A fluorescent off-on NBD-probe for F- sensing: Theoretical validation and experimental studies

Article abstract of DOI:10.1039/c3ob41886c

The design, synthesis and fluoride sensing ability of a 7-nitro-2,1,3-benzoxadiazole (NBD) based chemodosimeter is reported. Theoretical calculations were used to design a more applicable off-on response, by choosing NBD as the accurate fluorophore. React

Full text of DOI:10.1039/c3ob41886c

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
A fluorescent off–on NBD-probe for F− sensing:
theoretical validation and experimental studies†
Cite this: Org. Biomol. Chem., 2014,
12, 2143
Arundhati Roy, Avdhoot Datar,‡ Dnyaneshwar Kand,‡ Tanmoy Saha‡ and
Pinaki Talukdar*
The design, synthesis and fluoride sensing ability of a 7-nitro-2,1,3-benzoxadiazole (NBD) based chemo-
dosimeter is reported. Theoretical calculations were used to design a more applicable off–on response,
by choosing NBD as the accurate fluorophore. Reaction of the NBD-probe with 300 equivalents of
tetrabutyl ammonium fluoride (TBAF) exhibited a response time of 80 minutes and the reaction was
selective to F− and sensing of the ion was marked by a 110-fold enhancement of green fluorescence. The
off–on fluorescence characteristics of the probe enabled its application in live-cell imaging of intracellular
Received 16th September 2013,
Accepted 29th January 2014
DOI: 10.1039/c3ob41886c
www.rsc.org/obc
F
− ions.
Biological importance of the fluoride ion (F−) in preventing
enamel demineralization, dental and skeletal fluorosis, osteo-
Introduction
Biologically important species such as reactive oxygen species porosis treatment, etc. encouraged us to design a new fluo-
(ROS), biothiols, anions, metal ions and transition metals, are rescent probe for selective detection of the ion.9 As reported,
detected by various analytical techniques which include ratiometric10 and on–off probes11 are limited to determining
reverse-phase HPLC,1 gas chromatography,2 UV-vis,3 fluo- fluoride content in water, and fluorescent off–on probes12 are
rescence spectroscopy,4 etc. Among these, fluorescence-based essential for live-cell imaging which may help in determining
techniques have gained more interest in recent years because the onset of diseases caused by excess fluoride.13 Fluoride
of their high selectivity, sensitivity and straightforward mediated deprotection of aryl silyl ethers results either in a
measurement protocol.4b,5 Moreover, fluorescent probes can ratiometric or on–off or off–on response depending on the
be used in live-cell imaging which allow real time analysis of choice of the aryl fluorophore. For example, probe 1 (Fig. 1)
different analytes and various biological events.6 Fluorescent consisting of the triisopropylsilyl (TIPS) group connected to
off–on probes are usually preferred over either on–off or ratio- the dipyrrometheneboron difluoride (BODIPY) fluorophore11
metric probes for in vivo imaging of biologically relevant provided an on–off response. On the other hand, the fluoro-
species.
phore alteration to coumarin resulted in an off–on response by
Recently, theoretical calculations have been used in the probe 2.12b When a similar tert-butyldimethylsilyl (TBDMS)
development of fluorescent off–on probes.7 The relatively in- group was connected to the benzoimidazole fluorophore in the
expensive method has provided fruitful information on fluo- design of probe 3, F− sensing resulted in the ratiometric
rescence properties of the probe and the resulting species response.14 Another type of chemodosimeter consisting of a
generated during the sensing, prior to any experimental work. 4-(hydroxymethyl) phenol linker between the fluorophore and
In recent times, we have successfully applied time dependent
density functional theory (TDDFT) to design off–on probes for
sensing biothiols, arylthiols, etc.8
Department of Chemistry, Indian Institute of Science Education and Research, Pune,
India. E-mail: ptalukdar@iiserpune.ac.in; Fax: (+) 91 20 2589 9790;
Tel: (+) 91 20 2590 8098
†Electronic supplementary information (ESI) available: Additional experimental
procedures, crystal structures, theoretical calculations and 1H-, 13C-NMR spectra.
