The first example of molecularly imprinted nanogels with aldolase type I activity

Article abstract of DOI:10.1002/chem.200800675

The molecular-imprinting approach was used to obtain a nanogel preparation capable of catalysing the cross-aldol reaction between 4-nitrobenzaldehyde and acetone. A polymerisable proline derivative was used as the functional monomer to mimic the enamine-b

Full text of DOI:10.1002/chem.200800675

FULL PAPER
DOI: 10.1002/chem.200800675
The First Example of Molecularly Imprinted Nanogels with Aldolase Type I
Activity
Davide Carboni,[a] Kevin Flavin,[a] Ania Servant,[a] Veronique Gouverneur,[b] and
Marina Resmini*[a]
Dedicated to Professor Keith Brocklehurst on the occasion of his 70th birthday
Abstract: The molecular-imprinting ap-
proach was used to obtain a nanogel
preparation capable of catalysing the
cross-aldol reaction between 4-nitro-
benzaldehyde and acetone. A polymer-
isable proline derivative was used as
the functional monomer to mimic the
nanogels were prepared with dimen-
sions similar to those of an enzyme and
with the advantage of solubility and
flexibility previously unattainable with
monolithic polymers. Following tem-
plate removal and estimation of active-
site concentrations, the kinetic charac-
terisation of both imprinted and non-
imprinted nanogels was carried out
with catalyst concentrations between
0.7 and 3.5 mol%. Imprinted nanogel
AS147 was found to have a kcat value
of 0.25”10À2 minÀ1, the highest value
ever achieved with imprinted polymers
À
catalysing C C bond formation. Com-
parison of the catalytic constants for
both imprinted nanogel AS147 and
non-imprinted nanogel AS133 gave a
ratio of kcat 147/kcat 133 =18.8, which is in-
dicative of good imprinting efficiency
and highlights the significance of the
template during the imprinting process.
This work represents a significant dem-
onstration of the superiority of nano-
gels, when the molecular-imprinting ap-
proach is used, over “bulk” polymers
for the generation of catalysts.
A
type I enzymes. The diketone template
used to create the cavity was designed
to imitate the intermediate of the aldol
reaction and was bound to the func-
tional monomer using a reversible co-
valent interaction prior to polymeri-
sation. By using a high-dilution poly-
merisation method, soluble imprinted
Keywords: aldol reaction · enzyme
catalysis · enzyme mimics · molecu-
lar imprinting · nanostructures
Introduction
sults.[1] Molecular imprinting is an attractive approach for
the generation of recognition sites in macromolecular sys-
tems in which a template molecule is used in a casting pro-
cedure. Valuable results have been obtained with “bulk”
polymers in applications in which strong binding to the tem-
plate is required, but the development of polymeric matrices
with specific catalytic activity has proved to be a bigger
challenge. A variety of chemical reactions have been shown
to be catalysed by imprinted polymers,[2] but the rate accel-
erations and turnovers have been, with few exceptions,[3] dis-
appointing. Most of the data reported in the literature con-
cern bond breakage, in particular hydrolytic reactions with
The design and synthesis of catalytic systems capable of em-
ulating enzyme activity while overcoming some of the pro-
teinꢀs inherent limitations is a challenge that has attracted
scientists for a long time. Among the different enzyme
mimics studied, catalytic antibodies and imprinted polymers
have shown considerable potential and led to significant re-
[a] D. Carboni, Dr. K. Flavin, A. Servant, Dr. M. Resmini
School of Biological and Chemical Sciences
Queen Mary, University of London
Mile End Road, London E1 4NS (UK)
Fax : (+44)7882-3294
À
activated substrates. The only examples of catalytic C C
bond formation by imprinted polymers are a Diels–Alder
condensation,[4] a Pd-catalysed cross-coupling reaction,[5] an
example of class II aldolase[6] and a dimerisation by a perox-
idase-like polymer.[7]
E-mail: m.resmini@qmul.ac.uk
[b] Dr. V. Gouverneur
Chemistry Research Laboratory
Oxford University
The development of catalytic microgels, first reported by
Resmini et al.[8,9] and subsequently by Wulff and his
group,[10] represented a significant advance in the field of
Mansfield Road, Oxford OX1 3A (UK)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2008, 14, 7059 – 7065
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enzyme mimics. The application of the imprinting approach
to flexible polymeric matrices, such as the microgels, pio-
neered by Wulff,[11] allowed the preparation of materials
showing higher activities relative to the “bulk” polymers.
This is the result of a combination of the solubility and flexi-
bility in addition to the higher surface-to-volume ratio.
