Synthesis of O-Sulfated Human Syndecan-1-like Glyco-polypeptides by Incorporating Peptide Ligation and O-Sulfated Glycopeptide Cassette Strategies

Article abstract of DOI:10.1021/acs.orglett.0c02243

A successful synthesis of O-sulfated syndecan-1-like (Q23-E120) glyco-polypeptide was accomplished. The synthesis features the integration of an O-sulfated carbohydrate-bearing glycopeptide cassette with efficient protein ligation strategies, overcoming the acid lability of carbohydrate sulfates as a major hurdle in solid-phase peptide synthesis. Crucial to the synthesis is the microwave-assisted Ag(I) ligation, which afforded the ligation product in improved overall yield. This O-sulfated syndecan-1 (Q23-E120) is the longest O-sulfated glyco-polypeptide prepared to date.

Full text of DOI:10.1021/acs.orglett.0c02243

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Letter
Synthesis of O‑Sulfated Human Syndecan-1-like Glyco-polypeptides
by Incorporating Peptide Ligation and O‑Sulfated Glycopeptide
Cassette Strategies
Tianlu Li, Weizhun Yang, Sherif Ramadan, and Xuefei Huang*
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ABSTRACT: A successful synthesis of O-sulfated syndecan-1-like (Q23−
E120) glyco-polypeptide was accomplished. The synthesis features the
integration of an O-sulfated carbohydrate-bearing glycopeptide cassette
with efficient protein ligation strategies, overcoming the acid lability of
carbohydrate sulfates as a major hurdle in solid-phase peptide synthesis.
Crucial to the synthesis is the microwave-assisted Ag(I) ligation, which
afforded the ligation product in improved overall yield. This O-sulfated
syndecan-1 (Q23−E120) is the longest O-sulfated glyco-polypeptide
prepared to date.
roteoglycans are a class of complex glycoproteins
typical proteoglycan, which is expressed in high levels by
Pcommonly present on the surface mammalian cells and epithelial tumors.9 Syndecan-1 affects mesenchymal tumor cell
in the extracellular matrix.1 They play roles in many important
proliferation, adhesion, migration, and motility and modulates
genes involved in cell growth regulation. To overcome the
challenges in the synthesis of syndecan-1-like glyco-polypep-
tide 1, multiple strategies have been investigated. A successful
method has finally been established by devising a tetrapeptide
carrying the O-sulfated glycan as a cassette for peptide
synthesis. The backbone of the glycopeptide was extended
with several powerful ligation strategies including native
chemical ligation (NCL),10 serine/threonine ligation
(STL),11 and Ag(I)-mediated ligation12,13 under microwave
assistance, leading to glyco-polypeptide 1 with an acid-labile O-
sulfate. Our study lays the groundwork toward the chemical
synthesis of highly demanding O-sulfated glycoprotein
molecules.
The synthetic design of syndecan-1 glyco-polypeptide 1 was
based on several considerations. To improve the convergence
of the synthesis, peptide ligation strategies were explored. We
selected STL because the syndecan-1 sequence is rich in serine
and threonine residues. In addition, the NCL method was
chosen because it is orthogonal to STL, enabling further
peptidyl chain elongation. Thus we designed the strategic
disconnections at the G50−A51 for NCL followed by
desulfurization and at the S89−T90 for STL, leading to
three fragments 2−4 with similar lengths (Figure 1).
biological events, including growth factor binding, anti-
inflammation, and tumor metastasis. Proteoglycans contain
one or more sulfated glycosaminoglycan chains covalently
attached to the core protein backbone through serine hydroxyl
groups in serine−glycine dipeptides.2 It has been increasingly
appreciated that both the core protein and the glycosamino-
glycan can significantly impact the biological activities of
proteoglycans.3,4 Because of the high structural heterogeneity
of the glycan chains attached to proteoglycans isolated from
nature, it is important to develop synthetic methodologies
toward homogeneous proteoglycans.
