Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells

Article abstract of DOI:10.1021/acs.orglett.5b02910

Density functional theory (DFT) was applied to study the thermodynamics and kinetics of reversible thiol-Michael addition reactions. M06-2X/6-31G(d) with the SMD solvation model can reliably predict the Gibbs free energy changes (ΔG) of thiol-Michael addition reactions with an error of less than 1 kcal·mol^-1 compared with the experimental benchmarks. Taking advantage of this computational model, the first reversible reaction-based fluorescent probe was developed that can monitor the changes in glutathione levels in single living cells.

Full text of DOI:10.1021/acs.orglett.5b02910

Letter
pubs.acs.org/OrgLett
Theoretical and Experimental Investigation of Thermodynamics and
Kinetics of Thiol-Michael Addition Reactions: A Case Study of
Reversible Fluorescent Probes for Glutathione Imaging in Single
Cells
Jianwei Chen,†,‡ Xiqian Jiang,†,‡ Shaina L. Carroll,†,¶ Jia Huang, and Jin Wang*
$
,†,§
†
§
Department of Pharmacology, and Center for Drug Discovery, Dan L. Duncan Cancer Center, and Cardiovascular Research
Institute, Baylor College of Medicine, Houston, Texas 77030, United States
¶
Department of Chemistry, Rice University, Houston, Texas 77251, United States
Sciclotron LLC., Sugar Land, Texas 77479, United States
$
*
S Supporting Information
ABSTRACT: Density functional theory (DFT) was applied
to study the thermodynamics and kinetics of reversible thiol-
Michael addition reactions. M06-2X/6-31G(d) with the SMD
solvation model can reliably predict the Gibbs free energy
changes (ΔG) of thiol-Michael addition reactions with an
−1
error of less than 1 kcal·mol compared with the experimental
benchmarks. Taking advantage of this computational model,
the first reversible reaction-based fluorescent probe was
developed that can monitor the changes in glutathione levels
in single living cells.
ichael addition reactions have recently gained increasing
Scheme 1. General Scheme for the Mechanism of Thiol-
Michael Addition Reactions
a
M
interest in many different fields, including bioconjugation
chemistry, design of irreversible small molecule inhibitors, and
1
−3
development of molecular imaging probes.
An example of a
Michael addition commonly used for bioconjugation is the thiol-
maleimide reaction. This reaction is considered one of the “click”
1
reactions due to its fast reaction rate and aqueous compatibility.
Some investigational and approved drugs, including afatinib and
neratinib, also contain a Michael acceptor moiety, which can
irreversibly react with the cysteine residue in the active site to
2
achieve inhibition of the targeted proteins. In addition, taking
advantage of the reversibility of thiol Michael addition reactions,
a
our group recently reported the first fluorescent probe for
Compounds 1-X are the GSH probes investigated in this study.
3
quantitative glutathione (GSH) imaging in living cells.
Due to the broad applications of thiol-Michael addition
reactions, it is highly desirable to improve our understanding of
the reaction mechanisms and predict the reactivities between
chemistry, it predicted the energies of thiol Michael additions
with substantial inaccuracies. Rowley and co-workers suggested
6
4
−6
that range-separated DFT functionals can improve the accuracy
thiols and Michael acceptors using computational chemistry.
5
in calculating the energies of thiol Michael additions. In both
A generally accepted reaction mechanism for thiol-Michael
addition begins with deprotonation of the thiol, followed by
conjugate addition of the thiolate to the β-position of the Michael
acceptor to form an enolate (Scheme 1). Houk and co-workers
tested a series of density functional theory (DFT) based methods
to calculate the activation energies and Gibbs free energies of the
conjugate additions of MeSH to six α,β-unsaturated ketones and
concluded that M06-2X, along with two other DFT methods,
Houk and Rowley’s studies, the accuracies of the DFT methods
were compared to high level ab initio calculations as benchmarks
instead of experimental values. Rosenker et al. studied the
energetics of thiol addition and elimination reactions to bicyclic
1
enones in organic solvents using H NMR and DFT calculations
and found excellent agreement between experiments and
4
theory. Flanagan et al. measured the addition reaction kinetics
−1
gives results within 1 kcal·mol of the CBS-QB3 benchmark
6
values. It should be noted that despite the fact that the B3LYP
Received: October 7, 2015
DFT functional has been widely used in computational
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b02910
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
between a series of acrylamides and GSH in phosphate buffers
Table 1. Experimental and Calculated Thermodynamic and
Kinetic Parameters for GSH Probes 1-X
a
(
pH 7.4) and found that the calculated activation energies are
2
7
well correlated with the measured reaction rates (R = 0.915).
experiments
calculations
However, due to the low propensity of thiol elimination from the
adducts for the bicyclic enones and the acrylamides, the reverse
reactions were not investigated and the equilibrium constants of
these reactions cannot be accurately measured.
