Mechanistic study of the reaction of thiol-containing enzymes with α,β-unsaturated carbonyl substrates by computation and chemoassays

Article abstract of DOI:10.1002/cmdc.201000020

We investigated the reactions between substituted α,β- unsaturated carbonyl compounds (Michael systems) and thiols by computations as well as chemoassays. The results give insight into variations in the underlying mechanisms as a function of the substitution pattern. This is of interest for the mechanisms of inhibition of the SARS coronavirus main protease (SARS-CoV M^pro) by etacrynic acid derivatives as well as for the excess toxicity of substituted α,β-unsaturated carbonyl compounds. This study compares possible reaction courses including 1,4-addition followed by a ketonization step, and underscores the importance of a base-catalyzed step for the reactivity of thiol groups in enzymes. Phenyl and methyl substituents at the Michael system decrease the reactivity of the electrophilic compound, but chlorophenyl substituents partly recover the reactivity. Computations also indicate that electron-pushing substituents lead to a change in the reaction mechanism. The conformation of the Michael system is also found to significantly influence reactivity: the s-cis conformation leads to higher reactivity than the s-trans conformation. The computed data explain the trends in measured inhibition potencies of substituted α,β-unsaturated carbonyl compounds and of reaction rates in chemical assays. They also indicate that the reversibility of inhibition does not stand in contrast to the formation of a new covalent bond between inhibitor and protease.

Full text of DOI:10.1002/cmdc.201000020

DOI: 10.1002/cmdc.201000020
Mechanistic Study of the Reaction of Thiol-Containing
Enzymes with a,b-Unsaturated Carbonyl Substrates by
Computation and Chemoassays
Alexander Paasche,[a] Markus Schiller,[b] Tanja Schirmeister,[b] and Bernd Engels*[a]
We investigated the reactions between substituted a,b-unsatu-
rated carbonyl compounds (Michael systems) and thiols by
computations as well as chemoassays. The results give insight
into variations in the underlying mechanisms as a function of
the substitution pattern. This is of interest for the mechanisms
of inhibition of the SARS coronavirus main protease (SARS-CoV
Mpro) by etacrynic acid derivatives as well as for the excess tox-
icity of substituted a,b-unsaturated carbonyl compounds. This
study compares possible reaction courses including 1,4-addi-
tion followed by a ketonization step, and underscores the im-
portance of a base-catalyzed step for the reactivity of thiol
groups in enzymes. Phenyl and methyl substituents at the Mi-
chael system decrease the reactivity of the electrophilic com-
pound, but chlorophenyl substituents partly recover the reac-
tivity. Computations also indicate that electron-pushing sub-
stituents lead to a change in the reaction mechanism. The con-
formation of the Michael system is also found to significantly
influence reactivity: the s-cis conformation leads to higher re-
activity than the s-trans conformation. The computed data ex-
plain the trends in measured inhibition potencies of substitut-
ed a,b-unsaturated carbonyl compounds and of reaction rates
in chemical assays. They also indicate that the reversibility of
inhibition does not stand in contrast to the formation of a new
covalent bond between inhibitor and protease.
Introduction
In 2002, a hitherto unknown plague emerged from
the Guangdong province in southern China. Within a
few weeks it affected more than 8000 people in 25
countries.[1,2] This atypical form of pneumonia had an
overall fatality rate of 10–15%. It was termed severe
acute respiratory syndrome (SARS).[3] The etiological
agent for the epidemic was identified as the previ-
Figure 1. Two examples of inhibitors containing Michael systems as the electrophilic war-
head. The arrows indicate the positions attacked or thought to be attacked by a cysteine
residue of the enzyme.
ously unknown coronavirus SARS-CoV, which con-
tains the largest viral RNA genome known so far.[4]
The SARS-CoV main protease (SARS-CoV Mpro) was
found to be a suitable target for antiviral drugs be-
cause it controls viral replication and transcription.[5] Further-
more, the structure of the SARS-CoV Mpro is highly conserved
among other coronaviruses, which makes it attractive for ra-
tional drug design.[6]
Another approach starts from substituted etacrynic acid,[10,11]
which possesses an a,b-unsaturated ketone moiety as an elec-
trophilic building block (Figure 1). While the vinylogous esters
lead to irreversible inhibition, the etacrynic acid derivatives
show only reversible inhibition of proteases.[12] The underlying
reason for the difference is yet unknown.
The perspective of limiting or preventing the replication of
the SARS coronavirus led to the development of various inhibi-
tors against the SARS-CoV Mpro. Because of its similarity to pi-
cornavirus 3C proteases, it was thought that SARS-CoV Mpro
could be targeted by a drug that inhibits picornavirus 3C pro-
tease activity: AG7088[7] (Ruprintrivirꢀ, Figure 1) was in clinical
trials against the common cold, and was proposed as a prom-
ising lead structure against coronaviral infections as well.
AG7088 itself was found to be inactive in enzyme assays with
SARS-CoV Mpro; however, modifications to the peptidomimetic
scaffold lead to significant inhibition potencies.[8,9] The electro-
philic building block of these active-site-directed inhibitors
consists of an a,b-unsaturated ester function, which undergoes
nucleophilic attack by the thiol group of the cysteine residue.
The rational design of improved inhibitors requires detailed
knowledge about the inhibition process and especially about
the interplay between the various effects. Of particular interest
[a] A. Paasche, Prof. Dr. B. Engels
Institut fꢀr Physikalische und Theoretische Chemie
Universitꢁt Wꢀrzburg, Am Hubland, 97074 Wꢀrzburg (Germany)
Fax: (+49)931-31-85331
E-mail: bernd@chemie.uni-wuerzburg.de
[b] Dr. M. Schiller, Prof. Dr. T. Schirmeister
Institut fꢀr Pharmazie und Lebensmittelchemie
Universitꢁt Wꢀrzburg, Am Hubland, 97074 Wꢀrzburg (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000020.
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B. Engels et al.