Herein we report, for the first time, the preparation of
molecularly imprinted nanogels containing a proline deriva-
tive, which show high catalytic activity, turnover and enan-
tioselectivity in the cross-aldol reaction between 4-nitroben-
zaldehyde (1) and acetone (2; Scheme 1) and follow the en-
specific products that could not be otherwise obtained. We
have created a library of polymerisable amino acid deriva-
tives that can be used alone or in combination as functional
monomers in the imprinting approach. Previous examples
have included the use of polymerisable arginine and tyrosine
to obtain microgels for carbonate hydrolysis.[8,9]
Results and Discussion
The formation of the complex between the template and the
functional monomer is a key step in molecular imprinting
and its stability plays a significant role in determining the
specificity of the cavities in the polymer (Scheme 2). In this
work the reversible covalent approach was chosen, making
use of the enol form of diketone 4 as the template and the
polymerisable proline derivative 5 as the functional mono-
mer. These two molecules react to form the corresponding
enaminone 6, which is designed to mimic the intermediate
of the cross-aldol reaction. This complex contains the sty-
rene functional group that will allow incorporation of this
unit into the nanogel structure.
The proline-catalysed reaction between aldehyde 1 and
acetone proceeds by a two-step mechanism. The first step
involves the formation of the activated enamine 7 between
the proline derivative and acetone. This subsequently reacts
with 1 to give intermediate 8, which is then hydrolysed to
the final aldol product 3. Scheme 3 shows the analogy be-
tween intermediate 8 and enaminone 6 and its resonance
form 9. Following polymerisation, acidic hydrolysis of the
enaminone leads to complete cleavage of the template leav-
ing a series of cavities containing proline-analogue groups
Scheme 1. Cross-aldol reaction between 4-nitrobenzaldehyde (1) and ace-
tone (2) leading to the corresponding b-hydroxyketone 3.
amine-based mechanism characteristic of aldolase type I en-
zymes. A novel method for active-site titration that involves
reaction of the catalyst with a substrate and leads to forma-
tion of an easily detectable product was developed; this
allowed accurate calculation of the catalytic parameters,
thus providing important information on the polymer
A
The aldol condensation is a powerful reaction in organic
À
chemistry for the formation of C C bonds, which gives rise
to at least one new chiral centre bearing a hydroxyl group
that can be further transformed. Control of the stereoselec-
tivity of this reaction is important for its practical applica-
tions in synthesis. The use of
natural aldolase or artificial
enzymes, like catalytic anti-
bodies,[12] gives important ad-
vantages, such as high stereo-
specificity and efficiency, but is
limited by the ranges of pH,
Scheme 2. Formation of the template–monomer intermediate 6 with quantitative yield in DMF at 408C.
temperature and organic sol-
vents. As part of our research
in the field of enzyme mimics,
and based on our extensive ex-
perience of catalytic antibod-
ies,[13] we are focused on devel-
oping polymeric nanostruc-
tured catalysts that can mimic
enzyme-like active sites. The
aim of our work is not to chal-
lenge the effectiveness of en-
zymes and organocatalysts in
their activity, enantioselectivity
and substrate scope, but in-
stead to use the imprinting
technology to complement
these catalysts by targeting
Scheme 3. Analogy between resonance structure 9 and intermediate 8 of the catalysed reaction; R=CH=CH2
or a polymeric matrix.
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Nanogels with Aldolase Type I Activity
FULL PAPER
located in specific positions designed to react with the ace-
tone to form the activated enamine.
but prepared in the absence of the template) was synthes-
ised under identical conditions. An additional set of control
polymers containing only the acrylamide and cross-linker
but not the proline derivative were also prepared.
The design of functional monomer 5, which contains a
benzenesulfonamide group, was based on the structure of a
successful proline analogue,[14] selected from the large pool
of organocatalysts available in the literature,[15] and deriva-
tised with a double bond that would make it suitable for
polymerisation. The rationale for this work was based on
the theory that the imprinting approach combined with the
acidity of the sulfonamidic proton would lead to increased
selectivity and catalytic activity as a result of stronger hydro-
gen bonding in the transition state and a more rigid environ-
ment. The choice of this molecule was determined by a com-
bination of factors, notably the evaluation of the data avail-
able in the literature regarding small organic catalysts, the
solubility and the feasibility of transforming the molecule
into a polymerisable derivative.
GPLC measurements were carried out on the nanogel sol-
utions in DMSO; polymethylmethacrylate (PMMA) stand-
ards in DMSO were used to create a calibration curve. This
allows a more accurate estimate of the molecular mass com-
pared with the commonly used linear polystyrene standards.
