Obstacles and solutions for chemical synthesis of syndecan-3 (53–62) glycopeptides with two heparan sulfate chains

Article abstract of DOI:10.1016/j.carres.2016.10.005

Proteoglycans play critical roles in many biological events. Due to their structural complexities, strategies towards synthesis of this class of glycopeptides bearing well-defined glycan chains are urgently needed. In this work, we give the full account o

Full text of DOI:10.1016/j.carres.2016.10.005

Accepted Manuscript
Obstacles and solutions for chemical synthesis of syndecan-3 (53–62) glycopeptides
with two heparan sulfate chains
Weizhun Yang, Keisuke Yoshida, Bo Yang, Xuefei Huang
PII:
S0008-6215(16)30350-0
10.1016/j.carres.2016.10.005
CAR 7274
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 1 September 2016
Revised Date: 18 October 2016
Accepted Date: 19 October 2016
Please cite this article as: W. Yang, K. Yoshida, B. Yang, X. Huang, Obstacles and solutions for
chemical synthesis of syndecan-3 (53–62) glycopeptides with two heparan sulfate chains, Carbohydrate
Research (2016), doi: 10.1016/j.carres.2016.10.005.
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ACCEPTED MANUSCRIPT
Obstacles and Solutions for Chemical Synthesis of Syndecan-3 (53-62)
Glycopeptides with Two Heparan Sulfate Chains
Weizhun Yang,† Keisuke Yoshida,† Bo Yang,† Xuefei Huang*
Department of Chemistry, Michigan State University, 578 S. Shaw Lane, East Lansing,
Michigan 48824, USA
xuefei@chemistry.msu.edu
†These authors contributed equally to the work.
Abstract:
Proteoglycans play critical roles in many biological events. Due to their structural complexities,
strategies towards synthesis of this class of glycopeptides bearing well-defined glycan chains are
urgently needed. In this work, we give the full account of the synthesis of syndecan-3
glycopeptide (53-62) containing two different heparan sulfate chains. For assembly of glycans, a
convergent 3+2+3 approach was developed producing two different octasaccharide amino acid
cassettes, which were utilized towards syndecan-3 glycopeptides. The glycopeptides presented
many obstacles for post-glycosylation manipulation, peptide elongation, and deprotection.
Following screening of multiple synthetic sequences, a successful strategy was finally
established by constructing partially deprotected single glycan chain containing glycopeptides
first, followed by coupling of the glycan-bearing fragments and cleavage of the acyl protecting
groups.
Graphical abstract
Keywords:

ACCEPTED MANUSCRIPT
Carbohydrates, Glycopeptides, Glycosylation, Protecting groups, Synthesis design

ACCEPTED MANUSCRIPT
Introduction
Many naturally existing proteins contain glycan chains due to post-translational
modifications.1-2 Glycans can have broad effects on the parent peptides and proteins, ranging
from tertiary structure stabilization, enhanced stability against proteolysis, to altered
physiochemical properties and biological functions.2-4 As the expression of glycans is not
directly controlled by genes, glycoproteins and glycopeptides isolated from nature often exist as
a heterogeneous mixture containing various glycans attached to the same peptide/protein
backbone. Therefore, to decipher the structure and activity relationship, it is critical that
glycoproteins and glycopeptides bearing homogeneous glycan chains can be synthesized.
Structurally, native glycoproteins are classified into two major types: N-glycans and O-
glycans.2,5 In N-glycans, carbohydrate residues are covalently linked to an asparagine residue in
the protein backbone through an N-acetyl glucosamine. O-glycans can be further divided into
two main classes, i.e., the mucin type and the glycosaminoglycan family proteoglycans. The
mucin type O-glycans contain an N-acetyl galactosamine linkage to serine or threonine, while the
proteoglycan family glycopeptides share the general structure of glycosaminoglycan chains
connected to a serine of the core protein typically through a tetrasaccharide linker consisted of
glucuronic acid-β-1,3-galactose-β-1,3-galactose-β-1,4-xylose.6 Many creative chemical7-23 and
chemoenzymatic24-30 methods have been developed to synthesize glycopeptides bearing
sophisticated structures of N-glycans and mucin type O-glycans.31-38 Great successes have been
achieved in these areas with molecules approaching the sizes and complexities of native
glycoproteins produced via total synthesis.7-9 In comparison, methodologies for the preparation
of glycopeptides carrying homogenous glycosaminoglycan are under developed. Much synthetic
work related to proteoglycans has been on the synthesis of glycosaminoglycan
oligosaccharides39-44 45-68 and the tetrasaccharide linker.69-79 Recently, we have begun to develop
a strategy towards the proteoglycan family glycopeptides with syndecan-3 as the target.80 With
the highly complex structure, many obstacles were encountered during the synthesis. Herein, we
provide the full details for the successful synthesis of syndecan-3 (53-62) glycopeptides bearing
two different heparan sulfate glycan chains.

