 12 with ethylene diamine,[36] followed by treatment
with acetic anhydride, furnished peracetylated trisaccharide
15 in 90% yield. Hydrazine hydrate could not be used to
remove the phthaloyl groups as a result of a side reaction of
the anomeric azido function. The azido function was smoothly
reduced hydrogenolytically on palladium black, and the
resulting glycosyl amine was coupled to Cbz-protected b-
alanine with HBTU[37] as coupling reagent. O-Deacetylation
utilizing Zemplen conditions gave glycoconjugate 17, which
was used as a primer in subsequent glycosyltransferase-
catalyzed[38] glycosylations.
Application of fluorescent sLex derivatives as cell-staining
reagents: The cell-staining utility of the fluorescently labeled
sLex conjugates was then demonstrated. First, stable CHO-K1
cell lines expressing either L-selectin or E-selectin were
generated. Full length L-[44] and E-selectin[45] were amplified
with primers based on the published sequences by using the
reverse transcriptase (RT) product as the template. These
were subcloned in pcDNA.3, a mammalian expression vector
with CMV promoter, and Neo gene as the selectable marker.
In CHO-K1, the selectins were expressed on the cell surface
with normal transmembrane topology. After transfection,
cells incorporating the expression vector were selected by
G418 resistance. For further selection, the individual colonies
were grown in duplicate plates. One of the plates was used for
Enzymatic glycosylations and attachment of the fluorophor:
Treatment of primer 17 with b-1,4-galactosyltransferase (b-
1,4-GalT) and 2.6 equivalents of UDP-Gal gave pentasac-
charide 18 in quantitative yield (Scheme 4). Similarly, two
sialic acid residues were introduced with a-2,3-sialyltransfer-
ase (a-2,3-SiaT) to give heptasaccharide 19 (92% yield).
Subsequent addition of two fucose residues employing a-1,3-
fucosyltransferase V (a-1,3-FucT V) then afforded nonasac-
charide 20 (see above, 85% yield). Alkaline phosphatase
(AP) was added to all three glycosylation reactions in order to
OH
HO
HO
HO
O
OH
O
b)
a)
HO
O
17
O
H
NH-Cbz
N
OH
OH
HO
HO
O
NHAc
O
18
OH
OH
HO
CO2H
OH
O
HO
O
O
AcHN
HO
OH
OH
HO
O
d), e)
c)
2
20
HO
O
OH
O
H
HO
O
N
NH-Cbz
CO2H
O
N
N
O
OH
B
F
O
N
OH
O
O
O
AcHN
NHAc
21
F
HO
O
HO
OH
OH
HO
19
Scheme 4. a) UDP-Gal (2.6 equiv), b-1,4-GalT, AP, quant.; b) CMP-NeuAc (3.6 equiv), a-2,3-SiaT, AP, 92%; c) GDP-Fuc (3 equiv), a-1,3-FucT V, AP,
85%; d) H2/Pd-C; e) 21, Et3N, DMF, H2O, 89% (two steps).
164
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Sialyl Lewis X Derivatives
162±171
the cells were washed, they were fixed and visualized under a
fluorescence microscope. The antibody was detected at
570 nm by using TRITC-conjugated anti-mouse IgG.
After the expression of the cell-surface E-selectin was
established, the cells were incubated with the BODIPY-
labeled mono- and divalent sLex conjugates. The cells were
washed again, fixed, and visualized under a fluorescence
microscope. BODIPY-labeled conjugates were detected by
intrinsic fluorescence at 508 nm.
OH
HO
CO2H
O
OH
O
OH
O
O
H
N
O
O
AcHN
HO
O
O
N
B
N
OH
NHAc
OH
H
HO
F
O
N
H3C
O
F
OH
23
OH
HO
a)
OH
HO
CO2H
O
OH
O
OH
O
O
O
OH
AcHN
HO
O
NH2
O
OH
O
N
H
NHAc
Figure 2 shows the cell-staining experiments performed
with the CHO-K1 cells expressing E-selectin, while Figure 3
shows similar experiments conducted with L-selectin. The
HO
H3C
O
OH
OH
HO
22
O
NH
H
HN
H
O
b)
O
S
N
O
O
24
O
OH
NH
H
HO
HN
CO2H
O
OH
O
OH
O
O
H
H
O
O
AcHN
HO
O
N
O
S
N
OH
NHAc
OH
H
HO
O
H3C
O
OH
25
OH
HO
Scheme 5. a) 21, Et3N, DMF, 83%; b) 24, Et3N, DMF, 65%.
ELISA, employing monoclonal antibodies for either L-selec-
tin (mAb CD62L) or E-selectin (mAb CD62E), respectively.
In the ELISA assay, individual CHO-K1 cell lines expressing
L- or E-selectin were identified. The cell lines with maximal
OD450 were used for fluorescence activated cell sorting
(FACS) analysis, and the top 0.1% of cells showing expression
were collected (Figure 1). These cells were grown in MEM
(minimum essential medium) medium containing 5% fetal
Figure 2. CHO-K1 cells expressing E-selectin: a) under transmitted light;
b) stained with BODIPY-labeled sLex monomer 23; c) cell stained with
CD62E mAb followed by TRITC-conjugated anti-mouse IgG; d) under
transmitted light; e) stained with BODIPY-labeled sLex dimer 2; f) stained
with CD62E mAb followed by TRITC-conjugated anti-mouse IgG. CHO-
K1 cells negative for E-selectin expression did not exhibit staining with 2,
23, or CD62E mAb (data not shown).
1
calf serum, G418 (100 mgmL ), and 1% l-glutamine, and
finally selected by limited dilution method.
In another set of experiments, HUVEC cells were stimu-
lated to produce cell-surface E-selectin by treatment with
lipopolysaccharide (LPS) and/or interferon-1b (IFN-1b) fol-
lowing previously published procedures.[2d, 12] Expression of
E-selectin on the cell surface was verified by staining the
activated cells with mAb CD62E, as described above. After
staining of HUVEC cells expressing E-selectin is shown in
Figure 4. At the present levels of selectin expression, the cell-
staining pattern observed is similar for the mAb and the sLex
derivatives for all cell lines in-
vestigated. Thus, the usefulness
of the labeled sLex derivatives
in localizing E- and L-selectin
on various cell surfaces has
been established by these ex-
periments. Though E-selectin
expression has been visualized
with fluorescent sLex-based li-
gands previously, we are un-
aware of other reports of this
nature involving the detection
of L-selectin.
In the binding analysis of
carbohydrate ligands for the
Figure 1. FACS analysis of the TRITC-stained CHO-K1 stable cell lines: a) cells expressing E-selectin were
stained with CD62E mAb followed by TRITC-conjugated anti-mouse IgG; b) cells expressing L-selectin were
stained with CD62L mAb followed by TRITC-conjugated anti-mouse IgG. 0.1% of the maximally intense cells
were sorted out.
selectins, it is necessary to ver-
ify that mimetic structures have
access to the appropriate car-
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165

C.-H. Wong et al.
FULL PAPER
Conclusion
A short and efficient synthesis of fluorescently labeled
bivalent sLex-b-alanine conjugate 2 has been demonstrated
with a combined chemical and enzymatic approach. The key
features of the synthetic strategy were the transformation of
unprotected orthoester 13 into selectively protected galacto-
syl azide 14, and subsequent regioselective diglycosylation to
give trisaccharide 12. The b-alanine spacer introduced sub-
sequently facilitated the incorporation of molecular probes,
and also allows the possible formation of numerous neo-
glycoconjugates.[40] Glycosyltransferase-catalyzed elongation
of the carbohydrate branches proceeded in excellent yields,
despite the presence of the nonnatural Cbz-b-alanine group.
Commencing with galactose pentaacetate 3, fluorescently
labeled conjugate 2 was obtained in only 15 steps and an
overall yield of 20%. To demonstrate the utility of labeled
sLex-derivatives as tools in localizing cell-surface selectins, the
sLex conjugates were subjected to a cell-staining assay. Similar
cell-staining patterns of the sLex derivatives and anti-selectin
mAbs on the surface of activated HUVEC cells and CHO-K1
cells was observed. As such, the usefulness of small molecular
sLex-derivatives as cell-staining reagents has been established.
Furthermore, these results may lead to the development of a
fluorescence-based selectin binding assay in the near future.
Figure 3. CHO-K1 cells expressing L-selectin: a) under transmitted light;
b) stained with BODIPY-labeled sLex monomer 23; c) cell stained with
CD62L mAb followed by TRITC-conjugated anti-mouse IgG; d) under
transmitted light: e) stained with BODIPY-labeled sLex dimer 2; f) stained
with CD62L mAb followed by TRITC-conjugated anti-mouse IgG. CHO-
K1 cells negative for L-selectin expression did not exhibit staining with 2,
23, or CD62L mAb (data not shown).
Experimental Section
General methods: b-d-Galactopyranosyl azide (4),[25, 32, 42] 3,4,6-tri-O-
acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl bromide (6),[27] 1,2-O-
((1RS)-1-ethoxyethylidene)-b-d-galactopyranose (13),[32±34] and monoam-
monium GDP-Fuc[43] were prepared according to published procedures.
1,2,3,4,6-Penta-O-acetyl-b-d-galactopyranose (3), N-hydroxysuccinimido-
biotin (24), UDP-Gal, b-1,4-GalT, and alkaline phosphatase (type VII-N,
from bovine intestinal mucosa, P-2276) were purchased from Sigma (St.
