Direct chemoselective synthesis of glyconanoparticles from unprotected reducing glycans and glycopeptide aldehydes

Article abstract of DOI:10.1039/b911676a

Chemoselective oxime coupling was used for facile conjugation of unprotected, reducing glycans and glycopeptide aldehydes with core?shell gold nanoparticles carrying reactive aminooxy groups on the organic shell.

Full text of DOI:10.1039/b911676a

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Direct chemoselective synthesis of glyconanoparticles from unprotected
reducing glycans and glycopeptide aldehydesw
Mikkel B. Thygesen, Kasper K. Sørensen, Emiliano Clo and Knud J. Jensen*
Received (in Cambridge, UK) 16th June 2009, Accepted 21st September 2009
First published as an Advance Article on the web 1st October 2009
DOI: 10.1039/b911676a
Chemoselective oxime coupling was used for facile conjugation
of unprotected, reducing glycans and glycopeptide aldehydes
with core–shell gold nanoparticles carrying reactive aminooxy
groups on the organic shell.
Covalently bioconjugated nanoparticles, e.g. gold nano-
particles (AuNPs), are emerging as tools for detection and
modulation of specific interactions between biomolecules.
Such systems have been utilized in applications involving
e.g. oligonucleotide–oligonucleotide,1 peptide–protein,2,3
glycan–protein,4,5 glycan–glycan,6 and antibody–antigen7
interactions. Glyconanoparticles are a particularly attractive
platform due to the inherent multivalent presentation required
for most glycan binding events coupled with facile detection.8
Scheme 1 Strategy for nanoparticle functionalization and detection
Here, a protective shell, typically consisting of a monolayer of
of protein–glycan interactions. Pg = trimellitoyl (Trim).
hydrophilic oligomers such as oligo(ethylene glycol) (OEG), is
important for optimally presenting the glycan ligands as well
functionalized AuNPs from aggregating in aqueous solution
and (ii) allow quantification of ligand density (Scheme 2A).
For this purpose we applied a 4-carboxy-phthaloyl protecting
group (here abbreviated ‘‘Trim’’ for trimellitoyl), which allows
deprotection of aminooxy groups under extremely mild,
aqueous conditions without compromising nanoparticle integ-
rity and which enables quantification by HPLC. Self-assembly
in the absence of the Trim group (i.e. with fully unprotected
bifunctional linker) resulted in extensive aggregation. Thus,
OEG linker 1,5 based on tetra(ethylene glycol) and carrying a
tritylated thiol and a free aminooxy group, was protected
using trimellitic anhydride with microwave heating13 in 91%
yield. The trityl group was then removed followed by
self-assembly of OEG linker 2 onto the surface of citrate-
AuNPs (diameter B12 nm). The functionalized nanoparticles,
3, were then thoroughly washed, and a comparison of the
visible spectrum with that of citrate-AuNPs (see ESIw) showed
a shift in surface plasmon band maximum, lSP, of 4 nm,
indicative of thiol self-assembly.5 Release of the Trim group as
the phthalazinedione 5 by treatment of AuNPs 3 with aqueous
hydrazine (1 mM) was quantified by HPLC. After 24 h of
reaction there was no further increase in the concentration of
released 5, yielding aminooxy-terminated core–shell AuNPs 4
with a ligand density, i.e. average number of reactive linkers/
nanoparticle, of B435, corresponding to a surface coverage,
i.e. average number of reactive linkers/nm2 gold surface, of
0.88 (see ESIw). The obtained density is comparable to that
obtained in previous studies involving similar nanoparticle
cores.2,5,14
as encapsulating the nanoparticle core to reduce non-specific
interactions and associated aggregation of nanoparticles. Gold
glyconanoparticles (glyco-AuNPs) have so far mainly been
synthesized either (i) by in situ formation of AuNP cores in the
presence of thiol-modified glycans via modifications of the
Brust–Schiffrin protocol,9 or (ii) by introduction of
thiol-modified glycans onto preformed AuNP cores via
self-assembly10 on citrate-stabilized AuNPs11 (citrate-AuNPs).
Here we present the realization of a novel concept for
synthesis of glyco-AuNPs where unmodified glycans are
anchored via a single chemoselective reaction onto the outer
nanoparticle shell of reactive core–shell AuNPs. Only a few
examples of coupling of glycans to functionalized nano-
particles have thus far been reported, and these cases require
multi-step synthetic modifications of the glycans prior to
conjugation.12 We report now that oxime coupling on terminal
aminooxy groups allows anchoring of aldehyde-containing
biomolecules, specifically unprotected, reducing glycans,
peptide aldehydes, and glycopeptide aldehydes, under mild
conditions and in aqueous solution thus allowing broad access
to glyco-AuNPs (Scheme 1).
