Automated glycopeptide assembly by combined solid-phase peptide and oligosaccharide synthesis

Article abstract of DOI:10.1039/c3cc48761j

Current strategies for the synthesis of glycopeptides require multiple manual synthetic steps. Here, we describe a synthesis concept that merges solid phase peptide and oligosaccharide syntheses and can be executed automatically using a single instrument.

Full text of DOI:10.1039/c3cc48761j

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Automated glycopeptide assembly by
combined solid-phase peptide and
oligosaccharide synthesis†
Mattan Hurevicha and Peter H. Seeberger*ab
Cite this: Chem. Commun., 2014,
50, 1851
Received 16th November 2013,
Accepted 11th December 2013
DOI: 10.1039/c3cc48761j
www.rsc.org/chemcomm
Current strategies for the synthesis of glycopeptides require multiple
manual synthetic steps. Here, we describe a synthesis concept that
merges solid phase peptide and oligosaccharide syntheses and can
be executed automatically using a single instrument.
Most human proteins are glycosylated. The glycan structure as well
as the exact location and linkage to the protein influence the
glycoprotein function. Establishing structure–activity relationships
of glycoproteins is difficult since these molecules are typically
obtained as heterogeneous mixtures of glycoforms. Contrary,
synthetic glycopeptides are homogeneous and can serve to
elucidate the biological significance of specific glycoprotein
fragment.1 In addition, synthetic glycopeptides have been
explored as diagnostic tools and vaccine candidates.1d,2
Fig. 1 Strategies for glycopeptide synthesis. Routes A and B utilize gly-
cosylated amino acids as building blocks for the assembly of glycopeptides
by SPPS. Route C requires only monomeric building blocks for automated
assembly of glycopeptides by SPPS and SPOS.
Glycopeptide synthesis has been extensively developed in recent
years.3 O-Glycopeptides are usually synthesized via two main routes
based on early work by Kaifu and Osawa.4 One strategy incorporates
glycosylated amino acid building blocks in solid phase peptide Moreover, glycosylated amino acids may epimerize during SPPS
synthesis (SPPS) (Fig. 1A).1b,c,5 Alternatively, a shortened glycan is and the purity of the synthesized glycopeptide needs to be
grafted onto the peptide backbone and after completion of peptide ascertained.7 Consequently, a method that minimizes the
synthesis, the glycan is further elongated by chemical or chemo- synthetic effort involved in glycopeptide synthesis is sought.
enzymatic methods (Fig. 1B).6 In both strategies, glycosylated amino An alternative synthetic route based on oligosaccharide assembly
acids are required for glycopeptide assembly by SPPS. The synthesis on a solid supported peptide platform was outlined but was not
of glycosylated amino acids is time consuming, as it typically applied in automated practice.8 This route is attractive as it may
requires multistep solution phase procedures. Most building blocks circumvent the use of glycosylated amino acids.
are not commercially available or extremely expensive to purchase in
Recent advances in automated solid phase oligosaccharide
large quantities. Lack of accessibility limits the use of these building synthesis (SPOS)9 set the stage to combining both solid phase
blocks in solid phase glycopeptide synthesis wherein large peptide and oligosaccharide assembly platforms to prepare glyco-
amounts of building blocks are used for each coupling step. peptides. Here, we describe an automated, stepwise solid-phase
approach to glycopeptide synthesis that relies exclusively on mono-
saccharide and amino acid building blocks (Fig. 1C).
a Max-Planck-Institute of Colloids and Interfaces, Department of Biomolecular
The choice of a linker to tether the growing glycopeptide to
the solid support is the crucial strategic decision for syntheses
that require a multitude of protective groups. Photo-cleavable
linkers are compatible with a variety of functional groups and
were used for previous automated solid phase glycopeptide
syntheses.10 By using a photochemical flow reactor, inherent
problems with cleavage efficiencies have been overcome.11
¨
Systems, Am Muhlenberg 1, 14476 Potsdam, Germany.
E-mail: peter.seeberger@mpikg.mpg.de
b Freie Universitat Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,
14195 Berlin, Germany
† Electronic supplementary information (ESI) available: Detailed description of
building block synthesis and characterization, automated synthesis protocols and
modules, HPLC characterization of intermediates and NMR, HPLC and MS
analysis of the final compound. See DOI: 10.1039/c3cc48761j
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Fig. 2 Automated glycopeptide synthesis workflow. (I) Solid phase peptide
synthesis. (II) Selective hydroxyl deprotection. (III) Solid phase oligosaccharide
synthesis. (IV) Amine deprotection. (V) Photo-cleavage. PG1 = tBu or Trt,
R = amino acid side chain (protected), LG = SEt, OC(NPh)CF3, R1 = Bn, Ac,
Lev, Bz, benzylidene.
Since the automated assembly of oligosaccharides is instrumen-
tally more demanding than the more mature peptide assembly, a
recently developed oligosaccharide synthesizer that also accommo-
