Synthesis of Chondroitin Sulfate A Bearing Syndecan-1 Glycopeptide

Article abstract of DOI:10.1021/acs.orglett.7b02271

Syndecan-1 chondroitin sulfate glycopeptide was synthesized for the first time using the cassette approach. The sequence of glycosylation to form the octasaccharide serine cassette was critical. The glycopeptide was successfully assembled via a 2+ (3 + 3)

Full text of DOI:10.1021/acs.orglett.7b02271

Letter
pubs.acs.org/OrgLett
Synthesis of Chondroitin Sulfate A Bearing Syndecan‑1 Glycopeptide
Sherif Ramadan,†,‡ Weizhun Yang,† Zeren Zhang,† and Xuefei Huang*,†,§,∥
§
∥
†Department of Chemistry, Department of Biomedical Engineering, and The 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
S
* Supporting Information
ABSTRACT: Syndecan-1 chondroitin sulfate glycopeptide
was synthesized for the first time using the cassette approach.
The sequence of glycosylation to form the octasaccharide
serine cassette was critical. The glycopeptide was successfully
assembled via a 2+ (3 + 3) glycosylation strategy followed by
peptide chain elongation.
hondroitin sulfates (CSs) are polyanionic complex poly-
saccharides consisting of a disaccharide repeating unit of
D-glucuronic acid (GlcA) β-1,3-linked to 2-acetamido-2-deoxy-D-
galactosamine (GalNAc). CSs belong to the glycosaminoglycan
family and exist as a part of chondroitin sulfate proteoglycans
(CSPGs) in nature. In CSPG, CSs are covalently linked to the
hydroxyl group of a serine residue in the core protein through a
tetrasaccharide linker of GlcA-β-1,3-galactose (Gal)-β-1,3-Gal-β-
1,4-xylose.1−3 A representative example of CSPG is syndecan-1,
which interacts with a large variety of biological molecules such
as growth factors, chemokines, amyloid β, and cell adhesion
molecules.1−6 As a result, it plays important roles in mediating
cell binding, signaling, proliferation, and cytoskeletal organ-
ization.
C
CS and CSPG on average contain one O-sulfate per dis-
accharide unit, which is installed by O-sulfotransferases in
nature.7 The major types of CS have a 4- or 6-O-sulfo group on
the GalNAc residue, which are referred to as CS-A and CS-C,
respectively. Other sulfate variants are also known.1,3,8 Enzymatic
sulfation reactions are often not complete, leading to high struc-
tural heterogeneities of naturally existing CSs. This significantly
hinders the efforts to thoroughly understand the biological
functions of CS/CSPG. Although CS oligosaccharides8−10
and the tetrasaccharide linkage region11 have been prepared, to
date, the synthesis of CSPG glycopeptide bearing a homoge-
neous CS glycan chain has not been achieved. This paper
addresses this critical need by providing a synthesis of the CSPG
syndecan-112 glycopeptide 1, which bears a CS-A tetrasaccharide
on a core protein sequence (corresponding to amino acid residues
45−53 of human syndecan-1) with two additional glycine
residues at its N-terminal for future sortase-mediated ligation.
In nature, syndecan-1 can bear CS chains on multiple possible
serine locations. A successful completion of the synthesis of
CSPG 1 lays important groundwork for the synthesis of other
CSPGs.
To access CSPG glycopeptide 1, a cassette approach13 was
chosen, in which the glycosyl amino acid would be prepared as a
cassette for peptide elongation because although a short peptide
can be glycosylated chemically,14 it is often challenging to
directly glycosylate a large peptide acceptor. CS-A octasacchar-
ide-linked serine 2, containing the full tetrasaccharide linkage
region, galactosamine (GalN) β-linked GlcA, and O-sulfation,
was designed as a cassette with the two sites for O-sulfation
selectively protected as levulinoyl (Lev) esters.
With the large size of the glycan chain in 2, there are many
potential reaction sequences for glyco-assembly. The first route
investigated was through 3 + 2 + 3 glycosylation15 to access the
Received: July 24, 2017
© XXXX American Chemical Society
A
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octasaccharide with strategic disconnections at C/D and E/F
linkages. Whereas most published CS syntheses take advantage
of glycosyl trichloroacetimidates as building blocks,8−10 thiogly-
cosides can be an attractive alternative due to their higher
stabilities.16 The amine moiety of GalN was protected as a
trichloroacetyl (TCA) amide, which could direct β stereo-
selectivity8−10 and reduce the potential complications from the
formation of side products/intermediates arising from the use of
acetamide bearing building blocks.17,18 Thus, ABC trisaccharide 3,
DE disaccharide 4, and FGH trisaccharide 519 were designed first
to access octasaccharide 2.
the desired pentasaccharide. Extensive efforts in varying the
protective groups on either 3 or 4 failed to improve the gly-
cosylation yield. It should be noted that both AB and CD link-
ages are between glucosyl and GalN units. The much higher
glycosylation yield in using donor 11 to glycosylate disaccharide 4,
compared with the reaction between 3 and 4, suggests that it is
difficult to use a trisaccharide donor to form the CD linkage,
presumably due to the reduced reactivity of an oligosaccharide
glycosyl donor (e.g., 3) vs the corresponding monosaccharide
(e.g., 11).