CCDC 955810. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c3ob41886c
‡Equal contributions of authors.
Fig. 1 Structures of fluoride selective fluorescent probes 1–3.
Org. Biomol. Chem., 2014, 12, 2143–2149 | 2143
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Organic & Biomolecular Chemistry
absorption and emission spectra,19 they do not affect the
qualitative position of the spectral bands and also have no
bearing on the calculation of oscillator strengths. Since com-
puting the oscillator strengths at the absorption and emission
geometries is sufficient to predict fluorescence outcomes, cal-
culations of fine structure of the spectrum by including
vibrational effects were not considered. Some selected para-
meters for the vertical excitation (UV-vis absorption) and the
fluorescence emission are shown in Tables 1 and 2 respect-
ively. With TDDFT calculations based on the optimized
ground state (S0) geometry, the main allowed electronic tran-
sition observed for 4 was S0→S1 (HOMO → LUMO, f = 0.4323)
and for 4a was S0→S1 (HOMO → LUMO) with oscillator
strength f = 0.2690 (Table 1). From the HOMO and LUMO con-
tours, electronic transitions for 4, 4a, 5 and 5a were assigned
as n, π* or π, π* transitions. S0→S1 transitions for 4 and 4a
were characterized as π→π* transitions (Fig. S1†). In order to
investigate the emission of 4 and 4a, the geometry of the
singlet excited state (S1) was optimized and the excitation
based on the S1 geometry was calculated. The TDDFT calcu-
lations indicate an emissive S1 state; an allowed S1→S0 tran-
sition with f = 0.3820 and 0.2029 for 4 and 4a respectively was
observed and thus the radiative S1→S0 decay is possible
(Table 2). S1→S0 transitions for 4 and 4a were characterized as
π*→π transitions (Fig. S2†).
Similarly, TDDFT calculation based on the optimized
ground state (S0) geometry, electronic S0→S1 transition for 5
was not allowed (HOMO → LUMO, f = 0.0000). S0→S1 tran-
sition was characterized as π→π* and π→n transition
(Fig. S3†). Similarly, S0→S2 transition with f = 0.0000 was not
allowed. The first allowed transition is S0→S3 with f = 0.4471.
As shown in Fig. S2,† it is clear from the distributions of the
MOs that the HOMO and LUMO are localized at different
locations for 5. The HOMO is located on the phenyl moiety,
whereas the LUMO is mainly localized on the NBD fluoro-
phore. Consequently, the excitation from the S0 to S1 state
involves an intramolecular charge transfer to the NBD unit. To
investigate the emission of 5 the geometry of the singlet
excited state (S1) was optimized and the excitation based on
the S1 geometry was calculated. Absence of an emissive S1 state
(S1→S0 transition with f = 0.0000) suggests that the radiative
S1→S0 decay is not possible. S0→S1 transition was character-
ized as π*→π and n→π transition (Fig. S4†). S2→S0 transition
with f = 0.0000 was not allowed. The first allowed transition is
S3→S0 with f = 0.3807. As shown in Fig. S4,† the distributions
Fig. 2 Schematic presentation of cascade reaction based fluoride
sensing (A). Structures of ratiometric probe 4 and released fluorophore
4a (B). Structures of proposed off–on probe 5 and released fluorophore
5a (C).
the silyl group is also reported for sensing the F− ion (Fig. 2A).
For example, probe 4, reported by Kim and co-workers, exhibi-
ted a ratiometric response upon treatment with the F− ion
(Fig. 2B).15 Although a novel feature which involves F− ion pro-
moted silyl deprotection followed by decarboxylation strategy
was incorporated in the design of probe 4, the ratiometric
response limited its applicability in live-cell imaging. However,
F− ion mediated push–pull deprotection chemistry of the
probe encouraged us to develop the fluorophore having off–on
characteristics. Herein, we report the use of the TDDFT
method to rationalize the ratiometric response of probe 4 and
the use of the computational tool to design a new probe 5 for
providing an off–on response during F− ion sensing (Fig. 2C).
