Improved methodology for the preparation of water-soluble maleimide-functionalized small gold nanoparticles

Article abstract of DOI:10.1021/la302168g

Improved methodology to prepare maleimide-functionalized, water-soluble, small (<3 nm) gold nanoparticles using a retro-Diels-Alder strategy that we developed for similar organic-soluble AuNP's is described. Importantly, our results suggest that a recent paper by Zhu, Waengler, Lennox, and Schirrmacher describing a similar strategy gave results inconsistent with the formation of the titled maleimide-modified AuNP (Zhu, J.; Waengler, C.; Lennox, R. B.; Schirrmacher, R. Langmuir2012, 28, 5508) as the major product, but consistent with the major product being an adduct derived from the hydrolysis of maleimide formed under the conditions used for the required deprotection of the maleimide. Our methodology provides an efficient and accessible route to pure maleimide-modified small AuNP's that circumvents the formation of the hydrolysis product. The maleimide-modified small AuNP's are versatile because they are soluble in water and in a wide range of organic solvents and their reactivity can now be properly exploited as a reactive moiety in Michael addition for bioconjugation studies in aqueous solution.

Full text of DOI:10.1021/la302168g

Article
pubs.acs.org/Langmuir
Improved Methodology for the Preparation of Water-Soluble
Maleimide-Functionalized Small Gold Nanoparticles
Pierangelo Gobbo and Mark S. Workentin*
Department of Chemistry and the Centre for Materials and Biomaterials Research, Western University Canada, London, Ontario
N6A 5B7, Canada
S
* Supporting Information
ABSTRACT: Improved methodology to prepare maleimide-function-
alized, water-soluble, small (<3 nm) gold nanoparticles using a retro-
Diels−Alder strategy that we developed for similar organic-soluble AuNP's
is described. Importantly, our results suggest that a recent paper by Zhu,
Waengler, Lennox, and Schirrmacher describing a similar strategy gave
results inconsistent with the formation of the titled maleimide-modified
AuNP (Zhu, J.; Waengler, C.; Lennox, R. B.; Schirrmacher, R. Langmuir
2012, 28, 5508) as the major product, but consistent with the major
product being an adduct derived from the hydrolysis of maleimide formed
under the conditions used for the required deprotection of the maleimide.
Our methodology provides an efficient and accessible route to pure
maleimide-modified small AuNP's that circumvents the formation of the
hydrolysis product. The maleimide-modified small AuNP's are versatile
because they are soluble in water and in a wide range of organic solvents and their reactivity can now be properly exploited as a
reactive moiety in Michael addition for bioconjugation studies in aqueous solution.
utility.7 Recently, Zhu, Waengler, Lennox, and Schirrmacher
described an approach to preparing stable water-soluble
INTRODUCTION
■
Maleimide functionalities at the interface of small hydrosoluble
maleimide-tethered gold nanoparticles with an average core
nanoparticles are of interest in biological applications because
size of ca. 3 nm.8 The goal was to synthesize small, stable
of the ability of the maleimide moiety to react through Michael
maleimide water-soluble AuNP's through a route involving less-
addition with nucleophiles, especially amine and thiols. This
elaborate ligands that are easier and faster to synthesize and are
general methodology is extensively exploited to label peptides,
proteins, DNA strands, and cells.1,2 Thus, having a maleimide
based on tri- or tetra-ethylene glycol. Because of the smaller
size of the Au core and the simplicity of the ligands, these
group at the interface of a AuNP would allow for the
nanoparticles were expected to be easier to make and
exploitation of this type of reactivity in the use of the
characterize, allowing the effective number of functionalities
present at the nanoparticle interface to be calculated to provide
nanoparticle in applications such as drug or substrate delivery
or as an optical marker for diagnostics in biological systems.3,4
the ability for more careful control of the number of potential
Such water-soluble AuNP's are a desirable platform because of
bioconjugation sites. Their approach utilized a strategy that our
their chemical stability, the biocompatibility of the gold core,
group developed for the preparation of small organic-soluble
and the ability to tune the functionality at the interface through
maleimide AuNP's in the same size regime,9 which itself was
the reactivity of the maleimide. Indeed, interfacial maleimide
