Drastically decreased reactivity of thiols and disulfides complexed by cucurbit[6]uril

Article abstract of DOI:10.1021/ol8013667

(Figure Presented) The cucurbit[6]uril (CB6) host forms stable complexes with 2-aminoethanethiol (cysteamine) and a derivative that contains a bulky terminal group attached to the amine group, as well as with the related disulfide cystamine. In these comp

Full text of DOI:10.1021/ol8013667

ORGANIC
LETTERS
2008
Vol. 10, No. 17
3721-3724
Drastically Decreased Reactivity of
Thiols and Disulfides Complexed by
Cucurbit[6]uril
Lidia Strimbu Berbeci, Wei Wang, and Angel E. Kaifer*
Center for Supramolecular Science and Department of Chemistry, UniVersity of Miami,
Coral Gables, Florida 33124-0431
akaifer@miami.edu
Received June 17, 2008
ABSTRACT
The cucurbit[6]uril (CB6) host forms stable complexes with 2-aminoethanethiol (cysteamine) and a derivative that contains a bulky terminal
group attached to the amine group, as well as with the related disulfide cystamine. In these complexes, the thiol or the disulfide group is
encapsulated inside the host cavity. The CB6-complexed thiols show drastically decreased reactivity with several oxidants, while the CB6-
bound disulfide also exhibits hindered reactivity with reducing agents, such as dithiothreitol.
The thiol-disulfide interconversion equilibrium has consid-
erable biological importance.1 Disulfide bridges, formed by
cysteine’s thiols, constitute an important structural element
for the stabilization of a number of protein folded states.2
Thiol-disulfide interchange reactions play a key role in
maintaining an appropriate redox balance in living tissues,
and current thinking links these reactions to levels of
oxidative stress,3 effective platelet function,4 and even cancer
chemoprevention.5 It is thus important to understand any
factors that may affect the reactivity of biologically relevant
thiols and disulfides.
The growing interest in the host family of the cucurbit-
[n]urils (CBn, see structures in Figure 1) is well docu-
mented.6-8 While most of this rapidly expanding body of
work focuses on the larger cavity hosts CB7, CB8, CB10,
and related compounds, the much better known CB6 also
has very interesting properties.
In this work, we focus our attention on the binding
interactions between the CB6 host and the amino acid
L-cysteine, the main source of thiol groups in proteins, the
related thiol cysteamine (2-aminoethanethiol), and its cor-
responding disulfide, cystamine. We also investigate a
(1) Gilbert, H. F. AdV. Enzymol. 1990, 63, 69.
(2) Kim, P. S.; Baldwin, R. L. Annu. ReV. Biochem. 1990, 59, 631.
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(4) Essex, D. W. Antioxid. Redox Signaling 2004, 6, 736.
(5) Ryu, E. K.; Choe, Y. S.; Byun, S. S.; Lee, K.-H.; Chi, D. Y.; Choi,
Y.; Kim, B.-T. Bioorg. Med. Chem. 2004, 12, 859.
(6) Kim, Lee; J, W.; Samal, S.; Selvapalam, N.; Kin, H.-J.; Kim, K.
Acc. Chem. Res. 2003, 36, 621
(7) Lagona, J.; Mukhopadhyay, P.; Chakrabarti, S.; Isaacs, L. Angew.
Chem., Int. Ed. 2005, 44, 4844
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10.1021/ol8013667 CCC: $40.75
Published on Web 08/07/2008
2008 American Chemical Society

