A new approach for the synthesis of O-glycopeptides through a combination of solid-phase glycosylation and fluorous tagging chemistry (SHGPFT)

Article abstract of DOI:10.1039/c3ob42430h

Glycoproteins and glycopeptides play important roles in various physiological and pathophysiological processes. Efficient preparation of glycopeptides with a specific structure is one of the pivotal areas in current chemistry research. In this article, a new SHGPFT approach to the synthesis and efficient purification of O-glycosylated peptides is developed by combining a solid-phase glycosylation and a light-fluorous glycosyl donor protocol. The desired product is finally isolated from the side products in the cleaved mixture by an efficient fluorous solid-phase extraction (F-SPE) step.

Full text of DOI:10.1039/c3ob42430h

Organic &
Biomolecular Chemistry
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A new approach for the synthesis of
O-glycopeptides through a combination of
solid-phase glycosylation and fluorous tagging
chemistry (SHGPFT)†
Cite this: Org. Biomol. Chem., 2014,
12, 1892
Bo Liu,a Fa Zhang,a Yan Zhanga and Gang Liu*a,b,c
Glycoproteins and glycopeptides play important roles in various physiological and pathophysiological
processes. Efficient preparation of glycopeptides with a specific structure is one of the pivotal areas in
current chemistry research. In this article, a new SHGPFT approach to the synthesis and efficient purifi-
cation of O-glycosylated peptides is developed by combining a solid-phase glycosylation and a light-
fluorous glycosyl donor protocol. The desired product is finally isolated from the side products in the
cleaved mixture by an efficient fluorous solid-phase extraction (F-SPE) step.
Received 5th December 2013,
Accepted 19th January 2014
DOI: 10.1039/c3ob42430h
www.rsc.org/obc
Glycoproteins and glycopeptides play vital roles in physiologi- acids or oligosaccharides as building blocks during solid-
cal processes, such as cell adhesion, cell differentiation, phase glycopeptide synthesis is currently the most popular
and cell growth.1 Most of the approved protein-based drugs, method for the synthesis of glycopeptides,7 while it suffers
representing a quarter of the new drugs, are glycoproteins.2 from cumbersome operation, low efficiency, and an apparently
However, progress toward understanding the functions of gly- variable synthetic outcome from one target product to
coproteins and glycopeptides and analyzing their structure– another.8 Thus, it is an effort-saving strategy to directly access
activity relationships (SARs) is restricted by their limited sup- the carbohydrate portion onto the solid support to assemble
plemental resources. This includes difficult separation from the glycopeptides.9
natural glycoforms because of their microheterogenicity at
To directly assemble carbohydrate in the solid phase, the
carbohydrate portions3 and uncontrollable glycosylation in the choice of a suitable resin and linker is important. Our group
biosynthetic approach.4 To overcome these obstacles, there is a has found that the use of a large excess of Lewis acid can lead
need for rapid and efficient synthetic approaches to access the to glycosylation of the free hydroxyl group of a single amino
target glycopeptides, especially methods for preparing pure acid on TentaGel resin, which is one of the most popular
and structurally well-defined glycopeptide libraries for vaccine resins used for solid-phase organic synthesis and on-bead
development and drug discovery.
screening.10 However, the presence of the large excess of Lewis
Solid-phase peptide synthesis (SPPS), originally developed acid restricts the use of acid-labile linkers. On the other hand,
by Merrifield,5 has been well applied in recent years by the strong basic conditions for cleavage of a base-sensitive linker
introduction of automation and progressive methodologies;6 could lead to β-elimination of glycopeptides and could also
however, efficient construction of glycopeptides via SPPS is isomerize the stereogenic centers of peptides.11 Consequently,
still challenging because of the complexity of hybrid peptide we decided to employ an aryl hydrazine safety-catch linker12
assembly and oligosaccharide growth plus the difficult purifi- for hybrid glycopeptides synthesis.
cation of the final glycopeptide. Using glycosylated amino
The isolation of glycopeptides away from chromatographi-
cally similar impurities (this commonly means a deletion
sequence on the saccharide segment or truncated peptides) is
also a problematic, time-consuming, high-cost, and low-yield
step. Fluorous chemistry has been widely used in biphasic
catalysis, synthesis, and separation of small organic molecules
and biomolecules,13 for instance, peptides,14 oligosacchar-
ides,15 and oligonucleotides.16 However, it has never been
used in the synthesis of glycopeptides on solid supports.
