Synthesis of an Fmoc-threonine bearing core-2 glycan: A building block for PSGL-1 via Fmoc-assisted solid-phase peptide synthesis

Article abstract of DOI:10.1016/j.carres.2010.05.004

Selectins (L, E, and P) are vascular endothelial molecules that play an important role in the recruitment of leukocytes to inflamed tissue. In this regard, P-Selectin glycoprotein-1 (PSGL-1) has been identified as a ligand for P-Selectin. PSGL-1 binds to

Full text of DOI:10.1016/j.carres.2010.05.004

Carbohydrate Research 345 (2010) 1541–1547
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of an Fmoc-threonine bearing core-2 glycan: A building block for
PSGL-1 via Fmoc-assisted solid-phase peptide synthesis
Venkata R. Krishnamurthy a,b, , Ann Dougherty a,b, , Medha Kamat a,b, Xuezheng Song c,
Richard D. Cummings c, Elliot. L. Chaikof a,b,d,
*
a Department of Biomedical Engineering, Emory University/Georgia Institute of Technology, USA
b Department of Surgery, Emory University, Atlanta, GA 30322, USA
c Department of Biochemistry, Emory University, Atlanta, GA 30322, USA
d School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Selectins (L, E, and P) are vascular endothelial molecules that play an important role in the recruitment of
leukocytes to inflamed tissue. In this regard, P-Selectin glycoprotein-1 (PSGL-1) has been identified as a
ligand for P-Selectin. PSGL-1 binds to P-Selectin through the interaction of core-2 O-glycan expressing
sialyl Lewisx oligosaccharide and the three tyrosine sulfate residues. Herein, we report the synthesis of
threonine-linked core-2 O-glycan as an amino acid building block for the synthesis of PSGL-1. This build-
ing block was further incorporated in the Fmoc-assisted solid-phase peptide synthesis to provide a por-
tion of the PSGL-1 glycopeptide.
Received 21 March 2010
Received in revised form 2 May 2010
Accepted 6 May 2010
Available online 12 May 2010
Keywords:
PSGL-1
Core-2 glycan
Glycopeptides
Solid-phase synthesis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
tial both for the study of the structure and function of P-selectin
and for the discovery of new anti-inflammatory agents.
Selectins (L, E, and P) are a family of proteins that mediate the
adhesion of leukocytes and platelets to vascular surfaces.1,2 They
are responsible for the initial steps of extravasation of leukocytes
during an inflammatory response by the recognition of the specific
carbohydrate-binding ligands.3 Inhibitors of selectins may act as
potential therapeutic reagents targeting diseases such as asthma,
psoriasis, rheumatoid arthritis, acute respiratory distress syn-
drome (ARDS), lupus, cancer metastasis, septic shock, or reperfu-
sion syndrome diseases.4 Although there are many candidates for
selectins as ligands, only P-selectin glycoprotein ligand-1 (PSGL-
1) has been demonstrated to mediate the adhesion of leukocytes
to endothelial cells.5 P-Selectin binds to the extreme N-terminal re-
gion of PSGL-1 through a properly positioned core-2 O-glycan
expressing sialyl Lewisx (sLex) oligosaccharide at Thr57 and at least
one of the three sulfated tyrosine residues at Tyr46, Tyr48, and
Tyr51.5,6 It has also been demonstrated that the truncated N-termi-
nal PSGL-1 monomer exhibits high affinity toward P-selectin.7
Therefore, the development of efficient synthetic methods for the
generation of truncated PSGL-1 glycosulfopeptides remains essen-
Leppanen et al. employed a chemoenzymatic method to synthe-
size a series of glycosulfopeptides and to define the important
structural moieties that mediate P-selectin and L-selectin interac-
tions with PSGL-1.7,8 Subsequent to that report, a number of efforts
have been directed at the synthesis of glycosulfopeptides with only
limited success in improving overall reaction efficiency and yield.9
As a consequence, many investigative groups continue to pursue
the synthesis of oligosaccharide components as intermediates in
the solid-phase synthesis of a truncated PSGL-1 monomer.10 In this
report, we describe the synthesis of an Fmoc-threonine bearing tri-
saccharide 1 (Fig. 1) and demonstrate its facile incorporation in so-
lid-phase peptide synthesis. The trisaccharide-linked threonine 1
represents the core-2 O-glycan on which sLex is to be constructed
through a combination of chemical and enzymatic approaches.11
Sialylation is generally challenging to install chemically, while effi-
cient sialyltransferases have been cloned and are readily available.
However, sialyltransferases generally have low efficiency toward a
fucosylated Lex structure; therefore, it is more efficient to install fu-
cose enzymatically after sialylation. While installing b-(1?4)-D-
galactose is relatively easier chemically, the highly efficient b-
(1?4)-galactosyltransferase is commercially available.
The synthesis of a similar Fmoc-threonine bearing core-2 as
building block for the peptide synthesis has been previously re-
ported.12 However, the oligosaccharide component was protected
* Corresponding author. Tel.: +1 404 727 8413; fax: +1 404 727 3396.
E-mail address: echaiko@emory.edu (Elliot. L. Chaikof).
Authors with equal contribution.
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.05.004

1542
V. R. Krishnamurthy et al. / Carbohydrate Research 345 (2010) 1541–1547
The synthesis of 10 began from the known galactose derivative
OAc
O
6.21 Hydrolysis of the acetates, followed by addition of benzalde-
hyde dimethylacetal in the presence of PTSA, generated the desired
benzylidene acetal 7 in an overall yield of 53%.12 The imidate 8,22
which was generated in two steps from commercially available
AcO
AcO
O
AcHN
AcO
AcO
OAc
O
O
b-D-galactose pentaacetate, was added to acceptor 7 to smoothly
O
AcO
generate the disaccharide 9. Finally, acid hydrolysis of acetal 9 in
refluxing acetic acid generated diol 10 in moderate 54% yield along
with 28% recovery of 9. All efforts aimed to improve the yield via
increasing reaction time led to the hydrolysis of the tert-butyl ester
in 10 (Scheme 2). After activation of the donor 5 using N-iodosuc-
cinimide at 0 °C, addition to acceptor 10 generated two products,
which can be separated by flash chromatography. The first product
was the desired trisaccharide 11, and the second product was
found to be an undesired tetrasaccharide 11a generated from gly-
cosylation of the donor 5 to both hydroxyl groups in 10. The struc-
ture of the trisaccharide 11 was confirmed by the combination of
1D proton and 2D gCOSY, TOCSY, gHSQC, gHMBC, and NOESY
NMR spectra. In the gHMBC NMR spectrum of 11 (see Supplemen-
tary data), the cross-peaks from 101.3 ppm (C-1 of the glucosamine
residue, A-C1) and 4.02 ppm (H-6 of the galactosamine residue, B-
H6) and from 4.70 ppm (H-1 of the glucosamine residue, A-H1) and
69.5 ppm (C-6 of the galactosamine residue, B-C6), suggest that the
O-6 of the galactosamine residue is glycosylated. The (1?6)-link-
age between the galactosamine and the glucosamine was further
confirmed by the NOESY spectrum, which displayed a cross-peak
between H-1 of the glucosamine residue and H-6 of the galactos-
amine residue. The combination of these 2D NMR analyses allowed
the complete structural assignment of 11 which is listed in Table 1.
