Tunable degradation of maleimide-Thiol adducts in reducing environments

Article abstract of DOI:10.1021/bc200148v

Addition chemistries are widely used in preparing biological conjugates, and in particular, maleimide-thiol adducts have been widely employed. Here, we show that the resulting succinimide thioether formed by the Michael-type addition of thiols to N-ethylm

Full text of DOI:10.1021/bc200148v

ARTICLE
pubs.acs.org/bc
Tunable Degradation of MaleimideꢀThiol Adducts in Reducing
Environments
Aaron D. Baldwin† and Kristi L. Kiick*,†,‡
†Department of Materials Science and Engineering, 201 DuPont Hall, University of Delaware, Newark, Delaware 19716, United States
‡Delaware Biotechnology Institute, 15 Innovation Way, Newark, Delaware 19716, United States
S Supporting Information
b
ABSTRACT: Addition chemistries are widely used in preparing
biological conjugates, and in particular, maleimideꢀthiol ad-
ducts have been widely employed. Here, we show that the
resulting succinimide thioether formed by the Michael-type
addition of thiols to N-ethylmaleimide (NEM), generally
accepted as stable, undergoes retro and exchange reac-
tions in the presence of other thiol compounds at physiolo-
gical pH and temperature, offering a novel strategy for controlled
release. Model studies (1H NMR, HPLC) of NEM conjugated
to 4-mercaptophenylacetic acid (MPA), N-acetylcysteine, or
3-mercaptopropionic acid (MP) incubated with glutathione showed half-lives of conversion from 20 to 80 h, with extents of
conversion from 20% to 90% for MPA and N-acetylcysteine conjugates. After ring-opening, the resultant succinimide thioether
did not show retro and exchange reactions. The kinetics of the retro reactions and extent of exchange can be modulated by the
Michael donor’s reactivity; therefore, the degradation of maleimideꢀthiol adducts could be tuned for controlled release of
drugs or degradation of materials at time scales different than those currently possible via disulfide-mediated release. Such
approaches may find a new niche for controlled release in reducing environments relevant in chemotherapy and subcellular
trafficking.
’ INTRODUCTION
In contrast to disulfide-based drug conjugation and polymer
network formation strategies, thiol-based Michael-type addition
reactions have emerged as a widely employed strategy for co-
valent conjugation of proteins, peptides, and drugs (to various
polymers and other molecules) by the reaction of free cysteine or
thiols with acrylamides, acrylates, vinyl sulfones, and maleimides.10
Maleimides have been commonly employed due to their specificity
to thiols, fast aqueous reaction kinetics, lack of byproducts, and the
stability of the thioether addition product.11 Maleimides have thus
been utilized in homobifunctional cross-linkers,12 heterobifunc-
tional cross-linkers,13 fluorescent labels,14,15 as well as in PEGyla-
tion reagents16 and cross-linking of hydrogels.17
While the wide use of maleimideꢀthiol conjugation reactions
have been motivated by the product stability, there have been
limited reports indicating that select succinimide thioethers can
undergo retro reactions at high temperatures (>300 °C)18 and in
some aqueous environments,19ꢀ23 although the mechanisms and
exact solution conditions for these retro reactions were not
elucidated in great detail. Such reports are very limited, however,
despite the fact that the reversibility of adducts of thiols with α,
β-unsaturated carbonyls such as ethacrynic acid and 4-hydroxyalkenals
has been long reported.24ꢀ27 We have thus recently explored the
Controlled release of drugs has been a key area of research in
