Oxo-ester mediated native chemical ligation on microarrays: An efficient and chemoselective coupling methodology

Article abstract of DOI:10.1039/c2cc30844d

We report a highly efficient and selective method for the coupling of peptides and glycoconjugates bearing N-terminal cysteines to activated surfaces. This chemoselective method generates stable amide linkages without using any thiol additives.

Full text of DOI:10.1039/c2cc30844d

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Oxo-ester mediated native chemical ligation on microarrays: an efficient
and chemoselective coupling methodologyw
a
a
Martin J. Weissenborn,z Roberto Castangia,z Johannes W. Wehner,b Robert Sardzı
´
k,a
Thisbe K. Lindhorst*b and Sabine L. Flitsch*a
Received 6th February 2012, Accepted 19th March 2012
DOI: 10.1039/c2cc30844d
We report a highly efficient and selective method for the coupling
of peptides and glycoconjugates bearing N-terminal cysteines to
activated surfaces. This chemoselective method generates stable
amide linkages without using any thiol additives.
Fig. 1 The native chemical ligation (NCL) performed on thioester-
Peptide and protein microarrays are well established in the
field of proteomics and provide platform technologies for drug
discovery and clinical diagnosis.1 Such arrays are mostly
generated by covalent coupling of pre-prepared ligands to
solid surfaces.2 The conjugation chemistry needs to be highly
chemoselective, site-specific and efficient to ensure correct
presentation of the ligand on the surface. Next to rather classical
chemical acylation methods, more specific bioorthogonal coupling
methods3 have been developed such as Staudinger ligation,4 ‘‘click
chemistry’’,5 and native chemical ligation (NCL).6,7
functionalised surfaces.
Fig. 2 The chemoselective coupling of Cys derivatives on PFP-
activated gold plates is a powerful method for oxo-ester mediated
NCL on surfaces.
NCL is particularly attractive as it can be carried out under
physiological conditions.8 NCL generally involves the reaction
of a thioester and a cysteine (Cys) derivative, such as an
N-terminal Cys-peptide. If the NCL is performed on solid
phase, either the Cys- or the thioester-peptide needs to be
immobilised.6,9 Starting with immobilised Cys requires thioester-
peptides, which are incompatible with Fmoc protecting groups in
solid phase peptide synthesis.10 The reverse coupling method
proceeds from thioester-functionalised surfaces 1 by coupling of
the Cys derivative via a reversible transthioesterification to give
thioester 2 and the native peptide 3 after irreversible rearrangement
(Fig. 1). The formation of 1 has been shown first by Lesaicherre
et al.11 albeit in only 1/10 of monoloyer coverage.8
selectivity of cysteines over other amines could be retained in
the coupling reaction.
All our experiments were conducted on gold arrays covered
with self-assembled monolayers (SAMs) formed by alkane
thiols,14 which are omega-functionalised with hexaethylene
glycol (OEG6) carboxylic acid, thus providing points for
chemical modification (4, Fig. 3). Any reaction on this surface
can be monitored directly and label-free using matrix-assisted
laser desorption/ionisation-time-of-flight mass spectrometry
(MALDI-ToF MS).15
In the first instance, a range of activated oxo-esters such as
para-nitrophenyl (pNP), N-hydroxysuccinimidoyl (NHS) and
2,3,4,5,6-pentafluorophenyl (PFP) esters were generated from
carboxy-functionalised surfaces and were reacted with Cys.
Analysis of the reactions by MALDI-ToF mass spectrometry
suggested that the coupling reactions were most efficient using
PFP activation and this activation method was therefore
chosen for subsequent studies.
As an alternative, we have therefore investigated NCL
coupling protocols starting from readily available activated
oxo-ester 5 on surfaces (Fig. 2) instead of using thioester
surface 1.12,13 A concern using such oxo-esters would be higher
reactivity and it was therefore important to investigate if
a School of Chemistry & Manchester Interdisciplinary Biocentre,
The University of Manchester, 131 Princess Street,
The coupling of Cys itself was then further investigated for
efficiency of coupling and selectivity over other amino acids.16,17
The coupling was tested at different Cys concentrations using
the standard NCL conditions (7.5 eq. tris(2-carboxyethyl)-
phosphine hydrochloride (TCEP), 5 M guanidine buffer, 75 mM
Na2HPO4, pH 7)18 and it was found that coupling was
observed even at a concentration of 2 mM of Cys, which
compared favourably to the 50 mM previously necessary for
Manchester M17DN, UK. E-mail: sabine.flitsch@manchester.ac.uk;
Fax: +44-(0)161-275-1311; Tel: +44-(0)161-306-5172
b Otto Diels Institute of Organic Chemistry, Christiana Albertina
University of Kiel, Otto-Hahn-Platz 3/4, 24098 Kiel, Germany.
E-mail: tklind@oc.uni-kiel.de; Fax: +49-431-880-7410
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc30844d
z These authors contributed equally to this work.
c
4444 Chem. Commun., 2012, 48, 4444–4446
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Table 1 Competitive assay employing N-Cys and N-amino peptides
and glycoamino acids
Fig. 3 (a) Coupling experiments with 4-mercaptophenylacetic acid
(MPAA). (b) MALDI-ToF MS analysis of 7. (c) MALDI-ToF MS
analysis of 7.
