Molecularly imprinted artificial esterases with highly specific active sites and precisely installed catalytic groups

Article abstract of DOI:10.1039/c8ob01584h

A difficult challenge in synthetic enzymes is the creation of substrate-selective active sites with accurately positioned catalytic groups. Covalent molecular imprinting in cross-linked micelles afforded such active sites in protein-sized, water-soluble nanoparticle catalysts. Our method allowed a systematic tuning of the distance of the catalytic group to the bound substrate. The catalysts displayed enzyme-like kinetics and easily distinguished substrates with subtle structural differences.

Full text of DOI:10.1039/c8ob01584h

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Molecularly imprinted artificial esterases with
highly specific active sites and precisely installed
catalytic groups†
Cite this: DOI: 10.1039/c8ob01584h
Received 5th July 2018,
Accepted 24th July 2018
Lan Hu
and Yan Zhao
*
DOI: 10.1039/c8ob01584h
rsc.li/obc
A difficult challenge in synthetic enzymes is the creation of sub- rationally in an enzyme-resembling water-soluble nanoparticle.
strate-selective active sites with accurately positioned catalytic The work represents our continued efforts in creating bio-
groups. Covalent molecular imprinting in cross-linked micelles mimetic catalysts from simple building blocks.2 The resulting
afforded such active sites in protein-sized, water-soluble nano- artificial enzymes displayed esterase-like catalysis in the hydro-
particle catalysts. Our method allowed a systematic tuning of the lysis of activated esters and, more importantly, were able to
distance of the catalytic group to the bound substrate. The cata- distinguish substrates with subtle structural differences. The
lysts displayed enzyme-like kinetics and easily distinguished sub- highlight of the method is the facile construction of the sub-
strates with subtle structural differences.
strate-specific active site, the installment of the catalytic group
at predetermined location, and the ability to fine-tune the dis-
tance of the catalytic group to the reactive group. Although
hydrolysis is used as the model reaction, the method is general
and should be useful in the design of other artificial enzymes
with efficient and selective catalysis.
The artificial esterase was constructed through micellar
imprinting developed in our laboratory.3 Molecular imprint-
ing4 is a powerful technique to create guest-complementary
binding sites,5 and has been used to create synthetic catalysts
for a number of reactions including hydrolysis, the Diels–Alder
reaction, and aldol reactions, sometimes with remarkable acti-
vities.4a,6 However, several inherent challenges7 make it
difficult to manipulate imprinted active sites with molecular
precision, thus limiting their usage in catalyst designs.
The synthesis of our nanoparticle catalysts is shown in
Scheme 1, involving double cross-linking of the template-con-
taining micelle of 1 (Scheme 1).3,8 The surface-cross-linking
was achieved using diazide 2 by the click reaction and the
core-cross-linking using DVB by AIBN-initiated free radical
polymerization. The nanoparticles are typically decorated with
sugar-derived ligand 3 for water-solubility.
The key design of our material is in the template: 4 has a
methacrylate and thus will polymerize with 1 and DVB to be
covalently attached to the micelle during the core cross-
linking. The molecule consists of two parts: the cyan moiety is
used as a photo-cleavable placeholder to introduce the cata-
lytic pyridyl group through amine 5a–c; the red moiety is
similar to the substrate (p-nitrophenyl hexanoate or PNPH) in
the hydrolysis. Through such a design, we can create an active
site highly specific to any substrate in principle, with a nearby
catalytic group installed at predetermined position and angle.
Enzymes routinely perform extremely challenging catalysis
such as nitrogen fixation and selective C–H activation with
remarkable efficiency under ambient conditions. They also
tend to be highly selective among substrates with similar
intrinsic chemical reactivity. The center of these feats is their
active sites, where functional groups convergent from the
folded peptide chain work cooperatively, sometimes with co-
factors, to achieve the desired catalytic functions. In recent
years, chemists have increasingly recognized the power of a
functional, active-site-like microenvironment to enhance the
catalyst’s activity and selectivity.1 For these biomimetic active
sites, features of molecular recognition are introduced, in
addition to catalytic groups, to facilitate the entry of the
desired substrate over structural analogues and, in turn, to
impart selectivity.