Recently, Lin et al. reported that a BODIPY-coumarin hybrid
probe for detection of the F− ion also supports the use of
theoretical calculations to validate off–on phenomena.10c
Results and discussion
Theoretical calculations
Theoretical calculations were used to rationalize the ratio- of the MOs for 5, the HOMO is located on the phenyl moiety,
metric outcome of reported probe 4 and predict the response whereas the LUMO is mainly localized on the NBD fluoro-
of the proposed probe 5 to the F− ion. At first, ground state phore. Consequently, the emission from the S1 to S0 state
geometry optimization of species 4, 4a, 5 and 5a was carried involves an intramolecular charge transfer from the NBD unit.
out in water medium with CAM-B3LYP/6-311G(d,p) basis sets16 For 5a the allowed electronic transition was S0→S1 (HOMO →
using the Gaussian 09 program.17 The polarizable continuum LUMO, f = 0.2590). S0→S1 transition was characterized as π→π*
model (PCM) was used in the calculation of the absorption and π→n transition. To investigate the emission of 5a the geo-
and emission spectra in water.18 Then, geometry optimized metry of the singlet excited state (S1) was optimized and the
structures were further used for TDDFT calculations with the excitation based on the S1 geometry was calculated. For 5a the
same level of theory. Although the vibrational effects play an presence of an emissive S1 state (an allowed S0→S1 transition
important role in determining the structural details of with f = 0.1611) suggests that the radiative S1→S0 decay is
2144 | Org. Biomol. Chem., 2014, 12, 2143–2149
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
Table 1 Selected parameters for the vertical excitation (UV-vis absorptions) of probes 4, 4a, 5 and 5a obtained by the TDDFT/CAM-B3LYP/6-311G
(d, p), based on the optimized ground state geometries (water was employed as a solvent in all the calculations)
Compound
Electronic transitionsa
Energy (eV)
3.69
λ (nm)
f b
Main configurationsc
CI coefficientsd
4
S0→S1
335.82
0.4323
HOMO → LUMO
HOMO−1 → LUMO
HOMO → LUMO
0.66959
0.19295
0.69759
0.69983
0.18351
0.15818
0.50113
0.39602
0.69821
0.69588
0.10261
4a
5
S0→S1
S0→S1
S0→S2
3.39
2.40
4.18
365.52
516.33
296.72
0.2690
0.0000
0.0000
HOMO → LUMO
HOMO−7 → LUMO
HOMO−7 → LUMO+1
HOMO−5 → LUMO
HOMO−5 → LUMO+5
HOMO−1 → LUMO
HOMO → LUMO
S0→S3
S0→S1
3.45
3.24
359.92
382.39
0.4471
0.2590
5a
HOMO → LUMO+1
a Only the main configurations are presented. b Oscillator strength. c H stands for HOMO and L stands for LUMO. d The CI coefficients are in
absolute values.
Table 2 Selected parameters for the fluorescence emission of probes 4, 4a, 5 and 5a obtained by the TDDFT/CAM-B3LYP/6-311G (d, p), based on
the optimized ground state geometries (water was employed as a solvent in all the calculations)
Compound
Electronic transitionsa
Energy (eV)
λ (nm)
f b
Main configurationsc
CI coefficientsd
4
4a
5
S1→S0
S1→S0
S1→S0
S2→S0
3.05
2.69
0.80
3.99
406.31
460.61
1543.28
310.97
0.3820
0.2029
0.0000
0.0000
HOMO → LUMO
HOMO → LUMO
HOMO → LUMO
HOMO−4 → LUMO
HOMO−4 → LUMO+1
HOMO−4 → LUMO+5
HOMO−1 → LUMO
HOMO → LUMO
0.698850
0.700760
0.702350
0.53601
0.42264
0.13881
0.700580
0.700210
S3→S0
S1→S0
3.00
2.62
413.63
473.75
0.3807
0.1611
5a
a Only the main configurations are presented. b Oscillator strength. c H stands for HOMO and L stands for LUMO. d The CI coefficients are in
absolute values.
possible. S0→S1 transition was characterized as π*→π and n→π
transition.
