Studies of reversible conjugate additions

Article abstract of DOI:10.1002/ejoc.201300358

Benzalcyanoacetamides were designed and synthesized as reversible thiol conjugate addition acceptors. These thia-conjugate additions can rapidly and reversibly achieve equilibrium under aqueous conditions at neutral pH. Kinetic studies show that electron-withdrawing groups at the 4-position of the phenyl ring of the benzalcyanoacetamides promote the conjugate addition at equilibrium. Dynamic thiol exchange of these conjugate acceptors is faster than singly activated α,β-unsaturated carbonyl compounds. These thia-conjugate additions can be assembled as potentially useful components in dynamic combinatorial chemistry. Benzalcyanoacetamides were studied with thiols as reversible thiol conjugate addition acceptors. These thia-conjugate additions can rapidly and reversibly achieve equilibrium under aqueous conditions at neutral pH, which verifies the ultimate use of these conjugate acceptors in dynamic combinatorial chemistry. Copyright

Full text of DOI:10.1002/ejoc.201300358

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201300358
Studies of Reversible Conjugate Additions
Ye Zhong,[a,b] Yufang Xu,[a] and Eric V. Anslyn*[b]
Keywords: Sulfur / Reversible reactions / Michael addition / Combinatorial chemistry
Benzalcyanoacetamides were designed and synthesized as
reversible thiol conjugate addition acceptors. These thia-con-
jugate additions can rapidly and reversibly achieve equilib-
rium under aqueous conditions at neutral pH. Kinetic studies
show that electron-withdrawing groups at the 4-position of
the phenyl ring of the benzalcyanoacetamides promote the
conjugate addition at equilibrium. Dynamic thiol exchange
of these conjugate acceptors is faster than singly activated
α,β-unsaturated carbonyl compounds. These thia-conjugate
additions can be assembled as potentially useful components
in dynamic combinatorial chemistry.
recently, Taunton created covalent-modifying enzyme inhib-
itors for selective reaction with active-site cysteine residues
by using conjugate acceptors derived from active methylene
unit 4.[7] Because thiol addition was found to occur in these
three studies in an equilibrium process, it is apparent that
the exchange of different thiols and conjugate acceptors
should occur; yet, this was not studied. Recently, our group
reported the mechanistic details of a reversible and rapid
exchange of thiols with β-sulfido-α,β-unsaturated carbonyl
compounds 5 in organic and aqueous media.[8] In this
study, component exchange was proven. With these results
in mind, we set out to verify that rapid component ex-
change of thiols and conjugate acceptors derived from
active methylene species would occur at neutral pH, and we
sought to study the substituent effects thereof. This study
verifies the utility of this reaction for ultimate use in DCC.
Introduction
Dynamic combinatorial chemistry (DCC) employs re-
versible covalent bonds to create equilibrium mixtures of
interchanging components that create larger assemblies,[1]
often with the goal of templating the receptors to specific
guest molecules.[2] The spontaneous multicomponent as-
sembly of large molecules with clefts and cavities avoids the
effort involved in multistep synthesis. Recently, the thia-
Michael addition has been used to construct dynamic com-
binatorial libraries (DCLs), but its utility is limited by slow
exchange and other competitive reactions at physiological
pH.[3] Thus, improving the kinetics of exchange could make
this reaction another tool to exploit in DCC.
An example of the use of the thia-Michael addition
comes from Greaney, for which he sought to conjugate
glutathione.[4] The exchange of components in his conjugate
acceptor reactions took minutes to hours to achieve equilib-
rium at pH 8.0. He used singly activated conjugate ac-
ceptors, such as common α,β-unsaturated carbonyls 1 (Rn:
hydroxy group or small collection of amino groups). Inter-
estingly, in the 1960s, conjugate acceptors derived from
active methylene acceptor units were studied and found to
rapidly add to thiols in an equilibrium process.[5] For exam-
ple, equilibrium constants ranging from 17 to 1300 m–1 for
thiol addition to 2-cyanoacrylates 2 were reported. Further,
Bernasconi studied methoxybenzylidenemalononitrile (3) as
a conjugate acceptor for thiolates in DMSO and found
equilibrium constants ranging from 5 to 1410 m–1.[6] Most
[a] Shanghai Key Laboratory of Chemical Biology, School of
Pharmacy, East China University of Science and Technology,
Shanghai 20237, P. R. China
Results and Discussion
[b] Department of Chemistry and Biochemistry, University of
Texas at Austin,
Our work focused upon the reversible conjugate addition
of thiols to benzalcyanoacetamides in CD3CN/D2O solu-
tions at pD 7.0. First, we found that the addition of thiols
to benzalcyanoacetamides occurred faster than we could re-
Austin, TX 78712, USA
E-mail: Anslyn@austin.utexas.edu
Homepage: http://anslyn.cm.utexas.edu/research/eva.html
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300358.