The data showed all preparations to have an average molec-
ular mass between 258 and 288 kDa. These values are in ac-
cordance with the value of 262 kDa obtained by Wulff et al.
for nanogel preparations with the same value of Cm and
measured by using membrane osmometry.[10] The average
particle size was measured for all of the nanogels (solutions
in DMSO containing 0.5 mgmLÀ1 of polymer) by using dy-
namic light scattering (DLS) and was found to be between
13 and 33 nm. These data were further confirmed by using
transmission electron microscopy (TEM), as illustrated in
Figure 1; the particles were stained with OsO4, a commonly
used oxidant that darkens particles by oxidising the double
bonds.
Template 4 was synthesised from the base-catalysed aldol
reaction between 1 and 2 to give a racemic mixture of 3 that
was further oxidised to the diketone. The reaction of forma-
tion of enaminone 6 was monitored by thin-layer chroma-
1
tography (TLC) and H NMR spectroscopy to identify the
experimental conditions leading to the complex formation
with high yields. Anhydrous DMF was found to be the best
solvent with complete formation of the enaminone product
obtained in 48 h at 408C, by using activated molecular
sieves under an inert atmosphere. Covalent complex 6 was
1
isolated, purified and fully characterised by using H, COSY
and 13C NMR spectroscopy and HRMS analyses. The exper-
imental data for the 13C spectra, supported by high-level
NMR prediction—obtained by DFT quantum chemical cal-
culations carried out with the parallel version of Gaussi-
an 03[16] (see the Supporting Information)—confirmed that
the nitrogen atom of proline specifically attacks the carbon-
yl group further away from the phenyl ring. Enaminone 6
has been shown to exist in a mixture of two geometric iso-
mers, E and Z, in a percentage of 55:45, as evaluated from
Figure 1. TEMimage of imprinted nanogels showing small size distribu-
tion (scale bar: 100 nm). The particles were stained by using OsO4 and
the average diameter of the particles is ꢀ20 nm.
1
the H NMR spectroscopic signal of the olefinic proton. The
complex was shown to be stable at 708C, the temperature at
which polymerisation occurs, and hydrolytic studies demon-
strated that the reaction is completely reversible by addition
of dilute HCl, therefore providing the experimental condi-
tions for the release of the template from the nanogels after
polymerisation.
The nanogels were prepared in DMF using high-dilution
radical polymerisation. This technique does not use surfac-
tants; instead stabilisation of the growing nanogels is ach-
ieved through steric control when the total monomer con-
centration is below the critical value, Cm, which is deter-
mined experimentally for each system.[17] Following an es-
tablished protocol, acrylamide-based nanogels (with Cm =0.5
and 80% cross-linker) were synthesised with a ratio of func-
tional monomer to acrylamide ranging from 1:1 to 1:5. After
removal of the template the isolated nanogels were dis-
solved in DMF and DMSO to give clear colloidal solutions.
For each imprinted-nanogel preparation the corresponding
non-imprinted polymer (containing the proline derivative,
The catalytic activity of the nanogels was investigated by
monitoring the formation of b-hydroxyketone 3 at l=
283 nm using reversed-phase HPLC. A series of experiments
were carried out at fixed aldehyde and catalyst concentra-
tions and with increasing equivalents of acetone to evaluate
the dependence of the initial rate (vi) on acetone concentra-
tion. Results showed that when using a large excess of ace-
tone (>1300 equiv) saturation of the catalyst is achieved.
This large excess of one reagent also allowed us to work
under pseudo-first-order conditions, thus simplifying the ki-
netic studies. To verify that there is no non-specific binding
of the aldehyde or the aldol product to the polymeric matrix
under the reaction conditions, experiments were carried out
by incubating the nanogel solutions with only the aldehyde
substrate or aldol product. Results after 24 h showed that
there was no change, thereby confirming that there is no al-
teration in initial substrate concentration and that product
inhibition is not present.
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M. Resmini et al.