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General structure of glycosaminoglycan family proteoglycans
glucuronic acid galactose galactose
HO
xylose
OH HO
OH
O
OH
O
ꢀO2C
HO
Core protein
O
HO
Glycosaminoglycan
chain
O
O
O
O
O
O
OH
OH
OH
n
Tetrasaccharide linker region
Syndecan-3 is a transmembrane proteoglycan highly expressed in neural cells with its
extracellular domain bearing heparan sulfate chains.6,81-82 Syndecan-3 is involved in a wide
range of biological events such as cell-cell interaction,83 skeletal muscle growth and repair,84-87
and viral infection.88 Our synthetic targets are syndecan-3 extracellular domain glycopeptides 1
and 2 (corresponding to amino acids 53-62),89-90 which contain typical structural features of
heparan sulfate proteoglycans including different heparan sulfate chains, the tetrasaccharide
linkers, 2-O sulfation, 6-O sulfation, glucosamine α linked to both glucuronic acid and iduronic
acid, and N-acetylation.
NHAc
O
HN
A
B
C
D
E
F
G
H
OR
O
OH
O
O
-O2C
NH
O
HO
OH
O
O
OH
O
-O2C
HO
OH
O
HO
O
HO
O
OH
O
-O2C
OH
O
HO
AcHN
AcHN
HO
O
O
OH
O
O
O
HO
HN
O
HO
RO
OH
OH
OH
-
O
1: R = SO3
Syndecan-3 glycopeptide
NH
2
: R = H
O
HO
OH
O
HN
-O2C
O
O
HO
HO
OH
O
HO
O
-O2C
OH
AcHN
O
HO
NH
O
HO
OH HO
O
OH
OH
HO
-O2C
HO
O
HO
O
OH
O
O
AcHN
O
O
O
O
O
O
HN
OH
OH
OH
O
NH
O
HN
O
HO
O-
Results and Discussion
Synthetic design and preparation of iduronic acid containing octasaccharide serine cassette
3

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In order to prepare syndecan-3 glycopeptides, we adapted a cassette approach91 where
iduronic acid containing octasaccharide serine cassette 3 and glucuronic acid containing 4 were
utilized as cassettes for glycopeptide assembly. Due to the large sizes of the glycan chains in
cassettes 3 and 4, we aim to develop a convergent route where oligosaccharide modules were
synthesized and then joined to improve the overall synthetic efficiency.
Our initial design towards the serine derivative 3 was a 2+3+2+1 approach making the
strategic disconnections of the octasaccharide at the B/C, E/F and G/H linkages. Although
uronic acid thioglycosyl and trichloroacetimidate donors have been successfully utilized in
glycosaminoglycan synthesis,92-93 in our experience, the corresponding hexose donors tend to
give higher glycosylation yields.94 Thus, idose and glucose building blocks were used for
constructing the glycosyl linkages, which would be followed by oxidation to uronic acids.95 The
synthesis started from the preparation of the non-reducing end AB disaccharide by reacting
donor 5 with acceptor 6.53 The glycosylation reaction was initiated by pre-activating idosyl
donor 5 with p-TolSOTf,96 which was formed in situ through reaction of AgOTf and p-TolSCl at
-78 °C (Scheme 1a). Following complete activation of the donor 5, acceptor 6 was added to the
reaction mixture together with a non-nucleophilic base tri-tbutyl-pyrimidine (TTBP).97
Unfortunately, no desired disaccharide 7 was obtained. Analysis of the reaction mixture showed
that most acceptor 6 was recovered (~ 63%) with the major side product as the 1,6-anhydro
idoside 8 (~ 60%). The 1,6-linkage in 8 was presumably formed by nucleophilic attack of the
anomeric center upon donor activation by 6-O due to the electron rich 6-O-p-methoxybenzyl
(PMB) moiety. To address this problem, we tested donor 9 bearing the 6-O-tbutyldimethylsilyl
(TBS) group in reaction with acceptor 6, which gave the 1,6-anhydro sugar 10 as the major side