Over the past decade, tremendous progress has been made
in glycoprotein synthesis, especially for N-linked glycoproteins,
with multiple full-sized glycoproteins having been synthesized.5
In comparison, proteoglycan synthesis has been under-
developed, with major hurdles in incorporating the acid-
sensitive sulfated glycans into proteins. To date, several
synthetic proteoglycan glycopeptides have been reported,
with the longest one bearing a decapeptide backbone.6,7
Therefore, strategies need to be developed to synthesize
proteoglycan glycopeptides with a more extended peptide
chain.
Herein we report the synthesis of O-sulfated syndecan-1-like
glyco-polypeptide 1 with 98 amino acid residues in the peptide
backbone corresponding to Q23−E120 of human syndecan-1.
Our target sequence 1 spans a functionally active region of the
ectodomain of syndecan-1, from the N-terminus Q23 (after the
proteolytic cleavage of the signal peptide) to the end of the
integrin binding site (E120).8 This is the longest O-sulfated
glyco-polypeptide prepared to date. Syndecan-1 is a proto-
Received: July 7, 2020
https://dx.doi.org/10.1021/acs.orglett.0c02243
© XXXX American Chemical Society
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A

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Figure 1. Retrosynthetic plan of human syndecan-1 ectodomain (Q23−E120) glyco-polypeptide 1.
Glycopeptide 2 contains the O-sulfated xylose moiety. Peptide
3 bears the thiozolidine (Thz)-protected cysteine at the N-
terminus for future NCL14 and a salicylaldehyde ester at the C-
terminus for STL with peptide 4.
carried out. Subsequently, the sulfated glycopeptide cassette
would be ligated to yield the glyco-polypeptide.
Scheme 2. Preparation of Glycopeptide Cassette 5
Our synthesis started from the preparation of glycopeptide
2. The acid sensitivity of O-sulfate prevents the glycopeptide
assembly via standard Fmoc-based solid-phase peptide syn-
thesis (SPPS) due to the need for strong acids for
deprotection. Thus we first explored the installation of the
O-sulfate post glyco-peptide assembly (Scheme 1A).15,16
Scheme 1. Synthetic Strategies toward Glycopeptides
Bearing a Sulfated Glycan
The preparation of tetrapeptide 5 started by the reaction of
t-butyldimethylsilyl (TBS)-protected xylosyl donor 6 with the
Fmoc serine 7 in 82% yield (Scheme 2). The simultaneous
removal of the TBS and the p-methoxybenzyl (PMB) groups
from 8 by trifluoroacetic acid (TFA) gave a free carboxylic acid
intermediate, which was coupled to tripeptide 9, generating
glycopeptide 10 in 91% yield for the two steps. The sulfation of
the free hydroxyl group in 10 with the Et3N·SO3 complex
proceeded smoothly, leading to the sulfated glycopeptide 11 in
82% yield, the Fmoc group of which was then removed by
piperidine, producing cassette 5. The successful preparation of
11 suggested that the aforementioned difficulty encountered in
the sulfation of glycopeptide fragment (Q23−G50) S2 was
likely due to the long peptide at the reducing end of the xylose,
which potentially shielded the free hydroxyl group of the
xylose, reducing its nucleophilicity.
To extend the glycopeptide, we first attempted to prepare
the peptide fragment (Q23−G46) bearing a free carboxylic
acid at the C-terminus for direct coupling to the cassette 5
(Scheme 3A). Aspartimide was found to form during the
peptide synthesis of fragment (Q23−G46). Thus pseudopro-
line20,21 dipeptide building blocks were utilized to minimize
this side reaction, leading to the peptide in good yield. (See the
Supporting Information for details.) With the peptide (Q23−
G46) in hand, its coupling to 5 was tested. Unfortunately, little
desired glycopeptide product was obtained with a variety of
peptide coupling agents, including 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-
fluorophosphate (HATU) and 1-ethyl-3-(3-dimethylamino-
Whereas many sulfation reactions of oligosaccharides have
been successfully performed, the direct sulfation of syndecan-1
glycopeptide fragment (Q23−G50) S2 encountered significant
difficulties (Table S1). The yields of the desired product were
negligible under either basic or acidic conditions, even with
heating or microwave irradiation.