Louis, MO). CMP-NeuAc (sodium salt) was purchased from Calbiochem
(San Diego, CA). 4,4-Difluoro-5,7-dimethyl-4-bora-[3a,4a]-diaza-s-inda-
cene-3-propionic acid succinimidyl ester (21) (BODIPY FL, SE) was
1
purchased from Molecular Probes (Eugene, OR). a-2,3-SiaT (3 UmL
)
and a-1,3-FucT V (2.16 UmL 1) were a kind donation from Cytel (San
Diego, CA). Flash chromatography (FC) was performed on Mallinckrodt
silica gel 60 (230 ± 400 mesh). Analytical thin-layer chromatography was
performed by 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 molybdo-
phoshate. Size-exclusion chromatography was performed on Bio-Gel P-2
Gel, fine and Bio-Gel P-4 Gel, fine (Bio-Rad Laboratories, 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 dH ˆ 7.26, D2O dH ˆ 4.80, [D6]DMSO dH ˆ 2.50) or internal
standard TMS (dH ˆ 0.00). 13C chemical shifts are referenced to the solvent
signal (CDCl3 dC ˆ 77.0, [D6]DMSO dC ˆ 39.5) or to [D6]DMSO (dC ˆ 39.5)
as external standard. High resolution mass spectra (HR-MS) were recorded
by using fast atom bombardment (FAB) method in a m-nitrobenzyl alcohol
matrix doped with NaI or CsI.
Figure 4. HUVEC cells expressing cell surface E-selectin: a) a cell under
transmitted light; b) cell stained with BODIPY-labeled sLex monomer 23;
c) cell stained with CD62E mAb followed by TRITC-conjugated anti
mouse IgG; d) another cell under transmitted light; e) cell stained with
BODIPY-labeled sLex dimer 2; f) cell stained with CD62E mAb followed
by TRITC-conjugated anti mouse IgG. HUVEC cells negative for
E-selectin expression did not exhibit staining with 2, 23, or CD62E mAb
(data not shown).
3,4-O-Isopropylidene-b-d-galactopyranosyl azide (5), 4,6-O-isopropyli-
dene-b-d-galactopyranosyl azide, and 2,3:4,6-di-O-isopropylidene-b-d-gal-
actopyranosyl azide: b-d-Galactopyranosyl azide (4) (0.97 g, 4.73 mmol)
was dissolved in DMF (10 mL) and 2,2-dimethoxypropane (20 mL) and
heated to 658C. p-Toluenesulfonic acid (90 mg, 0.473 mmol) was added and
the solution was stirred at 658C for 5 h. After the solution was cooled down
to rt, Et3N (660 mL, 4.73 mmol) was added and the mixture was stirred for
15 min. The mixture was concentrated to dryness and toluene was
evaporated twice from the residue in order to remove traces of Et3N. The
bohydrate binding site. This is the advantage of using small
molecular sLex conjugates rather than anti-selectin mAbs in
this type of experiment. Selectins with accessible carbohy-
drate recognition domains on the cell surface can be assessed
directly utilizing the described sLex constructs.
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Sialyl Lewis X Derivatives
162±171
residue was dissolved in MeOH/H2O (10:1) (40 mL) and boiled for 30 min
until TLC (hexane/ethyl acetate 1:2) showed the complete disappearance
of the intermediate product 6-O-(2-methoxy-2-propyl)-3,4-O-isopropyli-
dene-b-d-galactopyranosyl azide (Rf ˆ 0.45). The solution was concentrat-
ed and coevaporated twice with toluene. FC (80 g silica, hexane/ethyl
acetate 1:2, then ethyl acetate and finally ethyl acetate/methanol 9:1) gave
2,3:4,6-di-O-isopropylidene-b-d-galactopyranosyl azide (20 mg, 2%), then
4-H), 3.96 (dd, 1H, J ˆ 5.5, 7.1 Hz, Gal 3-H), 3.91 ± 3.87 (m, 2H, Gal 5-H,
GlcN 5-H), 3.84 (dd, 1H, J ˆ 7.8, 10.7 Hz, Gal 6'-H), 3.36 (ddd, 1H, J ˆ 3.1,
7.1, 8.8 Hz, after addition of D2O: dd, J ˆ 7.1, 8.8 Hz, Gal 2-H), 2.33 (d, 1H,
J ˆ 3.1 Hz, exchangeable, Gal 2-OH), 2.13 (s, 3H, C(O)CH3), 2.04 (s, 3H,
C(O)CH3), 1.87 (s, 3H, C(O)CH3), 1.46 (s, 3H, C(CH3)2), 1.25 (s, 3H,
C(CH3)2); 13C NMR (100 MHz, CDCl3): d ˆ 170.7, 170.1, 169.5, 134.3, 131.3,
123.7, 110.5 (CMe2), 98.2, 89.1, 78.2, 74.1, 73.4, 72.9, 71.9, 70.7, 68.8, 68.7,
61.8, 54.5, 27.9, 26.0, 20.8, 20.6, 20.4; HR-MS (pos. FAB, NBA/CsI) calcd for
5
(1.00 g, 86%) and 4,6-O-isopropylidene-b-d-galactopyranosyl azide
‡
(51 mg, 4%).
C29H34N4O14Cs [M ‡ Cs] m/z: 795.1126, found 795.1139.
Data for 8: Rf ˆ 0.23 (toluene/ethyl acetate 1:1); syrup; 1H NMR
(400 MHz, CDCl3): d ˆ 7.87 ± 7.85 (m, 2H, Pht), 7.76 ± 7.73 (m, 2H, Pht),
5.89 (dd, 1H, J ˆ 9.0, 10.7 Hz, GlcN 3-H), 5.52 (d, 1H, J ˆ 8.5 Hz, GlcN
1-H), 5.17 (dd, 1H, J ˆ 9.1, 10.2 Hz, GlcN 4-H), 4.40 (d, 1H, J ˆ 8.6 Hz, Gal
1-H), 4.35 (dd, 1H, J ˆ 8.5, 10.7 Hz, GlcN 2-H), 4.31 (dd, 1H, J ˆ 5.2,
12.3 Hz, GlcN 6-H), 4.20 (dd, 1H, J ˆ 2.3, 12.3 Hz, GlcN 6'-H), 3.96 ± 3.92
(m, 1H, Gal or GlcN 5-H), 3.93 (dd, 1H, J ˆ 2.1, 5.7 Hz, Gal 4-H), 3.92 ±
3.82 (m, 2H, Gal 6-H, 6-OH), 3.84 (dd, 1H, J ˆ 5.7, 6.9 Hz, Gal 3-H), 3.80 ±
3.76 (m, 1H, Gal or GlcN 5-H), 3.75 ± 3.69 (m, 1H, Gal 6'-H), 3.46 (dd, 1H,
J ˆ 6.9, 8.6 Hz, Gal 2-H), 2.10 (s, 3H, C(O)CH3), 2.04 (s, 3H, C(O)CH3),
1.87 (s, 3H, C(O)CH3), 1.27 (s, 3H, C(CH3)2), 0.87 (s, 3H, C(CH3)2);
13C NMR (100 MHz, CDCl3): d ˆ 170.8, 170.1, 169.5, 134.0, 132.0 (br),
123.4, 110.4 (CMe2), 99.8, 88.2, 81.8, 77.8, 75.0, 73.3, 71.9, 70.4, 68.9, 62.2,
62.1, 54.9, 27.5, 25.6, 20.7, 20.6, 20.5; HR-MS (pos. FAB, NBA/CsI) calcd for
Data for 5: Rf ˆ 0.21 (hexane/ethyl acetate 1:2); white crystals (ethyl
acetate/hexane); m.p. 113.5 ± 1148C; 1H NMR (400 MHz, CDCl3): d ˆ 4.51
(d, 1H, J ˆ 8.9 Hz, 1-H), 4.22 (dd, 1H, J ˆ 2.1, 5.5 Hz, 4-H), 4.11 (dd, 1H,
J ˆ 5.5, 7.2 Hz, 3-H), 4.04 ± 3.96 (m, 2H, 5-H, 6-H), 3.87 (m, 1H, 6'-H), 3.50
(ddd, 1H, J ˆ 3.2, 7.2, 8.9 Hz, 2-H), 2.50 (d, 1H, J ˆ 3.2 Hz, 2-OH), 2.18 (m,
1H, 6-OH), 1.53 (s, 3H, CH3), 1.37 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3): d ˆ 110.7 (CMe2), 89.7, 78.5, 75.2, 73.7, 73.1, 62.4, 28.0 (CH3), 26.2
‡
(CH3); HR-MS (pos. FAB, NBA/CsI) calcd for C9H15N3O5Cs [M ‡ Cs]
m/z: 378.0066, found 378.0077; anal. calcd for C9H15N3O5: C 44.08, H 6.17,
N 17.13; found: C 43.99, H 6.32, N 16.97.
4,6-O-Isopropylidene-b-d-galactopyranosyl azide: Rf ˆ 0.06 (hexane/ethyl
acetate 1:2); white crystals (ethyl acetate/hexane); m.p. 145 ± 1468C;
1H NMR (250 MHz, CDCl3): d ˆ 4.53 (d, 1H, J ˆ 8.0 Hz, 1-H), 4.21 (dd,
1H, J ˆ 1.1, 3.3 Hz, 4-H), 4.10 (dd, 1H, J ˆ 2.2, 13.0 Hz, 6-H), 3.99 (dd, 1H,
J ˆ 1.7, 13.0 Hz, 6'-H), 3.69 ± 3.57 (m, 2H, 2-H, 3-H), 3.48 (m, 1H, 5-H), 2.55
(brs, 2H, 2-OH, 3-OH), 1.48 (s, 3H, CH3), 1.47 (s, 3H, CH3); 13C NMR
(62.9 MHz, CDCl3) d ˆ 99.3 (CMe2), 90.1 (C-1), 72.8, 71.2, 68.4, 67.8, 62.3,
29.0 (CH3), 18.6 (CH3); anal. calcd for C9H15N3O5: C 44.08; H, 6.17; N,
17.13; found: C 44.26; H 6.24; N, 17.00.
