Probing the mechanism of thermally driven thiol-Michael dynamic covalent chemistry

Article abstract of DOI:10.1039/c8ob00397a

The kinetics and mechanism of the thermally activated dynamic covalent exchange of thiol-Michael adducts is investigated. A model system of thiol-Michael adducts between thiophenol and phenylvinylketone derivatives and adducts between 2-mercaptoethanol phenylvinylketone derivatives in N,N-dimethylformamide (DMF) at elevated temperatures is used to probe the underlying exchange mechanism. The kinetic data show negligible free Michael acceptor, which is consistent with the highly efficient thiol-Michael reaction being a "click"-like reaction that significantly favors the adduct form. At elevated temperatures of 90 °C in DMF the thiol-Michael adducts reach equilibrium after 24 h, although equilibration did not occur within 24 h at 60 °C or 75 °C, and negligible exchange occurs under ambient conditions. A kinetic model was developed to describe the dynamic covalent exchange and equilibration. The experimental and simulation kinetic data of dynamic covalent exchange are consistent with the thiol-Michael adducts undergoing a retro-Michael reaction, followed by subsequent addition of a free thiol to the liberated Michael acceptor. Kinetic analysis is consistent with the fragmentation, or retro-Michael reaction, being the rate-determining step in the dynamic covalent exchange. This suggests that the key step in dynamic covalent exchange is not enhanced by addition of free thiol or free Michael acceptor, since the addition reaction is much faster than the retro-Michael reaction. This fundamental study will guide the design of organic compounds, materials, and bioconjugates that utilize the thermally activated dynamic covalent thiol-Michael linkages.

Full text of DOI:10.1039/c8ob00397a

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Probing the Mechanism of Thermally Driven
thiol-Michael Dynamic Covalent Chemistry
Borui Zhang§, Progyateg Chakma§, Max P. Shulman, Jun Ke, Zachary A. Digby, and Dominik
Konkolewicz*
Department of Chemistry and Biochemistry, Miami University, 651 E High St, Oxford, OH,
45056, USA
*Corresponding Author: d.konkolewicz@miamiOH.edu
Abstract
The kinetics and mechanism of the thermally activated dynamic covalent exchange of thiol-
Michael adducts is investigated. A model system of thiol-Michael adducts between thiophenol
and phenylvinylketone derivatives and adducts between 2-mercaptoethanol phenylvinylketone
derivatives in N,N-dimethylformamide (DMF) at elevated temperatures is used to probe the
underlying exchange mechanism. The kinetic data show negligible free Michael acceptor which
is consistent with the highly efficient thiol-Michael reaction being a “click”-like reaction that
significantly favors the adduct form. At elevated temperatures of 90 °C in DMF the thiol-
Michael adducts reach equilibrium after 24 h, although equilibration did not occur within 24 h at
60 °C or 75 °C, and negligible exchange occurs under ambient conditions. A kinetic model was
developed to describe the dynamic covalent exchange and equilibration. The experimental and
simulation kinetic data of dynamic covalent exchange are consistent with the thiol-Michael
adducts undergoing a retro-Michael reaction, followed by subsequent addition of a free thiol to
the liberated Michael acceptor. Kinetic analysis is consistent with the fragmentation, or retro-

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Michael reaction, being the rate-determining step in the dynamic covalent exchange. This
suggests that the key step in dynamic covalent exchange is not enhanced by addition of free thiol
or free Michael acceptor, since the addition reaction is much faster than the retro-Michael
reaction. This fundamental study will guide the design of organic compounds, materials, and
bioconjugates that utilize the thermally activated dynamic covalent thiol-Michael bonds.
Introduction
Dynamic covalent chemistry is an important field of organic chemistry with significant
applications from equilibrium based synthetic reactions, complex molecular libraries to materials
science.1-9 Unlike kinetically controlled reactions, where transformations with the lowest
activation energy barrier dominate the process, a dynamic covalent process is governed by
thermodynamics.1 A dynamic covalent reaction typically involves two or more species that are in
chemical equilibrium with each other, and able to participate in forward and reverse direction
transformations to maintain this dynamic equilibrium.1 There are many species that participate in
dynamic covalent processes, including Diels-Alder adducts, boronic esters and boronates,
imines, disulfides, olefins and alkynes in the presence of a metathesis catalyst, and thiol-Michael
adducts to name a few.1, 10-19 Incorporation of these moieties capable of dynamic covalent
exchange can lead to well-defined organic molecules, self-directed assembly, complex
dynamically associated macrocycles, and polymers with interesting properties such as self-
healing or malleable properties.4, 5, 20-22 However, to optimize the organic compounds made by
dynamic covalent approaches, or the performance of organic materials containing dynamic
covalent groups, it is necessary to understand the mechanism of the underlying dynamic covalent
exchange.

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The thiol-Michael reaction is a highly efficient reaction, between a thiol nucleophile and an
electron poor alkene.23-26 Typically the reaction is catalyzed with a small amount of base or
nucleophile, although the reaction can proceed in solvent as well.27 The thiol-Michael reaction
has found applications from organic synthesis, materials chemistry and bioconjugation due to its
facile implementation and high yield of the desired organic products.7 A typical thiol-Michael
reaction is shown in Scheme 1A. The high yield, tolerance to functionality and simple
28
implementation have made the thiol-Michael reaction a “click”-like reaction,23,
and
interestingly this reaction also satisfies the dynamic covalent criteria under appropriate
stimulus.17, 29-34 Typically, elevated pH or elevated temperatures are used as stimuli for the
activation of thiol-Michael adducts towards dynamic covalent exchange. This is an interesting
and important feature of the thiol-Michael adduct, since it is essentially static under ambient
conditions, and typically only undergoes dynamic covalent exchange under appropriate stimulus.
This has important implications for applications ranging from bioconjugation to material
stability.29, 35, 36 This dynamic covalent exchange of two thiol-Michael adducts is given in Scheme
1B.
Nucleophilic/Basic
A
RS
Initiator
+
RSH
X
X
B
R2S
R2S
R1S
R1S
+
+
Thermal
or
X2
X1
X1
X2
pH
Stimulus
Scheme 1: A) thiol-Michael adduct formation reaction, B) Dynamic covalent exchange of thiol-
Michael adducts.

