Colorimetric probe for the detection of thiols: The dihydroazulene/ vinylheptafulvene system

Article abstract of DOI:10.1002/ejoc.201200887

A new application of the dihydroazulene/vinylheptafulvene (DHA/VHF) system as a chemodosimeter for thiols is reported herein. A color change visible to the naked eye can be detected in acetonitrile/water (4:1) in the presence of cysteine and alkanethiols but not with other amino acids. The higher reactivity of DHA towards cysteine compared with VHF has been demonstrated. The easy synthesis of the main core and its versatility in terms of structural and physicochemical changes are essential elements for developing a new family of thiol-specific probes with higher selectivity and sensitivity than the reported prototype. Can the dihydroazulene/vinylheptafulvene (DHA/VHF) photo/thermoswitch system be used as a chemodosimeter for thiols? A new application for this versatile system has been discovered.

Full text of DOI:10.1002/ejoc.201200887

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FULL PAPER
DOI: 10.1002/ejoc.201200887
Colorimetric Probe for the Detection of Thiols: The Dihydroazulene/
Vinylheptafulvene System
Martina Cacciarini,*[a,b] Eduardo A. Della Pia,[b] and Mogens B. Nielsen[b]
Keywords: Sensors / Thiols / Molecular recognition / Chromophores
A new application of the dihydroazulene/vinylheptafulvene
(DHA/VHF) system as a chemodosimeter for thiols is re-
ported herein. A color change visible to the naked eye can
be detected in acetonitrile/water (4:1) in the presence of cys-
teine and alkanethiols but not with other amino acids. The
higher reactivity of DHA towards cysteine compared with
VHF has been demonstrated. The easy synthesis of the main
core and its versatility in terms of structural and physico-
chemical changes are essential elements for developing a
new family of thiol-specific probes with higher selectivity
and sensitivity than the reported prototype.
cyano group of a malononitrile moiety has recently been
reported by Zhao and co-workers for the detection of
Introduction
The key role that cysteine, homocysteine, and glutathione thiols.[2b]
play in biological systems has stimulated the development
In this context we became interested in investigating the
of numerous optical probes for thiols in recent years.[1,2] behavior of the dihydroazulene/vinylheptafulvene system
Abnormal levels of cysteine are associated with many syn- (DHA/VHF, 1/2; Scheme 1) towards thiols, attracted by its
dromes, from neurotoxicity to edema, slow growth in chil- peculiar structural features. DHA 1 contains two cyano
dren, liver damage, skin lesions, and hair depigmentation.[3] groups at C-1, whereas VHF 2 has the two cyano groups
In addition, altered levels of cysteine have been discovered conjugated to an unsaturated moiety. DHA 1 and VHF 2
in the late stages of HIV infection and other diseases associ- represent a photo/thermoswitch system. Thus, DHA 1 is
ated with the loss of skeletal muscle mass.[4] Recently, the a yellow photochromic compound that undergoes a light-
high potential of cysteine-reactive small molecules in drug induced 10-electron retro-electrocyclization to the red VHF
discovery as inhibitors of cysteine protease enzymes has 2 (Scheme 1 and Figure 1, first vial on the left).[7] The back-
been pointed out.[5]
reaction to the parent DHA can be realized by a thermally
Most of the optical probes reported to date for thiols are induced ring-closure and, more recently, by treatment with
chemodosimeters, which are molecules that react with the mild Lewis acids that enhance the rate of the thermal con-
thiol functionality through a specific and irreversible reac- version of VHF into DHA.[8] The significant structural dif-
tion leading to the formation of a covalent bond between ference between DHA and VHF, as reflected in their dif-
the probe and the thiol group, and producing an observable ferent colors and hence electronic properties, has made the
signal.[6] Of the various detection techniques, the most con- system interesting as a light-controlled molecular switch for
venient and cheapest method is the detection of such a sig- molecular electronics.[9] The system is particularly attractive
nal by the naked eye through a color change.