Nonetheless, in comparison to those found in enzymes,
synthetic models of active sites cannot be easily tuned to
accommodate substrates of different size and shape. Creation
of a three-dimensional nanospace specific to a guest molecule
is already a difficult challenge, let alone its multifunctionaliza-
tion with molecular level precision for efficient chemical
catalysis.
In this work, we report a method to introduce molecular
recognition features and catalytic functionalities readily and
Department of Chemistry, Iowa State University, Ames, Iowa 50011-3111, USA.
E-mail: zhaoy@iastate.edu; Fax: +1-515-294-0105; Tel: +1-515-294-5845
†Electronic supplementary information (ESI) available: Experimental details,
fluorescence titration curves, and additional figures. See DOI: 10.1039/
c8ob01584h
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Scheme 1 Preparation of artificial esterase MINP-DMAP by micellar imprinting of template 4.
Compared to our earlier examples,2c,d this design is expected
to yield strong substrate-selectivity in the catalysis as a result
of the shape-selectivity of the active site.
Synthesis and characterizations of MINPs followed pub-
lished procedures.3,8 Generally, the surface- and core-cross-
1
linking were monitored by H NMR spectroscopy. The surface-
cross-linking has been verified by mass spectrometry after the
1,2-diol in the cross-linked 2 was cleaved.9 Dynamic light scat-
tering (DSL) afforded the molecular weights of the nano-
particles and their size (∼5 nm with ligand 3). The DLS size
has been confirmed by transmission electron microscopy
(TEM).10
UV irradiation of the MINP with covalently attached 4 led to
photolytic cleavage of the well-known o-nitrobenzyl linkage to
free the template.8b To confirm the removal of the template,11
we studied the binding of guest 6 by MINP-COOH, the inter-
mediate nanoparticle to the final MINP-DMAP. We used the
acid because the carboxylate was soluble enough in water for
us to perform the titration. The guest is expected to fit within
the binding site formed by the removal of the template due to
its similarity to 4. The carboxyl group of 6 should also hydro-
Fig. 1 ITC curve obtained at 298 K from titration of MINP-COOH with 6
in 10 mM HEPES buffer (pH = 7). MINP-COOH = 80 µM in the cell. The
concentration of 6 in the syringe was 1.0 mM.
The above procedures are expected to yield an active site
gen-bond with the carboxyl group of MINP-COOH through the inside the hydrophobic core of the MINP that is highly specific
carboxylic acid dimer.8b Isothermal titration calorimetry (ITC) for binding the substrate (PNPH). The active site also contains a
gave an association constant of Ka = (1.85 0.22) × 104 M−1 in powerful nucleophilic catalyst (DMAP or 4,4-dimethylamino-
HEPES buffer (Fig. 1). The titration also revealed that the pyridine)13 in the proximity of the bound ester (Scheme 1). We
average number of the binding site per nanoparticle was N = expect the strong nucleophile would attack the ester bound in the
0.98 0.06. The number agreed well with the amount of tem- active site, followed by the removal of the p-nitrophenyl leaving
plate used in the MINP preparation. Because each MINP was group and the breakdown of the positively charged tetrahedral
estimated by dynamic light scattering to have ∼50 cross-linked intermediate by water to regenerate the catalyst. Unlike enzymes
surfactants. A 50 : 1 ratio of [1]/[4] is expected to afford an that can only use biologically available nucleophiles, our syn-
average of one template per nanoparticle.3 The ITC binding thetic catalysts can use more powerful groups such as DMAP.
thus confirmed a clean removal of the template under the Template 4 was designed so that the pyridyl nitrogen would point
photolysis condition, consistent with an earlier report.8b
to the carbonyl for the intended nucleophilic catalysis. By chan-
MINP-COOH was then derivatized through amide coupling ging the length of the tether (n = 1–3) in the amine (5a–c), we can
using EDCI. To ensure high-yielding conversion, a 10-fold tune the distance of the catalytic pyridyl to the substrate bound
excess of EDCI and the amine was used. The condition8b has in the active site. Product inhibition is not a concern because the
been shown to successfully convert a carboxylic group inside products (hexanoic acid and p-nitrophenol) are more hydrophilic
the MINP pocket to the corresponding amide.12
than the substrate and should migrate into the aqueous solution.
