Ring Substituent Effects on the Thiol Addition and Hydrolysis Reactions of N-Arylmaleimides

Article abstract of DOI:10.1021/acs.joc.5b02036

Maleimide groups are used extensively in bioconjugation reactions, but limited kinetic information is available regarding their thiol addition and hydrolysis reactions. We prepared a series of fluorogenic coumarin maleimide derivatives that differ by the

Full text of DOI:10.1021/acs.joc.5b02036

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Article
Ring substituent effects on the thiol addition
and hydrolysis reactions of N-arylmaleimides
Yingche Chen, Kelvin Tsao, Élise De Francesco, and Jeffrey W. Keillor
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b02036 • Publication Date (Web): 23 Nov 2015
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Ring substituent effects on the thiol addition and hydrolysis reactions
of N-arylmaleimides
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Yingche Chen, Kelvin Tsao, Élise De Francesco, Jeffrey W. Keillor*
Department of Chemistry and Biomolecular Sciences
University of Ottawa
10 Marie-Curie, Ottawa, Ontario K1N 6N5
Canada
*Corresponding author: jkeillor@uottawa.ca
Keywords
maleimide, hydrolysis, thiol addition, kinetics, mechanism
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TOC Graphic
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O
O
O
N
H
N
SR'
N
O
O
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O
R
O
O
O
R'SH
H2O
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N
N
H
O
O
O
N
O
O
R
N
H
N
H
R = H, Me, Et, iPr, OMe, OEt, OiPr
N
-O2C
R
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Abstract
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Maleimide groups are used extensively in bioconjugation reactions, but limited
kinetic information is available regarding their thiol addition and hydrolysis reactions.
We prepared a series of fluorogenic coumarin maleimide derivatives that differ by the
substituent on their maleimide C=C bond. Fluorescenceꢀbased kinetic studies of the
reaction with betaꢀmercaptoethanol (BME) yielded the second order rate constants (k2)
while pHꢀrate studies from pH 7ꢀ9 gave baseꢀcatalysed hydrolysis rate constants (kOH).
Linear freeꢀenergy relationships were studied through the correlation of log k2 and log
kOH to both electronic (σ+) and steric (Esnorm) parameters of the C=C substituent. These
correlations revealed the thiol addition reaction is primarily sensitive to the electronic
effects, while steric effects dominate the hydrolysis reaction. These mechanistic studies
provide the basis for the design of novel bioconjugation reactants or fluorogenic labelling
agents.
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Introduction
Bioorthogonal reactions are used extensively in chemical biology.1ꢀ3 In general,
these are based on the chemoselective reactions between functional groups that are not
prevalent in living systems. Some of the most commonly used bioorthogonal reactions
include azideꢀalkyne cycloaddition,1,2 tetrazine ligation,1 the Staudinger ligation2 and
aldehyde/ketone condensations.2 Other reactions have been developed for selectively
targeting the functional groups of naturally occurring amino acids such as Lys, Tyr and
Cys.3
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Maleimides are one of the most commonly used functional groups for performing
bioconjugation reactions with thiols,4ꢀ6 owing to the rapid Michael addition reaction7 that
occurs in good yield, under physiological conditions. Our group has exploited this thiol
addition reaction in the development of enzyme inhibitors8 as well as siteꢀspecific
fluorogenic protein labelling agents that comprise a phenyl ring bearing two maleimide
groups that are meta to each other, positioning the reactive C=C bonds around 10 Å
apart.9ꢀ13 The design of this dimaleimide moiety allows it to react efficiently with the two
thiol groups of an αꢀhelical peptide tag (‘dC10α’) bearing two Cys residues separated by
two turns of the αꢀhelix, about 10 Å.11 Although the rarity of this dicysteine motif
confers substantial selectivity for the use of this labelling reaction in biological milieu,11
the high intracellular concentration of glutathione (GSH), a thiolꢀcontaining tripeptide,
posed a considerable challenge for the intracellular application of our method.
Ultimately we were able to circumvent this problem by attenuating the reactivity of our
dimaleimide labelling agents with adventitious thiols such as GSH, through judicious
maleimide ring substituent effects.13
Over the course of that work, we were struck by the relatively limited detailed
kinetic data available for maleimide reactions, which is especially surprising if one
considers how frequently these reactions are used for bioconjugation. As discussed
below, the hydrolysis of Nꢀalkylmaleimides has been studied thoroughly,14,15 and their
thiol addition reaction has also been examined.4,16 However, less attention has been
given to the kinetics of the hydrolysis17 and thiol addition18 reactions of Nꢀ
arylmaleimides. Furthermore, even fewer studies11,19 have examined the kinetic effects
of substituents on the C=C double bond of the maleimide ring. Herein we present
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systematic kinetic studies of both the thiol addition and hydrolysis reactions of Nꢀ
arylmaleimides that reveal the maleimide ring substituent effects for each of these
relevant reactions. The rate data and mechanistic conclusions of this study will be useful
for the design of future probes and the optimisation of protocols for their application to
bioconjugation reactions.