as it is simple to synthesize on a large scale,[10] and it exhib-
Michael addition is one of the possible reactions that is its both tunable optical properties (and hence colors) and
often exploited by such optical probes, taking advantage of switching abilities if properly functionalized.[11] To the best
the strong nucleophilicity of the sulfur atom in the thiol of our knowledge, it has never been explored for its effective
moiety.[1] In addition, the reactivity of thiols towards the recognition capabilities. We therefore became interested in
elucidating how the two isomers would behave in the pres-
ence of a thiol. Here we show that the system can indeed
be employed as a chemodosimeter in which a thiol like cys-
[a] Department of Chemistry, University of Florence,
teine can conveniently be detected by color changes visible
to the naked eye. Moreover, it should be mentioned that
as thiols are often used as anchoring groups in molecular
electronics for adhering molecular wires and switches to
gold electrodes,[12] it is important to establish the reactivity
of DHA and VHF towards free thiols in order to explore
the system within this field as part of our ongoing efforts.[9]
Via della Lastruccia 3–13, 50019 Sesto F.no (Florence), Italy
Fax: +39-055-4573531
E-mail: martina.cacciarini@unifi.it
Homepage: http://www.unifi.it/dipchimica/CMpro-v-p-557.
html
[b] Department of Chemistry, University of Copenhagen,
Universitetsparken 5, 2100 Copenhagen Ø, Denmark
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200887.
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M. Cacciarini, E. A. Della Pia, M. B. Nielsen
FULL PAPER
ture[2b] for being the last step in the synthesis of luciferin, a
common substrate for firefly luciferase[14] and it has re-
cently been used as a biocompatible condensation reaction
in living cells.[15]
Scheme 1. DHA/VHF system.
Scheme 2. Proposed compounds formed in the presence of Cys 3
or the Cys methyl ester 6.
To shed light on which isomer of the DHA/VHF system
is reactive towards thiols, we studied the change in the reac-
tion mixture over time by UV/Vis spectroscopy. UV/Vis
spectra were recorded at room temp. in acetonitrile/water
(4:1) after adding an excess of Cys 3 (50 equiv.) to a
5.5ϫ10–2 mm solution containing only DHA 1 and to a
5.5ϫ10–2 mm solution containing only VHF 2. The absorp-
tion spectra of DHA 1 in the presence of Cys 3, with the
cuvette kept in the dark, show clear broadening and a de-
crease in the characteristic DHA absorption at 356 nm; a
small redshift of the absorption is observed after 24 h, but
Figure 1. Color changes of a 1.0 mm solution of DHA/VHF (left)
in CH3CN/H2O (4:1) upon addition of 10 equiv of EtSH, Cys, Ser,
DTT and Cys-OMe (at t = 1, 10 min, 17 h, 41 h).
Results and Discussion
Upon dissolving solid cysteine 3 (Cys) in a freshly pre- then a small blueshift is observed in the following 24 h (Fig-
pared 1 mm solution of DHA/VHF 1/2[13] in acetonitrile/ ure 2, solid lines). The scattering observed in the UV/Vis
water (4:1), a color change from orange to a slightly yellow curve recorded after 48 h (Figure 2, blue line) corresponds
solution was observed with the naked eye over time (see to the precipitation of a white solid, most likely due to both
Figure 1). In contrast, no obvious color change was de- cysteine aggregation and further reactions of 4, as con-
tected upon addition of other amino acids such as serine or firmed below in NMR studies. Two days after the addition
alanine. This simple experiment suggests that the DHA/ of Cys 3 to DHA 1, the slightly yellow solution was irradi-
VHF system reacts with the thiol group and can be used as ated at 357 nm for 20 min; this resulted only in a stronger
a dosimeter to detect Cys and other important biological yellow color, no red color typical of the parent VHF 2 was
thiols.
observed.