Next, the trisaccharide 11 was acetylated with acetic anhydride in
pyridine in the presence of catalytic DMAP, and the acetylated
derivative was subsequently taken to the Troc deprotection step.
Thus, addition of freshly activated zinc dust in the presence of ace-
tic anhydride as a solvent reduced both the azide and NH-Troc
group along with concomitant protection of both amines as corre-
sponding acetates to generate 12 in an overall yield of 47%. Finally,
hydrolysis of the tert-butyl ester on the threonine residue 12 was
accomplished by treatment with trifluoroacetic acid in dichloro-
methane at room temperature affording the trisaccharide-linked
threonine 1 in 72% yield (Scheme 3).
AcHN
AcO
O
O
OH
NHFmoc
1
Figure 1. Fmoc-threonine bearing core-2 O-glycan as a building block for PSGL-1.
as an O-benzoate that requires more severe basic conditions for re-
moval of the benzoyl protecting groups, thereby rendering them
less suitable for use in glycopeptide synthesis.13 Our intention
was to synthesize an appropriate building block where the protect-
ing groups in core-2 are fully compatible with solid-phase peptide
synthesis, including cleavage conditions necessary for further
enzymatic reactions required to obtain the full-length PSGL-1.
Thus, we designed Fmoc-threonine bearing core-2 with acetyl pro-
tecting groups 1, as a building block for PSGL-1. In the Fmoc proto-
col, the iterative removal of the N -Fmoc group with piperidine,14
a
together with the coupling reagents HBTU/HOBt15 for peptide
elongation, can be achieved without affecting the O-acetyl groups
of the trisaccharide or inducing b-elimination of the glycan.16 The
use of electron-withdrawing O-acetyl protecting groups stabilizes
the glycosidic linkage during TFA cleavage of the peptide from so-
lid support, and the acetyl groups can be subsequently removed by
treatment with dilute sodium methoxide in methanol or hydrazine
hydrate.8,17
2. Results and discussion
The synthesis of 1 commenced with the preparation of donor 5.
Condensation of glycosamine hydrochloride with p-anisaldehyde
generated an aldimine that was then peracylated to obtain 2.18
The aldimine was hydrolyzed, and the resulting amine 3 was pro-
tected with Troc-Cl, generating 4.19 It was decided to use a thiogly-
coside for the glycosylation due to its inherent stability and mild
reaction conditions. Therefore, the anomeric acetate was selec-
tively converted to thioether 520 via reaction with ethanethiol in
the presence of BF3ꢀOEt2 and 4 Å molecular sieves (Scheme 1).
Next, we investigated the incorporation of the trisaccharide-
linked threonine residue 1 into an Fmoc-based SPPS strategy using
Rink amide functionalized resin. The iterative amino acid
coupling using the HBTU/HOBt method provided the glycosylated
a
OAc
O
OH
O
AcO
OAc
O
OAc
a, b
HO
HO
AcO
OH
c
AcO
AcO
N
OAc
NH2.HCl
NH2.HCl
3
OMe
2
OAc
O
OAc
O
e
AcO
AcO
AcO
AcO
SEt
d
OAc
NHTroc
NHTroc
5
4
Scheme 1. Synthesis of donor 5. Reagents and conditions: (a) p-anisaldehyde 4 M NaOH, 1 h, 71%; (b) Ac2O, DMAP (cat.), 16 h, 80%; (c) 5 M HCl, acetone, 0.5 h, 92%; (d) Troc-
Cl, pyridine, DIPEA, 16 h, 80%; (e) EtSH, BF3ꢀOEt2, 4 Å MS, CH2Cl2, 2 h, 80%.

V. R. Krishnamurthy et al. / Carbohydrate Research 345 (2010) 1541–1547
1543
Ph
O
OAc
O
AcO
AcO
O
a, b
c
O
OAc
AcO
AcO
N3
O
HO
O
N3
O
CCl3
O
O
O
O
AcO
O
NH
NHFmoc
8
NHFmoc
6
7
Ph
OH
OH
O
AcO
AcO
OAc
O
O
O
O
AcO
OAc
O
d
O
N3
AcO
O
O
O
AcO
N3
AcO
O
O
O
NHFmoc
O
NHFmoc
10
9
Scheme 2. Synthesis of disaccharide 10. Reagents and conditions: (a) NaOMe, MeOH, 3 h; (b) benzaldehyde dimethyl acetal, PTSA, MeNO2, 2 h, 53%; (c) TMSOTf, 8, CH2Cl2, 4 Å
MS, CH2Cl2, 0 °C, 1 h, 61%; (d) 80% HOAc, 70 °C, 2 h (10: 54%, 9: 28%).
Table 1
Structural assignment of trisaccharide 11
Residue
Gal (C)
Nucleus
1
2
3
4
5
6
1H
4.74
102.1
5.26
68.6
5.03
68.8
5.61
67.0
3.97
71.3
4.19/4.08
61.5
13C
Gal-N3 (B)
1H
4.99
99.3
3.54
58.8
3.98
77.9
4.13
68.5
3.99
69.3
4.02/3.78
69.5
13C
GlcNTroc-1,6 (A)
1H
4.70
101.3
3.63
56.4
5.28
72.1
5.04
70.9
3.69
71.9
4.24/4.14
62.2
13C
OAc
OAc
OAc
O
OAc
O
TrocHN
AcO
AcO
AcO
AcO
O
O
O
O
TrocHN
TrocHN
AcO
a
OH
10
AcO
AcO
+
OAc
O
OAc
O
O
O
O
O
AcO
AcO
N3
N3
AcO
AcO
O
O
O
O
O
O
NHFmoc
11a
NHFmoc
11
OAc
O
OAc
O
AcO
O
AcO
AcO
O
AcHN
AcO
AcHN
AcO
AcO
OAc
O
b,c
d
AcO
O
AcO
OAc
O
O
O
AcO
AcHN
O
AcO
AcO
O
O
AcHN
AcO
O
O
O
OH
NHFmoc
1
NHFmoc
12
Scheme 3. Synthesis of trisaccharide-linked Fmoc-threonine 1. Reagents and conditions: (a) 5, NIS, TfOH, 4 Å MS, CH2Cl2, 0 °C, 12 h (11: 51%, 11a: 19%); (b) Ac2O, DMAP,
pyridine, 1 h, 47%; (c) Zn dust, Ac2O–AcOH–THF, 3 h, 47%; (d) TFA, CH2Cl2, 2 h, 72%.

