A tryptophan-containing fluorescent intramolecular complex as a designer peptidic proton sensor

Article abstract of DOI:10.1039/c6cp01912a

Pyrene and tryptophan groups judiciously placed on a novel molecular scaffold, namely, bispidine exhibited fluorescence due to the formation of an unprecedented emissive intramolecular complex in polar solvents. Upon protonation, the emission signal from the pyrene unit enhances at the expense of the emission signal from the complex. The probe demonstrates good sensitivity, excellent selectivity, and adequate reversibility towards proton sensing. The present design based on the bispidine scaffold opens up newer opportunities for the design of novel bispidine-peptide sensors.

Full text of DOI:10.1039/c6cp01912a

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A tryptophan-containing fluorescent
intramolecular complex as a designer
peptidic proton sensor†
Cite this: Phys.Chem.Chem.Phys.,
2016, 18, 15046
V. Haridas,* Anita Yadav, Sakshi Sharma and Siddharth Pandey*
Pyrene and tryptophan groups judiciously placed on a novel molecular scaffold, namely, bispidine
exhibited fluorescence due to the formation of an unprecedented emissive intramolecular complex in
polar solvents. Upon protonation, the emission signal from the pyrene unit enhances at the expense of
the emission signal from the complex. The probe demonstrates good sensitivity, excellent selectivity,
and adequate reversibility towards proton sensing. The present design based on the bispidine scaffold
opens up newer opportunities for the design of novel bispidine-peptide sensors.
Received 22nd March 2016,
Accepted 4th May 2016
DOI: 10.1039/c6cp01912a
www.rsc.org/pccp
amino acid proximal to another fluorophore is a simple strategy
to spin out molecules with unique photophysical properties. We
Introduction
The design of stimuli-responsive molecules is an emerging chose tryptophan (Trp), since it has an inherent fluorescence, and
theme as they can be transformed into smart materials and the choice of pyrene (Pyr) is primarily because it is well studied and
8
useful biochemical tools. Biology provides innumerable examples has well separated excitation and emission wavelengths. These
of molecular recognition of small molecules to even the size two fluorophores were combined with a rigid bicyclic scaffold,
of protons with elegance, superiority and dexterity that are namely bispidine. Bispidine contains two piperidine rings in
1
unmatchable with any synthetic systems. The majority of these chair–chair conformation with nitrogens at a distance of
feats in natural systems are accomplished by the amino acid B2.9 Å. The chair–chair conformation with proximal nitrogen
side chain and their inbuilt chirality that provides a unique atoms makes bispidine architecturally an ideal framework to
2
topology. The design and development of sensitive and effi- spatially display the attached fluorophores at close proximity.
cient proton sensing fluorescent probes are important as these We functionalized one side of bispidine with Trp by an amide
designer probes can provide vital information about the chemical linkage, while Pyr was anchored to the other nitrogen through a
3
–6
environment. Cellular pH plays a crucial role in biochemical methylene spacer. This design was envisaged by us since it
events; hence protons are one of the most important targets. allows the protonation of nitrogen on the side of Pyr while
Fluorescence based techniques are powerful analytical methods keeping the Trp-appended nitrogen unaffected thus leading to
for in vivo and in vitro sensing and imaging. The design and differences in the spectroscopic signatures.
development of sensitive and efficient proton sensing fluorescent
probes are important research topics, since these designer probes
4
–6
can provide vital information about the chemical environment.