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The dynamic nature of the thiol-Michael adduct has been known for over 50 years,30, 31
although it has only recently gained significant attention.23, 34 The pH or base driven thiol-
Michael adduct exchange has been studied as a dynamic covalent process,17, 37, 38 in the context of
bioconjugation processes,35, 38, 39 and polymer materials,29, 32, 40 or in a theoretical context.34 In
general the thermally driven dynamic thiol-Michael exchange has received significantly less
attention, except for the early studies,30, 31 and recent work from the past two years.29, 32, 41, 42 The
thermally driven thiol-Michael dynamic covalent chemistry has been used in polymer
materials,29, 32, 41, 42 however, the details of this process remain understudied. This is a particularly
interesting topic since the reaction appears to be essentially static under ambient temperature,
although capable of significant exchange at elevated temperatures. Studying both the kinetics of
this reaction and the process of this reaction has the potential to guide the development of the
next generation of bioconjugation reactions and synthetic materials. Here, a model system of
thiophenol (TPSH) and mercaptoethanol (MESH) adducts with phenylvinylketone (PVK) based
Michael acceptors in N,N-dimethylformamide (DMF) is used to study the nature of the thermally
activated dynamic covalent exchange of thiol-Michael adducts. This model system is shown in
Scheme 2.
OH
OH
OH
S
X
S
ka-ME-PVK
kf-ME-PVK
kf-TP-PVK
ka-TP-PVK
+
+
+
+
O
O
O
HS
HS
HS
HS
X
X
ME-PVK
TPSH
PVK
MESH
TPSH
TP-PVK
MESH
Scheme 2: Dynamic covalent exchange of thiophenol (TPSH), thiophenol-phenylvinylketone
adduct (TP-PVK), mercaptoethanol (MESH), mercaptoethanol-phenylvinylketone adduct (ME-
PVK), and phenylvinylketone (PVK). Rate coefficients of addition (ka) and fragmentation of the
adducts (kf) are used to describe the data.

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Experimental
Materials
All materials were purchased from commercial suppliers unless otherwise specified. All
materials were used as received unless otherwise specified.
Nuclear Magnetic Resonance (NMR)
All nuclear magnetic resonance experiments were performed on a Bruker 500 MHz NMR
spectrometer.
High-resolution mass spectrometry (HRMS)
The HRMS data were measured on a Thermo Scientific LTQ Orbitrap XL mass spectrometer
equipped with an Ion Max ESI source. It was operated in positive mode at 60,000 resolution,
using direct infusion from a syringe at 5 ul/min.
Synthesis of phenylvinylketone (PVK)
O
O
TEA
Cl
CHCl3, RT
The synthesis of PVK was adapted from the literature.43, 44 A solution of 3-chloropropiophenone
(3.00 g, 17.79 mmol, 1.0 equiv) in chloroform (50.00 g) was cooled to 0 °C. To this stirred
solution, triethyl amine (6.00 ml, 43.05 mmol, 2.4 equiv) was added dropwise. The reaction
mixture was stirred for 18h at room temperature, followed by washing with 0.1 M HCl aq. (2 ×
50 ml), distilled water (2 × 50 ml), saturated NaHCO3 aq. (2 × 50 ml), and brine (1 × 50 ml). The
organic layer was dried over Na2SO4 and concentrated under reduced pressure, giving the product
1
PVK as colorless oil in 99% yield. The product was confirmed by H NMR spectroscopy,
agreeing with the literature.44, 45 1 H NMR (500 MHz, CDCl3) δ 7.97–7.95 (m, 2H), 7.59–7.56 (t,

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1H, J = 7.67 Hz), 7.50–7.46 (t, 2H, J =7.84 Hz), 7.20-7.14 (m, 1H), 6.47-6.43 (dd, 1H, J = 1.61,
17.03 Hz), 5.95-5.92 (dd, 1H, J = 1.64, 10.59 Hz) ppm.
Synthesis of para-chlorophenylvinylketone (PVK-Cl)
The synthesis of PVK-Cl followed the same process as PVK, except that 3,4'-
dichloropropiophenone was used instead of 3-chloropropiophenone. The product was confirmed
by 1H NMR spectroscopy, agreeing with the literature.45 1H NMR (500 MHz, CDCl3) δ 7.95-7.84
(d, 2H, J = 8.57 Hz), 7.50-7.42 (d, 2H, J = 8.63 Hz), 7.18-7.06 (dd, 1H, J = 10.57, 17.52 Hz),
6.49-6.40 (dd, 1H, J = 1.65, 17.12 Hz), 5.99-5.92 (dd, 1H, J = 1.58, 10.58 Hz) ppm.
Synthesis of para-methoxyphenylvinylketone (PVK-OMe)
3-Chloro-1-(4-methoxyphenyl)-1-propanone was synthesized according to literature.46 The
product was used for next step without any purification. Para-methoxyphenylvinylketone was
synthesized following the same procedure as PVK, except 3-Chloro-1-(4-methoxyphenyl)-1-
propanone was used instead of 3-chloropropiophenone. The product was confirmed by 1H NMR
1
spectroscopy, agreeing with the literature.45 H NMR (500 MHz, CDCl3) δ 7.99-7.95 (d, 2H),
7.21-7.14 (dd, 1H, J = 10.52, 17.22 Hz), 6.98-6.94 (d, 2H), 6.45-6.39 (dd, 1H, J = 1.79, 17.18
Hz), 5.89-5.85 (dd, 1H, J = 1.85, 10.51 Hz), 3.98-3.82 (s, 3H) ppm.
Synthesis of 1-phenyl-3-(phenylthio)propan-1-one (TP-PVK)
O
O
SH
5% TEA
DMF, RT
S
+