ACCEPTED MANUSCRIPT
product. To reduce the remote participation by the 6-O moiety, the tbutyldiphenylsilyl (TBDPS)
group was examined next as the protective group (donor 11). Gratifyingly, the TBDPS ether was
sufficiently bulky, which effectively suppressed the 1,6-anydro sugar formation leading to 61%
yield of the desired disaccharide 13 along with 7% of the epimer 14 (Scheme 1b). The Ac and
Bz groups in 13 were then exchanged with levulinate (Lev) as sites for future O-sulfation
producing disaccharide donor 15 (Scheme 1c). The TBDPS moiety from 15 was removed and
the free hydroxyl group oxidized by 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and
[bis(acetoxy)iodo]benzene (BAIB)92 followed by benzyl ester formation with phenyl
diazomethane95 (donor 16).
With the AB module in hand, we moved onto the preparation of the CDE and FG
modules. The α
-linked disaccharide acceptor 1853 was successfully glycosylated by the idosyl
donor 17 producing CDE trisaccharide 19 in 85% yield (Scheme 2a). The FG di-galactoside 20
was synthesized by reacting di-Lev galactosyl donor 2198 with galactoside 2298 followed by
hydrazine acetate treatment to selectively remove the Lev groups (Scheme 2b). Trisaccharide 19
was then joined with di-galactoside acceptor 20 forming CDEFG pentasaccharide module 24 in
65% yield (Scheme 2c). The azide moiety in 24 was converted to acetamide and the product
was further transformed to acceptor 25. The non-reducing end hydroxyl group of 25 was

ACCEPTED MANUSCRIPT
protected with Lev generating CDEFG pentasaccharide donor 26. The hindered 2’-OH in 25 did
not undergo acylation under the reaction condition as it was flanked by two bulky glycan rings.
Merging the AB and CDEFG modules turned out to be highly problematic. Reaction of
donor 16 with pentasaccharide acceptor 25 failed to lead to any desired heptasaccharide 27
(Scheme 3a). To test the possibilities of acetamide99 or the electron withdrawing benzyl ester
groups negatively impacting glycosylation, donor 15 and idose/azide bearing thioglycosyl
trisaccharide acceptor 28 were examined, which did not lead to successful glycosylation either.
Several side products due to acceptor activation were observed from these reactions. To avoid
acceptor activation, hexasaccharide acceptor 30 was prepared by reacting 26 with xylosyl serine
29 followed by Lev removal (Scheme 3b). Although the yield was not high (~20%), sufficient
quantity of hexasaccharide 30 was acquired. However, reaction of 16 with 30 again did not
provide the desired octasaccharide 31 (Scheme 3c). These unsuccessful attempts suggest that it
is difficult to form the BC glycosyl linkage using oligosaccharide building blocks.

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To overcome the aforementioned difficulty, a second generation approach was designed
to avoid the late stage formation of the B/C linkage by adapting a 3+2+3 strategy using building
blocks consisted of ABC trisaccharide, DE disaccharide and FGH trisaccharide. To form the key
B/C linkage, glycosylation of idosyl acceptor 33 by glucosamine donor 32 was carried out
(Scheme 4a). Gratifyingly, this reaction proceeded smoothly, forming the BC disaccharide 34 in
80% yield with the α anomer as the sole stereoisomer isolated. The stereochemistry of the newly
formed glycosidic linkage was confirmed by NMR analysis (1JC1’-H1’ = 169 Hz).100 The low
reactivities of the oligosaccharide building blocks such as 16, 26 and 28 are presumably due to
electron withdrawing power and/or steric hindrance associated with the additional glycan
rings.101-103 Protective group manipulation of 34 produced disaccharide acceptor 35, which was
glycosylated by the idosyl donor 12 giving ABC trisaccharide 36 (Scheme 4b). The DE
disaccharide 40 was formed by reaction of azido glucoside donor 37 with glucoside 38 followed
by protective group adjustment (Scheme 4c). In order to avoid the remote participation by the 6-
O-PMB moiety in future glycosylation, the PMB group in 39 was replaced with TBDPS
(disaccharide 40). The reducing end FGH trisaccharide module was prepared by glycosylating
the xylosyl serine 29 with galactoside donor 4198 leading to disaccharide 42 in 81% yield