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Results and Discussion
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Design and synthesis
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Historically, many kinetic studies of maleimide reactions have been conducted
spectrophotometrically, following the decrease in absorbance at 302 nm upon the reaction
of Nꢀalkylmaleimides.4,14ꢀ16,20,21 However, the aromatic group of Nꢀarylmaleimides
makes the absorbance change at 302 nm more difficult to follow,18 which led us to
explore the suitability of a fluorometric assay for kinetic studies of the reactions of this
class of compounds. Kanaoka first reported that a maleimide group can suppress the
latent fluorescence of a pendant fluorophore,22 and many other fluorogenic maleimide
derivatives have been prepared.23,24 Although originally it was suggested22,25 that the
maleimide quenching mechanism may involve an nꢀπ* transition of the maleimide
group, more recently we have shown10,12 that the quenching mechanism is based on
photoinduced electron transfer (PeT) from the excited state of the fluorophore to the
LUMO of the adjacent maleimide.26 Coumarin was shown many years ago to be
efficiently quenched by maleimides,27ꢀ30 and has the advantage of being a particularly
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synthetically accessible fluorophore.31,32
Furthermore, the high quantum yield,
photostability and hydrophilicity of 7ꢀdiethylaminoꢀ3ꢀcarboxylcoumarin derivatives are
qualities that are particularly well suited for long kinetic studies in aqueous solution.12
This body of evidence inspired us to design a series of Nꢀarylmaleimides bearing a 7ꢀ
diethylaminocoumarinꢀ3ꢀylcarboxamide moiety at the meta position, anticipating that the
cyan fluorescence of the pendant coumarin should be restored13 upon reaction of the
maleimide group (Scheme 1).
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Scheme 1: Coumarin maleimide substrates S1-S8 and their hydrolysis and thiol
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addition reactions studied herein.
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O
O
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N
H
N
SR'
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N
O
O
O
R
O
O
O
R'SH
H2O
N
H
N
N
O
O
R
O
O
-
N
H
NH CO2
R
R = H (S1)
OMe (S5)
OEt (S6)
OiPr (S7)
Br (S8)
CF3 (S9)
Me (S2)
Et (S3)
iPr (S4)
N
O
O
Our goal was therefore to prepare a diversified series of coumarin maleimides that
differed only in the maleimide substituent (R, Scheme 1). Recently, aminoꢀ33 and arylꢀ
substituted34 maleimides have been prepared by direct displacement or catalytic crossꢀ
coupling from bromomaleimides. In a similar fashion, we exploited the reactivity of
bromomaleimides in a convergent synthetic approach where diversely substituted maleic
anhydrides are added to a common primary amine at the end of the synthetic route. The
synthesis of the key amine intermediate was also based on a convergent approach,
involving the synthesis of the 7ꢀdiethylaminocoumarin 3ꢀcarboxylate fluorophore, and
the appropriately functionalized 1,3ꢀdiaminobenzene linker.
The required maleic anhydrides are either commercially available or were
prepared as shown in Scheme 2. Specifically, Nꢀphenylmaleimide was first subjected to
dibromination prior to elimination of HBr to give the monobromo maleimide 2 in
excellent yields. The bromide of 2 was displaced by ROH in low to moderate yield, prior
to ringꢀopening hydrolysis to give Nꢀphenylmaleamic acids 6-8 in very good yields.
Aniline was then eliminated from these maleamic acids in a ring closing reaction that
provided alkoxy substituted derivatives 9-11 in moderate to excellent yields.
Alternatively, alkyl substituted anhydrides 14-15 were prepared by condensation of the
initial aldehydes with glyoxylic acid, leading to lactones 12-13 in moderate yields, prior
to oxidation to the desired anhydrides with DessꢀMartin periodinane (DMP).
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Scheme 2: Synthesis of maleic anhydride intermediates
O
7
8
9
O
O
PhN
O
Et3N
THF
0 °C, 2 h
98%
Br2
CCl4
reflux, 1 h
91%
PhN
O
PhN
O
Br
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Br
Br
1
2
O
R
O
O
R
1) KOH, ROH,
r.t., 1 h
2) HCl
Ph
Et3N, ROH
reflux, 1 h
9-56%
Ac2O/HOAc (1:1)
80 °C, 4 h
O
PhN
O
N
H
O
76-85%
HO2C
R
51-94%
3 R = OMe
4 R = OEt
5 R = OiPr
6 R = OMe
7 R = OEt
8 R = OiPr
9 R = OMe
10 R = OEt
11 R = OiPr
OH
O
morpholine HCl,
O
H
glyoxilic acid
dioxane, H2O
100 °C, o.n.
27-28%
O
O
DMP
R
R
R
CH2Cl2
r.t., 2.5 h
33-49%
O
O
12 R = Et
13 R = iPr
14 R = Et
15 R = iPr
The diaminophenyl linker moiety was prepared asymmetrically in order to
facilitate the protecting group strategy envisaged for the final conjugation procedure. To
that end, metaꢀnitroaniline was first protected with two Boc groups, in quantitative yield.
The nitro group was then reduced by palladiumꢀcatalyzed hydrogenation to give primary
amine 18 (Scheme 3).
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Scheme 3: Synthesis of linker intermediates
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NBoc2
NH2
NBoc2
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9
Boc2O, DMAP, Et3N
THF
H2, Pd/C, MeOH
THF
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NH2
NO2
NO2
reflux, 1 h
100%
r.t., o.n.
25%
18
16
17
Coumarin fluorophore 19 was prepared as described previously,13 adapting a
condensation reaction used to prepare similar coumarin analogues.32 Coupling with
aniline linker 18 was accomplished by first converting acid 19 to its acid chloride. The
resulting amide 20 was then deprotected quantitatively, giving key intermediate 21 as the
final synthetic precursor (Scheme 4). The synthesis of 21 by an alternative route has also
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been reported very recently elsewhere.
Final substrates S1-S9 were prepared by the
reaction of 21 with the appropriate maleic anhydride. None of the synthetic steps were
stringently optimized, since substrates S1-S9 were prepared in sufficient yields to allow
the kinetic studies central to this study.
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Scheme 4: Synthesis of final coumarin maleimide substrates
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O
O
O
O
1) POCl3, DCE,
94 °C, 4 h
2) 18, CH3CN, pyr.,
r.t., o.n.