To investigate the source of the color change, mass spec-
Thus, the original DHA 1 had undergone complete reac-
trometric analysis (MALDI-TOF) of the solution was per- tion with Cys 3 during the 2 d in the dark. The UV/Vis
formed. Two peaks at m/z = 361 and 315 were observed absorption spectrum recorded after irradiation (Figure 2,
(see the Supporting Information). The signal at m/z = 361 dotted line) revealed, however, the presence of a new species
agrees with the 4,5-dihydro-1,3-thiazolyl structure 4 [M + with an absorption maximum at 402 nm, which is blue-
H]+ suggested in Scheme 2 and can be explained by the re- shifted relative to the absorption band of the parent VHF
action of the thiol group of Cys 3 with one of the cyano 2 (λmax = 478 nm, Figure 3). Moreover, a characteristic ab-
groups of DHA 1 followed by a condensation/cyclization sorption at 293 nm is observed[2b] and the spectrum has
that also involves the amino group of Cys 3. The mass spec- been ascribed to the proposed structure of compound 5 in
trum also shows a signal at m/z = 315, which may corre- which the original cyano group of VHF 2 has been replaced
spond to the loss of CO2H from 4; this decarboxylation by a less electron-withdrawing group, provoking a blueshift
likely occurs during the MALDI ionization. The formation of more than 70 nm. In contrast, this new group does not
of a 4,5-dihydro-1,3-thiazole ring by reaction of a cyano really affect the absorption maximum of 4, which has an
group with a 1,2-aminothiol moiety is known in the litera- absorption maximum just slightly shifted by 2–3 nm. Next
2
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Colorimetric Probe for the Detection of Thiols
Figure 3. Absorption spectra of the reaction between VHF 2
(5.5ϫ10–2 mm) and Cys (3; 50 equiv.) in CH3CN/H2O (4:1) upon
irradiation at 366 nm at room temp. for 2 d (recorded, from top to
bottom, at t = 0, 4, 21, 26, 54 h).
Figure 2. Absorption spectra of the reaction in the dark between
DHA (1; 5.5ϫ10–2 mm) and Cys (3; 50 equiv.) in CH3CN/H2O
(4:1) at 25 °C for 2 d (solid lines, recorded, from top to bottom, at
t = 0, 4, 8, 12, 16, 20, 24, 48 h). The dotted line shows the absorp-
tion spectra of the final solution (48 h) after selective irradiation at
357 nm for 20 min.
An accurate description of the 1H NMR spectra depicted
1
in Figure 4 is certainly merited. The H NMR spectra of
Cys methyl ester 6 (spectrum 1) and DHA 1 (spectrum 2)
we studied the absorption properties of a solution of VHF are shown for direct comparison. Spectra 3, 4 and 5 were
2 and Cys 3 (50 equiv.) constantly irradiated by a UV lamp recorded for the reaction between DHA 1 and Cys 6 in
at 366 nm for 48 h. As is evident from Figure 3, the inten- the dark after 5 , 24, and 48 h, respectively. In spectrum 5,
sity of the VHF absorption decreases somewhat, possibly recorded after 48 h, selected signals typical of DHA 1 are
due to some decomposition, but possibly also due to almost undetectable, and signals from the modified DHA
Michael attack on the dicyanoethylene unit {see below for 7, present as two main diastereomers, are dominant. In par-
experiments on 2-[2-(cyclohepta-2,4,6-trienyl)-1-phenyle- ticular, the singlet arising from 3-H at δ = 7.1 ppm of 1
thylidene]malononitrile, a VHF precursor (see S10 in the (spectrum 2, Figure 4) is slightly shifted and transformed
Supporting Information)}, but the spectrum presented in into two singlets almost overlapping at δ = 7.05 ppm (spec-
Figure 2 (dotted line), tentatively assigned to VHF 5, is not trum 5), the doublet arising from the protons of the phenyl
observed. In other words, it seems that only a cyano group ring at δ =7.8 ppm in 1 (spectrum 2) is split into two dou-
in DHA 1 is able to react with Cys 3 to form a 4,5-dihydro- blets at δ = 7.7 and 7.6 ppm (spectrum 5), and the multiplet
1,3-thiazole unit. Indeed, the cyano groups on the ma- arising from 8a-H at δ = 3.8 ppm in 1 is shifted to δ =
lononitrile moiety 1 are better electrophiles than those on 3.3 ppm and again split into two multiplets in spectrum 5.