peptide coupling. By addition of imidazole12,17 we were able to
further lower the Cys concentration necessary to 0.25 mM.
Thiophenol and 4-mercaptophenylacetic acid (MPAA) were
less effective.
With a robust coupling protocol in hand, we investigated
the selectivity of Cys coupling over other amino acids. Inter-
estingly, when the activated surfaces 5 were reacted with
glycine (Gly) or phenylalanine (Phe) in the presence of thiol
MPAA as additive, only thioester 8 was formed and no
conversion to the respective amide was observed (Fig. 3).
The same reaction with Cys and MPAA, however, showed
the Cys amide 7 as the sole product. These experiments suggest
that on the surface O- to S-transesterification (5 to 8) is fast.
The thioester 8 consequently undergoes a reaction with Cys via
transthioesterification which rearranges to the amide 7. This
difference in acylation rates was also observed in competition
experiments. When Gly, Phe and Cys were added to the
reaction mixture in equimolar amount in the presence of
MPAA only the Cys-product 7 was formed.
2 mM each on PFP activated SAMs. The SPR slide was
subsequently interrogated with the mannose binding lectin
concanavalin A (ConA) followed by the N-acetylglucosamine
(GlcNAc) binding lectin wheat germ agglutinin (WGA).19
There was strong ConA and no WGA binding observed which
showed that exclusively the Cys-Man 13 had coupled to the
surface, but not 14.
After these promising results with small amino acids and
peptides, the coupling method was tested on more complex
bioconjugates. Of particular interest was the coupling of
glycoconjugates, which are used in glycoarrays20 and which
can often only be obtained in small quantities. Efficient
coupling strategies are therefore of great importance to the
field. The cysteine derivatives such as 13 are easily obtained
from aminoethyl glycosides 15. In competition with 15,
cysteine derivative 13 was exclusively coupled to surfaces
giving the product 26 as shown in MALDI-ToF MS analysis.
To test this coupling strategy further, we investigated
multivalent glycoconjugates, which have become important
tools to probe multivalent binding events on cell surfaces.21 The
required trivalent glycoclusters22 were synthesised as shown in
Scheme 1. Trivalent glycoclusters 21 and 23 were synthesized
Interestingly, similar selectivities were observed in the
presence of imidazole (instead of the thiol MPAA).12 Competitive
studies with mixtures of b-alanine (b-Ala), Phe, Gly and Cys
(1 mM each) also showed exclusively the Cys product even at
1 : 25 ratio of Cys : Gly (Fig. S17, ESIw). All subsequent reactions
were therefore performed with imidazole as additive.
Similar selectivities were observed with longer peptides
containing either cysteine or glycine at their N-termini
(Table 1). When mixtures of peptides 9 and 10, 11 and 10,
and 11 and 12, respectively, were applied to the surface, only
the products 24 or 25 were observed.
As an alternative and more sensitive method of analysis,
surface plasmon resonance (SPR) was employed. 13 and 14
were applied in a competitive assay at a concentration of
c
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Scheme 1 Synthesis of trivalent glycoclusters 21 and 23.
starting from the known building block 17.23 For the preparation
of the trivalent wedge 19 Fmoc-glycine (16) was coupled in
a HBTU/DIPEA-mediated reaction with 17 followed by
deprotection of the tBu esters with formic acid yielding
compound 19 in 85% over two steps. Mannosides were
introduced via peptide coupling following published chemistry24
giving 20 in a satisfying yield of 64%. Fmoc deprotection with
piperidine in DMF gave the trivalent cluster mannoside 21 in
89% yield. An amount of 21 was further functionalized with
Boc-L-Cys(Trt)-OH in another peptide coupling reaction yielding
22 in 58%. This step was followed by one step removal of the
Boc and Trityl protecting groups using TFA in DCM under
addition of TES followed by a treatment with basic ion exchange
resin Amberlyst A-21 following a literature protocol.25 After
size exclusion and reversed phase chromatography the Cys-
terminated ligand 23 was obtained in 65% yield.
The trivalent cluster mannosides manno-Cys 23 and manno-
Gly 21 were then compared in coupling experiments to the
gold array surfaces. From reaction mixtures containing 4 mM
each of 21 and 23 the manno-Cys product 27 was formed
exclusively (Table 1).
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Complex bioconjugates containing N-terminal cysteines can
be efficiently coupled to surfaces through amide bond formation
via native chemical ligation starting with activated oxo-esters on
the surface. These surface oxo-esters are easily prepared from
their carboxylic acids. Our studies have shown that the ligation in
the presence of imidazole is highly selective for cysteine derivatives
over competing amines.
This work was supported by the Royal Society (Wolfson
Award to SLF), the European Commission (MJW), the
EPSRC and the Evonik Foundation (JWW).
22 H. A. Shaikh, F. D. Sonnichsen and T. K. Lindhorst, Carbohydr.
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Notes and references
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4446 Chem. Commun., 2012, 48, 4444–4446
This journal is The Royal Society of Chemistry 2012