OH
N
NBoc2
TFA
CH2Cl2
r.t., 2 h
71%
H
N
O
19
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N
O
91%
20
O
O
O
O
1) substituted maleic anhydride,
acetone, r.t., 3 h
N
N
N
H
NH2
H
2) HMDS, ZnCl2, tol/DMF,
135 °C, 2 h
N
O
O
N
O
O
R
5-44%
21
R = H (S1)
Me (S2)
Et (S3)
OMe (S5)
OEt (S6)
OiPr (S7)
Br (S8)
Key intermediate
iPr (S4)
1) trifluoromethylmaleic anhydride,
CH2Cl2 (anh.), r.t., o.n.
2) (CO)2Cl2, DMF/DCM (1:50),
40 °C, o.n.
O
O
N
N
H
N
O
O
O
9%
CF3
S9
Scheme 5:
Synthesis of model compounds for product studies
O
1) substituted maleic anhydride,
acetone, r.t., 3 h
N
2) HMDS, ZnCl2,
NH2
O
tol/DMF, 135 °C, 2 h
58-84%
R
R = Me (M2)
Et (M3)
OMe (M5)
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Product studies
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Given the dense functionalization of the coumarin maleimide substrates used in
this work, product studies were first carried out to confirm their predicted reactivity. To
this end, model compounds were designed with simpler structures in order to facilitate
structural analysis by NMR. Although model compounds M2 and M3 are known,35ꢀ37
they were prepared for this study as shown in Scheme 5, by reacting pꢀtoluidine with the
appropriate maleic anhydride to give model substrates M2, M3, and M5.
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Thiol Addition
These model substrates were allowed to react with one equivalent of betaꢀ
mercaptoethanol (BME) in d6ꢀDMSO in an NMR tube at room temperature overnight
(see Experimental), and subsequent NMR analysis confirmed the formation of the
expected thiol addition products. A singlet at 2.34 ppm, corresponding to the remote pꢀ
methyl substituent of the toluidine moiety of all three model compounds, remained
unchanged in the formation of product, and was therefore used as an integration standard.
By this approach, the conversion of each reaction was determined, as shown in Table 1.
The regioselectivity of each reaction was also determined. For compound M2, the
maleimide methyl substituent R appeared as a singlet at 1.64 ppm in the “geminal”
product (G) and as a doublet at 1.25 ppm in the “vicinal” product (V, see Scheme 6). For
the ethyl derivative M3, the analysis was not quite as straightforward, but elucidation of
coupling patterns in an additional COSY spectrum allowed the ethyl CH2 group of G
(1.8ꢀ2.0 ppm) to be distinguished from that of V (1.6ꢀ1.7 ppm) (see Supporting
Information). Additional geminal coupling (J = 18.4 Hz) between the succinimide ring
protons of G, appearing as doublets at 3.25 and 2.66 ppm, confirmed the integration of
the assigned peaks. For the methylꢀsubstituted maleimide, BME added preferentially to
the less substituted carbon, and even more so for the ethylꢀsubstituted model compound,
suggesting steric hindrance may account at least partially for the observed
regioselectivity.
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Table 1: Product studies of model thiol addition reaction with BME (DMSO, 25 ºC,
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overnight).
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Model compound
R
Conversion
Regioselectivity
(V:G)
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(%)
92
Me
Et
58:42
M2
M3
M5
80
84:16
OMe
50
0:100
Scheme 6
Regioselectivity of model thiol addition reaction
OH
H
H
S
H
O
O
O
S
H
OH
BME
N
N
R
N
R
R
+
O
O
DMSO
O
vicinal product
geminal product
(V)
(G)
For the methoxy derivative M5, only one addition product was observed, after
50% conversion. The presence of geminally coupled succinimide protons at 2.90 ppm
and 3.48 ppm confirmed it was the G regioisomer that was formed exclusively. This
probably reflects the strong electron donating character of the methoxy group through
resonance with the maleimide double bond, increasing the electron density of the βꢀ
carbon (Scheme 7). This resonance delocalisation is also reflected in the increased
shielding of the maleimide proton of M5, whose chemical shift is 6.01 ppm, compared to
6.77 and 6.75 ppm for the methyl and ethyl derivatives.
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Scheme 7
Resonance model and regioselectivity of M5 thiol addition
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Finally, the effect of solvent on the regioselectivity of the thiol addition was
examined, by repeating the experiment with methyl model compound M2, in the presence
of increasing amounts of D2O. Interestingly, the V:G ratio shifted from 58:42 in 100%
DMSO to 55:45 at 10% D2O and 48:52 in 20% D2O. This trend suggests that in the 95%
water conditions of our kinetic studies (see below), the geminal addition product may be
formed exclusively. However, we were unable to measure this directly, as we were
unable to increase the concentration of D2O above 20%, while maintaining the
concentrations necessary for NMR analysis, due to the limited solubility of the model
compound.
Hydrolysis
Model compounds M2, M3 and M5 were also subjected to hydrolysis and
product analysis. As described in detail in the Experimental section, model compounds
were hydrolyzed in the presence of 1% NaOH and the resulting product mixture was
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extracted and analyzed by NMR. Hꢀ and CꢀNMR indicated that only one hydrolysis
product was formed for each of these reactions. Twoꢀdimensional HMBC experiments
showed a correlation between the carboxylate carbon of the maleamic acid product (see
Scheme 1) and the methyl protons of M2 or the methylene protons of M3, indicating
these two model compounds reacted exclusively through the carbonyl adjacent to the
substituted carbon (see Supporting Information). After the reaction of compound M5, the
methoxy proton in the sole hydrolysis product showed a correlation peak with the
substituted alkene carbon, whose chemical shift (160.3 ppm) is consistent with an
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adjacent carboxylic acid rather than an adjacent anilide (expected 170 ppm). This
indicates the reaction occurred with similar chemoselectivity.