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A 25ml glass vial equipped with a magnetic stir bar was charged with PVK (0.2 g, 1.51 mmol, 1
equiv), thiophenol (TPSH) (0.1667 g, 1.51 mmol, 1 equiv), 5% TEA (15 mg) and
dimethylformamide (DMF) (1.4260 g). The reaction was allowed to continue at room
temperature (25-30 °C) for 2 days until all the alkene protons were consumed as monitored by 1
H NMR, giving the TP-PVK adduct in DMF as light yellow solution. The product was
confirmed by 1H NMR spectroscopy, agreeing with the literature.47 1H NMR (500 MHz, CDCl3)
δ 7.93-7.91 (dd, 2H, J = 1.45, 8.62 Hz), 7.60-7.55 (t, 1H, J = 7.45 Hz), 7.49-7.44 (t, 2H, J = 7.79
Hz), 7.40-7.36 (m, 2H), 7.33-7.28 (t, 2H, J = 7.78 Hz), 7.23-7.18 (t, 1H, J = 7.33 Hz), 3.33 (m,
4H) ppm.
Synthesis of 3-((2-hydroxyethyl)thio)-1-phenylpropan-1-one (ME-PVK)
O
O
5% TEA
DMF, RT
OH
SH
S
+
HO
A 25mL glass vial equipped with a magnetic stir bar was charged with PVK (0.2 g, 1.51 mmol, 1
equiv), 2-mercaptoethanol (MESH) (0.1182 g, 1.51 mmol, 1 equiv), 5% TEA (15 mg) and
dimethylformamide (DMF) (1.3775 g). The reaction was allowed to continue at room
temperature (25-30 °C) for 2 days, giving the ME-PVK adduct in DMF as colorless solution.
1
The product was confirmed by H NMR spectroscopy, agreeing with the literature.48 1 H NMR
(500 MHz, CDCl3) δ 7.99-7.97 (dd, 2H, J = 1.48, 8.52 Hz), 7.63-7.58 (t, 1H, J = 7.44 Hz), 7.52-
7.48 (t, 2H, J = 7.70 Hz), 3.80-3.75 (t, 2H, J = 6.85 Hz), 3.37-3.31 (t, 2H, J = 7.18 Hz), 2.98-2.94
(t, 2H, J = 7.13 Hz), 2.78-2.73 (m, 2H) ppm.
Synthesis of 1-(4-methoxyphenyl)-3-(phenylthio)propan-1-one (TP-PVK-OMe)

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A 25mL glass vial equipped with a magnetic stir bar was charged with PVK-OMe (0.53 g,
0.00326 mol), thiophenol (TPSH) (0.3780 g, 0.003423 mol), few drops of 5% TEA in DMF
solution. The reaction was allowed to continue at room temperature (25-30 °C) for 1 day giving
the TP-PVK-OMe adduct. The product was purified by dissolving the reaction mixture in
dichloromethane (10 mL) and then washed with water (3 x 50 mL), saturated sodium bicarbonate
(2 x 50 mL), brine (50 mL). The organic solution was dried over magnesium sulfate and the
solvent was removed to give solid product. (0.55049 g, 0.00202 mol, 62% yield). The product
was confirmed by 1H NMR, 13C NMR and HRMS. 1 H NMR (500 MHz, CDCl3) δ 7.91-7.88 (m,
2H), 7.38-7.36 (m, 2H), 7.31-7.28 (m, 2H), 7.21-7.18 (t, 1H, J = 7.35 Hz), 6.93-6.90 (m, 2H),
13
3.87 (s, 3H), 3.33-3.30 (m, 2H), 3.27-3.24 (m, 2H) ppm. C NMR (126 MHz, CDCl3) δ 196.7,
163.7, 137.0, 136.0, 130.3, 129.7, 129.4, 129.1, 129.0, 127.5, 127.2, 126.2, 113.8, 55.5, 38.0,
28.1 ppm. (Figure S1). HRMS (ESI, M + H+) calcd. for C16H17O2S+: 273.0944, found: 273.0937.
Synthesis of 3-((2-hydroxyethyl)thio)-1-(4-methoxyphenyl)propan-1-one (ME-PVK-OMe)
A 25mL glass vial equipped with a magnetic stir bar was charged with PVK-OMe (0.53 g,
0.00326 mol), 2-mercaptoethanol (MESH) (0.26740 g, 0.003423 mol), few drops of 5% TEA
solution. The reaction was allowed to continue at room temperature (25-30 °C) for 1 day giving
the ME-PVK-OMe adduct. The product was purified by dissolving the reaction mixture in
dichloromethane (10 mL) and then washed with water (3 x 50 mL), saturated sodium bicarbonate
(2 x 50 mL), brine (50 mL). The organic solution was dried over magnesium sulfate and the
solvent was removed to give yellow oil. (0.336 g, 0.0014 mol mol, 43% yield). The product was
confirmed by 1H NMR,13C NMR and high resolution mass spectrometry (HRMS), agreeing with
1
the literature.48 H NMR (500 MHz, CDCl3) δ 7.93-7.90 (m, 2H), 6.93-6.90 (m, 2H), 3.85 (s,
3H), 3.77-3.74 (t, 2H, J= 5.80 Hz), 3.23-3.20 (t, 2H, J = 7.20 Hz), 2.93-2.90 (t, 2H, J = 7.11 Hz),