In our previous development of GSH probe ThiolQuant
Green (TQG), we identified TQG, which had an appropriate
b
c
d
e
e
e
e
X
Kd
kf
kr
ΔG
ΔG1
ΔG2
ΔG3
H
0.42
0.51
1.35
1.43
0.25
0.052
0.10
0.15
0.18
1.29
4.7
−4.7
−4.6
−4.0
−3.9
−5.0
−4.9
−4.4
−4.8
−4.0
−4.7
−2.6
−3.1
−3.2
−5.0
−6.0
−35.5
−34.4
−34.4
−34.1
−30.0
F
11.5
35.7
46.6
6050
Br
NO2
OH
equilibrium constant K when reacting with GSH. Unfortunately,
d
a
both the forward and reverse reaction rates between TQG and
GSH are slow; thus, the probe only allows one-point measure-
ments and is unsuitable for following the changes in GSH levels
K , k , and k are the dissociation equilibrium constant, the second-
d
f
r
order forward reaction rate constant, and the first-order reverse
b
reaction rate constant of reaction 1 in Scheme 1, respectively. Units
3
c
−1 −1
d
−6 −1
e
in single cells. Our goal in this study is 2-fold: to evaluate the
are in mM. Units are in M s . Units are in 10 s . Units are in
−1
kcal·mol . Refer to Scheme S3 in the SI for definitions of ΔG , ΔG ,
accuracies of DFT calculations in predicting the equilibrium
constants and reaction kinetics for thiol-Michael addition
reactions in aqueous environment using experimental values as
benchmarks; and to accelerate the reaction rate of our GSH
probe through systematic structural variation and apply the
newly developed probe to monitor GSH level changes in single
cells for the first time.
1
2
and ΔG .
3
we found that the calculated ΔG in the gas phase (−8.38 kcal·
−1
mol ) deviated significantly from the experimental values in
−1
water (−3.97 kcal·mol ) and concluded that the solvation
A series of TQG analogues were synthesized (1-X, Scheme 1).
All the GSH probes showed absorption maxima around 488 nm
energies are important to accurately predict the energies of
Michael addition reactions. We reoptimized all the reactant and
product structures in water using the same DFT functional with
the SMD solvation model. As shown in Table 1, accounting for
the solvation energies resulted in the calculated Gibbs free
(
representative spectra of 1-OH are shown in Figure 1). Upon
energies ΔG in excellent agreement with the experimental
1
values ΔG. In Houk’s study, most of the experimental ΔG values
for thiol-enone Michael addition reactions were estimated to be
−
1
≤
−4.6 kcal·mol due to the sensitivity limit of NMR
measurements, which renders it impossible to evaluate the
accuracy of the calculated ΔG values. In our study, all the model
reactions were carefully chosen in order to provide precisely
4
measurable ΔG values. It should be noted that the calculated ΔG
−1
for 1-Br related reactions has the largest error (0.8 kcal·mol ),
which could be due to the relatively small basis set used. We
attempted to recalculate the bottom-of-well electronic energies
using a large basis set 6-311G(2d,p) and other DFT methods at
the M06-2X/6-31G(d) geometries and found that the calculated
ΔG values have large deviations from the experimental
benchmarks (refer to SI for details). Therefore, we concluded
that M06-2X/6-31G(d) with the SMD solvation model can
reliably predict the Gibbs free energy changes of Michael
addition reactions, at least in water, with an error of less than 1
kcal·mol− (Table 1).
Figure 1. UV−vis and fluorescence spectra of GSH probe 1-OH (λ =
ex
4
85 nm) and 1-OH-GSH (λ = 405 nm) in PBS. The spectra of 1-OH-
ex
GSH were obtained by measuring the mixture of 1-OH (15 μM) and
GSH (80 mM) in PBS.
