Chemical synthesis of syndecan-3 glycopeptides bearing two heparan sulfate glycan chains

Article abstract of DOI:10.1002/anie.201404625

Despite the ubiquitous presence of proteoglycans in mammalian systems, methodologies to synthesize this class of glycopeptides with homogeneous glycans are not well developed. Herein, we report the first synthesis of a glycosaminoglycan family glycopeptid

Full text of DOI:10.1002/anie.201404625

Angewandte
Chemie
DOI: 10.1002/anie.201404625
Glycopeptide Synthesis
Chemical Synthesis of Syndecan-3 Glycopeptides Bearing Two
Heparan Sulfate Glycan Chains**
Keisuke Yoshida, Bo Yang, Weizhun Yang, Zeren Zhang, Jicheng Zhang, and Xuefei Huang*
[
6]
Abstract: Despite the ubiquitous presence of proteoglycans in
mammalian systems, methodologies to synthesize this class of
glycopeptides with homogeneous glycans are not well devel-
oped. Herein, we report the first synthesis of a glycosamino-
glycan family glycopeptide containing two different heparan
sulfate chains, namely the extracellular domain of syndecan-3.
With the large size and tremendous structural complexity of
these molecules, multiple unexpected obstacles were encoun-
tered during the synthesis, including high sensitivity to base
treatment and the instability of glycopeptides with two glycan
chains towards catalytic hydrogenation conditions. A success-
ful strategy was established by constructing the partially
deprotected single glycan chain containing glycopeptides
first, followed by union of the glycan-bearing fragments and
cleavage of the ester-type protecting groups. This work lays the
foundation for preparing other members of this important class
of molecules.
one glycosaminoglycan chain. To address this, we have
begun to establish a viable route towards homogeneous
glycopeptides bearing multiple heparan sulfate moieties with
syndecan-3 as the target. Serious difficulties were encoun-
tered since the presence of multiple glycan chains significantly
increases the structural complexity and instability of the
target molecules. Herein, we report the lessons we have
learned and the eventual establishment of a successful
approach to access these highly complex molecules.
Our synthetic target is the syndecan-3 extracellular
domain glycopeptide (1), which displays the typical structural
features of heparan sulfate proteoglycans, including a peptide
backbone, different heparan sulfate chains, the full tetrasac-
charide linkers, 2-O sulfation, 6-O sulfation, glucosamine a-
linked to both glucuronic acid and iduronic acid, and N-
acetylation. In order to prepare this molecule, we adapted
[
29]
a cassette approach in which the glucuronic acid containing
[
28]
octasaccharide cassette 2 and iduronic acid cassette 3 were
produced first and then incorporated into the glycopeptide
(Figure 1).
G
lycopeptides and glycoproteins play important roles in
many biological events, such as cellular proliferation, neuron
[
1]
development, and inflammation. There are two major
classes of glycopeptides/glycoproteins: N-linked glycans and
O-linked glycans. O-glycans can be further divided into two
main classes: the mucin type and the glycosaminoglycan
family proteoglycans. Many innovative strategies have been
designed to synthesize glycopeptides bearing N-glycans and
There are multiple possible reaction sequences to connect
the glycosyl units in the octasaccharide modules. After much
exploration, we established a 3+2+3 strategy that involves
building blocks consisting of the ABC trisaccharide, the DE
disaccharide, and the FGH trisaccharide to access the
octasaccharide modules 2 and 3. To prepare the ABC
trisaccharide, the glucoside donor 4 was pre-activated with
[
2,3]
mucin type O-glycans,
with successful preparation of
[
30]
molecules approaching the complexity of native glycopro-
p-TolSCl/AgOTf and subsequently glycosylated disacchar-
[
4,5]
[28]
teins.
ide 5 to generate the ABC trisaccharide 6 in 85% yield
In sharp contrast to peptides bearing N-glycans and mucin
type O-glycans, glycosaminoglycan glycopeptide syntheses
are much less developed, despite the ubiquitous presence and
many important biological functions of this class of glycans.
Synthesis in this area has mainly focused on the glycosami-
(Scheme 1). The 3+2 glycosylation between trisaccharide 6
and the DE disaccharide 5 went smoothly to produce
pentasaccharide 7. Pentasaccharide 7 reacted with the
[6]
[28]
trisaccharide serine unit 8 to generate the octasaccharide
cassette 9 in an excellent 87% yield. The TBDPS silyl ether
groups in 9 were removed by HF/pyridine to expose the three
[
7–23]
noglycan oligosaccharides
linker.
or the tetrasaccharide
Recently, we reported the first synthesis of
a proteoglycan family glycopeptide; a syndecan-1 glycopep-
[24–27]
[31]
primary hydroxyls, which were oxidized to carboxylic acids
and subsequently converted to methyl esters to give com-
pound 12. The two azide groups in 12 were transformed into
N-acetyl moieties through a one-pot reduction/acetylation
procedure with zinc, copper sulfate, and acetic anhydride to
afford octasaccharide 2.
[
28]
tide bearing one heparan sulfate chain. An additional level
of complexity of proteoglycans is that many carry more than
[
+]
[+]
[+]
[
*] K. Yoshida, B. Yang, W. Yang, Z. Zhang, J. Zhang, X. Huang
Department of Chemistry, Michigan State University
A serious challenge in the assembly of heparan sulfate
glycopeptides is the compatibility of the protecting group
removal conditions with the sulfated glycopeptide. Owing to
the high sensitivity of sulfates to acid, commonly used acid-
cleavable amino acid side chain protecting groups such as Boc
and trityl are to be avoided. Furthermore, caution needs to be
taken since the glycoside–serine linkage is prone to base-
578 S. Shaw Lane, East Lansing, MI 48824 (USA)
E-mail: xuefei@chemistry.msu.edu
+
[
] These authors contributed equally to the work.
[**] This work was supported by the National Science Foundation (CHE
1111550) and the National Institute of General Medical Sciences
NIH (R01GM072667).
[
28,32]
promoted b-elimination.
The sequence of deprotection
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201404625.
Angew. Chem. Int. Ed. 2014, 53, 9051 –9058
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
9051