the field of polymeric biomaterials, owing to the need to sustain
release in order to expand the therapeutic window and efficacy
of known drugs. Many chemical degradation approaches have
been used to control materials-based drug delivery including
chemical hydrolysis,1 enzymatic degradation,2 and disulfide
exchange.3 The rate of nonspecific chemical hydrolysis depends
mainly on aqueous pH and temperature, as well as on the
hydrophobicity of the environment around the hydrolytically
labile group. Enzymatic degradation, in contrast, occurs speci-
fically at enzyme-recognized peptide sequences, although rates
of degradation are dependent on the local enzyme concentra-
tion, activity, and accessibility to the substrate. Disulfide
exchange is sensitive to reducing environments and has found
use in drug delivery systems due to the weak reducing capacity
of blood (ca. 2ꢀ20 μM glutathione) compared with that of
reductive cellular compartments or highly reductive and hy-
poxic tumor tissues (ca. 0.5ꢀ10 mM glutathione).4ꢀ9 Disulfide
bonds have relatively short half-lives (<1 h) in highly reductive
environments, while maintaining a degree of stability in
circulation.5,8 Although control of the cleavage kinetics is
possible with variations in reducing agent concentration, only
limited control of kinetics has been achieved by varying the
disulfide’s neighboring chemical substituents (half-lives ranging
from 8 to 45 min).8
Received: March 24, 2011
Revised:
July 30, 2011
Published: August 25, 2011
r
2011 American Chemical Society
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Scheme 1. Proposed Exchange of Synthesized Maleimide
Thiol Adducts (1) with Glutathione in Aqueous Solutions
∼4, retarding the maleimideꢀthiol addition reaction; therefore,
the solution was stirred overnight at room temperature. The crude
product was diluted to 5 mL with solvent A and purified using RP-
HPLC. Purified fractions were collected and freezeꢀdried, with
approximately 90% yield for all reactions. 1a (Supporting Infor-
mation Figure S1) 1H NMR (MeOD): δ 0.96 (t, 3H), 2.63ꢀ2.69
(dd, 1H), 3.17ꢀ3.24 (dd, 1H), 3.39 (q, 2H), 3.61 (s, 2H),
4.13ꢀ4.17 (dd, 1H), 7.29 (d, 2H), 7.48 (d, 2H). ESI-MS: 338.1
+
(M-1H+2Na)+, 338.0 calc’d (C14H14NO4SNa2 ). 1b (Figure S2)
1H NMR (MeOD): δ 1.16 (t, 3H), 2.03 (d, 3H), 2.42ꢀ2.55 (m,
1H), 2.93ꢀ2.99 (m, 0.5H), 3.15ꢀ3.28 (m, 2H), 3.52ꢀ3.58 (m,
2.5H), 3.96ꢀ4.00 (dd, 1H), 4.67ꢀ4.75 (m, 1H). ESI-MS: 289.1
(M+H)+, 289.1 calc’d (C11H17N2O5S+). 1c (Figure S3) 1H NMR
(MeOD): δ 1.14 (t, 3H), 2.44ꢀ2.2.50 (dd, 1H), 2.69 (t, 2H),
2.91ꢀ2.99 (m, 1H), 3.09ꢀ3.23 (m, 2H), 3.53 (q, 2H), 3.91ꢀ3.94
(dd, 1H). ESI-MS: 276.0 (M-1H+2Na)+, 276.0 9 calc’d (C9H12-
reversibility of maleimideꢀthiol conjugation reactions as a potential
controlled degradation mechanism, which may have similar and
complementary applications to the disulfide-mediated release of drugs
in reducing environments. Scheme 1 illustrates the likely mechanism
for the covalent bond transfer from the initial succinimide thioether
compound (1) to a stable glutathione conjugate (3) in the presence
of excess reductant. We have found that the rate and extent of
this exchange, which is governed by the rate of the retro-addition, can
be modulated by increasing or decreasing the Michael donor’s
reactivity.
NO4SNa2 ). 3 (Figure S4) 1H NMR (D2O): δ 1.03 (t, 3H), 2.14
+
(m, 2H), 2.49 (m, 2H), 2.56ꢀ2.64 (m, 1H), 2.91ꢀ2.97
(dd, 0.5H), 3.06ꢀ3.28 (m, 2.5H), 3.45 (q, 2H), 3.89 (t, 1H),
3.93 (s, 2H), 3.94ꢀ4.00 (td, 1H), 4.60 (dt, 1H). ESI-MS: 455.2
(M+Na)+, 455.1 calc’d (C16H24N4O8SNa+).