The present methodology is based on water-soluble
citrate-AuNPs to allow installation of the reactive OEG shell
under aqueous conditions. The prerequisite is
a novel
protecting group approach able to (i) prevent the intermediate,
IGM-Bioorganic Chemistry, Centre for Carbohydrate Recognition and
Signalling, Faculty of Life Sciences, University of Copenhagen,
Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
E-mail: kjj@life.ku.dk; Fax: +45 3533 2398; Tel: +45 3533 2430
w Electronic supplementary information (ESI) available: Experimental
procedures, quantifications, NMR and UV-Vis spectra, and DLS and
TEM measurements on AuNPs. See DOI: 10.1039/b911676a
The peptide aldehyde15 Fmoc-Glu-Glu-Gly-Gly-H was used
for a detailed study on the efficiency of oxime coupling
reactions on the OEG shell of gold nanoparticles. The design
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Scheme 2 Synthesis of aminooxy-terminated core–shell AuNPs 4via transient Trim protection (A), and chemoselective peptide, glycopeptide, and
glycan functionalization (B). Conditions: (a) trimellitic anhydride, pyridine, 180 1C, 30 min, 91%; (b) TFA, triethylsilane, CH2Cl2, quant.;
(c) citrate-AuNPs, H2O; (d) 1 mM hydrazine (aq), 24 h; (e) Fmoc-Glu-Glu-Gly-Gly-H (1.25 mM), various temperatures and reaction times, see
Table 1; (f) piperidine–H2O 1 : 1, 20 min; (g) Ac-Tyr-Gly-Ser(aGalNAc)-Glu-Gly-Gly-H (1.25 mM), 40 1C, 16 h; (h) conditions I: glycan (0.5 M),
40 1C, 16 h; or conditions II: glycan (5 mM), 4-anisidine (5 mM), 40 1C, 16 h.
of the peptide incorporates in a simple form (i) a C-terminal
aldehyde functionality, (ii) an Fmoc protecting group for
spectrophotometric quantification of oxime coupling yields,
and (iii) two carboxylate groups for aqueous solubility and net
negative charge.2,16 The feasibility of Fmoc cleavage
from AuNPs was initially confirmed in experiments with
piperidine–H2O 1 : 1 on peptide conjugated AuNPs 6, that
had been prepared from a pre-assembled peptide linker
conjugate of Fmoc-Glu-Glu-Gly-Gly-H and OEG linker 1 in
solution (data not shown). We then applied novel oxime
coupling with Fmoc-Glu-Glu-Gly-Gly-H on aminooxy-
terminated core–shell AuNPs in phosphate buffer
(Scheme 2B, pathway e) under various conditions. The
efficiency of the oxime coupling reaction was monitored by
Fmoc quantification (Scheme 2B, pathway f, and Table 1).
The coupling was essentially quantitative when the reaction
was performed at 40 1C for 16 h; however, at room temperature
(16 h) and under microwave heating at 60 1C (1 h) the reaction
did not proceed to completion. Coupling with an analogous
octa(ethylene glycol) linker provided similar results (data not
UV-Vis spectrum of AuNPs 6 (see ESIw). Transmission
electron microscopy (TEM) analyses confirmed that gold cores
were unchanged during installation and functionalization of
the reactive shell (see ESIw).
Using the established conditions for peptide oxime
coupling, we investigated glyco-AuNP generation with the glyco-
peptide aldehyde Ac-Tyr-Gly-Ser(aGalNAc)-Glu-Gly-Gly-H
containing the Tn antigen (Scheme 2B, pathway g).17 After
extensive washing of resulting AuNPs 8 with phosphate buffer
to remove excess glycopeptide, a colorimetric assay with the
aGalNAc-binding protein Helix aspersa agglutinin (HAA)
was performed by monitoring the changes in lSP relative to
control protein (BSA). The surface plasmon band shift values,
DlSP, of lectin-induced aggregation followed apparent first
18
order kinetic profiles in DlSP
.
Glycopeptide-functionalized
AuNPs 8 displayed a dramatic shift, DlSP, of 16 nm in the
presence of 5 mM HAA relative to BSA (o1 nm) (Fig. 1A),
confirming the correct assembly of glycopeptide AuNPs 8.