Preliminary evaluation of different nanogels indicated
that preparation AS147, containing 10% functional mono-
mer, was the one with the highest catalytic activity and was
therefore fully characterised together with the correspond-
ing non-imprinted polymer AS133. Initial experiments car-
ried out using 2.5 mgmLÀ1 of imprinted nanogel proved too
fast for the HPLC monitoring protocol and initial rates
could not be determined accurately. Experiments performed
by varying the concentration of catalyst at constant substrate
concentration showed linear dependence of the initial rate,
as would be expected if catalysis were the result of the pres-
ence of the nanogels (Figure 2). From the data we chose a
Analysis by weighted non-linear regression of the vi
versus substrate (aldehyde 1) concentration ([S]0) data
shows good adherence to the Michaelis–Menten saturation
model (Figure 4) and provided the values for the kinetic pa-
*
Figure 4. Adherence of the initial rates, vi, for AS147 ( ) to the Michae-
lis–Menten equation is shown in comparison with the non-imprinted
À1
^
nanogel AS133 ( ). Both reactions were carried out with 0.25 mgmL
of nanogel in 20% DMSO in DMF at 258C with different concentrations
of 1 ([S]). The inset graph shows the linearity of the Hanes–Woolf plot.
rameters for AS147 of
V
max =1.92”10À7 (S.E.Æ1.58”
10À8)mminÀ1 (S.E.=standard error) and Km =7.15”10À3
(S.E.Æ1.14”10À3)m. Comparison of the AS147 data with
the corresponding non-imprinted AS133 data, shown in
Figure 4, demonstrates a significantly higher activity for the
imprinted nanogel. Kinetic data for the non-imprinted nano-
gel can also be fitted with a hyperbola, giving the following
Figure 2. Graph of initial rates, vi, versus catalyst concentration for the
imprinted polymer AS147, for which the concentration of aldehyde was
kept constant at 2 mm.
catalyst concentration (0.25 mgmLÀ1) that, in the substrate
range studied, showed good catalytic acceleration while al-
lowing accurate kinetic parameters to be obtained.
kinetic
parameters:
V
max =9.17”10À9
(S.E.Æ5.42”
10À10)mminÀ1 and Km =3.34”10À3 (S.E.Æ5.48”10À4)m. Both
nanogel preparations contain the proline analogue and the
only difference is that AS147 was incubated with the tem-
plate prior to the polymerisation. The difference in the
value of Vmax can be therefore taken as a measure of the im-
printing efficiency.
Kinetic experiments were performed by using
0.25 mgmLÀ1 of AS147, AS133 or AS134 (20% DMSO in
DMF containing 2.72m acetone) at 258C, and initial rates
(vi) were obtained by monitoring product 3 formation as a
function of time. Figure 3 shows the data for the imprinted
polymer AS147 at different aldehyde concentrations.
Imprinted polymers showing catalytic activity are often
described as enzyme mimics and the kinetic parameters, kcat
and Km, used in enzymology, are cited to characterise their
activity. The Michaelis–Menten saturation model can be ap-
plied only if two important requirements are fulfilled: 1) the
“initial” (steady-state) rate (vi) is measured; 2) the concen-
tration of substrate is considerably higher than the number
of active sites, so that the steady-state will be promptly es-
tablished, with the concentration of the catalyst–substrate
complex remaining essentially constant with time and the
substrate concentration approximating to its initial value.
Imprinted polymers can be described as “analogous to
polyclonal antibodies”, with their cavities containing a com-
bination of different binding and catalytic sites with differ-
ent characteristics. Interestingly, in our experience,[8,9] such
theoretical heterogeneity appears to be contradicted by the
observed functional homogeneity of the kinetic data, which
do not deviate from the single-site saturation model in the
substrate concentration range investigated, as evidenced by
the linearity of the Hanes–Woolf plot.
Figure 3. Determination of initial rates, vi, for the aldol reaction between
1 and 2 catalysed by AS147. The graph shows formation of the aldol
&
product 3 as a function of time with varying concentrations of 1 (
=
!
~
^
*
2 mm, =4 mm, =6 mm, =8 mm and =10 mm), while the concen-
tration of acetone is kept constant at 2.72m. The continuous lines repre-
sent the linear regression fitting. Reactions were carried out with
0.25 mgmLÀ1 of AS147 in a solution of 20% DMSO in DMF.
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Nanogels with Aldolase Type I Activity
FULL PAPER
As reported in the literature,[18] a mixture of catalysts with
different kinetic parameters would lead to plots of [S]/v
versus [S] following a markedly curved concave downwards
line. Data shown in Figure 3 confirm that AS147 is a nano-
gel preparation with high catalytic activity and displaying
functional homogeneity.
