Glycopeptide synthesis on an ionic liquid support

Article abstract of DOI:10.1021/ol501133u

An ionic liquid-supported synthetic method for the construction of glycopeptides in high yields is reported. This method avoids the use of large excesses of reagents and chromatographic purification and, therefore, represents a useful addition to existing approaches for the ionic liquid-supported synthesis of oligosaccharides and peptides.

Full text of DOI:10.1021/ol501133u

Letter
pubs.acs.org/OrgLett
Glycopeptide Synthesis on an Ionic Liquid Support
Changgeng Li,†,‡ Zhenxing Zhang,†,‡ Qian Duan,§ and Xuebing Li*,†
†CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences,
Chaoyang District, Beijing 100101, China
‡University of Chinese Academy of Sciences, Shijingshan District, Beijing 100049, China
§School of Materials Science and Engineering, Changchun University of Science and Technology, Weixing Road, Changchun 130022,
China
S
* Supporting Information
ABSTRACT: An ionic liquid-supported synthetic method for
the construction of glycopeptides in high yields is reported.
This method avoids the use of large excesses of reagents and
chromatographic purification and, therefore, represents a
useful addition to existing approaches for the ionic liquid-
supported synthesis of oligosaccharides and peptides.
fficient methods for the synthesis of glycopeptides are
good chemical stability, low molecular weight, and well-defined
Eindispensable for both elucidating their biological sig- structures, and the versatility of IL supports in this regard has
recently been demonstrated by their use for the rapid
construction of various molecules including peptides7 and
oligosaccharides.8
nificance and producing therapeutic reagents of relevant
diseases. The challenge associated with the construction of
these molecules lies in being able to exercise a high level of
control over both carbohydrate and peptide chemistries, which
require complex protection and coupling strategies as well as
tedious product purification processes. Standard solid-phase
peptide synthesis (SPPS) is currently the most commonly used
approach for the construction of glycopeptides and involves the
use of preprepared glycosylated amino acids as building blocks.1
The key advantage of SPPS over conventional liquid-phase
synthesis is the simple resin-washing process used for product
isolation, which allows for the synthesis to be conducted with a
high level of automation.2 Although a number of complex
glycopeptides have been successfully constructed by SPPS,3 the
heterogeneous conditions required for SPPS can result in low
reaction rates, and it can be particularly difficult to monitor the
progress of reactions conducted by SPPS using conventional
spectroscopic techniques. Several novel liquid-phase strategies
have been developed to address the issues associated with
SPPS. According to one such strategy, the reactions themselves
can be conducted under homogeneous conditions, whereas the
purification of the product can be achieved by a simple phase
separation. Prominent examples of this approach include the
use of functional supports such as soluble polymers,4 fluoride,5
or ionic liquids (ILs)6 as platforms for conducting the chemical
synthesis. A common feature of these supports is their tunable
solubility in different solvents, which allows the supported
reactants to undergo efficient homogeneous reactions in one
solvent and simple product purification processes in another
solvent without the need for column chromatography. ILs, in
particular, possess several attractive properties in terms of their
use as functional supports for chemical synthesis, including
Herein, we report for the first time the synthesis of
glycopeptides using an IL support. In a similar manner to
SPPS, our approach involved the use of a solid−liquid phase
product separation step to increase the overall efficiency of the
synthetic process. However, our approach avoided the use of
large excesses of the reagents, which is one of the major
problems associated with SPPS, especially for the large-scale
synthesis of glycopeptides involving expensive glycosylated
amino acid building blocks. We have demonstrated the
feasibility of our method for the synthesis of several O-
glycopeptides and shown that this strategy can be used for not
only the stepwise linear elongation of both peptide and glycan
chains but also the convergent assembly of glycopeptide
fragments.
The design of our method for the IL-based synthesis of
glycopeptides was based on three essential considerations. First,
the overall synthetic strategy was based on the use of a 9-
fluorenylmethoxycarbonyl (Fmoc) for the temporary protec-
tion of the hydroxyl or amine groups of the building blocks,
which were used in conjunction with glycosyl trichloro-
acetimidate donors (2 in Scheme 1; 20 and 23 in Scheme 2).
