Synthesis of LewisX-O-Core-1 threonine: A building block for O-linked LewisX glycopeptides

Article abstract of DOI:10.1016/j.carres.2017.10.002

Lewis^X (Le^X) is a branched trisaccharide Galβ1→4(Fucα1→3)GlcNAc that is expressed on many cell surface glycoproteins and plays critical roles in innate and adaptive immune responses. However, efficient synthesis of glycopeptides bearing Le^X remains a major limitation for structure-function studies of the Le^X determinant. Here we report a total synthesis of a Le^X pentasaccharide 1 using a regioselective 1-benzenesulfinyl piperidine/triflic anhydride promoted [3 + 2] glycosylation. The presence of an Fmoc-threonine amino acid facilitates incorporation of the pentasaccharide in solid phase peptide synthesis, providing a route to diverse O-linked Le^X glycopeptides. The described approach is broadly applicable to the synthesis of a variety of complex glycopeptides containing O-linked Le^X or sialyl Lewis^X (sLe^X).

Full text of DOI:10.1016/j.carres.2017.10.002

Accepted Manuscript
X
X
Synthesis of Lewis -O-core-1 threonine: A building block for O-linked Lewis
glycopeptides
Mohammed Y.R. Sardar, Venkata R. Krishnamurthy, Simon Park, Appi R.
Mandhapati, Walter J. Wever, Dayoung Park, Richard D. Cummings, Elliot L. Chaikof
PII:
S0008-6215(17)30550-5
10.1016/j.carres.2017.10.002
CAR 7456
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 29 August 2017
Revised Date: 7 October 2017
Accepted Date: 7 October 2017
Please cite this article as: M.Y.R. Sardar, V.R. Krishnamurthy, S. Park, A.R. Mandhapati, W.J. Wever,
X
D. Park, R.D. Cummings, E.L. Chaikof, Synthesis of Lewis -O-core-1 threonine: A building block for O-
X
linked Lewis glycopeptides, Carbohydrate Research (2017), doi: 10.1016/j.carres.2017.10.002.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of LewisX-O-Core-1 threonine: A building block for O-linked
LewisX glycopeptides
Mohammed Y. R. Sardar1,2, Venkata R. Krishnamurthy1,2, Simon Park1,2, Appi R. Mandhapati1,2,
Walter J. Wever1,2, Dayoung Park1,2, Richard D. Cummings1 & Elliot L. Chaikof1,2,3*
1Department of Surgery, Center for Drug Discovery and Translational Research, Beth Israel
Deaconess Medical Center, Harvard Medical School, 110 Francis Street, Suite 9F, Boston,
Massachusetts 02215, USA.
2Wyss Institute of Biologically Inspired Engineering, Harvard University, 110 Francis Street,
Suite 9F, Boston, Massachusetts 02115, USA.
3Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139
*Corresponding author
Elliot L. Chaikof, M.D., Ph.D.
Department of Surgery
Beth Israel Deaconess Medical Center
110 Francis St, Suite 9F
Boston, MA 02115
E-mail: echaikof@bidmc.harvard.edu
Phone: 617-632-9581

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2
Abstract
Lewis X (LeX) is a branched trisaccharide Gal
β1→4(Fucα1→3)GlcNAc that is expressed
on many cell surface glycoproteins and plays critical roles in innate and adaptive immune
responses. However, efficient synthesis of glycopeptides bearing LeX remains a major limitation
for structure-function studies of the LeX determinant. Here we report a total synthesis of a LeX
pentasaccharide 1 using a regioselective 1-benzenesulfinyl piperidine/triflic anhydride promoted
[3+2] glycosylation. The presence of an Fmoc-threonine amino acid facilitates incorporation of
the pentasaccharide in solid phase peptide synthesis, providing a route to diverse O-linked LeX
glycopeptides. The described approach is broadly applicable to the synthesis of a variety of
complex glycopeptides containing O-linked LeX or sialyl LewisX (sLeX).

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Introduction
The structural diversity of glycoproteins underlies a variety of molecular recognition
events, which are critical to many biological functions, including innate and adaptive immune
responses. LewisX (LeX) is a branched trisaccharide Gal
β1→4(Fucα1→3)GlcNAc that is present
on a variety of cell surface glycoproteins, including CD15,1–5 carcinoembryonic antigen-related
cell adhesion molecule (CEACAM),6–9 and stage specific embryonic antigen-1 (SSEA-1).3,4,10–13
Glycoproteins expressing LeX have been identified as early mediators of the immune response
against invading pathogens,6,14,15 and are pivotal in immunomodulation,6,16–19 regulation of the
growth and angiogenesis of tumor cells,1,4 embryogenesis of the central nervous system3,10,11
and promotion of the formation of a spermatozoa reservoir and subsequent release from the
oviduct.20–22 DC-SIGN (dendritic cell specific ICAM grabbing non-integrin) is a counter-receptor
to many LeX bearing glycoproteins, including CEACAM-1, Mac-1 and ICAM3, as well as
unidentified LeX glycoproteins on invading pathogens and neutrophils.2,7,9,23–27 In turn, the
interaction of dendritic cells and neutrophils enables antigen-presenting cells to stimulate T cells
and modulate adaptive immunity. Despite the critical role of DC-SIGN in the recognition of
invading pathogens, human immunodeficiency virus (HIV), hepatitis C virus, and Mycobacterium
tuberculosis, also use DC-SIGN to escape the immune response.17,28,29 A recent study has also
demonstrated that terminal LeX on cell surface glycoproteins regulates neutrophil chemotaxis,
transepithelial migration, and post-migratory function.19 Hence, blocking the carbohydrate
binding site of DC-SIGN may provide a strategy to impede the immunological evasion of a
number of infectious pathogens.14,30–32 Moreover, blocking the interaction of LeX glycoproteins
with their respective counter receptors holds therapeutic potential for many diseases, including
rheumatoid arthritis, inflammatory bowel diseases, metabolic syndrome, cancer, HIV, as well as
many other chronic disorders of innate immunity. LeX-antigens (CD15) are also expressed on
cancer stem cells and may be potentially used to develop antitumor vaccines.