Thiol 1 is a cysteamine derivative that was designed to
force the binding approach of CB6 to the guest from the
thiol end. As in the case of cysteamine, the two peaks
corresponding to the methylene protons between the thiol
and amine groups (labeled a and b in Figure 2), shift to higher
Figure 1. Structures of cucurbit[n]uril hosts and thiol and disulfide
guests used in this work.
Figure 2.
1H NMR spectrum (500 MHz, D2O/0.20 M NaCl, pD
derivative of cysteamine (compound 1), which has a bulky
group attached to the amine group. The structures of all these
compounds are shown in Figure 1.
2.0) of thiol 1 (a) in the absence and (b) in the presence of 2.0
equiv of CB6.
CB6 was prepared as previously reported.9 The synthesis
of compound 1 is described in the Supporting Information.
Disulfide 2 was prepared by oxidation of 1 (vide infra). All
other guests are commercially available. We carried out the
majority of the experiments in 0.2 M NaCl/D2O solution
adjusted to pD 2 to enhance the solubility of CB6 and to
ensure protonation of the amine functional groups on the
guests.
field as the concentration of CB6 increases. The signal for
protons c, on the other side of the amine group, experiences
CB6-induced shifts to lower fields, as do the d, e, and f
protons. Again, the CB6-induced chemical shifts of the c
protons were used to determine the corresponding K value,
which was found to be 7.3((1.0) × 104 M-1 (see the
Supporting Information). This inclusion complex is suf-
ficiently stable to be observed in MALDI-TOF mass
spectrometric experiments (m/z 1,268). We were initially
surprised by the CB6-induced shifts to lower field on protons
e and f, which appear to be far removed from the cavity
portal in the complex. However, semiempirical PM3 com-
putations with the CB6·1 complex yielded the minimized
stucture shown in the graphical sbstract, in which the guest
adopts a bent conformation that brings these protons
relatively close to the host portal and its rim of carbonyls, a
finding that is consistent with the observed NMR complex-
ation-induced shifts.
Oxidation of thiol 1 with trichloronitromethane in aceto-
nitrile solution yields disulfide 2, which was easily isolated
after recrystallization from ethanol. A comparison of the 1H
NMR spectra of both compounds reveals that protons a and
b, which resonate at 2.83 and 3.24 ppm, respectively, in thiol
1, appear at lower field (3.00 and 3.42 ppm) in 2 (see the
Supporting Information). Addition of excess CB6 to a D2O
solution containing 2 does not cause any significant shifts
in the proton resonances of the disulfide, suggesting the
absence of binding interactions between these two com-
pounds. This was anticipated, given the size of the 3,5-
dimethoxybenzene terminal groups in 2, which prevent
access by CB6 to the central binding site in the disulfide.
On the other hand cystamine, lacking any bulky terminal
groups, forms a stable inclusion complex with CB6. The two
The 1H NMR spectrum of cysteamine exhibits two triplets
at 2.83 and 3.20 ppm, which correspond to the methylene
protons adjacent to the thiol and amine functional groups,
respectively. In the presence of CB6 both peaks broaden and
shift to higher fields, clearly indicating the formation of an
inclusion complex (see the Supporting Information). The
CB6-induced shift is more pronounced for the protons
adjacent to the thiol group, suggesting that they are inserted
more deeply in the host cavity. The chemical exchange
between free and CB6-bound cysteamine is fast on the NMR
time scale and we only observed a set of proton signals,
shifting gradually to higher field as the host concentration
increases. The chemical shift of the methylene adjacent to
the amino group was measured as a function of added CB6
concentration. Optimization of the fitting of these data to a
1:1 binding isotherm yields an equilibrium association
constant (K) of 5.6((2.0) × 103 M-1 at 25 °C (see the
Supporting Information). In stark contrast to cysteamine,
L-cysteine is not bound by CB6, as evidenced by the
complete lack of CB6-induced changes, under identical
1
experimental conditions, on the H NMR spectrum of the
amino acid.
(9) Mock, W. L.; Shih, N.-Y. J. Org. Chem. 1986, 51, 4440.
(10) Lamourex, G. V.; Whitesides, G. M. J. Org. Chem. 1993, 58, 633.
(11) Lees, W. J.; Whitesides, G. M. J. Org. Chem. 1993, 58, 642.
3722
Org. Lett., Vol. 10, No. 17, 2008

methylene triplets of cystamine resonate at 3.02 and 3.40
ppm (Figure 3). Upon addition of 0.4 equiv of CB6 a new
Figure 4. Energy-minimized (B3LYP/3-21G method) structure of
the cystamine-CB6 inclusion complex.
oxidation. In aqueous solution at neutral pH this reaction
was completed in ca. 1 h at 25 °C. Figure 5A shows the
Figure 3.
1H NMR spectra (500 MHz, D2O/0.20 M NaCl, pD 2.0)
of 1.0 mM cystamine (a) in the absence and in the presence of (b)
0.4, (c) 0.8, and (d) 1.0 equiv of CB6. The asterisk labels the
residual acetone peak.
set of peaks at 2.32 and 3.21 ppm were observed. The
chemical exchange between free and bound cystamine is slow
in the NMR time scale, which allows the simultaneous
observation of both sets of peaks. As more CB6 is added,
the methylene peaks corresponding to the bound guest grow
at the expense of free guest peaks. The complexation is
quantitative at the millimolar concentration levels used in
these experiments. When 1.0 equiv of CB6 or more is
present, only the peaks corresponding to bound cystamine
were observed. We performed dilution experiments with a
solution containing 1.0 mM CB6 and 1.0 mM cystamine, in
which only methylene resonances corresponding to the
inclusion complex were observed. However, even after 10-
fold dilution, no resonances corresponding to the free guest
were observed. If we assume that a maximum of 10% of
the complex may undergo dissociation and remain undetected
1
by H NMR spectroscopy under these conditions, we can
estimate that the minimum K value must be 106 M-1.
A computational investigation of the cystamine-CB6
complex, using DFT methods (B3LYP/3-21G), leads to the
energy-minimized structure shown in Figure 4, in which the
disulfide bond, as expected, is located in the center of
the host cavity, while the ammonium groups at the ends
of the guest interact, via ion-dipole forces, with the rims of
carbonyl oxygens on the cavity portals.
The formation of such a stable inclusion complex with
the disulfide bond included and seemingly protected close
to the middle of the host cavity led us to question the
reactivity of the S-S linkage toward reductive cleavage. The
reagent of choice for R-S-S-R cleavage is dithiothreitol
(DTT).10,11 Indeed treatment of cystamine with a slight
excess of DTT (1.25 equiv) leads to the production of
cysteamine and the cyclic disulfide that results from DTT
Figure 5. Time evolution of the cystamine and cysteamine
concentrations. (A) Reaction of 1.0 mM cystamine with 1.25 mM
DTT in D2O in the absence and in the presence of 1.05 equiv of
CB6. (B) Reaction of 2.0 mM cysteamine with 4.0 mM FeCl3 in
D2O in the absence and in the presence of 1.25 equiv of CB6.
time evolution of the cystamine and cysteamine concentra-
tions recorded in this reaction. However, if the same reaction
Org. Lett., Vol. 10, No. 17, 2008
3723