1
NMR Analysis of MPA-NEM Retro Reactions. H NMR
spectroscopy with W5 water suppression28 was used to monitor
retro reactions of maleimide conjugates (Scheme 1). Samples of
MPA were dissolved at a concentration of 3 mg/mL in 0.2 M
phosphate buffer pH 7.4 with 10% D2O. High buffer concen-
trations were needed (relative to those employed in the HPLC
experiments below) in order to maintain a constant pH
throughout the experiment, owing to the high concentration
of MPA necessary for NMR investigation. After addition of
compounds, the pH was adjusted to 7.4 if necessary. A molar
equivalent of 2 was added to each NMR tube and the spectrum
was recorded. Some samples were ring-opened by incubation
at pH 8.0 @ 37 °C until ring-opening was complete (∼5 days).
Molar equivalents of GSH or glycine were added to ring-
opened and non-ring-opened samples. Samples were incu-
bated at 37 °C and spectra were recorded at time zero and
at 24 hr.
’ EXPERIMENTAL PROCEDURES
Chemicals and General Methods. N-Ethylmaleimide (2),
4-mercaptophenylacetic acid (MPA), N-acetyl-L-cysteine (AcCys),
3-mercaptopropionic acid (MP), and glycine were purchased
from Sigma-Aldrich (St. Louis, MO) and used without further
purification. All other reagents including glutathione (GSH) and
oxidized glutathione (GSSG) were purchased from Fisher Scien-
tific (Pittsburgh, PA). Crude reactions were purified via reverse-
phase chromatography on a Delta600 HPLC (Waters, Milford,
MA) equipped with analytical (3.5 μm particle size, 4.6 ꢁ 75
mm) and preparative (5 μm particle size, 19 ꢁ 150 mm) Waters
Symmetry300 C18 columns. Analytical linear gradients from
0% to 75% of solvent B were run over 20 min at 1 mL/min,
where solvent A is 0.1% trifluoroacetic acid (TFA) in water and
solvent B is 0.1% TFA in acetonitrile. Preparative-scale experi-
ments utilized similar elution profiles determined from the
analytical experiments, although with flow rates of 5 mL/min.
HPLC Evaluation of Reaction Kinetics. Synthesized conju-
gates (1) were dissolved at a concentration of 0.1 mM in 50 mM
phosphate buffers (pH 7.4) containing 10 mM GSH (and
5.0 mM, 0.5 mM, and 0.05 mM for GSSG). Lower buffer
concentrations were employed to permit quantitative detection
by HPLC, and these lower concentrations were sufficient to
maintain a constant pH throughout the experiment. The kinetics
of succinimide ring-opening were measured by monitoring
reactions incubated without reductant. The pH values of all
samples were verified and adjusted to 7.4 if needed before
incubation at 37 °C. 150 μL samples were collected periodically
and added to 150 μL of 0.5% formic acid solution to reduce the
pH and quench the retro and ring-opening reactions. Samples
were stored at ꢀ20 °C until analyzed. RP-HPLC injections were
carried out under the above-defined conditions and areas of
peaks were integrated to calculate conversion curves. The
identities of the compounds present in each peak were deter-
mined using LC-MS or MS.
1
Peaks were collected and analyzed via ESI-MS and H NMR.
1H NMR spectra were acquired under standard quantitative
conditions at ambient temperature on a Bruker DRX-400
NMR spectrometer (Billerica, MA). The spectra of all purified
compounds were recorded in either deuterated methanol or deu-
terium oxide.
SynthesisofSuccinimideThioetherCompounds.Methanol-
soluble mercapto-acids, MPA and MP (conjugates 1a and 1c),
were dissolved at a concentration of 100 mg/mL in methanol and
reacted with molar equivalents of 2. A catalytic amount of
triethylamine (0.01ꢁ) was added to the reaction mixture. The
reaction was stirred for 30 min at room temperature. The crude
product was diluted to 20 mL with solvent A and purified using
RP-HPLC. Water-soluble thiol compounds AcCys and GSH
(conjugates 1b and 3) were dissolved at a concentration of
50 mg/mL in deionized water. A molar equivalent of 2 was
dissolved in MeOH (75 mg/mL) and added to the thiol solution.