ACCEPTED MANUSCRIPT
(Scheme 4d). This yield was significantly higher than that for the formation of 30, again
suggesting higher reactivity of monosaccharide building blocks compared to the corresponding
oligosaccharide donor. The Lev group in 42 was removed, which subsequently underwent
glycosylation with galactoside donor 21 followed by hydrazine treatment to generate FGH
trisaccharide 44.
Scheme 4.
OAc
O
a)
OPMB
OAc
O
AgOTf, pꢀTolSCl, ꢀ78°C, then
STol
OBn
O
STol
TBSO
BnO
TBSO
STol
OBn
O
N3
BnO
PMBO
TTBP
O
N3
34
80%
BzO
32
33
OBz
HO
OLev
(a) Mg(OMe)2, DCM, ꢀ20°Cꢀ0°C
(b) LevOH, EDCꢀHCl, DMAP
(c) DDQ, DCM/H2O
(d) HF/Pyridine
(e) TBDPSCl, imidazole
OTBDPS
O
STol
OBn
O
HO
BnO
N3
O
BzO
35
44%
OLev
OTBDPS
b)
TBDPSO
O
STol
OBn
STol
OBn
O
O
BnO
N3
AgOTf, pꢀTolSCl, ꢀ78°C, then
35, TTBP
OBn
O
TBDPSO
O
O
BzO
36
PMBO
OLev
PMBO
OLev
52%
12
c)
OBn
O
OBn
O
AgOTf, pꢀTolSCl, ꢀ78°C, then
TBSO
OPMB
TBSO
BnO
OPMB
O
STol
N3
O
BnO
BnO
O
HO
BnO
TTBP
N3
39
75%
STol
STol
37
BzO
38
BzO
OBn
(1) DDQ, DCM/H2O
(2) HF/Pyridine
(3) TBDPSCl,
O
HO
BnO
OTBDPS
O
N3
O
BnO
imidazole, DCM
STol
40
46%
BzO
Ph
d)
Ph
O
O
O
29, TTBP, AgOTf,
pꢀTolSCl, ꢀ78°C,
O
O
O
O
BzO
O
O
RO
81%
STol
LevO
41
OBz
OBz
OBz
FmocHN
NH2NH2, HOAc
90%
CO2Bn
42: R = Lev
43: R = H
Ph
Ph
O
O
O
21, TTBP, AgOTf, pꢀTolSCl,
ꢀ78°C; NH2NH2, HOAc
O
O
OBz
O
O
O
BzO
O
O
HO
70%
OH
OBz
FmocHN
CO2Bn
44
With the three modules prepared, their union was tested next (Scheme 5). Under the pre-
activation condition, glycosylation of disaccharide DE acceptor 40 by ABC trisaccharide donor

ACCEPTED MANUSCRIPT
36 led to pentasaccharide 45 in an excellent 93% yield. Pentasaccharide 45 was a competent
donor, successfully glycosylating the FGH trisaccharide module 44 and producing the
octasaccharide module 46. The successful assembly of 46 indicates that the C/D and E/F
linkages are suitable strategic linkage points for constructing the octasaccharide module. In order
to prepare the compound 3, glycosyl serine 46 was de-silylated followed by oxidation of the
three newly freed hydroxyl groups to carboxylic acids, methyl ester formation and acetylation.
Elevated reaction temperature and excess acetic anhydride were necessary for acetylation due to
the low reactivity of the free hydroxyl group in 46.
Assembly of the glucuronic acid containing octasaccharide serine cassette 4
With the knowledge gained in preparing octasaccharide 3, the octasaccharide serine
cassette 50 was synthesized following the same 3+2+3 approach (Scheme 6). The glucoside
donor 47, pre-activated by p-TolSCl/AgOTf, glycosylated disaccharide 40 generating ABC
trisaccharide 48 in 85% yield. The 3+2 glycosylation between trisaccharide 48 and disaccharide
40 went smoothly producing pentasaccharide 49, which subsequently glycosylated the
trisaccharide serine unit 44 leading to the octasaccharide cassette 50 in an excellent 87% yield.
The successful preparation of octasaccharide 50 demonstrated the generality of the 3+2+3 route.
The TBDPS silyl ether groups in 50 were removed by HF/pyridine to expose the three primary
hydroxyls, which were oxidized to carboxylic acids92 and subsequently converted to methyl

