Solid phase synthesis of glycopeptides using Shoda's activation of unprotected carbohydrates

Article abstract of DOI:10.1039/c3cc43458c

An expedient and simple protocol to access S-linked glycopeptides by Fmoc SPPS using unprotected carbohydrates is reported. The utility of the method was demonstrated with the solid phase synthesis of a MUC1 fragment (20 mer) containing two glycosylation sites that were substituted with S-linked glycans. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3cc43458c

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Solid phase synthesis of glycopeptides using Shoda’s
activation of unprotected carbohydrates†
Alexandre Novoa,a Sofia Barluenga,ab Christelle Serbaab and Nicolas Winssinger*ab
Cite this: Chem. Commun., 2013,
49, 7608
Received 9th May 2013,
Accepted 26th June 2013
DOI: 10.1039/c3cc43458c
www.rsc.org/chemcomm
An expedient and simple protocol to access S-linked glycopeptides by 1,3-dimethylimidazolinium chloride) to obtain glycosyl azides and
Fmoc SPPS using unprotected carbohydrates is reported. The utility of pyridyl thioglycosides.12 The simplicity of this reaction coupled to the
the method was demonstrated with the solid phase synthesis of a expanding range of commercially available and biologically relevant
MUC1 fragment (20 mer) containing two glycosylation sites that were oligosaccharides prompted us to investigate this reaction for the
substituted with S-linked glycans.
synthesis of S-linked glycopeptides. However, preliminary experiments
with benzyl mercaptan or N-acetylcysteine methyl ester in lieu of the
Protein glycosylation plays a pivotal role in numerous biological 2-mercaptopyridine under the reported conditions (glycan (1.0 eq.);
functions,1 and there is a genuine need for robust methods to access DMC (>3.0 eq.); PySH (>5.0 eq.); H2O/MeCN) did not afford the
homogeneous glycopeptides.2 A strategy that has attracted signifi- desired glycosylation product. We reasoned that the failure of the
cant attention is the substitution of the anomeric oxygen in O-linked latter reactions was due to the higher basicity of these thiols compared
glycosides by sulfur.3,4 These S-linked glycopeptides are known to be to 2-mercaptopyridine resulting in competition with the glycan for
tolerated as functional surrogates and are more stable chemically as the nucleophilic addition to the DMC activator. While Shoda and
well as biologically (less prone to glycosidase cleavages5) than their co-workers always used the glycan as the limiting reactant, glycosylation
O-linked counterparts. A natural example of a S-linked glycopeptide in solid phase would require the thiol to be the limiting component.
was recently identified bringing cysteine into the fold of naturally Based on a possible competing reaction of the thiol for DMC, it
glycosylated residues.6 Furthermore, this specific S-linked glycoside appeared essential to pre-activate the glycan prior to its addition to
was found to be essential for the antimicrobial activity of the the polymer-bound thiol for glycosylation. We thus set out to gain
identified glycopeptide. From a synthetic perspective, the exceptional information on the kinetics of glycoside activation and its hydrolysis to
nucleophilicity of thiols offers unique opportunities to construct the identify a window of opportunity wherein the DMC would be fully
strategic bond between the glycan and the peptide.3 Most strategies consumed but a significant concentration of the activated glycan would
to access S-linked glycopeptides have been leveraged on conjugate remain available. Using glucose and thiophenol as starting materials,
addition or nucleophilic displacement reactions with 1-thioglycans.7 the reaction was monitored by 13C NMR to gain a qualitative insight
More recently, several alternative approaches based on free radical into the reaction progress.
thiol–ene coupling,8 desulfurative rearrangement,9 Ferrier reaction10
Performing the reaction in D2O–CD3CN (1/1) with glucose
and opening of 1,6-anhydrosugars11 have also been used. While (1.0 eq.), DMC (1.0 eq.) and Et3N (3.0 eq.) at room temperature
these methods have provided robust access to S-linked glycopeptides resulted in the formation of the DMC-activated product (2, Fig. 1) that
and their precursors, they typically involve multistep synthesis in disappeared within 15 min indicating a short half-life for the desired
order to prepare the required glycoside or amino acid components. activated intermediate under these conditions. The same reaction in
Herein we report a method using Shoda’s glycosylation with readily the presence of PhSH (1 eq.) showed a rapid disappearance of the
available unprotected (oligo)saccharides, which is compatible with PhSH signal that reappeared after 24 h without notable glycosylation
SPPS using the Fmoc strategy.