The unbuffered MeOH/aqueous solution had a final pH of
’ RESULTS AND DISCUSSION
NMR Analysis of MPA-NEM Retro Reactions. Our first
experiments sought to validate that retro Michael-type additions
were in fact a significant reaction route for select succinimide
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Figure 1. 1H NMR of aromatic protons of (A) MPA, (B) after addition of NEM to MPA, (C) immediately after addition of GSH to (NEM+MPA) and
(D) after incubation of (C) @ 37 °C for 24hrs. Evident in (D) are free MPA and small amounts of ring-opened 1a (7.14, 7.32 ppm) and GS-MPA mixed
disulfide (7.45, 7.18 ppm).
thioethers under reducing conditions. These experiments in-
volved 1H NMR analysis of the reaction of 4-mercaptophenyla-
cetic acid (MPA) with N-ethylmaleimide (NEM, 2) to yield (1a),
with subsequent incubation with glutathione (GSH). The for-
mation of the Michael-type adduct and its degradation via retro
reactions were easily observed in the NMR experiment.
incubation of conjugate 1a in the absence of reducing agents or in the
presence of other nucleophiles such as glycine yielded only the ring-
opened substituent (data not shown), clearly indicating that the
equilibrium lies toward the Michael adduct and that formation of
detectable quantities of free MPA only occurred when exogenous
thiols were added to the reaction. Therefore, select succinimide
thioethers should be sensitive to variations in reducing environments
near physiological conditions.
1
Figure 1A shows the H NMR spectrum for MPA in solution;
this spectrum presents chemical shifts centered at 6.88 and 7.18
ppm, which result from the reduced MPA, as well as minor
contributions from the oxidized form centered at 7.14 and 7.41
ppm. The chemical shifts of the thiophenyl aromatic protons are
sensitive to the identity of the thiol substituents and thus
provided a facile means to follow the addition and retro reactions
(Figure 1B,C). A molar equivalent of 2 was added to the solution
of MPA; upon addition of 2, the aromatic protons shift downfield
(to peaks centered at 7.18 and 7.36 ppm), indicating the
production of the conjugate 1a (Figure 1B). The resonances
resulting from the small fraction of oxidized MPA (centered at
7.14 and 7.41 ppm) remained unchanged. A molar equivalent of
GSH was added, resulting in immediate reduction of the
unreacted MPA, indicated by a shift in the aromatic protons
from 7.14 and 7.41 ppm (oxidized) to 6.88 and 7.18 ppm
(Figure 1C). No other changes in the spectrum for conjugate
1a were immediately apparent. After 24 h of incubation at 37 °C,
however, MPA was liberated from 1a as indicated by the increase
in intensity of the MPA aromatic protons (centered at 6.88 and
7.18 ppm, Figure 1D). Under these conditions, a minor amount
of ring-opening of the 1a also occurred (resonances centered at
7.14 and 7.32 ppm), but the low intensity of these resonances
indicates this occurs at a significantly slower rate than the retro
reaction. Hydrolysis of the succinimide ring could occur on either
carbonyl with respect to the thioether; however, the thiophenyl
protons of either species were not distinct. Hydrolysis of
the succinimide ring before incubation with GSH in these
NMR experiments hindered the retro reaction as no exchange was
observed over seven days incubation (data not shown). Furthermore,
HPLC Evaluation of Reactions Kinetics. We next sought to
determine the selectivity of these retro and thiol-exchange reac-
tions, as well as to obtain a quantitative measure of the time scales
of these reactions, at biologically relevant concentrations of reduc-
tant. Such analysis required the quantification of small quantities of
multiple compounds; HPLC and LC-MS were thus used to
permit both quantification and identification. Various addi-
tion products were synthesized by reacting 2 with MPA (1a),
N-acetylcysteine (1b), and 3-mercaptopropionic acid (1c).