dates SPPS was used for the synthesis (see ESI†).9c
The general workflow (Fig. 2) relies on four distinct phases
for iterative step by step automated glycopeptide synthesis.
Attachment of a Fmoc-protected amino acid on the amino-
Fig. 3 Glycosylating agents 1–4 were used for the automated synthesis
of glycopeptides 5–7.
functionalized solid support-bound linker was followed by standard synthesis of peptides carrying multiple glycosylation sites. In
peptide elongation using HBTU as an activator and piperidine as a the final step of the synthesis, the glycopeptide was removed
deprotection reagent (Fig. 2, I). Amino acid side chain functional from the solid support by irradiation.11
groups were permanently masked as benzyl ethers while either tert-
Three glycopeptides were prepared to illustrate the new
butyl (t-Bu) or trityl (Trt) ethers were used as temporary protecting automated synthesis approach (Fig. 3). Monosaccharide building
groups. The amino terminus of the peptide remained protected as blocks 1–4 that carry different protecting groups as well as
an Fmoc carbamate during glycosylation.
anomeric leaving groups were utilized to demonstrate the
Removal of the temporary t-Bu or Trt protecting groups from variability of the approach. Commercially available amino acids
threonine or serine was achieved under mild acidic conditions were used without any modifications. The target glycopeptides
using catalytic amounts of trimethylsilyl trifluoromethanesulfonate were selected to challenge our approach in different ways.
(TMSOTf) (Fig. 2, II). TMSOTf is also employed to activate glycosyl Glycopeptide 5 requires formation of a 1,2-cis-glycosidic linkage
trichloroacetimidates and N-phenyl trifluoroacetimidates in the on a peptide backbone. Glycopeptide 6 requires elongation of
oligosaccharide synthesizer.9c
With a solid support bound peptide carrying a free side chain multiple side chain glycans (Fig. 3).
the glycan side chain while peptide 7 requires incorporation of
hydroxyl group in hand, an automated glycosylation sequence
To install the cis-glycosidic linkage between the threonine/
was initiated to place the desired glycan portion on the peptide serine hydroxyl group and the GalNAc moiety on glycopeptide
(Fig. 2, III). Elongation of the peptide was simply possible by 5, a-selective glycosylating agent 1 was used.12 After coupling
Fmoc removal from the amino terminus and standard SPPS Fmoc-Thr-(t-Bu)-OH to the solid support, the t-Bu group was
(Fig. 2, IV). Repeating steps I–IV allows for the automated removed using TMSOTf. The free hydroxyl group was glycosylated
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with 1 in the presence of TMSOTf. Cleavage from the resin and Research Council (ERC Advanced Grant AUTOHEPARIN to P.H.S.)
HPLC analysis revealed that the glycosylation reaction had The authors would like to thank Dr Mark Schlegel and
proceeded with good selectivity (4: 1, major a). The a-anomeric Dr Claney Lebev Pereira for the fruitful discussion.
configuration of 5 was confirmed by comparison of the NMR
spectra of 5 with that of Fmoc-Thr(Ac3GalN3)-Ot-Bu.12b
After establishing that automated peptide glycosylation is
possible, glycopeptide 6 bearing a (Galb1-3GalNAcb-O) disaccharide
Notes and references
1 (a) J. R. Allen, C. R. Harris and S. J. Danishefsky, J. Am. Chem. Soc.,
was prepared. Commercially available amino acids were used along
with glycosylating agents 2 (prepared in three steps from a known
intermediate)13 and 3.14 The tetrapeptide backbone of 6 was
assembled using SPPS. Selective removal of the t-Bu group was
followed by glycosylation with glycosyl imidate 2 and TMSOTf
as an activator at À10 1C. The glycosylation reaction proceeded
with high conversion and yielded one major product. Removal
of the levulinoyl protecting group was followed by formation of
a second glycosidic linkage using galactosyl thioglycoside 3
activated by N-iodosuccinimide (NIS) and triflic acid (TfOH).
Progress of the automated synthesis of 6 was monitored by
cleaving small amounts of material from the solid support at
different stages of the synthesis followed by HPLC-MS (see ESI†).
Deprotection of the galactosamine by reduction of the azide
using AcSH,15 and benzylidene removal using TFA were carried
out manually after completion of the automated synthesis.
Finally, glycopeptide 6 was cleaved from the resin under UV
irradiation. The desired product 6 was obtained in 5.7%
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1
isolated yield after HPLC purification. H-NMR coupling con-
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b-linkages were formed.
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were employed twice. Placement of threonine on the solid
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4 was attached using the NIS–TfOH activator mixture. Fmoc
removal and repetition of the assembly cycle gave glycopeptide
7 after light-induced cleavage from the solid support in 14%
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We gratefully thank the Max Planck Society, the Minerva Stiftung
(post-doctoral Minerva fellowship to M.H.) and the European
52, 5862.
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Chem. Commun., 2014, 50, 1851--1853 | 1853