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2.77-2.74 (t, 2H, J = 5.94 Hz), 2.50 (s, 1H) ppm. 13 C NMR (126 MHz, CDCl3) δ 196.7, 163.7,
130.4, 129.6, 113.8, 60.5, 55.5, 38.5, 35.8, 26.0 ppm. (Figure S2). HRMS (ESI, M + H+) calcd.
for C12H17O3S+: 241.0893, found: 241.0887.
Synthesis of 1-(4-chlorophenyl)-3-(phenylthio)propan-1-one (TP-PVK-Cl)
The synthesis of TP-PVK-Cl followed the same process as TP-PVK-OMe. The isolated yield
1
13
1
was 45% and the product was confirmed by H NMR, C NMR and HRMS. H NMR (500
MHz, CDCl3) δ 7.86-7.83 (m, 2H), 7.43-7.41 (m, 2H), 7.38-7.36 (m, 2H), 7.32-7.29 (m, 2H),
7.23-7.19 (m, 1H), 3.33-3.25 (m, 4H) ppm. 13 C NMR (126 MHz, CDCl3) δ 197.0, 139.8, 135.6,
134.8, 129.6, 129.5, 129.1, 129.0, 126.4, 38.4, 27.9 ppm. (Figure S3). HRMS (ESI, M + H+)
calcd. for C15H14ClOS+: 277.0448, found: 277.0438.
Synthesis of 1-(4-chlorophenyl)-3-((2-hydroxyethyl)thio)propan-1-one (ME-PVK-Cl)
The synthesis of TP-PVK-Cl followed the same process as ME-PVK-OMe. The isolated yield
1
13
was 67% and the product was confirmed by H NMR, C NMR and HRMS, agreeing with the
literature.48 1 H NMR (500 MHz, CDCl3) δ 7.88-7.87 (m, 2H), 7.43-7.41 (m, 2H), 3.77-3.75 (t,
2H, J= 5.88 Hz), 3.26-3.23 (t, 2H, J = 7.14 Hz), 2.94-2.91 (t, 2H, J = 7.13 Hz), 2.77-2.74 (t, 2H, J
= 5.91), 2.47 (s, 1H) ppm. 13 C NMR (126 MHz, CDCl3) δ 197.1, 139.9, 134.8, 129.5, 129.0,
60.6, 38.9, 35.7, 25.8 ppm. (Figure S4). HRMS (ESI, M + H+) calcd. for C11H14ClO2S+: 245.0398,
found: 245.0398.
Typical Kinetic Analysis
O
O
SH
OH
elevated temperature
SH
S
S
+
+
HO
O
O
SH
elevated temperature
SH
OH
S
S
+
+
HO

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For 1:1 ratio, to the TP-PVK adduct solution was added 2-mercaptoethanol (MESH) (0.1182 g,
1.51 mmol, 1 equiv) and DMF (0.1182 g). To the ME-PVK adduct solution was added
thiolphenol (TPSH) (0.1667 g, 1.51 mmol, 1 equiv) and DMF (0.1667g). 0.25 mL of each two
mixtures was diluted into vial containing 1mL DMF respectively. A small volume of each
diluted was 0.1 mL of each diluted samples as 0 h point. The rest of the diluted solutions were
capped, protected by argon during the reaction and heated to 60, 75 or 90 °C, or kept at room
temperature of 20 °C, for 24 h with 0.1 mL samples taken at different length of time. Fraction of
each adduct was estimated using the ratio of integrals in the region 7.44-7.1 ppm to the integral
of peaks at 3.37-3.31 ppm. In both the system initiated from TP-PVK adduct or the ME-PVK
adduct, the integral in the region 7.44-7.1 ppm is 5 protons. However, the TP-PVK adduct has an
integral of 4 protons in the region 3.37-3.31 ppm, while the ME-PVK adduct has an integral of 2
protons in the region 3.37-3.31 ppm. Therefore, the mole fraction of TP-PVK and ME-PVK can
be estimated as follows:
I3.37-3.31ppm − 2
fTP-PVK
=
(1)
(2)
2
fME-PVK =1− fTP-PVK
A similar procedure was used for the dynamic exchange of para-substituted phenylvinylketone
derivatives. A similar procedure was performed where the ratio of MESH to TP-PVK adduct was
varied by changing the initial concentration of MESH. Since the protons in the region 7.44-7.1
ppm all derive from the TPSH, not the PVK derived aromatic protons, in integral 7.44-7.1 ppm
was assigned to 2.5 or 20 when the ratio of TPSH to ME-PVK was 0.5 or 4 respectively. In these
systems equations 1 and 2 are still used to calculate fTP-PVK and fME-PVK
.
Results and Discussion