ACCEPTED MANUSCRIPT
esters. The two azide groups were transformed to N-acetyl moieties through a one pot
reduction/acetylation procedure104 to afford octasaccharide 4.
Scheme 6
.
OTBDPS
O
OTBDPS
O
OBn
O
AgOTf, pꢀTolSCl,
ꢀ78°C, then 40,
TTBP
AgOTf, pꢀTolSCl,
ꢀ78°C, then 40,
TTBP
PMBO
BnO
O
PMBO
BnO
STol
BnO
OTBDPS
O
OBz
OBz
47
N3
O
48
STol
BnO
85%
BzO
OTBDPS
OBn
O
O
PMBO
BnO
O
BnO
OTBDPS
OBz
N3
O
OBn
O
O
44, TTBP, AgOTf,
pꢀTolSCl, ꢀ78°C
O
BnO
49
BnO
BzO
OTBDPS
O
80%
N3
O
BnO
OTBDPS
O
STol
OBn
O
OBz
PMBO
BnO
O
BnO
OTBDPS
OBz
Ph
Ph
OBn
O
N3
O
BnO
O
O
BzO
O
O
O
O
BnO
OTBDPS
OBz
BzO
N3
O
BnO
O
O
O
O
O
50
87%
O
O
O
OH
OBz
OBz
OBn
FmocHN
OBn
MeO2C
PMBO
BnO
O
O
O
BnO
O
OBz
CO2Me
O
(a) HF/pyridine
Ph
Ph
OBn
O
AcHN
(b) TEMPO, BAIB
(c) MeI, K2CO3, DMF
(d) Zn, CuSO4 (sat.),
HOAc/Ac2O/THF
O
O
BnO
O
O
O
BnO
O
BzO
4
CO2Me
O
OBz
AcHN
O
42%
O
BzO
O
O
O
O
O
BnO
O
OH
OBz
OBz
OBn
FmocHN
O
Obstacles in glycopeptide synthesis
The glycopeptide assembly was explored next. With the high sensitivity of O-sulfates to
acid and the propensity of the glycopeptide to undergo base promoted β-elimination,105-107
common amino acid side chain protective groups such as Boc and trityl should be avoided and
the sequence and condition of deprotection need to be optimized.
To test the deprotection condition, octasaccharide 4 was treated with piperidine108 to
remove the N-terminal Fmoc followed by coupling with glycine 51 and catalytic hydrogenolysis
to produce glycopeptide 52 (Scheme 7a). The next step was transesterification using
NaOMe/MeOH (~ pH 9.5) to remove the Ac and Bz groups. Although this condition was
previously successfully applied to an iduronic acid containing octasaccharide module 54,
multiple fragments from backbone cleavages at non-reducing ends of glucuronic acids of 53

ACCEPTED MANUSCRIPT
were observed based on mass spectrometry analysis. The higher lability of glycopeptide 53 to
base treatment was possibly because the 4-O can coordinate with Na+ ion thus bringing the
NaOMe closer to the acidic axial proton on the adjacent C-5 center (Scheme 7b). This in turn
can facilitate the removal of the H-5 arranged in 1,2-cis geometry to the 4-O. Neighboring group
assisted glycan cleavages are known.51,109
The base sensitivity of the glycopeptide suggests a less basic yet stronger nucleophile
such as hydrazine78,110 would be needed for deacylation. A model glycopeptide 55 was treated
with hydrazine, which successfully cleaved the Bz and Lev groups without affecting the sulfates
or the glycan/peptide linkage. In order to apply the hydrazine reaction to syndecan-3
glycopeptides, the carboxylic acid moieties of uronic acids cannot be protected as esters during
the hydrazine treatment to prevent the potential hydrazide formation. This consideration led to
the design of a new route, where the full length glycopeptide 56 was to be assembled from two
fragments: glycopeptides 57 and 58, which could be prepared through peptide coupling. The