‡
C29H34N4O14Cs [M ‡ Cs] m/z: 795.1126, found 795.1142.
Data for 9: Rf ˆ 0.40 (toluene/ethyl acetate 1:1); syrup; 1H NMR
(400 MHz, CDCl3): d ˆ 7.86 ± 7.82 (m, 4H, Pht), 7.76 ± 7.71 (m, 4H, Pht),
5.86 (dd, 1H, J ˆ 9.0, 10.7 Hz, GlcN 3-H), 5.78 (dd, 1H, J ˆ 9.1, 10.8 Hz,
GlcN 3-H), 5.45 (d, 1H, J ˆ 8.5 Hz, GlcN 1-H), 5.44 (d, 1H, J ˆ 8.5 Hz,
GlcN 1-H), 5.17 (ªtº, 1H, J ˆ 9.5 Hz, GlcN 4-H), 5.14 ('t', 1H, J ˆ 9.5 Hz,
GlcN 4-H), 4.33 ± 4.26 (m, 4H, 2GlcN 2-H, 2GlcN 6-H), 4.18 ± 4.14 (m, 2H,
2GlcN 6'H), 4.09 (d, 1H, J ˆ 8.7 Hz, Gal 1-H), 3.91 (dd, 1H, J ˆ 2.7, 11 Hz,
Gal 6-H), 3.89 (ddd, 1H, J ˆ 2.3, 5.1, 10.2 Hz, GlcN 5-H), 3.84 (ddd, 1H,
J ˆ 2.3, 4.2, 10.1 Hz, GlcN 5-H), 3.74 ± 3.65 (m, 4H, Gal 3-H, 4-H, 5-H, 6'-
H), 3.32 (dd, 1H, J ˆ 6.8, 8.7 Hz, Gal 2-H), 2.10 (s, 6H, 2C(O)CH3), 2.03 (s,
3H, C(O)CH3), 2.02 (s, 3H, C(O)CH3), 1.857 (s, 3H, C(O)CH3), 1.855 (s,
3H, C(O)CH3), 1.22 (s, 3H, C(CH3)2), 0.79 (s, 3H, C(CH3)2); 13C NMR
(100 MHz, CDCl3): d ˆ 170.8, 170.7, 170.1, 169.5, 134.3, 134.0, 123.6, 110.3
(CMe2), 99.6, 98.4, 87.6, 81.5, 77.7, 77.3, 73.9, 73.1, 71.9, 71.8, 70.6, 70.4, 68.83,
68.77, 62.2, 61.8, 54.9, 54.4, 27.4, 25.5, 20.8, 20.7, 20.6, 20.4; HR-MS (pos.
2,3:4,6-Di-O-isopropylidene-b-d-galactopyranosyl azide: Rf ˆ 0.49 (hex-
ane/ethyl acetate 1:2); syrup; 1H NMR (250 MHz, CDCl3): d ˆ 4.80 (d, 1H,
J ˆ 8.6 Hz, 1-H), 4.48 (dd, 1H, J ˆ 1.4, 2.8 Hz, 4-H), 4.16 (dd, 1H, J ˆ 2.2,
13.1 Hz, 6-H), 4.04 (dd, 1H, J ˆ 1.6, 13.1 Hz, 6'-H), 3.91 (dd, 1H, J ˆ 8.6,
9.4 Hz, 2-H), 3.56 (dd, 1H, J ˆ 2.8, 9.4 Hz, 3-H), 3.44 (ddd, 1H, J ˆ 1.4, 1.6,
2.2 Hz, 5-H), 1.50 (s, 6H, 2CH3), 1.473 (s, 3H, CH3), 1.466 (s, 3H, CH3);
13C NMR (62.9 MHz, CDCl3): d ˆ 111.5 (CMe2), 98.7 (CMe2), 89.2 (C-1),
78.4, 72.0, 69.0, 66.2, 62.7, 29.0 (CH3), 26.5 (CH3), 26.4 (CH3), 18.5 (CH3).
6-O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-3,4-
O-isopropylidene-b-d-galactopyranosyl azide (7), 2-O-(3,4,6-tri-O-acetyl-
2-deoxy-2-phthalimido-b-d-glucopyranosyl)-3,4-O-isopropylidene-b-d-gal-
actopyranosyl azide (8), and 2,6-bis-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
imido-b-d-glucopyranosyl)-3,4-O-isopropylidene-b-d-galactopyranosyl
azide (9): Procedure A: A solution of 5 (60 mg, 0.244 mmol) and 2,4,6-
collidine (43 mL, 0.324 mmol) in nitromethane (3.5 mL) was mixed with
powdered molecular sieves (4 Š) (ca. 300 mg) and stirred under argon for
1.5 h at rt. Freshly dried AgOTf (76 mg, 0.295 mmol) was added and the
‡
FAB, NBA/CsI) calcd for C49H52N5O23Cs [M ‡ Cs] m/z: 1211.2107, found
1211.2148.
2-O-Acetyl-6-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyra-
nosyl)-3,4-O-isopropylidene-b-d-galactopyranosyl azide (10): A solution of
7 (460 mg, 0.694 mmol) and acetic anhydride (656 mL, 6.94 mmol) in
pyridine (20 mL) was stirred for 12 h at rt. The mixture was concentrated to
dryness and coevaporated with toluene (3 Â 5 mL) and diethyl ether (3 Â
5 mL). The resulting oil was dried in vacuo to give 10 (474 mg, 97%) which
was used in the next step without further purification: Rf ˆ 0.55 (toluene/
ethyl acetate 1:1.5); syrup; 1H NMR (400 MHz, CDCl3): d ˆ 7.89 ± 7.81
(brm, 2H, Pht), 7.75 ± 7.71 (m, 2H, Pht), 5.82 (dd, 1H, J ˆ 9.1, 10.7 Hz,
GlcN 3-H), 5.50 (d, 1H, J ˆ 8.5 Hz, GlcN 1-H), 5.20 (dd, 1H, J ˆ 9.1,
10.2 Hz, GlcN 4-H), 4.79 (dd, 1H, J ˆ 6.8, 8.2 Hz, Gal 2-H), 4.35 (dd, 1H,
J ˆ 4.3, 12.4 Hz, GlcN 6-H), 4.33 (dd, 1H, J ˆ 8.5, 10.7 Hz, GlcN 2-H), 4.21
(dd, 1H, J ˆ 2.3, 12.3 Hz, GlcN 6'-H), 4.17 (d, 1H, J ˆ 8.2 Hz, Gal 1-H),
4.09 ± 4.02 (m, 3H, Gal 3-H, 4-H, 6-H), 3.91 ± 3.81 (m, 3H, GlcN 5-H, Gal
5-H, 6'-H), 2.13 (s, 3H, C(O)CH3), 2.06 (s, 3H, C(O)CH3), 2.04 (s, 3H,
C(O)CH3), 1.87 (s, 3H, C(O)CH3), 1.49 (s, 3H, C(CH3)2), 1.24 (s, 3H,
C(CH3)2); 13C NMR (100 MHz, CDCl3): d ˆ 170.7, 170.1, 169.5, 169.4, 134.3,
131.3, 123.6, 110.8 (CMe2), 98.4, 86.6, 75.9, 73.9, 73.2, 71.9, 71.4, 70.6, 69.0,
68.8, 61.8, 54.5, 27.4, 25.9, 20.8, 20.6, 20.5; HR-MS (pos. FAB, NBA/CsI)
yellowish mixture was cooled to
208C. A solution of 6 (146 mg,
0.293 mmol) in nitromethane (3.5 mL) was added dropwise during 15 min
to the reaction mixture. After 3 h at 208C, the mixture was allowed to
warm up slowly to rt. After a total reaction time of 8 h, the mixture was
diluted with acetonitrile, filtered through Celite, and evaporated to give a
syrup (324 mg). FC (25 g silica, toluene/ethyl acetate 1:1, then ethyl
acetate) gave 9 (24 mg, 9% based on 5), then 7 (100 mg, 62%), 8 (11 mg,
7%), and finally unreacted 5 (12 mg, 20%).
Procedure B: Powdered molecular sieves (4 Š) (ca. 150 mg) was added to a
solution of 5 (60 mg, 0.244 mmol), 2,4,6-collidine (47 mL, 0.353 mmol), and
freshly dried AgOTf (83 mg, 0.322 mmol) in dichloromethane (3 mL). The
suspension was stirred under argon for 1 h at rt and then cooled to 208C.
A solution of 6 (146 mg, 0.293 mmol) was added dropwise during 15 min to
the reaction mixture in dichloromethane (350 mL). After 1 h at 208C, the
mixture was allowed to warm up to rt and stirred for another 40 min. After
dilution with dichloromethane, the mixture was filtered through Celite and
evaporated. FC (25 g silica, toluene/ethyl acetate 1:1, then ethyl acetate)
gave 9 (83 mg, 32% based on 5), 7 (59 mg, 37%), 8 (18 mg, 11%), and
unreacted 5 (12 mg, 20%).