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The dynamic covalent system between thiophenol (TPSH), thiophenol-phenylvinylketone
adduct (TP-PVK), mercaptoethanol (MESH), mercaptoethanol-phenylvinylketone adduct (ME-
PVK), and phenylvinylketone (PVK) given in Scheme 2 can be represented through an overall
equilibrium (KTP-ME-PVK). This TP-ME-PVK system is chosen as a model system to determine the
behavior of the thiol-Michael dynamic equilibrium since it enables a range of analyses including
Hammett analysis.49 In all cases, the NMR spectra show negligible signal in the vinyl region (5-
6.5 ppm),50 which indicates that the concentration of free PVK is very low, and it is likely a
reactive intermediate in this system with a low, steady state concentration below the detection
limit of NMR. Since the free PVK cannot be quantified in these experiments, the overall
equilibrium is given below in terms of TPSH, MESH, TP-PVK, ME-PVK.
[TPSH]eq[ME-PVK]eq
kf-TP-PVKka-ME-PVK
KTP-ME-PVK
=
=
ka-TP-PVKkf-ME-PVK [TP-PVK]eq[MESH]eq
where the subscript eq refers to the equilibrium concentration.
Typical NMR exchange data are given in Figure S5 are given for the TP-ME-PVK system
starting from both the TP-PVK adduct with 1 equivalent of MESH (Figure S5A) and starting
from the ME-PVK adduct with 1 equivalent of TPSH (Figure S5B). These NMR data can be
processed to extract the mole fraction of ME-PVK adduct and the mole fraction of TP-PVK
adduct. These two fractions are plotted consistently in all Figures in this paper, and mole fraction
specifically refers to the mole fraction of TP-PVK and mole fraction of ME-PVK, with the sum
of these two mole fractions equaling 1. The TP-PVK adduct with 1 equivalent of MESH in
DMF, or the ME-PVK adduct with 1 equivalent of TPSH in DMF, were heated at 90 °C for 24h
with kinetic samples taken at allotted time point over 8 h, and the final point at 24h. Figure 1
gives the time evolution of the TP-PVK adduct and the ME-PVK adduct. The first clear
conclusion is that the system under these conditions reached equilibrium after 24 h, with the [TP-

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PVK]eq /([TP-PVK]eq +[ME-PVK]eq) ≈ 0.55 and [ME-PVK]eq /([TP-PVK]eq +[ME-PVK]eq) ≈
0.45 regardless of whether the system was initiated from the TP-PVK adduct or the ME-PVK
adduct. However, since the reaction is a dynamic covalent equilibrium process, involving
forward and reverse reactions across 5 species, a simple first or second order kinetic model
cannot be used to describe the system. Instead a kinetic model that explicitly accounts for
forward and reverse reactions was developed as outlined in the supporting information.
A sensitivity analysis of the kinetic model was performed to evaluate the confidence of
estimated parameters. As indicated in Figure S6- Figure S13, the kinetic model is very sensitive
to the fragmentation rate coefficient (kf) corresponding to the initial adduct. Changes in the
fragmentation rate coefficient (kf) of the adduct formed in exchange (not initialized from) lead to
minimal change in the fitted data, except at very long times. Similarly, variations in absolute
value and ratio of the addition rate coefficients lead to minimal changes in the predicted kinetic
evolution.
The sensitivity analysis and the fact that the system is initialized from both the TP-PVK and
ME-PVK adducts indicates that there is a high confidence over the estimated values kf-TP-PVK
(which can be estimated from the reaction initialized from TP-PVK) and kf-ME-PVK (which can be
estimated from the reaction initialized from ME-PVK). Further, knowledge of the overall
equilibrium constant KTP-ME-PVK obtained from the experiment and the estimated values of kf-TP-PVK
and kf-ME-PVK gives the ratio ka-TP-PVK/ ka-ME-PVK for systems that reached equilibrium. These values
are reported in Table 1. Figure S6 shows that the absolute values of the addition rate coefficients
is not important in the kinetic evolution, as long as the ratio of the two addition rate coefficients
is the same.

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However, for system that did not reach equilibrium, KTP-ME-PVK is not known. The data
obtained for the TP-PVK and ME-PVK indicates that the equilibrium constant is close to 1,
therefore for systems that did not reach equilibrium, but rather only a small fraction of the initial
adducts have exchanged, a value of KTP-ME-PVK = 1 is used for these simulations. The sensitivity
analysis in Figures S6-S13 suggests that these systems far from are dominated by the
fragmentation rate coefficeints (kf) and small changes in addition rate coefficients for either
adduct (ka) will not lead to substantial changes in the kinetic profiles.
Table 1 gives the kinetic parameters used to describe the experimental dynamic covalent
exchange data in Figure 1. Figure 1 also includes the theoretical (Th) data predicted from the
kinetic model with parameters given in Table 1, rows 1 and 2. In general the kinetic model
provides a good description of the experimental data with discrepancies between the theoretical
and experimental data being less than the typical error of NMR analysis of 1-5% and up to
10%.51-53 The good agreement between experimental and kinetic modeling data suggest that the
transformation pathway in Scheme 2 is a plausible pathway for dynamic covalent exchange of
thiol-Michael adducts. However, to rule out alternative pathways such as direct attack by the free
thiol, further kinetic studies are needed.

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ME-PVK (TP-PVK-In)
TP-PVK (TP-PVK-In)
1
0.8
0.6
0.4
0.2
0
ME-PVK (ME-PVK-In)
TP-PVK (ME-PVK-In)
TP-PVK (TP-PVK-In)-Th
ME-PVK (TP-PVK-In)-Th
TP-PVK (ME-PVK-In)-Th
TP-PVK (ME-PVK-In)-Th
0
2e+4 4e+4 6e+4 8e+4
time (s)
Figure 1: Kinetics of thiol-Michael dynamic covalent exchange over 24h, and simulations of the
data at 90 °C. System initialized from the thiophenol-phenylvinylketone adduct (TP-PVK-In) or
the mercaptoethanol-phenylvinylketone adduct (ME-PVK-In).
Figures 2 and 3 show the fitted and experimental data with different ratios of the initial
adduct (TP-PVK or ME-PVK) with different ratios of the free thiol (MESH or TPSH). Note that
the same parameters are used to fit all experiments in this TP-ME-PVK system as given in Table
1 rows 1 and 2. The experimental kinetic data in Figure 2 indicates that the initial ratio of
MESH:TP-PVK dictates the equilibrium position (after 24h), with higher ratio of MESH:TP-
PVK leading to less TP-PVK at equilibrium. The dotted lines give the equilibrium mole fraction
of TP-PVK or ME-PVK based on the initial ratio of MESH:TP-PVK and the mean equilibrium
constant KTP-ME-PVK = 0.66, taken as the average of Table 1 rows 1 and 2. The predicted
equilibrium concentrations (Eq) of TP-PVK and ME-PVK (dotted lines in Figure 2) are in good
agreement with the experimentally observed equilibrium values.