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is the distinction between effects that influence the reversible
versus the irreversible step. The former is influenced by nonco-
valent interactions between inhibitor and enzyme. The latter
may also be influenced by such effects, as they may orient the
inhibitor in the right arrangement. However, it also relies on
the chemical reactivity of the electrophilic warhead which is in-
fluenced by substituent effects. Such information can be pro-
vided by chemoassays. To elucidate such effects for the inhibi-
tion potency of three-membered heterocycles we employed a
thiophenol assay. It proved to be a good model for the active
site of cysteine proteases, as it represents a rather acidic thiol
(pKa =7.4–7.8) with half the concentration being deprotonated
at the pH used (pH 7.6).[13c,d] In the work reported herein this
assay was applied to gain insight into the reactivity of the
studied etacrynic acid derivatives. To analyze the inherent reac-
tivity of electrophiles against thiol-containing enzymes Schultz
and co-workers[14] and Schꢂꢂrmann and co-workers[15] devel-
oped assays based on glutathione. They were employed to
quantify the electrophilic reactivity of SN2 electrophiles such as
aliphatic, allylic, and benzylic halides as well as a-halogenated
carbonyls. Because these assays were also used to study the
reactivity of a,b-unsaturated carbonyls acting as Michael ac-
ceptors, they are of significant interest to the goals of the pres-
ent study.
trophilic warhead. The resulting information also provides in-
sight into the excess toxicity of unsaturated carbonyls acting
as Michael acceptors.
Results and Discussion
Model systems
Inhibition mechanisms in which covalent bonds are formed
can be formally divided into two steps (Scheme 1).[19] The first
step consists of the formation of a noncovalent enzyme–inhibi-
tor complex (EI). Within this complex, the attacking enzyme
Scheme 1. Two-step model for the irreversible inhibition of proteases:
E=enzyme, EI=noncovalent enzyme–inhibitor complex, EꢀI=covalently
and irreversibly blocked enzyme.
moiety is oriented in such a way that the covalent bond (EꢀI)
can be formed, e.g., by attack of a nucleophilic center of the
enzyme at the electrophilic center of the inhibitor. The result-
ing reaction represents the second step of the overall inhibi-
tion. Within this step the bond formation is mainly influenced
by enthalpic effects, as all entropic contributions which are as-
sociated with the formation of the enzyme–inhibitor complex
are already included in the first step.[13a] Furthermore, entropy
effects are quite uniform for similar reactions and hence do
not change the relative trends.
To disentangle the various effects that determine the reactiv-
ity of Michael systems, the reactions were also modeled by
quantum chemical computations. Earlier theoretical investiga-
tions[16,17] predict that the reaction of a thiolate with a Michael
system leads to quite stable covalent bonds between the thio-
late and the a,b-unsaturated carbonyl moiety. Thus, they pre-
dict an irreversible inhibition of thiol-containing enzymes by
inhibitors containing such electrophiles as warheads. However,
the theoretical model systems used completely neglected sol-
vent effects and did not consider substituent effects. Addition-
ally, they assumed the attack of a negatively charged thiolate
moiety, while the catalytic active cysteine residue within the
SARS-CoV Mpro[18] or other enzymes represents a neutral thiol.
Computations are challenging because various reaction paths
are conceivable, and a strong influence of the environment
can be expected.
The work presented herein focuses on the second step. In
the present context it involves the formation of a covalent
bond between a thiol residue (e.g., of the cysteine residue of
the SARS-CoV Mpro) and the electrophilic building block of the
inhibitor. Models that aim to describe this step in all details
must account for the influence of the protein and the solvent
environment. This can be achieved with quantum mechanical/
molecular modeling (QM/MM) approaches that take enzyme
and solvent explicitly into account. They are even able to de-
scribe effects such as regio- or stereoselectivity of the inhibi-
tion process.[20] However, as shown recently by investigations
into the inhibition of papain-like proteases,[13] valuable insights
into the influence of the substitution pattern of the warhead
are already possible from simpler models. These mimic all im-
portant parts of the considered system by truncated model
compounds in combination with a continuum model such as
COSMO.[21] These models approximate the cysteine residue by
a methyl thiolate, while the environment is modeled either by
two water molecules (pKs ꢁ15) to mimic a low proton-donat-
ing ability of the environment, or by two ammonium ions (pKs
ꢁ9.3) to simulate a higher proton-donating ability. These ex-
plicit solvent molecules were employed in combination with
the continuum model COSMO which accounts for the overall
polarizability of the solvent and the protein environment.
From the inhibitor, only the electrophilic warhead and its di-
rectly attached substituents were taken into account. Informa-
Hence, we developed model systems which can be used to
extract the influence of the substitution pattern on the inhibi-
tion mechanism. The computed models include the addition
step of the thiol to the Michael system via a base-catalyzed
mechanism. Possible addition mechanisms include reaction
courses that run across enol intermediates and also consider
direct addition pathways. For the former, the subsequent
enol–keto tautomerization was also taken into account, as the
corresponding activation barriers are expected to be similar or
even higher than the barriers of the addition step.
The work is organized as follows: after a brief description of
the theoretical and experimental approaches the computed re-
action courses are discussed as a function of the different sub-
stitution patterns. After describing the experimental results, ex-
periment and theory are compared in order to gather insight
as to how the substitution pattern influences the potency of
inhibitors that possess substituted Michael systems as an elec-
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Mechanistic Study of Thiol-Containing Enzymes
tion about the kinetics and thermodynamics of the irreversible
step of inhibition were obtained through potential energy sur-
faces of the inhibition process which were computed by em-
ploying the B3LYP/TZVP//BLYP/TZVP level of theory. Because
such approaches are considerably less expensive than QM/MM
computations they allowed a scan of a large number of sub-
stituents to test changes in the inhibition potencies of epox-
ides and aziridines. Such model calculations are also important
to distinguish between influences of the enzyme environment
and inherent effects resulting from the electronic structure of
the inhibitor itself. That the prediction of the computations
could be proven experimentally underscores the reliability of
the simple approach.[13]
To transfer such models to the situation in thiol-containing
enzymes in reaction with etacrynic acid derivatives, the follow-
ing approximations were adopted (see Figure 2). The thiol-con-
taining residue of the enzyme is mimicked by methyl thiol.
Possible proton-accepting groups (e.g., the histidine residue of
the SARS-CoV Mpro) are assumed to play the role of the proton
acceptor in the base-catalyzed process. To evaluate the impor-
tance of base catalysis, such groups are modeled by an ammo-
nia molecule, which has been shown to be a good approxima-
tion.[13] This is shown by comparison between simpler QM[13a,b]
and more elaborate QM/MM[20a] approaches. The former used
an ammonium ion, whereas the latter employed a histidine
group.