.
Angewandte
Communications
Figure 1. Syndecan-3 glycopeptide (1) and the octasaccharide cassettes 2 and 3.
and reagents applied thus needs to be carefully designed and
The failure of the previously established acyl removal
strategy prompted us to examine alternatives. We envisioned
that a less basic yet strong nucleophile such as hydrazine
established.
Previously, we showed that the ester protecting groups
Ac, Bz) on glycopeptide 13 (Figure 2) could be successfully
removed under transesterification conditions by using
[34]
(
could potentially remove the Ac and Bz groups without
damaging the glycopeptide linkage. To incorporate hydrazi-
nolysis, the full-length glycopeptide 17 was designed, which
would be assembled from glycopeptides 18 and 19 (Figure 3).
The uronic acid groups in the glycan chains of 18 and 19 are
protected as methyl esters, which can be converted into free
carboxylic acids by mild base treatment, thus setting the stage
for hydrazinolysis to cleave all of the acyl protective groups.
Synthesis of glycopeptide 18 started with the acetylation
of 3. Subsequent conversion of azides into acetamides, Lev
group removal by hydrazine acetate, and sulfation afforded
octasaccharide 20 (Scheme 3). The Fmoc group in 20 was
removed and the resulting free amine was coupled to
dipeptide 21 to produce glycopeptide 22 in 56% yield over
two steps. Selective removal of the benzyl ester in glycopep-
[28]
NaOMe. The free C terminus of the glycopeptide 13 was
crucial to prevent base-promoted b-elimination of the glycan
chain. This route was applied to the glucoside-containing
octasaccharide cassette 15, which was produced from octa-
saccharide module 2 (Scheme 2). However, NaOMe treat-
ment of 15 at room temperature led to multiple products as
a result of backbone cleavage at the glucuronic acid sites, with
only trace amounts of the desired product obtained. Lowering
the pH value or reaction temperature led to incomplete
removal of the Bz groups. The high lability of glycopeptide 15
to base treatment compared to glycopeptide 13 is possibly due
[
11,33]
to neighboring group assisted glycan cleavages
(
Scheme S1 in the Supporting Information).
9
052 www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 9051 –9058

