6570
B. Tautges et al. / Tetrahedron Letters 56 (2015) 6569–6573
agents through conjugation to a gadolinium chelate MRI contrast
agent. In the presence of NADH in cells these probes produced con-
trast enhancement greater than 20%, opening up an attractive new
class of imaging agents capable of characterizing the concentra-
tions of redox active species in vivo.19,20 In this work we address
the issue of developing spiropyran molecular switches that
demonstrate thiol response with a functional handle for future
use with MRI contrast agents.
Li and co-workers have demonstrated that glutathione respon-
siveness requires two nitro groups on the chromene ring of a
spiropyran switch,21 and in our previous work the linker to the
gadolinium chelate occupies one of these positions. In this study
we investigated two other positions for functional modification
of spiropyrans and characterize their effect on switching by deter-
mining the response, selectivity, and reversibility toward thiol
sensing of these spiropyrans. We found that adding functionality
to the indoline portion of a dinitro spiropyran had little effect on
the thiol response of these spiropyrans. This work demonstrates
thiol responsive spiropyran isomers ready for conjugation and
overcomes the difficult task of adding functionality without signif-
icantly altering thiol response.21,22
relative responses for these spiropyrans in the presence of
equimolar amounts of Cys, Hcy, GSH, and GSSG. All three spiropy-
rans exhibit a response to the sulfhydryl-containing amino acids as
illustrated by the strong absorbance increase centered around
500 nm. The disulfide GSSG was used as a control and the lack of
response to GSSG supported the necessity of the sulfhydryl moiety
for ring opening. Similar to previous literature reports, spiropyran
1 displays a similar response for all three thiols with cysteine and
GSH perfectly overlapping, indicating the same extent of switching
response for these two primary thiols (0.551 AU, 0.548 AU, at kMC
respectively). The response to homocysteine was slightly lower
(0.513 AU at kMC) and minimal response to GSSG (0.060 AU at
kMC) was observed; these results recapitulate literature observa-
tions (Fig. 2A).21 Spiropyrans 5 and 6 show much greater variability
between the three primary thiols. For all three isomers Cys
produced the greatest changes in absorbance, resulting in final
absorbances of 0.51 AU, 0.71 AU and 0.75 AU at the peak absor-
bance wavelength of the MC form (kMC for spiropyrans 1, 5, and
6 respectively). Repeating these assays demonstrated that minor
fluctuations in absorbance profile in response to analytes could
be expected, resulting in no significant selectivity for thiols with
spiropyran 6. The aggregate data is discussed further in the next
section. There is also a markedly higher baseline absorbance for
spiropyran 5 after 5 min of visible light irradiation compared to
the other two spiropyrans, 1 and 6, suggesting that spiropyran 5
has a greater population of the MC form at equilibrium compared
to spiropyrans 1 and 6 (Fig. 2B). Longer visible light irradiation
times were tested in an attempt to drive conversion to the closed
form, but after 20 min of visible light irradiation this high baseline
still persisted (SI, Fig. S1).
Results and discussion
Spiropyrans are a well known class of molecular switching com-
pounds that can be converted between the closed spiropyran form
(SP) or the open merocyanine form (MC) in response to external
stimuli (Scheme 2).23 In this work we synthesized switches for
antioxidant sensing, the presence of thiols induces ring opening;
this can be reverted back to the closed form using visible light. This
conversion can be easily observed by UV/Vis spectroscopy with the
MC form absorbing strongly in the visible region while the SP form
absorbs strongly in the UV region. Measurement of the MC absor-
bance peak was performed to monitor switching from SP to MC
form induced by response to various amino acids.
Synthesis of spiropyrans 5 and 6 proceeded from the commer-
cially available 3-aminobenzyl alcohol 2, which was oxidized to
the hydrazine under Sandmeyer conditions. Utilizing continuous
extraction over 24 h provided 50% mass recovery, and the crude
aryl hydrazine underwent Fischer indole synthesis to generate
indoles 3 and 4 as a mixture. The overall yield of both indole iso-
mers was 32% over two steps, a significant improvement on previ-
ously reported yields over the same two steps.24 Indoles 3 and 4
were methylated with iodomethane and the crude enamines were
subjected to condensation with 2-hydroxy-3,5-dinitrobenzalde-
hyde in refluxing ethanol (24 h, 80 °C) to yield spiropyrans 5 and
6 (Scheme 1) in four steps with 2.5% and 1.5% overall yield. Spiro-
pryan 1 (Fig. 1) was prepared as reported by Buback and co-work-
ers25 (Supporting information).
Spiropyran 1, featuring two nitro groups in the 6- and 8-posi-
tions of the chromene moiety, was reported in the literature to
respond to primary thiols such as Cys, Hcy, and GSH, however,
no switching to the MC form was reported in the presence GSSG.21
We used this spiropyran as reference to test for positional effects of
the introduction of linkers. Hydroxyl groups, which can readily be
converted to the alkyne for conjugation to azide species, were
placed in different positions on the indole ring of spiropyran 1 to
form spiropyrans 5 and 6. Spiropyrans 5 and 6 were successfully
synthesized and structure identified by accurate mass spec-
troscopy, proton and carbon NMR spectroscopy (Supporting
information).
Selectivity
Building upon this initial characterization of thiol responsive-
ness, spiropyrans 1, 5, and 6 were evaluated for their thiol selec-
tivity by incubating with GSH, Cys, Hcy, and other nucleophilic
amino acids. As expected, all three spiropyran solutions shifted
to an orange color in the presence of Cys, GSH, and Hcy indicating
a ring opening to the MC form, which is supported by the
appearance of
a strong absorbance centered around 500 nm
(kMC) (Fig. 3A). Figure 3 represents the ratio of (A À A0)/A0 for
the kMC for each isomer respectively. Based on these ratios all
three isomers showed selectivity for thiols over GSSG and other
nucleophilic amino acids without primary thiols (p <0.01, Fig. 3).
Spiropyrans 1 and 6 demonstrated poor selectivity among the
three primary thiols GSH, Hcy, and Cys (Fig. 3). In contrast,
spiropyran 5 exhibits the greatest response to cysteine at the
kMC with a 5.29 fold increase from the baseline prior to incuba-
tion with thiol and reduced responsiveness to GSH (4.08 fold,
p <0.05), further reduced response to Hcy (3.82 fold, p <0.01) indi-
cating some level of selectivity over primary thiol (Fig. 3B). Like
spiropyran 1, spiropyran 5 had no response to amino acids lack-
ing a primary thiol (Fig. 3B). The addition of the hydroxyl in this
position seems to afford some degree of cysteine selectivity over
the other sulfhydryl containing amino acids (GSH p <0.05, Hcy
p <0.01).
While the values of A À A0/A0 appear to be smaller for
Spiropyran 5, it is important to note that this ratio may not be
reflective of the response of the switch. While final absorbance
values between spiropyrans 5 and 6 were similar in magnitude
after incubation with Cys (Student t test, p = 0.1727) the higher
baseline of spiropyran 5 reduced the A À A0/A0 values. The posi-
tion of the linker placed on spiropyran 6 had little effect on
selectivity, and spiropyran 6 behaved similarly to spiropyran 1
(Fig. 3C).
Following the synthesis, switching ability of spiropyrans 1, 5,
and 6 in response to Cys, Hcy, GSH and GSSG was investigated
by UV/Vis spectroscopy. Representative data from single samples
of spiropyrans 1, 5, and 6 are shown in Figure 2 to illustrate the