Data for 7: Rf ˆ 0.30 (toluene/ethyl acetate 1:1); syrup; 1H NMR (400 MHz,
CDCl3): d ˆ 7.90 ± 7.82 (brm, 2H, Pht), 7.74 ± 7.72 (m, 2H, Pht), 5.80 (dd,
1H, J ˆ 9.1, 10.7 Hz, GlcN 3-H), 5.52 (d, 1H, J ˆ 8.5 Hz, GlcN 1-H), 5.21
(dd, 1H, J ˆ 9.1, 10.2 Hz, GlcN 4-H), 4.37 ± 4.31 (m, 2H, GlcN 6-H, 2-H),
4.26 (d, 1H, J ˆ 8.8 Hz, Gal 1-H), 4.21 (dd, 1H, J ˆ 2.4, 12.3 Hz, GlcN 6'-H),
4.05 (dd, 1H, J ˆ 2.6, 10.7 Hz, Gal 6-H), 4.02 (dd, 1H, J ˆ 2.2, 5.5 Hz, Gal
‡
calcd for C31H36N4O15Cs [M ‡ Cs] m/z: 837.1231, found 837.1251.
2-O-Acetyl-6-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyra-
nosyl)-b-d-galactopyranosyl azide (11):
A solution of 10 (459 mg,
0.651 mmol) in HOAc/H2O 80:20 (9 mL) was stirred for 10 h at 60 ±
708C. The mixture was concentrated and coevaporated with toluene (3 Â
5 mL). Purification by FC (45 g silica, hexane/ethyl acetate 1:6) gave 11
(322 mg, 74%): Rf ˆ 0.28 (hexane/ethyl acetate 1:6); white solid (lyophi-
lized from benzene); 1H NMR (400 MHz, CDCl3): d ˆ 7.88 ± 7.85 (m, 2H,
Pht), 7.78 ± 7.74 (m, 2H, Pht), 5.78 (dd, 1H, J ˆ 9.1, 10.7 Hz, GlcN 3-H), 5.49
(d, 1H, J ˆ 8.5 Hz, GlcN 1-H), 5.14 (dd, 1H, J ˆ 9.1, 10.2 Hz, GlcN 4-H),
4.88 (dd, 1H, J ˆ 8.8, 9.8 Hz, Gal 2-H), 4.36 (d, 1H, J ˆ 8.8 Hz, Gal 1-H),
Chem. Eur. J. 2000, 6, No. 1
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0601-0167 $ 17.50+.50/0
167

C.-H. Wong et al.
FULL PAPER
4.33 (dd, 1H, J ˆ 2.2, 12.1 Hz, GlcN 6-H), 4.29 (dd, 1H, J ˆ 8.5, 10.7 Hz,
GlcN 2-H), 4.22 (dd, 1H, J ˆ 5.3, 12.4 Hz, GlcN 6'-H), 3.98 (dd, 1H, J ˆ 7.3,
10.3 Hz, Gal 6-H), 3.97 (m, 1H, Gal 4-H), 3.92 (ddd, 1H, J ˆ 2.4, 5.3,
10.2 Hz, GlcN 5-H), 3.86 (dd, 1H, J ˆ 5.9, 10.3 Hz, Gal 6'-H), 3.59 (ddd,
1H, J ˆ 1.2, 5.9, 7.2 Hz, Gal 5-H), 3.57 (m, 1H, Gal 3-H), 3.08 (brd, 1H, J ˆ
4.6 Hz, Gal OH), 2.87 (brd, 1H, J ˆ 8.4 Hz, Gal OH), 2.15 (s, 3H,
C(O)CH3), 2.13 (s, 3H, C(O)CH3), 2.05 (s, 3H, C(O)CH3), 1.87 (s, 3H,
C(O)CH3); 13C NMR (100 MHz, CDCl3): d ˆ 170.9, 170.8, 170.1, 169.5,
134.4, 131.3, 123.7, 98.2, 87.8, 74.8, 72.1, 72.0, 70.5, 68.8, 67.8, 67.3, 61.8, 54.4,
20.9, 20.8, 20.6, 20.4; HR-MS (pos. FAB, NBA/CsI) calcd for
deprotected trisaccharide azide (Rf ˆ 0.45, iPrOH/1m NH4OAc 2:1) was
stirred with pyridine (10 mL) and acetic anhydride (5 mL) at rt for 18 h.
The reaction mixture was evaporated and the residue was coevaporated
with toluene (3 Â 10 mL) and MeOH (2 Â 5 mL) and purified by FC (80 g
silica, CH2Cl2/MeOH, 16:1) to yield 15 (185 mg, 90%): White solid; Rf ˆ
0.30 (CH2Cl2/MeOH, 16:1); 1H NMR (400 MHz, CDCl3): d ˆ 6.10 (d, 1H,
J ˆ 8.3 Hz, GlcN NH), 5.56 (d, 1H, J ˆ 7.9 Hz, GlcN NH), 5.48 (dd, 1H, J ˆ
9.2, 10.7 Hz, GlcN 3-H), 5.44 (dd, 1H, J ˆ 9.2, 10.7 Hz, GlcN 3-H), 5.37 (d,
1H, J ˆ 3.7 Hz, Gal 4-H), 5.09 ± 5.02 (m, 4H), 4.83 (d, 1H, J ˆ 8.3 Hz), 4.49
(d, 1H, J ˆ 8.9 Hz), 4.41 (dd, 1H, J ˆ 12.3, 2.5 Hz), 4.26 (dd, 1H, J ˆ 12.3,
4.5 Hz), 4.15 (dd, 1H, J ˆ 12.3, 2.4 Hz), 4.04 (dd, 1H, J ˆ 12.3, 3.7 Hz),
3.91 ± 3.83 (m, 3H), 3.74 ± 3.56 (m, 4H), 3.34 (ddd, 1H, J ˆ 7.9, 7.9, 10.7 Hz,
GlcN 2-H), 2.135 (s, 3H, C(O)CH3), 2.130 (s, 3H, C(O)CH3), 2.126 (s, 3H,
C(O)CH3), 2.09 (s, 3H, C(O)CH3), 2.025 (s, 9H, 3C(O)CH3), 2.019 (s, 3H,
C(O)CH3), 1.95 (s, 3H, C(O)CH3), 1.91 (s, 3H, C(O)CH3); 13C NMR
(100 MHz, CDCl3): d ˆ 171.13, 170.69, 170.61, 170.43, 170.40, 169.58,
169.51, 169.46, 100.19, 99.66, 88.07, 75.99, 74.89, 71.77, 71.71, 71.68, 71.12,
69.94, 69.05, 68.57, 68.51, 67.26, 61.82, 61.03, 56.06, 55.34, 23.30, 23.27, 20.89,
20.75, 20.64, 20.57; HR-MS (pos. FAB, NBA/NaI) calcd for C38H53N5O23Na
‡
C28H32N4O15Cs [M ‡ Cs] m/z: 797.0919, found 797.0909; anal. calcd for
C28H32N4O15: C 50.60, H 4.85, N 8.43; found: C 50.84, H 4.91, N, 8.11.
2-O-Acetyl-3,6-bis-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glu-
copyranosyl)-b-d-galactopyranosyl azide (12): Procedure A: Powdered
molecular sieves (4 Š) (ca. 400 mg) was added to a solution of 11 (185 mg,
0.278 mmol), 2,4,6-collidine (66 mL, 0.497 mmol), and freshly dried AgOTf
(116 mg, 0.452 mmol) in dichloromethane (3.9 mL). The suspension was
stirred under argon for 1.5 h at rt and then cooled to 238C. A solution of 6
(204 mg, 0.409 mmol) in dichloromethane (3.9 mL) was added dropwise
during 10 min to the reaction mixture. After stirring for 2 h at 238C, the
mixture was allowed to warm up to rt overnight, diluted with acetonitrile,
filtered through Celite and evaporated. FC (100 g silica, hexane/ethyl
acetate 1:4) gave 12 (254 mg, 84%): Rf ˆ 0.43 (hexane/ethyl acetate 1:4);
‡
[M ‡ Na] m/z: 970.3029, found 970.3011; anal. calcd for C38H53N5O23: C
48.15, H 5.64, N 7.39; found: C 48.02, H 5.85, N, 7.21.