Angewandte
Chemie
Scheme 1. TBDPS=tert-butyldiphenylsilyl, Tf=trifluoromethanesulfonyl, Bn=benzyl, Bz=benzoyl, PMB=para-methoxybenzyl, Fmoc=9-fluorenyl-
methyloxycarbonyl, TEMPO=2,2,6,6-tetramethylpiperidin-1-yloxyl, BAIB=bis(acetoxy)iodobenzene, DMF=N,N-dimethylformamide, THF=tetra-
hydrofuran.
Figure 2. The structure of glycopeptide 13.
Scheme 2. HATU=O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate, DIPEA=N,N-diisopropylethylamine.
Angew. Chem. Int. Ed. 2014, 53, 9051 –9058
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
9053

.
Angewandte
Communications
Figure 3. The full-length glycopeptide 17 is assembled from glycopeptides 18 and 19.
Scheme 3.
tide 22 under hydrogenation in the presence of NH OAc
generated glycopeptide 18 with a free carboxylic acid
terminus (Scheme 3).
piperidine to give the free amine bearing glycopeptide 19
(Scheme 4).
With glycopeptides 18 and 19 in hand, these two frag-
4
The synthesis of glycopeptide 19 began with the hydro-
ments were joined together in a reaction promoted by HATU
to produce glycopeptide 28, which bears two different glycans.
To remove the benzyl ethers, PMB, and benzylidene groups,
glycopeptide 28 was subjected to global hydrogenation with
Pearlmanꢀs reagent or Pd/C under atmospheric pressure of
hydrogen in mixed solvents of CH Cl and methanol. How-
genation of octasaccharide 2 in the presence of NH OAc to
4
afford glycopeptide 23, which contains a free C-terminal
carboxylic acid. Coupling 23 with tripeptide 24 gave glyco-
peptide 25, which was treated with piperidine and then joined
to tripeptide 26. The resulting product 27 was deprotected by
2
2
9
054 www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 9051 –9058

Angewandte
Chemie
Scheme 4.
Scheme 5.
ever, this reaction failed to yield any of the desired product 17
and decomposition products were observed in mass spec-
(
Scheme 5). Given that it is possible that the partially
trometry analysis.
deprotected glycopeptides formed during the hydrogenation
of 28 could undergo aggregation, thereby preventing access to
the palladium catalyst, colloidal Pd nanoparticles were tested
for the hydrogenation of 28 since these nanoparticles could
Owing to the unexpected difficulty in the hydrogenation
of 28, an alternative route was envisioned in which hydro-
genation was to be performed on glycopeptides 22 and 27,
which bear a single heparan sulfate chain. The iduronic acid
containing nonsulfated glycopeptide 29 was tested first and
was prepared in a manner analogous to the preparation of 22.
Catalytic hydrogenation of compound 29 under slightly acidic
conditions (pH ꢀ 5.5) with Pearlmanꢀs catalyst went smoothly
and gave the desired product 30 in quantitative yield
[35]
remove benzyl ethers from solid phase bound glycans.
Various solvents, pH values, and reaction temperatures;
elevated hydrogen pressure; and low acidity triflic acid
[36]
conditions for benzyl removal were also examined. How-
ever, none of these endeavors yielded the desired compounds
Angew. Chem. Int. Ed. 2014, 53, 9051 –9058
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
9055

.
Angewandte
Communications
Scheme 6.
Scheme 7.
(
Scheme 6A). In a similar manner, sulfated glycopeptide 22
and glycopeptide 27 were successfully hydrogenated to give
1 and 32 in quantitative yield (Scheme 6B). The reason for
the methyl esters in 33 were cleaved by LiOH (pH 9.5), which
was followed by hydrazine treatment. The hydrazinolysis
procedure successfully removed all of the acyl protecting
groups, thereby affording the fully deprotected glycopeptide
1a.
3
the increase in sensitivity of the glycopeptides bearing two
heparan sulfate chains to hydrogenation is not clear.
The two partially deprotected glycopeptides 30 and 32
were joined through HATU-mediated coupling to afford
glycopeptide 33 (Scheme 7). To complete the deprotection,
With the successful preparation of the nonsulfated
glycopeptide 1a, we moved on to the synthesis of the sulfated
glycopeptide 1. The coupling of glycopeptides 31 and 32
9
056 www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 9051 –9058