Employment of fluorous chemistry in solid-phase synthesis of
glycopeptides should give all the advantages of both solid-
aInstitute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union
Medical College, 2A Nanwei Rd., Xicheng Dist., Beijing 100050, P. R. China
bTsinghua-Peking Center for Life Sciences, Tsinghua University, Haidian Dist.,
Beijing 100084, P. R. China
cDepartment of Pharmacology and Pharmaceutical Sciences, School of Medicine,
Tsinghua University, Haidian Dist., Beijing 100084, P. R. China.
E-mail: gangliu27@biomed.tsinghua.edu.cn; Fax: +86 010 63167165;
Tel: +86 010 63167165
†Electronic supplementary information (ESI) available: General methods, experi-
mental procedure, spectroscopic data of products. See DOI: 10.1039/c3ob42430h
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phase synthesis and fluorous chemistry. Obviously, this its acidic and/or basic liability to the glycomoiety and side-pro-
approach will be very complicated because it integrates tection groups of peptides. Third, no matter how structurally
peptide chemistry, solid-phase synthesis, carbohydrate chem- variable and complex the desired glycopeptides are, only the
istry, and fluorous chemistry.
F-tagged glycosyl donor in the final assembly step requires
Herein, we report the first attempt at solid-phase synthesis special preparation, so this approach is applicable to a wide
of O-linked glycopeptides through the combination of a solid- range of glycopeptide syntheses. Moreover, each component of
phase hybrid glycopeptidation procedure and the fluorous the saccharide portion and/or the peptide motif could be
tagging method (SHGPFT) (Scheme 1). In the SHGPFT glycosy- diversified with other homologous building blocks; therefore,
lation steps, the deprotection of a temporary protected sac- this SHGPFT method provides the possibility of efficient con-
charide 1 on the resin to afford an acceptor is followed by struction of a pure glycopeptide library with determinate struc-
glycosylation with a nonfluorous glycosyl donor 2. The cycle is tures. Finally, because only the final target glycopeptides(s)
repeated to build the anticipated oligosaccharide moiety 3 on contains the F-tag, the pure desired product can be obtained
the resin. This solid-phase synthesis procedure has the advan- after a simple F-SPE, even if the solid-phase synthetic outcome
tage of simply washing away all unreacted reagents such as the is not perfect.
catalyst of the solid-phase glycosylation and the inactive mono-
To test this SHGPFT method, the glycopeptide 9 was
saccharide 4 from the recycling of excessive donor 2. In the employed as the desired molecule, in which the Ser is placed
last cycle, a light-fluorous tagged glycosyl donor 5, which was in the middle of the peptide sequence to simulate as closely as
developed by our group,17 was introduced to give 6. As a result, possible the glycosylated site in natural glycopeptides
the desired glycopeptide 8 with a fluorous tag was afforded as (Scheme 2). To complete the synthesis of 9, three key building
well as truncated sequences without the fluorous tag on the blocks were pre-synthesized (Scheme 2) including the penta-
resin and with other inclusions such as the fluorous monosac- peptide that contains an O-linked saccharide moiety on the
charide 7 in the solution. Following simple filtration and clea- resin 10; the non-fluorous tagged glycosyl donor 11; and the
vage of the glycopeptides under mild conditions through an fluorous tagged glycosyl donor 12. The monosaccharide 11 was
aryl hydrazine linker, the designed pure product 8 was designed to elongate the carbohydrate chains for the route.
obtained simply by fluorous solid-phase extraction (F-SPE).