The reaction required for the elongation of the peptide and
glycan chains would involve the repeated deprotection of these
reactive groups, and the sequence of Fmoc-removal reaction
could be achieved under mildly basic conditions without
affecting the acid-labile O-glycosidic bonds.9 It was envisaged
Received: April 19, 2014
Published: May 20, 2014
© 2014 American Chemical Society
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Scheme 1. IL-Supported Synthesis of Oligosaccharides Using
the Fmoc-Protection and Trichloroacetimidate-Based
Glycosylation Strategies
Scheme 2. IL-Supported Stepwise Synthesis of
Glycopeptides
a
a
a
The yields for compounds 5−10 represent the overall yields for the
glycosylation and deprotection reactions (2 steps). Glycosylation
conditions: donor 2 (2.5 equiv), TMSOTf (0.15 equiv), and MS (4
Å) in DCM (a DCM/MeCN mixture was used for the synthesis of 3)
at −20 °C for 1 h. Deprotection conditions: Et3N in DCM at rt for 2 h.
Cleavage of the IL support: TFA and H2O in DCM at rt for 6 h to give
oligosaccharide 11, which was purified by gel filtration chromatog-
raphy (eluent: 1:1 DCM/MeOH). Phase separation procedure:8e Upon
completion of reaction, the mixture was concentrated under vacuum to
give a residue, which was dissolved in a DCM/IPE mixture (1:4 v/v).
The polar DCM was then removed by rotary evaporation, which
allowed for the IL-supported substrate to precipitate from the
nonpolar IPE. The precipitate was collected by filtration and washed
with diethyl ether to yield the product. Abbreviations: TMSOTf,
trimethylsilyl trifluoromethanesulfonate; DCM, dichloromethane; MS,
molecular sieve; rt, room temperature; IPE, isopropyl ether.
a
The product isolation steps were achieved by phase separation, unless
otherwise noted. Reaction conditions: (a) amino acid 12 (2.0 equiv),
EDC·HCl (2.0 equiv), and DMAP (0.2 equiv) in MeCN at rt for 7 h;
(b) piperidine (10.0 equiv) in DCM at rt for 30 min; (c) Fmoc-
protected amino acid (2.0 equiv) or glycosylated serine 15 (1.5 equiv),
HOBt (2.0 equiv), HBTU (2.0 equiv), and DIPEA (2.5 equiv) in
MeCN at rt for 8 h; (d) TFA and H2O in a mixture of DCM and
anisole at rt for 1 h gave glycopeptides 18 and 25, which were isolated
by HPLC; (e) HF·Py in Py at 0 °C to rt for 12 h; (f) donor 20 or 23
(2.5 equiv), TfOH (0.4 equiv), and MS (4 Å) in DCM at −30 °C for 1
h. Abbreviations: EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbo-
diimide; DMAP, 4-dimethylaminopyridine; HOBt, 1-hydroxybenzo-
triazole; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; DIPEA, N,N-diisopropylethylamine; TfOH,
trifluoromethanesulfonic acid; Py, pyridine.
that the use of highly reactive trichloroacetimidates,10 with an
ester protecting group on their C2-amino moiety, would enable
the glycosylation reaction to occur in high yield and in a
stereospecific manner to form the β-isomer exclusively (i.e.,
neighboring group participation).8e,f Second, benzyl ethers and
acetyl esters can be used as permanent protecting groups and
would be compatible with the chemistries required for the
sequential Fmoc-deprotection steps, as well as the glycosylation
and amino acid coupling strategies. Finally, we designed the IL
support 1 bearing a p-alkoxybenzyl alcohol spacer (Supporting
Information Scheme S1) so that it could be tethered to the
growing glycan or peptide chain. This spacer was structurally
very similar to the Wang linker,11 which has been used
extensively in SPPS and is stable under all of the reaction
conditions required during the synthesis of glycopeptides. The
Wang linker can be selectively cleaved with trifluoroacetic acid
(TFA) when needed.
for the trichloroacetimidate-based glycosylation and the Fmoc-
protection strategies by synthesizing a series of β1,6-linked
glucosamine oligomers (compounds 5−11 in Scheme 1).