Encouraged by these results, we applied oxime coupling for
anchoring of glycans with subsequent colorimetric detection of
lectin interactions. We have previously demonstrated that
carbohydrate oximes, when presented on AuNPs, may be
recognized by lectins with a very high degree of specificity.5
Thus, aminooxy-terminated core–shell AuNPs 4 were reacted
with unprotected, reducing glycans (maltose, lactose, and
N-acetyllactosamine) under two different sets of conditions
to promote glycan oxime formation: (i) a vast excess (0.5 M) of
glycan (conditions I),19 or (ii) nucleophilic catalysis by
4-anisidine with 5 mM glycan (conditions II),20 both at 40 1C
for 16 h (Scheme 2B, pathway h). After extensive washing of
AuNPs 9–11 with phosphate buffer to remove excess glycans,
colorimetric assays were performed directly by addition of
shown). In
a
control experiment, thiolytic release5 of
the organic shell after oxime coupling, and analysis by
HPLC-MS confirmed that peptide oxime bonds were indeed
formed on the OEG shell (data not shown). Additionally,
oxime coupling with the peptide aldehyde was corroborated by
dynamic light scattering measurements, which showed an
increase in hydrodynamic radius, as well as by a spectrum
subtraction of AuNPs before (6) and after (7) Fmoc deprotection,
which clearly revealed a characteristic Fmoc signal in the
Table 1 Oxime coupling with Fmoc-Glu-Glu-Gly-Gly-H on aminooxy-
terminated core–shell AuNPs
various lectins and monitoring of DlSP
glucose-binding lectin Concanavalin
.
The terminal
Ligand density Surface coverage Yield
(peptides/AuNP) (peptides/nm2) (%)a
A
(Con A) from
Entry Conditions
Canavalia ensiformis and the terminal galactose-binding lectin
Erythrina cristagalli agglutinin (ECA) were used to assess
glycan functionalization. The apparent first order rate
constant, kobs, and the maximal surface plasmon band shift,
DlSP,max, were used to describe the aggregation process
(see ESIw).18 Maltose-functionalized 9(conditions I) and
1
2
3
25 1C, 16 h
60 1Cb, 1 h
40 1C, 16 h
157
162
427
0.32
0.33
0.86
36
37
98
a
Calculated from Fmoc quantification relative to Trim quantification.
By microwave heating.
b
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to conjugation and allows direct evaluation of e.g. glycan–protein
interactions with high specificity. Additionally, the methodo-
logy allows quantitation of the ligands installed in the shell.
We envision that the methodology may be applied for
synthesis of glyco-AuNPs of complex glycans and glyco-
peptide aldehydes, as well as in a parallel format to facilitate
rapid screening of biomolecular interactions.
Support from the Danish Natural Science Research Council
and the University of Copenhagen (M. B. T.) is acknowledged.
We thank Lise Arleth for DLS measurements.
Notes and references
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Fig.
1
Colorimetric detection assays for HAA (K) and BSA
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Con A (B) and ECA (C) with glyco-AuNPs 9–11. Legend: 4
(Â, control), 95 (’), 9(I) (m), 9(II) (.), 10(I) (n), 10(II) (,), 11(II)
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9(conditions II) displayed similar DlSP,max of B30 nm
and kobs of B3 hÀ1 with Con A (5 mM), essentially identical
to previously reported maltose oxime glyco-AuNPs
(9, DlSP,max B33 nm and kobs B2.5 hÀ1, ligand density
B300 glycans/nanoparticle5), indicating that glycan oxime
formations proceeded to high degrees of conversion under
both conditions (Fig. 1B). The detection limit for Con A was
B100 nM (see ESIw). Lactose-functionalized AuNPs,
10(conditions I and II), were completely devoid of a shift in
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of ECA (5 mM) the selectivity was completely opposite; lactose
AuNPs, 10(conditions I and II), showed DlSP,max of B14 nm
and kobs of B1 hÀ1, whereas maltose AuNPs 9(conditions I)
were completely devoid of a shift (DlSP,max o1 nm) (Fig. 1B).
Interestingly, oxime coupling with the less reactive N-acetyl-
lactosamine and subsequent lectin recognition was comparable
to that of lactose, as judged by the similar profiles of
11(conditions II) and 10(conditions I and II). Control
experiments with aminooxy-terminated AuNPs 4 showed only
minute shifts in lSP of B3 nm and o1 nm upon treatment
with Con A and ECA, respectively (Fig. 1A and B).
In summary, we have developed a novel method for anchoring
of unmodified, reducing glycans and glycopeptide aldehydes
directly to AuNPs with a reactive core–shell architecture.21
This approach avoids synthetic modifications of glycans prior
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This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 6367–6369 | 6369