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Kinetic studies
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Hydrolysis
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Although our primary interest is the fluorogenic thiol addition reaction of
maleimide derivatives, it is also relevant to consider the stability of maleimides in
aqueous solution, since maleamic acids do not show the same propensity for thiol
addition reactions.38 Previously it has been shown that Nꢀphenylmaleimide undergoes
hydrolysis 25 times more rapidly than Nꢀethylmaleimide (pH 7.0, 30 ºC).17 However, at
near neutral pH, Nꢀarylmaleimides are still stable enough to be used for conjugation
reactions, based on their much faster thiol addition reactions.11,18,24,39
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We sought to study the effect of maleimide ring substituents on their hydrolytic
activity, by measuring the rate constants of hydrolysis for our series of substrates, from
pH 5.0 to 10.5. We immediately observed that our most electrophilic substrate, S9, was
highly prone to degradation in aqueous solution, leading to a multitude of products. This
is consistent with McLeod’s report19 that an Nꢀaryl trifluoromethylmaleimide reacts
completely with NꢀAcꢀCysꢀOH in <3 minutes in DMSO; however, we deemed that the
kinetic investigation of S9 in water would be highly impractical, so it was not studied
further. In contrast, pseudoꢀfirst order rate constants of hydrolysis (kobs) were easily
measured for substrates S1-S7 (see Experimental), although the limited solubility of S6
prevented its study below pH 7.7.
Three things are evident from the resulting plot of log kobs vs pH (Figure 1). First,
the pHꢀrate profile of each substrate appears to plateau from pH 5 to ~7. The pH
independence over this range suggests the hydrolysis reaction may include a mechanistic
pathway that involves the attack of neutral water on maleimide. Varying the
concentration of buffer led to statistically insignificant variations in the measured rate
constants, suggesting no buffer catalysis occurred. Second, the slope of the basic limb of
the pHꢀrate profile can be fit to a slope of 1, indicating the reaction of one equivalent of
hydroxide above pH 7. Third, the second order rate constants for reaction with hydroxide
(kOH) calculated for each substrate (see Experimental) differ significantly from one
another, suggesting the hydrolysis reaction is sensitive to maleimide ring substituent
effects (see below).
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Figure 1. pHꢀrate profile for the hydrolysis of A) substrates S1-S4 and B) substrates
S5-S7 (50 mM aqueous buffer, 5% DMSO, 22 ºC). Solid lines are for fitted
second order rate constants, as described in Experimental.
A
B
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-1.0
-2.0
-3.0
-4.0
-5.0
-6.0
-7.0
-8.0
-1.0
-2.0
-3.0
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-8.0
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S7
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pH
pH
The second order rate constants for hydroxideꢀcatalyzed hydrolysis are shown in
Table 2. Very few rate constants have been reported for the hydrolysis of Nꢀaryl
maleimides. Salhany studied18 the hydrolysis of an unsubstituted maleimide bearing an
eosin fluorophore on its ring nitrogen, showing a linear pHꢀrate profile from pH 7 to 10.2
(25 ºC). Although no secondꢀorder rate constant was reported, halfꢀlives corresponding
to log kobs values of ꢀ4.00 (pH 7.0) and ꢀ2.21 (pH 9.0) allow a rough comparison to the
corresponding unsubstituted maleimide S1 (see Figure 1) that indicate the latter is an
order of magnitude less reactive. It is not surprising that the substituent on the maleimide
nitrogen would influence its rate of hydrolysis; Kanaoka documented this effect
17
thoroughly in a study of a series of six paraꢀsubstituted Nꢀphenylmaleimides.
At pH
8.0, the reported halfꢀlives correspond to log kobs values from ꢀ3.46 (pꢀNMe2) to ꢀ2.77 (pꢀ
NO2) (30 ºC), which are also 1ꢀ2 orders of magnitude greater than that of S1. The σpara
values of the substituents of Kanaoka’s six substrates were used to provide a Hammett ρ
value of +0.35 for the series, consistent with nucleophilic attack on the maleimide
carbonyl being rateꢀlimiting. 17
It is also instructive to compare our second order rate constants with kOH values
measured for Nꢀalkyl maleimides. Matsui reported15 kOH values for a series of
maleimides bearing H or alkyl groups on the maleimide nitrogen. These include Nꢀ
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ethylmaleimide (NEM), whose kOH values were reported to be 34.7 Mꢀ1sꢀ1 at 20 ºC and
46.8 Mꢀ1sꢀ1 at 30 ºC, which are comparable to that of S1 (Table 2). Furthermore,
Matsui’s pHꢀrate plot gives a log kobs value of ꢀ5 for NEM at pH 7 (30 ºC), which is also
very similar to that measured for S1 (Figure 1). From these comparisons it would appear
that not all Nꢀarylmaleimides are hydrolyzed more rapidly than all Nꢀalkylmaleimides.
Finally, by plotting the log kOH values against the Taft polar parameter σ*, the authors
measured a ρ* value of 0.55 (30 ºC) for the series, consistent with attack by hydroxide
being rateꢀlimiting for their Nꢀalkylmaleimides.
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Table 2. Second Order Rate Constants for Hydrolysis and BME Addition Reactions of
NꢀArylmaleimides
Substrate
S1
kOH (Mꢀ1sꢀ1)
67 ± 17
24 ± 4
k2 (Mꢀ1sꢀ1)
8.9 ± 0.7
4.4 ± 0.2
2.2 ± 0.2
1.3 ± 0.1
1.3 ± 0.1
N.D.a
kOH / k2 Ratio
R
H
7.6
5.6
1.1
0.8
40.5
ꢀ
Me
S2
Et
iPr
2.5 ± 0.5
1.1 ± 0.3
45 ± 5
S3
S4
OMe
OEt
OiPr
S5
41 ± 7
S6
15 ± 1
N.D.b
ꢀ
S7
a
Not Determined: S6 is not soluble in aqueous solution at pH 7.4, used for the thiol
addition reactions. b Not Determined: The hydrolysis of S7 dominates at pH 7.4, used for
the thiol addition reactions.