2.[16]
Most of the signals of the diastereomers 7 are overlapping;
To validate the hypothesis described above for the reac- nevertheless, it is possible to select some more signals that
tion between DHA 1 and Cys 3 in the dark, NMR experi- are diagnostic of the presence of two main products. In fact,
ments were performed under these conditions. The reac- the two doublets of doublets at δ ≈ 5.2–5.3 ppm can be as-
tions between DHA 1 and Cys 3 and between DHA 1 and cribed to CH-α of the amino acid in the two different dia-
Cys methyl ester 6 (both in an NMR tube; [DHA] = 23 mm stereomers, and two methoxy groups can also be detected in
in CD3CN/D2O (4:1); [DHA]/[Cys] ≈ 1:1.2) were monitored the region of δ ≈ 3.8 ppm (see the Supporting Information).
for 2 d, the latter allowing easier spectral interpretation. When using an excess of Cys, as in the UV/Vis studies, a
1
The H NMR spectra are rather complicated, but it is pos- white precipitate, insoluble in most common organic sol-
sible to detect the presence of two main products in the vents, formed, which completely removed the DHA deriva-
diagnostic region (δ = 5.2–5.3 ppm) of CH-α of the amino tive 4 or 7 from solution in a few days.
acid (Figure 4). In fact, both DHA 1 and Cys 3/6 are chiral
Cys methyl ester 6 (1 equiv.) was also used in the reaction
molecules, and – although DHA 1 was used as a racemic with DHA/VHF on a preparative scale aiming at isolating
mixture – Cys was chosen as a single enantiomer of the the products 7 and 8 (Scheme 2). The presence of several
natural l series. DHA derivatives 4 and 7 described in diastereomers of 7 with similar or overlapping Rf values
Scheme 2 have three stereocenters, namely C-1 and C-8a on inevitably affected a proper purification, as reflected in the
the “DHA” core and CH-α of the amino acid. Although 1H NMR spectrum reported in the Supporting Infor-
the third stereocenter in l-Cys is well defined, the others mation. The mass spectrum recorded with the micrOTOF-
are not and can theoretically give rise to four different dia- Q II spectrometer interfaced to an HPLC instrument exhib-
stereomers.
its a peak at m/z = 375 (see the Supporting Information),
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M. Cacciarini, E. A. Della Pia, M. B. Nielsen
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Figure 4. 1H NMR spectra in CD3CN/D2O (4:1) recorded for the reaction of DHA 1 (23 mm) with Cys methyl ester 6 (1.2 equiv.). From
bottom to top: only 6, only 1, 1 + 6 at t = 5, 24, 48 h.
in agreement with the calculated value for [M + H]+ of the in the synthesis of DHA 1[10] that still presents two cyano
proposed structure 7.[17] Moreover, when the NMR tube groups conjugated to a double bond (see the Supporting
was exposed to light, conversion of the different dia- Information). In this case we can detect the Michael ad-
stereomers of 7 into a main single product, tentatively as- dition of Cys 3 to the double bond through the disappear-
1
signed to 8, was detected together with decomposition ance of selected and characteristic signals both in the H
products (see the Supporting Information). Indeed, the and 13C NMR spectra (see the Supporting Information).
characteristic signal of 8a-H of a “DHA” form at δ = This observation also suggests the preferred Michael ad-
3.45 ppm disappears, the two CH-α signals of the amino dition of Cys 3 to VHF 2 in the total absence of DHA 1,
acid at δ = 5.24 and 5.32 ppm merge into one signal at δ = as did the UV studies (Figure 3).
4.6 ppm, and the AB part of the ABX system CH-α/CH2-
From the above experiments, we can conclude that in the
S at δ = 3.6 ppm appears much simpler than before. More- dark the first transformation involves one cyano group on
over, the two singlets of the methoxy groups merge into one. DHA 1, which reacts with the 1-amino-2-mercapto moiety
The 13C NMR spectrum supports the proposed structure of Cys to give a 4,5-dihydro-1,3-thiazole ring, proved to be
of compound 7, present as two main diastereomers. All the responsible for the color change. Thereafter, DHA deriva-
1
signals are double (1:1 ratio in accordance to the H NMR tive 4 can still react with Cys producing an insoluble white
spectrum) and by comparison with the 13C NMR spectrum solid. As for VHF 2, we cannot demonstrate neither reactiv-
of the parent DHA 1, disappearance of one cyano group ity nor inertness to Cys, but we showed that 2-[2-(cy-
(resonance in the characteristic region at δ = 115 ppm) and clohepta-2,4,6-trienyl)-1-phenylethylidene]malononitrile
appearance of the signals of four new carbon atoms at δ ≈ undergoes Michael addition with Cys. Efforts to perform
170–175 ppm, in accordance with the formation of an S– the reaction on a preparative scale were not successful in
C=N group and the presence of an ester, is clearly detected terms of the purification of the single diastereomers, but
(see the Supporting Information). 2D NMR experiments proved a quantitative transformation of DHA 1 into DHA
were performed, and the HMBC spectrum revealed corre- 7 and demonstrated the faster reactivity of DHA 1 com-
lation peaks between those new carbon atoms and both the pared with VHF 2, even in the presence of light. Hence we
CH-α and the CH2-S protons of the amino acid, which sup- can state that the color change of the DHA/VHF system in
ports the formation of the 4,5-dihydro-1,3-thiazole ring (see the presence of Cys reported in Figure 1 is provoked by the
the Supporting Information).