Figure 2. Sketch of the model systems used to compute the potential
energy surfaces shown in Figure 4. The varied internal coordinates are also
given.
enol form of the 1,4-addition is obtained. A comparison be-
tween both pathways was computationally performed by
Weinstein and co-workers,[22] who studied the addition of am-
monia to a,b-unsaturated carbonyl compounds. They comput-
ed a direct addition as well as the 1,4-addition. However, they
did not consider the base-catalyzed reactions discussed in
Figure 2. In contrast, for the direct addition they assumed an
intermolecular proton transfer from the ammonium group to
C2. For the 1,4-addition an intramolecular proton transfer from
the nitrogen atom to the carbonyl oxygen was considered.
They found that in such cases the 1,4-addition is favored over
direct addition. Both reactions are catalyzed by a single water
molecule, but 1,4-addition remains the favorable reaction
The similarity may result because the pKa value of ammonia
(9.3) is not too different from that of histidine residues in thiol
proteases (pKa 8–9).[20] Additionally, to investigate the influence
of the substitution pattern of the a,b-unsaturated carbonyl de-
rivates on the reaction mechanisms, we modeled the reactions
of compounds 1–11 shown in Figure 3.
The possible reaction paths of
the overall addition of thiols to
Michael systems are depicted in
Scheme 2. For the reactions
under physiological conditions,
thiols are present in protonated
(R-SH) and deprotonated (R-Sꢀ)
form, but as discussed below,
only the reactivity of the thiolate
seems to be sufficiently high for
an efficient attack at the a,b-un-
saturated
carbonyl
moiety.
Hence, the proton must be
transferred to a proton acceptor,
that is, a base-catalyzed reaction
has to take place. For the result-
ing thiolate the reaction can pro-
ceed along two pathways. In the
direct addition mechanism the
thiolate attacks C3 (Figure 2) and
at the same time the proton is
transferred from the base to C2
(direct E2-like addition). If the
proton is instead transferred to
the carbonyl oxygen atom, the Figure 3. List of a,b-unsaturated carbonyl compounds (Michael systems) studied in this work.
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871

B. Engels et al.
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tween keto and enol forms
depend on the molecular struc-
ture of the solvent.[27]
Due to the strong depend-
ence on the actual number of
water molecules, computation of
the activation energy for this
complicated process is too in-
volved for the goal of the pres-
ent work which is to study the
influence of various substitution
patterns. Nevertheless, since the
corresponding barriers are simi-
lar or perhaps even higher than
the barriers of the addition step,
the ketonization can be expect-
ed to influence the inhibition
potency of a given inhibitor sig-
nificantly.
In our model the influence of
this part of the reaction path is
estimated by the enolate inter-
mediate Eꢀ and the enol cation
E+ (see Scheme 2). The enolate
Eꢀ represents the most impor-
tant intermediate if the reaction
takes place in basic solution
(first deprotonation, then addi-
tion). The cation E+ is important
for an acidic environment in
which the deprotonation and
Scheme 2. Conceivable reaction mechanisms for the addition of methylthiol to a,b-unsaturated carbonyl com-
pounds.
course. For acrolein the barrier of the 1,4-addition is
~70 kJmolꢀ1 lower than the corresponding barrier of the direct
addition (96 kJmolꢀ1 vs. 27 kJmolꢀ1). For acrylic acid the differ-
ence is only ~5 kJmolꢀ1 (59 kJmolꢀ1 vs. 54 kJmolꢀ1), but the
1,4-addition is still favored. Tezer and Ozkan[23] came to similar
conclusions.
the protonation steps interchange. More information about
our approach is given in the Supporting Information.
The determination of relative energies that are not biased
due to hydrogen bonding networks is quite complicated. Be-
cause we are only interested in trends that arise from the sub-
stitution pattern it is necessary to compute relative energies
without the influence of the network. A suitable reference for
all energy considerations is E0, which represents the energy of
the model system in which all reactants and all explicitly ac-
counted solvent molecules are infinitely separated so that no
hydrogen bonding network exists. Because the s-trans form is
slightly lower in energy, E0 is defined as:
If the reaction proceeds along the 1,4-addition the final
product is only reached after a ketonization of the enol form.
In the studies mentioned above the influence of possible barri-
ers of this step was not considered. Experimentally, the barrier
of the acid-catalyzed ketonization of acetone enol in water
was determined to DH0° =40 kJmolꢀ1. The energy difference
between the enol and the keto form was measured to DH0 =
ꢀ43 kJmolꢀ1.[24] Comparison with gas-phase data show that
DH0 depends little on the surroundings, but the actual course
of the reaction and the kinetics are strongly influenced.[25]
Recent computations reveal that the barrier of the ketonization
strongly depends on the nature of a water bridge, which facili-
tates the proton transfer. If the bridge consists of three water
molecules the computed barrier height is ~73 kJmolꢀ1, while a
value of >210 kJmolꢀ1 is computed for the direct transfer. A
similar value was also computed by Lien and co-workers.[26] If
more than three water molecules are added, the water bridge
is solvated itself and the barrier increases again. Notably, the
computations did not consider proton catalysis. Further work
also showed that in some cases the energy differences be-
E0 ¼ Eðreactant, s-trans formÞ þ Eðmethyl thiolÞ þ EðNH Þ þ E
ðNH4þÞ
ð1Þ
3
The energies of ammonia and the ammonium ion are neces-
sary for the comparison with the computed potential energy
surface (PES). Relative energies with respect to E0 which also
do not account for the influence of the hydrogen bonding net-
work arising due to the explicit solvent molecules are abbrevi-
ated with E0(X). As an example, the relative energy of the enol
form E (see Scheme 2) without considering the influence of
the hydrogen bonding network is given as:
DE0ðEÞ ¼ E0ꢀ½EðEÞ þ EðNH Þ þ E þÞꢂ
ð2Þ
ðNH4
3
872
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Mechanistic Study of Thiol-Containing Enzymes
Corresponding computations were performed for all other
intermediates depicted in Scheme 2. The actual formulas are
given in the Supporting Information. The resulting energies
computed for compounds 1–11 are summarized in Table 1.