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Thiol Addition
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Kinetic studies of the thiol addition reactions of substrates S1-S7 were carried out
at pH 7.4 and 37 ºC, in order to increase the relevance to in vivo labelling applications.
Similarly, BME was used as a model thiol, owing to its structural resemblance to
biological thiols and its aqueous solubility. Kinetic reactions were performed under
pseudoꢀfirst order conditions, using 25 µM substrate and 10ꢀ100 eq of BME, allowing
first order rate constants to be measured from the monoꢀexponential increase in
fluorescence (see Experimental). Second order rate constants (k2) were then measured
from the plots of kobs vs [BME]. Under these conditions, S6 was not sufficiently soluble
to allow kinetic studies (see above), and substrate S7 reacted so slowly that it was
difficult to discern above background hydrolysis. However, for substrates S1-S5, the
thiol addition reaction was typically >100ꢀfold faster than background hydrolysis, and
easily measured.
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The second order rate constants (k2) thus obtained for substrates S1-S5 are shown
in Table 2. The unsubstituted maleimide S1 had the highest rate constant for thiol
addition, and can be compared with a few values reported in the literature. For example,
Bednar showed4 the reactivity of neutral BME with NEM is insignificant, but the thiolate
of BME reacts with a rate constant of 1.8 × 105 Mꢀ1sꢀ1 at 25 ºC. The pKa of BME is
9.61;40 correcting for the proportion of BME thiolate at pH 7.4 leads to a predicted rate
constant for the reaction with NEM of 1.2 × 103 Mꢀ1sꢀ1. This is lower than the value of 9
× 103 Mꢀ1sꢀ1 reported18 for the reaction of Cys with Nꢀeosinꢀmaleimide at pH 7.4 (25 ºC),
but very similar to the value of 1.1 × 103 Mꢀ1sꢀ1 that we reported11 previously for the
reaction of GSH with a diꢀNꢀarylmaleimide fluorogen at pH 7.5 (20 ºC). It is noteworthy
that all of these rate constants are still two orders of magnitude higher than that measured
for the reaction of BME with S1 (8.9 Mꢀ1sꢀ1 from Table 2); however, the structural
differences in the thiols and the maleimide Nꢀsubstituent between all of these studies
makes a direct comparison impossible.
It is also possible to note significant substituent effects for the thiol addition
reaction, in that the k2 rate constants vary by nearly an order of magnitude from S1 down
to S5. However, these substituent effects are less pronounced than those observed for
hydrolysis, for which the kOH values vary by nearly two orders of magnitude.
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Furthermore, it appears that a given substituent can have a different effect on the
hydrolysis and the thiol addition reaction. This is evident from the ratio of rate constants
shown in Table 2. If the substituents had analogous effects on both reactions, one would
expect the ratio of rate constants to be constant or to vary smoothly. However, while the
methoxy substituent of S5 decreases its k2 value relative to unsubstituted S1, it has very
much less effect on the value of kOH relative to S1. This suggests the mechanisms for the
two reactions may show very different dependence on the substituents.
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Linear free energy relationships
Comparison of the first four entries of hydrolysis rate constants in Table 2 (S1-
S4) reveals a significant effect of the steric bulk of the alkyl substituent on the hydrolytic
reactivity. Further comparison of these values to those of the next three entries (S5-S7)
suggests that the alkoxy substituents may promote hydrolysis relative to the alkyl
substituents. However, it is difficult at first glance to separate the steric from the
electronic effects of the substituents, implying that if a linear free energy relationship
(LFER) exists for these rate data, it probably involves both steric and electronic
parameters for the ring substituents. As mentioned above, different trends can be noted
for the thiol addition rate constants, but these substituent effects probably involve both
steric and electronic components, also. From this it appears that the first step of more
extended mechanistic analysis is the choice of appropriate substituent parameters.
In both hydrolysis and thiol addition reactions, maleimides react as electrophiles.
Furthermore, the substituents of substrates S1-S9 are directly attached to the conjugated π
system of the maleimide ring. These observations led us to propose that the Hammett σ+
parameter may be an appropriate measure of the electronic effect of the substituents
studied herein, including alkoxy substituents that can act as electron donating groups
41
through resonance delocalisation.
DFT calculations were performed on substrates S1-
S9, providing density maps and energy levels for the LUMO of each molecule (see Table
S1, Supporting Information). These energy levels correlate very well with the
corresponding σ+ values (see Figure S1, Supporting Information), as demonstrated
recently for other systems,42 providing some confirmation that this substituent parameter
is appropriate for measuring electronic effects.
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However, plotting log kOH and log k2 values against σ+ values alone (Supporting
Information, Figures S2 and S3) clearly illustrates the poor correlation between the
measured rate constants and the isolated electronic substituent effects. Given the
proximity of the maleimide ring substituent to the site of nucleophilic attack, this is not
surprising. In particular, the nucleophilic attack of thiolate on the substituted alkene
carbon, giving rise to the G regioisomer discussed above, resembles the ester hydrolysis
reaction chosen by Taft43 for which he proposed the use of steric parameters. In Taft’s
model reaction, the steric effects of substituents on the C=O group of methyl esters were
found to influence reaction rate in addition to their electronic effects, so it is reasonable to
presume that this may also be true for thiolate attack on substituted carbon of the C=C
bond in the maleimide ring. Steric parameter values were therefore either taken directly
from the literature44 or calculated (see Supporting Information), then normalized to H and
subsequently used in the twoꢀparameter fitting of log kOH and log k2 values (see Table 3
and Experimental).