N3-Benzyloxycarbonyl-N1-[3,6-bis-O-(2-acetamido-3,4,6-tri-O-acetyl-2-de-
oxy-b-d-glucopyranosyl)-2,4-di-O-acetyl-b-d-galactopyranosyl]-b-alanine
amide (16): Trisaccharide 15 (104 mg, 110 mmol) was dissolved in anhy-
drous MeOH (4 mL) and, after addition of dry 10% palladium on carbon
catalyst (4 spatula tips), vigorously stirred under a hydrogen atmosphere
(1 atm) at 08C for 30 min. The mixture was filtered, evaporated, and
coevaporated with anhydrous THF (2 Â 4 mL). The crude glycosyl amine
(101 mg, white solid, Rf ˆ 0.26, CH2Cl2/MeOH 9:1), Cbz-b-Ala-OH (43 mg,
193 mmol), and HOBt (30 mg, 193 mmol) were dissolved in anhydrous THF
(0.65 mL) and iPr2NEt (66 mL, 386 mmol) and HBTU (73 mg, 193 mmol)
were added. The solution was stirred at rt for 22 h. Then the reaction
mixture was diluted with ethyl acetate (30 mL) and washed with 0.5n HCl,
sat aq NaHCO3, and brine. The organic layer was dried (Na2SO4),
evaporated, and purified by FC (65 g silica, CH2Cl2/MeOH 95:5 to 9:1) to
give 16 (83 mg, 67%) as a white solid: Rf ˆ 0.45 (CH2Cl2/MeOH 9:1);
1H NMR (400 MHz, CDCl3/[D6]DMSO 2:1): d ˆ 8.60 (d, 1H, J ˆ 7.2 Hz,
NH), 7.76 (d, 1H, J ˆ 9.0 Hz, NH), 7.67 (d, 1H, J ˆ 9.6 Hz, NH), 7.36 ± 7.25
(m, 5H, C6H5), 6.69 (t, 1H, J ˆ 5.5 Hz, CH2NH), 5.33 (d, 1H, J ˆ 3.6 Hz,
Gal 4-H), 5.18 (dd, 1H, J ˆ 9.7, 10.1 Hz, GlcN 3-H), 5.10 ± 4.97 (m, 5H),
4.91 (t, 1H, J ˆ 9.7 Hz, GlcN 4-H), 4.90 (t, 1H, J ˆ 9.7 Hz, GlcN 4-H), 4.77
(d, 1H, J ˆ 8.3 Hz), 4.69 (d, 1H, J ˆ 8.6 Hz), 4.17 ± 4.08 (m, 3H), 4.01 ± 3.84
(m, 4H), 3.74 (dd, 1H, J ˆ 12.7, 2.5 Hz), 3.71 ± 3.64 (m, 2H), 3.64 ± 3.58 (m,
1H), 3.55 (dd, 1H, J ˆ 12.6, 8.1 Hz), 3.47 ± 3.29 (m, 2H, CH2NH), 2.60 ±
2.51, 2.44 ± 2.36 (2m, 2H, CH2CH2NH), 2.070, 2.059, 2.055, 2.020, 1.994,
1.969, 1.963, 1.960, 1.920, 1.820 (10s, 30H, 10C(O)CH3); 13C NMR
(100 MHz, CDCl3/[D6]DMSO 2:1): d ˆ 171.79, 168.88, 168.82, 168.74,
168.59, 168.47, 168.41, 168.17, 168.08, 167.77, 154.93, 135.70, 126.99, 126.40,
126.17, 99.35, 99.17, 77.20, 76.88, 76.36, 74.82, 71.43, 71.03, 69.77, 68.48, 67.62,
67.12, 67.05, 66.66, 64.30, 60.51, 60.07, 52.55, 51.66, 35.42, 34.17, 21.51, 21.48,
19.40, 19.34, 19.30, 19.25, 19.20, 19.17, 19.10; HR-MS (pos. FAB, NBA/CsI)
1
white foam; H NMR (400 MHz, CDCl3): d ˆ 8.00 ± 7.81 (partly brm, 4H,
Pht), 7.78 ± 7.75 (m, 4H, Pht), 5.75 (dd, 1H, J ˆ 9.1, 10.7 Hz, GlcN 3-H), 5.65
(dd, 1H, J ˆ 9.1, 10.8 Hz, GlcN 3-H), 5.51 (d, 1H, J ˆ 8.5 Hz, GlcN 1-H),
5.34 (d, 1H, J ˆ 8.5 Hz, GlcN 1-H), 5.18 (dd, 1H, J ˆ 9.1, 10.2 Hz, GlcN
4-H), 5.07 (dd, 1H, J ˆ 9.1, 10.2 Hz, GlcN 4-H), 4.89 (dd, 1H, J ˆ 8.9,
9.7 Hz, Gal 2-H), 4.34 (dd, 1H, J ˆ 4.6, 12.4 Hz, GlcN 6-H), 4.30 (dd, 1H,
J ˆ 8.5, 10.7 Hz, GlcN 2-H), 4.28 (dd, 1H, J ˆ 8.4, 10.8 Hz, GlcN 2-H),
4.22 ± 4.09 (m, 3H, GlcN 6-H, 2GlcN 6'-H), 4.11 (d, 1H, J ˆ 8.9 Hz, Gal
1-H), 4.03 (dd, 1H, J ˆ 4.6, 11.4 Hz, Gal 6-H), 3.91 ± 3.85, 3.56 ± 3.52
(eachm, each3H, Gal 3-H, 4-H, 5-H, 6'-H, 2GlcN 5-H), 2.64 (brm, 1H, Gal
OH), 2.14 (s, 3H, C(O)CH3), 2.10 (s, 3H, C(O)CH3), 2.05 (s, 3H,
C(O)CH3), 2.04 (s, 3H, C(O)CH3), 1.86 (s, 3H, C(O)CH3), 1.85 (s, 3H,
C(O)CH3), 1.46 (s, 3H, C(O)CH3); 13C NMR (100 MHz, CDCl3): d ˆ 170.8,
170.7, 170.1, 169.5, 169.2, 168.8, 134.5, 131.3, 123.7, 98.1, 87.5, 80.2, 75.1,
72.04, 71.99, 70.8, 70.1, 69.0, 68.8, 68.7, 68.2, 67.7, 61.9, 61.7, 54.5, 54.1, 20.8,
20.7, 20.63, 20.59, 20.43, 20.38, 19.8; HR-MS (pos. FAB, NBA/CsI) calcd for
‡
C48H51N5O24Cs [M ‡ Cs] m/z: 1214.1978, found 1214.1947.
Procedure B: A suspension of 14 (81 mg, 0.328 mmol), 2,4,6-collidine
(164 mL, 1.25 mmol), and powdered molecular sieves (4 Š) (ca. 600 mg) in
dichloromethane (4.7 mL) was stirred under argon for 1 h at rt. After
addition of freshly dried AgOTf (316 mg, 1.23 mmol), the mixture was
cooled to 308C and a solution of 6 (408 mg, 0.819 mmol) in dichloro-
methane (4.7 mL) was added dropwise during 5 min. The mixture was
stirred for 1 h at 308C, slowly warmed up to rt (2 h), and stirred for
another 20 h at rt. After dilution with MeOH (5 mL) the suspension was
filtered and evaporated. Purification by FC (100 g silica, hexane/ethyl
acetate 1:4) gave 12 (247 mg, 70%).
2-O-Acetyl-b-d-galactopyranosyl azide (14): A solution of 13 (1.14 g,
4.55 mmol) and TMS-N3 (6 mL, 45.5 mmol) in THF (2 mL) was stirred at rt
for 12 h and then refluxed (heating bath with 908C) for 22 h. After addition
of 80% aqueous HOAc (10 mL), the mixture was stirred at ca. 808C for 1 h
in order to cleave the TMS ethers. The solution was concentrated and
coevaporated several times with toluene. FC (80 g silica, CH2Cl2/MeOH
4:1) gave 14 (1.01 g, 90%): White crystals (acetonitrile); m.p. 154 ±
155.58C; Rf ˆ 0.50 (CH2Cl2/MeOH 4:1); 1H NMR (400 MHz, [D6]DMSO):
d ˆ 5.17 (d, 1H, J ˆ 5.8 Hz, 3-OH), 4.88 (d, 1H, J ˆ 4.2 Hz, 4-OH), 4.88 (dd,
1H, J ˆ 8.9, 9.8 Hz, 2-H), 4.76 (t, 1H, J ˆ 5.5 Hz, 6-OH), 4.54 (d, 1H, J ˆ
8.9 Hz, 1-H), 3.73 (ddd, 1H, J ˆ 3.9, 3.9, <1 Hz, 4-H), 3.61 ± 3.52 (m, 4H,
3-H, 5-H, 6-H, 6'-H), 2.05 (s, 3H, C(O)CH3); 13C NMR (100 MHz,
‡
calcd for C49H66N4O26Cs [M ‡ Cs] m/z: 1259.3020, found 1259.3053; anal.
calcd for C49H66N4O26: C 52.22, H 5.90, N 4.97; found: C 51.93, H 6.05, N
5.01.
N3-Benzyloxycarbonyl-N1-[3,6-bis-O-(2-acetamido-2-deoxy-b-d-glucopyr-
anosyl)-b-d-galactopyranosyl]-b-alanine amide (17):
A solution of 16
(71 mg, 63.0 mmol) in anhydrous MeOH (3 mL) was treated with a solution
of NaOMe in anhydrous MeOH (0.1n, 1.2 mL) and stirred for 18 h at rt.
The solution was neutralized with cation-exchange resin (AG 50W-X2, Bio-
Rad Laboratories, pyridinium form), filtered (water was used to rinse the
resin), and evaporated to give 17 (43 mg, 86%) as a white foam: Rf ˆ 0.23
(MeCN/H2O 4:1); 1H NMR (400 MHz, D2O): d ˆ 7.44 ± 7.35 (m, 5H, C6H5),
5.13 ± 5.07 (m, 2H, CH2C6H5), 4.86 (d, 1H, J ˆ 9.1 Hz), 4.67 (d, 1H, J ˆ
8.4 Hz), 4.53 (d, 1H, J ˆ 8.5 Hz), 4.13 (d, 1H, J ˆ 3.2 Hz), 3.94 ± 3.84 (m,
3H), 3.82 ± 3.60 (m, 8H), 3.56 ± 3.51 (m, 1H), 3.50 ± 3.35 (m, 7H), 2.56 ± 2.44
(m, 2H, CH2CH2NH), 1.99 (s, 6H, 2C(O)CH3); 13C NMR (100 MHz, D2O):
d ˆ 177.66, 177.40, 177.04, 160.67, 138.87, 131.22, 130.78, 130.04, 105.14,
103.83, 85.09, 82.14, 78.33, 78.11, 77.86, 76.22, 76.03, 72.26, 72.05, 71.57,
70.92, 70.59, 69.30, 63.08, 62.88, 58.13, 57.81, 39.13, 38.21, 24.61, 24.58; HR-
ˆ
[D6]DMSO): d ˆ 169.7 (C O), 87.3, 77.8, 71.4, 70.8, 68.1, 60.3, 20.9 (CH3);
‡
HR-MS (pos. FAB, NBA/NaI) calcd for C8H13N3O6Na [M ‡ Na] m/z:
270.0702, found 270.0707; anal. calcd for C8H13N3O6: C 38.87 H 5.30, N
17.00; found: C 39.00, H 5.26, N 17.12.