One of the key issues in comparing different imprinted
polymers is that the catalytic parameters reported are often
estimates, calculated using the total functional monomer
content instead of the actual concentration of active sites—
the functional molarity—in the preparation.
lytic constants, kcat 147/kcat 133 =18.8, provides an indication of
the imprinting effect. The requirements for an efficient cata-
lyst are 1) strong binding to the transition state and 2) weak
binding to the substrate in the ground state.[19] A direct mea-
surement of the efficiency of the imprinting strategy can be
obtained by comparing the values of kcat/Km, which repre-
sent the rate constant for the overall reaction. For AS147
kcat/Km =0.35 minÀ1 mÀ1
and
for
AS133
kcat/Km =
0.04 minÀ1 mÀ1, which demonstrate how the imprinting strat-
egy has led to a more efficient catalyst.
The enantioselectivity of the nanogels described in this
work originates from the chirality of the functional mono-
mer used. Experimental data confirmed that racemisation
did not occur during the synthesis of the proline-containing
monomer and, therefore, the enantioselectivity is expected
to be retained in the polymeric matrix. To determine the
enantioselectivity of the nanogels, reaction mixtures were
analysed by chiral HPLC using a Diacel Chiralcel OJ-H
column. The preliminary data for imprinted polymer AS147
show an enantiomeric excess of 62%, whereas non-imprint-
ed polymer AS133 gives an enantiomeric excess of 60%.
This result is not unexpected given that both nanogels con-
tain the optically active proline derivative and that the tem-
plate design was not targeting enantioselectivity. The inter-
esting result is that the presence of the polymeric matrix
and the imprinting with the template allow the enantioselec-
tivity to be retained. A more detailed investigation of the ef-
fects of nanogel composition on the enantioselectivity will
be required to further optimise these results.
In this work, active-site titration was achieved by treating
the nanogel preparations with a solution of 4-nitrophenyl
acetate (1 equiv, 558C, 5 d). This reagent selectively acetyl-
A
corresponding N-acetyl proline derivative and releasing
4-nitrophenolate, which is easily detected by using HPLC. It
was found that given the low concentration of polymer used
in the solutions, HPLC monitoring offered increased accura-
cy in the determination of the concentration of 4-nitrophe-
nolate compared with UV-visible spectroscopy. This value
corresponds to the concentration of proline units available.
Results of the titration show that when using nanogel solu-
tions of 0.25 mgmLÀ1 the following active-site concentra-
tions are available: AS147=79 mm and AS133=69 mm. As
expected, AS134, which does not contain any proline deriva-
tive, does not show any reactivity with 4-nitrophenyl acetate.
These values represent 54 and 47%, respectively, of the the-
oretical concentration of proline monomer in each prepara-
tion, when the corresponding yields are taken into account.
The data for the imprinted nanogel AS147 has been further
confirmed by carrying out a set of rebinding experiments
using template 4. A solution of polymer (0.25 mgmLÀ1) in
20% DMSO in DMF with diketone 4 (1.1 equiv) was react-
ed for 48 h at 408C. HPLC measurements allowed determi-
nation of the concentration of leftover diketone, and by dif-
ference, the concentration of template that had reacted with
the proline side chain contained in the nanogels. For AS147
the value of 77 mm was obtained, which is in accordance
with the data previously obtained.
Conclusion
We have successfully imprinted acrylamide-based nanogels
using a covalent approach, by formation of a reversible en-
aminone, to obtain nanogel preparations that show remark-
able catalytic activity, turnover and enantioselectivity. The
low concentrations used, together with the good solubility
and high imprinting efficiency make these materials a valua-
ble alternative to biochemical catalysts. The data presented
in this paper demonstrate the superiority of nanogels, when
the molecular-imprinting approach is used, over “bulk”
polymers for the generation of catalysts. This work repre-
sents a significant advance in the field of enzyme mimics
and confirms the potential of the imprinting approach. The
strategic design of the template molecule offers the invalu-
able opportunity to generate “ad hoc” catalysts with tailored
specificities and that are able to give access to a range of
molecules otherwise difficult to obtain.
It is important to note that the kinetic experiments pre-
sented in Figure 4, carried out with concentrations of 1 from
2 to 10 mm and 0.25 mgmLÀ1 of nanogel (equivalent to
77 mm active sites) clearly fulfil the requirements of the
A
centration ranging from 3.5 to 0.7 mol%, values that are
among the lowest in the literature covering this field. The
accurate value of the kinetic constant, kcat, can be calculated
from Vmax/[active site]=0.25”10À2 minÀ1. This value, al-
though still below the activity of the best aldolase enzymes,
is the highest ever achieved with imprinted polymers cata-
À
lysing C C bond formation. Moreover, given that the titra-
Experimental Section
tion experiments confirmed that both imprinted and non-im-
printed nanogels contain a similar concentration of proline
active sites, the large difference in rate acceleration ob-
served between AS147 and AS133 can be taken as a true in-
dication of successful imprinting. The ratio of the two cata-
Instrumentation: 1H (270 MHz), 13C (67 MHz) and COSY NMR spectra
were recorded by using a JEOL EX-270 instrument and 1H (400 MHz),
COSY and 13C NMR (100 MHz) spectra were recorded by using
a
1
Bruker 400 MHz instrument. H NMR peak multiplicities are reported as
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M. Resmini et al.