These thio-acids, which exhibit different thiol pKa values
(6.6,29 9.5,30 10.3,31 respectively), were selected to determine
how the rate of exchange may vary with the pKa of the thio-acid.
0.1 mM of 1 was incubated in 10 mM GSH or 5.0 mM, 0.5 mM,
and 0.05 mM GSSG (see below) in phosphate buffer at pH 7.4
and 37 °C; succinimide ring-opening hydrolysis rates were
determined from solutions of 1 in buffer without addition of
GSH. Rates of formation of the product 3 (Scheme 1), under
these various conditions, were determined by monitoring
changes of peak area in the HPLC experiment; the identity of
the chemical species present in each fraction was confirmed by
LC-MS (data not shown).
Figure 2 shows a typical set of traces obtained upon incubation
of 1a with excess GSSG, showing the location of all peaks and
expanded regions highlighting relevant peaks. Arrows indicate
the direction of peak growth or recession with increasing time.
At time zero, a single peak was observed for compound 1a.
Over time, the peak for 1a decreased in intensity, while peaks for
compounds 3, GS-MPA mixed disulfide, and 1aRO (ring-opened 1a)
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Figure 2. HPLC traces of degradation of 1a in 50 mM phosphate buffer with 0.5 mM GSSG pH 7.4 @ 37 °C over a period of 6 days; masses obtained
from LC-MS experiments are displayed for (M+H)+. The peak area for 1a decreases with time as peak areas for 3, GS-MPA (mixed disulfide), and 1aRO
increase with time, indicating the occurrence of the retro Michael-type reaction. Arrows indicate the direction of peak area growth or decline with time.
Peak 3 consists of two equal peaks representing two diastereomers, while the two peaks of 1aRO evolve from ring-opening on either side of the
succinimide with respect to the thioether.
Scheme 2. Complete Reaction Cycle of 1 in Solution with Oxidized or Reduced Thiols
Scheme 3. Simplified Reaction Cycle and Kinetics Constants
for the Reaction of 1
all increased in intensity. The loss of 1 occurred concomitantly
with the generation of 3. Peaks for 3 and 1aRO are split into two
equal peaks. Peak 3 is split as a result of the two diastereomers
formed from the Michael addition of GSH to 232 (verified by the
equivalence of masses determined from LC-MS of the two
different peaks), while 1aRO is split depending on the side of
succinimide ring-opening in relation to the thioether (also verified
by the equivalence of masses determined from LC-MS of the two
different peaks). Scheme 2 illustrates the likely reaction cycle for an
initial conjugate 1 in the presence of excess thiols, showing ring-
opening of conjugates, as well as the exchange with GSH or GSSG.
Either GSH or GSSG causes exchange, with 5.0 mM GSSG yielding
identical results as 10 mM GSH (Supporting Information, Figure
S5). This observation suggests that the formation of 2 is the rate-
limiting step in the overall reaction to 3 and is significantly slower
than the disulfide exchange of the free thio-acid with excess GSSG;
therefore, the oxidation state of GSH does not significantly impact
the rate of formation of 3.
Throughout these experiments, no measurable amount of 2
was detected, confirming the widely known fact that the equilib-
rium greatly favors the succinimide thioether under the experimental
conditions. Thus, by neglecting the kinetics of formation of 2 (and
the thiol-exchange of GSSG to yield GSH as our above experiments
validate), Scheme 2 can be simplified to the combination of a
consecutive and parallel reactions (Scheme 3). Thus, pseudo first-
order rate constants, k1 and k3, can be defined for the ring-opening
of 1 and 3, and k2 can be defined as the pseudo first-order rate
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Figure 3. Relative HPLC measured concentrations for 1a (panel A) and 1b (panel B) over time, with constructed curves using derived rate constants
and eqs 5ꢀ8 for (9) 1 (a or b), (0) 1RO, (b) 3, and (O) 3RO
.