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A
B
1
1
0.8
0.6
0.4
0.2
0
0.6
0
8e+3
4 MESH-Ex
1 MESH-Ex
0.5 MESH-Ex
4 MESH-Th
1 MESH-Th
0.5 MESH-Th
4 MESH-Eq
1 MESH-Eq
0.5 MESH-Eq
0
2e+4 4e+4 6e+4 8e+4
time (s)
4 MESH-Ex
0.4
1 MESH-Ex
0.5 MESH-Ex
4 MESH-Th
1 MESH-Th
0.5 MESH-Th
4 MESH-Eq
1 MESH-Eq
1
0.8
0.6
0.4
0.2
0
0
0
8e+3
0.5 MESH-Eq
0
2e+4 4e+4 6e+4 8e+4
time (s)
Figure 2: Kinetics and equilibrium position of dynamic covalent exchange experiments (Ex)
using three ratios of mercaptoethanol (MESH) to the TP-PVK adduct at 90 °C. Kinetic
simulations (Th) and predicted equilibrium (Eq) presented. A) Gives the mole fraction of TP-
PVK, B) gives the mole fraction of ME-PVK. Insets of A and B give data for the first 2 hours of
reaction.
In addition, the kinetic model (Th) provides an acceptable agreement with the experimental
values, especially since no additional fitting was performed. An important observation is that in
the early phase of the reaction (first two hours, Inset Figure 2A and Inset Figure 2B), there is
essentially no experimentally observed difference in the kinetic evolution of TP-PVK and ME-

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PVK adduct mole fractions, despite the ratio of MESH:TP-PVK being changed by almost an
order of magnitude. This provides evidence that the exchange of adducts occurs through a
fragmentation (or retro-Michael reaction) pathway, followed by subsequent addition of the
Michael acceptor with a free thiol. If direct reaction of the free thiol, or its associated thiolate, on
the adduct was significant, increases in the free thiol concentration should increase in the rate of
exchange, which is in contrast to the early phase kinetic data with different free thiol
concentrations.
Figure 3 provides similar data to Figure 2, except that the ratio of TPSH:ME-PVK is varied
from 0.5 to 4 equivalents of thiol to adduct. Again at 24h, the concentration of TP-PVK and ME-
PVK agrees well with the values predicted from the mean equilibrium constant KTP-ME-PVK = 0.66
for each system studied (dotted line for each system). Further, there is acceptable agreement
between the kinetic model predictions (Th, dashed lines), using parameters from Table 1 rows 1
and 2, and the experimental data (Ex) for each system. The agreement is notable especially
considering that no further fitting was performed in describing these data. The early phase data
of each system (Figure 3A inset and Figure 3B inset) indicate that there is minimal difference in
the rate of initial exchange with the different free thiol concentrations. This again provides
evidence that the retro-Michael fragmentation of adducts is involved and rate determining in the
exchange of adducts. This similarity in early phase of the kinetics occurs over almost an order of
magnitude in thiol-concentration, which is inconsistent with a process where the free thiol, or its
thiolate, reacts with a thiol-Michael adduct. Overall the data in Figures 1-3 and the kinetic
modeling corroborate that the thermally driven thiol-Michael reaction is consistent with the
mechanism proposed in Scheme 2.

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A
B
4 TPSH-Ex
1 TPSH-Ex
0.5 TPSH-Ex
4 TPSH-Th
1 TPSH-Th
0.5 TPSH-Th
4 TPSH-Eq
1 TPSH-Eq
0.5 TPSH-Eq
0.4
0
1
0.8
0.6
0.4
0.2
0
0
8e+3
0
2e+4 4e+4 6e+4 8e+4
time (s)
4 TPSH-Ex
1 TPSH-Ex
0.5 TPSH-Ex
4 TPSH-Th
1 TPSH-Th
0.5 TPSH-Th
4 TPSH-Eq
1 TPSH-Eq
0.5 TPSH-Eq
1
0.8
0.6
0.4
0.2
0
1
0.6
0
8e+3
0
2e+4 4e+4 6e+4 8e+4
time (s)
Figure 3: Kinetics and equilibrium position of dynamic covalent exchange experiments (Ex)
using three ratios of thiophenolethanol (TPSH) to the ME-PVK adduct at 90 °C. Kinetic
simulations (Th) and predicted equilibrium (Eq) presented. A) Gives the mole fraction of TP-
PVK, B) gives the mole fraction of mercaptoethanol- ME-PVK. Insets of A and B give data for
the first 2 hours of reaction.
To further understand the underlying thermally activated thiol-Michael dynamic covalent
process, the kinetics of exchange were studied at 4 different temperatures 90, 75, 60 °C and room
temperature (363, 348, 333, and 293 K). Figure 4 gives the kinetic evolution of each elevated
temperature system, as well as experimentally fitted data. No measurable exchange of thiol-

