An Enzyme Model Which Mimics Chymotrypsin and N-Terminal Hydrolases

Article abstract of DOI:10.1021/acscatal.0c02121

Enzymes are the most efficient and specific catalysts to date. Although they have been thoroughly studied for years, building a true enzyme mimic remains a challenging and necessary task. Here, we show how a three-dimensional geometry analysis of the key catalytic residues in natural hydrolases has been exploited to design and synthesize small-molecule artificial enzymes which mimic the active centers of chymotrypsin and N-terminal hydrolases. The optimized prototype catalyzes the methanolysis of the acyl enzyme mimic with a half-life of only 3.7 min at 20 °C, and it is also able to perform the transesterification of vinyl acetate at room temperature. DFT studies and X-ray diffraction analysis of the catalyst bound to a transition state analogue proves the similarity with the geometry of natural hydrolases.

Full text of DOI:10.1021/acscatal.0c02121

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Research Article
An Enzyme Model Which Mimics Chymotrypsin and N‑Terminal
Hydrolases
a
́
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Jose J. Garrido-Gonzalez, M Mercedes Iglesias Aparicio, Miguel Martínez García, Luis Simon,
́
́
Francisca Sanz, Joaquín R. Moran,* and Angel L. Fuentes de Arriba*
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ABSTRACT: Enzymes are the most efficient and specific catalysts to date. Although they have been thoroughly studied for years,
building a true enzyme mimic remains a challenging and necessary task. Here, we show how a three-dimensional geometry analysis
of the key catalytic residues in natural hydrolases has been exploited to design and synthesize small-molecule artificial enzymes which
mimic the active centers of chymotrypsin and N-terminal hydrolases. The optimized prototype catalyzes the methanolysis of the acyl
enzyme mimic with a half-life of only 3.7 min at 20 °C, and it is also able to perform the transesterification of vinyl acetate at room
temperature. DFT studies and X-ray diffraction analysis of the catalyst bound to a transition state analogue proves the similarity with
the geometry of natural hydrolases.
KEYWORDS: artificial hydrolase, enzyme mimic, chymotrypsin, N-terminal hydrolase, organocatalysis, hydrogen bond, oxyanion hole,
transesterification
Chemists have tried to mimic hydrolases for a long time.
Especially remarkable are the contributions of Lehn in
enantioselective thiolysis reactions6 as well as those of
Breslow7 and Cram8 in the hydrolysis of nitrophenyl esters,
with reaction rate increases up to 7.5 × 105 and 1011,
respectively. While most of these enzyme models rely on the
formation of inclusion complexes of activated esters inside
cyclodextrins, cucurbiturils, cavitands, and other macro-
cycles,7,9 artificial enzymes which try to mimic the geometry
of the catalytic triad of serine or cysteine proteases are much
scarcer. One of the reasons is their challenging synthesis: each
of the reactive groups in the active site corresponds to different
domains of the protein backbone chain and are only brought
INTRODUCTION
■
Enzymes are the only catalysts that operate in biochemistry;
however, they have also found important applications in
industry since, to date, many processes cannot be carried out
economically with synthetic catalysts.1 The highly selective 11-
hydroxylation of 11-deoxycortisol to yield cortisol by 11-β-
hydroxylase is a good example.2
In particular, hydrolytic enzymes, which are a family of more
than 200 enzymes responsible for lipid, sugar, and peptide
hydrolysis, have found important applications in fine chemical
production, the food and detergent industry, paper manufac-
turing, chemical degradation processes, biomass conversion,
and the pharma industry.3 However, their low stability, high
cost, and risk of denaturalization under extreme pH,
temperature, or salt conditions make them tricky substances
to work with. Hence, there is an increasing interest in
developing artificial hydrolases for industrial, academic, and
therapeutic purposes. Suh has recently outlined the importance
of artificial proteases as new catalytic drugs4 for amyloid
diseases.5
Received: May 13, 2020
Revised: August 13, 2020
Published: August 31, 2020
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© XXXX American Chemical Society
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Figure 1. (A) General mechanism of hydrolytic enzymes. (B) Active center of bovine γ-chymotrypsin (PDB 1AB9). (C) Active center of human
aspartylglucosaminidase (PDB 1APY). (D) Catalytic features of the hydrolase mimic designed in this work.