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Table 3. Electronic (σ+) and steric parameter (Esnorm) values used herein for the
substituents (R) of substrates S1-S7.
Esnorm b
σ+ a
0.00
Substrate
R
H
0.00
ꢀ1.24
ꢀ1.31
ꢀ1.71
ꢀ0.25
ꢀ0.34
ꢀ0.82 c
S1
S2
S3
S4
S5
S6
S7
Me
ꢀ0.31
ꢀ0.29
ꢀ0.28
ꢀ0.78
ꢀ0.81
Et
iPr
OMe
OEt
OiPr
ꢀ0.85
b
a Taken from reference 41. Taken from reference 44.
c Calculated from extrapolated values (see Supporting Information).
Twoꢀparameter fitting of hydrolysis rate data of substrates S1-S7 gave an
electronic reaction constant of ρ = ꢀ0.1 ± 0.5 and a steric reaction constant of δ = 0.9 ±
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0.2, leading to the plane shown in Figure 2 and the correlation of observed to predicted
values shown in Figure S4 (Supporting Information). Although this correlation is
significantly better than that obtained for fitting with electronic parameters alone (not
shown), it still only accounts for ~80% of the measured data points. Nevertheless, it is
clear that the effect of ring substituent on the hydrolysis reaction is largely steric and
minimally electronic. Assuming that hydroxide attack is rateꢀlimiting, as LFER studies
of the hydrolysis of other maleimides have shown 15,17 (see above), the significant δ value
suggests the nucleophilic attack on at least one imide carbonyl may be hindered by the
substituent, perhaps owing to the trajectory of the attack on the delocalised π* system.
Indeed, the results from our product studies indicate that the imide carbonyl that
undergoes hydrolysis is the carbonyl adjacent to the substituted carbon, which may be the
most sensitive to the steric bulk of that substituent. The fact that this carbonyl reacts
preferentially may be due to the relief of steric strain between the carbonyl and the
adjacent coꢀplanar substituent upon formation of a tetrahedral intermediate during
nucleophilic attack. The tetrahedral intermediate formed by attack on the carbonyl distal
from the substituent would not benefit from this driving force due to ground state
destabilization. Finally, the small ρ value measured for the hydrolysis reaction suggests
that hydroxide may be a strong enough nucleophile to be insensitive to the electron effect
of the substituent on the C=C bond.
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Figure 2: Plane fitted to the correlation of log kOH values with both electronic (σ+) and
steric (Esnorm) substituent parameters, yielding respective reaction constants of
ρ = ꢀ0.1 ± 0.5 and δ = 0.9 ± 0.2.
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S5
S6
S1
S7
S2
S3
S4
For the rate data from the thiol addition reaction of substrates S1-S5, twoꢀ
parameter fitting gave an electronic reaction constant of ρ = 1.1 ± 0.4 and a steric
reaction constant of δ = 0.2 ± 0.4. The plane defined by these fitted values is shown in
Figure 3, and the correlation of predicted to measured rate constants is shown in Figure
S5 (Supporting Information). The fitted values of these parameters suggest that the thiol
addition reaction is unlike the hydrolysis reaction, in that it is less sensitive to the steric
effect of the substituent, and more sensitive to its electronic effect. The smaller steric
sensitivity of thiolate addition compared to hydrolysis may be due to the greater
polarisability of the larger Sꢀ nucleophile compared to the smaller Oꢀ nucleophile. The
greater electronic sensitivity of thiolate addition can also be explained by the direct
conjugation of the substituents with the reactive C=C bond as shown above in Scheme 7.
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However, the rough correlation shown in Figure 3 cautions against overꢀ
interpretation of the data. Moreover, as discussed above, the thiol addition reaction leads
to the formation of two regioisomers. The rate constants for the formation of each of
these regioisomers would presumably be affected differently by any given substituent,
but our kinetic experiments, based on the appearance of total product, only inform us of
the sum of the rate constants. Although knowledge of the ratio of these regioisomers
would have allowed the correction of the observed second order rate constants into
relative rate constants for geminal and vicinal addition, we were unable to directly
measure this ratio under the conditions of the kinetic experiments (5% DMSO) due to
solubility issues (see above). This uncertainty with respect to regioselectivity underlines
the intrinsic approximation in using only one pair of substituent parameters for substrates
that may undergo two different (regioisomeric) reactions. However, taking into account
the solventꢀdependent regioselectivity that we were able to measure, we can conclude, at
least qualitatively, that thiol addition is more sensitive to electronic than steric effects.
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Figure 3: Plane fitted to the correlation of log k2 values with both electronic (σ+) and
steric (Esnorm) substituent parameters, yielding respective reaction constants of
ρ = 1.1 ± 0.4 and δ = 0.2 ± 0.2.
9
S1
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Conclusions
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In summary, a series of ringꢀsubstituted maleimide derivatives was prepared and the
reactivity of these derivatives towards baseꢀcatalyzed hydrolysis and thiol addition was
studied. Product studies indicate the hydrolysis reaction proceeds in a chemoselective
manner, favouring the reaction of the carbonyl adjacent to the substituted carbon. The
regioselectivity of the thiol addition reaction was found to depend upon the nature of the
substituent. Twoꢀparameter substituent effects indicate the hydrolysis rate constant is
almost entirely dependent on the steric effect of the substituent, while the thiol addition
reaction shows predominant dependence on the electronic effect of the substituent. These
kinetic data and trends in reactivity should prove useful for the design of substituted
maleimide derivatives for a variety of bioconjugation applications.
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Experimental Section
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General
7
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9
All reagents and solvents for reactions were used as received unless otherwise stated.