reacting with GSH in a phosphate-buffered saline at pH 7.4
(
PBS), the absorption and fluorescence peaks of these GSH
probes shift hypsochromically (Figure 1). The equilibrium
constants between the probes 1-X and GSH were measured by
incubating the probes with a series of concentrations of GSH
1
(
0.1−80 mM) in PBS under anaerobic conditions for 24 h to
We also measured the kinetic parameters of both forward and
reverse thiol-Michael addition reactions. The forward reaction
ensure equilibrium had been established. It should be noted that
due to the reversible nature of the reactions, the ratiometric
spectrometric changes of these probes are GSH concentration-
rate constants k were determined by monitoring the time-
f
dependent absorption changes of the GSH probes 1-X (479 nm)
and the GSH adducts 1-X-GSH (405 nm) when reacting with
GSH in PBS (SI, Figure S2). The pseudo first-order rate
dependent instead of time-dependent as we demonstrated
3
previously. The K values for the reaction between the probes
d
1
-X and GSH were calculated based on the corresponding
absorption changes in different concentrations of GSH solutions
Supporting Information (SI), Figure S1). As shown in Table 1,
constants k ′ were calculated based on a monoexponential global
f
fitting of the decay and growth of the absorbance at 479 and 405
(
nm, respectively. The second order rate constants k were
f
the K values are in the range of 0.25−1.43 mM (please refer to
calculated based on k ′ (Table 1). The reverse reaction rate
d
f
the SI for the detailed procedure to calculate the K values).
constants k were measured using pre-equilibrated mixtures of 1-
d
r
In order to calculate the K values, we employed the M06-2X
X and GSH with addition of 5,6-dihydro-2H-pyran-2-one as a
GSH scavenger to initiate the retro-Michael addition process (SI,
d
4
,6
DFT method following Houk’s previous work. To simplify the
calculations, methylthiol was used to substitute GSH. The Gibbs
free energies of thiol-Michael addition reactions (reaction 1 in
Scheme 1) were initially calculated by optimizing the geometries
of reactants and products in the gas phase with frequency
analyses at the M06-2X/6-31G(d) level of theory. Unfortunately,
Figure S2). The first order rate constants k were calculated based
r
on a monoexponential global fitting of the decay and growth of
the absorbance at 405 and 479 nm, respectively (Table 1). It is
interesting to note that a faster forward reaction rate is always
associated with a faster reverse reaction rate (Table 1).
B
DOI: 10.1021/acs.orglett.5b02910
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Organic Letters
Letter
Precisely predicting the solution-phase reaction rates using
computational chemistry is nontrivial due to the difficulty of
predicting absolute free energies of solvation for ions and
inaccurate estimation of the pre-exponential factors in the
8
Arrhenius equation. In other studies, the M06-2X functional has
been applied to compare the energy barriers of different reaction
9
pathways and to predict the kinetic isotope effects. We
attempted to locate the transition state structures of the Michael
addition reactions using M06-2X/6-31G(d) with the SMD
solvation model, but to no avail. This may be because the attack
of the thiolate on the enones has a very small enthalpy barrier,
resulting in difficulty in identifying the transition state on the
potential energy surface. Based on the Hammett’s linear free-
energy relationship, a more exothermic reaction in the rate-
determining step (RDS) has a lower activation energy barrier.
Previous studies established that thiolate conjugate addition
(
reaction 2 in Scheme 1) is the RDS in Michael addition
6
reactions. Therefore, in order to qualitatively compare the
reaction rates between the GSH probes, we calculated the Gibbs
free energy changes (ΔG in Table 1) for the thiolate conjugate
Figure 3. Confocal images and ratio map of HeLa cells stained with 1-
2
OH-AM. Fluorescent images were acquired with (A) λ = 405 nm, λ =
addition reactions. Plotting ΔG versus log k afforded a fair linear
ex
em
2
f
2
418−495 nm; and (B) λ = 488 nm, λ = 499−695 nm. (C) Bright field
relationship (SI, Figure S3, R = 0.84). Among the GSH probes
investigated, compound 1-OH shows the fastest forward reaction
rate. This may be due to the hydrogen bonding between the
hydroxyl and the carbonyl groups, which stabilizes the enolate
ex
em
image. (D) The ratio map was calculated by dividing the fluorescence
intensity values for the 405 nm channel by the 488 nm channel at each
corresponding pixel. The ratio values are proportional to the GSH
concentrations. In the rainbow scale bar, red and blue represent high and
low GSH concentrations, respectively.
10
intermediate (SI, Figure S4). Regarding the reverse reactions,
the enolate intermediate should be formed based on the principle
of microscopic reversibility. Thurlar and co-workers provided
computational analysis for the reaction mechanisms of α,β-
elimination of esters and thioesters to support a stepwise first-
monitor GSH dynamics, a GSH-ester solution (100 μM) was
added to the imaging plate to transiently increase the intracellular
level of GSH, and the same cells were imaged again. Based on the
ratiometric images in Figure 4A, we observed an increase in the
GSH level in all cells imaged as expected. In a similar experiment,
an N-ethylmaleimide (NEM) solution (100 μM) was used as a
GSH scavenger and a decrease in the ratio was observed in
accordance with the GSH concentration decrease (Figure 4B).
Therefore, 1-OH-AM can be a powerful tool to monitor the
GSH level changes in single cells upon biological stimulation.