Angewandte
Chemie
Scheme 8.
afforded compound 34 (Scheme 8). The sulfated glycan chain
turned out to be very sensitive to base since pH 9.5 LiOH led
to partial chain cleavage. Instead, the methyl ester removal
was performed at pH 9.0 with frequent monitoring by mass
spectrometry, followed by hydrazinolysis to afford glycopep-
tide 1 in 61% yield.
[5] I. Sakamoto, K. Tezuka, K. Fukae, K. Ishii, K. Taduru, M.
Maeda, M. Ouchi, K. Yoshida, Y. Nambu, J. Igarashi, N. Hayashi,
T. Tsuji, Y. Kajihara, J. Am. Chem. Soc. 2012, 134, 5428 – 5431.
6] J. R. Bishop, M. Schuksz, J. D. Esko, Nature 2007, 446, 1030 –
[
[
1037.
7] S. U. Hansen, G. J. Miller, C. Cole, G. Rushton, E. Avizienyte,
G. C. Jayson, J. M. Gardiner, Nat. Commun. 2013, 4, 2016.
In conclusion, a successful strategy was developed for the
assembly of syndecan-3 glycopeptides bearing two heparan
sulfate chains. Many obstacles were encountered during the
syntheses and the previous synthetic route established for
syndecans-1 with one glycan chain could not be directly
applied to glycopeptide 1. The reason for this is that as the size
of the molecule increases, unique reactivity and stability
problems emerge. To overcome these challenges, the hydro-
genation reaction was performed on glycopeptides bearing
a single glycan chain followed by union of the partially
deprotected fragments. The final deprotection required the
cleavage of all of the ester protective groups, which was
accomplished by mild base treatment followed by hydrazin-
olysis. The hydrazinolysis procedure was critical to ensure
complete removal of the benzoyl moieties without undesired
b-elimination or cleavage of the highly sensitive glycan chain.
The knowledge gained in the current study will be valuable in
the synthesis of other glycosaminoglycan family glycopep-
tides bearing multiple heparan sulfate chains.
[8] G. J. Sheng, Y. I. Oh, S.-K. Chang, L. C. Hsieh-Wilson, J. Am.
Chem. Soc. 2013, 135, 10898 – 10901.
[
9] Y. Xu, S. Masuko, M. Takeiddin, H. Xu, R. Liu, J. Jing, S. A.
Mousa, R. J. Linhardt, J. Liu, Science 2011, 334, 498 – 501.
10] Y.-P. Hu, S.-Y. Lin, C.-Y. Huang, M. M. L. Zulueta, J.-Y. Liu, W.
Chang, S.-C. Hung, Nat. Chem. 2011, 3, 557 – 563.
11] G. Tiruchinapally, Z. Yin, M. El-Dakdouki, Z. Wang, X. Huang,
Chem. Eur. J. 2011, 17, 10106 – 10112.
12] P. Czechura, N. Guedes, S. Kopitzki, N. Vazquez, M. Martin-
Lomas, N.-C. Reichardt, Chem. Commun. 2011, 47, 2390 – 2392.
[
[
[
[13] S. Arungundram, K. Al-Mafraji, J. Asong, F. E. Leach, I. J.
Amster, A. Venot, J. E. Turnbull, G. J. Boons, J. Am. Chem. Soc.
2009, 131, 17394 – 17405.
[
[
14] F. Baleux, L. Loureiro-Morais, Y. Hersant, P. Clayette, F.
Arenzana-Seisdedos, D. Bonnaffꢁ, H. Lortat-Jacob, Nat. Chem.
Biol. 2009, 5, 743 – 748.
15] J. Chen, Y. Zhou, C. Chen, W. Xu, B. Yu, Carbohydr. Res. 2008,
343, 2853 – 2862.
[16] J. Tatai, P. Fꢂgedi, Tetrahedron 2008, 64, 9865 – 9873.
[17] T. Polat, C.-H. Wong, J. Am. Chem. Soc. 2007, 129, 12795 – 12800.
[
[
18] C. Noti, J. L. de Paz, L. Polito, P. H. Seeberger, Chem. Eur. J.
006, 12, 8664 – 8686.
2
19] J. D. C. Codꢁe, B. Stubba, M. Schiattarella, H. S. Overkleeft,
C. A. A. van Boeckel, J. H. van Boom, G. A. van der Marel, J.
Am. Chem. Soc. 2005, 127, 3767 – 3773.
Received: April 23, 2014
Published online: June 30, 2014
[
[
[
20] J. L. de Paz, M. Martin-Lomas, Eur. J. Org. Chem. 2005, 1849 –
Keywords: glycopeptides · glycosylation · proteoglycans ·
sulfation · total synthesis
.
1858.
21] R.-H. Fan, J. Achkar, J. M. Hernandez-Torres, A. Wei, Org. Lett.
2005, 7, 5095 – 5098.
22] L. Poletti, M. Fleischer, C. Vogel, M. Guerrini, G. Torri, L. Lay,
Eur. J. Org. Chem. 2001, 2727 – 2734.
[
1] B. G. Davis, Chem. Rev. 2002, 102, 579 – 601, and references
therein.
[23] M. Petitou, J.-P. Herault, A. Bernat, P.-A. Driguez, P. Duchaus-
soy, J.-C. Lormeau, J.-M. Herbert, Nature 1999, 398, 417 – 422.
[24] K. Aꢃt-Mohand, C. Lopin-Bon, J. C. Jacquinet, Carbohydr. Res.
2012, 353, 33 – 48, and references therein.
[25] T.-Y. Huang, M. M. L. Zulueta, S.-C. Hung, Org. Lett. 2011, 13,
1506 – 1509.
[2] C. Unverzagt, Y. Kajihara, Chem. Soc. Rev. 2013, 42, 4408 – 4420.
[3] P. Siman, A. Brik, Org. Biomol. Chem. 2012, 10, 5684 – 5697.
[4] P. Wang, S. Dong, J.-H. Shieh, E. Peguero, R. Hendrickson,
M. A. S. Moore, S. J. Danishefsky, Science 2013, 342, 1357 – 1360.
Angew. Chem. Int. Ed. 2014, 53, 9051 –9058
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 9057