To overcome these setbacks, we designed a new 2 + (3 + 3)
synthetic route, wherein the difficult CD linkage was formed
earlier in the synthesis. Preactivation-based glycosylation of
glucosyl donor 11 and disaccharide 12 gave the CDE
trisaccharide 13 in 77% yield (Scheme 3a). Glycosylation between
Scheme 3. Synthesis of (a) CDE Trisaccharide Donor 13 and
(b) Hexasaccharide 17
Synthesis started with preparation of the DE disaccharide 4
by a glycosylation reaction between donor 6 and acceptor 7
(Scheme 1). Donor 6 was preactivated with p-TolSCl/AgOTf20
Scheme 1. Preparation of DE Disaccharide 4
at −78 °C. Upon complete activation of donor (5 min), accep-
tor 7 was added together with a non-nucleophilic base tri-t-
butylpyrimidine.21 Disaccharide 8 was obtained in an excellent
yield of 92%. Protective group manipulation of 8 gave TBDPS-
protected disaccharide 4. It was necessary to switch the PMB in 8
to TBDPS, as activation of 8 as a glycosyl donor in a subsequent
glycosylation reaction gave the 1,6-anhydrosugar 10 as a major
side product. The TBDPS group in 4 was chosen because it can
be selectively removed and the resulting free primary hydroxyl
groups can be oxidized after glyco-assembly to generate the
corresponding GlcA.
To prepare the ABC trisaccharide (Scheme 2), glucoside
donor 11 was preactivated for 5 min at −78 °C, and then dis-
accharide acceptor 4 was added over 2 min in DCM to generate
the ABC trisaccharide 3 in 81% yield. Formation of the CD gly-
cosyl linkage was tested next. However, glycosylation of donor 3
and disaccharide acceptor 4 gave very low yield (<10%) of
donor 13 and FGH trisaccharide acceptor 5,19 promoted by
N-iodosuccinimide/TfOH, proceeded smoothly (Scheme 3b),
forming the CDEFGH hexasaccharide 14 in an excellent yield
(95%). The free hydroxyl group at C2 on hexasaccharide 14 was
acetylated with Ac2O to produce 15, which was treated with
HF/pyridine to form the triol hexasaccharide 16 in 92% yield.
The regiostereoselectivity in the glycosylation of 13 and 5 was
confirmed by comparing the chemical shift difference of H2″ in
14 vs 15. Selective oxidation of the two primary hydroxyl groups
in triol 16 was tested first using TEMPO and [bis(acetoxy)-
iodo]benzene.22 However, this reaction was slow, with multiple
partial oxidation side products presumably due to the slow
hydration of the aldehyde intermediates. Similar phenomena
have been observed in synthesis of a Staphylococcus aureus asso-
ciated trisaccharide.23 Switching the oxidation protocol to a two-
step, one-pot oxidation (TEMPO, NaOCl followed by NaClO2)24
Scheme 2. Preparation of ABC Trisaccharide Donor 3
B
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solved this problem and produced the desired product 17 in 87%
yield after methyl ester formation.
To access the AB module, donor 18 was preactivated by
p-TolSCl/AgOTf and then glycosylated acceptor 19, producing
the desired disaccharide 20 in 55% yield (Scheme 4a). When 20
to CS synthesis.28,29 However, when 26 was treated with
Bu3SnH/AIBN, the reaction gave several byproducts containing
one or two residual chlorine atoms, which were difficult to
separate. As an alternative, we tested the Zn−Cu couple reduc-
tion method.30,31 When octasaccharide 26 was treated with
Zn−Cu couple in acetic acid, one or both of the benzylidene
rings were cleaved, generating a complex mixture. To overcome
this, octasaccharide 26 was first subjected to 80% aqueous acetic
acid followed by acetylation of the tetraol, affording octasac-
charide 27 (Scheme 5). Subsequent Zn−Cu couple mediated
reduction of TCA led to the octasaccharide cassette 2 in 70% yield.
The Lev groups in 2 were removed by hydrazine acetate, and the
resulting diol 28 was sulfated to provide the octasaccharide 29 in
81% yield, which set the stage for peptide chain elongation.