The temporary protecting Lev group is suitable to be removed
There are several beneficial features of this SHGPFT on this designed solid support with an employed aryl hydra-
method. First, the incomplete coupling of a glycosyl-amino zine linker. All the glycosyl donors were transformed into a tri-
acid to the growing peptide chain on the solid support gener- chloroacetimidate form because of its ideal properties for
ally induces large amounts of impurity.8 This is always the solid-phase glycosylation.18
major reason for sequence deficiency of the peptide motif in
Glucopyranosyl trichloroacetimidate 11 was prepared from
the preparation of glycopeptides. Purification of the desired 13 after introduction of the Lev group and transformation of
product from the deletion sequence is time-consuming. This the thioglycoside. After glycosylation of Fmoc-Ser-OAll19 with
SHGPFT protocol greatly simplifies the purification procedure 11, followed by Pd(PPh3)4-catalyzed deprotection of the allyl
by means of the F-tag of the fluorous phase. Second, the stable group, 16 was obtained (Scheme 3). The preparation of com-
aryl hydrazine linker under reaction conditions eliminates the pound 10 with the aryl hydrazine linker was finished by the
risk of destroying the structure of the glycopeptide because of prepared glycosylSer 16 with
a standard SPPS protocol
(Scheme 4).
The peptide chain assembly was achieved using a Gly–Gly
spacer functionalizing TentaGel amino resin, which was
further equipped with an aryl hydrazine linker (loading
capacity: 0.186 mmol g−1). The assembly of the peptide chain
was performed according to a standard Fmoc-based protocol.
Loading of the first amino acid (Phe) was realized with a BOP/
Scheme 1 Schematic overview of our SHGPFT approach for a hybrid
Scheme 2 Retrosynthesis of our target pentapeptides with tri-
glycopeptide synthesis.
saccharides.
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Scheme 5 (a) DMAP, Et3N, DCM 90%; (b) i. NBS, TMSOTf, H2O, DCM;
ii. Cl3CCN, DBU, DCM, 0 °C; two-step yield 57%.
Scheme 3 (a) LevOH, DCC, DMAP, DCM 92%; (b) i. NBS, TMSOTf, H2O,
DCM; ii. Cl3CCN, DBU, DCM, 0 °C, two-step yield 62%; (c) Fmoc-Ser-
OAll, TMSOTf, DCM, 0 °C, 4 Å MS, 64%; (d) Pd(PPh3)4, morpholine,
DCM 96%.
To complete this new strategy, we required a fluorous glyco-
sylated donor at the end of the carbohydrate chain elongation.
The preparation of fluorous donor 12 was achieved from thio-
glycoside 13 by installation of a fluorous-tagged benzoyl
chloride17b and transformation of thioglycoside 20 into a
trichloroacetimidate 12 (Scheme 5).
After that, the pentapeptide with disaccharide 22 on the
resin was reacted with fluorous donor 12 by the solid-phase
glycosylation protocol used before to provide resin 23
(Scheme 6). The final product was released from resin 23 in a
two-step protocol, using N-bromosuccinimide as the oxidizing
agent and subsequent addition of H2O (5%) in THF solution.
The crude mixture was analyzed by HPLC (Fig. 1a). The
desired protected glycopeptide 24 was detectable along with
the unglycosylated non-fluoride material 22′ and minor
amounts of side products. We herein leave an incomplete
coupling result in reaction step c in Scheme 6 of 12 for one
time assembly (Fig. 1a) because we want to demonstrate the
separating efficacy by F-SPE. The yield of 24 after executing the
Scheme 4 Solid-phase synthesis of pentapeptide with a monosaccharide
motif.
HOBt/DIPEA cocktail of reagents to make the condensation
complete, while the standard DIC/HOBt protocol was used to
extend the other peptide chain. After each incorporation of a
Fmoc-protected amino acid, the reaction support was treated
with Ac2O in DCM (15%) to cap the trace unreacted amino
group. With the protected pentapeptide with monoglucose 19
in hand, the Lev group on the carbohydrate motif was selec-
tively removed by a solution of N2H4–Py–HOAc that resulted in
10 as the glycosyl acceptor on the solid support (Scheme 4).
The solid-phase O-glycosylation of 10 was then performed
under the optimal condition (10 equiv. acceptor in the pres-
ence of 20 equiv. TMSOTf) in advance to offer 21. This con-
dition did not affect the linker or any protecting groups. After
the glycosylation, the reaction mixture was quenched with
Et3N, and then the excess of the donor in the residue was
recovered in the form of anomeric hydroxy glucose (86%). The
unreacted free hydroxyl on the first saccharide fragment was
then capped using acetic anhydride in pyridine (1 : 3 v/v). Sub-
sequently, by removal of the Lev group of 21 under the above
conditions, a second acceptor was assembled onto the solid
support, i.e., a pentapeptide with disaccharide 22 on the resin
was successfully obtained (Scheme 6).