1544
V. R. Krishnamurthy et al. / Carbohydrate Research 345 (2010) 1541–1547
peptide 13 on solid support. Subsequent treatment of 13 with
9.5:0.2:0.2:0.1 TFA–EDT–TES–H2O cleaved the peptide from resin
without damaging the glycan. These conditions also cleaved the
tert-butyl ester and trityl protecting groups, thereby, yielding the
desired glycopeptide 14 (Scheme 4) in 50% yield (determined by
HPLC). Product 14 was characterized by MALDI-TOFMS and re-
versed-phase HPLC, both of which confirmed the expected glyco-
peptide. The second product 14a (43% yield determined HPLC)
was identified as a variant of glycopeptide 14 in which the C-termi-
nal cysteine underwent a disulfide linkage with ethanedithiol, the
reagent used in the final cleavage step of glycopeptide 14 from the
resin.23,24
3.2. Preparation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-p-
methoxybenzylidineamino-b- -glucopyranose (2)
D
Glucosamine hydrochloride (5 g, 23.3 mmol) was dissolved in
24 mL of 1 M aq NaOH to give a colorless solution. p-Anisaldehyde
(2.85 mL, 23.3 mmol) was added by syringe under intense stirring,
to form a turbid solution. After several min of agitation a white
precipitate was formed. The system was kept in an ice bath for 1 h
to ensure complete precipitation. The solid was then collected by fil-
tration and washed with water (2 ꢁ 20 mL) and a mixture of
1:1 MeOH–Et2O (2 ꢁ 20 mL). The precipitate was dried under vac-
uum to yield 2-deoxy-p-methoxybenzylidineamino-D-glucopyran-
In summary, the Fmoc-threonine bearing core-2 O-glycan 1 was
successfully synthesized, and the GlcNAcb1,6-Gal NAc linkage was
verified by 2D NMR methods. This building block can be readily
incorporated into solid-phase peptide synthesis to provide a por-
tion of the PSGL-1 glycopeptide that can be further modified by
chemoenzymatic means to obtain the full-length PSGL-1.
ose (4.93 g, 71%) as a white solid. The precipitate was dissolved in
27 mL of pyridine and cooled to 0 °C. To this was added catalytic
DMAP (50 mg, 4.1 mmol) and Ac2O (15 mL, 157.7 mmol). The solid
slowly went into solution, and the reaction mixture was left at room
temperature overnight. The starting material dissolved slowly over
time to give a clear solution. After 16 h, the reaction mixture was
poured into ice forming a white crystalline solid. The crystals were
collected by filtration, washed with water (2 ꢁ 10 mL) and ether
(2 ꢁ 10 mL), and dried under vacuum to afford the title compound
218 (6.21 g, 80%) as a white crystalline solid. The NMR data for 2
matched those previously reported.18 1H NMR (CDCl3): dH 1.88
(3H, s), 2.01 (3H, s), 2.03 (3H, s), 2.10 (3H, s, 3.44 (1H, t, J 9.6 Hz),
3.84 (3H, s), 3.97 (1H, ddd, J 2.4 Hz, 4.8 Hz, 9.6 Hz), 4.12 (1H, dd, J
2.1 Hz, 12.6 Hz), 4.37 (1H, dd, J 4.5 Hz, 12.3 Hz), 5.14 (1H, t,
J 9.6 Hz), 5.42 (1H, t, J 9.3 Hz), 5.94 (1H, d, J 8.1 Hz), 6.91 (2H, d, J
8.7 Hz), 7.64 (2H, d, J 8.4 Hz), 8.15 (s, 1H).
3. Experimental
3.1. General
All reagents were purchased from commercial sources and used
as received, unless otherwise indicated. All solvents were dried and
distilled by standard protocols. All reactions were performed under
an inert atmosphere of argon or nitrogen, unless otherwise indi-
cated. For peptide synthesis, Rink amide resin (resin loading:
0.4 mmol/g), DMF (Biotech grade), and piperidine were purchased
from Novabiochem. HOBt, PyBOP, and all Fmoc-protected amino
acids were purchased from Peptides International. 1H and 13C
NMR spectra were recorded with an Inova 600-MHz spectrometer.
High-resolution ESI-Mass spectra (HRESIMS) were recorded by The
Emory University Mass Spectrometry Center using a JEOL JMS-
SX102 instrument. Optical rotation values were recorded using a
Perkin–Elmer polarimeter.
3.3. Preparation of 2-amino-1,3,4,6-tetra-O-acetyl-2-deoxy-b-D-
glucopyranosyl hydrochloride (3)
Compound 2 (5 g, 10.8 mmol) was dissolved in 25 mL of reflux-
ing acetone, and to this solution was added dropwise 2.5 mL of 5 N
HCl. After 30 min, a thick white precipitate formed, and the system
was cooled to room temperature. The precipitate was filtered and
OAc
O
AcO
AcO
O
AcHN
AcO
AcO
Fmoc-NH
Rink Amide
OAc
O
O
O
1
AcO
AcHN
AcO
a,b,c
O
H
N
(tBu)Glu-HN
Glu (tBu)-Pro-Pro-Glu(tBu)-Cys(Trt)-CONH
O
13
OAc
O
AcO
O
AcO
AcHN
AcO
d
AcO
OAc
O
O
O
AcO
AcHN
O
AcO
H
N
FmocHN-Glu-HN
Glu-Pro-Pro-Glu-Cys-CONH2
O
14
Scheme 4. Incorporation of trisaccharide-linked threonine 1 into Fmoc-assisted solid-phase peptide synthesis. Reagents and conditions: (a) Fmoc-AA-OH; HBTU/HOBt; (b)
20% piperidine in DMF; (c) repeat steps a and b for subsequent amino acid coupling; (d) 9.5:0.1:0.2:0.2 TFA–H2O–EDT–Et3SiH, 2 h.

V. R. Krishnamurthy et al. / Carbohydrate Research 345 (2010) 1541–1547
1545
washed with acetone (10 mL) and ether (2 ꢁ 25 mL). This was sub-
sequently dried under vacuum overnight to provide 318 (3.45 g,
92%) as a white powder. The NMR data for 3 matched those previ-
ously reported.18 1H NMR (DMSO-d6): dH 2.03 (3H, s), 2.05 (3H, s),
2.09 (3H, s), 2.23 (3H, s), 3.62 (1H, t, J 9.3 Hz), 4.03–4.11 (2H, m),
4.25 (1H, dd, J 3.9 Hz, 12 Hz), 4.99 (1H, t, J 9.3 Hz), 5.42 (1H, t, J
9.9 Hz), 5.97 (1H, d, J 8.7 Hz), 8.93 (br s, 2H).
–NH), 7.25–7.82 (13H, m, -Fmoc Ar); HRESIMS: Calcd for
36H40N4O9 [M+Na], m/z 695.7140; found 695.2688.