The s-trans and the s-cis isomers are depicted in Figure 5
below. More information is given in the Supporting Informa-
tion.
which r(OꢀH) is varied together with the r(SꢀC). The corre-
sponding PES (Figure 4B) indeed shows that r(OꢀH) smoothly
adjusts. More information is given in the Supporting Informa-
tion.
The PES of the addition reaction (Figure 4A) gives the quali-
tative picture of a two-step mechanism. As the first step, de-
protonation of the thiol reactant R occurs leading to a zwitter-
ionic state that consists of a thiolate anion H3C-Sꢀ which inter-
acts with an ammonium ion (NH4+···ꢀS-CH3). In the following
this state is abbreviated as Sꢀ. It lies 12 kJmolꢀ1 higher than
the reactants. The deprotonation process has a barrier (TS1)
that is computed to 18 kJmolꢀ1. Figure 4A shows that the
shape of the potential energy curve describing the deprotona-
tion step is nearly independent from the second reaction coor-
Computations
In the first model the inhibitor is represented by acrolein (1),
which represents the simplest Michael system. The PES for the
reaction branch up to the enol form is depicted in Figure 4A.
It was computed as a function of the distances between the
center of methyl thiol and the C3 center of the inhibitor (r(Sꢀ dinate RCS for RCS >3 ꢃ. For smaller distances the barrier flat-
C), see Figure 2), and between the sulfur and the proton of the
ammonium ion (r(SꢀH)).
tens. Nevertheless, such paths remain energetically less favora-
ble than deprotonation paths with RCS >3 ꢃ because they start
from considerably higher energies. The approach of the pro-
tonated thiol to the Michael system is repulsive (Figure 4A).
The subsequent conjugated addition reaction at the C3 atom
of the inhibitor proceeds to the enol intermediate E without
passing a second transition state (TS2), that is, the prior depro-
tonation step is the rate-determining step. During this reaction
step, the carbonyl oxygen atom of acrolein (1) is simultaneous-
ly protonated by the proton donor ammonium. This is also
shown by the PES that is obtained if r(SꢀH) and r(OꢀH) are
varied (Figure 4B). The whole reaction to the enol possesses
an exothermicity of only ꢀ45 kJmolꢀ1. For the addition of the
deprotonated thiolate to the Michael system, a reaction
energy of ꢀ57 kJmolꢀ1 is predicted. The reverse reaction is, in
both cases, easily possible.
Both coordinates were varied independently, as the relation-
ship between them was unknown. The S–H distance (r(SꢀH),
see Figure 2) ranges from 1.0 to 3.0 ꢃ, while the C3–S distance
(r(SꢀC), see Figure 2) varied between 1.7 and 3.7 ꢃ. For each
point of the PES, r(SꢀC) and r(SꢀH) were kept fixed at certain
values while all other internal degrees of freedom were opti-
mized. Transition states were identified as maximum points on
the potential curves. Frequency analysis of transition states
confirmed their nature.
In this PES the r(OꢀH) distance (see Figure 2) was not treat-
ed as an independent variable. It represents the main coordi-
nate of the protonation of the emerging intermediate, but
during the optimization of the remaining internal degrees of
freedom this coordinate smoothly adjusted for varying distan-
ces r(SꢀC) and r(SꢀH). To investigate the influence of r(OꢀH) in
greater detail we also computed a two-dimensional PES for
An analysis of selected bond parameters within the Michael
system at the characteristic points of the PES is given in
Table 1. Summary of the relative energies [Eq. (2)] computed for the various intermediates shown in Scheme 2 without considering hydrogen bonding
networks.
Compd
Structure
R3
E [kJmolꢀ1
]
E
R1
H
R2
H
R4
H
Conformer
R
Sꢀ
TS2
Eꢀ
30
33
50
44
43
31
25
20
41
42
NF[b]
NF[b]
NF[b]
NF[b]
35
E+
K
[a]
1
2
3
4
5
6
7
H
H
H
H
H
H
H
s-trans
s-cis
s-trans
s-cis
s-trans
s-cis
s-trans
s-cis
s-trans
s-cis
s-trans
s-cis
s-trans
s-cis
–
0
11
0
2
0
ꢀ4
0
3
0
11
0
ꢀ2
0
ꢀ7
0
0
ꢀ4
0
43
54
43
45
43
39
43
46
43
54
43
41
43
36
43
43
39
43
43
–
–
ꢀ20
ꢀ19
ꢀ8
ꢀ13
ꢀ10
ꢀ19
ꢀ18
ꢀ19
ꢀ6
ꢀ8
54
53
55
39
ꢀ5
10
103
ꢀ47
[a]
Me
H
H
H
H
H
H
H
59
58
59
–
–
82
72
78
97
97
16
ꢀ51
ꢀ57
ꢀ52
ꢀ31
ꢀ58
ꢀ70
Ph
H
[a]
[a]
PhCl2
PhCl2
OMe
NMe2
H
[a]
–
Me
H
58
–
–
–
–
–
[a]
[a]
[a]
[a]
H
[a]
8
9
Ph
Ph
–
H
Me
Me
–
H
67
76
66
–
–
ꢀ34
ꢀ118
ꢀ35
s-trans
s-cis
s-cis
64
52
33
NF[b]
94
0
ꢀ7
45
[a]
10
11
Ph
PhCl
CH2OR
H
H
H
H
OPh
76
138
ꢀ51
[a]
s-trans
0
1
[a] The connection between Sꢀ and enolate Eꢀ contains no additional barrier. [b] No stationary point found.
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873

B. Engels et al.
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bond. The product of the conjugated addition shows the typi-
cally observed bond lengths referring to a sulfur–carbon bond
C1ꢀS, a C2ꢀC3 single bond, a C1=C2 double bond, and a C1ꢀ
O single bond.
The exothermicity of the reaction strongly depends on the
proton donor molecule in the vicinity of the inhibitor’s carbon-
yl oxygen atom. If the ammonia molecule is replaced by a
water molecule the inhibitor is not protonated during the ad-
dition reaction, and the exothermicity lowers by ~40 kJmolꢀ1
.