together when the protein folds in its tertiary structure (Figure
1B). Ema and Sakai10 and Schafmeister11 have reported small-
molecule enzyme mimics able to simulate the catalytic triad of
hydrolytic enzymes. Although large acceleration rates were
obtained, their systems are limited to highly activated vinyl
trifluoroacetate esters. Several research groups have tried to
mimic the three-dimensional arrangement of the catalytic
groups in chymotrypsin by using polymer imprinting
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Figure 2. Hydrolase mimics synthesized in this work.
techniques12 or by attaching the catalytic groups onto
polymeric materials13 or micelles;14 however, the results are
still not optimized enough to be applicable in industry. Connal
has recently highlighted the state of the art of synthetic
catalysts inspired by hydrolytic enzymes which is still far from
reproducing reactions of native enzymes on esters, amides, and
proteins under mild conditions.15 In our opinion, a rational
understanding of the geometry of the active center of
hydrolytic enzymes, on the basis of an analysis of the X-ray
structure of hydrolases, could be a promising starting point to
design a true enzyme mimic. The synthesis and testing of small
molecules which try to mimic the catalytic groups of the active
center of hydrolases could shed some light on the importance
of the geometry of the catalytic groups of the active site to
achieve high levels of catalysis. Herein, we wish to report our
findings.
RESULTS AND DISCUSSION
■
Kinetic and Computational Studies. Taking inspiration
from natural chymotrypsin and N-terminal hydrolases, we
synthesized enzyme mimic 1 (Figure 2, see the Supporting
Information for its preparation). Catalyst 1 combines an
oxyanion-hole mimic, based on an isophthalic acid moiety, a
nucleophilic hydroxyl group, and a basic dimethylamine group.
The isophthalic acid spacer, with two NHs and an aromatic
C−H that can also behave as an H-bond donor, has been
successfully used in the literature as an oxyanion-hole mimic.22
The oxyanion hole and hydroxyl group are separated by two
carbon atoms (as in the chymotrypsin catalytic triad; Figure
1B). Concurrently, we hope that the 1,2-amino alcohol moiety
plays the same role as terminal threonine in N-terminal
hydrolases, in which −NH2 and −OH groups are in a 1,2-
disposition (Figure 1C). 1,2-Amino alcohols have already
shown transacylation activity with activated esters.23
Among all the different families of proteolytic enzymes, N-
terminal hydrolases16 are a superfamily of enzymes which lack
the histidine−aspartate combination of the catalytic triad,17
being replaced by a terminal −NH2 group which stems from a
terminal serine, threonine, or cysteine (Figure 1C). This fact
makes them attractive candidates for the development of
artificial enzymes, since the combination of a simple 1,2-amino
alcohol with a suitable oxyanion hole could provide a decent
enzyme mimic. Although one would expect the amino group to
be protonated in aqueous solution, the protein environment
reduces the pKa of the α-amino group to a value close to
histidine’s first pKa (6.2).18 On the other hand, the usual
hydrophobic pocket may not even be necessary, since
chymotrypsin excludes water from the active center during
its reactions.19 According to Warshel, the main factor to
explain enzymatic catalysis comes from a preorganized polar
environment which is complementary with the guest transition
state and which is mainly obtained through H bonds.20 In fact,
site-directed mutagenesis studies have shown that most of the
catalytic activity of chymotrypsin (1010) can be explained with
the H bonds of the oxyanion hole (104) and the catalytic triad
(106).21 Therefore, a similar array of H bonds from a small
synthetic molecule may provide catalysis comparable to that of
natural enzymes.
The classical mechanism of hydrolytic enzymes through the
well-known catalytic triad requires an acylation step of a serine
or cysteine residue (II → IV, Figure 1A) and subsequent
hydrolysis of the acyl intermediate by H2O (IV → VI, Figure
1A). It has been observed for both natural and artificial
enzymes that the acylation step is faster than the acyl
intermediate hydrolysis,10,23,24 although these results may be
biased, as most examples use activated esters as substrates,
which possess a good leaving group that is not available for the
second step. This difference in the reaction rates causes a lack
of turnover,25 as once the hydroxyl group of the enzyme mimic
is acylated (IV, Figure 1A) it does not undergo further
reaction.