C
3.7. Preparation of 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl
trichloroacetimidate (8)
Galactose pentaacetate (0.20 g, 0.51 mmol) was dissolved in
DMF (2 mL) and flushed with argon, hydrazine acetate (0.047 g,
0.51 mmol) was added, and the reaction was monitored by TLC.
Upon completion, the reaction mixture was diluted with EtOAc
(25 mL) and washed with water (10 mL). The EtOAc layer was
dried and concentrated under reduced pressure to give a yellow
residue (140 mg). This was subsequently dissolved in anhyd
CH2Cl2 (2 mL) and flushed with argon. To this solution were added
K2CO3 (0.081 g, 0.59 mmol) and trichloroacetonitrile (0.11 mL,
1.11 mmol). The reaction mixture was stirred at room temperature
for 8 h. Upon completion, the mixture was filtered through Celite
and concentrated. Subsequent purification by chromatography on
silica gel (1:1 EtOAc–hexanes) afforded the imidate 822 (0.161 g,
82%) as a white foam. The NMR data for 8 matched those previ-
ously reported.22 1H NMR (400 MHz): dH 2.01 (3H, s), 2.02 (3H,
s), 2.03 (3H, s), 2.17 (3H, s), 4.08 (1H, dd, J 6.7 Hz, 11.3 Hz), 4.17
(1H, dd, J 6.6 Hz, 11.3 Hz), 4.44 (1H, m), 5.36 (1H, dd, J 10.8 Hz,
3.4 Hz), 5.43 (1H, dd, J 10.8 Hz, 3.0 Hz), 5.56 (1H, dd, J 3.0 Hz,
1.4 Hz), 6.61 (1H, d, J 3.4 Hz), 8.67 (1H, s).
3.4. Preparation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranose (4)
Compound 3 (1.5 g, 4.3 mmol) was dissolved in 20 mL of pyridine
and cooled to 0 °C. To this were added DIPEA (0.75 mL, 4.3 mmol)
and Troc-Cl dropwise over a period of 30–45 min. The reaction was
allowed to stir at room temperature overnight. After 16 h, the reac-
tion was neutralized by adding 15 mL of MeOH. The solvents were
removed under reduced pressure, and the resulting residue was
purified by chromatography over silica gel (1:1 EtOAc–hexanes) to
obtain the title compound 419 (1.7 g, 80%) as white foam. The NMR
data for 4 matched those previously reported.19 1H NMR (CDCl3):
dH 2.01–2.21 (9H, m, 3 ꢁ OAc), 3.65 (1H, s, –OH), 4.01–4.29 (4H,
m), 4.61–4.83 (2H, m), 5.09–5.48 (4H, m).
3.5. Preparation of ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-N-(2,2,2-
trichloroethoxycarbonyl)amino-1-thio-D-glucopyranoside (5)
a
Compound 4 (1.68 g, 3.45 mmol) was dissolved in dichloro-
methane (10 mL), and 4 Å molecular sieves (1.6 g) were added.
The reaction mixture was cooled to ꢂ10 °C, to which ethanethiol
(0.51 mL, 6.9 mmol) and BF3ꢀEt2O (0.87 mL, 6.9 mmol) were added
in succession. After stirring at room temperature for 2 h, the sus-
pension was filtered with Celite, washed with satd aq NaHCO3
(10 mL), and concentrated under reduced pressure. The resulting
oil was purified by chromatography over silica gel (1:1 EtOAc–hex-
anes) to yield 520 (1.37 g, 80%) as a white foam. The NMR data for 5
matched those previously reported.20 1H NMR (CDCl3): dH 1.24 (3H,
t, J 6.4 Hz), 2.04 (9H, 3 ꢁ s), 2.69 (2H, m), 3.68 (1H, m), 3.75 (1H, dd,
J 10.3 Hz, 1H, H-2), 4.10 (1H, dd, J 12.3 Hz, 2.2 Hz), 4.22 (1H, dd, J
12.3 Hz, 5.1 Hz, H-6), 4.60 (1H, d, J 10.3 Hz, H-l), 4.78 (2H, dd, J
12.1 Hz), 5.05 (1H, dd, J 9.6 Hz), 5.19 (1H, d, J 9.1 Hz, –NH), 5.21
(1H, dd, J 9.6 Hz, H-3).
3.8. Preparation of N -(fluoren-9-ylmethoxycarbonyl)-O-[O-
(2,3,4,6-tetra-O-acetyl-b-
4,6-O-benzylidene-2-deoxy-
D
-galactopyranosyl)-(1?3)-2-azido-
-galactopyranosyl]- -threonine
a-D
L
tert-butyl ester (9)
Trichloroacetimidate 8 (42.7 mg, 0.087 mmol), acceptor deriva-
tive 7 (36.5 mg, 0.054 mmol), and freshly activated 4 Å molecular
sieves (150 mg) were added together. The solids were dissolved
in CH2Cl2 (2 mL), flushed with argon, and stirred for 2 h at 0 °C.
TMSOTf (2 lL, 8.7 lmol) was added dropwise. The reaction mix-
ture was stirred at 0 °C for 1 h, and then quenched with Hunig’s
base (diisopropylethylamine, DIEA). The reaction mixture was fil-
tered through Celite, concentrated, and dried. Column chromatog-
raphy on silica gel (1:5 acetone–toluene) afforded the disaccharide
912 (30 mg, 61%) as a clear oil. The NMR data for 9 matched those
previously reported.12 1H NMR (CDCl3): dH 1.33 (3H, d, J 6.4 Hz),
1.51 (9H, s), 1.99 (3H, s), 2.07 (3H, s), 2.17 (3H, s), 2.36 (3H, s),
3.74 (1H, s), 3.80 (1H, dd, J 3.6 Hz, 10.8 Hz), 3.95 (1H, t, J 6.4 Hz),
4.06–4.16 (3H, m), 4.20–4.32 (4H, m), 4.40–4.52 (3H, m), 4.81
(1H, d, J 7.6 Hz), 5.04 (1H, dd, J 3.2 Hz, 10.4 Hz), 5.13 (1H, d, J
3.6 Hz), 5.32 (1H, dd, J 7.6 Hz, 10.2 Hz), 5.42 (1H, d, J 2.8 Hz),
5.57 (1H, s), 5.76 (1H, d, J 9.6 Hz), 7.15–7.45 (7H, m), 7.54 (2H,
dd, J 1.9 Hz, 7.9 Hz), 7.64 (2H, dd, J 3.2 Hz, 7.2 Hz), 7.78 (2H, d, J
7.6 Hz); HRESIMS: Calcd for C50H58N4O18 [M+Na], m/z 1026.0013;
found, 1025.3638.