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In nature, glycans are either linked to proteins or lipids and their functions are
increasingly best understood in the context of its aglycone component. Thus, LeX containing
glycans linked to model glycopeptides provide an important tool to study proteins that may
interact with LeX in context of the aglycone moiety. An important challenge, however, in the
generation of high affinity glycopeptide ligands is the lack of efficient and scalable chemical
synthetic schemes. For example, undesired regio- and stereoselectivity during key glycosylation
steps have limited the development of an efficient chemical synthesis of P-selectin glycoprotein
ligand-1 (PSGL-1) analogues.33,34 Additionally, the chemical incorporation of N-
acetylglucosamine remains a significant impediment due to the formation of inert oxazoline
during the activation step35,36 A common strategy to bypass oxazoline formation is to employ a
phthalimide protecting group, which has been used for the synthesis of LeX containing
analogues in other reports.37–45 However, the conditions for the removal of the phthalimide
protecting group are incompatible with an Fmoc group, essential to generating a glycopeptide.
Moreover, the fucopyranosyl glycosidic linkage in LeX is highly acid-sensitive and any
modification involving a stronger acid results in degradation of the fucosyl residue.46 Thus, there
is great need to develop efficient schemes for the facile synthesis of LeX bearing O-glycans.
While glycopeptides containing LeX are responsible for diverse biological functions, structural
and conformational information of these glycans in participating molecular interactions are
poorly understood. Hence, it is critical to establish a synthetic platform for glycopeptides
containing LeX. Lack of facile synthesis of complex O-glycans still impedes the generation
glycopeptide despite seminal contributions by research groups including Danishefsky, Wong,
and Kunz.46–50
The mucin type T-antigen (Galβ1→3GalNAcα-O-Ser/Thr) is one of the most abundant
O-glycan motifs of many complex glycoproteins and is the precursor for the synthesis of the
branched core 2 O-glycan on which LeX can be found within a pentasaccharide (LeX-C2-O-
pentasaccharide). The availability of a LeX-C2-O-pentasaccharide-bearing threonine amino

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acid 1 (Fig. 1) will be a very useful precursor towards the synthesis of a variety of glycopeptides
for biological studies. In this report, we describe the synthesis of C2-O-LeX pentasaccharide 1
as a glycopeptide building block by a convergent [3+2] glycosylation between a LeX donor 2 and
a Core-1 diol acceptor 3.
Figure 1. Retrosynthesis of C2-O-LeX pentasaccharide.
Results and Discussion
Galactosyl thioglycoside donor 8, fucosyl imidate donor 13, and Troc-protected
glucosamine diol acceptor 19 were chosen as suitable building blocks for LeX donor 2 (Fig. 2).
Orthogonality of the protecting groups and ease of synthesis provided the rationale for selecting
building blocks 8, 9, and 15. In particular, the NHTroc protecting group was chosen as it
displays β
-selectivity and its deprotection conditions are compatible with NHFmoc chemistry.51
Figure 2. Building blocks for LeX donor 2.

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The synthesis of 8 commenced from commercially available D-galactose 4. As detailed
elsewhere,
D-galactose 4 was converted to galactose thioglycoside 5 by initial acetylation using
pyridine-acetic anhydride and catalytic DMAP, followed by substitution of the anomeric OAc
using BF3OEt2, PhSH.52 Deacetylation of 5 under Zémplen conditions afforded the intermediate
tetraol, which was converted in situ using PhCH(OMe)2 and catalytic camphor sulfonic acid to
the 4,6-O-benzylidine acetal protected 6.53 Regioselective cleavage was carried out using
triethylsilane and trifluoroacetic acid to afford the 6-O-benzyl protected galacoside donor 7. The
free hydroxyl in 7 was subsequently acetylated using acetic anhydride and pyridine to obtain
donor 8 (Scheme 1).53 Of note, TES/TFA was used in the regioselective ring opening of 6,
which lead to a significantly higher yield, as compared to previously reported approaches that
have used BH
3
.NMe
3
-AlCl .
3
Scheme 1. Synthesis of galactose donor 5: a) Py, Ac2O, DMAP(cat): b) PhSH, BF3 OEt2, CH2Cl2, rt, 12h:
c) 0.2 (N) NaOMe, MeOH, 5h: d) PhCH(OMe)2, cat CSA, CH3CN, 80°C: e) TES, TFA, CH2Cl2, RT 3h: f)
Py, Ac2O, DMAP(cat), rt, 3h: TES = triethylsilane, TFA = trifluoroacetic acid, DMAP = 4-
dimethylaminopyridine.
D
-Glucosamine was selectively protected using TrocCl and sodium bicarbonate as an
initial step to synthesize acceptor 15. The Troc protected glucosamine was then converted to
tetracetylated -glucosamine 11 by pyridine and acetic anhydride. Compound 11 was then
converted to thioglycoside 12 by treatment with BF3-OEt2 and PhSH. Deacetylation under
D