based on approaches used to install maleimide on other types
can react with amine or thiol functionalities present on
of materials.10−12 In fact, the strategy has long been used as a
liposomes or cell membranes, allowing the nanoparticle to be
click-type reaction in macromolecular synthesis.13 The strategy
immobilized on the biosystem while preserving all of the
optothermal properties of the original nanoparticle.5 Despite
involves incorporating the maleimide moiety onto a AuNP as
its furan Diels−Alder adduct and then liberating the free
maleimide when desired by taking advantage of the propensity
of this adduct to undergo an efficient retro-Diels−Alder
reaction with the loss of furan at elevated temperatures
(Scheme 1). This strategy must be employed because AuNP's
of these types and sizes are prepared either by direct synthesis
the numerous potential biological applications that these water-
soluble maleimide AuNP's can have, until recently the only
hydrosoluble maleimide-functionalized nanoparticles synthe-
sized were larger than 15 nm and their synthesis involved a
difficult place-exchange reaction from citrate-protected or from
CTAB-protected (cetyltrimethyl ammonium bromine) gold
nanoparticles with very bulky and complex polyethylene glycol-
based ligands.5,6 Monomaleimide-functionalized gold nano-
particles derived from phosphine-protected gold cores have
been reported but are not very stable and thus are of limited
Received: May 28, 2012
Revised: July 16, 2012
Published: August 10, 2012
© 2012 American Chemical Society
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Scheme 1. Synthesis of Maleimide-Functionalized Gold Nanoparticles
or by a place-exchange reaction that requires the thiol derivative
of the functionality that you wish to install on the AuNP. In
either method, a maleimide-functionalized thiol cannot be used
directly because of its (desired) susceptibility to Michael
additions and also because the ligand would simply react with
itself. To avoid this and other complications, in our original
report we prepared the dodecanethiol-derivatized maleimide-
furan Diels−Alder adduct and introduced this ligand by a place
exchange onto dodecane thiol-protected AuNP's ca. 2.2 nm in
size. When desired, the free maleimide-AuNP's could be
liberated by heating to 100 °C in toluene, which resulted in the
loss of the furan that could then be removed to yield organic-
soluble maleimide-modified AuNP's with only a small increase
in the core size.9 We subsequently utilized maleimide-AuNP's
to prepare AuNP networks through a Diels−Alder strategy9
and use it as a reaction template to prepare a wide variety of
organic-soluble AuNP's using Diels−Alder and 1,3-dipolar
cycloaddition reactions and Michael additions.14−16
This strategy was utilized by Lennox and Schirrmacher to
prepare water-soluble maleimide-AuNP's with the difference
being that both the ligand in the starting (base) AuNP and the
tether used for the furan-protected maleimide−thiol used in the
place exchange were based on PEG ligands to make them
water-soluble. This represents an important breakthrough
because many applications using AuNP, especially those for
biological systems, require water solubility and the ones we
originally prepared were of limited utility because they were
soluble only in organic solvents. Having a water-soluble
maleimide-modified AuNP is especially relevant because of
the wide use of Michael addition reactions of maleimide in
bioconjugation-type studies. In this regard, the contribution of
Lennox, Schirrmacher, and co-workers was important. How-
ever, because of our experience in this area, when their work
appeared we were perplexed by aspects of the characterization
of the maleimide-AuNP that differed from that previously
published for the organic-soluble version and other derivatives.
In particular, the maleimide-AuNP (after the removal of furan)
was reported to have its olefinic protons 0.5 ppm upfield from
the olefinic protons in the furan-maleimide adduct, whereas in
all other studies and indeed for model maleimide compounds
these protons are downfield from the olefinic protons of the
furan-maleimide adduct. This is a dramatic solvent effect and
perhaps suggests that what they prepared was not the
maleimide-AuNP. We were surprised by the apparent low
reactivity towards Michael addition given the high apparent
loading of maleimide at the interface because unlike the
Michael addition reactions we reported for maleimide-AuNP in
organic solvents that require high pressure to proceed
efficiently, this type of reaction should be rapid and efficient
in aqueous and protic solvents.