Experimental
Synthesis of compounds 1–3
Combining fluorophores with peptides, nucleotides or polymers
has considerably enhanced the performance of optical sensing
systems. The proton ‘‘activatable probes’’ allow the researcher to All reagents employed in the synthesis were used without
control and manipulate fluorescence output signals by altering further purification. All solvents were distilled or dried using
7
the chemical environment.
an appropriate drying agent prior to use. Reactions were
In this paper, we report the design, synthesis and demon- monitored by thin layer chromatography (TLC). Silica gel G
stration of a pyrene-based fluorescent intramolecular exciplex (Merck) was used for TLC and column chromatography. The
sensor for protons. We envisioned that positioning a fluorescent silica gel columns were generally made from the slurry in
hexane, hexane/ethyl acetate or chloroform. The Fisher-Johns
melting point apparatus was used for recording the melting
Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas,
point. IR spectra were recorded on a Nicolet, Prot ´e g ´e 460
New Delhi, 110016, India. E-mail: haridasv@chemistry.iitd.ac.in,
1
spectrometer as KBr pellets. H NMR spectra were recorded
sipandey@chemistry.iitd.ac.in
1
13
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cp01912a on a Brucker-DPX-300 ( H, 300 MHz; C, 75 MHz) spectrometer
1
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using tetramethylsilane ( H) as an internal standard. Coupling Na
PCCP
1
2
SO
4
and evaporated to yield 0.180 g of crude compound.
1
constants are in Hz and the H NMR data are reported as s Column chromatography over silica gel (100–200 EtOAc : hexane,
singlet), d (doublet), br (broad), br d (broad doublet), t (triplet), 6 : 4), gave 0.100 g of the pure product 2.
(
q (quartet), and m (multiplet). HRMS were recorded using a AB
Sciex, 1011273/A model by the ESI-technique.
Materials
Preparation of compound 3. To a well stirred and ice cooled
solution of C (0.286 g, 0.91 mmol), in methanol (10 mL), were
added 0.050 g of Pd/C and H gas was purged for 4 hours. The
2
progress of reaction was monitored by TLC. The reaction
mixture was filtered through a sintered funnel and washed
with methanol. Methanol was evaporated under reduced pressure
to yield 0.205 g of the product Bisp NH (Boc) as yellow oil.
The following solvents were used as received: 2,2,2-trifluoro-
ethanol (extra pure, 99.8%, Acros Organics), acetonitrile (LC-MS
Chromasolv, Z99.9%, Sigma-Aldrich), chloroform (ACS spectro-
photometric grade, Z99.8%, Sigma-Aldrich), propylene carbonate
(Chromasolv for HPLC, 99.7%, Sigma-Aldrich), N,N-dimethyl-
formamide (Chromasolv plus for HPLC, Z99.9%, Sigma-Aldrich),
acetone (Chromasolv for HPLC, Z99.9%, Sigma-Aldrich), dimethyl-
sulfoxide (Chromasolv plus for HPLC, Z99.9%, Sigma-Aldrich),
ethylene glycol (spectrophotometric grade, Z99%, Sigma-Aldrich),
ethylacetate (HPLC, 99.8%, Spectrochem), tetrahydrofuran (Lichro-
solv chromatography for HPLC, 99.7%, Merck) and ethanol (99.9%,
Merck). The following salts are used: cobalt chloride (98%,
Qualigens), sodium chloride (99.9%, Qualigens), potassium chloride
Yield: quantitative
To an ice-cooled solution of Bisp NH (Boc) (0.205 g, 0.91 mmol)
in 60 mL of dry acetonitrile were added triethylamine (0.5 mL,
2 2
3.64 mmol) and Br–CH –CO–NH–CH –pyrene (0.320 g, 0.91 mmol).
The reaction mixture was left stirred overnight at room temperature.
The reaction mixture was evaporated and dissolved in 80 mL of
dichloromethane, washed with 2N H SO , saturated aqueous
(99.8%, Qualigens), ammonium chloride (99.8%, Qualigens),
2
4
cesium chloride (99.8%, Qualigens), calcium chloride (98%,
Merck) and copper(II) sulfate 5-hydrate (99%, Merck). Sodium
hydroxide (98%) and nitric acid (ACS, 69%), were purchased
from Fischer scientific and Merck, respectively. Proton sensing
experiments were carried out by simple addition of conc. nitric
acid to the solution.