An alternative strategy to O-sulfate is to use protected sulfate
esters such as trichloroethyl (TCE) ester or dichlorovinyl
(DCV) ester (Scheme 1B),17,18 which are known to have
enhanced acid stabilities. Such sulfate esters have been
employed to install aryl sulfates to synthesize tyrosine sulfated
peptides.19 Recently, we utilized DCV ester to prepare
syndecan-4 glyco-decapeptide.4 Unfortunately, although the
glycopeptide S5 with DCV-protected sulfate ester was
obtained, the glycopeptide unexpectedly decomposed during
the deprotection of the DCV sulfate (Scheme S1).
The difficulties encountered prompted us to explore an
alternative cassette approach (Scheme 1C). A short glyco-
tetrapeptide (S47−G50) 5 was prepared as a cassette (Scheme
2), on which glycan manipulation and sulfation would be
B
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with N-hydroxysuccinimide (NHS) as the additive (entry 3),12
the corresponding NHS ester was observed as a major product
within 0.5 h, which gradually underwent hydrolysis, indicating
the poor reactivity of the amine. Switching the reaction solvent
to DMSO gave rise to the desired product, albeit with a low
yield (<10%) (entries 4−6), which was not further improved
with the varied stoichiometry of either 5 or 5a.
Scheme 3. Glycopeptide Cassette Approach
To promote this peptide coupling, we decided to test
microwave irradiation to the Ag(I) ligation reaction. Under
microwave conditions at 50 °C for 20 min in THF/DMF, 30%
of the desired product 13 was observed based on HPLC
analysis (entry 7). Other solvent systems, including the acidic
aqueous buffered condition,24 did not improve the reaction
(entries 8 and 9). DMSO appeared to be the best solvent
(entries 10−14). The effect of various Ag(I) salts was
investigated next, among which the best result was obtained
with AgNO3 in DMSO, leading to 75% yield of the desired
glycopeptide 13 (entry 7). It is worth mentioning that
prolonged treatment should be avoided, which led to
aspartimide product hardly separable from the desired product
(Figure S15).
propyl)carbodiimide (EDC) (Table S2). As an alternative, we
explored the Ag(I)-mediated ligation13 to facilitate this peptide
bond formation, where Ag(I) selectively activated the C-
terminal thioester reacting with the N-terminal free amine of
the glycopeptide cassette (Scheme 3B). The mild condition of
Ag(I)-mediated ligation can potentially reduce the side
reactions such as the loss of the O-sulfate or the elimination
of the glycosyl chain often observed in sulfated glycopeptide
synthesis.6,22
The Ag(I)-mediated ligation was investigated between
Q23−G46 thioester 12 and the glycopeptide cassette 5. The
DMF/THF mixed-solvent system was used in our initial trial.23
With AgNO3 as the activator in the presence of 3-hydroxy-
In parallel, STL was carried out with peptide fragment 3
salicylaldehyde ester and fragment 4 bearing an N-terminal
threonine. The ligation yielded the desired product, the
cysteine of which was deprotected, yielding peptide 14
(Scheme 4).
i
1,2,3-benzotriazin-4-one (HOOBt) as an additive and Pr2NEt
as a base, the formation of product 13 was not detected by
electrospray ionization mass spectrometry (ESI-MS), whereas
the thioester 12 gradually hydrolyzed (Table 1, entry 1).
Elongation of the reaction time led to an increase in the side
product aspartimide formation from 12. To enhance the
nucleophilicity of the N-terminal amine to favor peptide
coupling, we removed the electron-withdrawing Bz protection
from the xyloside to give tetrapeptide 5a. However, that did
not improve the yield of the desired product (entry 2). When
the reaction was performed in an aqueous NaHCO3 solution
Scheme 4. Preparation of Fragment 14 via STL
a
Table 1. Exploration of Ag(I)-Mediated Ligation
entry
room temperature, 24 h
solvent (v/v)
cassette
Ag(I)
additive
product (%)
1
2
3
4
5
6
DMF/THF (1/2)
5
AgNO3
AgNO3
AgNO3
AgNO3
AgNO3
AgNO3
HOOBt
HOOBt
NHS
HOOBt
HOOBt
HOOBt
n.d.
n.d.
n.d.