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Zémplen conditions afforded triol 17, which was converted in situ to a 4,6-O-benzylidene acetal
14 by PhCH(OMe)
15.
2
and catalytic CSA.54 Regioselective ring opening of 14 afforded acceptor
The approach of selecting either galactosylation or fucosylation of diol 15 was influenced
by prior studies by Ellervik, Cao, and colleagues.37,53 While the galactosylation of a similar diol
acceptor resulted in a (1
ratio favoring the (1 3) linked glycan. Hence, the synthesis of thioglycoside 2 was initiated by
assembling Gal (1 4)-GalNAc 16 using iterative glycosylation. Preactivation of thioglycoside
→4) linked glycan, exclusively, fucosylation was associated with a 3.6:1
→
β
→
donor 8 at -70°C using BSP in the presence of triflic anhydride, followed by the addition of
acceptor 15 afforded thioglycoside 16 in 76% yield (Scheme 3). It was imperative to quench
the excess promoter with 1-octene before addition of the acceptor in the reaction mixture.55
This low temperature pre-activation strategy enabled selective activation of thioglycoside in 8
without activation of thioglycoside in 15. As a consequence, byproducts arising from
oligomerization of 15 were minimized and overall yield of the desired disaccharide 16 improved.
The outcome of the glycosylation reaction between donor 8 and acceptor 15 was in agreement
with previous reports53,56 and afforded the
β(1→4) linked product, which was confirmed by
COSY and HSQC NMR spectra (Supporting Information). Specifically, the coupling constant of
the anomeric proton (4.50 ppm, J = 8.0 Hz) and the downfield 13C chemical shift (78.0 ppm) of
glucosamine C4 were consistent with β(1→4) linkage formation. The resulting disaccharide 16
was readily fucosylated using fucosyl imidate donor 957 and activated by TMS-OTf catalyst at -
20°C to obtain the LeX thioglycoside 2 in 72% yield. Sterically crowded 3-OH group of
glucosamine is known to be a poor acceptor and excess fucose donor was required to drive the
reaction to completion.58 The
α(1→3) linkage was confirmed by COSY and HSQC NMR
spectra, as demonstrated by the coupling constant of the fucosyl anomeric proton (5.15 ppm, J
= 3.6 Hz).

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8
OH
OAc
OAc
O
c
86%
a, b
O
O
HO
HO
AcO
AcO
AcO
AcO
OH
OAc
NHTroc
SPh
NHTroc
91%
(2 steps)
NH2HCl
10
11
12
OBn
O
OH
Ph
O
e
d
f
O
O
O
HO
HO
HO
HO
SPh
SPh
NHTroc
SPh
NHTroc
HO
78%
84%
(2 steps)
NHTroc
14
15
13
OBn
AcO
OBn
AcO
NH
O
OBn
OBn
O
OBn
O
CCl3
8
, g
O
O
OBn
O
13
O
O
BnO
h
SPh
NHTroc
SPh
NHTroc
AcO
AcO
O
HO
OAc
76%
OAc
72%
O
16
OBn
OBn
2
BnO
Scheme 3. Synthesis of sLeX thioglycoside: a) TrocCl, NaHCO3, H2O, 0°C: b) Py, Ac2O, DMAP(cat), c)
PhSH, BF3 OEt2, CH2Cl2, 12h, rt: d) 0.1 (N) NaOMe, MeOH, 2h: e) PhCH(OMe)2, cat CSA, CH3CN: f)
TES, TFA, CH2Cl2, RT 3h: g) BSP, Tf2O, octene, 4 Å MS, CH2Cl2, -60°C, 1h: h) TMS-OTf, 4 Å MS,
CH2Cl2, -20°C, 1h: CSA = (1S)-(+)-10-camphorsulfonic acid, BSP = 1-benzeneylsulfinylpiperidine, MS =
molecular sieves.
We performed a [3+2] glycosylation between the LeX thioglycoside and the Core-1 diol33
using 1-benzenesulfinylpiperidine (BSP), Tf2O promoter59, and 2,4,6-tri-tert-butylpyrimidine
TTBP at -60°C. Unfortunately, the yield was poor and a significant amount of hydrolyzed donor
was recovered (Table 1, entry 1). With the presumption that the reaction temperature was too
low for such bulky donor and acceptor, we explored promoters suitable for activation at higher
temperature. Dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST)60 was found to
be compatible with room temperature activation. Unfortunately, the yield continued to be poor
and a new regioisomer was formed, as observed in 1H NMR (Table 1, entry 2). Furthermore,
de-fucosylated tetrasaccharide was observed by MALDI-TOF, highlighting the acid lability of the
highly armed fucopyranoside. As a consequence, we investigated the NIS/TfOH61,62 promoter
and performed the glycosylation at a lower temperature. While yield improved, regioselectivity
and cleavage of fucose, though minimized, continued to be problematic (Table 1, entry 3).
Similar reaction conditions with a different solvent system (DCM-toluene) did not improve the
regioselectivity. While unexpected, we have previously observed the formation of a similar 1→4