a
3.6. Preparation of N -(fluoren-9-ylmethoxycarbonyl)-O-(2-
azido-4,6-O-benzylidine-2-deoxy-a-D-galactopyranosyl)-L-
threonine tert-butyl ester (7)
The galactose derivative 6 (2.99 g, 0.84 mmol) was dissolved in
MeOH (84 mL), and the flask was flushed with argon. To this solu-
tion was added a freshly prepared 1 M NaOMe solution in MeOH
(1 mL). The reaction mixture was stirred at room temperature for
3 h. Upon completion of the reaction the mixture was neutralized
with Amberlite IR-120 (H+) resin. The reaction mixture was filtered
through Celite, and the solvent was removed under reduced pres-
sure to afford the crude diol. The diol was redissolved in nitrometh-
ane (70 mL), and flushed with argon. Benzaldehyde dimethyl acetal
(1.27 mL, 8.42 mmol) was added, followed by PTSA (0.12 g,
0.63 mmol). The reaction mixture was stirred for 2 h, neutralized
with TEA, azeotroped with toluene, and dried. Column chromatog-
raphy on silica gel (1:5 acetone–toluene) gave acetal 712 (103 mg,
53%) as a white foam. The NMR data for 7 matched those previously
reported.12 1H NMR (CDCl3): dH 1.31 (3H, d, J 6.4 Hz, Thr -CH3), 1.51
(9H, s, Bu), 2.31 (1H, d, J 4.0 Hz, –OH), 3.57 (1H, dd, 2-H), 3.78 (1H, s,
Fmoc CH), 4.07 (1H, dd, J 10.7 Hz, 3-H), 4.16 (1H, ddd, J 3.6 Hz,
7.1 Hz, 7.2 Hz, 5-H), 4.22–4.30 (4H, m, 4-H, 6-H2 and Thr CH2),
4.34 (1H, dd, J 2.5 Hz, 7.6, Hz, Thr -CH), 4.43 (2H, m, Fmoc CH2),
5.10 (1H, d, J 3.6 Hz, 1-H), 5.57 (1H, s, PhCH), 5.75 (1H, d, J 9.1 Hz,
a
3.9. Preparation of N -(fluoren-9-ylmethoxycarbonyl)-O-[O-
(2,3,4,6-tetra-O-acetyl-b-
deoxy- -galactopyranosyl]-L
D
-galactopyranosyl)-(1?3)-2-azido-2-
-threonine tert-butyl ester (10)
a
-D
Benzylidene acetal 9 (2.45 g, 2.44 mmol) was dissolved in a
mixture of HOAc (88 mL) and H2O (22 mL) and stirred at 80 °C
for 2 h. The reaction mixture was cooled to room temperature
and azeotroped with toluene (2 ꢁ 45 mL). The combined organic
layer was dried, concentrated, and purified by chromatography
over silica gel (1:1 EtOAc–hexanes) to afford the diol 1012
(54 mg, 54%) as a white amorphous solid. The NMR data for 10
matched those previously reported.12
NMR (CDCl3): dH 1.33 (3H, d, J 6.7 Hz, Thr -CH3); 1.51 (9H, s, –
NHBoc), 2.00 (3H, s, OAc), 2.06 (3H, s, OAc), 2.10 (3H, s, OAc),
½
a 2D2 + 66.0 (c 1, CHCl3); 1H
ꢃ

1546
V. R. Krishnamurthy et al. / Carbohydrate Research 345 (2010) 1541–1547
2.17 (3H, s, OAc), 3.57 (1H, dd, J 3.3 Hz, 10.5 Hz, H-2), 3.81 (1H, d, J
7.6 Hz, H-5), 3.93 (1H, s, H-50), 3.95 (2H, m, 6-H2), 4.02 (1H, dd, J
2.9 Hz, 10.5 Hz, H-3), 4.11 (1H, d, J 5.3 Hz, H-60), 4.17 (1H, dd, J
4.1 Hz, 11.7 Hz), 4.21 (1H, d, J 2.5 Hz, H-4), 4.26 (1H, J 7.1 Hz,
7.2 Hz), 7.31 (2H, ddd, J 2.4 Hz, 7.2 Hz, 7.2 Hz, -Fmoc Ar), 7.41
(2H, ddd, J 3.7 Hz, 7.2 Hz, 7.2 Hz, -Fmoc Ar), 7.63 (2H, d, J 7.8 Hz,
-Fmoc Ar), 7.77 (2H, d, J 7.8 Hz, -Fmoc Ar); 13C NMR (CDCl3): dC
19.2, 20.7, 20.8, 20.9, 21.1, 28.2, 47.3, 55.6, 56.2, 59.1, 59.6, 60.8,
61.8, 62.2, 67.0, 67.5, 68.5, 69.0, 69.2, 69.8, 71.4, 72.0, 72.5, 72.6,
73.4, 74.5, 75.4, 75.6, 83.3, 97.6, 96.0, 98.7, 101.2, 102.1, 120.2,
125.3, 125.4, 125.5, 127.2, 127.3, 127.9, 128.4, 129.2, 154.2, 155.0,
155.8, 169.4, 169.6, 170.1, 170.3, 170.6, 170.7, 170.8; HRESIMS:
Calcd for C73H90Cl6N6O36N6Cl6 [M+Na]+, m/z 1859.3425; found,
1859.34469. Calcd for [M+K]+, m/z 1875.31645; found, 1875.31979.
a
7.6 Hz, Fmoc -CH2), 4.30 (1H, d, J 8.4 Hz, Thr -CH ), 4.45 (1H, s,
Fmoc -CH), 4.47 (1H, dd, J 1.5 Hz, 6.1 Hz, Thr -CHb), 4.76 (1H, d, J
7.6 Hz, H-10), 5.04 (1H, dd, J 3.3 Hz, 10.5 Hz, H-30), 5.08 (1H, d, J
3.8 Hz, H-1), 5.30 (1H, dd, J 8.1 Hz, 10.5 Hz, H-20), 5.41 (1H, d, J
3.3 Hz, H-40), 5.70 (1 H, d, J 9.5 Hz, –NH), 7.31 (2H, at, J 7.6 Hz,
7.1 Hz, Fmoc -Ar), 7.40 (2H, dt, J 3.8 Hz, 7.1 Hz, Fmoc -Ar), 7.63
(2H, d, J 6.7 Hz, Fmoc -Ar), 7.77 (2H, d, J 7.2 Hz, Fmoc -Ar), 13C
NMR (CDCl3): dC 19.2, 20.7, 20.8, 20.9, 28.2, 47.3, 58.7, 59.2, 61.8,
62.9, 67.5, 68.5, 69.4, 69.7, 70.8, 71.5, 76.0, 77.0, 77.2, 77.4, 78.0,
83.2, 99.4, 102.1, 120.2, 124.5, 125.4, 127.2, 127.3, 127.9, 141.5,
143.9, 144.1, 156.9, 169.4, 169.8, 170.2, 170.3, 170.7; HRESIMS:
Calcd for C43H53N4O18 [M+Na]+, m/z 937.3325; found, 937.3335.