Dichloromethane, methanol and tetrahydrofuran were dried by a solvent purification
system. All reactions were performed under an inert atmosphere (e.g. N2) in ovenꢀdried
glassware unless otherwise stated.
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Reactions were monitored by thin layer chromatography (TLC) using silica gel preꢀ
coated aluminum plates. Components were visualized by illumination with a shortꢀ
wavelength ultraꢀviolet light or longꢀwavelength visible light after which staining was
performed with KMnO4 solution followed by heating. Flash column chromatography was
performed on silica gel (40ꢀ63 µm mesh) using ethyl acetate/nꢀhexane or
acetonitrile/dichloromethane as eluting solvents.
Nuclear magnetic resonance (NMR) spectra were recorded in deuterochloroform with
tetramethylsilane (TMS) as internal reference at ambient temperature unless otherwise
stated. Spectra were obtained at 400 or 500 MHz for 1H and at 100.6 MHz for 13C.
EIꢀMS spectra were recorded for both low resolution and high resolution mass spectra
using a magnetic sector mass analyzer. ESIꢀMS spectra were recorded using a
quadrupoleꢀtime of flight mass analyzer.
Ultraviolet absorption spectra and fluorescence spectroscopic studies were performed on
a UV/vis microtiter plate reader.
Synthesis
Compounds 1-4, 6, 7, 9, and 10 were synthesized according to a literature procedure45
and isopropoxymaleic anhydride (11) was synthesized according to a similar procedure
through intermediates 5 and 8. Alkyl substituted maleimides (12-15) were prepared by an
aldol reaction, followed by the oxidation of the alcohol to the ketone.46
3-Isopropoxy-1-phenyl-1H-pyrrole-2,5-dione (5): To a suspension of 3ꢀbromoꢀ1ꢀ
phenylꢀ1Hꢀpyrroleꢀ2,5ꢀdione (2) (253 mg, 1 mmol) in isopropyl alcohol (4 mL), was
added triethylamine (500 µL, 3.6 mmol). The mixture formed was heated to reflux and
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stirred for 1 h. The reaction was cooled and the solvents were removed by evaporation.
5
6
7
8
The residue was purified by column chromatography to obtain compound 5 as yellow oil
1
(20 mg, 9 % yield). H NMR (400 MHz, CDCl3): δ (ppm) 7.45 (m, 2 H), 7.31 (m, 3H),
13
5.45 (s, 1H), 4.51 (m, 1H), 1.46 (s, 3H), 1.44 (s, 3H); C NMR (100 MHz, CDCl3): δ
9
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(ppm) 169.6, 158.9, 131.3, 129.0, 128.8, 127.7, 127.2, 126.4, 126.1; LRMS (EI) m/z (%):
231.1 (M+, 63%); HRMS (EI): calcd for C13H13NO3: 231.0895, found: 231.0888.
2-Isopropoxy-4-oxo-4-(phenylamino)but-2-enoic acid (8): KOH (300 mg, 5.4 mmol)
in H2O (7.2 mL) was added to a solution of 5 (825 mg, 3.6 mL) in isopropyl alcohol (7.2
mL), and the mixture formed was stirred at ambient temperature for 1 h. After the starting
material was totally consumed, the organic solvents were evaporated. The residue was
acidified with concentrated HCl at 0 ºC. The precipitate was filtered out and dried as
crude product 8 (679 mg, 76% yield) and used in the following steps without further
purification.
3-Isopropoxyfuran-2,5-dione (11): 2ꢀIsopropoxyꢀ4ꢀoxoꢀ4ꢀ(phenylamino)butꢀ2ꢀenoic
acid (8) (679 mg, 2.73 mmol) was heated at 80 ºC in a mixture of HOAc and Ac2O (1:1,
18 mL) for 4 h. Ac2O and HOAc were distilled off in vacuo and the residue was purified
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by silica gel column chromatography to obtain 11 (400 mg, 94%) as a colourless oil. H
NMR (400 MHz, CDCl3): δ (ppm) 5.62 (s, 1H), 4.51 (m, 1H), 1.46 (s, 3H), 1.43 (s, 3H);
13C NMR (100 MHz, CDCl3): δ (ppm) 129.8, 128.7, 126.1, 97.9, 78.6, 27.0, 21.2;
HRMS (EI): m/z calcd for C7H8O4 ([M+]): 156.0423, found: 156.0436.
4-Ethyl-5-hydroxyfuran-2(5H)-one (12)47 : Solid morpholine hydrochloride (3.4 g, 27.5
mmol) was added to a solution of glyoxylic acid monohydrate (2.3 g, 25 mmol) in 20 mL
of dioxane. To this suspension was added dropwise 3 mL of H2O, after which all the solid
material was dissolved. A solution of butyraldehyde (2.36 mL, 26.25 mmol) in 5 mL
dioxane was added via syringe and the colourless solution was stirred at ambient
temperature for 3 h, then heated to 100 ºC and stirred overnight. The reaction mixture
was evaporated, extracted 3 times with Et2O, and the combined organic layers were then
washed with saturated NaHCO3 (aq) and brine, dried over MgSO4 and concentrated. The
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residue was purified with column chromatography to obtain compound 12 as a colorless
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oil (850 mg, 27 %). H NMR (400 MHz, CDCl3): δ (ppm) 6.00 (s, 1H), 5.83 (s, 1H),
13
4.85 (s, 1H), 2.44 (bs, 2H), 1.20 (t, J = 7.4 Hz, 3H); C NMR (100 MHz, CDCl3): δ
(ppm) 171.3, 116.8, 100.0, 99.0, 21.0, 10.9; LRMS (EI) m/z (%): 127.0 ([MꢀH]ꢀ, 13.4
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%); HRMS (EI): calcd for C6H7O3: 127.0401, found: 127.0377.