NaHCO solution and finally with water. The organic layer was
3
2 4
dried over anhydrous Na SO , filtered and evaporated to yield
0.300 g of the crude compound. Column chromatography over
silica gel (100–200 EtOAc : hexane, 6 : 4) gave 0.250 g of the pure
product 3.
Preparation of compound 1. To a well stirred and ice cooled
solution of 3 (0.100 g, 0.201 mmol), in dry CH Cl (2 mL), was
2
2
Methods
added TFA (0.4 mL, 4.83 mmol) and stirred at room tem-
perature for 4 h. The reaction mixture was subjected to vacuum The stock solutions of compounds 1, 2, and 3 were prepared in
to remove CH Cl
and TFA to afford 80 mg of the product Bisp ethanol and stored in an amber glass vial at 4 ꢀ 1 1C. The
NH (pyrene) as yellow oil. required amounts of compounds 1, 2, and 3 to prepare stock
2
2
To an ice-cooled solution of Boc-Trp-OH (0.061 g, 0.201 mmol) solutions were weighed using a Denver Instrument balance
in 80 mL of dry dichloromethane, were added N-hydroxy- having a precision of ꢀ0.1 mg. An appropriate amount of solutions
succinimide (0.030 g, 0.241 mmol) and DCC (0.050 g, 0.241 mmol) of compounds 1, 2, and 3 from the stock was transferred to the
2
and stirred for 10 min. Bisp NH (pyrene) (0.080 g, 0.201 mmol) 1 cm quartz cuvette. Ethanol was evaporated under a gentle
and triethylamine (0.1 mL, 0.804 mmol) were added. The reaction stream of high purity nitrogen gas.
mixture was stirred overnight, filtered and washed the filtrate with
N H SO , saturated aqueous NaHCO solution and finally with with variable bandwidth and a Peltier-temperature controller
water. The organic layer was dried over anhydrous Na SO was used for acquisition of the UV-vis molecular absorbance
A Perkin-Elmer Lambda 35 double beam spectrophotometer
2
2
4
3
2
4
and evaporated to yield 0.100 g of crude compound. Column data. Steady-state emission and excitation spectra were acquired
chromatography over silica gel (100–200 EtOAc : hexane, 8 : 2), on a model FL 3-11, Fluorolog-3 modular spectrofluorimeter with
gave 0.50 g of the pure product 1.
single Czerny–Turner grating excitation and emission mono-
Preparation of compound 2. To a well stirred and ice cooled chromators having a 450W Xe arc lamp as the excitation source
solution of 3 (0.160 g, 0.322 mmol), in dry CH Cl (2 mL), was and a PMT as the detector. This spectrofluorimeter was purchased
2
2
added TFA (1 mL, 12.44 mmol) and stirred at RT for 4 h. The from Horiba-Jobin Yvon, Inc. The temperature was controlled
reaction mixture was subjected to vacuum to remove CH Cl using a Thermo NESLAB RTE7 circulating chiller bath having
and TFA to afford 128 mg of the product Bisp NH (pyrene) as a stability of ꢀ0.01 1C. Since the use of pyrene and pyrene-
yellow oil. appended compounds is usually known to result in high
2
2
9
To an ice-cooled solution of Boc-Leu-OH (0.074 g, 0.322 mmol) fluorescence quantum yields, the blank signal was o0.1% of
in 80 mL of dry dichloromethane, were added N-hydroxy- the sample signal in all cases. This is also considered to be a
1
0
succinimide (0.050 g, 0.386 mmol) and DCC (0.080 g, 0.386 mmol) necessary condition for fluorescence lifetime measurements.