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9
linkage.33 We speculate that the intermediate oxazolinium ion reacts with the 6-OH group via
the exocyclic carbon and upon intramolecular rearrangement forms the 1→4 linked product. It
was apparent that the stability of the fucose and regioselectivity were both dependent on
reaction temperature. Therefore, we attempted the glycosylation reaction at -40°C using
NIS/TfOH activation with improved yield and regioselectivity. Anticipating that the presence of
base might facilitate the formation of an inert oxazoline, we explored glycosylation BSP-Tf2O
activation without TTBP and observed optimal yield with only a single regioisomer (Table 1,
entry 6).
Scheme 4. [3+2] Glycosylation: a) BSP, Tf2O, 4 Å MS, CH2Cl2, -60°C, 1h, 84%: b) NIS, TfOH, 4 Å MS,
CH2Cl2, -40°C, 1h, 81%.
Table 1
Entry Promoter
Temperature Solvent
Yield Selectivity*
1
BSP, TTBP, Tf2O
- 60°C
DCM
48%
20:1
2
3
DMTST
r.t.
DCM
DCM
40%
54%
3:1
4:1
NIS, TfOH
0°C
4
5
NIS, TfOH
NIS, TfOH
0°C
DCM/Toluene(3:1) 52%
4:1
- 40°C
DCM
DCM
81%
84%
10:1
6
BSP, Tf2O
- 60°C
20:1
*ratio between 1
→
6 and 1
→4 linked regioisomer

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Structural assignment of the final pentasaccharide was made by a combination of 1-D
(1H and 13C) and 2-D (COSY, HSQC, and HMBC) NMR spectroscopy (Supporting information).
13C NMR spectra of the Core-1 diol and pentasaccharide 1 revealed a significant downfield shift
of C6 of GalN3Thr, while C4 was unchanged indicating the formation of 1
Furthermore, HMBC correlation between H6 protons of GalN3 and C1 of GlucNAc confirmed the
formation of 1
6 linkage. Additionally, a large coupling constant of GlcNHTroc H1 (4.62 ppm, J
= 10.2 Hz) confirmed the formation of the -linkage.
→6 linkage.
→
β
In conclusion, we were successful in synthesizing a protected LeX bearing Core-1-O-
threonine pentasaccharide, and the anomeric linkage was assigned by 2-D NMR. Major
synthetic hurdles involving efficient development of LeX and regio- and enantioselective [3+2]
glycosylation were resolved. The synthetic methodology described herein can serve as a
strategy to afford various glycoamino acid analogues in high yield. Furthermore, the protected
C2-O-LeX pentasaccharide 1 will serve as an important building block in future efforts directed at
the synthesis of a variety of target glycoproteins to probe the biological roles of LeX in the
context of diverse aglycone peptide sequences.
3. Experimental
3.1 General
All reactions were performed under inert atmosphere of nitrogen or argon, unless
otherwise noted.
D-Galactose, D-glucosamine hydrochloride, and L-fucose were purchased
from Carbosynth LLC, CA. All other reagents were purchased from commercial sources and
used directly. All solvents were dried and distilled following standard protocols. All glycosylation
reactions were performed in oven dried round bottom flasks. Proton nuclear magnetic
resonance (1H NMR) and 13C NMR spectra were recorded with a Varian 400-MHz spectrometer.
High Resolution Mass spectra (HRMS) were acquired using an Orbitrap Lumos MS (Thermo
Fisher Scientific, CA). Thin layer chromatography (TLC) was performed on silica gel matrix,

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with a 254 nm fluorescent indicator, and flash column chromatography purification performed on
Silica Gel 60 (Sigma-Aldrich Corporation, WI).
3.4. Phenyl 2,3,4-tri-O-acetyl-6-O-benzyl-1-thio- β-D-galactopyranoside (8)
TFA (5.3 mL, 68 mmol) and triethylsilane (10.9 mL, 68.0 mmol) were added to a solution
of galactose diol 6 (4.68 gm, 13.0 mmol) in CH2Cl2 (50 mL) at 0°C and stirred at room
temperature for 3 h. The reaction was monitored by TLC and upon completion of the reaction,
concentrated in vacuo. The crude residue was dissolved in pyridine (50 mL), followed by the
addition of Ac2O (30 mL) and DMAP (245 mg, 2.0 mmol), which was stirred at room temperature
for 12 h. The reaction mixture was concentrated and dissolved in EtOAc (100 mL), washed with
satd aq NaHCO3 (2 x 50 mL), brine (100 mL), dried over Na2SO4, filtered, and concentrated in
vacuo. The crude residue was purified by column chromatography over silica gel using 30%
EtOAc in hexanes to obtain a white solid (5.4 gm, 85%). The NMR spectra for 8 matched those
previously reported.53 1H NMR (400 MHz; CDCl3)
δ 7.52-7.49 (2H, m), 7.33-7.26 (8H, m), 5.49
(1H, dd, J 0.8, 3.2 Hz), 5.23 (1H, t, J 10.0 Hz), 5.05 (1H, dd, J 3.2, 10.0 Hz), 4.73 (1H, d, J 10.0
Hz), 4.54 (1H, d, J 11.6 Hz), 4.42 (1H, d, J 11.6 Hz), 3.89 (1H, td, J 1.2, 6.0 Hz), 3.60 (1H, dd, J
6.0, 9.6 Hz), 3.50 (1H, dd, J 6.4, 9.6 Hz), 2.08 (3H, s), 2.03 (3H, s), 1.97 (3H, s).
3.8. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-α/β-D-
glucopyranoside (11)
To a solution of D-glucosamine hydrochloride (20 g, 92.8 mmol) in H2O (250 mL) satd aq
NaHCO3 (250 mL) and 2,2,2-trichloroethyl chloroformate (14.05 mL, 102 mmol) were added and
stirred vigorously for 18 h at room temperature. The reaction mixture was filtered through a
fritted funnel and the white solid was dried under vacuum overnight. A solution of the solid in
pyridine (100 mL) was cooled to 0°C, to which Ac2O (100 mL) was added slowly, followed by
DMAP (500 mg, 41 mmol). The solution was warmed to room temperature and stirred for 12 h.
The solution was then diluted with CH2Cl2 (200 mL), washed with 1M HCl (3 x 100 mL), satd aq