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Michael adducts was observed in the NMR spectra at room temperature. Parameters used to fit
the data are given in Table 1. One key observation in Figure 4A and Figure 4B is that even after
24, the system has not approached equilibrium for the data obtained at 75 and 60 °C, since the
mole fraction of TP-PVK and ME-PVK is not the same when initialized from TP-PVK or ME-
PVK. This is due to the clearly slower rate of exchange at the lower temperatures of 75 and 60
°C, compared to the data obtained at 90 °C. Due to the highly efficient thiol-Michael adduct
forming reaction, with rate coefficients of ca. 1000 M-1s-1 for thiol-Maleimide coupling,54, 55 it
seems likely that the fragmentation is the rate determining step in the dynamic exchange. This is
consistent with the kinetic modeling and the experimental data in Figures 1-3. Therefore, the lack
of equilibrium established even after 24h at the lower temperatures is likely due to a slow rate of
fragmentation, or retro-Michael reaction. Fitting these experimental data with the kinetic model,
yields fragmentation rate coefficients for each adduct (TP-PVK and ME-PVK) for each
temperature studied. These rate coefficients given in Figure 4C as an Arrhenius like plot of log10
of the rate coefficient vs 1/Temperature. The slope of the Arrhenius plot can be used to estimate
the activation energy of the fragmentation, or retro-Michael, reaction. The activation energy of
TP-PVK is estimated as: Ea-TP-PVK = 75 kJ/mol while activation energy of ME-PVK is estimated
as: Ea-ME-PVK = 63 kJ/mol. These values of the activation energy are similar to each other, within
the uncertainty of the underlying experiments, and suggest a relatively slow process with a high
activation energy barrier, consistent with the observed data. The pre-exponential factors (A) of
the Arrhenius equation are: ln(ATP-PVK/1 s–1) = 13.8 ln(AME-PVK/1 s–1) = 9.3.

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A
ME-PVK (363 K)
TP-PVK (363 K)
ME-PVK (348 K)
TP-PVK (348 K)
ME-PVK (333 K)
TP-PVK (333 K)
ME-PVK (363 K)-Th
TP-PVK (363 K)-Th
ME-PVK (348 K)-Th
TP-PVK (348 K)-Th
ME-PVK (333 K)-Th
TP-PVK (333 K)-Th
1
0.8
0.6
0.4
0.2
0
0
2e+4 4e+4 6e+4 8e+4
time (s)
ME-PVK (363 K)
TP-PVK (363 K)
ME-PVK (348 K)
TP-PVK (348 K)
ME-PVK (333 K)
TP-PVK (333 K)
ME-PVK (363 K)-Th
TP-PVK (363 K)-Th
ME-PVK (348 K)-Th
TP-PVK (348 K)-Th
ME-PVK (333 K)-Th
TP-PVK (333 K)-Th
B
1
0.8
0.6
0.4
0.2
0
0
2e+4 4e+4 6e+4 8e+4
time (s)
C
k
f-TP-PVK
1x10-5
1x10-6
1x10-7
f-ME-PVK
k
0.0027
0.003
0.0033
1/T (K-1)
Figure 4: Experimental and simulated (Th) kinetics of dynamic covalent exchange experiments
at temperatures 363, 348 and 333 K. A) Initialized from TP-PVK, B) Initialized from ME-PVK,
C) Arrhenius plot of fitted adduct fragmentation rate coefficients.

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Figure 4C also extrapolates the Arrhenius plot to 1/T = 1/298 K, giving a predicted
fragmentation rate coefficient of kf ≈ 10–7 s-1 for either adduct under ambient conditions. This
would correspond to a half-lifetime of exchange at this temperature of t = ln(2)/kf ≈ 7 × 106 s ≈
½
80 days. Therefore, under ambient conditions of T = 25 °C there would be essentially no
exchange of thiol-Michael adducts observed even after ca. 2 months due to the very slow rate of
retro-Michael reaction, which is consistent with the experimental data. However, it is important
to note that elevated pH can induce retro-Michael reactions and promote dynamic exchange even
at ambient temperature.33
In addition to temperature and concentration based kinetic experiments, a Hammett analysis
can be performed to provide mechanistic insights. Figure 5 compares the dynamic exchange
kinetics for a range of para substituted (X group) vinyl ketone adducts. In particular, the methoxy
group (OMe) and the chloro group (Cl) are used to generate a system with an electron donating
(OMe) group and a electron withdrawing group (Cl) relative to the unsubstituted (H) system. The
data in Figure 5A and 5B clearly indicate that the withdrawing Cl group accelerates the rate of
dynamic exchange, whereas the donating OMe group decelerates the dynamic exchange. These
kinetic data are described using the kinetic model using parameters outline in Table 1. The
sensitivity analysis in Figures S6- Figure S13 indicate that the kinetic model is most sensitive to
the fragmentation rate coefficient (kf) of the adduct used to initialize the system. Therefore, the
increasing trend kf-TP-PVK-X and kf-ME-PVK-X with electron withdrawing character (increasing
Hammett σp parameter) can be reliably used to construct a Hammett plot.49 Figure 5C gives a
Hammett plot of kf-TP-PVK-X and kf-ME-PVK-X against the Hammett σp parameter,49 giving a strong
positive slope and good linear fit. The Hammett slopes are 1.6 for kf-TP-PVK-X and 1.5 for kf-ME-PVK-X

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which are indicative of strong negative charge character in the retro-Michael reaction’s rate
determining step.

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A
ME-PVK-OMe
TP-PVK-OMe
ME-PVK-H
TP-PVK-H
ME-PVK-Cl
1
0.8
0.6
0.4
0.2
0
TP-PVK-Cl
ME-PVK-OMe-Th
TP-PVK-OMe-Th
ME-PVK-H-Th
TP-PVK-H-Th
ME-PVK -Cl-Th
TP-PVK-Cl-Th
0
2e+4 4e+4 6e+4 8e+4
time (s)
ME-PVK-OMe
TP-PVK-OMe
ME-PVK-H
TP-PVK-H
ME-PVK-Cl
B
1
0.8
0.6
0.4
0.2
0
TP-PVK-Cl
ME-PVK-OMe-Th
TP-PVK-OMe-Th
ME-PVK-H-Th
TP-PVK-H-Th
ME-PVK-Cl-Th
TP-PVK-Cl-Th
0
2e+4 4e+4 6e+4 8e+4
time (s)
0.4
0.2
0
C
-0.2
-0.4
-0.6
TP-PVK-X
ME-PVK-X
-0.3 -0.2 -0.1
0
σp
0.1 0.2 0.3
p
Figure 5: Experimental and simulated (Th) kinetics of dynamic covalent exchange experiments
at 90 °C with different substituents. A) Initialized from TP-PVK, B) Initialized from ME-PVK,
C) Hammett plot of fitted adduct fragmentation rate coefficients across the substituents.