To complement previous studies, we decided to tackle the a
priori, more challenging acyl intermediate hydrolysis with
enzyme mimic 1 (Figure 2). Hence, by aiming at the rate-
limiting step of the mechanism, we should have a fast
estimation of the possibilities of this scaffold to be used in
hydrolysis or transesterification reactions. To test the catalytic
activity of compound 1, acetylation of its hydroxyl group was
first performed in Ac2O, and after isolation of acetylated
catalyst 1, it was transferred to an NMR tube and dissolved in
deuterated methanol. Catalyst 1 was designed to be soluble in
chloroform, which is a solvent that can mimic the hydrophobic
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environment of the hydrolase active site. However, to speed up
and simplify the study, as pseudo-first-order kinetics can be
applied, deuterated methanol was used as the solvent and the
and amino groups in a 1,3-amino alcohol arrangement could
be a key aspect to circumvent this problem. An extensive
conformational search for a model compound (see the
Supporting Information) suggested that the equatorial
disposition of all the catalytic groups, required to make the
proton transport feasible, is the most stable conformation by
more than 4.6 kcal/mol over the most stable axial disposition
(Figure 3). This geometry provides a distance of 2.75 Å
1
deacetylation kinetics were followed by H NMR at 20 °C.
According to kinetic experiments (see the Supporting
Information), deacetylation of 1a by CD3OD took place with
a half-life of 187 min (Table 1, entry 1). This result was slower
Table 1. Half-Life (min) of the Methanolysis Reaction of
Acetylated Catalysts 1a−7a
a
entry
catalyst
t1/2 (min)
1
2
3
4
5
6
7
1a
2a
3a
4a
5a
6a
7a
187
165
87
17
12
Figure 3. Most stable conformation of a model of catalysts 3−7
showing the equatorial arrangement of catalytic groups.
15
3.7
a
Ca. 10 mg of the acetylated catalyst 1a−7a was dissolved in 400 μL
between OH and NH2, which is close to the 2.92 Å between
the same groups in N-terminal hydrolase human aspartylglu-
cosaminidase or the 3.02 Å between OH and imidazole in
bovine γ-chymotrypsin. In this enzyme one of the NHs in the
oxyanion hole is 3.09 Å from the serine OH, while in the
model of Figure 3 this distance is around 2.66 Å.
Thus, this motif was incorporated in catalyst 3. Pleasingly,
kinetic studies showed that the catalyst 3 skeleton is almost
twice as good as that of catalyst 2, with a half-life of 87 min
(Table 1, entry 3).
Next, a series of modifications were introduced in the
catalyst 3 structure to improve its catalytic activity. While the
replacement of the dimethylamino group for a more basic
pyrrolidine entailed a 5-fold decrease in half-life (Table 1, entry
4), the introduction of a second NH group via an isophthalic
acid moiety to construct the oxyanion-hole motif allowed
reducing the half-life to 12 min (Table 1, entry 5).
To our surprise, increasing the acidity of the second NH in
the oxyanion hole of catalyst 6 did not reduce the reaction rate
(Table 1, entry 6). Probably the rigidity of the dihydronaph-
thalene scaffold prevents the formation of the required short H
bond between the acetate carbonyl group and the aromatic
isophthalic NH, favoring the formation of some kind of dimer.
Indeed, the 1H NMR spectrum of 6a showed a shielding of 0.4
ppm for the isophthalic NH in comparison with the same NH
in free catalyst 6 (Figure 4).
of CD3OD, and 1H NMR spectra were recorded periodically at 20 °C.
The half-life was determined by H NMR integration.
1
than expected, as the biological conversion of ethyl acetate to
ethanol takes place in only 5−10 min.26 Probably, the free
rotation of the methylene groups generates nonproductive
conformations in the catalyst structure due to the establish-
ment of intramolecular H bonds between the basic dimethyl-
amine nitrogen and the oxyanion hole, which hamper catalyst
activity.