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NaHCO3 (2 x 100 mL), brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo,
to afford glucosamine 11 as a white foam (44.3 g, 91%). The NMR spectra for 11 matched
those previously reported.54 1H NMR (400 MHz; CDCl3)
δ: 6.23 (1H, d, J 3.4 Hz), 5.28 (1H, dd,
J 10.7, 9.6 Hz), 5.20 (1H, t, J 9.8 Hz), 5.14 (1H, d, J 9.3 Hz, NHTroc), 4.82 (1H, d, J 12.1 Hz),
4.62 (1H, d, J 12.1 Hz), 4.28 (1H, dd, J 12.4, 4.0 Hz), 4.20 (1H, ddd, J 10.7, 9.4, 3.8 Hz), 4.06
(1H, dd, J 12.5, 2.4 Hz), 4.06-4.01 (1H, m), 2.20 (3H, s), 2.09 (3H, s), 2.04 (6H, 2xs).
3.9. Phenyl 3,4,6-tri-O-acetyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-
glucopyranoside (12)
Glucosamine 11 (14.5 g, 27.8 mmol) was dissolved in anhydrous CH2Cl2 (150 mL) and
cooled to 0°C. Thiophenol (6.8 mL, 66 mmol) and BF3•OEt2 (6.6 mL, 55.4 mmol) were added
and the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was
diluted in CH2Cl2 (100 mL), washed with water, aqueous NaHCO3 (saturated) (2 x 100 mL),
dried over MgSO4, filtered, and concentrated. Purification by column chromatography over
silica gel using 30% EtOAc in hexanes as eluent afforded thioglycoside 12 (13.7 gm, 86 %) as a
white foam. The NMR spectra for 12 matched those previously reported.54 1H NMR (400 MHz;
CDCl3)
δ 7.55-7.48 (2H, m), 7.35-7.28 (3H, m), 5.32 (1H, d, J 9.2 Hz), 5.28 (1H, t, J 9.8 Hz),
5.02 (1H, t, J 9.7 Hz), 4.86 (1H, d, J 10.3 Hz), 4.79 (1H, d, J 12.0 Hz), 4.72 (1H, d, J 12.0 Hz),
4.23 (1H, dd, J 12.4, 5.3 Hz), 4.16 (1H, dd, J 12.4, 2.5 Hz), 3.77-3.65 (2H, m), 2.07 (3H, s), 2.00
(3H, s),1.99 (3H, s).
3.10. Phenyl 4,6-O-benzylidene-2-deoxy-1-thio-2-(2,2,2-trichloroethoxycarbonyl-amino)-
-glucopyranoside (14)
Glucosamine thioglycoside 12 (5.2 gm, 9.1 mmol) was dissolved in MeOH (100 mL) and
β-
D
cooled to 0°C. A freshly prepared 0.2 (N) solution of NaOMe in MeOH (100 µL) was added
dropwise and the reaction mixture stirred for 5h at room temperature. The reaction was then
quenched with acetic acid (300 µL) before concentrating in vacuo. The resulting deacetylated

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crude was dissolved in MeCN (100 mL), after which PhCH(OMe)2 (5.5 mL, 36.7 mmol) and CSA
(464 mg, 2.0 mmol) were added and the reaction mixture stirred for 4h at 80°C. The reaction
was subsequently quenched by the addition of Et3N (2 mL), concentrated in vacuo, and purified
by column chromatography over silica gel using 30% EtOAc in hexanes to obtain 14 as a white
solid (4.1 gm, 84 %). The NMR spectra for 14 matched those previously reported.54 1H NMR
(400 MHz; CDCl3)
δ 7.53-7.45 (4H, m), 7.40-7.30 (6H, m), 5.54 (1H, s), 5.29 (1H, d, J 7.5 Hz,
1H), 4.91 (1H, d, J 10.2 Hz), 4.82 (1H, d, J 12.0 Hz), 4.71 (1H, d, J 12.0 Hz), 4.37 (1H, dd, J
10.6, 4.6 Hz), 4.08-3.96 (1H, m), 3.79 (1H, t, J 10.3 Hz), 3.57-3.40 (3H, m), 2.90 (1H br s).
3.11 Phenyl 6-O-benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxycarbonyl-amino)-β-D-
glucopyranoside (15)
Trifluoroacetic acid (1.42 mL, 18.5 mmol) was added to a solution of 14 (2 gm, 3.7
mmol) and trimethylsilane (2.95 mL, 18.5 mmol) in CH2Cl2 (30 mL) at 0°C The reaction mixture
.
was then warmed to room temperature and stirred for 3 h. The reaction mixture was
subsequently diluted in chloroform (20 mL), washed with water, satd aq NaHCO3 (2 x 20 mL),
dried over MgSO4, filtered and concentrated. Purification was performed by column
chromatography over silica gel using 40% EtOAc in hexanes as eluent, which afforded
thioglycoside 15 as a white foam (1.54 gm, 78%). [α D
]
24 28.8 (C = 1.0, CHCl3), Rf 0.42
(hexane/EtOAc, 1:1). 1H NMR (400 MHz; CDCl3)
δ
7.49-7.30 (10H, m, 2 Ph), 5.64 (1H, d, J =
5.2 Hz, NHTroc), 5.58 (1H, d, J = 7.6 Hz, H-1), 4.78 (1H, d, J = 12.0 Hz, Troc-CH2), 4.71 (1H, d,
J = 12.0 Hz, Troc-CH2), 4.62 (1H, d, J = 11.8 Hz, PhCH2), 4.55(1H, d, J = 11.8 Hz, PhCH2), 4.31
(1H, m, H-4), 4.12 (1H, m, H-2), 3.82 (1H, dd, J = 4.8, 10.8 Hz, H-6), 3.75 (1H, dd, J = 4.4, 10.0
Hz, H-6), 3.71-3.64 (2H, m, H-3, H-5 ), 3.34 (1H, br s, OH), 3.33 (1H, br s, OH). 13C NMR (100.5
Hz, CDCl3)
δ 155.7, 137.6, 136.6, 131.9, 130.1, 129.1, 128.5, 128.4, 127.9, 127.8, 88.8 (C-1),
74.8, 73.7 (C-5), 73.1 (C-3), 72.8, 71.1 (C-4), 69.9 (C-6), 55.5 (C-2), ESIHRMS m/z calcd for
C22H24Cl3NO6SNa [M + Na]+ 558.0288, found 558.0276.