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Y. Zhong, Y. Xu, E. V. Anslyn
SHORT COMMUNICATION
1
Figure 1. (a) Partial H NMR spectrum of 8 upon the addition of 2-ME in CD3CN (1 mL). The peaks corresponding to Ha, Hc and He
were integrated in order to determine the equilibrium constants. (b) 1H NMR spectrum 15 min after the addition of D2O (100 µL). The
α hydrogen (He) was deuterated by D2O in this solvent system, so the peaks at 4.1 ppm disappeared and peaks for adjacent Ha/Hb
changed to singlets.
1
cord a H NMR spectrum. Further, although addition was position of the conjugate acceptors, and different conjugate
clear in the NMR spectra, the products could not be iso- acceptors (differing in the A and B groups) likewise ex-
lated, but rather only the starting reactants were isolable. change.
Therefore, the complexes must dissociate upon dilution dur-
ing the workup procedure.
To study the conjugate addition itself and the reactivity
of various conjugate acceptors, we first studied the addition
of 2-mercaptoethanol (2-ME) to benzalmalononitrile (6),
methylbenzalcyanoacetate (7), and benzalcyanoacetamide
(8) by using 1H NMR spectroscopy in CD3CN. An equilib-
rium constant for the addition of 2-ME to 8 was measured
as 161 m–1 through integration of the Ha and He resonances
(Figure 1, a). Upon addition of D2O, the α hydrogen atoms
disappeared within 15 min (Figure 1, b).
Scheme 1. Dynamic exchange of different thiols as well as dif-
ferently substituted 2-cyanoacrylates.
Our first study involved the addition of 4-mercaptobenz-
oic acid to an equilibrated solution of 2-ME and 2-chloro-
1
However, within 30 min, the same reaction with the use benzal cyanoacrylamide (9). The H NMR spectrum taken
of 2-ME and 6 in an acetonitrile/water (5:6) mixture led to after 0.5 h showed that the resonance of the product from
hydrolysis to give malonitrile, which thereby limits the util- the addition of 2-ME had decreased and that there was an
ity of 6. Similarly, after 12 h, 7 also showed significant hy- increase in the resonance attributable to the product re-
drolysis to methyl cyanoacrylate. However, the conjugate sulting from the addition of 4-mercaptobenzoic acid (Fig-
acceptor derived from cyanoacetamide (8) was stable to hy- ure 2). We then changed the sequence of the addition of
drolysis, and thus, our subsequent studies concentrated on the thiols, adding 4-mercaptobenzoic acid first and 2-ME
these derivatives, although methyl cyanoacrylate was stable second. The 1H NMR spectrum appeared identical, and
enough to be used in some studies.
this indicates that the same equilibrium mixture of products
After confirming reversible conjugate addition of a single and starting thiols and conjugate acceptors results. Simi-
thiol, we moved to testing component exchange in a larly, the two conjugate acceptors, methylbenzalcyanoacet-
CD3CN/D2O (5:6) mixture at pD 7.0. This solvent mixture ate and benzalcyanoacetamide, were added in different se-
proved optimal for keeping all the components soluble quences to 2-ME, and exactly the same 1H NMR spectrum
while maximizing the amount of water. Scheme 1 presents was recorded (see the Supporting Information). Equilib-
the basic idea in which different thiols exchange at the β rium constants for the single addition of thiols are given in
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Studies of Reversible Conjugate Additions
1
Figure 2. H NMR spectra of reactions in CD3CN/D2O (5:6) in phosphate buffer, pD 7.0. (a) Partial spectrum of the reaction of 9 with
4-mercaptobenzoic acid. (b) Spectrum 30 min after the addition of 2-ME to (a). (c) Partial spectrum of the reaction of 2-ME with 9.
(d) Spectrum 30 min after the addition of 4-mercaptobenzoic acid to (c). Spectra (b) and (d) are essentially identical.
Table 1. The values suggest that electron-withdrawing sub-
Because our ultimate goal was to have reactions amena-
stituents on the phenyl ring, such as chloride, favor the ble to DCC in pure water, we set out to improve the water
products at equilibrium.
solubility of the conjugate acceptors. In this regard, we syn-
thesized two derivatives of benzalcyanoacetamide in which
the amide nitrogen atom was derived from 2-(2-amino-
ethoxy)ethanol and 3-aminopropane-1,2-diol (Scheme 2).