Herein, we report an efficient method for preparing small,
2.5-nm-core-size water-soluble maleimide-AuNP using a differ-
ent synthesis approach but still utilizing the furan-maleimide
adduct to prepare the AuNP's and liberating the maleimide-
AuNP through the retro-Diels−Alder reaction. Characterization
confirms the formation of the target maleimide-AuNP,
1
particularly the H NMR spectroscopy that verifies the olefinic
protons in the expected chemical shift region. Furthermore, we
show that when the deprotection of the furan-maleimide AuNP
adduct is carried out in the presence of water the initially
formed interfacial maleimide moieties are hydrolyzed at these
temperatures to yield an interfacial polyammonium/maleic acid
salt adduct. The method we describe outlines how to prepare
small (<3 nm) maleimide-AuNP's that are soluble in both water
and a host of other organic solvents. The latter allows for their
preparation under conditions that avoid water and the resulting
secondary hydrolysis reaction of the maleimide moiety. The
versatile maleimide-AuNP's we describe are easy to prepare and
suitable for bioconjugation studies.
RESULTS AND DISCUSSION
■
Our approach to preparing maleimide-AuNP's is shown in
Scheme 1 and follows the strategy that we reported for organic-
soluble maleimide-AuNP's of the same size, with the difference
being that the thiolate ligands are PEGylated. This approach
first involved the synthesis of methyl-terminated triethylene
glycol thiol (Me-EG3-SH) monolayer-protected AuNP's (Me-
EG3-AuNP's). The methyl-terminated EG3 ligands were
selected as the base ligand for the nanoparticles because unlike
hydroxyl-terminated (HO-EG3-AuNP) species they are soluble
in both water and a wider selection of organic solvents, making
the resulting maleimide gold nanoparticles more versatile. Me-
EG3-AuNP was then subjected to place-exchange reaction
conditions in the presence of the Diels−Alder furan-protected
maleimide tetraethylene glycol thiol (Pt-maleimide-EG4-SH).
This reaction was carried out in a mixture of dry methanol and
acetone as the solvent. It is important to note that during this
step the maleimide must be protected; otherwise, it will
undergo a Michael addition with the thiols present in solution.
The final step in the preparation of the desired maleimide-
AuNP was the deprotection of the maleimide at the AuNP
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interface through the retro-Diels−Alder reaction at 100 °C in
toluene (Scheme 1).
The required Me-EG3-SH and Pt-maleimide-EG4-SH ligands
were synthesized starting from readily available triethylene
glycol monomethyl ether (Me-EG3-OH) and tetraethylene
glycol (HO-EG4-OH), respectively (Scheme 2). The maleimide
transmission electron microscopy (TEM) images (Supporting
Information). 1H NMR spectroscopy showed the expected
broadened peaks at 3.34, 3.58, and 3.66 ppm, corresponding to
the Me-EG3-SH ligands (Figure 1, spectrum i). Thermogravi-
Scheme 2. Synthesis Paths for the Preparation of Me-EG3-
SH (Left) and Pt-maleimide-EG4-SH (Right)
ligand was synthesized with one more ethylene glycol unit than
in the base ligand to lower the steric hindrance on the
maleimide functionality and make it easier to react through
Michael addition or other cycloaddition reactions.14−16 To
synthesize Me-EG3-SH, Me-EG3-OH was tosylated to trans-
form the hydroxyl group into a good leaving group. The
tosylated Me-EG3-OH was then converted to its corresponding
thioacetate via an SN2 reaction. The thiol was obtained through
basic hydrolysis of the thioacetate functionality (Scheme 2).
The same strategy was employed to synthesize Pt-maleimide-
EG4-SH. In this case, both hydroxyl groups of HO-EG4-OH
were tosylated, then one of the tosylated extremities of the
molecule was reacted with furan-protected maleimide (3,6-
endoxo-Δ4-tetrahydrophthalimide), and then the other tosy-
lated extremity was converted to the thioacetate and
subsequently hydrolyzed under basic conditions to generate
the desired thiol, Pt-maleimide-EG4-SH. A detailed synthesis
procedure and a characterization of the ligands are reported in
the Supporting Information.
1
Figure 1. H NMR spectra (recorded in D2O) of (i) Me-EG3-AuNP,
(ii) Pt-maleimide-EG4-AuNP, (iii) maleimide-EG4-AuNP, and (iv) the
hydrolysis product of maleimide-EG4-AuNP. * indicates residual H2O.
metric analysis (TGA) revealed that 38.3% of the AuNP's are
composed of the PEGylated ligands, corresponding to 2.13
μmol of Me-EG3-SH per milligram of AuNP (Figure 2).