Table 1. Pseudo-First-Order Rate Constants and Respective
Half-Lives for Retro and Ring-Opening Reactionsa
constant for the retro and exchange reaction of 1 (to yield 3) in the
presence of a large excess of a thiol compound (10 mM GSH or
5 mM GSSG). Using the simplified Scheme 3, reaction rate equa-
tions (eqs 1ꢀ4)
retro reaction
k2 (hꢀ1
ring-opening
k1 or k3 (hꢀ1
half-life (h)
conjugate
)
half-life (h)
)
d½1ꢂ
¼ ꢀ k1½1ꢂ ꢀ k2½1ꢂ
ð1Þ
ð2Þ
ð3Þ
ð4Þ
dt
1a
1b
1c
3
0.0371 ( 0.0038 19 ( 2 0.0033 ( 0.0002 (k1) 211 ( 15
0.00207 ( 0.0002 337 ( 27 0.0044 ( 0.0003 (k1) 157 ( 12
d½1RO
ꢂ
n/a
n/a
0.0032 ( 0.0002 (k1) 215 ( 11
0.0076 ( 0.0007 (k3) 92 ( 2
¼ k1½1ꢂ
dt
--
--
a Standard deviations for all values were calculated from the standard
d½3ꢂ
dt
error from fit.
¼ k2½1ꢂ ꢀ k3½3ꢂ
eqs 5ꢀ8. As clearly illustrated in the figure, the fits show
exceptional agreement with collected data for all compounds.
1c did not exhibit measurable retro and exchange reactions
under these experimental conditions; therefore, only k3 was
determined. The fractional concentrations of 1 and 1RO as a
function of time decreased and increased, respectively, as ex-
ponential decay functions with respect to k1 and k2, as shown in
Figure 3. The dependence of the fractional concentrations of
3 and 3RO as a function of time were more complex, owing to the
consecutive reaction mechanism, as shown by an initial increase
and subsequent decrease in the fractional concentration of 3;
the fractional concentration of 3RO increased over time with a
delayed onset. The extent of formation of 1RO and 3RO was
directly related to the relative reaction rates of k1 and k2. In the
reactions of 1a, k2 > k1; therefore, the major product at equili-
brium is 3RO. Conversely, for 1b k1 > k2; therefore, 1RO is the
major product. Retro and exchange rates for 1a were an order of
magnitude greater than those for 1b, with rate constants of
0.0371 ( 0.0038 versus 0.00207 ( 0.0002 hꢀ1 and respective
half-lives of 19 ( 2 versus 337 ( 27 h, while half-lives for ring-
opening reactions of compounds 1 were the same order of
magnitude (ca. 102) for all compounds. In comparison, half-lives
for the glutathione-mediated cleavage of disulfide bonds under
similar conditions range from 8 to 45 min (with pseudo first-
order rate constants of 5 to 0.9 hꢀ1),8 indicating that the use
of maleimideꢀthiol adducts rather than disulfides may have
advantages for producing more stable, yet degradable
d½3RO
dt
ꢂ
¼ k3½3ꢂ
can be defined and converted to integrated rate laws (eqs 5ꢀ8)
þ k2Þt
½1ꢂ ¼ ½1ꢂ0eꢀðk
ð5Þ
1
ꢀ
ꢁ
k1½1ꢂ0
þ k2Þt
½1ROꢂ ¼
½3ꢂ ¼
1 ꢀ eꢀðk
ð6Þ
ð7Þ
1
k1 þ k2
k2½1ꢂ0
ꢀ
ꢁ
eꢀðk
ꢀ eꢀk t
þ k2Þt
1
3
k3 ꢀ k2 ꢀ k1
!
þ k2Þt
eꢀðk
ꢀ 1
eꢀk t ꢀ 1
1
3
k3k2½1ꢂ0
½3ROꢂ ¼
þ
ð8Þ
k3 ꢀ k2 ꢀ k1
ꢀk1 ꢀ k2
k3
Fractional concentrations of 1, 1RO, 3, and 3RO measured by
HPLC (relative to the initial concentration) were plotted as a
function of time for 1a (Figure 3A) and 1b (Figure 3B). Equa-
tion 7 was employed to fit to the data for 3 (converted from 1a and
1b (R2 > 0.98)), yielding k1, k2, and k3 with values shown in
Table 1, with their corresponding half-lives. Standard deviations
for all values were calculated from the standard error from the fit.