Structures of this type are very similar to those of the major
components of many microbial surface antigens,12 and
considerable synthetic efforts have been directed toward the
development of strategies capable of providing access to these
biologically important molecules.13 In this study, we used an
“IL-supported glycosyl acceptor” strategy8e,f for the oligosac-
charide synthesis. The IL support 1 was attached to the
reducing end of the growing glycan chain at the anomeric
center via a glycosylation reaction with imidate 2. The
triethylamine-mediated removal of the Fmoc group followed
by the glycosylation of the resulting alcohol with donor 2 in the
presence of the promoter TMSOTf resulted in the formation of
The construction of glycan chains is generally considered to
be technically more challenging than that of peptides. With this
in mind, we focused initial efforts on exploring the
compatibility of the IL support 1 with the conditions required
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the target oligosaccharides 5−10. All of these reactions were
conducted in dichloromethane under homogeneous conditions
and afforded the desired products in good yields without using
a large excess of the donor. Importantly, the IL-supported
reactants were highly soluble in polar solvents, such as
dichloromethane, acetonitrile, and methanol, which allowed
for the progress of these reactions to be readily monitored by
TLC, NMR, and MS analyses. The product purification steps
were performed in accordance with a previously reported phase
separation procedure,8e which was based on the poor solubility
of the IL-bound glycans in nonpolar solvents such as isopropyl
ether and diethyl ether. The resulting intermediates 3−10
exhibited sufficient purity to be progressed directly into the
subsequent reactions. Finally, the IL support was cleaved from
compound 10 with TFA to give the partially protected
oligosaccharide 11 in an overall yield of 29% following gel
filtration purification. The structure of 11 was verified by NMR
that the hexapeptide backbone was monoglycosylated with a β-
linkage. These results therefore confirmed that this method
allowed for the successful construction of a glycopeptide with a
shorter glycan branch via the incorporation of a glycosylated
amino acid to the IL-supported stepwise peptide assembly
process.
To demonstrate the utility of our IL-based method for the
construction of more complex glycopeptides bearing multiple
sugar units, we conducted two consecutive glycosylation
reactions using the IL-bound glycopeptide acceptor 19 with
the glycosyl donors 20 and 23. Glycopeptide 19 was obtained
from 17 following the selective cleavage of the TBS group by
treatment with a HF·pyridine complex. The N-2,2,2-trichloro-
ethoxycarbonyl (Troc)-protected imidates 20 and 23 were
selected as the donors instead of the N-phthaloyl (Phth)-
protected imidate 2, because the Troc group is more amenable
to the conditions required for the glycopeptide synthesis than
the Phth group.15 The TBS group on the C6 hydroxyl of donor
20, which is orthogonal to the Fmoc group used to protect the
amines, was employed for temporary protection that could be
removed as necessary to allow for the introduction of additional
sugar units at this position. Both glycosylation reactions
proceeded smoothly in the presence of a TfOH promoter to
afford the desired glycopeptide 25 in 16% yield, following the
removal of the IL support and purification of the cleavage
product by HPLC. The 13C NMR spectrum of 25 (125 MHz,
in DMSO-d6) contained three unique anomeric C signals (δ
around 100 ppm) and three CCl3 peaks characteristic of the
Troc group (δ around 96 ppm), confirming that the
trisaccharide side chain had been successfully grafted onto the
peptide backbone. HPLC analysis indicated that there had been
no discernible isomerization of the product during the
glycosylations and the IL-support removal process. This
convenient and high-yielding synthesis of 25 on an IL support
demonstrates that our method is highly effective for
glycopeptide construction.