ACCEPTED MANUSCRIPT
uronic methyl esters of 56 can be converted to free carboxylic acids by mild base treatment for
subsequent hydrazinolysis.
OBz
O3SO
OBz
O3SO
O
O
O
O
O
O
LevO
BzO
OH
OH
OBz
OLev
OBz
O
O
O
O
O
O
H
H
H
N
H
H
OH
H
N
N
N
N
N
N
N
N
N
N
N
H
H
AcHN
H
H
O
H
O
O
O
O
O
O
55
OH
O
OH
O
glycan 1
glycan 2
OH
O
O
O
O
H
H
N
H
H
N
H
N
N
N
OMe
N
H
N
N
N
AcHN
H
H
H
O
O
O
O
O
56
OH
OH
O
MeO2C
HO
HO
O
O
HO
glycan 1
OH
O
CO2Me
O
OBz
O
HO
OH
AcHN
OH
O
OH
O
OH
O
CO2Me
O
HO
OBz
O
BzO
OBz
O
AcHN
O
O
O
O
HO
OH
OBz
OBz
ꢀ
OSO3
O
OH
O
CO2Me
glycan 2
MeO2C
HO
OH
OH
O
O
HO
O
HO
OH
OH
O
OH
OH
O
OBz
CO2Me
O
AcHN
O
BzO
O
O
ꢀO3SO
O
O
AcHN
O
O
O
OBz
OAc
HO
OBz
OBz
OBn
glycan 2
glycan 1
O
OBn
H
N
O
O
O
O
OH
H
H
N
H
N
N
H
AcHN
N
H2N
+
OMe
N
H
N
H
N
H
O
O
O
O
O
57
58
OBn
OBn
O
glycan 2
glycan 1
OBn
O
O
H
N
O
O
H
H
OBn
O
N
OH
FmocHN
+
AcHN
N
+ H2N
+
H2N
OH
OH
O
N
OMe
N
FmocHN
H
O
H
O
O
OBn
In order to synthesize glycopeptide 57, glycosyl serine 3 was transformed to
octasaccharide 59 through conversion of azides to acetamides, Lev group removal by hydrazine
acetate and sulfation of the free hydroxyl groups.111 The Fmoc group in octasaccharide 59 was
removed and the resulting amine was coupled with dipeptide 60 to produce glycopeptide 61.
The tyrosine hydroxyl group in the side chain of peptide 61 was protected with Bn, which can be

ACCEPTED MANUSCRIPT
deprotected under hydrogenation conditions. The benzyl ester in glycopeptide 61 was
selectively removed under hydrogenation in the presence of NH4OAc112 leading to free
carboxylic acid glycopeptide 57 (Scheme 8).
Similarly, hydrogenation of octasaccharide 4 in the presence of NH4OAc112 afforded
glycopeptide 62 containing the free C-terminal carboxylic acid (Scheme 9). Peptide elongation
with tripeptide 63 gave glycopeptide 64. Treatment of 64 with piperidine followed by coupling
with tripeptide 65 and Fmoc deprotection led to the free amine bearing glycopeptide 58.
Scheme 9.
OBn
MeO2C
O
Ph
PMBO
O
BnO
O
Ph
OBn
O
BnO
CO2Me
O
HATU, 2,4,6ꢀcollidine, 63, DMF
O
O
OBz
O
BnO
O
AcHN
O
O
CO2Me
O
BnO
OBz
O
62%
OBn
O
O
BzO
OBz
O
AcHN
O
O
O
O
BnO
OH
OBz
OBz
O
O
H
4: R = Bn
62: R = H
H2, Pd/C, CH2Cl2/MeOH,
NH4OAc
N
H2N
OR
72%
OMe
N
FmocHN
H
O
O
63
OBn
MeO2C
PMBO
BnO
O
Ph
O
BnO
O
Ph
OBn
CO2Me
O
O
O
OBz
O
BnO
O
O
AcHN
O
O
CO2Me
O
BnO
OBz
O
O
O
(a) piperidine, CH2Cl2
(b) 65, HATU, 2,4,6ꢀcollidine, DMF
(c) piperidine, CH2Cl2
BzO
OBz
O
AcHN
O
O
O
O
BnO
OH
OBz
OBz
OBn
58
46% over three steps
OBn
64
O
O
H
O
O
N
FmocHN
H
N
H
OH
N
N
H
OMe
FmocHN
N
O
H
65
O
O