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optimal for the catalysis. The butylene oxide tether in 5c prob-
ably is too long and might have interfered with the substrate
binding or catalysis (see below for additional discussion).
Fig. 2 Absorbance at 400 nm as a function of time for the hydrolysis of
PNPH in a 25 mM HEPEs buffer (pH 8.0) at 40 °C. The data sets corres-
pond to hydrolysis in the absence of catalysts (△) and catalyzed by
□
◊
MINP-DMAP-5a ( ) and DMAP( ), respectively. [PNPH]
= 50 µM.
[MINP-DMAP-5a] = [DMAP] = 15 µM.
MINP-DMAP-5a and MINP-DMAP-5b, our best catalysts, both
showed remarkable selectivity for the intended substrate
(PNPH) over other activated ester analogues. The observed rate
constants for 7–9 were 14–40 times slower in comparison to
that of PHPH. Thus, the active site was highly selective as
designed. Branching on the alkyl chain (in 7), change of the
substituent on the phenyl ring (in 8), and an increase of hydro-
philicity on the carbonyl side (in 9) could all be distinguished
with ease. In contrast, molecular DMAP in Table 1 afforded
much slower kinetics and much lower selectivity in the hydro-
lysis—this should reflect the intrinsic reactivity of the sub-
strate as DMAP in solution in Fig. 1 gave essentially the same
reaction rate as the hydrolysis in buffer. Take PNPH and the
hydrophilic 9 as an example: with molecular DMAP, the ratio
of the rate constants of the two was 1.4/1 but, with
MINP-DMAP-5b, became as high as 23/1.
All the MINPs displayed enzyme-like Michaelis–Menten
kinetics described by v0 = Vmax[S0]/(Km + [S0]), in which v0 is
the initial velocity, S0 the initial substrate concentration, Vmax
the maximum velocity at a particular enzyme concentration,
and Km the Michaelis constant that measures the binding
affinity of the substrate to the enzyme (ESI†).15 As shown by
Table 2, MINP-DMAP-5a and MINP-DMAP-5b behaved very
similarly but MINP-DMAP-5c was more different. The rate
acceleration factor (kcat/kuncat) ranged from 11 000 to 17 000 for
PNPH at pH 8.
Fig.
2 shows the hydrolysis of PNPH catalyzed by
MINP-DMAP-5a in aqueous buffer (pH 8). The reaction was
monitored by the formation of p-nitrophenoxide with a strong
absorption at 400 nm. Our experiments show that the ester
hydrolyzed negligibly in pH8 buffer, with or without molecular
DMAP. Protonated DMAP has a pKa of 9.7.13a At lower pH, the
pyridyl nitrogen is protonated and loses its catalytic activity.
The high activity of MINP-DMAP-5a indicates that the pyridyl
behaved very differently inside the cross-linked micelle.
Enzymes frequently use the microenvironment of the active
site to shift the pKa of acidic/basic groups.14 Because ionic
groups are better solvated by water than hydrocarbon, it is
more difficult to protonate an amine (or deprotonate a car-
boxylic acid) in a hydrophobic pocket. In addition, positive
charges nearby are often used by enzymes to hinder the proto-
nation of an amine. We can easily imagine both principles in
operation within the positively charged MINP.
Table 1 summarizes the rate constants of the different
MINPs. In addition to PNPH, we examined three other acti-
vated esters (7–9) to understand the selectivity of our catalysts
as a result of the shape and hydrophobicity of the active site.
As shown by entry 1, the hydrolysis of PNPH occurred in the
order of MINP-DMAP-5a ≈ MINP-DMAP-5b > MINP-DMAP-5c.