follows: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q
(quartet), m (multiplet). Low-resolution mass spectrometry (LRMS) data
were obtained by using a reversed phase HPLC HP Agilent 1200 system
combined with a Bruker Daltonics Esquire 3000 Plus mass spectrometer
possessing an MSD Trap. High-resolution mass spectral data was ob-
tained at the EPSRC National Centre, Swansea (UK) with ZQ4000
nano-electrospray. Enantiomeric excesses were measured with a Dionex
P680 HPLC pump with UVD 3400 detector and using a Daicel Chiralcel
OJ-H chiral column. TEMmeasurements were performed with a JEOL
1200 EX instrument (120 kV) with a beam at 908 on a 300 mesh copper
grid.
1H+2H; CH2-CH2-CH-C=O
+
-CH2-CH2-CH-C=O); 13C NM R
(67 MHz, CDCl3 +CD3OD, 258C, TMS): d=173 (-NH-CH-CONH-), 142
(CAr-SO2-), 141 (CAr-CH=CH2), 136 (-CH=CH2), 127 (Ar, 2C), 126 (Ar,
2C), 116 (-CH=CH2), 62 (-NH-CH-CONH-), 46 (CH2-CH2-NH-CH-), 29
(-CH2-NH-CH-CH2-), 24 ppm (-CH2-CH2-NH-CH-); HRMS (ESI): m/z
calcd for C13H17O3N2S: 281.0954 [M+H]+; found: 281.0953.
1-(4-(4-Nitrophenyl)-4-oxobut-2-en-2-yl)-N-(4-vinylphenylsulfonyl)pyrro-
lidine-2-carboxamide (6): Compound 5 (141 mg, 0.51 mmol) dissolved in
dry dimethylformamide (4 cm3) was added to 4 (105 mg, 0.51 mmol)
under a nitrogen atmosphere. Activated molecular sieves (0.4 nm) were
added to the mixture, which was stirred for 64 h at 408C. After 64 h, the
reaction was shown to be complete by using TLC analyses (dichlorome-
thane/methanol 8:2). The reaction mixture was purified by flash chroma-
tography (dichloromethane/methanol 95:5), without any previous work
up. A quantity of template–monomer complex was recovered (167 mg,
0.36 mmol, 71%). The desired compound was obtained pure as a mixture
of the E and Z geometric isomers. The compounds were characterised by
using 13C NMR spectroscopy. The 1H NMR and COSY-NMR spectra
4-Hydroxy-4-(4-nitrophenyl)butan-2-one (3): p-Nitrobenzaldehyde (2 g,
13.2 mmol) was dissolved in acetone (24 cm3, 18.7 g, 322.3 mmol) in an
ice bath. NaOH (2.6 cm3, 0.613 mmol, 0.24m) in aqueous solution was
added and the mixture was stirred for 20 min. TLC analyses with petro-
A
was complete. The acetone was removed by rotary evaporation and the
mixture was extracted three times with dichloromethane. The organic
layer was dried over MgSO4, filtered and concentrated under vacuum.
The crude product was purified by flash chromatography (petroleum
1
clearly confirmed the formation of the enaminone. M.p. 1068C; H NM R
([D6]DMSO, 400 MHz, 258C, TMS) (mixture of E and Z isomers; see the
Supporting Information): p-nitro-Ar: d=8.22 (d, J=8.00 Hz, 2H; Ar-H),
8.01 ppm (d, J=8.00 Hz, 2H; Ar-H); p-nitro-Ar: 8.14 (d, J=8.00 Hz,
2H; Ar-H), 7.83 ppm (d, J=8.00 Hz, 2H; Ar-H); benzenesulfonamide-
Ar : 7.72 (d, J=8.00 Hz, 2H; Ar-H), 7.53 ppm (d, J=8.00 Hz, 2H; Ar-
H); benzenesulfonamide-Ar: 7.67 (d, J=8.00 Hz, 2H; Ar-H), 7.28 ppm
(d, J=8.00 Hz, 2H; Ar-H); styrenic: 6.78 (dd, JCIS =12.00 Hz, JTRANS
16.00 Hz, 1H; CH2 =CH), 5.93 (d, JTRANS =16.00 Hz, 1H; CH2 =CH),
5.36 ppm (d, JCIS =12.00 Hz, 1H; CH2 =CH); styrenic: 6.61 (dd, JCIS
ether/diethyl ether 3:7).