reaction of DHA 1 with Cys and by the formation of a 4,5-
Because NMR studies on VHF 2 in the presence of Cys dihydro-1,3-thiazole ring.
3 were always affected by the presence of traces of DHA
Fluorescence measurements to monitor the transforma-
1, we investigated the reactivity of 2-[2-(cyclohepta-2,4,6- tion of DHA 1 into DHA 4 were performed at 77 K (Fig-
trienyl)-1-phenylethylidene]malononitrile, an intermediate ure 5) to retard the photoswitch of DHA 1 into VHF 2 and
4
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Colorimetric Probe for the Detection of Thiols
of 4 into 5. Indeed, compound 4, like its parent DHA 1,[18]
does not show any significant emission at room temperature
due to its photoconversion into 5. DHA 1 shows a broad
Conclusions
We have reported herein a new important application of
the DHA/VHF system 1/2 as a naked-eye probe for thiols.
It has been shown that DHA 1 reacts with cysteine to pro-
duce a visible color change. In fact, even when present in
only traces in a deep-orange solution of VHF 2, DHA 1
still reacts with thiols, shifting the equilibrium of the DHA/
VHF system towards its side in the presence of light. This
reactivity provokes an evident and visible color change in
solution and a redshift in the emission of DHA 1 after reac-
tion with Cys 3 that allows our system to be classified as a
new chemodosimeter for the detection of thiols. The simple
synthesis of the scaffold, its versatility in terms of structural
changes and consequent chemical and optical properties, as
demonstrated by the numerous derivatives reported in the
last few years,[20] lead us to believe that the system has
much promise. Herein, its value as a thiol-specific probe has
been demonstrated for the parent system 1/2, which opens
up access to a new family of probes for thiols, and we ex-
pect to improve sensitivity, selectivity, and the properties of
the sensing system.
emission band at 77 K in the 400–700 nm range (λmax
=
475 nm, Figure 5 and the Supporting Information). After
reaction with Cys 3, DHA 1 is converted into compound 4
and, upon excitation at 356 nm, the emission maximum
shifts to a higher wavelength (λmax = 499 nm, Figure 5 and
the Supporting Information). As for the absorption spec-
trum, the first excitation peak of 4 (268 nm) does not signif-
icantly differ from that of DHA 1, whereas the second
broader peak shifts towards a higher wavelength (ca.
367 nm). Irradiation of 4 with UV light provokes the trans-
formation into 5 and results in a new emission peak at a
higher wavelength (λmax = 532 nm, see the Supporting In-
formation). Interestingly, although VHF 2 is non-emit-
ting,[18] compound 5 shows a weak emission at room tem-
perature (λmax = 520 nm) that is strongly enhanced at 77 K.
The similarity between the excitation and the absorption
spectra of the converted compound 5 supports the hypothe-
sis that the fluorescence originates from the S1–S0 transition
that is not observed in VHF 2.[18]
Experimental Section
General: All solvents and reagents were purchased commercially
and used without further purification. NMR spectra used for the
characterization of products were recorded with Bruker 300 and
500 MHz spectrometers. The NMR spectra are referenced to the
solvent. MALDI-TOF MS were recorded with a Bruker Daltonics
mass spectrometer by using 2,5-dihydroxybenzene as the matrix.
Spectra were calibrated by using a peptide standard solution.