These nanoparticles, having PEG ligands with terminal
methylether units at the solution interface, have the greater
advantage of being soluble in both organic and water solutions.
In particular, these Me-EG3-AuNP's are soluble in water,
Me-EG3-AuNP was synthesized using a modified one-phase
synthesis method.17 Briefly, the gold salt, HAuCl4·3H2O, was
dissolved in a mixture of methanol and glacial acetic acid. Me-
EG3-SH was dissolved in this solution, and finally a fresh
solution of NaBH4 was slowly added. The mixture was stirred
overnight at room temperature. Purification of the resulting
nanoparticles involved extracting them from the aqueous phase
using toluene. Successively, unreacted Me-EG3-SH was washed
away from a film of nanoparticles inside a round-bottomed flask
using cyclohexane, in which the AuNP's are not soluble.
Subsequently, the AuNP's were purified by dialysis. Through
control of the gold−thiol ratio, it is possible to control the size
of Me-EG3-AuNP. In this work, a ratio of 1:3 gold/thiol was
employed to yield 2.0
AuNP's were characterized by UV−vis spectroscopy and
0.3 nm Me-EG3-AuNP's. These
Figure 2. TGA of Me-EG3-AuNP (−), Pt-maleimide-EG4-AuNP (---),
and maleimide-EG4-AuNP (···).
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methanol, ethanol, tetrahydrofuran, dichloromethane, chloro-
form, toluene, ethyl acetate, acetone, dimethylformamide, and
acetonitrile. They can be repeatedly dried and redissolved in
these solvents with little to no degradation or aggregation
(none was apparent).
though they are still readily redissolvable in water. The
solubility in toluene is of crucial importance because toluene
is a solvent suitable for the deprotection of maleimide without
interference from competing reactions of maleimide (vide
infra).
Pt-maleimide-EG4-AuNP was synthesized using a place-
exchange reaction. In a typical synthesis, 100 mg of Me-EG3-
AuNP is mixed with 37.1 mg of Pt-maleimide-EG4-SH for 15
min in 20 mL of dry methanol and 2.5 mL of acetone. The
solvent was then evaporated under vacuum, and the resulting
film was washed with cyclohexane (in which it is not soluble) to
get rid of excess Pt-maleimide-EG4-SH and exchanged Me-EG3-
SH. Pt-maleimide-EG4-AuNP's were then purified further by
dialysis to remove any unbound ligands. The furan-protected
nanoparticles, Pt-maleimide-EG4-AuNP's, were characterized by
1H NMR and UV−vis spectroscopy, TGA, and TEM. The
nanoparticles synthesized under these conditions are found to
maintain their size of 2.0 0.3 nm according to TEM images
(Supporting Information). TGA confirmed the presence of the
protected maleimide ligand (Figure 2). The first weight loss
accounting for 3.9% takes place between 100 and 170 °C and is
related to the loss of furan. This feature in the TGA is
diagnostic of the incorporation of the protected maleimide,
confirms the reversibility of the Diels−Alder reaction at around
100 °C, and can be used to determine the amount of maleimide
ligand incorporated onto the AuNP's. Using the percentage
mass loss of furan (3.9%) as an indication of the amount of
furan-protected maleimide incorporated onto the AuNP's
allows us to estimate that 30% of ligands on the Pt-
maleimide-EG4-AuNP's were the required furan-protected
maleimide ligands. This loss of furan is then followed by the
loss of triethylene glycol monomethyl ether ligands and the
deprotected maleimide ligand. These data can be compared
with previous work carried out in our laboratories on small
maleimide organic-soluble nanoparticles.9
To carry out the retro-Diels−Alder reaction to liberate the
desired maleimide moiety at the interface, Pt-maleimide-EG4-
AuNP's were dissolved in toluene and the solution was stirred
overnight at 100 °C. The solvent was then evaporated under
vacuum, and the AuNP film was washed with cyclohexane as
described previously. Maleimide-EG4-AuNP was characterized
by 1H NMR and UV−vis spectroscopy, TGA, and TEM. TEM
images and UV−vis spectroscopy revealed a slight increase in
the nanoparticle size to 2.5
0.3 nm, similar to what we
observed during the preparation of our original organic-soluble
maleimide-AuNP's.9 TGA (Figure 2) of the resulting
maleimide-EG4-AuNP's did not show the first weight loss
between 100 and 170 °C present in the Pt-maleimide-EG4-
AuNP's, confirming that this was most likely due to the loss of
furan in the latter. According to the mass loss measured, on 1
mg of maleimide-EG4-AuNP's there are 1.30 μmol of Me-EG3-
SH and 0.50 μmol of Pt-maleimide-EG4-SH, corresponding to a
28% maleimide ligand. TGA showed the presence of two
ligands in essentially the same ratio as was obtained with the Pt-
maleimide-EG4-AuNP's.