(3a) concurring speculation that thiols react with DMC to form an
Recently Shoda and co-workers reported the remarkable activation intermediate that decomposed slowly over the course of the reaction.
of unprotected glycosides in aqueous solutions with DMC (2-chloro- While this addition may be reversible for more acidic nucleophiles,
more basic thiols are sequestered from the reaction mixture. Cooling
a
´
´
Institut de Science et Ingenierie Supramoleculaires (ISIS – UMR 7006),
the reaction in the absence of thiol to À15 1C preserved the DMC-
activated intermediate, however, a complete conversion was not
possible due to competing hydrolysis. Performing the reaction by
pre-activation at À15 1C using 3.0 eq. of DMC and 9.0 eq. of Et3N for
40 min followed by the addition of thiol afforded the desired product
´
´
Universite de Strasbourg – CNRS, 8 allee Gaspard Monge, F67000 Strasbourg, France
b Department of Organic Chemistry, University of Geneva, 30 quai Ernest Ansermet,
CH-1211 Geneva 4, Switzerland. E-mail: Nicolas.Winssinger@unige.ch
† Electronic supplementary information (ESI) available: Synthetic procedures and
physical characterization of products. See DOI: 10.1039/c3cc43458c
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Table 1 Solid phase S-glycosylation of a tripeptide
Conversion
(with 3 iterations) configuration 9a–h
Anomeric
Entry Saccharide
1
D-Glucose (without 7-100%
preactivation)
D-Glucose
2
3
4
5
6
7
8
9
8a-95% (>99%)
8b-45% (>99%)
8c-30% (>99%)
8d-50% (>99%)
8e-75% (>99%)
8f-65% (>99%)
8g-55% (>99%)
8g-60% (>99%)
b
b
a
b
b
b
b
b
D-Galactose
D-Mannose
L-Fucose
Fig. 1 Thioglycosylation followed by 13C NMR. (A) 13C NMR of D-glucose recorded in
1,4-dioxane-D8–D2O (1/1) at À10 1C; (B) addition of DMC (3.0 eq.) and Et3N (9.0 eq.),
recorded after 20 min of activation; (C) addition of PhSH (1.0 eq.), recorded after
2 h of reaction.
Gal-b-1,4-Glc
Gal-b-1,4-Glc
Gal-a-1,6-Glc
Gal-a-1,4-Glc
with >50% yield. Notably, these conditions also worked with benzyl
mercaptan (3b) and N-acetylcysteine methyl ester (3c). Encouraged by
the fact that pre-activation of the glycan provided a solution to the
glycosylation of more basic thiols, we screened different solvent
combinations (H2O–ethylene glycol; H2O–1,4-dioxane; H2O–DMSO;
H2O–DMF) and different bases (Et3N, iPr2EtN, 2,6-lutidine, DBU). The
best results were obtained with H2O–1,4-dioxane at À10 1C. Using
3.0 eq. of DMC and 9.0 eq. of Et3N, complete consumption of DMC
was observed after 30 min affording >80% of the activated glucose (2)
(Fig. 1, the decrease in the signal to noise ratio following addition of
excess reagents precluded a more quantitative analysis). Under these
conditions, the hydrolysis half-life of 2 was >100 min suggesting there
was a clear window of opportunity to add this activated sugar to a
polymer-bound thiol in order to achieve glycosylation of a polymer-
bound substrate. Gratifyingly, performing the reaction on resin 4
(Scheme 1) with 30 min pre-activation at À10 1C led to desired
conversion based on LC/MS analysis of the reaction.13
We next turned our attention to the glycosylation of cysteine in a
model tripeptide (Fmoc-Cys-Phe-Phe) with different mono and disac-
charides (Table 1). Using the previously identified conditions but
without pre-activation (direct addition of all reagents) led exclusively
to the side-product arising from the reaction of cysteine with DMC
(7, entry 1). However, the same conditions using 30 min pre-activation
afforded >90% conversion to the desired glycosylation product 8
(entry 2). Reiteration of the reaction for a second cycle yielded a
complete conversion to the desired product. All monosaccharides
(Gal, Man, Fuc) performed well in the glycosylation following two or
three iterations (entries 3–5). Prolonging the glycosylation from 1 h to