Curves to plot the fractional concentrations of the other
compounds as a function of time were constructed, as shown
in Figure 3, by input of the appropriate rate constants into
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ring-opening became significant and limited the overall conver-
sion to 3 for conjugates 1b and 1c (evidenced by the reduction in
turnover of these compounds at later time points), which could
be used to tailor initial release and long-term stability for specific
conjugates.
The manipulation of the Michael-type addition has very
limited precedent in literature, and there are no previous reports
to our knowledge that seek to selectively utilize the retro reaction
for a specific use. In studies by Lewis et al., in which a
bis-maleimide was used to conjugate a chelating agent to a
monoclonal antibody, it was determined that the resulting adduct
was cleaved to some extent when exposed to fresh human
serum.19 Later, Alley et al. described mass spectrometry experi-
ments that illustrated that an antibodyꢀdrug conjugate linked by
maleimideꢀthiol chemistries had exchanged with rat serum
albumin in vivo to yield albuminꢀdrug adduct.20 Concurrently,
Lin et al. described studies indicating that maleimideꢀcysteine
adducts disappeared in cells while similar iodoacetate adducts
were stable.21 The commonality in these reports stems from the
use of the resulting succinimide thioether in environments
containing reduced or oxidized thiol species, and although the
mechanism underlying these previous observations was not
discussed or determined, the reaction mechanism could proceed
as suggested in Scheme 1, as supported by our experiments. More
recently, Baker and co-workers have described the bromination
of maleimides for reversible conjugation of thiols as a bioconju-
gate technique.35ꢀ37 The process of bromination and subsequent
elimination of HBr by the addition of a protected cysteine was
found to be more rapid than the addition to maleimides. This
bromomaleimide conjugate was found to be reversible by
adding reducing agents such as GSH36 or TCEP37 with complete
conversion within 4 h for incubation with excess GSH. In
contrast, 70 h was required for 85% conversion of 2a, indicating
that the reduced reactivity of maleimides compared with bro-
momaleimides correlates with the rate of the retro reaction.
Figure 4. Fraction of 3 generated from (9) 1a, (b) 1b, and (2) 1c
(at the baseline) in the presence of (ꢀ) 10 mM, (--) 1.0 mM, and (
)
3 3
0.1 mM GSH.
bioconjugates. Indeed, in vivo blood circulation stability has
been shown to be as little as 4 h for disulfide-based immuno-
toxin-antibody conjugates, with circulation stability directly
dependent on the kinetics of reduction by glutathione.7,33,34
Accordingly, the decreased rate of exchange of the maleimideꢀ
thiol adduct with glutathione would increase compound circula-
tion stability while maintaining cleavage sensitivity in highly
reducing environments.
The release data from HPLC experiments illustrate the extent
of turnover of the initial succinimide thioether under various
reducing conditions, which is relevant to the use of these
strategies for the liberation of drug conjugates or materials
degradation. Thus, using these treatments, variations in the
overall generation of 3 as a function of time (for different
compounds and reductant concentrations) are presented in
Figure 4 (calculations from obtained data are shown in the
Supporting Information, Figures S6ꢀS8). 1a supported the
greatest rate of conversion of the initial adduct, with conversion
of nearly 85% after 70 h, followed by 1b with much slower
kinetics and substantially lower conversion, and then by 1c, for
which no measurable conversion was observed. The rate of
exchange of the initial adduct is clearly impacted by the thiol
pKa, with higher pKa decreasing the rate (Figure 4); the use of a
Michael donor with sufficiently high pKa (∼10.3) can eliminate
any impact of the retro reaction, as illustrated by the lack of
conversion of 1c.