ACCEPTED MANUSCRIPT
14
3.12 Phenyl 3,4,6-Tri-O-acetyl-6-O-benzyl-
β-
D-galactopyranosyl-(1
→4)-6-O-benzyl-2-
deoxy-1-thio-2-(2,2,2-trichloroethoxycarbonyl-amino)-β-D-glucopyranoside (20)
A mixture of phenyl thiogalactopyranoside 8 (244 mg, 0.5 mmol), BSP (126 mg, 0.6
mmol), and 4 Å molecular sieves (300 mg) was dried under vacuum for 0.5 h. Freshly distilled
CH2Cl2 (10 mL) was added, and the reaction mixture stirred for 0.5 h at room temperature, then
cooled to -65°C and stirred for an additional 5 min before the addition of Tf2O (120 L, 0.64
µ
mmol). The reaction mixture was stirred for 30 min at −65 to −60°C and then was cooled to
−78°C and stirred for 5 min before 1-octene (0.6 mL, 4 mmol) was added and stirring continued
for an additional 5 min. A solution of thioglucopyranoside acceptor 15 (288 mg, 0.54 mmol) in
CH2Cl2 (5 mL) was added dropwise, and the reaction mixture stirred for 1.5 h at -60°C before it
was quenched with Et3N (0.5 mL) and warmed to room temperature. The reaction mixture was
filtered through Celite, concentrated, and purified by chromatography over silica gel using 30%
24
EtOAc in hexanes as eluent to afford disaccharide 16 as a white foam (348 mg, 76 %). [α D
]
-
54.2 (C = 1.0, CHCl3), Rf 0.34 (hexane/EtOAc, 2:1). 1H NMR (400 MHz, CDCl3)
δ
7.53-7.22
(15H, m, 3 Ph), 5.36 (1H, d, J 2.8 Hz, H-4’), 5.19 (1H, s, NHTroc), 5.15 (1H, dd, J 3.8, 10.6 Hz,
H-2’), 4.95 (1H, dd, J 3.6, 10.4 Hz, H-3’), 4.92 (1H, d, J 10.0 Hz, H-1), 4.80 (1H, d, J 11.8 Hz,
PhCH2), 4.71 (1H, d, J 12.0 Hz, PhCH2), 4.65 (1H, d, J 11.8 Hz, PhCH2), 4.52 (1H, t, J 11.8 Hz,
TrocCH2), 4.50 (1H, d, J 8.0 Hz, H-1’), 4.39 (1H, d, J 12.4 Hz, TrocCH2), 4.27 (1H, s, OH), 3.89
(1H, t, J 8.0 Hz, H-3), 3.84 (1H, t, J 6.4 Hz, H-5’), 3.73-3.61 (3H, m, H-6, H-4), 3.54-3.48 (1H, t,
J 7.2 Hz, H-5, H-6’), 3.46-3.39 (2H, m, H-2, H-6’), 2.05 (3H, s, CH3), 1.98 (3H, s, CH3), 1.97(3H,
s, CH3); 13C NMR (100.5 Hz, CDCl3)
δ 170.0, 169.9, 169.2, 153.9, 138.0, 137.0, 132.7, 132.5,
128.9, 128.5, 128.4, 128.0, 128.9, 128.8, 128.7, 101.2 (C-1’), 86.0 (C-1), 80.9 (C-5), 78.0 (C-4),
74.5 (C-3), 73.6 (PhCH2), 73.3 (C-5’), 72.4 (NHTroc), 70.9 (C-3’), 69.0 (C-2’), 68.2 (C-6), 67.3
(C-6’), 67.2 (C-4’), 56.6 (C-2), 20.7 (CH3), 20.6 (CH3), 20.5 (CH3); ESIHRMS m/z calcd for
C41H46Cl3NO14SNa [M + Na]+ 936.1597, found 936.1599.