3,6-Bis-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-d-glucopyranosyl)-
2,4-di-O-acetyl-b-d-galactopyranosyl azide (15): A solution of 12 (235 mg,
217 mmol) in n-butanol (30 mL) and ethylene diamine (6 mL) was stirred
under Ar at 908C for 24 h. The solution was evaporated and the residue was
coevaporated with toluene (2 Â 5 mL) and MeOH (2 Â 5 mL). The crude
‡
MS (pos. FAB, NBA/CsI) calcd for C33H50N4O18Cs [M ‡ Cs] m/z:
923.2174, found 923.2132.
168
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0601-0168 $ 17.50+.50/0
Chem. Eur. J. 2000, 6, No. 1

Sialyl Lewis X Derivatives
162±171
N3-Benzyloxycarbonyl-N1-{3,6-bis-O-[b-d-galactopyranosyl-(1,4)-2-acet-
amido-2-deoxy-b-d-glucopyranosyl]-b-d-galactopyranosyl}-b-alanine
amide (18): Trisaccharide 17 (37.4 mg, 47.3 mmol), UDP-Gal (75 mg,
123 mmol), and MnCl2 ´ 4H2O (1m in H2O) (23.5 mL, 23.5 mmol) were
dissolved in HEPES buffer (50mm, pH 7.0) (4.7 mL) and b-1,4-galactosyl-
transferase (55 mL, 2.75 U) and alkaline phosphatase (7.5 mL, 37.6 U) were
added. The mixture was gently shaken at 378C for 12 h. The precipitate
formed was removed by centrifugation (23700 g) and the supernatant
purified by size-exclusion chromatography (Bio-Gel P-4, 2.5 Â 95 cm,
50mm NH4HCO3). Product containing fractions were pooled and lyophi-
lized. To remove contaminant UDP-Gal, the crude product was dissolved
in a small amount of H2O, applied to an anion-exchange column (Dowex-1
X8, HCO3 form) and eluted with H2O. Lyophilization gave 18 (66.5 mg,
purity 79%, corresponding to 53 mg pure 18, quant.) as a white fluffy
powder contaminated with a small amount of HEPES buffer (21%): Rf ˆ
0.56 (iPrOH/1m NH4OAc 2:1); 1H NMR (400 MHz, D2O): d ˆ 7.43 ± 7.33
(m, 5H, C6H5), 5.12 ± 5.06 (m, 2H, CH2C6H5), 4.85 (d, 1H, J ˆ 9.0 Hz), 4.68
(d, 1H, J ˆ 8.2 Hz), 4.54 (d, 1H, J ˆ 8.1 Hz), 4.44 (d, 1H, J ˆ 7.9 Hz), 4.41
(d, 1H, J ˆ 7.9 Hz), 4.13 (d, 1H, J ˆ 3.1 Hz), 3.95 ± 3.35 (m, 31H), 2.55 ± 2.43
(m, 2H, CH2CH2NH), 1.97 (s, 6H, 2C(O)CH3); 13C NMR (100 MHz, D2O):
d ˆ 177.65, 177.36, 177.00, 160.68, 138.88, 131.23, 130.78, 130.03, 105.27,
105.04, 103.78, 85.16, 82.15, 80.76, 80.44, 77.77, 77.19, 77.00, 74.90, 74.83,
74.65, 73.37, 71.58, 70.96, 70.89, 70.58, 69.29, 63.46, 62.41, 62.25, 57.66, 57.31,
39.14, 38.20, 24.63, 24.60; HR-MS (pos. FAB, NBA/CsI) calcd for
99.34, 83.57, 80.52, 76.37, 76.00, 75.78, 75.63, 75.56, 75.39, 73.69, 72.65, 72.47,
72.43, 70.03, 69.92, 68.95, 68.84, 68.45, 68.03, 67.41, 63.37, 62.22, 60.24, 56.75,
56.28, 52.42, 51.11, 40.41, 40.25, 37.34, 36.28, 31.08, 25.04, 24.55, 23.02, 22.92,
22.78, 16.02, 15.93; MALDI-MS (H20, neg.) calcd for C79H124N6O52Na [M ‡
‡
Na] m/z: 2011.7, found 2011.
N3-(4,4-Difluoro-5,7-dimethyl-4-bora-[3a,4a]-diaza-s-indacene-3-propion-
yl)-N1-{3,6-bis-O-[(5-acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-
2-ulopyranosylonic acid)-(2,3)-b-d-galactopyranosyl-(1,4)-[a-l-fucopyra-
nosyl-(1,3)]-2-acetamido-2-deoxy-b-d-glucopyranosyl]-b-d-galactopyrano-
syl}-b-alanine amide (2): A mixture of 20 (21.7 mg, 10.9 mmol), 10%
palladium on carbon catalyst (one spatula tip), MeOH (0.5 mL), and H2O
(0.5 mL) was vigorously stirred under an hydrogen atmosphere (1 atm) at rt
for 30 min. The mixture was filtered through Celite and evaporated. The
crude nonasaccharide amine (19.3 mg, Rf ˆ 0.07, iPrOH/1m NH4OAc 2:1)
was dissolved in DMF (300 mL) and H2O (100 mL) and stirred with 21
(7.4 mg, 19 mmol) and Et3N (11 mL, 79.5 mmol) for 1 h at rt. Ten drops of a
solution of NH3 in MeOH (saturated at 08C) were added and stirring was
continued for 15 min. The mixture was evaporated and purified by size-
exclusion chromatography (Bio-Gel P-2, 2.5 Â 70 cm, 100mm NH4HCO3)
to give, after lyophilization, 2 (20.7 mg, 89%) as an orange solid: Rf ˆ 0.35
(iPrOH/1m NH4OAc 2:1); 1H NMR (400 MHz, D2O): d ˆ 7.44 (s, 1H),
7.05 ± 7.02 (m, 1H), 6.31 ± 6.27 (m, 2H), 5.10 (d, 1H, J ˆ 3.9 Hz, Fuc 1-H),
5.07 (d, 1H, J ˆ 3.9 Hz, Fuc 1-H), 4.67 (d, 1H, J ˆ 8.5 Hz), 4.53 ± 4.46 (m,
1H), 4.50 (d, 1H, J ˆ 7.5 Hz), 4.46 (d, 1H, J ˆ 7.7 Hz), 4.11 (d, 1H, J ˆ
2.6 Hz), 4.09 ± 4.03 (m, 2H), 3.96 ± 3.31 (m, 49H), 3.17 ± 3.10 (m, 2H), 2.77 ±
2.70 (m, 2H), 2.70 ± 2.56 (m, 2H, CH2CH2NH), 2.55 ± 2.45 (m, 2H), 2.49 (s,
3H, Ar-CH3), 2.23 (s, 3H, Ar-CH3), 2.00, 1.97, 1.96 (eachs, 12H,
4C(O)CH3), 1.78 (t, 2H, J ˆ 12.1 Hz), 1.13, 1.11 (eachd, 6H, J ˆ 6.6 Hz,
‡
C45H70N4O28Cs [M ‡ Cs] m/z: 1247.3231, found 1247.3319.
N3-Benzyloxycarbonyl-N1-{3,6-bis-O-[(5-acetamido-3,5-dideoxy-d-gly-
cero-a-d-galacto-non-2-ulopyranosylonic acid)-(2,3)-b-d-galactopyrano-
syl-(1,4)-2-acetamido-2-deoxy-b-d-glucopyranosyl]-b-d-galactopyranosyl}-
b-alanine amide (19): Pentasaccharide 18 (37.5 mg, 79% purity, 26.7 mmol),
CMP-NeuAc (55 mg, 76 mmol), MnCl2 ´ 4H2O (1m in H2O) (29 mL,
29 mmol), and Triton X-100 (11.6 mg in 387 mL H2O) were dissolved in
HEPES buffer (100mm, pH 7.0) (5.4 mL) and a-2,3-sialyltransferase
(193 mL, 0.58 U) and alkaline phosphatase (3.5 mL, 17.4 U) were added.