HRMS were recorded with a Q-Tof mass spectrometer with PEG
as the reference mass. HPLC–MS analysis was performed by using
a Dionex UltiMate 3000 RSCLC System U-HPLC machine cou-
pled with a Bruker micrOTOF-Q II mass spectrometer. Solution
absorption spectra were recorded with a Cary 50-Bio Varian UV/
Vis spectrophotometer with pure solvent as the baseline. Fluores-
cence spectra were recorded by using a FluoroLog-3 spectropho-
tometer at 77 K and an excitation and emission slit width of 3 nm.
The emission and excitation spectra were corrected for instrumen-
tal function. UV/Vis and fluorescence spectra were recorded by
starting with a 5.5ϫ10–2 mm solution of DHA 1 in CH3CN/H2O
(4:1) after addition of 50 equiv. of Cys 3. DHA 1 was prepared
according to a literature procedure.[10]
Figure 5. Fluorescence spectral changes over time of DHA 1
(5.5ϫ10–2 mm) upon addition of Cys 3 (50 equiv.). The spectra
were recorded at 77 K with λex = 356 nm (from top to bottom: only
1, then 1 + 3 at t = 0, 4, 8, 20, 24, 28, 40, 44, 48 h).
Synthesis of Compound 7: l-Cys-Me 6 (75 mg, 0.44 mmol) was
added to a solution of DHA 1 (108 mg, 0.42 mmol) in CH3CN/
H2O (4:1, 20 mL). The mixture was stirred at room temp. for 2 d
and then concentrated to dryness, redissolved in CH2Cl2 (25 mL),
Experiments performed with dithiothreitol (DTT),[19] an washed with water (2ϫ 20 mL), and dried with MgSO4. Evapora-
tion of the solvent gave an orange oil that was purified by flash
column chromatography in the dark (SiO2, from 70% CH2Cl2 in
important antioxidant agent, and ethanethiol, chosen as an
example of a simple alkanethiol, support the general, but
lower, reactivity of DHA 1 towards thiols as well as the
ability to detect the reaction with the naked eye (Figure 1).
Nevertheless, because both these thiols lack the amino
moiety, the reaction mechanism is different, although not
yet investigated in detail. Preliminary experiments again
suggested that nucleophilic attack on the cyano group is
preferred to a Michael addition on the malononitrile sys-
tem.
heptane to 100% CH2Cl2) to give compound
7 (120 mg,
0.32 mmol, 76%) as a 1:1 mixture of two inseparable diastereomers.
1H NMR (500 MHz, CDCl3): δ = 7.76–7.68 (m, 2 H, Ph), 7.67–
7.61 (m, 2 H, Ph), 7.51–7.32 (m, 6 H, Ph), 6.94 (s, 1 H, 3-H), 6.93
(s, 1 H, 3-H), 6.65–6.53 (m, 2 H, 4-H or 5-H), 6.46 (dd, J = 6.0,
1.1 Hz, 1 H, 6-H), 6.44 (dd, J = 6.0, 1.1 Hz, 1 H, 6-H), 6.37–6.32
(m, 2 H, 5-H or 4-H), 6.29–6.23 (m, 2 H, 7-H), 5.96 (d, J = 4.0 Hz,
1 H, 8-H), 5.94 (d, J = 4.0 Hz, 1 H, 8-H), 5.32 (dd, J = 9.7, 5.6 Hz,
1 H, CH-α), 5.24 (dd, J = 9.4, 8.6 Hz, 1 H, CH-α), 3.84 (s, 3 H,
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M. Cacciarini, E. A. Della Pia, M. B. Nielsen