Thus, the location of the pyridyl group inside the MINP active
site did affect the hydrolysis, as expected. Since the active site
was created from template 4, which has an ethylene oxide
tether between the substrate-like moiety and the phenyl group,
we did anticipate MINP-DMAP(5a) or MINP-DMAP(5b) to be
Earlier, we saw that MINP-DMAP-5c was the least active
catalyst among the three for PNPH. The Michaelis–Menten
study revealed that the culprit was the weak binding of the
substrate, as shown by the largest Km value of this catalyst
among the three. The Vmax and kcat (=Vmax/[enzyme concen-
Table 1 Kinetic data for the hydrolysis of activated estersa
Rate constant k × 104 (s−1
)
Entry
Ester
MINP-DMAP(5a)
MINP-DMAP(5b)
MINP-DMAP(5c)
DMAP
1
2
3
4
PNPH
54.8 0.1
1.4 0.1
2.3 0.4
3.9 0.1
56.7 3.0
2.0 0.3
1.6 0.4
2.5 0.3
42.7 1.8
0.50 0.17
0.14 0.01
0.04 0.01
0.35 0.03
7
8
9
—
—
—
a The reaction rates were measured in 25 mM HEPES buffer (pH 8.0) at 40 °C. The background hydrolysis of PNPH in the same buffer was very
slow, with an estimated rate constant of 0.15 × 10−4 s−1. [PNPH] = 50 µM. [MINP-DMAP] = 15 µM.
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Table 2 Michaelis–Menten parameters for the hydrolysis of PNPH catalyzed by MINP-DMAP catalysts and some natural enzymesa
Entry
Catalyst
Vmax (µM s−1
)
Km (µM)
kcat (s−1
)
kcat/Km (M−1 s−1
)
1
2
3
4
5
MINP-DMAP-5a
MINP-DMAP-5b
MINP-DMAP-5c
BCA
0.85 0.07
0.98 0.01
1.28 0.02
0.033 0.003
7.33 0.83
405 12
0.17 0.01
0.196 0.001
0.259 0.003
0.0042 0.0004
0.73 0.08
420
440
387
18
442
1
663 10
230 40
670 80
α-Chymotrypsin
1090
a The reaction rates were measured in 25 mM HEPEs buffer (pH 8.0) at 40 °C in duplicates.
tration]) values of MINP-DMAP-5c were actually the highest have high selectivity among structural analogues. The cross-
among the three MINP catalysts. linked micelle is a powerful platform to prepare strong and
For comparison purposes, we included data for bovine car- selective receptors for many types of molecules including
bonic anhydrase (BCA) and α-chymotrypsin, which are fre- small-molecule drugs,8c carbohydrates,18 and peptides.10,19
quently used in the literature to catalyze the hydrolysis of para- This work shows that the micellar imprinting method could
nitrophenyl esters16 (note that these enzymes were not natural be used rationally to create well-defined, substrate-specific
esterases designed to hydrolyze the activated esters). As our active sites and accurately installed catalytic groups. A notable
data shows, the MINP-DMAPs were far more active than BCA feature of our catalysts is the ability to fine-tune the position of
and ∼40% as efficient as α-chymotrypsin, evident from the the catalytic group with respect to the functional group to be
catalytic efficiency values (kcat/Km).15 Many artificial zinc transformed. This could be an extremely useful feature in the
enzymes have been produced to mimic carbonic anhydrase design of future artificial enzymes.
and their activities were often evaluated in their catalytic
hydrolysis of p-nitrophenyl acetate (PNPA).17 The catalytic
efficiency of our DMAP-MINPs (kcat/Km = 390–440 M−1 s−1
)
Conflicts of interest
compared favorably with the reported values (kcat/Km = 3–180
M−1 −1) for these artificial enzymes, especially considering
There are no conflicts to declare.
s
that PNPH was less reactive than PNPA in the background
hydrolysis.
Acknowledgements
Many reported synthetic esterases displayed low turnovers
(10–50) in the hydrolysis of p-nitrophenyl esters, due to
product inhibition.17 When a large excess (500 equiv.) of
PNPH was used, MINP-DMAP-5b, our most efficient catalyst,
was found to function even at high turnovers (Fig. 3). The turn-
over number (TON) was calculated to be 390 at 600 min. The
much higher turnovers observed suggest that product inhi-
bition was much less of a problem in our catalyst than those
reported in the literature.
Although many artificial enzymes have been reported
including ones that could hydrolyze activated esters,1 creation
of tailor-made active sites for arbitrary substrates remains a
difficult challenge, making it difficult for synthetic catalysts to
We thank NSF (DMR-1464927 and CHE-1708526) for support-
ing this research.
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