One of the great challenges in CS glycopeptide assembly is the
acid sensitivity of O-sulfates and the propensities of the glycosyl
serine linkages and the glycan chains to undergo β-elimination
under basic conditions. Thus, common acid-sensitive amino acid
side chain protective groups such as Boc and trityl as well as
strongly basic conditions for deprotection should be avoided.
The Fmoc group in 29 was removed, and the resulting free amine
30 was coupled with tetrapeptide 31 using O-(7-azabenzotriazol-
1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HATU) (Scheme 6). Subsequent catalytic hydrogenolysis was
Scheme 4. Synthesis of AB Disaccharide Donor
was treated with Et3SiH/TFA25 to selectively open the
benzylidene ring for future sulfation, the 4-O-PMB group was
cleaved. To avoid this complication, we tried the glycosylation
of donor 21 with acceptor 22, which smoothly formed the
disaccharide 23 in 80% yield. Cleavage of the p-methoxybenzy-
lidene ring in 23 using 60% aqueous TFA led to diol 24, which
then underwent oxidation, methyl esterification, and acetylation,
affording the AB disaccharide donor 25 in 80% yield over three
steps (Scheme 4b).
Scheme 6. Synthesis of Glycopeptide 1
With the AB and CDEFGH modules in hand, formation of the
octasaccharide was explored. Glycosylation of the hexasaccharide
acceptor 17 by disaccharide donor 25 produced octasaccharide
26 in 70% yield (Scheme 5). The successful assembly of 26
indicates that BC and EF linkages are suitable strategic linkage
points for constructing the octasaccharide cassette.
The next task was the conversion of the NHTCA in octa-
saccharide 26 to an acetamide and introduction of the O-sulfates.
Radical reduction of NHTCA using tributyltin hydride (Bu3SnH)/
AIBN26,27 is a popular method that has been successfully applied
Scheme 5. Synthesis of the Octasaccharide Cassette
performed with Pearlman’s catalyst, which went smoothly to
generate the carboxylic acid 32. Coupling of the carboxylic acid
32 and peptide 33 was promoted by HATU in the presence of
diisopropylethyl amine (DIPEA) as the base. During this reac-
tion, we observed a β-elimination side product32 due to cleav-
age of glycan from the glycopeptide. The extent of elimination
could be reduced by lowering amounts of HATU and DIPEA
from 3 equiv typically applied in peptide coupling to 1.5 equiv,
leading to the glycopeptide 34 (60% yield).
Deprotection of 34 was first performed using LiOH at pH 9.
Even when the reaction was carried out at 0 °C, partial cleavage of
the sulfated glycan chain was observed. Switching to the reagent
combination of H2O2/LiOH28,31,33 at pH 9 avoided the glycan
cleavage problem and hydrolyzed the methyl esters as monitored
by mass spectrometry. As removal of the benzoyl moieties was
very slow under H2O2/LiOH, hydrazine was found to be a
suitable agent to lead to the desired glycopeptide 1 in 71% yield
(Scheme 6).
C
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In conclusion, a strategy was successfully established for the
first synthesis of a CS glycopeptide. Many obstacles were
encountered in the synthesis of this highly complex structure. For
glycan chain assembly, we found that it was difficult to form the
CD linkage using trisaccharide donors. Rather this linkage was
best assembled using a monosaccharide glycosyl donor, which
gave the desired product in a high yield. A 2 + (3 + 3) route was
developed to successfully prepare the octasaccharide cassette.
Subsequently, suitable reaction conditions were identified for
peptide chain elongation and deprotection to produce the CS-A
bearing syndecan-1 glycopeptide 1. The successful establishment
of a synthetic route to CS glycopeptide will now greatly facilitate
understanding of their important biological functions.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b02271.
(23) Hagen, B.; van Dijk, J. H. M.; Zhang, Q.; Overkleeft, H. S.; van der
Experimental procedures and analytical data and NMR
spectra of new products (PDF)
́
Marel, G. A.; Codee, J. D. C. Org. Lett. 2017, 19, 2514.
(24) Huang, L.; Teumelsan, N.; Huang, X. Chem. - Eur. J. 2006, 12,
5246.
AUTHOR INFORMATION
■
(25) DeNinno, M. P.; Etienne, J. B.; Duplantier, K. C. Tetrahedron Lett.
Corresponding Author
*E-mail: xuefei@chemistry.msu.edu.
ORCID
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Xuefei Huang: 0000-0002-6468-5526
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(31) Ait-Mohand, K.; Mirault, A.; Jacquinet, J.-C.; Lopin-Bon, C. Org.
Biomol. Chem. 2016, 14, 7962.
(32) Yang, W.; Yoshida, K.; Yang, B.; Huang, X. Carbohydr. Res. 2016,
We are grateful for the financial support from National Science
Foundation (CHE 1507226) and the National Institute of General
Medical Sciences, NIH (R01GM072667, U01GM116262).
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