<10
<10
<10
DMF/THF (1/2)
aq. NaHCO3, pH 8
DMSO
DMSO
DMSO
5a
5a
5a
5a
5
b
microwave conditions: 50 °C, 20 min
7
8
9
10
11
12
13
14
DMF/THF (1/2)
DMSO/dioxane (10/1)
DMSO/acetate buffer (pH 5.5) (1/1)
DMSO
DMSO
DMSO
DMSO
DMSO
5
5
5
5
5
5
5
5
AgNO3
AgNO3
AgNO3
AgSbF6
AgSCN
AgPF6
HOOBt
HOOBt
HOOBt
HOOBt
HOOBt
HOOBt
HOOBt
HOOBt
30
16
21
33
16
53
32
75
c
AgBF4
AgNO3
a
Reaction was performed with cassette 5 or 5a in excess (3 equiv). The concentration of peptide thioester 12 was 3−5 mM at room temperature
and 1 mM under microwave conditions. The formation of 13 was calculated based on the HPLC integration of UV absorbance at 220 nm. n.d. =
b
c
not detected. Glycopeptide 5a was added in 0.67 equiv. No iPr2NEt was added.
C
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For the union of peptide 14 and glycopeptide 13, we
resorted to NCL chemistry. To activate glycopeptide 13 for
NCL, hydrazinolysis of the C-terminus methyl ester of 13 was
the next key step.25 Surprisingly, the treatment of 13 with a
hydrazine/MeOH mixture6 resulted in a slow reaction and a
major side product (around 30%) with an increase of 15 Da in
molecular weight (Table 2, entry 1). This indicated that the
hydrazide product 15 but also removed the benzoyl protective
groups from the xylose ring.
The acyl hydrazide 15 was subjected to nitrous acid
treatment for hydrazide activation.25 Caution was taken to
perform the reaction at −15 °C, pH 3 to prevent the loss of O-
sulfate from 15 due to acid treatment (Figure 2A). The
resulting acyl azide intermediate was incubated in situ with 4-
mercaptophenylacetic acid (MPAA) to form the thioester
followed by NCL with peptide fragment 14 (C51−E120) in
one pot. The desired ligation product 16 was obtained with
high conversion, as judged by HPLC and ESI-MS (Figure 2A).
The reaction was highly reproducible, with two batches giving
35 to 36% yields for the three steps from acyl hydrazide 15.
With 16 in hand, P−B desulfurization26 was performed, which
successfully produced the syndecan-1-like glyco-polypeptide 1.
The structure and the purity of product 1 were supported by
1H NMR, HPLC, and MS analysis (Figure 2 and Figures S9−
S14).
a
Table 2. Optimization of Hydrazinolysis
entry
MeOH/H2O
time (h)
major side product formed
1
2
3
4
MeOH only
80/20
80/1
12
12
0.5
4.5
side-chain acyl hydrazide
hydrolysis of methyl ester
none
80/1
side-chain acyl hydrazide
In conclusion, we successfully synthesized the syndecan-1-
like glyco-polypeptide corresponding to Q23−E120 bearing an
O-sulfated carbohydrate. This sulfated glycopeptide has the
longest polypeptide chain prepared to date. Our approach
features an effective integration of the glycopeptide cassette
bearing a short tetrapeptide with high-efficiency protein
chemical ligation technologies (Ag(I)-mediated ligation,
NCL, and STL). The rationally designed glycopeptide cassette
enabled the facile glycan manipulation and incorporation of
acid-labile O-sulfates, whereas the microwave-assisted Ag(I)-
mediated ligation was critical in overcoming the low
nucleophilicity of the free N-terminus of the sulfated
glycopeptide cassette in peptide bond formation. Both Ag(I)-
mediated ligation and NCL were compatible with the O-
sulfated glycan. Whereas glyco-polypeptide 1 only bears a
a
Concentrations: peptide 13 (10 mM), N2H4·H2O (4 M).