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Table 1: Summary of rate coefficients and obtained equilibrium constants used in all
simulations. a X is the substituent para on the benzene ring of PVK. b Equilibrium not observed
experimentally in 24 h, and value KTP-ME-PVK = 1 used in simulation. Estimated based on
c
equilibrium constant of 1.
Initial Adduct T (°C)
Xa
H
KTP-ME-PVK kf-TP-PVK (s-1) kf-ME-PVK (s-1) ka-TP-PVK/ ka-ME-PVK (-)
1.6×10-5
1.6×10-5
4.8×10-6
4.8×10-6
1.7×10-6
1.7×10-6
4.8×10-6
4.8×10-6
3.0×10-5
3.0×10-5
1.3×10-5
1.3×10-5
4.0×10-6
4.0×10-6
2.0×10-6
2.0×10-6
4.3×10-6
4.3×10-6
2.3×10-5
2.3×10-5
TP-PVK
ME-PVK
TP-PVK
ME-PVK
TP-PVK
ME-PVK
TP-PVK
ME-PVK
TP-PVK
ME-PVK
90
90
75
75
60
60
90
90
90
90
0.74
0.58
1 b
1.7
2.1
H
H
1.2 c
1.2 c
0.9 c
0.9 c
1.1 c
1.1 c
1.7
H
1b
H
1b
H
1b
OMe
OMe
Cl
1b
1b
0.79
0.83
Cl
1.6
This negative charge character seems counterintuitive due to the neutral nature of all compounds
shown in Scheme 2. However, close inspection of the proposed mechanism of the thiol-Michael
adduct formation reaction shows a chain reaction in thiolate and an anionic thioether-enolate
intermediate.23 This thiol-Michael adduct formation reaction is given in Scheme 3. The anionic
intermediate RS-PVK-X– is likely to explain the strong negative charge character suggested by
the Hammett analysis in Figure 5C. Of particular relevance to this analysis is the principle of
microscopic reversibility, which states that if a reaction pathway is followed in the forward in a
chemical processes, the microscopic reverse of this pathway is also traversed in the reverse
direction.56 Therefore, if the anionic intermediate RS-PVK-X– is an intermediate in the adduct
forming thiol-Michael reaction, it is also an intermediate in the retro-Michael reaction. In the
conjugation reaction thiolate attack is the rate determining step, implying loss of thiolate is also
significant in the elimination process.57 The Hammett analysis in Figure 5 suggests that
formation of the RS-PVK-X– intermediate, with its strong anionic character, is a major
contributor to the rate-determining step of the dynamic exchange of thiol-Michael adducts. These
results combined suggest that dynamic exchange of thiol-Michael adducts is primarily dictated

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by the fragmentation, or retro-Michael reaction. Therefore, dynamic properties in materials and
bioconjugates based on the thiol-Michael adduct will be primarily dictated by the rate of the
unimolecular retro-Michael reaction. This implies that the rate of dynamic exchange is unlikely
to be significantly accelerated by the addition of large quantities free thiol or free-Michael
acceptor, although this could lead to a change in material composition, especially if a large
excess of thiol or Michael acceptor were used. Additionally, this implies that a material
containing a thermally labile thiol-Michael adduct can be activated to dynamic exchange, even in
the absence of free thiol or free Michael acceptor, since the intrinsic retro-Michael reaction can
drive the dynamic exchange.
R
R
R
S
S
S
R S
R S
R S
R S
R SH
R SH
O
O
O
O
X
X
X
X
RS-PVK-X–
PVK-X
RS-PVK-X
Scheme 3: Proposed thiol-Michael and retro thiol-Michael reaction mechanism.
Conclusions
A mechanistic study of the thermally activated dynamic covalent chemistry of thiol-Michael
adducts was performed using a model system of thiophenol/mercaptoethanol dynamic
equilibrium with phenylvinylketone based Michael acceptors in N,N-dimethylformamide. The
dynamic equilibrium of thiol-Michael adducts was consistent with a retro-Michael reaction
followed by subsequent addition of a free thiol to the generated Michael adduct. The reaction
rate was primarily dictated by the rate of the retro-Michael reaction, or fragmentation of thiol-
Michael adducts. The early phase kinetics of dynamic thiol-Michael exchange was essentially
independent of the thiol concentration, suggesting that direct reaction of a free thiol with the

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thiol-Michael adduct was negligible. Additionally, these data are consistent with the retro-
Michael step being rate determining in the dynamic exchange of thiol-Michael adducts.
Temperatures of at least 90 °C were needed to observe equilibration in 24 h, for thiol-
phenylvinylketone adducts, and least 60 °C were needed in DMF to observe greater than 10%
exchange of thiol-phenylvinylketone adducts in 24h. Hammett analysis indicated that a donating
group on the Michael acceptor decreased the rate of dynamic exchange, while withdrawing
group accelerated the reaction, suggesting that the retro-Michael reaction goes through an
intermediate with strongly anionic character. This work provided insights into the dynamic
thermally driven thiol-Michael reaction, and can facilitate the optimization of the reaction in
dynamic covalent libraries and (bio)materials that utilize dynamic bonding.
Notes
§ Authors contributed equally.
Acknowledgements
We thank Hannah Lacy for contribution to the modeling of the reaction kinetics. We thank
Theresa Ramelot for experimental assistance with the HRMS data. Acknowledgment is made to
the Donors of the American Chemical Society Petroleum Research Fund (57243-DNI7) for
support of this research.
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