Two other observations recommending the use of these
strategies for tailoring the degradation of succinimide thioethers
are indicated from these data. Importantly, the data in Figure 4
illustrate that the rate of thiol exchange varies with the concen-
tration of reductant. 1a showed the greatest dependence on
reductant concentration with the total production of 3 ranging
from 30% to 90% under these conditions. In contrast, conversion
of 1b showed little dependence, and that of 1c showed no
dependence, on reductant concentration; Michael donors can
thus be selected for application on the basis of desired sensitivity
to reductant. Additionally, the conversion to 3 can be impacted
by the inactivation of 1 by ring-opening of certain adducts. The
minimum half-lives for the retro reaction were approximately 19
h for 1a and 337 h for 1b, while those for inactivation of 1 by ring-
opening were approximately 200 h. Thus, the inactivation by
’ CONCLUSIONS
We have confirmed that succinimide thioethers undergo
reversible addition in solutions and that exchange with nearby
free thiols or disulfides can be manipulated under relevant
physiological conditions. Reverse reactions of these kinds have
not been reported for other Michael-type addition products such
as thiolꢀacrylate conjugates, most likely due to the reduced
Michael acceptor reactivity of acrylates compared to maleimides.11
Glycine, substituted for GSH in control experiments, did not
induce the reverse reaction under these conditions, although it
has been shown that maleimides can be Michael acceptors for
amine donors;38,39 these observations suggest that the retro reac-
tionmaynotbesubstantiallyaffectedbyamine-bearingcompounds
present in vivo. Our data also indicate that ring-opening of the
succinimide before the addition of GSH stabilizes the conjugate
and will inhibit the liberation of free thio-acid and conversion to 3.
Hence, purposely ring-opening a succinimide thioether will stabi-
lize bioconjugates for in vivo or in vitro assays if retro reactions
are not desired, and ring-opening that would occur in vivo could be
employed to dictate a lifetime over which a conjugated drug may be
cleaved by GSH.
Importantly, thiophenyl conjugates were also sensitive to the
reducing environment, with only approximately 30% converted
at low reductant concentration within three days, versus 90% at
high reductant concentration, potentially allowing for targeted
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Bioconjugate Chemistry
ARTICLE
release/delivery. The slower 2w?>relative to disulfide-mediated
release (e.g., half-lives >20 h for succinimide thioethers com-
pared to minutes for disulfide-mediated release) may also allow
for longer-term delivery of drugs in reducing environments. We
note that the exchange and retro reactions kinetics discussed here
were modulated by altering the thiol serving as the Michael
donor; in many bioconjugates such alteration of the thiol
reactivity would not be possible, particularly given that alkylthiols
(i.e., cysteine) of natural proteins and peptides are common
Michael donors in bioconjugation reactions. In cases in which
Michael donor reactivity on proteins/peptides would be desir-
able, post-translational modification of natural proteins or pep-
tides or non-natural amino acid incorporation could be em-
ployed for the addition of suitable thiols.40 Other possibilities not
investigated here rely on modulating the maleimide stability and
reactivity as has been accomplished by addition of cyclohexyl or
benzyl moieties to the nitrogen group to reduce the susceptibility
of the maleimide ring to hydrolysis prior to addition reactions.41
Albeit there are many different possibilities for tailoring retro
reactions for use as delivery mechanisms, our observations ex-
pressed here could be exploited for both systemic and local
administration of bioconjugated drugs or for imparting degrada-
tion sites in polymeric backbones or cross-linked biomaterials.
Studies to test these potential opportunities are underway.
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’ ASSOCIATED CONTENT
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Supporting Information. Experimental procedures, cha-
b
racterization details, and data analysis are provided. This material
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: +1 302 831 0201. Fax: +1 302 831 4545. E-mail:
kiick@udel.edu.
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’ ACKNOWLEDGMENT
This work was supported in part by the Nemours Foundation,
the National Science Foundation (DGE-0221651) and the
National Institutes of Health (5-P20-RR016472-10). The con-
tents of the manuscript are the sole responsibility of the authors
and do not necessarily reflect the official views of the National
Institutes of Health nor of the National Center for Research
Resources. The authors would like to thank Professor J. M. Fox
and research group members for assistance with LC-MS experi-
ments, as well as Dr. S. Bai for assistance with water suppression
1H NMR techniques.
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