a
3.11. N -(Fluoren-9-ylmethoxycarbonyl)-O-{O-(2,3,4,6-tetra-O-
acetyl-b-
acetyl-2-deoxy -b-
acetyl-2-deoxy-
D
-galactopyranosyl)-(1?3)-O-[2-acetamido-3,4,6-tri-O-
-glucopyranosyl-(1?6)]-2-acetamido-4-O-
-galactopyranosyl- -threonine tert-butyl
D
a-
D
L
ester (12)
a
3.10. N -(Fluoren-9-ylmethoxycarbonyl)-O-{O-(20,30,40,60-tetra-
The trisaccharide 11 (61 mg, 0.04 mmol) was dissolved in 3:2:1
THF–AcOH–Ac2O (10 mL). Zinc powder (1 g) activated in 2% aq
CuSO4 was added, and the reaction mixture was stirred at room
temperature for 1 h. The solid was filtered off through a pad of Cel-
ite, and the residue was diluted with CH2Cl2, and then filtered
through a pad of Celite. The CH2Cl2 layer was subsequently washed
with 10% aq HCl (25 mL), collected, and dried over anhyd Na2SO4.
The solvents were removed under reduced pressure, and the resi-
due was subsequently dissolved in pyridine (1 mL) and cooled to
0 °C. Excess Ac2O (3 mL) was added to this solution, followed by
the addition of a catalytic amount of DMAP (10 mg). The reaction
mixture was allowed to warm to room temperature and stir for
an additional 3 h. After 3 h the solvents were removed under re-
duced pressure, and the resulting oil was purified by column chro-
matography (6:1 EtOAc–hexanes) over silica gel to yield 12 as a
O-acetyl-b-
D
-galactopyranosyl)-(1?3)-O-[3,4,6-tri-O-acetyl-2-
-glucopyranosyl-
-threonine
deoxy-2-2,2,2-trichloroethoxycarbonylamino)-b-
(1?6)]-2-azido-2-deoxy-
tert-butyl ester (11)
D
a-D-galactopyranosyl-L
Donor
5 (130 mg, 0.24 mmol) and acceptor 10 (180 mg,
0.20 mmol) were mixed with freshly activated 4 Å molecular sieves
(500 mg). The reaction mixture was suspended in CH2Cl2 (8.0 mL)
and stirred at room temperature for 2 h, then cooled to 0 °C. N-
Iodosuccinimide (0.09 g, 0.40 mmol) was added, and the suspen-
sion was stirred for 15 min. Trifluoromethanesulfonic acid
(3.5 lL, 0.04 mmol) was added, and stirring was continued over-
night at room temperature. After 12 h, the reaction mixture was fil-
tered through Celite into an aqueous solution of sodium thiosulfate
with disappearance of the dark red color as the solution became
colorless. The organic phase was separated, dried, and concen-
trated under reduced pressure. Subsequent purification by chro-
matography over silica gel (3:2 EtOAc–hexane), afforded the
desired trisaccharide 11 (142 mg, 51% yield) and tetrasaccharide
white amorphous solid (34 mg, 54%). ½a D22
ꢃ
+38.3 (c 1, CHCl3); 1H
NMR (CDCl3): dH 1.33 (3H, d, J 6.6 Hz, Thr -CH3); 1.51 (9H, s, –
NHBoc), 2.04 (24 H, 8 s, OAc), 3.46 (2H, m), 3.55 (1H, dd, J 3.6 Hz,
10.2 Hz, Gal-N3 H-2), 3.68 (1H, d, J 8.4 Hz), 3.91 (2H, m), 4.20–
4.00 (8H, m), 4.28 (5H, m), 4.39 (1H, m), 4.49 (1H, dd, J 7.2 Hz,
10.2 Hz), 4.57 (1H, d, J 11.4 Hz), 4.73 (2H, t, J 7.8 Hz, Gal H-1),
4.87 (1H, d, J 12.0 Hz, GlcNTroc-1,6 H-1), 5.05–5.00 (4H, m), 5.18
(1H, dd, J 7.8 Hz, 10.2 Hz), 5.36 (2H, m), 5.42 (1H, d, J 2.4 Hz),
5.45 (1H, d, J 7.8 Hz), 5.65 (1H, d, J 9.6 Hz, Gal H-4), 7.32 (2H,
ddd, J 3.0 Hz, 7.8 Hz, 7.8 Hz, -Fmoc Ar), 7.41 (2H, ddd, J 4.2 Hz,
7.8 Hz, 7.8 Hz, -Fmoc Ar), 7.63 (2H, d, J 7.2 Hz, -Fmoc Ar), 7.77
(2H, d, J 6.6 Hz, -Fmoc Ar); HRESIMS: C61H79N3O28 Calcd for
[M+Na]+, m/z 1324.47478; found, 1324.47592. Calcd for [M+K]+,
m/z 1340.44872; found, 1340.45010.