1H NMR spectroscopy of the product (Figure 1, spectrum
iii) showed the disappearance (absence) of the peaks related to
the furan Diels−Alder adduct (6.60, 5.25, and 3.07 ppm) and a
new signal downfield at 6.86 ppm, as expected for olefin peaks
of the maleimide moiety. This result completely agrees with our
previous characterization of maleimide organic-soluble nano-
particles.9 The simplified formula for the maleimide-EG4-
AuNP's can be calculated from the TGA and TEM measure-
ments, assuming a spherical shape of the gold core.8 Maleimide-
EG4-AuNP's were found to have the simplified formula
Au483(Me-EG3-S)198(maleimide-EG4-S)77.
As stated above, the solubility of the Pt-maleimide-EG4-
AuNP's in toluene is a key component in the synthesis of
maleimide-EG4-AuNP's. Some attempts to deprotect the Pt-
maleimide-EG4-AuNP's were carried out by employing
acetonitrile, acetone, and water as reaction solvents, and all of
them resulted in a nanoparticle size growth characterized by a
change in the solution color from dark brown (typical of a
nanoparticle size between 2 and 3 nm) to ruby (typical of
nanoparticles with diameter of between 4 and 5 nm).
Performing the retro-Diels-Alder deprotection in water gave
very different results. After the reaction of Pt-maleimide-EG4-
AuNP in H2O at 100 °C overnight, there was no peak due to
the expected maleimide olefin protons at 6.86 ppm but a signal
appeared at 6.23 ppm, upfield with respect to the peak at 6.60
ppm of the alkene protons of the protected maleimide ligand.
The appearance of this upfield signal is also accompanied by
significant changes in the region between 3 and 4 ppm. The
peak at 3.07 ppm disappears, and three broad peaks (one at
3.15 ppm, one at 2.89 ppm, and one at 3.77 ppm) appear. This
is also consistent with that observed in the study by Lennox and
Schirrmacher.8 Because the peak they assigned to maleimide
was significantly upfield from where it was expected, we
suspected that it was due to a reaction of the liberated
maleimide with H2O under the same reaction conditions.
To explore this possibility, we performed a number of
control reactions. First, we took our fully characterized
maleimide-EG4-AuNP, prepared by deprotection in toluene,
1H NMR spectroscopy of the Pt-maleimide-EG4-AuNP's
(recorded in D2O as the solvent, with the residual H2O signal
used as a reference) shows the appearance of the three
broadened peaks related to the furan-protected maleimide
functional group (Figure 1, spectrum ii). The peak at 6.60 ppm
is related to the two alkene protons, the peak at 5.25 ppm is
related to the protons adjacent to the bridging oxygen of the
Diels−Alder adduct, and the peak at 3.07 ppm is related to the
two protons closer to the carbonyl groups.9 The relative
integration of the −CH3 peak due to the Me-EG3S ligands with
those of furan-protected maleimide confirms the 30%
incorporation of the desired ligand onto the particle.
Furthermore, the solubility of these nanoparticles is unchanged
with respect to that of the Me-EG3-AuNP's.