and stirred for 10 min. Bisp NH (pyrene) (0.128 g, 0.322 mmol) All absorbance and fluorescence data were acquired using
2
and triethylamine (0.2 mL, 1.74 mmol) were added. The reaction 1 cm quartz cuvettes. Spectral response from appropriate
mixture was stirred overnight and filtered, and the filtrate was blanks was subtracted before data analysis. The time-resolved
2 4 3
washed with 2N H SO , saturated aqueous NaHCO solution and fluorescence measurements were carried out using a Horiba-Jobin
finally with water. The organic layer was dried over anhydrous Yvon Fluorocube time-correlated single photon counting (TCSPC)
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fluorimeter. Compounds 1, 2, and 3 dissolved in acetonitrile and Boc-Leu-OH instead of Boc-Trp-OH. Control 3 is Pyr-conjugated
ethanol at room temperature were excited at 340 nm using a bispidine without any appended amino acid (Fig. 1).
UV-pulsed NanoLED-340 source having a pulse width of o1.0 ns.
Fluorescence emission spectra of 1 in acetonitrile (ACN,
The excited-state intensity decay of monomer and excimer emission polar-aprotic) and in ethanol (EtOH, polar-protic), respectively,
was collected using a Peltier-cooled red-sensitive TBX-04 PMT are collected by exciting at 265 nm, corresponding to the Trp
detection module at 376 nm and 480 nm, respectively. The unit, and at 340 nm, corresponding to the Pyr unit, respectively.
Em
temperature was controlled using a Peltier (TC125) having an While emission features characteristic of Trp (lmax E 330 nm
1
2
accuracy of ꢀ0.3 1C and a stability of ꢀ0.03 1C. The data were in EtOH) do not appear in the emission spectra of 1 (Fig. S1,
collected using a DAQ-MCA-3 Series (P7882) multichannel ESI†), well-documented vibronic coupling resulting in highly
analyzer. The count rate at the reference and emission detectors structured emission spectra with five peaks characterizing
was maintained at less than 2% of the NanoLED repetition rate. fluorescence emission from the Pyr moiety is clearly visible
1
3–15
All data were collected at least in triplicate starting from the (Fig. 2).
However, most interestingly, the emission spectra
sample preparation.
of 1 is highlighted by the appearance of a low-energy structure-
less broad band in the 450–600 nm region. This band is
attributed to the presence of an intramolecular complex involving
both the Trp and Pyr units on the molecular scaffold. It is
noteworthy that a fluorescent complex involving a Pyr unit and
Results and discussion
We synthesized bispidine with two orthogonal protecting groups. a Trp unit on the same molecular scaffold is unprecedented.
Boc-piperidone A was reacted with benzylamine and form- In order to explore whether this complex exists in the ground-
aldehyde under double Mannich reaction conditions yielding state, we acquired molecular absorbance spectra of 1 at 10 mM
bispidinone B. Reduction of the keto group by Wolff–Kishner and at a higher concentration of 50 mM under ambient condi-
1
1
reaction yielded the bis-protected bispidine C (Scheme 1).
tions (Fig. S2, ESI†). The absorbance spectra of 1 at both these
The Boc and benzyl protected bispidine was first debenzylated concentrations are devoid of any new spectral features in the low
by hydrogenolysis using Pd/charcoal as the catalyst followed energy regime; the absorbance spectra are representative of the
by reaction with a bromoderivative of pyrene to yield Pyr- uncomplexed 1 even at as high as 50 mM concentrations. The
conjugated bispidine. Deprotection of Boc by trifluoroacetic normalized absorbance spectra of 10 and 50 mM of 1 overlap and
acid, followed by coupling with Boc-Trp-OH using N-hydroxy- exhibit no differences. The absorbance spectral outcomes hint
succinimide and dicyclohexylcarbodiimide, yielded model at the possible absence of the complex in the ground state.
compound 1. The control compound 2 is obtained by appending Furthermore, in order to confirm that the complex formation is
Scheme 1 Synthesis of compounds 1, 2 and 3.
1
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Fig. 1 Structures of the bispidine-based compounds.