ACCEPTED MANUSCRIPT
Glycopeptides 57 and 58 were united in a peptide coupling reaction promoted by O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) to generate
glycopeptide 66 bearing two different glycans (Scheme 10). Glycopeptide 66 was hydrogenated
with Pearlman’s reagent or Pd/C under atmospheric pressure of hydrogen gas in mixed solvents
of CH2Cl2 and methanol to remove the benzyl ethers, PMB and benzylidene groups. However,
no desired product 56 was obtained. It is possible that the partially deprotected glycopeptides
formed during hydrogenation aggregated due to solubility changes preventing access to the
palladium catalyst. Colloidal Pd nanoparticles were tested next as the Wong group reported that
these nanoparticles could remove benzyl ethers from solid phase bound glycans.113 Various
solvents including tetrahydrofuan, methanol/water and hexafluoro-isopropanol, were also
examined to disrupt the potential aggregate. Other conditions tested included various pH values,
reaction temperature and elevated hydrogen pressure. However, the desired compound 56 was
not found with decomposition products observed in mass spectrometry analysis. The difficulty
with glycopeptide 66 was not unique as several other glycopeptides bearing two heparan sulfate
glycans decomposed as well when subjected to hydrogenation. Hojo, Nakahara and coworkers
reported a procedure using low acidity triflic acid to remove benzyl ethers from O-sulfated
glycopeptides.114 In our hands, this condition cleaved the O-sulfates from glycopeptide 66
without successful debenzylation.

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Successful synthesis of glycopeptide 1
Due to the unexpected difficulty in the hydrogenation of 66, an alternative was to
perform hydrogenation on glycopeptides 61 and 58 bearing a single heparan sulfate chain. The
iduronic acid containing non-sulfated glycopeptide 67 was tested first, which was prepared
analogously to 61. Catalytic hydrogenation of compound 67 under a slightly acidic condition
(pH ~ 5.5) using Pearlman’s catalyst went smoothly and gave the desired product 68 in
quantitative yield (Scheme 11). Similarly, sulfated glycopeptide 61 was successfully
hydrogenated yielding 69. The reason for the increase in fragility of glycopeptides bearing two
heparan sulfate chains towards hydrogenation is not clear.
The hydrogenation of glycopeptide 58 was performed next to generate amine 70 (Scheme
12). This reaction was slower than those of 61 and 67 and needed to be closely monitored to
prevent over-reduction. 70 was coupled with carboxylic acid 68 (2 eq) producing glycopeptide
71 in 46% yield. To complete the deprotection, the three methyl esters in 71 were cleaved by
LiOH (pH 9.5) followed by hydrazine to afford the fully deprotected glycopeptide 2 in 72% yield
over the two steps (Scheme 12).

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The synthesis of the sulfated glycopeptide 1 was tested next. When 2 eq of carboxylic
acid 69 was utilized in the HATU mediated coupling with amine 70, the desired product 56 was
observed, but contaminated with glycan 72 resulting from β-elimination of 69 (Scheme 13). The
separation of 72 from 56 turned out to be very difficult. When 1.5 equiv of glycopeptide 69 was
utilized, the coupling yield was much lower.

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To overcome the elimination problem, we adjusted the sequence for peptide chain
elongation by coupling carboxylic acid 69 with tripeptide 73 first (Scheme 14a). We envisioned
the smaller tripeptide 73 should be a better nucleophile than amine 70. Indeed, the reaction of
glycopeptide 69 with tripeptide 73 went smoothly with no β-elimination product 72, which led to
glycopeptide 75 after hydrogenation. This suggests the formation of 72 (Scheme 13) was not
because of the inherent instability of glycopeptide 56 under the peptide coupling condition.
Rather, it was possibly due to the enhanced lability of the activated ester 76 formed between 71
and HATU towards β-elimination (Scheme 14b), which could outcompete the desired amide
bond formation reaction with the more bulky and presumably less reactive amine 70.

