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Chemoenzymatic Synthesis and Fluorescent Visualization of Cell‐Surface Selectin‐Bound Sialyl Lewis X Derivatives

03 Jan 2000-Chemistry: A European Journal (John Wiley & Sons, Ltd)-Vol. 6, Iss: 1, pp 162-171

TL;DR: A short chemoenzymatic synthesis of a fluorescently labeled bivalent sLe(x) conjugate is presented, demonstrated for a HUVEC cell line expressing E-selectin and for CHO-K1 cells expressing either L- or E- selectin.

AbstractSialyl 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 cells can be detected through fluorescence of bound selectin ligands. Herein we present a short chemoenzymatic synthesis of a fluorescently labeled bivalent sLe x conjugate. The use of an amino-substituted monovalent sLe x to obtain fluorescent- and biotin-labeled sLe x derivatives is also described. The cell-staining utility of the fluorescent sLe x conjugates is demonstrated for a HUVEC cell line expressing E-selectin and for CHO-K1 cells expressing either L- or E-selectin.

Topics: Sialyl-Lewis X (59%), Selectin (51%)

Summary (1 min read)

Introduction

  • Leukocyte adhesion to the vascular endothelium is a defining event in the inflammatory response.
  • 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.
  • 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.
  • Monomeric labeled sLex derivatives were readily obtained through conjugation of 22 to molecular probes containing activated esters.

Results and Discussion

  • Synthetic route to divalent 2 : The structure GlcNAcb1,3(GlcNAcb1,6)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.
  • Thus, trisaccharide 12 was available in eight steps from penta-Oacetyl-b-d-galactopyranose (3) (Schemes 1 and 2).

CH2Cl2:

  • Enzymatic glycosylations and attachment of the fluorophor : Treatment of primer 17 with b-1,4-galactosyltransferase (b1,4-GalT) and 2.6 equivalents of UDP-Gal gave pentasaccharide 18 in quantitative yield (Scheme 4).
  • The cell-staining utility of the fluorescently labeled sLex conjugates was then demonstrated.
  • 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.

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 galactosyl azide 14, and subsequent regioselective diglycosylation to give trisaccharide 12.
  • The b-alanine spacer introduced subsequently facilitated the incorporation of molecular probes, and also allows the possible formation of numerous neoglycoconjugates.[40].
  • 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.

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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 (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 cells can be detected through fluorescence of bound selectin ligands.
Herein we present a short chemoenzymatic synthesis of a fluorescently labeled
bivalent sLe
x
conjugate. The use of an amino-substituted monovalent sLe
x
to obtain
fluorescent- and biotin-labeled sLe
x
derivatives is also described. The cell-staining
utility of the fluorescent sLe
x
conjugates is demonstrated for a HUVEC cell line
expressing E-selectin and for CHO-K1 cells expressing either L- or E-selectin.
Keywords: chemoenzymatic synthe-
sis ´ oligosaccharides ´ regioselec-
tive glycosylation ´ selectin ´ sialyl
Lewis x
Introduction
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 (sLe
x
),
[2]
sialyl Lewis a (sLe
a
),
[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]
sLe
x
deriva-
tives have suggested that the selectin ± ligand interaction may
be multivalent in nature.
Bivalent sLe
x
derivative 1, previously reported by this
laboratory, inhibits binding of HL-60 cells to immobilized
E-selectin five times more efficiently than sLe
x
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]
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-
O
OH
HO
OH
HO
O
CO
2
H
OH
AcHN
OH
HO
O
O
HO
OH
R
O
O
O
O
NHAc
O
O
O
OH
HO
OH
HO
O
CO
2
H
OH
AcHN
OH
HO
O
O
H
3
C
HO
OH
OH
OH
O
NHAc
OH
O
H
3
C
OH
HO
O
OH
HN
H
N
O
N
B
N
F
F
O
HN
NH-Cbz
O
1: R = OEt
2: R =
20: R =
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 sLe
x
derivative
22 has been previously reported by this laboratory.
[18]
Mono-
meric labeled sLe
x
derivatives were readily obtained through
conjugation of 22 to molecular probes containing activated
esters.
The utility of labeled sLe
x
-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
FULL PAPER
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000 0947-6539/00/0601-0162 $ 17.50+.50/0 Chem. Eur. J. 2000, 6,No.1
162
First publ. in: Chemistry - A European Journal 6 (2000), 1, pp. 162-171
Konstanzer Online-Publikations-System (KOPS)
URL: http://www.ub.uni-konstanz.de/kops/volltexte/2007/4394/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-43941