FULL PAPER
Mrozek, A. Ajayaghosh, J. Daub, “Optoelectronic Molecular
Switches Based on Dihydroazulene-Vinylheptafulvene (DHA-
VHF)” in Molecular Switches (Ed.: B. L. Feringa), Wiley-
VCH, Weinheim, 2001, pp. 63–106; c) M. B. Nielsen, S. L. Bro-
man, M. Å. Petersen, A. S. Andersson, T. S. Jensen, K. Kilså,
A. Kadziola, Pure Appl. Chem. 2010, 82, 843–852.
CH3), 3.77 (s, 3 H, CH3), 3.68–3.52 (m, 4 H, 2 CH2S), 3.45 (dt, J
= 3.9, 2.0 Hz, 1 H, 8a-H), 3.42 (dt, J = 3.9, 2.0 Hz, 1 H, 8a-H)
ppm. 13C NMR (125 MHz, CDCl3): δ = 174.7, 173.9, 170.5, 170.4,
145.0, 140.24, 140.18, 132.2, 132.2, 132.0, 131.1, 131.0, 130.4,
130.3, 129.5, 129.4, 128.94, 128.98, 126.80, 126.77, 126.6, 126.4,
121.33, 121.2, 119.7, 119.5, 117.0, 78.1, 77.6, 59.2, 59.0, 52.99,
52.96, 51.44, 51.38, 37.1, 36.6 ppm. MS (EI) of the HPLC peak
eluted at t = 8.71 min: 375.15 [M + H]+, 397.14 [M + Na]+, 771.26
[2 M + Na]+.
[8] C. R. Parker, C. G. Tortzen, S. L. Broman, M. Schau-Mag-
nussen, K. Kilså, M. B. Nielsen, Chem. Commun. 2011, 47,
6102–6104.
[9] a) S. Lara-Avila, A. V. Danilov, S. E. Kubatkin, S. L. Broman,
C. R. Parker, M. B. Nielsen, J. Phys. Chem. C 2011, 115,
18372–18377; b) S. L. Broman, S. Lara-Avila, C. L. Thisted,
A. D. Bond, S. Kubatkin, A. Danilov, M. B. Nielsen, Adv.
Funct. Mater. DOI:10.1002/adfm.20120089.
Supporting Information (see footnote on the first page of this arti-
cle): 1D and 2D NMR spectra of 7, HPLC/MALDI-TOF MS of
7, fluorescence spectra of 1, 4, and 5, MALDI-TOF MS spectrum
of vial of 1 + 3 after 24 h, 1H and 13C NMR spectra of the reaction
of 2-[2-(cyclohepta-2,4,6-trienyl)-1-phenylethylidene]malononitrile
with excess of Cys 3.
[10] S. L. Broman, S. L. Brand, C. R. Parker, M. Å. Petersen, C. G.
Tortzen, A. Kadziola, K. Kilså, M. B. Nielsen, ARKIVOC
2011, (ix), 51–67.
[11] a) S. L. Broman, M. Å. Petersen, C. G. Tortzen, A. Kadziola,
K. Kilså, M. B. Nielsen, J. Am. Chem. Soc. 2010, 132, 9165–
9174; b) M. Å. Petersen, S. L. Broman, K. Kilså, A. Kadziola,
M. B. Nielsen, Eur. J. Org. Chem. 2011, 1033–1039.
Acknowledgments
[12]
K. Nørgaard, M. B. Nielsen, T. Bjørnholm in Functional Or-
ganic Materials (Eds.: T. J. J. Müller, U. H. F. Bunz), Wiley-
VCH, Weinheim, 2007, pp. 353–392, and references cited
therein.
When DHA 1 is dissolved in the solvent mixture in the pres-
ence of daylight, conversion into VHF 2 starts immediately and
can be detected by a change in the color of solution from yel-
low to orange. VHF thermally reverts back to DHA and the
composition of the equilibrium mixture depends on both the
light intensity and the temperature. The exclusive presence of
VHF 2 occurs only after selective irradiation at 350 nm by
using a 150 W xenon arc lamp equipped with a monochroma-
tor for 1 h.
The authors acknowledge Mr. Dennis Larsen for HPLC–MS
analysis and Mr. Søren L. Broman for donating 2-[2-(cyclohepta-
2,4,6-trienyl)-1-phenylethylidene]malononitrile. Support from the
Danish Council for Independent Research – Natural Sciences (#10-
082088) and from Lundbeckfonden is acknowledged.
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Received: July 5, 2012
Published Online: ᭿
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20887
/KAP1
Date: 10-09-12 16:26:42
Pages: 7
Colorimetric Probe for the Detection of Thiols
Thiol Chemodosimeter
Can the dihydroazulene/vinylheptafulvene
(DHA/VHF) photo/thermoswitch system
be used as a chemodosimeter for thiols? A
new application for this versatile system
has been discovered.
M. Cacciarini,* E. A. Della Pia,
M. B. Nielsen .................................... 1–7
Colorimetric Probe for the Detection of
Thiols: The Dihydroazulene/Vinylhepta-
fulvene System
Keywords: Sensors / Thiols / Molecular
recognition / Chromophores
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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