Table 1 Ratio of the emission intensity of complex (475 nm)-to-pyrene
band I (376 nm) for 1 in different solvents at 25 1C
Solvent
I
Complex/IPyrI (ꢁ10ꢂ2
)
Polar-aprotic
Acetonitrile
Acetone
Ethylacetate
Propylene carbonate
Tetrahydrofuran
Dimethylformamide
Dimethyl sulfoxide
18.6 ꢀ 0.6
12.2 ꢀ 0.5
10.4 ꢀ 0.3
8.7 ꢀ 0.3
8.2 ꢀ 0.3
6.3 ꢀ 0.2
4.6 ꢀ 0.2
Polar-protic
2,2,2-Trifluoroethanol
21.6 ꢀ 0.6
15.5 ꢀ 0.5
3.7 ꢀ 0.1
Ethanol
Ethylene glycol
Chlorinated
Dichloromethane
Chloroform
17.5 ꢀ 0.5
10.7 ꢀ 0.3
clear that the indole unit must be present to observe this
intramolecular fluorescent complex. Apart from ACN and EtOH,
the low-energy structureless broad emission band also appears
when 1 is dissolved in several other protic, aprotic, and chlori-
nated polar solvents (Fig. S4, ESI†). However, an estimation
of the complex-to-pyrene band I fluorescence intensity ratio
(IComplex/IPyrI representing I475nm/I376nm, listed in Table 1) reveals
that this intramolecular fluorescent complex formation depends
on the viscosity of the solvent; fluorescence from the complex is
less in more viscous solvents.
Fig. 2 Normalized fluorescence emission spectra of 1 at 25 1C. Emission
spectra of controls 2 and 3 under identical conditions are shown in insets.
Arrows highlight the presence and absence of the emission band corres-
ponding to the intramolecular complex in 1 and 2/3, respectively.
It is noted that when the electron/charge donor and acceptor
form part of the same molecule, intramolecular charge-transfer
states leading to detectable fluorescence are sometimes termed
1
6–20
intramolecular in nature, the fluorescence spectra of 1 are ‘‘exciplex’’ states.
In the case of intramolecular donor–
acquired at five different concentrations in the range 1–50 mM acceptor systems with a flexible linkage, the conformations
in ethanol under ambient conditions. The results presented in having face-to-face geometry were initially termed ‘‘exciplex
Fig. S3 (ESI†) reveal the normalized fluorescence spectra of 1 to conformations’’. However, in a seminal work it was shown that
be independent of the concentration of the compound; the a sandwich conformation was not a prerequisite for fluores-
2
1
normalized fluorescence scans at five different concentrations cence in such systems. Along with structural and geometrical
of 1 overlap with no statistical differences. This implies the factors a medium effect is also known to contribute to the
complex formation to be intramolecular in nature.
thermodynamics and the kinetics of the intramolecular charge
2
2
In order to decipher the role of the Trp unit on the molecular transfer process. Rapid photoinduced long-range electron
scaffold to form this fluorescent complex, emission spectra transfer followed by emissive charge recombination was earlier
from two control compounds, 2 and 3 (Fig. 1), were acquired demonstrated for the donor–acceptor systems with rigidly
23,24
under identical conditions. Contrary to that observed for 1, no extended bridges.
It was recognized that the steric require-
low-energy structureless fluorescence is observed for any of the ments were perhaps not so restrictive in forming polar exciplexes;
control compounds in ACN and EtOH, respectively (Fig. 2). It is the appearance of exciplex fluorescence is hypothesized to be due
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to conformationally close chromophores. We believe that in our state, then equal and opposite pre-exponential factors may not be
case the conformational proximity between donor Trp and excited- recovered as a result of the fitting protocol. Based on the fact that
state acceptor Pyr results in the formation of an intramolecular the fit of the intensity decay at 480 nm using a single exponential is
fluorescent complex.
not too inadequate (and the absence of the ground-state complex
We explored the effect of dissolved O on the fluorescence in the absorbance spectral data, vide supra), we lean toward the
2
emission from the complex. In EtOH, for lex = 265 nm, the possibility of rapid formation of the complex in the excited-state.