Scheme 6 Solid-phase glycosylation and F-SPE purified the cleavage
residue: (a) i. TMSOTf, 0 °C, DCM; ii. Ac2O : Py = 1 : 3, 1 h; (b) NH2NH2,
Py–AcOH; (c) 12, TMSOTf, 0 °C, DCM; (d) i. NBS–Py, 15 min; ii. H2O,
THF, 4 h; (e) 22’ is the cleavage product of resin 22.
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and L. L. Kiessling, Science, 2001, 291, 2357; (d) D. P. Gamblin,
E. M. Scanlan and B. G. Davis, Chem. Rev., 2009, 109,
131–163.
2 T. J. Boltje, T. Buskas and G.-J. Boons, Nat. Chem., 2009, 34,
611.
3 P. M. Rudd and R. A. Dwek, Crit. Rev. Biochem. Mol. Biol.,
1997, 32, 1.
4 R. P. Matthew and C. R. Bertozzi, Chem. Soc. Rev., 2005, 34,
58.
5 R. B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149.
6 (a) Chemical Approaches to the Synthesis of Peptides and
Proteins, ed. P. Lloyd-Williams, F. Albericio and E. Giralt,
CRC LLC, NY, 1997; (b) T. Conroy, K. A. Jolliffe and
R. J. Payne, Org. Biomol. Chem., 2009, 7, 2255; (c) M. Amblard,
J.-A. Fehrentz, J. Martinez and G. Subra, Mol. Biotechnol.,
2006, 33, 239.
7 (a) T. Reipen and H. Kunz, Synthesis, 2003, 2487;
(b) C. Brocke and H. Kunz, Synlett, 2003, 2052;
(c) M. A. Walczak, J. Hayashida and S. J. Danishefsky, J. Am.
Chem. Soc., 2013, 135, 4700; (d) N. Yamamoto,
A. Takayanagi, A. Yoshino, T. Sakakibara and Y. Kajihara,
Chem.–Eur. J., 2007, 13, 613; (e) N. Shao, J. Xue and Z. Guo,
J. Org. Chem., 2003, 68, 9003; (f) N. Shao and Z. Guo, Org.
Lett., 2005, 7, 3589.
Fig. 1 HPLC analysis profiles of glycosylation of 12 with 22: (a) crude
product and (b) fluorous fraction after F-SPE.
F-SPE protocol of the crude cleavage is 37% (one-step purifi-
cation: see ESI†). In fact, repeated step c in Scheme 6 of 12
assembly increased the yield of 24 (data not shown).
Notably, the recyclable fluorous glycosyl donor and fluorous
silica gel used in the whole procedure avoided unnecessary
costs for purification. Analysis of the fluorous fraction of F-SPE
by HPLC (Fig. 1b) indicated good purity (96%).
8 Y. Zhang, S. M. Muthana, D. Farnsworth, O. Ludek,
K. Adams, J. J. Barchi Jr. and J. C. Gildersleeve, J. Am.
Chem. Soc., 2012, 134, 6316.
9 (a) M. Hollósi, E. Kollát, I. Laxzkó, K. F. Medzihradszky,
J. Thurin and L. Otvös Jr., Tetrahedron Lett., 1991, 32, 1531;
(b) M. Meldal, F.-I. Auzanneau, O. Hindsgaul and
M. M. Palcic, J. Chem. Soc., Chem. Commun., 1994, 1849;
(c) H. Paulsen, A. Schleyer, N. Mathieux, M. Meldal and
K. J. Bock, J. Chem. Soc., Perkin Trans. 1, 1997, 6, 281;
(d) A. Schleyer, M. Meldal, R. Manat, H. Paulsen and
K. Bock, Angew. Chem., Int. Ed. Engl., 1997, 36, 1976;
(e) K. M. Halkes, C. H. Gotfredsen, M. Grøtli, L. P. Miranda,
J. O. Duus and M. Meldal, Chem.–Eur. J., 2001, 7, 3584;
(f) M. Mogemark, M. Elofsson and J. Kihlberg, Org. Lett.,
2001, 3, 1463; (g) M. Mogemark, F. Gårdmo, T. Tengel,
J. Kihlberg and M. Elofsson, Org. Biomol. Chem., 2004, 2,
1770.