ACCEPTED MANUSCRIPT
15
3.13 Phenyl 3,4,6-Tri-O-acetyl-6-O-benzyl-
-fucopyranosyl- -(1
amino)- -glucopyranoside (2)
β
-
D
-galactopyranosyl-(1
→
4)-[(2,3,4-tri-O-benzyl-
α
-
L
α
→3)]-6-O-benzyl-2-deoxy-1-thio-2-(2,2,2-trichloroethoxycarbonyl-
β-D
Trichloroacetimidate 957 (404 mg, 0.7 mmol), disaccharide acceptor 26 (340 mg, 0.37
mmol), and 4 Å freshly activated molecular sieves (300 mg) were stirred in freshly distilled
CH2Cl2 (10 mL) for 1 h at -30°C. TMSOTf (18.4 µL, 0.1 mmol) was added dropwise and the
reaction mixture stirred for 1 h at -15°C. Progress of reaction was monitored by TLC and upon
consumption of the acceptor, the reaction mixture was quenched with diisopropylethylamine (1
mL). The reaction mixture was filtered through Celite, concentrated and purified by
chromatography over silica gel with 35% EtOAc in hexanes to obtain LeX trisaccharide 2 (354
mg, 72 %) as a white foam. [α D
]
24 -28.4 (C = 1.0, CHCl3), Rf 0.42 (hexane/EtOAc, 2:1). 1H NMR
(400 MHz, CDCl3) 7.46-7.18 (m, 30H, 5 Bn, Ph), 5.49 (1H, d, J 7.2Hz, TrocNH), 5.39 (1H, d, J
δ
3.2Hz, H-4’), 5.21 (1H, d, J 10.0 Hz, H-1), 5.15 (1H, d, J 3.6 Hz, H-1’’), 5.01 (1H, dd, J 8.0, 10.0
Hz, H-2’), 4.96 (1H, d, J 11.8 Hz, PhCH2), 4.84 (1H, d J 11.4 Hz, PhCH2), 4.84 (1H, d, J 11.4
Hz, PhCH2), 4.75-4.69 (5H, m), 4.66 (1H, d, J 11.6 Hz, PhCH2), 4.66 (1H, d, J 11.6 Hz, PhCH2),
4.64 (1H d, J 7.2 Hz, H-1’), 4.57 (1H, d, J 6.4 Hz, H-5’’), 4.49 (1H, d, J 12.0 Hz, PhCH2), 4.43
(1H, d, J 12.4 Hz, TrocCH2), 4.27 (1H, d, J 12.4 Hz, TrocCH2), 4.17 (1H, t, J 8.8 Hz, H-3), 4.10
(1H, dd, J 4.7, 10.2 Hz, H-2’’), 3.92 (1H, t, J 8.8 Hz, H-4), 3.89 (1H, dd, J 2.4, 11.2 Hz, H-3’’),
3.82 (1H, dd, J 3.0, 11.4 Hz, H-6’), 3.76 (1H, dd, J 1.8, 11.0 Hz, H-6’), 3.58 (1H, s, H-4’’), 3.56-
3.50 (2H, m, H-5, H-6), 3.42 (1H, d, J 8.8 Hz, H-5’), 3.32 (1H, dd, J 5.2, 13.6 Hz, H-6), 3.30 (1H,
m, H-2), 1.96 (6H, s, 2CH3), 1.81 (3H, s, CH3), 1.19 (3H, d, J 6.4 Hz, CH3); 13C NMR (CDCl3)
δ
169.8, 169.7, 168.9, 153.5, 138.7, 138.6, 138.4, 137.8, 137.4, 133.1, 132.3, 128.9, 128.6, 128.5,
128.4, 128.4, 128.3, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.5, 127.1, 99.7
(C-1’), 99.0 (C-1’), 95.4 (Troc-CCl3), 84.6 (C-1), 80.3 (C-4), 79.3, 75.5 (C-3), 74.5 (C-3’), 74.3
(C-4’’), 74.1 (C-5), 73.5, 73.2, 72.8, 71.4, 71.1 (C-2’), 69.1, 67.9 (C-4’), 67.3, 66.6 (C-5’’), 66.4,

ACCEPTED MANUSCRIPT
16
57.8 (C-2), 20.7, 20.6, 20.5, 16.8 (C-6’’); ESIHRMS m/z calcd for C68H75Cl3NO18SNa [M + Na]+
1352.3584, found 1352.3583.
3.14 Nα-(Fluoren-9-ylmethoxycarbonyl)-O-(3,4,6-tri-O-acetyl-6-O-benzyl-
galactopyranosyl)-(1 4)-O-[(2,3,4-tri-O-benzyl- -fucopyranosyl)-(1 3)-6-O-benzyl-2-
deoxy-1-thio-2-(2,2,2-trichloroethoxycarbonyl-amino)- -glucopyranosyl]-(1 6)-[
(2,3,4,6-tetra-O-acetyl- -galactopyranosyl)-(1 3)-O-2-azido-2-deoxy-
galactopyranosyl]- -threonine tert-butyl ester (1)
β-D-
→
α-
L
→
β
-
D
→
β
-
D
→
α-D-
L
3.14.1 Method A: LeX thioglycoside 2 (340 mg, 0.26 mmol), Core-1 diol 3 (357 mg, 0.38
mmol), and 4 Å freshly activated molecular sieves (200 mg) were stirred in freshly distilled
CH2Cl2 (8 mL) for 1 h at room temperature. The reaction mixture was cooled to -60°C before
adding BSP (75 mg, 0.36 mmol) and Tf2O (60 µL, 0.36 mmol). Progress of the reaction was
monitored by TLC, and upon consumption of the donor, the reaction mixture was quenched with
diisopropylethylamine (2 mL). The reaction mixture was then filtered through Celite,
concentrated and purified by chromatography over silica gel with 60% EtOAc in hexanes to
obtain pentasaccharide 1 as a white foam (442 mg, 81%).
3.14.2 Method B: LeX thioglycoside 2 (340 mg, 0.26 mmol), Core-1 diol 3 (357 mg, 0.38
mmol), and 4 Å freshly activated molecular sieves (200 mg) were stirred in freshly distilled
CH2Cl2 (8 mL) for 1 h at room temperature. The reaction mixture was cooled to -40°C before
adding NIS (137 mg, 0.61 mmol) and TfOH (4.4 µL, 0.05 mmol). Progress of the reaction was
monitored by TLC and upon consumption of the donor, the reaction mixture was quenched with
diisopropylethylamine (2 mL). The reaction mixture was then filtered through Celite,
concentrated and purified by chromatography over silica gel with 60% EtOAc in hexanes to
obtain pentasaccharide 1 as a white foam (459 mg 84%). [α D
(hexane/EtOAc, 1:2). 1H NMR (400 MHz, CDCl3)
]
24 23.8 (C = 1.0, CHCl3), Rf 0.28
δ
7.77 (2H, d, J 7.6 Hz Fmoc H), 7.65 (2H, d, J
7.2 Hz, Fmoc H), 7.40 (2H, dt, J 7.6 Hz, 2.4 Hz, Fmoc H), 7.36-7.11 (27H, m, 5Bn, Fmoc), 5.64