I
Complex/IPyrI values are 0.16(ꢀ0.01) and 0.15(ꢀ0.01) for the The fit of the intensity decay at 376 nm (corresponding to the Pyr
unpurged and purged samples, respectively (purged samples unit) for 1 in ACN to a single exponential decay model is clearly
are the ones where most of the dissolved O is expelled by unacceptable; the use of a two-exponential decay model results in
0 min of gentle purging with the highest purity Ar gas, Fig. S5, considerably improved fitting (Fig. 3 and Table S1, ESI†). Apart
2
3
ESI†). It is clear that quenching of the fluorescence of the Pyr from supporting the presence of two excited species, this also
unit and that of the emissive complex by molecular oxygen, a implies measurable dissociation of the excited complex back to the
2
5,26
ubiquitous quencher, are fairly similar.
evaluation of this fluorescent complex for sensing and other decay times at 376 nm and at 480 nm, respectively, along with the
applications a favourable simplicity.
This renders the excited uncomplexed species. The similarities in the two recovered
presence of ꢂve pre-exponential factors (in excited complex decay)
Time-resolved fluorescence was employed to obtain insights further emphasize the existence and formation of complexes in the
into this unprecedented fluorescent complex formation involving excited state (Table 2).
the Trp unit. Excited-state emission intensity decay data were
The intensity decay data of 1 in EtOH are more complicated.
collected for 1 along with controls 2 and 3 in ACN and EtOH, The decay at 480 nm requires three exponentials (not only
2
respectively. For both the control compounds intensity decay at the reduced w is improved substantially from the 2- to the
376 nm (Pyr group emission) fit best to a single-exponential decay 3-exponential function, the two recovered decay times for the
model (Table 2) further confirming the absence of the fluorescent 2-exponential model are fairly similar and hence unacceptable,
complex for these two control compounds. For 1 in ACN, the Fig. S6 and Table S1, ESI†). The decay at 376 nm also fits better
intensity decay at 480 nm (corresponding to the complex) best fits to a 3-exponential model with similarities in the three recovered
to a double exponential decay model though the fit to a single decay times at 376 nm and at 480 nm, respectively. This emphasizes
exponential decay model is not too inadequate (Fig. 3). This may the presence of three excited state species, where the additional
imply the presence of two excited-state species for 1 – one species could be the ground-state complex exhibiting a different
corresponding to the uncomplexed and the other to the stable decay time. Alternatively, the polar-protic media on occasions
complex. However, a careful examination of the data presented in introduces an additional decay component due to the presence of
28–31
Table 2 reveals that the two pre-exponential factors obtained from H-bonded species in the excited-state.
the fit are not equal to each other in magnitude and opposite in The usefulness of the fluorescent complex formed by 1 is
sign. If the complex is only formed after the excitation of 1, the two amply demonstrated via the change in its signal in the presence
+
pre-exponential factors should be equal in magnitude and oppo- of H . The emission of 1 is found to have high sensitivity,
2
7
+
site in sign. However, in case the rate constant of the complex excellent selectivity, and good reversibility to H in both ACN
+
formation in the excited-state is too large (i.e., the complex and EtOH. As the H is added incrementally, the fluorescence
formation is faster than the time-resolution of the instrument) signal of the complex diminishes and that of the Pyr unit
and/or some population of the complex is present in the ground augments resulting in a significant decrease in IComplex/IPyrI
(Fig. 4). The clear appearance of an isoemissive point at
B453 nm is an indication that the complex is converted back
+
to the uncomplexed conformer by H in a reversible manner. As
Table 2 Recovered intensity decay parameters for 1, 2 and 3 at 25 1C.