A whitish solid was recovered (2.12 g,
10.14 mmol, 77%). 1H NMR (275 MHz, CDCl3, 258C, TMS): d=8.19 (d,
J=8.9 Hz, 2H; Ar-H), 7.51 (d, J=8.9 Hz, 2H; Ar-H), 5.26 (m, 1H; CH),
3.58 (d, J=3.45 Hz, 1H; OH), 2.84 (d, J=7.42 Hz, 2H; CH2), 2.20 ppm
(s, 3H; CH3); 13C NMR (100 MHz, CDCl3, 258C, TMS): d=209 (C=O),
150 (O2N-C-Ar), 147 (Ar-C-C-OH), 127 (Ar, 2C), 124 (Ar, 2C), 69 (Ar-
C-OH), 52 (CH2), 31 ppm (CH3).
=
=
1-(4-Nitrophenyl)butane-1,3-dione (4): b-Hydroxyketone
3
(1.99 g,
9.5 mmol) was dissolved in dichloromethane (30 cm3). Bu4NHSO4
(339 mg, 0.99 mmol) was subsequently added to the solution. An oxidis-
ing solution was prepared by adding K2Cr2O7 (1.149 g, 3.90 mmol) to
H2SO4 (35 cm3, 30%). The oxidising mixture was added to the reaction
mixture while stirring was applied. TLC analyses with petroleum ether/
diethyl ether (3:7) were used to monitor the reaction and showed com-
plete reaction after 20 min; the organic phase was then separated and the
aqueous phase was extracted twice with dichloromethane. The combined
organic layers were washed with saturated NH4Cl solution and dried
over MgSO4, filtered and concentrated under vacuum. The crude product
was purified by flash chromatography (petroleum ether/diethyl ether 3:7)
and recrystallised from cold diethyl ether. A yellow solid was recovered
(1.62 g, 7.82 mmol, 42%). 1H NMR (275 MHz, CDCl3, 258C, TMS): d=
15.89 (s, 1H; CH=C-OH), 8.27 (d, J=8.9 Hz, 2H; Ar-H), 8.00 (d, J=
8.9 Hz, 2H; Ar-H), 6.21 (s, 1H; CH), 2.21 ppm (s, 3H; CH3); 13C NM R
(67 MHz, CDCl3, 258C, TMS): d=196 (C=O), 179 (HO-C=CH), 150
(O2N-C-Ar), 140 (Ar-C-C-OH), 128 (Ar, 2C), 124 (Ar, 2C), 98 (HO-C=
CH-C=O), 26 ppm (CH3); IR (nujol): n˜ =1720 (C=O), 1219 cmÀ1 (C=
C-OH); HRMS (ESI): m/z calcd for C10H9NO4: 206.0459 [MÀH]+;
found: 206.0458.
12.00 Hz, JTRANS =16.00 Hz, 1H; CH2 =CH), 5.77 (d, JTRANS =16.00 Hz,
1H; CH2 =CH), 5.28 ppm (d, JCIS =12.00 Hz, 1H; CH2 =CH); enaminic
proton for major isomer: 5.58 ppm (s, 1H; C=O-CH=C-N); enaminic
proton for minor isomer: 5.43 ppm (s, 1H; C=O-CH=C-N); COSY-
NMR ([D6]DMSO, 400 MHz, 258C, TMS) (mixture of E and Z isomers;
see the Supporting Information); 13C NMR ([D6]DMSO, 67 MHz, 258C,
TMS) (mixture of E and Z isomers): d=(183.31/183.02), (175.45/175.12),
(163.27/162.82), (148.89/148.60), 144.34, (128.63, 127.53, 126.16, 126.05,
123.85, 123.68), 116.74, (93.05/92.33), (65.31/64.87), (50.00/49.62), (31.09/
30.89), (23.75/23.20), 17.87 ppm (see the Supporting Information);
HRMS (ESI): m/z calcd for C23H24O6N3S: 470.1380 [M+H]+; found:
470.1378.