The mixture was gently shaken at 378C for 20 h and another portion of
CMP-NeuAc (15 mg, 21 mmol) and a-2,3-sialyltransferase (100 mL, 0.3 U)
were added. Incubation was continued for further 22 h and the mixture was
filtered and evaporated. Size-exclusion chromatography (Bio-Gel P-4,
2.5 Â 95 cm, 100mm NH4HCO3) gave 19 (41.8 mg, 92%) as a white fluffy
powder after lyophilization: Rf ˆ 0.12 (iPrOH/1m NH4OAc 3:1); 1H NMR
(400 MHz, D2O): d ˆ 7.44 ± 7.34 (m, 5H, C6H5), 5.13 ± 5.06 (m, 2H,
CH2C6H5), 4.85 (d, 1H, J ˆ 8.9 Hz), 4.68 (d, 1H, J ˆ 8.3 Hz), 4.55 ± 4.48
(m, 1H), 4.57 (d, 1H, J ˆ 7.8 Hz), 4.50 (d, 1H, J ˆ 8.0 Hz), 4.13 (d, 1H, J ˆ
3.1 Hz), 4.11 (t, 1H, J ˆ 3.1 Hz), 4.08 (t, 1H, J ˆ 3.1 Hz), 3.96 ± 3.46 (m,
41H), 3.39 (m, 2H), 2.72 (dd, 2H, J ˆ 4.4, 12.4 Hz), 2.56 ± 2.44 (m, 2H,
CH2CH2NH), 2.00, 1.97 (eachs, 12H, 4C(O)CH3), 1.80 (t, 2H, J ˆ 12.2 Hz);
13C NMR (100 MHz, D2O): d ˆ 177.65, 177.40, 177.34, 176.99, 175.69, 160.69,
138.89, 131.23, 130.78, 130.02, 105.09, 104.97, 104.92, 103.80, 101.87, 85.18,
82.13, 80.61, 80.25, 77.87, 77.53, 77.20, 76.99, 75.38, 74.82, 74.63, 73.95, 72.01,
71.80, 70.94, 70.57, 70.50, 69.91, 69.28, 65.07, 63.42, 62.39, 62.22, 57.66, 57.30,
54.07, 41.83, 39.13, 38.17, 24.62, 24.58, 24.46; ESI-MS (H2O, neg.) calcd for
12
‡
2Fuc CH3); ESI-MS (H2O, neg.) calcd for
C
13CH130BF2N8O51 [M H]
84
m/z: 2128.8, found 2129.
5-[2-(4,4-Difluoro-5,7-dimethyl-4-bora-[3a,4a]-diaza-s-indacene-3-propio-
nylamino)ethylaminocarbonyl]pentyl (5-acetamido-3,5-dideoxy-d-glyc-
ero-a-d-galacto-non-2-ulopyranosylonic acid)-(2,3)-b-d-galactopyranosyl-
(1,4)-[a-l-fucopyranosyl-(1,3)]-2-acetamido-2-deoxy-b-d-glucopyranoside
(23): A solution of 22 (3 mg, 3.07 mmol), 21 (1.5 mg, 3.7 mmol), and Et3N
(1 mL, 7.2 mmol) in DMF (60 mL) was stirred for 2 h at rt. Two drops of a
solution of NH3 in MeOH (saturated at 08C) were added and stirring was
continued for 1 h. The mixture was evaporated and purified by size-
exclusion chromatography (Bio-Gel P-2, 2.5 Â 70 cm, 50mm NH4HCO3) to
give, after lyophilization, 23 (3.2 mg, 83%) as an orange solid: Rf ˆ 0.58
(iPrOH/1m NH4OAc 2:1); 1H NMR (400 MHz, D2O): d ˆ 7.45 (s, 1H), 7.04
(d, 1H, J ˆ 4.0 Hz), 6.35 (d, 1H, J ˆ 4.0 Hz), 6.30 (s, 1H), 5.07 (d, 1H, J ˆ
4.0 Hz), 4.50 (d, 1H, J ˆ 7.8 Hz), 4.41 (d, 1H, J ˆ 8.1 Hz), 4.08 (dd, 1H, J ˆ
3.1, 9.8 Hz), 3.95 ± 3.40 (m, 23H), 3.34 ± 3.21 (m, 4H), 3.17 (t, 2H, J ˆ
7.2 Hz), 2.75 (dd, 1H, J ˆ 4.6, 12.4 Hz), 2.66 (t, 2H, J ˆ 7.2 Hz), 2.50 (s,
3H, arom. CH3), 2.25 (s, 3H, arom. CH3), 2.07 ± 2.01 (m, 2H), 2.02 (s, 3H,
C(O)CH3), 1.97 (s, 3H, C(O)CH3), 1.80 (t, 1H, J ˆ 12.2 Hz), 1.47 ± 1.38 (m,
4H), 1.20 ± 1.11 (m, 2H), 1.15 (d, 3H, J ˆ 6.6 Hz); 13C NMR (125 MHz,
D2O): d ˆ 177.7, 176.0, 175.8, 174.8, 174.4, 162.5, 156.4, 147.4, 136.2, 133.9,
129.4, 125.6, 122.0, 117.2, 102.4, 101.7, 100.2, 99.4, 76.5, 76.0, 75.6, 74.1, 73.7,
72.7, 72.5, 71.0, 70.1, 70.0, 69.0, 68.9, 68.5, 68.1, 67.5, 63.4, 62.2, 60.4, 56.6,
52.5, 40.5, 39.6, 39.1, 36.4, 35.7, 29.0, 25.6, 25.5, 25.1, 23.0, 22.8, 16.1, 15.1,
‡
C67H103N6O44 [M H] m/z: 1695.6, found 1696.
N3-Benzyloxycarbonyl-N1-{3,6-bis-O-[(5-acetamido-3,5-dideoxy-d-gly-
cero-a-d-galacto-non-2-ulopyranosylonic acid)-(2,3)-b-d-galactopyrano-
syl-(1,4)-[a-l-fucopyranosyl-(1,3)]-2-acetamido-2-deoxy-b-d-glucopyrano-
syl]-b-d-galactopyranosyl}-b-alanine amide (20): Saccharide 19 (31 mg,
18.3 mmol), GDP-Fuc (34.5 mg, 55 mmol), and MnCl2 ´ 4H2O (1m in H2O)
(110 mL, 110 mmol) were dissolved in MES buffer (50mm, pH 6.0) (5.2 mL)
and a-1,3-fucosyltransferase (254 mL, 0.55 U) and alkaline phosphatase
(7.7 mL, 38.7 U) were added. The mixture was gently shaken at 378C for
48 h. The precipitate formed was removed by centrifugation (23700 g) and
the supernatant purified by size-exclusion chromatography (Bio-Gel P-4,
2.5 Â 95 cm, 100mm NH4HCO3). Lyophilization of product containing
fractions gave 20 (31 mg, 85%) as a white fluffy powder: Rf ˆ 0.31 (iPrOH/
1m NH4OAc 2:1); 1H NMR (500 MHz, D2O): d ˆ 7.44 ± 7.34 (m, 5H, C6H5),
5.15 ± 5.04 (m, 4H, CH2C6H5, 2Fuc 1-H), 4.68 (d, 1H, J ˆ 8.2 Hz), 4.55 ±
4.48 (m, 1H), 4.50 (d, 1H, J ˆ 7.9 Hz), 4.46 (d, 1H, J ˆ 7.8 Hz), 4.13 (d, 1H,
J ˆ 2.6 Hz), 4.08 ± 4.04 (m, 2H), 3.97 ± 3.33 (m, 49H), 2.73 (dd, 2H, J ˆ 4.5,
12.4 Hz), 2.60 ± 2.46 (m, 2H, CH2CH2NH), 2.00, 1.98, 1.97 (eachs, 12H,
4C(O)CH3), 1.78 (t, 2H, J ˆ 12.1 Hz), 1.14, 1.12 (eachd, 6H, J ˆ 6.6 Hz,
2Fuc CH3); 13C NMR (125 MHz, D2O): d ˆ 176.20, 175.75, 175.45, 175.09,
174.32, 159.05, 137.38, 129.58, 129.05, 128.18, 103.21, 102.29, 102.00, 100.19,
‡
11.3; ESI-MS (H2O, neg.) calcd for C53H80BF2N6O25 [M H] m/z: 1249.5,
found 1249.5.
Biotin-conjugated sialyl Lewis x (25): NHS-biotin 24 (1.0 mg, 2.8 mmol) and
dry triethylamine (70 mL, 5.1 mmol) were added to a solution of 22 (2.5 mg,
2.6 mmol) in dry DMF (330 mL). The reaction flask was covered in foil and
the reaction allowed to stir at rt for 24 hours. Solvent was evaporated under
reduced pressure, and the resulting residue was purified by size-exclusion
chromatography (Bio-Gel P-2, 2.5 Â 65 cm, 50mm NH4HCO3). Lyophiliza-
tion gave 25 as a white foam (2.0 mg, 65%). 1H NMR (400 MHz, D2O): d ˆ
8.29 (s, 1H), 4.94 (dd, 1H, J ˆ 3.7 Hz), 4.46 (dd, 1H, J ˆ 8.1, 5.1 Hz), 4.37 (d,
2H, J ˆ 7.9 Hz), 4.27 (dd, 1H, J ˆ 7.7, 4.3 Hz), 3.93 (dd, 1H, J ˆ 9.8, 3.1 Hz),
3.85 (d, 1H, J ˆ 10.2 Hz), 3.78 ± 3.68 (m, 9H), 3.62 (d, 1H, J ˆ 3.1 Hz),
3.57 ± 3.43 (m, 13H), 3.38 (dd, 1H, J ˆ 21.0, 13.1 Hz), 3.20 ± 3.17 (m, 6H),
2.84 (dd, 1H, J ˆ 13.2, 5.0 Hz), 2.65 ± 2.55 (m, 3H), 2.09 (dd, 5H, J ˆ 14.6,
7.2 Hz), 1.88 (s, 3H), 1.87 (s, 3H), 1.64 (t, 1H, J ˆ 12.3 Hz), 1.50 ± 1.41 (m,
4H), 1.26 ± 1.23 (m, 2H), 1.17 ± 1.12 (m, 4H), 1.01 (d, 3H, J ˆ 7.0 Hz);
13C NMR (125 MHz, D2O): d ˆ 176.3, 176.2, 174.2, 173.0, 170.2, 100.8, 100.1,
98.8, 97.8, 74.8, 74.5, 74.1, 72.6, 72.1, 71.1, 70.9, 69.5, 68.8, 68.6, 68.4, 68.4,
67.5, 66.9, 66.5, 65.8, 61.8, 61.3, 60.6, 59.5, 59.4, 55.0, 54.5, 50.9, 39.0, 38.9,
Chem. Eur. J. 2000, 6, No. 1
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169

C.-H. Wong et al.