Excitation was carried out using a 340 nm LED. Errors associated with
decay times and pre-exponential factors are rꢀ5%
+
compared to EtOH, the sensitivity to H in polar-aprotic ACN is
+
ꢂ5
higher (for [H ] = 10 M, the decrease in IComplex/IPyrI is B78%
in ACN versus B18% in EtOH, Fig. S7, ESI†). The decrease in
l
em
t
1
t
2
t
3
+
ꢂ5
2
2
IComplex/IPyrI is fairly linear till [H ] = 10 M [for ACN, R = 0.973
Compound
(nm) (ns) (ns) (ns)
a
1
a
2
a
3
w
4
ꢂ1
2
with slope E ꢂ2.1(ꢀ0.1) ꢁ 10 M and for EtOH, R = 0.990
Acetonitrile
1
3
ꢂ1
with slope E ꢂ4.5(ꢀ0.2) ꢁ 10 M ]. While within ACN, the
376
80
376
480
376 17.2
376 17.5
1.8 12.6
3.7 11.9
1.7 14.6
1.8 12.8
0.86
0.14
1.13
+
ꢂ5
4
ꢂ0.13
0.40
0.72
1.00
1.00
0.87
0.60
0.28
1.08 plateau in IComplex/IPyrI is reached for [H ] 410 M, in EtOH it
ꢂ3
1
+ 3.16 ꢁ 10
M
M
1.59
1.34
1.56
+
ꢂ4
is reached for a fold higher [H ] of 10 M (Fig. S7, ESI†).
+
[
H ]
Intensity decay data of 1 collected at 376 nm and 480 nm,
2
3
+
1.41 respectively, in the presence of sufficiently high [H ] (=3.16 ꢁ
ꢂ3
10
M, where I /I
attains a plateau) afford insight into
PyrI Complex
Ethanol
1
+
the possible mode of H recognition. In ACN, in the presence of
376
80
1.0 6.2 18.3
0.49
0.31 0.19 1.34
+
4
0.8 8.1 15.9
1.2 6.5 26.7
1.0 5.1 23.1
0.26 ꢂ0.24 0.49 1.18 [H ], the intensity decay at 376 nm and that at 480 nm again fit
ꢂ3
1
+ 3.16 ꢁ 10
376
480
376 22.5
376 23.6
0.51
0.55
1.00
1.00
0.11 0.38 1.06
0.27 0.19 1.03
1.15
best to a double-exponential decay model with recovered decay
+
[
H ]
+
times similar to those extracted when no [H ] was present
2
3
2.15 (Table 2 and Table S1, ESI†). However, for the decay at
1
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Fig. 3 Fit of excited-state intensity decay data of 1 in ACN at 25 1C. Residuals are provided below each panel. The top two panels show fits of the decays
at 376 nm and 480 nm, respectively, to a single exponential decay model, whereas the two lower panels show the fits of the same decays to the double
exponential decay model.
3
76 nm, the pre-exponential factors are very different in the
+
absence and the presence of H implying the differential
distribution of the species due to their H -induced interconver-
+
sion. Interestingly, the ꢂve pre-exponential factor recovered in
+
the decay at 480 nm in the absence of [H ] highlighting the
formation of the complex in the excited-state (vide supra) is no
longer observed as both the pre-exponential factors in the
+
+
presence of [H ] are +ve (Table 2). This indicates that [H ] has
hindered the formation of the fluorescent complex possibly by
protonating the Pyr-attached bispidine nitrogen. Decay para-
+
meters recovered in the presence of [H ] for 1 in EtOH show
similar trends as those in ACN and thus support this proposi-
tion (Table 2). We believe that the protonation at the Pyr side of
bispidine nitrogen changes the conformation of 1 from chair–
chair to boat-chair hence rendering it unsuitable for intra-
Fig. 4 Fluorescence emission spectra of 1 (10 mM) in EtOH in the
presence of varying [H ] at 25 1C.
3
2
+
molecular complex formation (Scheme 2). The fact that 1 is
ꢂ3
+
stable in the presence of 3.16 ꢁ 10 M [H ] is amply evident by
the mass spectroscopic data (Fig. S8, ESI†).