Preparation of the imprinted nanogels: The synthesis of the template–
monomer complex was performed in situ under an N2 atmosphere at
408C in a crimp cap Wheaton vial for 48 h. Following the addition of a
mixture of N,N’-methylenebisacrylamide, acrylamide and azobisisobutyr-
onitrile in DMF the polymerisation mixture—with Cm =0.5, 80% cross-
linker and a ratio of catalytic monomer/acrylamide of either 1:1 (AS147,
AS133, AS134) or 1:5 (AS141, AS142, AS143)—was put into an oven at
708C. After removal of the template, the isolated nanogels were analysed
to assess their solubility and it was shown to be good in DMSO, DMF or
a mixture of both. Each polymer preparation imprinted with the template
was complemented by the corresponding non-imprinted polymer and a
control polymer that did not contain functional monomer. The character-
isation of the nanogel particles were performed by using different tech-
niques that included DLS, with solutions containing 0.5 mgcmÀ3 of nano-
gel in DMSO, gel permeation liquid chromatography (GPLC) using a cal-
ibration curve obtained with PMMA and finally with TEM. The particles
were stained with OsO4, a strong oxidant widely used to darken particles
by oxidising the double bonds. All of the data obtained show that the
average particle size measures approximately 20 nm, with a very low dis-
persity, and with an average molecular weight of 260 kDa.
Active-site titration: Solutions of polymer (4.5 mgcmÀ3) were prepared in
20% DMSO in DMF (using AS147 (18.3 mg), AS133 (18.3 mg) and
AS134 (18.1 mg) dissolved in anhydrous DMF (2.8 cm3) and anhydrous
DMSO (0.8 cm3) in three different vials). A solution of 4-nitrophenylace-
tate (0.4 cm3, 26.46 mm) in DMF was added at t=0 min to the polymer
solutions to obtain a final concentration of 2.646 mm. As a reference, a
2.646 mm solution of 4-nitrophenylacetate on its own and a 2.646 mm so-
lution of 4-nitrophenylacetate in the presence of proline benzenesulfona-
(S)-N-(4-Vinylphenylsulfonyl)pyrrolidine-2-carboxamide (5): Dicyclohex-
ylcarbodiimide (963 mg, 4.67 mmol) was added to Fmoc-proline (1.284 g,
3.82 mmol) in dichloromethane (20 cm3) at 08C. After 1 h, 4-vinylben-
zenesulfonamide (0.697 g, 3.80 mmol; synthesis in the Supporting Infor-
mation) and dimethylaminopyridine (98 mg, 0.80 mmol) were added at
08C. The reaction mixture was stirred for 48 h and monitored by using
TLC analyses (dichloromethane/ethyl acetate 8:2). The reaction mixture
was filtered, concentrated under vacuum and purified by flash chroma-
tography (petroleum ether/ethyl acetate 6:4) to afford a white solid
(0.957 g, 1.90 mmol, 50%). The solid was added to a 30% aqueous am-
monia solution (30 cm3) and homogenised with tetrahydrofuran (8 mL).
The solution was allowed to react for 16 h and was extracted with diethyl
ether to remove any impurity. The aqueous phase was freeze-dried for
24 h and purified by flash chromatography (dichloromethane/methanol
95:5) to afford a white solid (0.746 g, 70%). M.p. 201–2068C; 1H NM R
(275 MHz, CDCl3 +CD3OD, 258C, TMS): d=7.81 (d, J=8.39 Hz, 2H;
Ar-H), 7.39 (d, J=8.39 Hz, 2H; Ar-H), 6.67 (dd, JCIS =10.92 Hz, JTRANS
=
18.07 Hz, 1H; CH2 =CH), 5.76 (d, JTRANS =18.07 Hz, 1H; CH2 =CH),
5.30 (d, JCIS =10.92 Hz, 1H; CH2 =CH), 4.00 (m, 1H; N-CH-C=O), 3.28
(m, 2H; N-CH2-CH2), 2.23 (m, 1H; CH2-CH2-CHCO), 1.89 ppm (m,
7064
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Chem. Eur. J. 2008, 14, 7059 – 7065

Nanogels with Aldolase Type I Activity
FULL PAPER
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lution was added to 10 mL of internal standard and 25 mL of this new so-
lution was injected into the HPLC instrument. The product concentration
was determined by using a calibration curve of the ratio of area of 4-ni-
trophenol out of area of internal standard (methyl-4-nitrobenzoate).
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and Queen Mary (studentship to D.C.) and by the EC by means of the
Marie Curie RTN NASCENT (MRTN-CT-2006-033873, for A.S. and
K.F.).
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Received: April 9, 2008
Published online: July 4, 2008
Chem. Eur. J. 2008, 14, 7059 – 7065
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7065