Synthesis of probe 5
Once the predicted fluorescence off–on response is validated
by an in silico method, synthesis of the probe 5 was carried out
from NBD-amine 5a.20 4-(tert-Butyldimethylsilyloxy)benzylalco-
hol 6 was synthesized first according to the reported protocol
from 4-hydroxybenzaldehyde.21 Alcohol 6 was then treated
with 4-nitrophenylchloroformate in the presence of DMAP in
CHCl3 at room temperature to generate the corresponding
active carbonate intermediate which was reacted subsequently
with 5a to provide probe 5 with overall 37% yield (Scheme 1A).
1
The structure of probe 5 was confirmed by H-, 13C-NMR spec-
troscopy, high resolution mass spectrometry and single crystal
X-ray diffraction studies (Scheme 1B). Superimposition of the
crystal structure (blue) and geometry optimized structure (red)
of 5 based on Si- and all N-atom alignment provided a RMSD =
0.39 Å (Scheme 1C). Excellent superimposition of two struc-
tures indicates that correct atom coordinates were used for the
TDDFT calculation.
Scheme 1 Synthesis of the probe 5 (A); crystal structure (B); and
overlay of crystal (blue) structure of 5 and its geometry optimized (red)
structure (C).
absorption band centered at λmax = 399 nm with a molar
extinction coefficient value, ε = 12 190 M−1 cm−1 in 9 : 1
Photophysical properties
Experimental validation of fluorescence off–on character- ethanol–HEPES buffer (10 mM, pH = 7.4) solution. However,
istics. Photophysical studies of the probe 5 displayed a strong the probe 5 exhibited negligible fluorescence intensity and low
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2143–2149 | 2145

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 4 Emission kinetics profile of probe 5 (10 μM) in 9 : 1 EtOH–HEPES
buffer (10 mM, pH = 7.4) solution at room temperature at λem = 535 nm
upon excitation of λex = 460 nm.
Fig. 3 Fluorescence lifetime decay profiles (λex = 460 nm) of probe 5
(
) and 5a ( ) in 9 : 1 ethanol–HEPES buffer (10 mM, pH = 7.4) moni-
tored at 535 nm. Prompt represents the laser profile (─).
this experiment, 300 equivalents of tetrabutylammonium fluo-
ride (TBAF) was added to probe 5 (10 μM) in 9 : 1 EtOH–HEPES
buffer (10 mM, pH = 7.4) solution at 30 s and enhancement of
fluorescence intensity at λ = 535 nm (λex = 460 nm) was
recorded in time (Fig. 4). A sharp enhancement of fluorescence
intensity was observed up to 60 minutes followed by flattening
of the enhancement. Therefore, a reaction time of 60 minutes
was used for further studies with ∼80% completion of the
reaction.
quantum yield, ΦF = 0.0495 (standard: NBD-NHMe in aceto-
nitrile ΦF = 0.38).22 NBD-amine 5a displayed a strong absorp-
tion band centered at λmax = 460 nm (ε = 13 113 M−1 cm−1),
intense fluorescence with λem = 535 nm (λex = 460 nm) and
ΦF = 0.36. These data confirm the predicted off–on character-
istics if probe 5 is subjected to chemical conversion to 5a.