Furthermore, the conjugated addition step possesses a barrier
(TS2) of ~3 kJmolꢀ1; that is, the deprotonation remains the
rate-determining step. While the energies change considerably,
the corresponding geometrical parameters resemble those
found before. They are summarized in the Supporting Informa-
tion. Without any proton donor, the conjugated addition be-
comes the rate-determining step with a barrier height of
24 kJmolꢀ1 for the second transition state (TS2). The reaction
energy of the complete reaction is +5 kJmolꢀ1; that is, the re-
action becomes endothermic. The geometric parameters again
resemble those found for the previous systems; these are also
given in the Supporting Information. The strong influence of
the proton donor underscores the importance of a proper ori-
entation of the carbonyl oxygen atom of the Michael system
toward a stabilizing group, such as the oxyanion hole.
The influence of the hydrogen bonding network is reflected
by the relative energies from the PES scan (Figure 4A) com-
pared with those calculated by Equation (2) (Table 1). If the in-
fluence of the hydrogen bonding network is switched off, the
energy difference between the reactant and the intermediate
Sꢀ increases to ~40 kJmolꢀ1. The enol form is predicted to lie
~ꢀ20 kJmolꢀ1 below the reactants. These values can be com-
pared with the relative energies for the ketonization step (eno-
late or enol cation) to get information about the complete 1,4-
addition mechanism. The enolate is found to lie ~10 kJmolꢀ1
below the Sꢀ intermediate. Hence, our computations predict
that for acrolein the proton transfer leading to the Sꢀ inter-
mediate represents the rate-determining step. This is in line
with previous findings.[22,23,26] For the addition of ammonium
to acrolein, previous computations also indicate that the 1,4-
addition mechanism takes place and that the addition step is
rate determining. This also supports that our approach is suffi-
ciently accurate.
Figure 4. Potential energy surfaces (PES) computed to characterize the reac-
tion branch up to enol formation for the acrolein model system 1. The inter-
nal coordinates are defined in Figure 2. A) PES obtained as a function of r(Sꢀ
H) and r(SꢀC); B) PES computed as a function of r(SꢀH) and the r(OꢀH) dis-
tance.
Table 2. The bond lengths within the nearly undisturbed Mi-
chael system, referring to a C–S distance of 3.7 ꢃ, agree well
with the experimentally determined counterparts,[28] reported
as 1.22 ꢃ for the C1ꢀO carbonyl bond, 1.49 ꢃ for the C1ꢀC2
single bond, and 1.35 ꢃ for the C2=C3 conjugated double
Substituents are known to influence the inhibition potency
of Michael-system-based inhibitors. In order to study such ef-
fects we investigated the influence of various substituents of
the Michael system on the relative position of the intermedi-
ates depicted in Scheme 2. The results obtained for the com-
pounds depicted in Figure 3 are summarized in Table 1. The s-
cis form of acrolein (1) (Figure 5) is ~10 kJmolꢀ1 higher in
energy than the s-trans form. The corresponding barriers are
very similar so that acrolein is expected to react in the s-trans
form. The addition of thiolate to the a,b-unsaturated carbonyl
compound with R1 =methyl possesses an additional barrier of
10–15 kJmolꢀ1, which was not present for acrolein. Together
with the energy necessary to form the thiolate, the total barrier
for reaching the enol form increases to ~60 kJmolꢀ1. The rela-
tive energy of the enolate is computed to ~50 kJmolꢀ1. Hence
Table 2. Geometric parameters of the model system acrolein (1) for se-
lected points on the PES given in Figure 4.
Bond[a]
Bond length [ꢃ]
TS1
R
Sꢀ
E
CꢀS
SꢀH
3.70
1.40
1.24
1.46
1.36
3.70
1.60
1.25
1.45
1.35
3.70
2.20
1.25
1.45
1.35
1.90
2.20
1.36
1.35
1.49
C1ꢀO
C1ꢀC2
C2ꢀC3
[a] The atom numbers are defined in Figure 2.
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ChemMedChem 2010, 5, 869 – 880

Mechanistic Study of Thiol-Containing Enzymes
formed for the direct E2-like addition in which the proton
donor was placed in the vicinity of C2 indicated considerably
lower barriers at ~60–70 kJmolꢀ1.[29] Hence, for R1 =methoxy
our computations indicate a direct addition. This switch in the
mechanism is in line with previous experimental findings of
Miyata et al.,[30] who explained stereospecific nucleophilic addi-
tions of thiols to derivatives of a,b-unsaturated carboxylic
acids by a fast protonation of the arising enolate at C2.
Figure 5. Definition of s-trans and s-cis isomers.
the ketonization is also predicted to be slower than for acrole-
in, but the addition of the thiolate remains the rate-determin-
ing step. The increase with respect to the unsubstituted acrole-
in can be explained by the +I effect of the methyl group. This
effect also explains why the enol cation becomes more stable
by about the same degree, but it is still considerably higher in
energy than the enolate. The barriers computed for the s-trans
and s-cis isomers are virtually identical which represents an ad-
ditional difference to the unsubstituted counterpart. In sum-
mary, the methyl substituent decreases the reactivity of the
a,b-unsaturated moiety, but the overall mechanism is not ex-
pected to change.
For compound 3 (R1 =phenyl) similar effects are observed.
In contrast to 2, however, the s-cis form becomes slightly more
stable than the s-trans form. Furthermore, while the addition
step for the s-trans form possesses a barrier (TS2=59 kJmolꢀ1),
no barrier for the s-cis form is computed. This could result
from mesomeric effects, but the phenyl group is rotated with
respect to the a,b-unsaturated moiety so that mainly the in-
ductive effect of the phenyl group remains. The size seems to
be similar to the effects observed for R1 =methyl. The differen-
ces to compound 2 result from steric effects which are mainly
present in the s-trans form. Going from compound 3 to 4 the
+I effects should be diminished due to the electron-withdraw-
ing effects of the chlorine substituents. Such effects are ob-
served mainly for the s-trans form, for which the barrier of the
addition step disappears. Furthermore, the enol and enolate
forms become more stable. The steric effects strongly increase
in going from compound 4 to 5. The s-cis form lies even
higher in energy, and the barriers of the addition step increase
as well as the enolate intermediate. In summary, 5 is expected
to have similar reactivity as 3; that is, the advantages gained
through the chlorine substituents are lost due to the additional
methyl group. This indicates that many of the effects are addi-
tive.