The choice of the solvent for the place-exchange reaction was
found to be of crucial importance to the final synthesis of
maleimide-AuNP's. Because the place-exchange reaction takes
place on the time scale of minutes, a solvent with a low vapor
pressure is required for the reaction. The use of the methanol−
acetone mixture as the solvent for the place-exchange reaction
guarantees better control over the reaction time because it can
be removed faster than water. Better control over the reaction
time allows better control of the number of protected
maleimide ligands that are introduced onto AuNP's. The
quantity of the furan-protected maleimide ligand strongly
influences the nanoparticle solubility. For example, when the
percentage of furan-protected maleimide−thiol was over 40%,
the resulting AuNP's were no longer soluble in toluene even
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and heated this sample in water to 100 °C (similar to the
the peaks of N-methylmaleimide at 2.94 and 6.80 ppm, related
respectively to the methyl protons and to the olefinic protons.
The main products of the reaction are represented by the peaks
at 2.55 and 6.30 ppm, which are the same as those observed
after heating the maleimide-EG4-AuNP's in water. On the basis
of the reactants (N-methylmaleimide and water) and the
deprotection conditions employed by Lennox, Schirrmacher,
1
and co-workers). H NMR spectroscopy shows the loss of the
signal of the olefinic protons in maleimide from 6.86 ppm, a
new peak growing in at 6.23 ppm, and the appearance of the
same peaks in the region between 3 and 4 ppm as observed
after the direct deprotection of Pt-maleimide-EG4-AuNP's in
water. The spectrum is shown in Figure 1(iv). This latter
spectrum suggests that this product arises from the hydrolysis
of maleimide by water under these conditions. But what could
be the hydrolysis product that would give rise to such a simple
1
resulting H NMR analysis, we suspected that the N-methyl
maleimide was hydrolyzed to methylamine (as methylammo-
+
nium, CH3NH3 ) and maleic acid (as hydrogen maleate,
HOC(O)C(O)O−) under these conditions. Indeed, the
large amount of water (the solvent) and the high temperature
favor the hydrolysis of N-methylmaleimide. To validate the
hypothesis of forming methylamine and maleic acid, small
amounts of authentic N-methylamine and subsequently
authentic maleic acid were added to the NMR tube containing
1
singlet in the H NMR spectrum at ca. 6.2 ppm and still be
associated with the AuNP? To address this issue, we carried out
a number of model reactions.
For simplicity, N-methylmaleimide was chosen as the first
model compound. Its 1H NMR spectrum was recorded in D2O,
and it revealed the expected signals at 2.94 ppm (CH3−N) and
6.80 ppm (olefinic protons of the maleimide). This sample was
then heated in nanopure water at 100 °C in a round-bottomed
flask for 48 h. After reaction, the pH of the solution had
changed from neutral to acidic. The solvent was then
the products of hydrolysis of the model compound. 1H and 13
C
NMR spectra recorded after every addition show an increase in
the corresponding peak intensities upon addition of the pure
reagent and, importantly, no new signals were recorded
(Figures SI 12 and SI 13). Note that because of the pH
change after the addition of excess base and then excess acid,
the peaks, especially those corresponding to the maleic acid,
shift slightly depending on the extent of salt formation but
remain in the 6.2−6.35 ppm range. Mass spectrometry also
revealed the presence of the molecular peaks of methylamine
and maleic acid. Finally, the Kaiser test for the detection of
primary amine was conducted for the reaction mixture and for a
control solution of N-methylmaleimide in water.18 The reaction
mixture becomes dark blue (positive test), indicating the
presence of a primary amine, and the N-methyl maleimide
control solution becomes yellow (negative test) (Figure SI 11).