Conclusions
A new and efficient hybrid strategy to synthesize and purify
O-glycopeptides was developed via a combination of the advan-
tages of the solid-phase glycosylation strategy with the super-
iority of fluorous chemistry. This approach not only introduces
a fluorous-tagged glycosyl donor and an aryl hydrazine safety-
catch linker into the solid-phase synthesis of O-linked glyco-
peptides for the first time, but also avoids generation of extra
costs with the help of solid-phase glycosylation and F-SPE
chemical recycling. This SHGPFT strategy will be a significant
advance in realizing automated O-linked glycopeptide syn-
thesis for nonspecialists. The synthesis of more complex
natural bioactive O-linked glycopeptides and construction of
glycopeptide libraries for new active material discovery by our
protocol is in progress.
10 N.-H. Yao, W.-Y. He, K. S. Lam and G. Liu, J. Comb. Chem.,
2004, 6, 214.
11 T. Buskas, S. Ingale and G.-J. Boons, Glycobiology, 2006, 16,
113R.
12 (a) C. R. Millington, R. Quarrell and G. Lowe, Tetrahedron
Lett., 1998, 39, 7201; (b) C. Peters and H. Waldmann, J. Org.
Chem., 2003, 68, 6053; (c) Y. Kwon, K. Welsh, A. R. Mitchell
and J. A. Camarero, Org. Lett., 2004, 6, 3801; (d) J. A. Camarero,
B. J. Hackel, J. J. de Yoreo and A. R. Mitchell, J. Org.
Chem., 2004, 69, 4145; (e) A. Shigenaga, J. A. Moss,
F. T. Ashley, G. F. Kaufmann and K. D. Janda, Synlett, 2006,
551.
Acknowledgements
This research was supported financially by the National 863
Program of China, no. 201202AA0303.
Notes and references
13 (a) W. Zhang, Chem. Rev., 2004, 104, 2531; (b) D. P. Curran,
Science, 2008, 321, 1645; (c) W. Zhang, Chem. Rev., 2009,
109, 749 and reference therein.
1 (a) H. Lis and N. Sharon, Eur. J. Biochem., 1993, 218, 1;
(b) R. Dwek, Chem. Rev., 1996, 96, 683; (c) C. R. Bertozzi
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 1892–1896 | 1895

View Article Online
Paper
Organic & Biomolecular Chemistry
14 P. C. de Visser, M. van Helden, D. V. Filippov, G. A. van
der Marel, J. W. Drijfhout, J. H. van Boom, D. Noort
K.-Y. Jung and A. D. Sercel, J. Org. Chem., 2005, 70,
7114.
and H. S. Overkleeft, Tetrahedron Lett., 2003, 44, 17 (a) F. Zhang, W. Zhang, Y. Zhang, D. P. Curran and G. Liu,
9013.
J. Org. Chem., 2009, 74, 2594; (b) Y. Zhang, B. Liu and
G. Liu, Mol. Diversity, 2013, 17, 613.
15 (a) E. R. Palmacci, M. C. Hewitt and P. H. Seeberger,
Angew. Chem., Int. Ed., 2001, 40, 4433; (b) F. R. Carrel 18 (a) L. Schofield, M. C. Hewitt, K. Evans, M. A. Siomos and
and P. H. Seeberger, J. Org. Chem., 2007, 73,
2058.
P. H. Seeberger, Nature, 2002, 418, 785; (b) P. H. Seeberger,
Chem. Soc. Rev., 2008, 37, 19.
16 (a) S. Tripathi, K. Misra and Y. S. Sanghvi, Org. Prep. Proced. 19 T. Vorherr and W. Bannwarth, Bioorg. Med. Chem. Lett.,
Int., 2005, 37, 257; (b) W. H. Pearson, D. A. Berry, P. Stoy, 1995, 5, 2661.
1896 | Org. Biomol. Chem., 2014, 12, 1892–1896
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