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To complete the glycopeptide synthesis, amine 77 was needed. Catalytic hydrogenation
of 64 catalyzed by Pd(OH)2 or Pd/C proceeded very slowly (Scheme 15a). With prolonged
reaction time, multiple side products due to methylation of the free N-terminus115 and reduction
of phenyl rings were formed, which could not be separated from the desired product. To address
this issue, an alternative route was explored by installing a protective group onto the N-terminus,
which is stable under hydrogenation condition and yet known to be removable under mild
conditions.
Several amine protective groups including trifluoroacetamide (TFA), 2-
trimethylsilylethoxycarbonyl (Teoc)116 and 2-pyridylethoxycarbonyl (Pyoc)117 were examined.
Global hydrogenolysis went smoothly on the TFA and Teoc protected glycosyl serine, while
Pyoc protected one gave multiple unidentified side products. After peptide coupling leading to
compounds 78 and 79, we focused on deprotection to generate the free amine. However, with
78, TFA could not be removed under basic or NaBH4 reductive condition118 without affecting the
methyl esters in the molecule. The Teoc group in 79 could not be cleaved by HF/pyridine.
Treatment of 79 with tetrabutylammonium fluoride (TBAF) led to the cleavage of glycan chain.
These unsuccessful attempts prompted us to re-examine the substrate for hydrogenation. After
many trials, a viable route was established by first removing the Fmoc from glycopeptide 64
(Scheme 15b). The resulting amine was successfully hydrogenated under a slightly acidic
condition (pH 6 with the addition of CCl3CO2H).119 No over-reduction or amine alkylation side

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products were observed. The usage of trichloroacetic acid was important as the reaction was not
successful with acetic acid.
With amine 77 in hand, its coupling with 75 was carried out with 1.3 eq of 75 and HATU
providing 56 in 58% yield (Scheme 16). No β-elimination side product 72 was observed from
this reaction, which was consistent with the formation of 74. Final deprotection of 56 was
performed under a mild basic condition. The sulfated glycan chain was very sensitive to base as
pH 9.5 LiOH led to partial chain cleavage. Instead, the methyl ester removal was performed at
pH 9.0 with frequent monitoring using mass spectrometry, which was followed by
hydrazinolysis to afford glycopeptide 1 in 82% yield.

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Conclusion
A successful strategy was developed for the assembly of syndecan-3 (53-62)
glycopeptides bearing two heparan sulfate chains. Many obstacles were encountered during the
syntheses of these highly complex molecules. For construction of the glycan chains, among
many potential routes, we found the difficult B/C glycosyl linkages should be formed early due
to higher reactivities of the monosaccharide building blocks. Furthermore, as the size of the
molecule grows larger, unique reactivity and stability problems can emerge as evident from the
instability of glycopeptides containing two heparan sulfate chains to the catalytic hydrogenation
condition. To overcome this obstacle, the hydrogenation reaction was performed on the
glycopeptide bearing a single glycan chain. Performing the hydrogenation reaction with addition
of trichloroacetic acid at pH6 was found to significantly improve the yield and suppress side
product formation. Another challenge encountered was the propensity of glycopeptide bearing
glycan chain at the C-terminus to undergo competing β-elimination during peptide elongation
reaction with larger and less reactive amine partners. This was overcome by varying the peptide
coupling sequences. Following the union of partially deprotected fragments, the final
deprotection of the glycopeptide required the cleavage of all ester protective groups, which was
accomplished by mild base treatment (pH 9.0) followed by hydrazinolysis. The hydrazinolysis
procedure was critical to ensure complete removal of all acyl protective groups without the
undesired β-elimination or cleavage of the highly sensitive glycan chain. Efforts are ongoing to

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extend the peptide backbone and glycan chains of glycopeptides as well as applying the strategy
to synthesis of other glycosaminoglycan family glycopeptides bearing multiple heparan sulfate
chains.
Acknowledgement: This work was supported by the National Science Foundation (CHE
1507226) and the National Institute of General Medical Sciences, NIH (R01GM072667, U01
GM116262 and U01 GM102137).
Supporting information: Complete experimental procedures and characterizations are provided,
including 1H and 13C NMR spectra of all new compounds.
Notes: The authors declare no competing financial interest.
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