It should be noted that GSH probes based on irreversible
11
order elimination from a conjugate base (E1cB) mechanism.
Based on our computational data, we found that plotting −ΔG3
versus log k , but not −ΔG , −ΔG , nor −ΔG , afforded an
r
1
2
4
2
excellent linear relationship (SI, Figure S5, R = 0.97), which
demonstrates that the formation of the enolate intermediates is
the RDS for retro-Michael addition reactions and supports an
E1cB mechanism.
With the extensive theoretical and experimental investigation
of Michael addition reactions, we identified 1-OH as an
improved GSH probe that has faster kinetics than TQG. As in
our previous study, we applied acetoxymethyl (AM) ester to
facilitate cell uptake of the probe, which is designated as 1-OH-
AM (Figure 2). The procedure to apply 1-OH-AM for GSH
reactions or reversible reactions with inappropriate K in aqueous
d
12−14
environment
can only reflect the difference in GSH levels in
bulk cell lysates or in different cells, but cannot follow the GSH
level changes in an individual cell. Furthermore, due to the
sluggish reverse reaction rate of TQG, it only allows one-point
measurements and is unsuitable for following the changes in
3
GSH levels in single cells. Kim et al. reported a GSH probe with
a similar structure but without the aqueous solubilizing
10
carboxylic acid group. We synthesized Kim’s GSH probe and
found it has little aqueous solubility. Kim et al. measured the
second-order rate constant between his probe and β-
Figure 2. Chemical structures of GSH probe (1-OH) and its cell-
permeable form (1-OH-AM).
−2
−1 −1
mercaptoethanol to be 6.98 × 10
M
s , which is only
∼
5% of the reaction rate for 1-OH. Therefore, due to the
hydrophobicity of Kim’s GSH probe, it reacts very slowly with
GSH both in the forward and reverse reactions and cannot be
used to monitor the GSH level changes in single cells.
measurements in cells is similar to that for TQG. As shown in
Figure 3, HeLa cells were incubated with 1-OH-AM (1 μM) for
3
0 min and imaged using a confocal microscope with both 405
In summary, we evaluated a small library of TQG analogues
and identified 1-OH as an improved GSH probe that allows for
the monitoring of changes in GSH levels in single cells. We
extensively measured the thermodynamic and kinetic parameters
for the reactions between GSH and the probes, which can serve
as experimental benchmarks to evaluate the accuracy of
computational methods. We found that M06-2X/6-31G(d)
with the SMD solvation model can precisely predict the Gibbs
free energy changes for the Michael addition reactions with an
and 488 nm excitations. The ratiometric images (Figure 3D)
were generated by dividing the fluorescence intensity values for
the 405 nm channel (Figure 3A) by the 488 nm channel (Figure
3
B) at each corresponding pixel. The ratio values are
proportional to the GSH concentrations.
Taking advantage of the reaction reversibility and fast reaction
kinetics of 1-OH, we were able to observe the GSH level changes
in single cells for the first time. To illustrate the ability of 1-OH to
C
DOI: 10.1021/acs.orglett.5b02910
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported in part by the National Institutes of
Health (R01-GM115622 to J.W.), the Welch Foundation (Q-
■
1
798 to J.W.), Cancer Prevention and Research Institute of
Texas (CPRIT R1104 to J.W.), the start-up funding from Baylor
College of Medicine (to J.W.), the Texas Advanced Computing
Center (TACC) for computational resources, and the Optical
Imaging and Vital Microscopy core at Baylor College of
Medicine.
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Figure 4. Time-lapsed ratiometric imaging of the changes of GSH levels
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(
error within 1 kcal·mol−1 when compared with the experimental
benchmarks. We also discovered that the reaction kinetics of the
Michael addition reactions can be qualitatively predicted based
on the Gibbs free energy changes of the thiolate conjugate
addition reactions. Although this strategy cannot accurately
predict the reaction rates, it serves as a convenient method for
qualitatively comparing the reaction kinetics of Michael addition
reactions without locating the transition states. In addition, our
calculations support an E1cB mechanism for the retro-Michael
addition reaction, in which the formation of the enolate anions is
the RDS. Therefore, this study not only provided a convenient
computational method to predict the thermodynamics and
kinetics of Michael addition reactions but also developed the first
probe that can monitor GSH level changes in single cells, which is
expected to be a powerful tool in redox biology studies.
(
1
(
(
(
6
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.5b02910.
Experimental details for organic synthesis, computational
studies, and cell imaging studies (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: wangj@bcm.edu.
Author Contributions
‡
These authors contributed equally to this work.
D
DOI: 10.1021/acs.orglett.5b02910
Org. Lett. XXXX, XXX, XXX−XXX