hydrazinolysis likely occurred on the side-chain amide residues
as well, such as Asn23, Asn34, Gln28, or Gln43, to form a side-
chain acyl hydrazide (Scheme S2). The addition of 20% water
to the reaction significantly mitigated the side reaction, which,
on the contrary, led to hydrolysis of the glycopeptide methyl
ester 13 (entry 2). After multiple trials, it was discovered that
the amount of water added was critical to providing an
expedited reaction rate while suppressing the formation of the
side-chain hydrazide side product (entries 3 and 4). With a
ratio of 80:1 MeOH/water (entry 3), an optimal outcome was
obtained (complete within 0.5 h, 77% isolation yield after
HPLC purification), which not only formed the desired
Figure 2. (A) Final assembly of syndecan-1-like glyco-polypeptide 1. (B) HPLC and (C) ESI-MS analysis of 1. (See Figure S9 for details.) (D) 1H
NMR analysis of glyco-polypeptide 1 was performed at 298 K in D2O. See Figures S10−S14 for 2D NMR spectra.
D
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(6) Yang, W.; Yoshida, K.; Yang, B.; Huang, X. Carbohydr. Res. 2016,
435, 180. and references cited therein
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4838.
xylose unit, it can provide valuable knowledge toward the
synthesis of O-sulfated glycopeptides approaching the
complexities of naturally existing proteoglycan.
(8) Mali, M.; Jaakkola, P.; Arvilommi, A. M.; Jalkanen, M. J. Biol.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c02243.
■
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Detailed experimental procedures and preparation and
characterization of the products (PDF)
AUTHOR INFORMATION
Corresponding Author
■
(16) Young, T.; Kiessling, L. L. Angew. Chem., Int. Ed. 2002, 41,
3449.
Xuefei Huang − Departments of Chemistry and Biomedical
Engineering, Institute for Quantitative Health Science and
Engineering, Michigan State University, East Lansing, Michigan
48824, United States; orcid.org/0000-0002-6468-5526;
Email: huangxu2@msu.edu
(17) Liu, Y.; Lien, I. F. F.; Ruttgaizer, S.; Dove, P.; Taylor, S. D. Org.
Lett. 2004, 6, 209.
(18) Ali, A. M.; Taylor, S. D. Angew. Chem., Int. Ed. 2009, 48, 2024.
(19) Bunschoten, A.; Kruijtzer, J. A. W.; Ippel, J. H.; de Haas, C. J.
C.; van Strijp, J. A. G.; Kemmink, J.; Liskamp, R. M. J. Chem.
Commun. 2009, 2999.
(20) Ullmann, V.; Raedisch, M.; Boos, I.; Freund, J.; Poehner, C.;
Schwarzinger, S.; Unverzagt, C. Angew. Chem., Int. Ed. 2012, 51,
11566.
(21) Wang, P.; Aussedat, B.; Vohra, Y.; Danishefsky, S. J. Angew.
Chem., Int. Ed. 2012, 51, 11571.
Authors
Tianlu Li − National Glycoengineering Research Center,
Shandong University, Qingdao, Shandong 266237, China;
Departments of Chemistry and Biomedical Engineering, Institute
for Quantitative Health Science and Engineering, Michigan
State University, East Lansing, Michigan 48824, United States
Weizhun Yang − Departments of Chemistry and Biomedical
Engineering, Institute for Quantitative Health Science and
Engineering, Michigan State University, East Lansing, Michigan
48824, United States
Sherif Ramadan − Departments of Chemistry and Biomedical
Engineering, Institute for Quantitative Health Science and
Engineering, Michigan State University, East Lansing, Michigan
48824, United States; Chemistry Department, Faculty of
Science, Benha University, Benha, Qaliobiya 13518, Egypt;
orcid.org/0000-0002-8639-4105
̈
(22) Sjolin, P.; Kihlberg, J. J. Org. Chem. 2001, 66, 2957.
(23) Inoue, M.; Shinohara, N.; Tanabe, S.; Takahashi, T.; Okura, K.;
Itoh, H.; Mizoguchi, Y.; Iida, M.; Lee, N.; Matsuoka, S. Nat. Chem.
2010, 2, 280.
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(26) Jin, K.; Li, T.; Chow, H. Y.; Liu, H.; Li, X. Angew. Chem., Int.
Ed. 2017, 56, 14607.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c02243
Author Contributions
T.L. and X.H. designed the project. T.L., W.Y., and S.R.
performed the synthesis. All authors were involved in preparing
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for the financial support from the National
Institute of General Medical Sciences, NIH (R01GM072667).
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