162±171
Chem. Eur. J. 2000, 6, No. 1 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000 0947-6539/00/0601-0163 $ 17.50+.50/0
163
The synthetic sLe
x
conjugates were shown to bind specifically
to each selectin, in a manner similar to that of anti-selectin
monoclonal antibodies (mAb).
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 sLe
x
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 sLe
x
glycosyl azides in
the formation of multivalent sLe
x
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.
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
O
AcO
OAc
OAcAcO
OAc
O
NPht
AcO
AcO
OAc
Br
O
O
O N
3
O
O
PhtN
AcO
AcO
OAc
O
HO
OH
N
3
HO
OH
O
O
O
OH
N
3
OH
O
O
O
OH
N
3
O
O
PhtN
AcO
AcO
OAc
O
NPht
AcO
AcO
AcO
O
O
O
O N
3
O
NPht
AcO
AcO
AcO
O
OH
a)-c)
+ +
3
4
5
6
7
37 %
62 %
8
11 %
7 %
9
32 %
9 %
CH
2
Cl
2
:
CH
3
NO
2
:
e)
d)
Scheme 1. a) HBr, HOAc, b) NaN
3
, Bu
4
NHSO
4
, EtOAc/NaHCO
3
soln;
c) NaOMe, MeOH, 92 % (three steps); d) dimethoxypropane, p-TsOH,
DMF; then Et
3
HN
TsO
ÿ
, MeOH, H
2
O, reflux 3 h, 86%; e) 6 (1.2 equiv),
AgOTf, collidine, ÿ 208C.
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 %,
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
1
H 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
O
O
O
OAc
N
3
O
O
PhtN
AcO
AcO
O
HO
HO
OAc
N
3
O
O
PhtN
AcO
AcO
O
HO
OAc
N
3
O
O
PhtN
AcO
AcO
O
O
NPht
AcO
AcO
AcO
OAc
OAc
OAc
12
10
11
7
a)
b)
c)
Scheme 2. a) Ac
2
O, pyridine, 97 %; b) 80 % HOAc, 74%; c) 6 (1.5 equiv),
AgOTf, collidine, CH
2
Cl
2
, ÿ 208C, 84%.
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]
In an alternative synthetic strategy, 14 was efficiently
obtained by making use of a 1,2-orthoester (Scheme 3).
O
HO
HO
OH
O
O
OEt
O
HO
OAc
N
3
HO
OH
O
AcO
OAc
N
3
O
O
AcHN
AcO
AcO
O
O
NHAc
AcO
AcO
OAc
OAc
O
AcO
OAc
O
O
AcHN
AcO
AcO
O
O
NHAc
AcO
AcO
OAc
OAc
H
N
NH-Cbz
O
O
HO
OH
O
O
AcHN
HO
HO
O
O
NHAc
HO
HO
OH
OH
H
N
NH-Cbz
O
3
12
13
14
15
16
17
a)-c)
e) f), g)
h), i)
j)
d)
Scheme 3. a) HBr, HOAc; b) Bu
4
NBr, EtOH, collidine; c) NaOMe,
MeOH, 87% (three steps); d) TMS-N
3
(10 equiv), THF, rt to reflux, then
80% HOAc, 90%; e) 6 (2.5 equiv), AgOTf, collidine, CH
2
Cl
2
, ÿ 308C,
70%; f) ethylene diamine; g) Ac
2
O, pyridine, 90 % (two steps); h) H
2
/Pd-C;
i) Cbz-b-Ala-OH, HBTU, HOBt, iPr
2
NEt, 67% (two steps); j) NaOMe,
MeOH, 86%.

FULL PAPER
C.-H. Wong et al.
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164
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.
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.
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
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 sLe
x
deriv-
ative 22 was described. Compound 22 was employed in the
preparation of sLe
x
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 sLe
x
derivative 23 and
biotinylated sLe
x
derivative 25 in 83% and 65% yields,
respectively. Thus, labeled monovalent sLe
x
derivatives were
also easily accessible by short chemoenzymatic routes.
Application of fluorescent sLe
x
derivatives as cell-staining
reagents: The cell-staining utility of the fluorescently labeled
sLe
x
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
O
OH
HO
OH
HO
O
HO
OH
O
O
O
O
NHAc
HO
O
O
OH
HO
OH
HO
OH
O
NHAc
OH
HO
O
OH
HO
OH
HO
O
CO
2
H
OH
AcHN
OH
HO
O
O
HO
OH
O
O
O
O
NHAc
HO
O
O
OH
HO
OH
HO
O
CO
2
H
OH
AcHN
OH
HO
O
OH
O
NHAc
OH
HO
H
N
NH-Cbz
O
H
N
NH-Cbz
O
O
N
B
N
F
F
O
N
O
O
c)
18
2
d), e)
21
17
a)
b)
19
20
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) H
2
/Pd-C; e) 21, Et
3
N, DMF, H
2
O, 89% (two steps).