4 h resulted in a small improvement in yield suggesting there is still
an active glycosyl donor in all cases after 1 h, however, it was overall
more efficient and faster to reiterate the reaction for 1 h cycles. Glyco-
sylation with disaccharides was also found to be efficient (entries 6–9)
affording complete conversion after two or three iterative cycles of
glycosylation. Performing the reaction with iPr2EtN rather than with
Et3N afforded exclusively the side product 7. In order to facilitate
1
characterization of the compounds by H NMR, the products were
acylated and the Fmoc exchanged for an acetate (9). In agreement with
Shoda and co-workers’s previous studies,12 b-glycosylation was obtained
as the major product in all cases except when using mannose.
To assess the utility of this procedure in the synthesis of more
complex glycopeptides, we next investigated the synthesis of mucin
(MUC1) analogs. Overexpression of MUC1 is often associated with
many forms of cancer.14 MUC1 has an extracellular domain that
includes O-glycosylated 20 amino acids (HGVTSAPDTRPAPGSTAPPA).
The role of this glycoprotein in cancer has instigated synthetic efforts
on the truncated version of the protein, in particular, its extracellular
domain.15 Standard Fmoc peptide synthesis was used to construct
the peptide using Mmt-protected cysteine at the position of the
glycosylated threonine of the native glycopeptide. The glycans
were introduced either sequentially after the introduction of each
cysteine or simultaneously following complete peptide synthesis
(Scheme 2). Deprotection of the Mmt to unmask the thiol was
achieved under mild acidic conditions (2% TFA in CH2Cl2 with 3%
Et3SiH) and glycosylation with either glucose or lactose was achieved
according to the optimized protocol with three glycosylation itera-
tions. For sequential glycosylation, the product was then acetylated
(Ac2O, pyridine, cat. DMAP) and the peptide synthesis continued until
the next cysteine. Reiteration of the Mmt deprotection–glycosylation
and acetylation allowed the introduction of a second carbohydrate
before completion of the peptide synthesis. This chemistry was used
Scheme 1 Solid phase S-glycosylation.
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Scheme 2 Glycosylation of the MUC1-like fragment.
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to prepare four permutations of MUC1 glycosylation (13–16).
For uniform glycosylation, the peptide can be synthesized and
glycosylated at multiple positions at the end of the peptide
synthesis. This approach, while more convergent, is restricted to
glycosylation with the same carbohydrate at all positions (com-
pounds 17 and 18). In all cases, the major product obtained from
crude cleavage was the desired product (see the inset in Scheme 2).
The acetylated glycans were conveniently deprotected using
ammonia in methanol.
In conclusion, a simple and expedient protocol to access
S-linked glycopeptides was developed. It was found to be essential to
pre-activate the carbohydrate with DMC (Shoda activation) in order
to avoid capping of the polymer-bound thiol with DMC. Optimiza-
tion of the pre-activation enabled a high yield in the glycosylation in
solid phase with a variety of mono and disaccharides. The procedure
is compatible with standard Fmoc-based SPPS and unprotected
sugars. The fact that no manipulation of the sugar is required in
the synthesis of this important class of glycopeptide should
greatly facilitate accessibility to non-specialists. The synthesis of
four different permutations of a doubly glycosylated 20 mer MUC1
fragment illustrates the ease and efficiency of this protocol.
This work was supported by a grant from the European
Research Council (ERC 201749).
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