Table 2 shows the equilibrium constants for reactions of
these compounds with 2-ME in CD3CN/D2O (5:6) at
pD 7.0, as found by integration of the peaks in the 1H
NMR spectra. Although our ultimate goal was to use the
conjugate acceptors in pure water (these compounds are
soluble in 90:10 water/acetonitrile), to compare the activi-
ties of the conjugate acceptors with 2-ME and 4-mercapto-
benzoic acid we used the previous solvent system. The
conjugate additions were still fast and reversible such that
the products could not be isolated. The trends in Table 2
reveal that in both studies, electron-withdrawing groups on
the phenyl ring increase the equilibrium constants for con-
jugate addition, whereas electron-donating groups decrease
Table 1. Equilibrium constants for the reaction of two thiols with
7, 8, and 9 in CD3CN/D2O (5:6) at pD 7.0.
Compound
K [ϫ10–3 m–1]
2-Mercaptoethanol
4-Mercaptobenzoic acid
7
8
9
0.09
0.26
1.06
0.06
0.12
0.25
Table 2. Equilibrium constants for the reactions of 10–14 and 15–
19 with 2-ME in CD3CN/D2O (5:6) at pD 7.0 in phosphate buffer.
Compound[a]
K [ϫ10–3 m–1]
Deviation [%][b]
10
11
12
13
14
15
16
17
18
19
0.43
0.26
0.86
0.94
3.1
0.39
0.27
0.80
0.78
2.7
3
0
8
8
6
3
1
8
3
6
[a] See Scheme 2 for structures of the compounds. [b] Standard de-
viation.
Scheme 2. Synthesis of 10–14 and 15–19.
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Y. Zhong, Y. Xu, E. V. Anslyn
SHORT COMMUNICATION
and hence increased product formation at equilibrium. This
was further revealed by examination of the Hammett plots
(Figure 3). Although these plots appear to be slighted
curved, the ρ values are near 1 (Table 3).
Table 3. Reaction conditions and ρ-values for the equilibrium con-
stants taken from Table 2.
Structure[a]
ρ
KH
r[b]
CA-A
CA-B
1.12
1.07
414
388
0.97
0.99
[a] See Scheme 2 for structures of the compounds. [b] Correlation
coefficient.
After confirming that the reaction between 2-ME and
the new conjugate acceptors was a rapid equilibrium pro-
cess, we again set out to explore dynamic thiol and conju-
gate acceptor exchange. After equilibrium was reached be-
tween 2-ME and 10–14 and 15–19, a second thiol was
added. Three thiols were explored: 4-mercaptobenzoic acid,
3-mercaptopropane-1,2-diol, and N-acetyl-l-cysteine. For
example, the 1H NMR spectrum of 17 with 4-mercapto-
benzoic acid was recorded 30 min after the addition of 2-
ME, and it showed the product of the second thiol conju-
gate addition with diminished product from the first thiol
addition (Figure 4). The spectra obtained were the same ir-
respective of the order of addition. Similar results were
found for the other two thiols (see the Supporting Infor-
mation). The results indicate dynamic component exchange
for all the thiols studied with both conjugate acceptors.
Conclusions
Benzalcyanoacetamides rapidly and reversibly add thiols
in mixtures of acetonitrile and water at neutral pH.
Hammett plots reveal that electron-withdrawing groups at
Figure 3. Hammett plot for (a) CA-A and (b) CA-B.
the values. Electron-withdrawing groups on the phenyl ring the 4-position of the phenyl ring facilitate conjugate ad-
led to increased electrophilicity of the conjugate acceptor, dition. The dynamic nature of these conjugate addition re-
Figure 4. 1H NMR spectra of dynamic thiol exchange in CD3CN/D2O (5:6) in phosphate buffer. (a) Partial spectrum of 17. (b) Spectrum
15 min after the addition of 4-mercaptobenzoic acid to (a). (c) Spectrum 30 min after the addition of 2-ME to (b).
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Studies of Reversible Conjugate Additions
actions implies that they are amenable for use in DCC and
that they are faster than thia-conjugate addition reactions
to singly-activated α,β-unsaturated carbonyl compounds.
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about the synthesis and characterization of all new compounds are
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1
ies of the H NMR and 13C NMR spectra for compounds 10–19.
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Acknowledgments
Thanks for financial support are due to the U. S. National Science
Foundation (NSF) (CHE-1212971) and the Welch and China
Scholarship Council. The authors thank Dr. Gururaj Joshi for
helpful discussions.
Received: March 8, 2013
Published Online: July 4, 2013
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