The observed difference in fluorescence of molecules 5 and
5a encouraged us to investigate further the quenching mech-
anism working in these two molecules. Fig. 3 represents the
fluorescence decay profile of probes 5 and 5a recorded in 9 : 1
ethanol–HEPES buffer (10 mM, pH = 7.4) with excitation at
460 nm. Emission at 535 nm for the probe 5 showed a mono-
exponential decay profile with τ = 1.303 ns. On the other hand,
amine 5a exhibited a mono-exponential decay profile with τ =
5.465 ns. From these data, radiative decay rate constant (kr)
and non-radiative decay rate constant (knr) for species 5 and 5a
were determined using the following equations:23
Selectivity studies. In the next stage, selectivity of probe 5
towards F− was examined in the presence of various competi-
tive anions. When probe 5 (10 μM) in 9 : 1 EtOH–HEPES buffer
(10 mM, pH = 7.4) solution was treated with 200 equivalents of
TBAF, a 110-fold enhancement in the fluorescence intensity
was observed after 1 h stirring at room temperature. Selectivity
of the probe 5 towards F− was evaluated by pre-treating it with
various competing anions e.g. Br−, I−, Cl−, ClO4−, PF6−, NO3
,
−
HSO4−, OAc−, and SO4 (2 mM) followed by reaction with F−
(2 mM). Under comparable reaction conditions, these anions
provided insignificant fluorescence enhancement compared to
the probe 5 (Fig. 5A, front row). The probe 5 showed fluo-
rescence intensity enhancements only after addition of F−
which proves the selectivity of the probe 5 towards F− in the
presence of other ions (Fig. 5A, back row). Naked eye monitor-
ing of the detection process was associated with the color
change from colorless to yellow only in the presence of the F−
ion and other anions did not provide any color change
(Fig. 5B).
2−
kr ¼ Φ=τ
ð1Þ
ð2Þ
knr ¼ ð1=τÞ ꢀ kr
The probe 5 displayed a kr value of 0.0379 ns−1 and a corres-
ponding knr = 0.727 ns−1. For amine 5a, calculated kr and knr
values were 0.0658 and 0.1169 ns−1, respectively. For probe 5,
the kr/knr = 0.052 confirmed a predominant non-radiative
pathway, responsible for its quenched fluorescence state. On
the other hand, kr/knr = 0.5628 calculated for 5a corroborate
with its strong fluorescence due to the radiative pathway as the
main channel (Table 3).
Quantitative off–on response. Reaction of the probe
5
(10 μM) with increasing concentrations of TBAF (0–3.5 mM)
resulted in the rapid enhancement of fluorescence intensity at
λem = 535 nm (λex = 460 nm) and with a saturation of fluo-
rescence intensity after 300 equivalents of F− added (Fig. 6A).
When images were taken under the hand-held UV lamp, the
cuvette containing probe 5 exhibited negligible blue fluo-
rescence (Fig. 6B). On the other hand, the reaction mixture
Sensing of the F− ion by probe 5
Determination of response time. Reactivity of probe 5
towards the F− ion was evaluated by fluorescence kinetics. In
containing probe
fluorescence.
5 and TBAF displayed strong green
Table 3 Time resolved fluorescence data for 5 and 5a
Probe
τ (ns)
kr (ns−1
)
knr (ns−1
)
kr/knr
Live-cell imaging of F− ions. The off–on response of the
probe 5 allowed us to investigate its application in cell
imaging. Live-cell imaging was carried out using the
5
5a
1.303
5.465
0.0379
0.0658
0.727
0.1169
0.052
0.5628
2146 | Org. Biomol. Chem., 2014, 12, 2143–2149
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 7 Confocal microscopic images of A549 (human epithelial lung
carcinoma) cells. Brightfield (A), fluorescence (B), and overlay (C) images
of A-549 cells pre-incubated with NaF (50 mM) for 3 h followed by incu-
bation with probe 5 (50 μM) for 7 h. Brightfield (D), fluorescence (E), and
overlay (F) images of A-549 cells incubated with probe 5 (50 μM) for 7 h.
Fig. 5 Relative fluorescence intensity of the probe 5 (10 μM) in 9 : 1
ethanol–HEPES buffer (10 mM, pH = 7.4) solution towards ranges of
anions (2 mM) followed by addition of TBAF (2 mM). All data were taken
after 1 h of addition of anions at room temperature at λem = 535 nm (A).
Photograph was taken under ambient light upon treating probe 5 with
respective analytes in 9 : 1 ethanol–HEPES buffer (10 mM, pH = 7.4)
solution (B).
Conclusions
In conclusion, we have successfully designed a NBD-based
off–on probe for sensing F− ions using theoretical calculations.