11a (53 mg, 19% yield). ½a D22
ꢃ
+45.4 (c 1, CHCl3); 1H NMR (CDCl3):
d 1.31 (3H, d, J 6.0 Hz, Thr -CH3), 1.51 (9H, s, –NHBoc), 1.98 (3H,
s, OAc), 2.00 (3H, s, OAc), 2.01 (3H, s, OAc), 2.02 (3H, s, OAc),
2.05 (3H, s, OAc), 2.08 (3H, s, OAc), 2.10 (3H, s, OAc), 3.54 (1H, d,
J 10.8 Hz, Gal-N3 H-2), 3.67 (1H, m), 3.74 (1H, m), 4.20–4.40 (12
H, m), 4.50–4.61 (2H, m, Fmoc CH), 4.67 (1H, d, J 8.4 Hz, Thr
CHb), 4.70 (1H, d, J 12.0 Hz, GlcNTroc-1,6 H-1), 4.74 (1H, d, J
7.8 Hz, Gal H-1), 4.83 (1H, d, J 12.6 Hz), 4.99 (1H, d, J 9.0 Hz, Gal-
N3 H-1), 5.11 (3H, m), 5.26 (1H, ddd, J 8.4 Hz, 12 Hz, 22.5 Hz, Gal
H-2), 5.28 (1H, d, J 3.0 Hz, GlcNTroc-1,6 H-3), 5.52 (1H, d, J
8.4 Hz), 5.61 (1H, d, J 9.6 Hz, Gal H-4), 6.07 (1H, d, J 9.6 Hz, Thr
-NH), 7.16 (1H, d, J 6.6 Hz), 7.32 (2H, dd, J 7.2 Hz, 7.8 Hz, -Fmoc
Ar), 7.43 (2H, m, -Fmoc Ar), 7.63 (2H, d, J 7.2 Hz, -Fmoc Ar), 7.77
(2H, d, J 7.8 Hz, -Fmoc Ar); 13C NMR (CDCl3): dC 19.2, 20.7, 20.8,
20.9, 20.9, 28.2, 47.3, 56.5, 58.7, 59.2, 61.5, 62.2, 67.0, 67.7,
68.6, 68.7, 69.2, 69.3, 70.9, 71.3, 72.5, 72.1, 74.6, 75.6, 83.4, 95.6,
99.2, 101.3, 102.2, 120.2, 125.1, 125.3, 127.2, 127.3, 127.9,
141.5, 143.9, 144.0, 154.2, 157.1, 158.7, 159.3, 169.5, 169.7,
169.9, 170.3, 170.4, 170.6, 170.8, 170.9; HRESIMS: Calcd for
a
3.12. N -(Fluoren-9-ylmethoxycarbonyl)-O-{O-(2,3,4,6-tetra-O-
acetyl-b-
acetyl-2-deoxy-b-
acetyl-2-deoxy-
D
-galactopyranosyl)-(1?3)-O-[2-acetamido-3,4,6-tri-O-
-glucopyranosyl-(1?6)]-2-acetamido-4-O-
-galactopyranosyl- -threonine (1)
D
a
-D
L
The protected trisaccharide 12 (27 mg) was dissolved in CH2Cl2
(4 mL) and cooled to 0 °C. Trifluoroacetic acid (2 mL) was added,
and the reaction mixture was allowed to warm slowly to room tem-
perature. After 2 h the solvent was concentrated under reduced
pressure, and the residue was loaded onto a reversed-phase HPLC
column with gradient eluting from 70:30 to 30:70 of H2O–CH3CN
at a flow rate of 6 mL/min. The fraction eluting at a retention time
of 58 min was collected and concentrated under reduced pressure
C
58H72Cl3N5O27 [M+K]+, m/z 1414.31144; found, 1414.31119.
Analytical data for 11a: ½a D22
ꢃ
+7.5 (c 1, CHCl3); 1H NMR (CDCl3):
dH 1.33 (3H, d, J 6.6 Hz, Thr –CH3), 1.50 (9H, s, –NHBoc), 2.04 (27H,
s, 9 ꢁ 3 OAc’s), 2.18 (3H, s), 3.54 (1H, dd, J 3.6 Hz, 10.8 Hz), 3.61
(1H, m), 3.70 (1H, d, J 7.2 Hz), 3.80 (1H, t, J 7.8 Hz), 3.95–4.4
(18H, m), 4.41 (1H, d, J 6.0 Hz), 4.48 (1H, dd, J 7.2 Hz, 9.6 Hz),
4.58 (1H, d, J 12.0 Hz), 4.73 (1H, d, J 19.2 Hz), 4.76 (1H, d, J
7.8 Hz), 4.86 (1H, d, J 12.0 Hz), 5.00 (1H, d, J 4.2 Hz), 5.10–5.03
(4H, m), 5.30 (2H, m), 5.41 (1H, d, J 3.0 Hz), 5.49 (1H, d, J 8.4 Hz),
5.65 (1H, d, J 9.6 Hz), 6.05 (1H, d, J 8.5 Hz, 1H), 7.17 (1H, d, J
to obtain 1 as a white amorphous solid (19 mg, 72% yield). ½a D22
ꢃ
+35.7 (c 1, CHCl3); 1H NMR (CDCl3): dH 1.21 (3H, d, J 6.6 Hz, Thr
-CH3), 2.04 (30H, s, 10 ꢁ 3 OAc’s), 2.48 (1H, br s, –OH), 3.43 (1H, t, J
8.4 Hz), 3.66–3.69 (2H, m), 3.79–3.83 (4H, m), 4.04–4.13 (5H, m),
4.22–4.29 (5H, m), 4.42 (1H, d, J 6.0 Hz), 4.56–4.51 (3H, m), 4.68
(1H, d, J 7.8 Hz), 4.84 (1H, m), 4.92 (1H, m), 4.97–5.03 (2H, m), 5.25

V. R. Krishnamurthy et al. / Carbohydrate Research 345 (2010) 1541–1547
1547
3. (a) Phillips, M. L.; Nudelman, E.; Gaeta, F. C. A.; Perez, M.; Singhal, A. K.;
Hakomori, S.; Paulson, J. C. Science 1990, 250, 1130–1132; (b) Walz, G.; Arrufo,
A.; Kolanus, W.; Bevilacqua, M.; Seed, B. Science 1990, 250, 1132–1135; (c)
McEver, R. P.; Moore, K. L.; Cummings, R. D. J. Biol. Chem. 1995, 270, 11025–
11028.
(2H, m), 5.32 (1H, d, J 3.6 Hz), 7.30 (2H, ddd, J 3.0 Hz, 7.8 Hz, 7.8 Hz, -
Fmoc Ar), 7.39 (2H, ddd, J 4.2 Hz, 7.8 Hz, 7.8 Hz, -Fmoc Ar), 7.50 (1H,
br s, –NH), 7.60 (2H, d, J 7.2 Hz, -Fmoc Ar), 7.76 (2H, d, J 6.6 Hz, -Fmoc
Ar); 13C NMR (CDCl3): dC 18.5, 20.7, 20.8, 20.9, 22.9, 23.1, 29.8, 47.4,
49.3, 49.4, 49.5, 49.7, 49.8, 50.0, 54.8, 58.5, 61.1, 62.1, 66.7, 66.9, 68.5,
68.6, 68.7, 68.8, 69.5, 70.7, 70.9, 72.0, 72.5, 76.1, 98.9, 99.0, 100.5,
101.0, 120.1, 125.0, 127.3, 127.4, 127.9, 141.5, 143.9, 144.0, 157.0,
157.2, 169.7, 169.8, 170.6, 170.8, 171.0, 171.1, 172.1; HRESIMS:
Calcd for C57H71N3O28 [M+H]+, m/z 1246.1769; found, 1246.4297.
4. Welply, J. K.; Keene, J. L.; Schmuke, J. J.; Howard, S. C. Biochim. Biophys. Acta
1994, 1197, 215–226.