Although Breslow and others have shown that more
structural flexibility provided improved outcomes in enzyme
mimics,27 in this case it is necessary to anchor the different
catalytic groups to a rigid scaffold in order to prevent
nonproductive conformations. This is a challenging task,
because if the distances between the different groups are not
appropriate, the catalyst will not show any catalytic activity.
After screening different possibilities, we chose a rigid template
based on a dihydronaphthalene scaffold to anchor the basic
group, nucleophilic hydroxyl group, and oxyanion-hole moiety
(Figure 1D). The presence of the aromatic ring confers
structural stability to the molecule and facilitates the synthesis.
With these premises in hand, catalyst 2 was prepared. For
the sake of synthetic simplicity, the oxyanion-hole role was
performed by a single NH. Under these conditions, a small
reduction in the half-life of 20 min was observed in comparison
with catalyst 1 (Table 1, entry 2).
To shorten the distance between this NH bond and the
carbonyl group, an oxyanion-hole mimic with a shorter
distance between both NHs was explored. In this regard, 2,6-
pyridinedicarboxylic acid may be a reasonable choice, since
aromatic C−N distances (1.33 Å) are shorter than aromatic
C−C distances (1.39 Å), the pyridine nitrogen nonbonding
lone pair should direct the NHs toward the cavity,29 and also
the pyridine ring should enhance the NH acidity. According to
our expectations, catalyst 7 showed a stronger intramolecular
Looking for alternative scaffolds, we envisaged a 1,3-amino
alcohol geometry. Although this disposition differs from the
1,2-amino alcohol scaffold in N-terminal hydrolases, water
molecules could be necessary in N-terminal hydrolases to
transport the proton between −NH2 and −OH groups,
preventing in this way the formation of a strained four-
membered ring.28 The greater distance between the hydroxyl
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1
Figure 4. H NMR spectra (6.6−10.4 ppm) showing the movement of NH signals of catalysts 6 and 7 after acetylation.
H bond between the acetate carbonyl group and the aromatic
NH, which was deshielded by 0.34 ppm in the presence of the
acetate group (Figure 4).
DFT studies of the reaction transition state supported this
observation, showing a short H bond of 2.8 Å between the
isophthalic NH and the carbonyl oxygen atom (Figure 5).
Kinetics studies corroborated this hypothesis, as the
methanolysis of 7a showed a half-life of only 3.7 min (Table
1, entry 7). Figure 6 collects the variation with time of the
molar fraction of the acetylated catalysts 1−7 prepared in this
study.
Transesterifications and Association Studies. Encour-
aged by this result, we next tested the ability of compound 7 to
catalyze the transesterification of vinyl acetate. Interestingly,
low catalyst loadings of 7 (<1 mol %) were enough to catalyze
the transesterification between vinyl acetate and methanol with
50% conversion after 68 h at 20 °C (Figure 7). No reaction
took place in the absence of catalyst after 100 h. Also,
compound 7a could be obtained after 3 days by mixing catalyst
7 in vinyl acetate, showing the participation of the acyl
intermediate in the reaction mechanism. To our knowledge
this is the first time that a small-molecule hydrolase mimic has
been able to catalyze the transesterification of vinyl acetate.
Figure 5. Calculated geometry of the transition state structure for the
methanolysis reaction of compound 7a. Selected distances between
heteroatoms are depicted in angstroms.
Next, to prove the enzyme-like similarities of compound 7,
the association constant between catalyst 7 and EtOAc was
measured by 1H NMR titration in CDCl3 at 20 °C. Successive
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from slow evaporation of a dichloromethane/methanol
solution.
Figure 6. Time-dependent methanolysis of acetylated catalysts 1−7.
Figure 8. (A) Reaction of compound 7 with phenylphosphonic acid
chloride. (B) X-ray diffraction structure of compound 7b. Selected
distances between heteroatoms are depicted in angstroms.