Based on TDDFT calculations of excitation and emission for
the probe and for the NBD-amine fluorophore, we rationalized
the predicted off–on response. Steady state and time resolved
fluorescence properties of these species further validated the
design. The NBD-probe exhibited
a response time of
80 minutes in the presence of 300 equivalents of TBAF and the
reaction was selective to only F−, even in the presence of
various interfering analytes. The F− ion sensing ability of the
probe was associated with a 110-fold enhancement in fluo-
rescence at λem = 535 nm and selectivity was achieved even in
the presence of competing anions. The sensing of F− ions was
marked by a visible color change from colorless to yellow and
green turn-on fluorescence. Cell permeability of the probe and
its ability to detect intracellular F− ions was confirmed by live
cell imaging.
Fig. 6 Fluorescence enhancement of the probe 5 (10 μM) in 9 : 1
EtOH–HEPES buffer (10 mM, pH = 7.4) solution with varied concen-
trations of TBAF (0–3.5 mM). Inset: fluorescence intensities at 535 nm
plotted versus concentration of TBAF (mM) added (A). Cuvette images of
probe 5 and after addition of TBAF under a handheld UV (λex = 365 nm)
lamp (B).
Experimental section
General methods
All reactions were conducted under a nitrogen atmosphere. All
the chemicals were purchased from commercial sources and
used as received unless stated otherwise. Solvents: petroleum
ether and ethyl acetate (EtOAc) were distilled prior to thin layer
and column chromatography. Column chromatography was
performed on Merck silica gel (100–200 mesh). TLC was
carried out with E. Merck silica gel 60-F-254 plates.
fluorescence microscopic technique with the A549 (human
lung carcinoma) cell line.12f No significant fluorescence was
observed when A549 cells were incubated with only 5 (50 μM
in 1 : 100 DMSO–DNEM v/v, pH = 7.4) at 37 °C for 7 h (Fig. 7E).
But, when A549 cells were pre-treated with NaF (50 mM in
1 : 100 H2O–DMEM, pH = 7.4) at 37 °C for 3 h followed by incu-
bation with probe 5 (50 μM in 1 : 100 DMSO–DNEM v/v, pH =
7.4) at 37 °C for 7 h, strong fluorescence was observed inside
these cells (Fig. 7B). These data indicate the cell permeability
of the probe 5 and its ability to detect intracellular F− ions.
Instrumentation and software
The 1H and 13C spectra were recorded on 400 MHz Jeol
ECS-400 (or 100 MHz for 13C) spectrometers using either
residual solvent signals as an internal reference on the δ scale
(CDCl3 δH, 7.24 ppm, δC 77.0 ppm). The chemical shifts (δ) are
reported in ppm and coupling constants (J) in Hz. The
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2143–2149 | 2147

View Article Online
Paper
Organic & Biomolecular Chemistry
following abbreviations are used: s (singlet), d (doublet). High- with a graphite monochromator. R = 0.0793 (for 5031 reflec-
resolution mass spectra were obtained from a MicroMass tions I > 2σ(I)), wR = 0.2393 which was refined against |F2| and
ESI-TOF MS spectrometer. All MALDI-MS measurements were S = 0.947 for 569 parameters and 10 720 unique reflections.
recorded on an Applied Biosystems 4800 Plus MALDI TOF/TOF The structure was obtained by direct methods using
analyzer. Absorption spectra were recorded on a PerkinElmer, SHELXS-97. All non-hydrogen atoms were refined isotropically.
Lambda 45 UV-Vis spectrophotometer. Steady state fluo- The hydrogen atoms were fixed geometrically in the idealized
rescence experiments were carried out in a micro fluorescence position and refined in the final cycle of refinement as riding
cuvette (Hellma, path length 1.0 cm) on a Fluoromax 4 instru- over the atoms to which they are bonded. μ = 0.148 mm−1
ment (Horiba Jobin Yvon). Time-resolved fluorescence
.