A ketonization across the enolate form is also quite unfavor-
able for compound 7. In this case, however, the electron-push-
ing properties of the N,N-dimethylamine group stabilize the
enol cation in such a way that the ketonization should be
easily possible. For compound 8 the addition step is also pre-
dicted to represent the rate-determining step, and the ketoni-
zation will proceed across the enol cation. Due to its high sta-
bility, the enol cation is expected to be formed directly. In gen-
eral, compound 8 represents an outlier due to its different
electronic structure. Compound 9 was characterized in order
to investigate, in combination with compound 3, how R3 =
methyl influences the reactivity. According to our computa-
tions this substitution should lower the reactivity because the
barriers associated with the 1,4-addition and the ketonization
step become higher. Compounds 10 and 11 were investigated
to distinguish experimentally between the reactivity of the s-
cis and s-trans forms. The computations predict that the s-cis
form (compound 10) reacts considerably faster than the s-trans
form. However, the difference results mainly from the ketoniza-
tion step; while it should proceed quite rapidly for 10, it
should not take place for 11.
The computations point to three different mechanisms,
which are depicted in Figure 6. For acrolein (1) the formation
of the thiolate is the rate-determining step, whereas for its ad-
dition to the a,b-unsaturated carbonyl, no additional energy
barrier has to be overcome. Compound 2 represents a proto-
Despite these variations, the overall mechanism remains the
same for compounds 1–5. In all cases the formation of the thi-
olate in combination with the addition to the Michael system
represents the rate-determining step. The ketonization process
has lower-lying intermediates and seems to proceed across the
enolate, which is considerably lower in energy than the enol
cation. This does not appear to be the case for the remaining
compounds. For compound 6 the barrier for the addition step
is similar to the other systems, but the necessary ketonization
process seems to be impossible. The enol is already considera-
bly less stable than the reactants (+55 kJmolꢀ1), and the eno-
late is predicted to be instable. The enol cation lies at ~+
100 kJmolꢀ1 so that a possible ketonization would represent
the rate-determining step. However, test computations per-
Figure 6. Sketch of the three different mechanisms found for the reaction of
a,b-unsaturated carbonyl compounds with thiols. Prototypical compounds
for the different situations are also shown.
ChemMedChem 2010, 5, 869 – 880
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875

B. Engels et al.
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Figure 7. 1H NMR reference spectrum of etacrynic acid (13) in buffer solution.
type for a mechanism in which the barrier of the addition step
represents the highest point on the total reaction path. This re-
action type is found for various alkyl-substituted compounds.
Finally, for compounds such as the a,b-unsaturated carboxylic
ester 6, the ketonization step is so unfavorable that a direct
addition is assumed to take place.
The decrease of the inhibitor and the formation of the prod-
uct could be quantified independently by integration of the
signals of the ethyl groups. Relevant signals are the triplet of
the methyl group of the etacrynic acid side chain (reactant:
d=1.11 ppm; product: d=0.86 ppm). The product increase
and reactant decrease followed an exponential curve as illus-
trated in Figure 9.
Quantification for the thiol was performed in the same way
using the decrease in the signal of the aromatic proton (d=
6.75 ppm, Figure 8) in ratio to the sum of the integration
values of the singlet of the methoxy group of the thiol reac-
tant and the product. With constant sums of the integration
values of the respective signals of reactant and product, it was
possible to calculate concentrations of both reactants etacrynic
acid [A] and 4-methoxythiophenol [B]. Considering the initial
concentrations [A]0 =1.25 mm and [B]0 =2.50 mm, the second-
order rate constant k2 could be calculated with the following
equation:
Experimental Results
All tested model compounds showed appropriate solubility
and stability in the reaction medium. The signals of the C=C
double bond could be monitored for each compound without
any overlap with signals originating from the thiol or other re-
1
action products, as illustrated by the H NMR spectra of eta-
crynic acid (13) (Figure 7).
For the reaction with 4-methoxythiophenol the signals of
the C=C double bond and the ethyl side chain of etacrynic
acid (13) were expected to be affected (Scheme 3).
Data analysis of the time-dependent NMR experiments
showed that after the first spectrum recorded (t=12 min)
>60% of the etacrynic acid had already reacted with the thiol.
Within 60 min the turnover was >95%. Figure 8 shows the de-
crease in the signals of the C=C double bond and the ethyl
group of etacrynic acid (13) as well as of the thiophenol.
½Aꢂ
½Bꢂ
½Aꢂ0
½Bꢂ0
ð3Þ
ln
¼ ln
þ ð½Aꢂ0 ꢀ ½Bꢂ0Þk2t
Plotting ln([A]/[B]) against time (t) resulted in a linear correla-
tion. From the slope, the second-order rate constant was calcu-
lated from ([A]0ꢀ[B]0)k2. For etacrynic acid (13) a second-order
rate constant of k2 =25.6mꢀ1 minꢀ1 was determined. For the
other compounds this calculation was not possible owing to
reaction rates that were too rapid. Therefore only the percent-
age turnover rates could be obtained.
Regarding the structural features of compounds 12 and 10,
these were expected to show a higher reactivity than etacrynic
acid (13). This could be confirmed by the NMR experiments.
For both compounds the double bond signals had already dis-
appeared completely in the first recorded spectra (t=12 min)
of the series. Because reference spectra of 12 and 10 without
thiophenol approved solubility and stability of the compounds,
Scheme 3. Model reaction of the NMR test series illustrated for etacrynic
acid (13).
876
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ChemMedChem 2010, 5, 869 – 880

Mechanistic Study of Thiol-Containing Enzymes
Figure 8. 1H NMR spectra monitoring the reaction of etacrynic acid (13) with 4-methoxythiophenol.
this means that addition of the 4-methoxythiophenol to the
Michael system had already reached completion within 12 min.
For compound 9 containing an endo-double bond, reactivity
was similar to that of etacrynic acid (13). After 12 min, a turn-
over rate of 88% was detected. The reaction reached near
quantitative completion (99%) after 42 min.