is carried out in the presence of 1.0 equiv of CB6, no
cysteamine (free or CB6-bound) formation was observed
after 50 h (Figure 5A and the Supporting Information).
Clearly the inclusion complexation of cystamine inside the
cavity of CB6 results in a pronounced stabilization effect
and decreased reactivity against reductive cleavage by DTT.
The kinetic stability of the CB6·cystamine complex suggests
that its reactivity versus thiol scrambling reactions should
also be strongly diminished. Cystamine readily reacts with
the methyl ester of L-cysteine to produce a mixture of thiols
and disulfides (see the Supporting Information for possible
product structures). However, the presence of 1 equiv of
CB6, under otherwise identical experimental conditions, fully
prevents this reaction from taking place in a period of 24 h.
Therefore, the reactivity of the CB6 complex in thiol
scrambling reactions is also greatly decreased.
the same experimental conditions, no product (disulfide 2)
was detected after a period of 40 min in the presence of
CB6.
In the field of supramolecular kinetics the primary goal is
the development of supramolecular catalysts.12-14 However,
the stabilization of reactive compounds is also of interest,15
and considerable work with cyclodextrin hosts has targeted
the stabilization and/or protection of included guests.16 We
must also note that unique molecular behavior is emerging
from recent work on molecular encapsulation.17,18 The CB6-
induced, kinetic stabilization effects reported here are
remarkable not only because of their magnitude, but also
because the same host affects very strongly the kinetics of
the forward and reverse reactions (the reductive cleavage of
cystamine and the oxidation of cysteamine, see Figure 5).
These kinetic effects are primarily attributed to reactant
stabilization by formation of an inclusion complex with the
CB6 host. We are currently investigating in more detail the
mechanistic effect of CB6 on the kinetics and thermodynam-
ics of these reactions.
After collecting these data on the reactivity of the
CB6·cystamine complex, we decided to investigate the effect
of CB6 complexation on the reactivity toward oxidation of
included thiol guests. For instance, cysteamine can be readily
oxidized to cystamine with FeCl3 in aqueous solution. As
an example, the time evolution of the cysteamine and
cystamine concentrations in a solution initially containing
2.0 mM cysteamine and 4 mM FeCl3 is shown in Figure
5B. The reaction is essentially complete in ca. 7 h. In contrast
to this, the presence of CB6 leads again to effective thiol
protection. No oxidation was detected for a period of ca.
24 h.
Acknowledgment. The authors are grateful to the NSF
(to AEK, CHE-0600795) for the generous support of this
work. W.W. acknowledges support from the University of
Miami in the form of a Maytag Graduate Fellowship. The
authors are grateful to Dr. Winston Ong for obtaining
preliminary data on the early stages of this work.
Supporting Information Available: Synthetic details for
compounds 1 and 2 and additional NMR spectroscopic data
as mentioned in the text. This material is available free of
charge via the Internet at http://pubs.acs.org.
We also ran additional experiments to investigate cys-
teamine oxidation using other oxidizing agents, such as
dissolved oxygen and Cl3CNO2. In an aqueous solution (pH
7) equilibrated with the laboratory atmosphere we monitored
cysteamine oxidation using 1H NMR spectroscopy and found
that it takes ca. 3 h for the cysteamine peaks to disappear
completely (as the cystamine peaks reach full development).
However, in the presence of a slight stoichiometric excess
of CB6 (1.25 equiv), only a trace of the disulfide was
observed after a period of 24 h. Similar results were obtained
for the oxidation of thiol 1. For instance, oxidation of 1 by
Cl3CNO2 is essentially complete in ca. 40 min, while, under
OL8013667
(12) Smaldone, R. A.; Moore, J. S. Chem. Eur. J. 2008, 14, 2650
(13) Kovbasyuk, L.; Kra¨mer, R. Chem. ReV. 2004, 104, 3161
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2004, 37, 113
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(15) Cram, D. J.; Tanner, M. E.; Thomas, R. A well-known example is
the isolation of cyclobutadiene inside a hemicarcerand host. Angew. Chem.,
Int. Ed. Engl. 1991, 30, 1024.
(16) Osa, T.; Suzuki, I. In ComprehensiVe Supramolecular Chemistry;
Atwood, J. L., Davies, J. E. D., MacNicol, D. D., Vo¨gtle, F., Eds.; Elsevier
Science: New York, 1996; Vol. 3, pp 367-400.
(17) Biros, S. M.; Rebek, J. Chem. Soc. ReV. 2007, 36, 93
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(18) Rebek, J. Angew. Chem., Int. Ed. 2005, 44, 2068
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