FULL PAPER
37.8, 35.0, 34.7, 27.5, 27.0, 26.9, 24.3, 24.2, 23.9, 21.5, 21.4, 21.2, 19.2, 14.4, 9.5;
a Falcon tube and washed three times with PBS for staining in solution by
using anti-selectin (either hL- or hE-) antibody as follows: the cells were
incubated for one hour in the blocking buffer (PBS containing 0.5% human
serum) at rt. The blocking buffer was removed by washing the cells in PBS.
Anti-selectin antibody diluted in the blocking buffer (1:500) was added and
the cells were incubated at rt for 45 min. These cells were washed four times
with PBS at room temperature, and TRITC-conjugated anti-mouse IgG
antibody was used as a secondary antibody (diluted 1:1000 in blocking
buffer). Incubation was carried out for one hour (in the dark) at room
temperature. The cells were washed again four times with PBS, finally
suspended in 400 mL of PBS containing 0.1% bovine serum albumine
(BSA), and 25mm HEPES for FACS analysis. 0.1% of the maximally
intense cells were sorted out (Vantage, TSRI core facility) and collected in
200 mL of fetal calf serum. These were then transferred to a T25-tissue
culture flask containing MEM medium and placed in a CO2 incubator at
378C for two weeks of growth. These cells were used for immunofluor-
escence microscopy.
‡
ESI-MS (H2O, neg.) calcd for C49H82N6O26S [M H] m/z: 1202.3, found
‡
1202; ESI-MS (H2O, pos.) calcd for C49H82N6O26S [M H‡2Na] m/z:
1248.3, found 1248.
RT-PCR and construction of the expression vector for hL- and hE-selectin:
Human homologue of L-selectin (hL-selectin) was amplified with total
RNA from human spleen tissue (Clontech, Palo Alto, CA) by RT-PCR.
Total RNA was used for reverse transcription (RT) by using reverse
transcriptase, following the supplierꢁs protocol (Life Technologies, Gai-
thersburg, MD). This RT product was used for PCR. For amplification of
L-selectin, the forward primer (5'-CGGAATTCATGATATTTCCATG-
GAAATGTCAG-3'; with internal Eco RI site underlined) and the reverse
primer (5'-GTTCTAGATTAATATGGGTCATTCATACTTCTC-3'; with
internal Xba I site underlined) were used in 100 mL reaction mixture by
using Pfu DNA polymerase (Stratagene, San Diego, CA) following the hot-
start method. After 30 cycles, the reaction mixture was analyzed by agarose
gel electrophoresis which showed the generation of a single major band of
about 1.06 kb. The fragment was purified by agarose gel electrophoresis,
followed by Geneclean. Subcloning in pcDNA.3 (Invitrogen, Carlsbad,
CA) was done with Eco RI and Xba I digestion, followed by ligation.
Transformation was carried out following usual procedure. The clone
containing the plasmid DNA was grown in LB-ampicillin containing
medium. Plasmid DNA was isolated and confirmed by double-stranded
sequencing at TSRI core facility.
Similarly, hE-selectin was amplified with the forward primer (5'-ATAA-
GAATGCGGCCGCTAATGATTGCTTCACAGTTTCTCTC-3'; with in-
ternal Not I site underlined) and the reverse primer (5'-GCTCTA-
GAAACTTAAAGGATGTAAGAAGGCTTTTG-3'; with internal Xba I
site underlined). The full length cDNA was amplified by using Pfu DNA
polymerase. The amplification yielded a major band of 1.825kb. This was
purified by agarose gel electrophoresis, followed by Geneclean. Subcloning
in pcDNA.3 was carried out by using Not I and Xba I. The clone containing
the plasmid DNA was grown in LB-ampicillin medium. The plasmid DNA
was isolated and verified by double-stranded sequencing in the TSRI core
facility.
Activation of HUVEC cells: HUVEC monolayers grown in endothelial
cell growth medium (Cell Applications, Inc., San Diego, CA) were seeded
at the second passage onto 35 mm glass coverslips treated with fibronectin
1
(treated with 20 mg mL for 1 ± 2 hrs). These were grown to confluency in
complete M199 culture medium in a 5% CO2 incubator at 378C. The cells
were activated for 4 h with LPS (100 ngmL 1) following the method of
Welply et al.[12] These were washed three times with PBS (Dulbecco
phosphate-buffered saline solution without calcium and magnesium salts;
Irvine Scientific, CA) and fixed in 1 ± 2% freshly prepared formaldehyde
(made in PBS without calcium and magnesium salts) for 35 ± 50 min at
room temperature. Before staining, the cells were washed twice with 0.1m
glycine in PBS and then incubated in blocking buffer (0.5% human serum
in PBS) for 45 min.
For experiments involving CHO-K1, cells (about 106 cells per plate) were
plated in a 100 cm plate containing fibronectin treated cover slips. The cells
were grown in MEM medium (containing 5% fetal calf serum and 1% l-
glutamine) until 80 ± 90% confluency on the cover slips was observed
under a light microscope. The cells were washed twice in PBS and then
fixed in formaldehyde, as above.
Transfection of cDNAs in CHO-K1 and isolation of stable cell lines by
ELISA: CHO-K1 cells were routinely grown in MEM medium containing
5% fetal calf serum and 1% glutamine. The plasmid DNAs for full length
hL-selectin or hE-selectin were used to transfect freshly grown CHO-K1
cells with Lifofectamine, following the procedure of the supplier (Life
Technologies). After 48hrs of transfection, cells were digested with trypsin
and replated on medium containing G418. After about two weeks,
individual colonies were isolated by trypsin digestion, and were grown in
48 well plates containing G418 in the medium. The cells were incubated at
378C and used for selection in an ELISA assay as follows: the G418
resistant transfected cells (about 104 cells) expressing selectin were added
into a polylysine-coated Falcon 96 well plate (a replica plate for each was
also made and saved) and incubated overnight at 378C. The medium was
removed and the cells were washed with PBS. The plates were blocked for
one hour with blocking buffer (PBS‡1% human serum [Sigma]) at room
temperature. These were washed three times with PBS. Primary antibody
(50 mL per well, Pharmingen, San Diego, CA) diluted in the blocking buffer
(1:500) was added and incubated for 2hrs at rt (or 48C overnight). The cells
were washed three times with PBS, and then sheep anti-mouse IgG-HRP
conjugated antibody (50 mL per well, Amersham) diluted in the blocking
buffer (1:1000) was added. The cells were incubated one hour at rt and then
washed three times with PBS. The color was developed by the addition of
TMB peroxidase substrate (50 mL per well; Pierce, Rockford, IL). The
reaction was quenched with 1m phosphoric acid (50 mL per well), and the
plates were read at OD450. The wells containing cells with maximal OD450
by ELISA were selected. The expression of selectins by these cells was also
verified by sandwich ELISA. Plates were coated with anti-selectin anti-
body, incubated overnight at 48C, and then used for ELISA as above. Two
clones for each (hL-selectin and hE-selectin) were selected. A control cell
line incorporating the vector only was selected by G418 resistance. The
cells with OD450 over this control cell line were considered positive in the
assay. Two clones for each of the selectin-expressing stable cell lines were
selected for final sorting by a cell sorter.
Immunofluorescence microscopy: For detection of E-selectin expression,
the cells were washed and then incubated for 1 h at room temperature in
50 mL of blocking buffer containing 1:100 dilution of affinity purified anti-
human CD62E monoclonal antibody (Pharmingen, San Diego, CA). These
were washed three times in PBS, and incubated for 45 min at room
temperature in 1:100 dilution of tetramethylrhodamine isothiocyanate
(TRITC)-conjugated anti-mouse IgG (Fab specific) in blocking buffer. For
study of the fluorescent synthetic ligands, the cells were incubated for
45 min at room temperature in BODIPY-conjugated oligosaccharide
(6.5 mm in PBS containing 1mm each of calcium and magnesium salts).
Cells were washed four times with PBS (containing calcium and magne-
sium) and mounted on microscope slides with approximately 20 mL of
mounting medium (Molecular Probes, Inc., Eugene, OR). These were
visualized by using a Nikon Microphot-FXA microscope. Pictures were
taken with a digital CCD camera and were processed with Adobe
Photoshop.
Acknowledgment
This work was kindly supported by the NSF and NIH. V.W. acknowledges a
fellowship from the Deutsche Forschungsgemeinschaft. Sequencing of
DNA, mass spectrometry, NMR spectroscopy, and FACS analysis were
done at the core facilities of TSRI. The authors kindly acknowledge
Arlene A. Hipolito and the laboratory of Dr. Shelley Halpain for help in
the immunofluorescence experiments. We thank Dr. Phil Trotter for his
help with FACS analysis.
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Received: April 26, 1999 [F 1746]
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