ACCEPTED MANUSCRIPT
17
(1H, d, J 9.2 Hz, FmocNH), 5.48 (1H, d, J 6.4 Hz, TrocNH), 5.41 (1H, d, J 3.2 Hz, H-4’), 5.37
(1H, d, J 3.6 Hz, H4’’’’), 5.29 (1H, dd, J 8.0 Hz, 10.4 Hz, H-2’), 5.15 (1H, br s, H-1’’’’), 5.04 (1H,
dd, J 3.4 Hz, 6.6 Hz, H-3’), 5.00-4.92 (3H, m, H-1, H-2, PhCH2), 4.86 (1H, d, J 9.2 Hz, H-1’’),
4.82-4.60 (12H, m, H-3’’’’, H-1’’’, 5PhCH2), 4.54 (2H, d, J 7.6 Hz, H-1’’’’,H-5’’’ ), 4.49 (1H, dd, J
6.0, 8.8 Hz, H-6), 4.47-4.40 (3H, m, FmocCHCH2, TrocCH2), 4.38 (1H, d, J 6.4 Hz, ThrH-2),
4.31-4.24 (4H, m, H-6, TrocCH2, Thr H-1, FmocCH), 4.16-4.07 (5H, m, H-3’’, H-2’’’, 2H-6’, H-5’ ),
3.99-3.86 (4H, m, H-5, H-3’’, H-3’’’, H-4’’, H-5’, H-4), 3.76 (1H, dd, J 3.4, 10.2 Hz, H-6a’), 3,71
(1H, s), 3.67 (1H, dd J 2.0, 10.0 Hz, H-6b’), 3.58 (1H, d, J 1.6 Hz, h-4’’’), 3.53-3.47 (3H, m, H-
6a’’’’, H-2, H-5’’’’), 3.39 (1H, d, J 7.2 Hz, H-5’’), 3.32 (1H, t, J 10.2 Hz, H-6b’’’;), 3.16 (1H, q, J 8.4
Hz, H-2’’), 2.14 (3H, s, OCOCH3), 2.11 (3H, s, OCOCH3), 2.01 (3H, s, OCOCH3), 1.99 (3H, s,
OCOCH3), 1.95 (3H, s, OCOCH3), 1.94 (3H, s, OCOCH3), 1.79 (3H, s, OCOCH3), 1.51 (9H, s,
OtBu), 1.25 (3H, d, J 6.0 Hz, Thr-CH3), 1.17 (3H, d, J 6.4 Hz, Fuc-CH3); 13C NMR (CDCl3)
δ
170.1, 169.7, 169.5, 169.1, 168.8, 156.8, 153.5, 143.8, 141.3, 138.6, 138.3, 137.4, 128.7, 128.5,
128.2, 128.0, 127.5, 127.1, 125.2, 119.9, 101.9 (C-1’’’’), 100.1 (C-1’), 99.9 (C-1’’), 99.7 (C-1), ,
97.8(C-1’’’), 95.5 (Troc-CCl3), 82.9, 80.2, 76.7 (C-5’’’’) 74.8 (C-3’’), 74.6 (C-5’’), 74.3 (C-5’), 74.0,
73.6, 73.3, 73.0, 71.4 (C-3’), 71.0 (C-3’’’’), 70.7, 69.1 (C-6), 68.4 (C-2’’’’), 68.0 (C-4’’’’), 67.4 (C-
6’), 66.8 (C-4’), 66.6 (C-6’’), 66.4 (C-6’’’’), 61.3, 59.4, 58.9 (C-2), 58.6 (C-2’’), 47.1 (FmocCH),
29.7, 28.0, 20.6, 19.1, 16.7 (C-6’’’); ESIHRMS m/z calcd for C105H122Cl3N5O36Na [M + Na]+
2156.6827, found 2156.6367.
Acknowledgments
We acknowledge support from the National Institute of Health (R01DK107405, U01GM116196,
RO1 HL128237, P41GM103694).

ACCEPTED MANUSCRIPT
18
Author contribution
M.Y.R.S., V.R.K., and E.L.C. jointly conceived the project, and directed the chemistry. M.Y.R.S.
was responsible for execution of the chemistry. All authors contributed to the preparation of the
manuscript.
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Synthesis of Lewis X thioglycoside by iterative glycosylation.
Regio- and stereoselective [3+2] glycosylation of the LeX donor to the Core 1 diol acceptor.
Synthesis of LewisX-O-Core-1 threonine pentasaccharide with an Fmoc-Thr handle to access
a wide array of LeX based glycopeptide analogues.