.
Angewandte
Communications
[
[
[
26] J.-i. Tamura, T. Nakamura-Yamamoto, Y. Nishimura, S. Mizu-
[31] L. J. Van den Bos, J. D. C. Codee, J. C. Van der Toorn, T. J.
Boltje, J. H. Van Boom, H. S. Overkleeft, G. A. Van der Marel,
Org. Lett. 2004, 6, 2165 – 2168.
[32] P. Sjçlin, M. Elofsson, J. Kihlberg, J. Org. Chem. 1996, 61, 560 –
565.
[33] J. Gao, D. A. Thomas, C. H. Sohn, J. L. Beauchamp, J. Am.
Chem. Soc. 2013, 135, 10684 – 10692.
[34] P. W. Glunz, S. Hintermann, L. J. Williams, J. B. Schwarz, S. D.
Kuduk, V. Kudryashov, K. O. Lloyd, S. J. Danishefsky, J. Am.
Chem. Soc. 2000, 122, 7273 – 7279.
moto, J. Takahashi, K. Sugahara, Carbohydr. Res. 2010, 345,
2115 – 2123.
27] K. Shimawaki, Y. Fujisawa, F. Sato, N. Fujitani, M. Kurogochi, H.
Hoshi, H. Hinou, S.-I. Nishimura, Angew. Chem. Int. Ed. 2007,
46, 3074 – 3079; Angew. Chem. 2007, 119, 3134 – 3139.
28] B. Yang, K. Yoshida, Z. Yin, H. Dai, H. Kavunja, M. H. El-
Dakdouki, S. Sungsuwan, S. B. Dulaney, X. Huang, Angew.
Chem. Int. Ed. 2012, 51, 10185 – 10189; Angew. Chem. 2012, 124,
1
0332 – 10336.
29] X.-T. Chen, D. Sames, S. J. Danishefsky, J. Am. Chem. Soc. 1998,
20, 7760 – 7769.
30] X. Huang, L. Huang, H. Wang, X.-S. Ye, Angew. Chem. Int. Ed.
004, 43, 5221 – 5224; Angew. Chem. 2004, 116, 5333 – 5336.
[
[
[35] O. Kanie, G. Grotenbreg, C.-H. Wong, Angew. Chem. Int. Ed.
2000, 39, 4545 – 4547; Angew. Chem. 2000, 112, 4719 – 4721.
[36] K. Kawahira, H. Tanaka, A. Ueki, Y. Nakahara, H. Hojo, Y.
Nakahara, Tetrahedron 2009, 65, 8143 – 8153.
1
2
9
058 www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 9051 –9058