measurements were carried on a TCSPC instrument (Horiba
Jobin Yvon, Fluoro 3PS). FT-IR spectra were obtained using a
NICOLET 6700 FT-IR spectrophotometer as KBr discs and
Acknowledgements
reported in cm−1. Melting points were measured using a We acknowledge IISER Pune, DAE (grant no. 2010/20/37C/6/
VEEGO melting point apparatus. All melting points were BRNS/2480) and DST-SERB (grant no. SR/S1/OC-65/2012) for
measured in open glass capillary and values are uncorrected. financial support. We thank Dr Anirban Hazra of IISER Pune
Crystal structures were recorded on a Bruker single crystal for valuable discussions and comments. A. R., T. S. thank
X-ray diffractometer. Geometry optimizations and emission UGC, A. D. thanks IISER Pune and D. K. thanks CSIR for
characteristics were carried out using the Gaussian 09 software research fellowships.
package.17 Superimpositions of crystal structure and geometry
optimized structure of 5 were obtained using VMD 1.9.1
software.
Notes and references
Synthesis of 4-((tert-butyldimethylsilyl)oxy)benzyl (7-nitro-
benzo[c][1,2,5]oxadiazol-4-yl)carbamate 5. Alcohol 6 (264 mg,
1.1 mmol) was dissolved in chloroform (15 mL) and then
4-dimethylaminopyridine (DMAP) (161 mg, 1.32 mmol) and
4-nitrophenylchloroformate were added to it and stirred at
room temperature for 3 h. TLC showed the consumption of
both starting materials at this stage. NBD-amine 5a (178 mg,
0.99 mmol), DMAP (161 mg, 1.32 mmol) dissolved in CHCl3
(5 mL) was added dropwise to the reaction mixture and
refluxed overnight. After the completion of the reaction, the
solvent was evaporated under reduced pressure and the yellow
residue was subjected to column chromatography over silica
gel (eluent: 5% EtOAc in petroleum ether) to furnish pale
yellow compound 5 (175 mg, 37%). M.p.: 134.5–135.5 °C;
IR (KBr): νmax/cm−1: 3415, 2940, 2857, 1740, 1565, 1514, 1454,
1365, 1326, 1258; 1H NMR (400 MHz, CDCl3): δ 8.57 (d, J =
8.24 Hz, 1H); 8.17 (d, J = 8.7 Hz, 1H), 7.32 (d, J = 8.72 Hz, 2H),
6.87 (d, J = 8.68 Hz, 2H), 5.25 (s, 2H), 0.99 (s, 9H), 0.21 (s, 6H);
13C NMR (100 MHz, CDCl3): δ 156.5, 151.9, 144.7, 143.0, 134.3,
133.9, 130.3, 126.9, 120.2, 110.4, 68.0, 25.2, 18.2, −4.4; HRMS
(ESI): calc. for C20H24N4O6Si [M − H]−: 443.1392; found:
443.1387.
1 (a) J. Rendl, S. Seybold and W. Börner, Clin. Chem., 1994,
40, 908; (b) C. E. Kibbey and M. E. Meyerhoff, Anal. Chem.,
1993, 65, 2189.
2 (a) P. R. Bérubé, P. D. Parkinson and E. R. Hall, J. Chroma-
togr., A, 1999, 830, 485; (b) W. C. Butts and W. T. Rainey,
Anal. Chem., 1971, 43, 538.
3 M. Vázquez, L. Fabbrizzi, A. Taglietti, R. M. Pedrido,
A. M. González-Noya and M. R. Bermejo, Angew. Chem., Int.
Ed., 2004, 43, 1962.
4 (a) R. M. Duke, E. B. Veale, F. M. Pfeffer, P. E. Kruger and
T. Gunnlaugsson, Chem. Soc. Rev., 2010, 39, 3936;
(b) T. Gunnlaugsson, M. Glynn, G. M. Tocci, P. E. Kruger
and F. M. Pfeffer, Coord. Chem. Rev., 2006, 250, 3094.
5 R. Martínez-Máñez and F. Sancenón, Chem. Rev., 2003, 103,
4419.