Scheme 2 Schematic representation of the proposed protonation induced conformational change.
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Fig. 6 Adjusted fluorescence of 1 (10 mM) in ethanol (1 and 0 depict
maximum and minimum of IComplex/IPyrI in the absence and presence of
ꢂ3
+
3
.16 ꢁ 10 M [H ] for the first addition, respectively), as a function of
ꢂ3 +
scanning time. Downward arrows indicate addition of 3.16 ꢁ 10 M [H ]
ꢂ3
ꢂ
and upward arrows depict the addition of 3.16 ꢁ 10 M [OH ]. Additions
+
ꢂ
of [H ] and [OH ] are carried out sequentially.
through intramolecular fluorescent complex formation. The
concept was demonstrated by appending a biologically relevant
unit such as Trp in the design, which gives rise to the formation
of an unprecedented fluorescent intramolecular complex with
pyrene. The potential of the bispidine framework to exist in
chair–chair, boat-chair, chair-boat, and boat–boat conforma-
tions can expand further the scope of the design.
Acknowledgements
This work was generously funded by a grant to S. P. from DST-
SERB, GoI [grant no. SB/S1/PC-80/2012]. A. Y. thanks CSIR, GoI
and S. S. thanks UGC, GoI for their fellowships. The authors
would like to thank DST-FIST for the instrument grant for the
mass spectrometer.
Fig. 5 Normalized fluorescence emission spectra (lex = 340 nm) of 1
(
10 mM) (A) in the presence of various salts (0.01 M) in ethanol at 25 1C (A),
ꢂ3
in the presence of 10 M NaOH in ethanol (B), and in ACN (C).
+
The excellent selectivity for H recognition by 1 is exhibited
Notes and references
by the fact that no change in the fluorescence signal is observed
+
+
+
2+
4
even in the presence of as high as 0.01 M NH , K , Na , Cu ,
1
2
3
M.-G. Ludwig, M. Vanek, D. Guerini, J. A. Gasser, C. E. Jones,
U. Junker, H. Hofstetter, R. M. Wolf and K. Seuwen, Nature,
2
+
2+
+
Ca , Co , and Cs , respectively (Fig. 5). It is noteworthy that
addition of 10 M NaOH to 1 in ACN as well as in EtOH also
ꢂ3
2003, 425, 93–98.
results in no change in the fluorescence from the complex.
F. Galindo, M. I. Burguete, L. Vigara, S. V. Luis, N. Kabir,
J. Gavrilovic and D. A. Russell, Angew. Chem., Int. Ed., 2005,
44, 6504–6508.
(a) J. Han, A. Loudet, R. Barhoumi, R. C. Burghardt and K. A.
Burgess, J. Am. Chem. Soc., 2009, 131, 1642–1643; (b) H. F.
Higginbotham, R. P. Cox, S. Sandanayake, B. A. Graystone,
S. J. Langford and T. D. M. Bell, Chem. Commun., 2013, 49,
+
Sensitive and selective fluorescence-based recognition of H by
1
is also accompanied by good reversibility. The IComplex/IPyrI
+
decreased as a result of [H ] addition and it is readily recovered
ꢂ
back upon addition of equal [OH ] (Fig. 6). For subsequent
cycles also, the decrease and recovery of the signal may be
considered adequate.
5061–5063.
4
D. Staneva, M. S. I. Makki, T. R. Sobahi, P. Bosch, R. M.
Abdel-Rahman, A. Asiri and I. Grabchev, J. Lumin., 2015,
162, 149–154.
Conclusions
In summary, we have presented bispidine as a promising scaffold
+
for the sensing of H based on fluorescence. The designer system
5 G. R. C. Hamilton, S. K. Sahoo, S. Kamila, N. Singh, N. Kaur, B. W.
+
described showed an unusual and unique ability to sense H
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1
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