The single-crystal X-ray diffraction analysis showed a perfect
fit of the phosphonate group inside the catalyst 7 oxyanion
hole (Figure 8B). The phosphoryl oxygen which mimics the
negatively charged oxygen of the transition state establishes
two H bonds of 2.89 and 3.13 Å with the pyridine aliphatic and
aromatic NHs, respectively. The pyridine oxyanion hole
showed a 4.70 Å distance between both NHs, which is in
good agreement with natural oxyanion holes.30
The shortest H bond of only 2.68 Å is set between the
pyrrolidinium group and one of the phosphate oxygen atoms.
Both phosphoryl oxygens showed similar O−P distances of
1.49−1.50 Å, which can be explained by a negatively charged
phosphonate group, which contributes to the short pyrrolidi-
nium H bond.
One of the most attractive features of the structure is the
similar distances established between the pyrrolidinium group
with both the phosphonoester oxygen (3.06 Å) and the
phosphoryl oxygen (2.68 Å), which may be a key factor in
transporting the proton from the nucleophile to the leaving
group.
Figure 7. Time-dependent transesterification of vinyl acetate
catalyzed with compound 7.
The conclusions obtained from the X-ray structure of
compound 7b agree with the observations detected in solution
1
by H NMR, which revealed the deshielding of the oxyanion-
additions of ethyl acetate aliquots to a solution of catalyst 7 in
CDCl3 and plotting of the signal shifts vs equivalents of ethyl
acetate gave an association constant of 0.79 M−1 (see the
Supporting Information). This value could explain the still low
reaction rate for transesterification reactions in comparison
with deacetylation reactions.
X-ray Studies. In order to show that catalyst 7 possesses a
well-preorganized polar environment complementary to the
transition state, its reaction with the transition state mimic
phenylphosphonic acid chloride was performed (Figure 8A).
Pleasingly, X-ray-quality crystals of the product were obtained
hole NHs upon acetylation of the catalysts. This implies that
the acetate carbonyl group is hydrogen-bonded by the
oxyanion-hole NHs in a way similar to that for the
phosphonate group which mimics the transition state of the
reaction.
Comparison with Natural Chymotrypsin. To prove the
similarity of catalyst 7 with the active site of a natural
hydrolase, the single-crystal X-ray diffraction structure of
compound 7b was superimposed with the active center of a
chymotrypsin phosphate (PDB 1GCD) (Figure 9). The
phosphate group was fixed for both compounds, which allowed
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Figure 9. Superimposition of the crystal structure of chymotrypsin phosphate (ribbon model) and compound 7b (ball and stick model), in which
the phosphate group is fixed for both compounds. Hydrogen bonds between the oxyanion-hole NHs and the phosphate oxygen are depicted as red
dashed lines for chymotrypsin phosphate and yellow dashed lines for compound 7b.
a comparison of the geometry of the catalytic groups in the
synthetic catalyst and the enzyme.
Experimental procedures, spectroscopic data, NMR
titrations, kinetic data, ORTEP diagrams, X-ray crystal
structure data, and modelization studies (PDF)
As can be observed in Figure 9, the geometry of compound
7b is fairly similar to that of the active center of chymotrypsin,
with the exception of the basic group, probably because the
chymotrypsin phosphate is a neutral compound, which
prevents the proximity of the imidazole ring due to the lack
of hydrogen bonding, while in compound 7b a strong H bond
is set between pyrrolidinium and phosphate groups. A better fit
is obtained for the oxyanion hole, with 2.90 Å distance
between the phosphoryl oxygen and both the NH of Ser195 and
the NH of catalyst 7. The second H bond of the oxyanion hole
between Gly193 and the phosphoryl oxygen is slightly shorter in
chymotrypsin. The rigid pyridinedicarboxamide spacer, with a
4.70 Å distance between both NHs, is probably responsible for
this fact, since in chymotrypsin, the oxyanion-hole distance
between the NHs is around 4.30 Å.