5-Hydroxy-4-isopropylfuran-2(5H)-one (13)48 : The above reaction was carried out
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with 3ꢀmethylbutanal, affording 13 as a colorless oil (983 mg, 28 % yield). H NMR
(400 MHz, CDCl3): δ (ppm) 6.07 (s, 1H), 5.79 (s, 1H), 2.72 (m, 1H), 1.19 (d, J = 6.8 Hz,
6H); 13C NMR (100 MHz, CDCl3): δ (ppm) 171.3, 116.2, 98.3, 25.5, 22.4; HRMS (ESI):
calcd for C7H10O3Na ([MNa+]): 165.0528, found: 165.0538.
3-Ethylfuran-2,5-dione (14)49 : DessꢀMartin periodinane (3.40 g, 8.02 mmol) was
suspended in anhydrous dichloromethane and stirred at room temperature for 10 min
before a solution of compound 12 (856 mg, 6.68 mmol) in 20 mL dichloromethane was
added. The resulting mixture was stirred at room temperature for 2.5 h. The reaction
mixture was then washed with saturated Na2S2O3 (aq) and brine. The organic layer was
dried over MgSO4 and concentrated. The residue was purified by column
chromatography as a colorless oil (413 mg, 49 %). 1H NMR (400 MHz, CDCl3): δ (ppm)
13
6.60 (t, J = 2.0 Hz, 1H), 2.57 (qd, J = 7.4, 2.0 Hz, 2H), 1.28 (t, J = 7.4 Hz, 3H); C
NMR (100 MHz, CDCl3): δ (ppm) 165.7, 164.0, 155.1, 128.0, 19.6, 11.0; LRMS (EI)
m/z (%): 126.0 (M+, 14.6 %); HRMS (EI): calcd for C6H6O3: 126.0317, found: 126.0311.
3-Isopropylfuran-2,5-dione (15)50 : The above reaction was carried out starting with 5ꢀ
hydroxyꢀ4ꢀisopropylfuranꢀ2(5H)ꢀone (13), affording 15 as a colorless oil (324 mg, 33 %
1
yield). H NMR50 (400 MHz, CDCl3): δ (ppm) 6.44 (d, J = 1.6 Hz, 1H), 2.9 (m, 1H),
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1.29 (s, 3H), 1.27 (s, 3H); C NMR50 (100 MHz, CDCl3): δ (ppm) 165.2, 163.9, 127.0,
26.6, 23.5, 20.5; LRMS (EI) m/z (%): 140.0 (M+, 2.9 %); HRMS (EI): calcd for C7H8O3:
140.0473, found: 140.0473.
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tert-Butyl (3-nitrophenyl)carbamate (17): To a solution of 3ꢀnitroaniline (4.00 g, 29.0
mmol) in THF (40 mL) was added Boc2O (19.0 g, 87.0 mmol), DMAP (26 mg, cat.) and
Et3N (10 mL, 73.0 mmol); this reaction mixture was heated to reflux for 3 h. The mixture
was evaporated to remove solvents, and the residue was dissolved in Et2O, washed with 1
N HCl (aq), NaHCO3 and brine, dried over MgSO4 and concentrated to obtain compound
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17 as a white solid (4.99 g, 14.8 mmol) in 51% yield. H NMR (400 MHz, CDCl3): δ
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(ppm) 8.19 (m, 1 H), 8.06 (t, J = 1.86 Hz, 1H), 7.54 (m, 2H), 1.45 (s, 18H); C NMR
(100 MHz, CDCl3): δ (ppm) 151.1, 148.4, 140.4, 134.4, 129.4, 123.5, 122.4, 83.8, 2 7.9.
HRMS (ESI): m/z calcd for C16H22N2O6Na ([MNa+]): 361.1376, found: 361.1360; m.p.:
70.0 °C.
tert-Butyl (3-aminophenyl)carbamate (18): tertꢀButyl (3ꢀnitrophenyl)carbamate (17)
(2.50 g, 7.40 mmol) was dissolved in MeOH/THF (16 mL/24 mL), Pd/C (625 mg) was
added to the solution and the mixture was stirred under H2 overnight. The Pd/C was
removed by filtering through celite, and the filtrate was evaporated to dryness. The
residue was purified by column chromatography to yield compound 18 as a white solid
(2.07 g, 6.72 mmol) in 91% yield. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.11 (t, J = 7.94
Hz, 1H), 6.60 (ddd, J = 8.03, 2.25, 0.88 Hz, 1H), 6.54 (ddd, J = 7.84, 1.96, 0.90 Hz, 1H),
13
6.47 (t, J = 2.06 Hz, 1H), 3.67 (s, 2H), 1.43 (s, 18H); C NMR (100 MHz, CDCl3): δ
(ppm) 152.1, 146.9, 140.2, 129.4, 118.1, 114.7, 114.3, 82.5, 27.9; HRMS (ESI): m/z
calcd for C16H24N2O4Na ([MNa+]): 331.1634, found: 331.1645; m.p.: 136.3 °C.
7-Diethylaminocoumarin-3-carboxylic acid (19) was synthesized according to a
literature procedure.51
Preparation of compound 20: Under nitrogen and anhydrous conditions, acid 19 (1.62
mg, 6.20 mmol) was dissolved in dichloroethane (60 mL). POCl3 (2.9 mL, 31.0 mmol)
was added and the solution was brought to reflux for 4 hours. Afterwards, the solution
was concentrated and the residue was dried under vacuum. Following this, the residue
was dissolved in pyridine (60 mL) and aniline linker 18 (2.967 g, 9.30 mmol) was added.
The mixture was stirred overnight at room temperature. The solvent was evaporated and
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