Compound 11 (s-trans isomer) was chosen for direct com-
parison with 10 (s-cis). In both compounds the s-cis or s-trans
conformation of the Michael system is fixed by a bicyclic struc-
ture. However, in contrast to compound 10, no reaction with
the thiophenol was detected for 11. Even after two days the
starting material was still present in almost full amount. The re-
sults of all NMR test series are summarized in Table 3, which
also contains data reported by Schultz et al.[14]
Discussion
The data measured in the present work are collected in Table 3
together with some selected values reported by Schultz and
colleagues.[14] Further experimental data are available from the
work of Schꢂꢂrmann and co-workers[15] and from the studies
of Schultz et al.[14] An understanding of the relationships be-
tween molecular structure and the reactivity against thiols is
important for the comprehension of excess toxicity of a,b-un-
saturated carboxylic derivates and the development of inhibi-
tors of cysteine-containing proteases. To achieve such an un-
derstanding a comparison of the experimental and computed
data (Table 1) was performed. Furthermore, this comparison
provides information about whether the chosen theoretical
Figure 9. Progress curves of the addition of 4-methoxythiophenol to eta-
crynic acid (13).
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877

B. Engels et al.
MED
experimentally. Indeed, as ex-
pected, 10 is very reactive. How-
ever, for 11 no reaction takes
place. The computations explain
the astonishing stability of 11
through very high energy barri-
ers for the ketonization step. Be-
cause the addition leading to
the enol is strongly endothermic,
no reaction will take place. That
our model can explain even this
unexpected behavior indicates
that it does indeed capture the
main aspects that govern the re-
activity of a,b-unsaturated car-
bonyl compounds with respect
to thiol-containing enzymes.
Table 3. Summary of experimental data; the results of this work are taken from the time-dependent NMR ex-
periments.
Compd
Turnover [%]
Time
k2 [mꢀ1 minꢀ1
]
RC50 [mm]
Reference
1
2
6
9
–
–
–
–
–
–
–
–
–
0.086
0.090
0.55
–
–
–
–
–
–
[14]
[14]
[14]
88
12 min
42 min
12 min
2 days
12 min
12 min
42 min
90 min
ND[a]
ND[a]
ND[a]
ND[a]
ND[a]
25.6
25.6
25.6
this work
this work
this work
this work
this work
this work
this work
this work
>99
>99
<5
>99
63
10
11
12
13
90
>99
–
–
[a] Not determined.
model captures the main effects that determine the reactivity
of a,b-unsaturated carbonyl compounds.
Experimental inhibition assays for SARS-CoV Mpro and related
enzymes show that some agents inhibit the thiol-containing
enzymes irreversibly, whereas for others only reversible inhibi-
tion is observed. This is surprising, as all agents possess a,b-un-
saturated carbonyl derivatives as electrophilic warheads. For
this unexpected finding our investigations provide two possi-
ble explanations. In some cases, for example, compound 5 or
9, the reaction energy is estimated to be only ~30 kJmolꢀ1. In
such cases the reverse reaction is much slower than the for-
ward reaction but may still occur. In other cases the reaction
energies are sufficiently high to impede any reverse reaction.
But also in such cases a reversible inhibition may occur be-
cause complicated multistep inhibition mechanisms must
occur to reach the final keto form. For all compounds, base
catalysis is necessary since only the thiolate is sufficiently reac-
tive for the addition. In most cases, the second step consists of
a 1,4-addition leading to the enol intermediate. The final keto
form is then obtained through a ketonization of the enol form.
This ketonization, as shown in previous investigations, is only
possible if the necessary proton transfer is catalyzed by an effi-
cient water bridge, for example. Because such catalysis cannot
take place in the active site of many enzymes, the reaction
might get stuck at the enol intermediate. Alternatively, if no
proton donor is available the reaction might become stuck in
an enolate intermediate, which is strongly stabilized by avail-
able oxyanion holes. In such cases our computations predict
considerably smaller reaction energies. Hence the reverse reac-
tion can take place easily, and only reversible inhibition is ob-
served. Particularly for derivatives of a,b-unsaturated carboxylic
acids the enol form is so high in energy that a direct addition
to the olefinic double bond becomes more favorable than 1,4-
addition. For direct addition an efficient proton donor is neces-
sary because the reaction proceeds in an E2-like manner. How-
ever, as shown by various Protein Data Bank (PDB) structures,
this role could be played by histidine residues, which would
also serve as the base in the first step of the overall reaction.
Therefore, providing that a histidine residue can serve as an ac-
ceptor and donor and that the electronic structure of the war-
head steers the reaction toward direct addition, the keto form
can be reached without the ketonization process. Only in such
According to our computations an alkyl substitution at C3
increases the reaction barrier of the addition step to the enol
form (+15 kJmolꢀ1) and also shifts the enolate intermediate to
higher energies (+16 kJmolꢀ1). This is reflected in the mea-
sured decrease of the turnover found in going from com-
pound 13 to 12 (Table 3). This trend is also manifested in the
work of Schꢂꢂrmann and co-workers in two examples.[15] Com-
paring 2-cyclopentene-1-one and 2-methyl-2-cyclopenten-1-
one, the RC50 values increase from 0.58 to 8.40, and in going
from methyl acrylate to methyl methacrylate, the value increas-
es from 0.42 to 74.1. Similar trends are also reported by Schultz
et al.[14]
As a second trend our computations predict that a terminal
alkyl substituent (3 vs. 9) will decrease the reactivity of the
a,b-unsaturated carbonyl compound again, as both the barrier
of the addition (+27 kJmolꢀ1) and the relative energy of the
enolate (+21 kJmolꢀ1) increase. This finding explains the de-
crease in the RC50 values going from 1-pentene-3-one to 4-
hexene-3-one.[15] A similar effect is also described by Schultz
et al.,[14] who observed that a methyl substitution at the olefin
moiety decreases activity.
The well-known experience that aldehydes are more reactive
than the corresponding ketones is reflected in the difference
computed between compounds 1 and 2. It is also reflected in
the investigations of Schultz et al.[14] Another important finding
of our computations is the prediction that derivatives of a,b-
unsaturated carboxylic esters are less reactive, because the ke-
tonization step is so strongly hampered that a direct addition
may take place. This is in line with the explanation provided
by Miyata et al.[30] for stereospecific nucleophilic additions of
thiols to derivatives of a,b-unsaturated carboxylic acids. An ex-
perimental proof of the predicted decrease in reactivity is
given by Schꢂꢂrmann and colleagues.[15] They found decreas-
ing RC50 values for 1-pentene-3-one (0.09) and methyl acrylate
(0.42).