Crystallographic data (CIF)
Accession Codes
CCDC 2031919 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Authors
■
́
Joaquín R. Moran − Organic Chemistry Department, University
of Salamanca, Salamanca E-37008, Spain; Email: romoran@
usal.es
́
Angel L. Fuentes de Arriba − Organic Chemistry Department,
CONCLUSIONS
■
University of Salamanca, Salamanca E-37008, Spain;
orcid.org/0000-0001-7424-8146; Email: angelfuentes@
usal.es
In conclusion, a detailed analysis of the key amino acid
residues responsible for catalysis in serine proteases and N-
terminal hydrolases has allowed us to simulate their active
center by using a rigid cyclohexane skeleton where the base,
nucleophilic hydroxyl group, and oxyanion hole have been
rationally positioned. This small-molecule artificial enzyme
catalyzes the deacetylation of enzyme mimics in less than 10
min and the transesterification of vinyl acetate at room
temperature. Investigations to extend this reactivity to peptide
bonds is currently ongoing in our laboratory and will be
reported in due course.
Authors
́
́
Jose J. Garrido-Gonzalez − Organic Chemistry Department,
University of Salamanca, Salamanca E-37008, Spain
Ma Mercedes Iglesias Aparicio − Organic Chemistry
Department, University of Salamanca, Salamanca E-37008,
Spain
Miguel Martínez García − Organic Chemistry Department,
University of Salamanca, Salamanca E-37008, Spain
́
Luis Simon − Chemical Engineering Department, University of
Salamanca, Salamanca E-37008, Spain; orcid.org/0000-
0002-3781-0803
ASSOCIATED CONTENT
■
Francisca Sanz − X-Ray Diffraction Service, University of
Salamanca, Salamanca E-37008, Spain
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.0c02121.
Complete contact information is available at:
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3673−3680. (c) Tabushi, I. Cyclodextrin Catalysis as a Model for
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Enzyme Action. Acc. Chem. Res. 1982, 15, 66−72. (d) Klock, C.;
Dsouza, R. N.; Nau, W. M. Cucurbituril-Mediated Supramolecular
Acid Catalysis. Org. Lett. 2009, 11, 2595−2598. (e) Purse, B. W.;
Rebek, J. Functional Cavitands: Chemical Reactivity in Structured
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(f) Soberats, B.; Sanna, E.; Martorell, G.; Rotger, C.; Costa, A.
Programmed Enzyme-Mimic Hydrolysis of a Choline Carbonate by a
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Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
(10) Ema, T.; Tanida, D.; Matsukawa, T.; Sakai, T. Biomimetic
Trifunctional Organocatalyst Showing a Great Acceleration for the
Transesterification Between Vinyl Ester and Alcohol. Chem. Commun.
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ACKNOWLEDGMENTS
This work was supported by Junta de Castilla y Leon
(European Regional Development Fund-SA069P17), the
University of Salamanca (Own Research Programs-KCEP/
■
́
̈
(11) Kheirabadi, M.; Çelebi-Olcu̧ m, N.; Parker, M. F. L.; Zhao, Q.;
̈
́
463AC01), MINECO (CTQ2017-87529-R), and Fundacion
Memoria de D. Samuel Solorzano Barruso (FS/8-2019). J.J.G.-
G. and A.L.F.d.A. gratefully acknowledge the University of
Salamanca for predoctoral and postdoctoral fellowships,
respectively. We also thank the NUCLEUS platform at
University of Salamanca, A. Lopez Garcia and J. A. Gonzalez
Ramos for IT support, V. Obregon from Bio-Oils Huelva, and
the University of Salamanca server housing service.
Kiss, G.; Houk, K. N.; Schafmeister, C. E. Spiroligozymes for
Transesterifications: Design and Relationship of Structure to Activity.
J. Am. Chem. Soc. 2012, 134, 18345−18353.
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Applications. Chem. Soc. Rev. 2011, 40, 2922−2942. (b) Li, S.;
Turner, A. P. F. Molecularly Imprinted Catalysts: Principles, Syntheses,
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Enzyme-Like Catalysis by Molecularly Imprinted Polymers. Chem.
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Molecular Imprinting: Perspectives and Applications. Chem. Soc. Rev.
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DEDICATION
■
This article is dedicated to Professor Josefa Anaya on ocassion
of her retirement.
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