5. (a) Wilkins, P. P.; Moore, K. L.; McEver, R. P.; Cummings, R. D. J. Biol. Chem. 1995,
270, 22677–22680; (b) DeLuca, M.; Dunlop, L. C.; Andrews, R. K.; Flannery, J. V.,
Jr.; Ettling, R.; Cumming, D. A.; Veldman, G. M.; Berndt, M. C. J. Biol. Chem. 1995,
270, 26734–26737; (c) Sako, D.; Comess, K. M.; Barone, K. M.; Camphausen, R.
T.; Cumming, D. A.; Shaw, G. D. Cell 1995, 83, 323–331; (d) Pouyani, T.; Seed, B.
Cell 1995, 83, 333–343.
6. (a) Cummings, R. D. Braz. J. Med. Biol. Res. 1999, 32, 519–528; (b) Moore, K. L.;
Stults, N. L.; Diaz, S.; Smith, D. F.; Cummings, R. D.; Varki, A.; McEver, R. P. J. Cell
Biol. 1992, 118, 445–456.
3.13. Glycopeptide 14
7. Leppanen, A.; Mehta, P.; Ouyang, Y.-B.; Ju, T.; Helin, J.; Moore, K. L.; van Die, I.;
Canfield, W. M.; McEver, R. P.; Cummings, R. D. J. Biol. Chem. 1999, 274, 24838–
24848.
The peptide 14 was synthesized manually on a Rink amide resin
using a standard Fmoc amino acid coupling strategy. Briefly, Fmoc-
Cys-Rink-amide resin (0.4 mg, 0.5 mmol/g) was loaded into a poly-
8. (a) Leppanen, A.; White, S. P.; Helin, J.; McEver, R. P.; Cummings, R. D. J. Biol.
Chem. 2000, 275, 39569–39578; (b) Leppanen, A.; Penttila, L.; Renkonen, O.;
McEver, R. P.; Cummings, R. D. J. Biol. Chem. 2002, 277, 39749–39759.
9. (a) Koeller, K. M.; Smith, M. E. B.; Huang, R. F.; Wong, C.-H. J. Am. Chem. Soc.
2000, 122, 4241–4242; (b) Huang, K. T.; Wu, B. C.; Lin, C. C.; Luo, S. C.; Chen, C.
P.; Wong, C.-H.; Lin, C. C. Carbohydr. Res. 2006, 341, 2151–2155; (c) Baumann,
K.; Kowalczyk, D.; Gutjahr, T.; Pieczyk, M.; Jones, C.; Wild, M. K.; Vestweber, D.;
Kunz, H. Angew. Chem., Int. Ed. 2009, 48, 1–6.
10. (a) Vohra, Y.; Buskas, T.; Boons, G.-J. J. Org. Chem. 2009, 74, 6064–6071; (b)
Baumann, K.; Kowalczyk, D.; Kunz, H. Angew. Chem., Int. Ed. 2008, 47, 3445–
3449; (c) Hanashima, S.; Castagner, B.; Esposito, D.; Nokami, T.; Seeberger, P. H.
Org. Lett. 2007, 9, 1777–1779; (d) Otsubo, N.; Ishida, H.; Kiso, M. Tetrahedron
Lett. 2000, 41, 3879–3882.
propylene centrifuge filter tube (0.22
equipped with a plastic cap. The resin was swelled by stirring
gently with CH2Cl2 (4 ꢁ 500 L) for 10 min and filtered. The cou-
pling reaction was performed with Fmoc amino acid (1.6 mol,
L, 0.2 M), HBTU (1.6 mol, 8 L, 0.2 M), HOBt (1.6 mol, 8 L,
0.2 M), and DIEA (2.4 mol, 8 L, 0.3 M). Fmoc deprotection was
performed twice with 20% piperidine in DMF (100 L). Cleavage
of the peptide and removal of the side chain protecting group from
the resin were accomplished by gently stirring in 500 L of
lm, Corning International)
l
l
8
l
l
l
l
l
l
l
l
l
11. For chemical synthesis of sialyl Lex, see: (a) Kameyama, A.; Ishida, H.; Kiso,
M.; Hasegawa, A. Carbohydr. Res. 1991, 209, C1–C4; (b) Nicolaou, K. C.;
Hummel, C. W.; Bockovich, N. J.; Wong, C.-H. J. Chem. Soc., Chem. Commun.
1991, 13, 870–872; (c) Nicolaou, K. C.; Hummel, C. W.; Iwabuchi, Y. J. Am.
Chem. Soc. 1992, 114, 3126–3128; for enzymatic synthesis of sialyl Lex, see:
(d) Huang, K. T.; Wu, B. C.; Lin, C. C.; Luo, S. C.; Chen, C.; Wong, C.-H.; Lin, C.
C. Carbohydr. Res. 2006, 341, 2151–2155; (e) Palcic, M. M.; Venot, A.; Ratcliffe,
R. M.; Hindsgaul, O. Carbohydr. Res. 1989, 190, 1–11; (f) Dumas, D. P.;
Ichikawa, Y.; Wong, C.-H.; Lowe, J. B.; Nair, R. P. Bioorg. Med. Chem. Lett. 1991,
1, 425–428.
1:2:2:95 of H2O–EDT–Et3SiH–TFA for 2 h. The solvent was evapo-
rated, and the yield of glycopeptide 14 was determined to be 50%
(yield was determined by HPLC). The yield of the second product
14a was determined to be 43% (yield was determined by HPLC).
The molecular weights of glycopeptide 14 and 14a were deter-
mined by MALDI-TOFMS analysis. Calcd for 14 C85H112N10O39
[M+Na]+, m/z 1952.6546; found, 1952.706. Calcd for 14a
87H116N10O39S3 [M+H], m/z 2021.66415; found, 2022.677. Calcd
S
C
12. Mathieux, N.; Paulsen, H.; Meldal, M.; Bock, K. J. Chem. Soc., Perkin Trans. 1
1997, 13, 2359–2368.
for [M+Na], m/z 2043.6461; found, 2044.685.
13. (a) Paulsen, H.; Schultz, M.; Klamann, J. D.; Waller, B.; Paal, M. Liebigs Ann.
Chem. 1985, 66, 2028–2048; (b) Reimer, K. B.; Meldal, M.; Kusumoto, S.;
Fukase, K.; Bock, K. J. Chem. Soc., Perkin Trans. 1 1993, 925–932.
14. Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404–3409.
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Acknowledgments
This work was supported by grants from the National Institutes
of Health and Juvenile Diabetes Research Foundation. The authors
would like to thank Complex Carbohydrate Research Center (CCRC,
University of Georgia) for 2D NMR analysis.
Supplementary data
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Supplementary data (1D and 2D NMR spectra) associated with
this article can be found, in the online version, at doi:10.1016/
j.carres.2010.05.004.
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