In many cases the computations predict that the s-cis forms
possess higher reactivity than their s-trans counterparts. Com-
pounds 10 and 11 were synthesized to prove this prediction
878
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ChemMedChem 2010, 5, 869 – 880

Mechanistic Study of Thiol-Containing Enzymes
which is the corresponding value for water. For cavity radii, stan-
dard settings were used. DFT is well known to describe many prop-
erties with an excellent cost–benefit value.[35] Nevertheless, this is
often based on an error compensation that does not work in all
cases,[36] and indeed in many cases multi-reference approaches are
necessary to obtain reliable potential energy surfaces.[37] However,
because we are only interested in trends, DFT should be sufficiently
accurate for the present work.
cases is an irreversible inhibition found. In other cases the reac-
tion proceeds only up to the enolate form and only reversible
inhibition is observed.
According to our computations such a direct addition is
more favorable for a,b-unsaturated carboxylic esters as provid-
ed in AG7088.[7] For alkyl or phenyl substituents the 1,4-addi-
tion is predicted to be more favorable. In line with this finding,
AG7088 performs an irreversible inhibition of the picornavirus
3C protease,[7] whereas agents including alkyl or phenyl sub-
stituents indeed inhibit the SARS-CoV Mpro reversibly.[12]
Chemoassays
Within the experimental test series, five different model com-
pounds were treated with 4-methoxythiophenol: etacrynic acid
(13, synthesized according to ref. [11]), its norethyl derivative (12,
for synthesis, see the Supporting Information), trans-(1-phenyl)but-
2-en-1-one (9, Sigma–Aldrich), 3-methylenechroman-4-one (10,
synthesized according to ref. [38]), and 6-chloro-4H-chromen-4-one
(11, Maybridge). The structures of these compounds are shown in
Figure 3.
Summary and Conclusions
The work presented herein involved the development of a sim-
plified model system to describe the reaction of a,b-unsaturat-
ed carbonyl derivates with thiol-containing enzymes. The com-
puted PES of the addition of thiols to acrolein is only possible
with a base-catalyzed process. It showed that such catalysis is
necessary for an efficient addition. The computations can also
explain various trends found in chemoassays which were per-
formed to estimate the excess toxicity of this important class
of compounds.
The choice of the substances reflected the various substitution pat-
terns of the Michael system treated computationally. Reactions of
inhibitors 9–13 with the model thiol 4-methoxythiophenol were
1
performed and followed by H NMR spectroscopy. Due to the aro-
matic and symmetric structure of the thiol, only few well-defined
signals in 1H NMR spectra were expected from the aromatic pro-
tons (d>6.7 ppm) as well as from the signal of the methoxy group
(d<3.8 ppm). For both groups an overlap with the characteristic
signals of the Michael systems (dꢁ5.5–6.5 ppm) of compounds 9–
13 could be ruled out.
Finally, our investigations provide an explanation for why,
depending on agent and enzyme, reversible or irreversible in-
hibition of thiol-containing active sites are observed despite
the fact that all agents possess a,b-unsaturated carbonyl deri-
vates as warheads.
For the NMR experiments stock solutions of the thiol (SH, 100 mm)
and the inhibitor model compounds 9–13 (I, 100 mm) were pre-
pared in [D6]DMSO. As reaction medium a phosphate buffer
(KH2PO4/K2HPO4, 100 mm, pH 7.6) in D2O was used. For the reac-
tion, 10 mL inhibitor stock solution, 770 mL phosphate buffer solu-
tion, and 20 mL thiol stock solution (SH) were subsequently trans-
ferred into an NMR tube and mixed well, reaching a final volume
of 800 mL and concentrations of [I]=1.25 mm and [SH]=2.50 mm.
With respect to the pKa value of the thiol and the pH used, the
concentrations refer to second-order reaction conditions.
Irreversible inhibition is observed only if the reaction steers
directly to the final keto form. The computations predict this
behavior for unsaturated carbonyl esters. If the reaction pro-
ceeds across the 1,4-addition, which is predicted for most de-
rivatives, the reaction will get stuck in the enol or in a stabi-
lized enolate because the necessary ketonization is strongly
hindered in the active sites of the proteins. In such cases the
inhibition is reversible because the enol is not sufficiently sta-
bilized with respect to the reactant to hinder the reverse reac-
tion.
1H NMR experiments were performed on a Bruker Avance 400
spectrometer at 400.13 MHz (258C, 64 scans per spectra) directly
after mixing the reaction partners. The mixing of the reactants was
regarded as the starting point of the reaction (t=0 min). Spectra
were recorded continuously for 60 min at first, afterward with in-
creasing intervals depending on the reactivity of the reactants. To
assure stability of the model compounds during the measurement,
reference spectra of a solution of the single model compounds in
buffer at the respective concentration were recorded before and
afterward.
Experimental Section
Computational methods
The points of the potential energy surfaces were computed with
density functional theory (DFT) employing the TURBOMOLE soft-
ware package[31] with standard settings. The PES scans were per-
formed with a resolution of 0.1 ꢃ for the respective grid points.
Optimization of the geometrical parameters (except the two char-
acteristic bond distances) that define the PES was performed with
the BLYP exchange correlation functional,[32] the resolution of iden-
tity approximation (RI),[33] and the triple zeta basis set TZVP,[34]
which includes three sizes of contracted functions and further p-
functions to take polarization into account. The electronic energies
of all obtained geometries were recalculated by additional single-
point calculations, employing Becke’s three-parameter hybrid func-
tional B3LYP[32] and TZVP basis set. As in previous computations,
solvent effects were taken into account within the conductor-like
screening model (COSMO),[21] which was used for all calculations
(geometry optimizations and single-point calculations). The dielec-
tric constant of the polarizable environment was set to 78.39,
Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft
(DFG) in the frameworks of the projects EN197/13-2, SCHI441/4-
2/5-3 and the SFB630 (projects A4 and C3).
Keywords: chemoassays · Michael systems · SARS · theory ·
toxicity
ChemMedChem 2010, 5, 869 – 880
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879

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Received: January 16, 2010
Revised: March 9, 2010
Published online on April 16, 2010
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