To understand better the process that leads to the hydrolysis
reaction on the AuNP's and to explain what might have
occurred in the earlier study, the protected maleimide
tetraethylene glycol thioacetate (Pt-maleimide-EG4-SAc) was
employed as a second model compound. A few milligrams of
Pt-maleimide-EG4-SAc were inserted into a J. Young NMR
tube and dissolved in D2O, and the temperature was increased
1
evaporated, and the reaction product was characterized by H
and 13C NMR spectroscopy and mass spectrometry. As shown
in Figure 3, 1H NMR spectroscopy reveals the disappearance of
1
to 100 °C. H NMR spectra were recorded after various time
intervals (Figure SI 14 in the Supporting Information). In
addition to the expected hydrolysis of the thioacetate moiety
(indicated by the signal due to acetic acid at 2.06 ppm), the
signals due to the protected maleimide were also monitored as
a function of time during heating in D2O. This study shows the
progressive deprotection of the maleimide functionality,
indicated by the appearance of a signal at 6.85 ppm that is
due to maleimide and then its progressive hydrolysis. After 8 h,
the intensity of the peaks of protected maleimide (3.11, 5.29,
and 6.60 ppm) markedly decreases, and this decrease is
accompanied by the appearance of the furan peaks (6.47 ppm
and 7.53 ppm), the peak related to the alkene protons of
maleimide (6.85 ppm), the supposed maleic acid/maleate peak
(6.31 ppm), and the peak of the protons alpha to the amine of
the resulting amine(ammonium) tetraethylene glycol (+H3N-
EG4-S-D) (3.2 ppm). After 24 h, the amount of starting
material is further decreased but the product of the hydrolysis
of maleimide (indicated by the peak at 6.31 ppm) becomes the
major product. After 48 h, the amounts of protected and
deprotected maleimide are negligible and the major product is
+
Figure 3. 1H NMR spectrum recorded in D2O showing the kinetics of
hydrolysis of N-methylmaleimide. Peaks A and B are relative to the
reactant, and peaks C and D are relative to the main products. * is the
H2O signal.
represented by maleic acid/maleate and H3N-EG4-SD.
The same experiment was performed using our maleimide-
EG4-AuNP, and it showed the same changes as described for
model ligand Pt-maleimide-EG4-SAc (Figure SI 15) with the
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loss of the maleimide signals at 6.86 ppm and the appearance of
the signal at 6.23 ppm. At the end of this experiment and to
confirm that the signal at 6.23 ppm was due to the maleic acid
product from the hydrolysis of maleimide, a small amount of
authentic maleic acid was added to the fully hydrolyzed AuNP's
and an increase in the peak at 6.23 ppm was recorded (Figure
SI 16). The addition of authentic N-methylmaleimide shows
the expected olefinic signal at 6.86 ppm. In total, these
experiments demonstrate that on heating in water to 100 °C
the protected maleimide undergoes the expected retro-Diels−
Alder reaction to form the desired maleimide, but this reacts
ASSOCIATED CONTENT
■
S
* Supporting Information
General experimental details, details of the synthesis and
characterizations of ligands, gold nanoparticles, model reac-
tions, and zeta potential measurements. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mworkent@uwo.ca.
Notes
+
under conditions that yield the corresponding R-NH3 and
maleate by the hydrolysis of maleimide.
The authors declare no competing financial interest.
These results highlight the importance of the choice of
solvent for the maleimide deprotection reaction. Water, even
though it is a green solvent and has a high boiling point, was
found to react irreversibly at high temperatures with the
maleimide functionalities present at the nanoparticle interface.
Hydrolysis causes the release of maleic acid and the formation
of amine functionalities at AuNP's, which can couple as their
salt. Our approach allows for the deprotection to be carried out
in toluene, circumventing the hydrolysis of the desired
maleimide functionality at the interface.
To demonstrate that maleimide-EG4-AuNP's react via the
Michael addition reaction for the possible application as a
bioconjugation template, the maleimide-EG4-AuNP's were
functionalized with cysteine. In this experiment, 50 mg of
maleimide-EG4-AuNP's was mixed with 0.8 mg of L-cysteine for
1 h in nanopure water. The sample was then purified by dialysis
to remove any unreacted nucleophile and to leave only the
cysteine-functionalized AuNP's. The 1H NMR spectrum
showed the complete disappearance of the peak at 6.86 ppm
due to maleimide and the appearance of broadened peaks at
3.17 and 3.25 ppm, expected for the Michael addition product,
which partially overlapped with the peak at 3.32 (Figure SI 17).
ACKNOWLEDGMENTS
■
This work is supported by the Natural Sciences and
Engineering Research Council of Canada (NSERC) under
the Discovery Grants program and Western University Canada.
We thank Professor J. P. Guthrie for helpful discussions and
Professor E. R. Gillies for access to her nanoparticle zeta
potential instrumentation.
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■
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CONCLUSIONS
■
In this article, we described the first synthesis method leading
to stable maleimide, water-soluble, small (<3 nm) gold
nanoparticles through a retro Diels−Alder strategy. The
amount of protected maleimide ligand at the interface
accounted for 30% of the total ligands. Attempts to incorporate
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