Convergent synthesis represents an efficient way by which to
generate peptides with a repetitive sequence and is generally
employed in SPPS for the construction of glycopeptides.16 To
determine whether our IL-based method was compatible with
this strategy, we investigated the block coupling of the
glycopeptide segments 27 and 28 using the method (Scheme
3). The monoglycosylated tripeptide 26, which was synthesized
as an intermediate in the construction of glycopeptide 18, was
used as a precursor to produce 27 and 28. Cleavage of the IL
support from 26 exposed the carbonyl group of the peptide
backbone (27), whereas removal of the Fmoc-protecting group
revealed the amino terminus (28). The block coupling of the
two precursors in the presence of a PyBOP activator afforded
glycopeptide 30 in 29% yield following the removal of the IL
support and purification of the crude product by HPLC (the
HBTU−HOBt activating conditions were ineffective for this
block coupling reaction because they led to the formation of a
guanidine side product17). The NMR and HR-ESIMS data for
30 corresponded well with the proposed bis-glycosylated
hexapeptide structure. This synthesis therefore verified the
feasibility of synthesizing glycopeptides by segment coupling on
an IL support.
1
spectroscopies (1H, 13C, COSY, and HSQC). The H NMR
spectrum of 11 (400 MHz, in CDCl3) showed characteristic
signals for the anomeric protons (δ = 5.5−4.9 ppm) with
coupling constants around 8.0 Hz, clearly indicating that 11
contained seven monomeric glycosyl units that were linked to
each other with β-configurations. HR-MALDI-TOF-MS
analysis of 11 provided further confirmation of the heptaglucos-
amine structure. Collectively, these results demonstrate that
relatively large and complex oligosaccharides can be efficiently
constructed using our IL-based method.
Although several IL-based approaches have been developed
for the construction of oligosaccharides,8 the glycan-assembly
strategy outlined above for the IL-supported synthesis of
glycopeptides warranted further investigation. To highlight the
potential of this particular strategy, we applied it to synthesize
the glycopeptide fragment of the serum response factor14 (18
in Scheme 2) as well as its glycosylated products 21 and 25.
Use of the Fmoc protocol was particularly preferable for these
IL-supported glycopeptide constructions, because the corre-
sponding tert-butoxycarbonyl (Boc) protocol7a,b requires the
use of strongly acidic conditions (TFA) for the recurring
cleavage of the Boc groups, and these conditions could have an
adverse effect on the acid-labile O-glycosidic bond and the
Wang linker. The esterification of the Fmoc-protected alanine
12 with the IL support 1 afforded the IL-bound amino acid 13,
which was used as a starting material for the glycopeptide
synthesis. In addition to the preprepared glycosylated serine 15
(Scheme S3), several commercially available amino acid
building blocks (12, 14, and 16) were incorporated in the
reaction cycle and assembled step by step using piperidine, for
the cleavage of the Fmoc-protecting groups, and a mixture of
HBTU and HOBt, as the coupling activator. The homogeneous
liquid-phase conditions allowed for these deprotection and
coupling reactions (each amino acid was added in only minor
excess) to be accomplished in good to excellent yields. Similar
to the IL-supported glycan synthesis, purification by phase
separation afforded the intermediates in high purity, and these
compounds were subsequently characterized by NMR and MS
analyses. Furthermore, HPLC analysis revealed that no
detectable racemization or epimerization had occurred during
the synthetic process. The desired glycopeptide 18 was
obtained in 31% yield following the removal of the IL support
from 17 by TFA. This acidic condition also resulted in the
concomitant cleavage of the tert-butyldimethylsilyl (TBS)
protecting group from the C6 hydroxyl of the glucosamine
residue. The NMR and HR-ESI-MS analyses of 18 suggested
In summary, we have developed a new approach for the
construction of glycopeptides, based on the IL-supported
synthesis methodology. The use of an IL support bearing a
Wang linker, combined with the glycosyl trichloroacetimidate
donor and Fmoc-protection strategies, facilitated the synthesis
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Scheme 3. IL-Supported Convergent Synthesis of a
Glycopeptide
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: lixb@im.ac.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the Ministry of Science
and Technology, China (973 Program, 2012CB518803) and
the National Natural Science Foundation, China (Grant
Number 81273381).
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