Sialyl Lewis X Derivatives 162±171
Chem. Eur. J. 2000, 6, No. 1 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000 0947-6539/00/0601-0165 $ 17.50+.50/0
165
O
NHAc
O
O
OH
HO
OH
OH
HO
O
CO
2
H
OH
AcHN
OH
HO
O
O
O
H
3
C
HO
OH
OH
O
N
H
O
NH
2
O
NHAc
O
O
OH
HO
OH
OH
HO
O
CO
2
H
OH
AcHN
OH
HO
O
O
O
H
3
C
HO
OH
OH
O
N
H
O
H
N
N
B
N
F
F
O
O
NHAc
O
O
OH
HO
OH
OH
HO
O
CO
2
H
OH
AcHN
OH
HO
O
O
O
H
3
C
HO
OH
OH
O
N
H
O
H
N
O
S
NH
HN
O
H
H
O
S
NH
HN
O
H
H
O
N
O
O
22
24
b)
25
23
a)
Scheme 5. a) 21, Et
3
N, DMF, 83%; b) 24, Et
3
N, 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
OD
450
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
calf serum, G418 (100 mgmL
ÿ1
), 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
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 sLe
x
conjugates. The cells were
washed again, fixed, and visualized under a fluorescence
microscope. BODIPY-labeled conjugates were detected by
intrinsic fluorescence at 508 nm.
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
Figure 2. CHO-K1 cells expressing E-selectin: a) under transmitted light;
b) stained with BODIPY-labeled sLe
x
monomer 23; c) cell stained with
CD62E mAb followed by TRITC-conjugated anti-mouse IgG; d) under
transmitted light; e) stained with BODIPY-labeled sLe
x
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).
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 sLe
x
derivatives for all cell lines in-
vestigated. Thus, the usefulness
of the labeled sLe
x
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 sLe
x
-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
selectins, it is necessary to ver-
ify that mimetic structures have
access to the appropriate car-
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.

FULL PAPER
C.-H. Wong et al.
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166
Figure 3. CHO-K1 cells expressing L-selectin: a) under transmitted light;
b) stained with BODIPY-labeled sLe
x
monomer 23; c) cell stained with
CD62L mAb followed by TRITC-conjugated anti-mouse IgG; d) under
transmitted light: e) stained with BODIPY-labeled sLe
x
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).
Figure 4. HUVEC cells expressing cell surface E-selectin: a) a cell under
transmitted light; b) cell stained with BODIPY-labeled sLe
x
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 sLe
x
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).
bohydrate binding site. This is the advantage of using small
molecular sLe
x
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 sLe
x
constructs.
Conclusion
A short and efficient synthesis of fluorescently labeled
bivalent sLe
x
-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
sLe
x
-derivatives as tools in localizing cell-surface selectins, the
sLe
x
conjugates were subjected to a cell-staining assay. Similar
cell-staining patterns of the sLe
x
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
sLe
x
-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.
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
purchased from Molecular Probes (Eugene, OR). a-2,3-SiaT (3 UmL
ÿ1
)
and a-1,3-FucT V (2.16 UmL
ÿ1
) 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 F
254
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.
1
H NMR chemical shifts are referenced to residual protic
solvent (CDCl
3
d
H
7.26, D
2
O d
H
4.80, [D
6
]DMSO d
H
2.50) or internal
standard TMS (d
H
0.00).
13
C chemical shifts are referenced to the solvent
signal (CDCl
3
d
C
77.0, [D
6
]DMSO d
C
39.5) or to [D
6
]DMSO (d
C
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.
3,4-O-Isopropylidene-b-dd-galactopyranosyl azide (5), 4,6-O-isopropyli-
dene-b-dd-galactopyranosyl azide, and 2,3:4,6-di-O-isopropylidene-b-dd-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 65 8C. 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, Et
3
N (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 Et
3
N. The

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Herein the authors present a short chemoenzymatic synthesis of a fluorescently labeled bivalent sLex conjugate.