6 M. Cametti and K. Rissanen, Chem. Commun., 2009, 2809.
7 (a) S. Ji, H. Guo, X. Yuan, X. Li, H. Ding, P. Gao, C. Zhao,
W. Wu, W. Wu and J. Zhao, Org. Lett., 2010, 12, 2876;
(b) L. Long, X. Li, D. Zhang, S. Meng, J. Zhang, X. Sun,
C. Zhang, L. Zhou and L. Wang, RSC Adv., 2013, 3, 12204.
8 (a) D. Kand, A. M. Kalle, S. J. Varma and P. Talukdar, Chem.
Commun., 2012, 48, 2722; (b) D. Kand, P. K. Mishra,
T. Saha, M. Lahiri and P. Talukdar, Analyst, 2012, 137,
3921.
X-Ray single crystal determination
Single crystal of 5 suitable for X-ray diffraction study was
grown as mentioned below.
9 (a) M. Cametti and K. Rissanen, Chem. Soc. Rev., 2013, 42,
2016; (b) P. A. Gale, Chem. Soc. Rev., 2010, 39, 3746;
(c) P. A. Gale, S. E. Garcia-Garrido and J. Garric, Chem. Soc.
Rev., 2008, 37, 151.
Crystal structure of probe 5 (CCDC 955810)
C20H24N4O6Si; compound 5 was crystallized from ethyl acetate
at room temperature. A yellow needle shaped crystal with 10 (a) X.-F. Yang, H. Qi, L. Wang, Z. Su and G. Wang, Talanta,
approximate dimensions 0.16 × 0.14 × 0.10 mm3 gave a Tri-
2009, 80, 92; (b) B. Zhu, F. Yuan, R. Li, Y. Li, Q. Wei, Z. Ma,
B. Du and X. Zhang, Chem. Commun., 2011, 47, 7098;
(c) X. Cao, W. Lin, Q. Yu and J. Wang, Org. Lett., 2011, 13,
6098; (d) M. Dong, Y. Peng, Y.-M. Dong, N. Tang and
Y.-W. Wang, Org. Lett., 2011, 14, 130; (e) M. R. Rao,
ˉ
clinic with space group P1; a = 6.2718(19) Å b = 18.621(6) Å c =
19.391(6) Å, α = 82.717(6)° β = 84.770(6)° γ = 86.359(6)°; V =
2233.9(12) Å3; T = 200 (2) K; Z = 4; ρcalc = 1.322 g cm−3; 2θmax
=
55.98°; MoKα λ = 0.71073 Å. A fine-focus sealed tube source
2148 | Org. Biomol. Chem., 2014, 12, 2143–2149
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
S. M. Mobin and M. Ravikanth, Tetrahedron, 2010, 66,
1728; (f) Y. Bao, B. Liu, H. Wang, J. Tian and R. Bai, Chem.
Commun., 2011, 47, 3957.
R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin,
R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin,
K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador,
J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas,
J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox,
GAUSSIAN 09 (Revision D.01), Gaussian, Inc., Wallingford
CT, 2009.
11 O. A. Bozdemir, F. Sozmen, O. Buyukcakir, R. Guliyev,
Y. Cakmak and E. U. Akkaya, Org. Lett., 2010, 12, 1400.
12 (a) S. Y. Kim and J.-I. Hong, Org. Lett., 2007, 9, 3109;
(b) P. Sokkalingam and C.-H. Lee, J. Org. Chem., 2011, 76,
3820; (c) D. Kim, S. Singha, T. Wang, E. Seo, J. H. Lee, 18 (a) B. Mennucci, Phys. Chem. Chem. Phys., 2013, 15, 6583;
S.-J. Lee, K. H. Kim and K. H. Ahn, Chem. Commun., 2012,
48, 10243; (d) B. Ke, W. Chen, N. Ni, Y. Cheng, C. Dai,
H. Dinh and B. Wang, Chem. Commun., 2013, 49, 2494;
(e) X.-F. Yang, Spectrochim. Acta, Part A, 2007, 67, 321;
(f) S. Y. Kim, J. Park, M. Koh, S. B. Park and J.-I. Hong,
Chem. Commun., 2009, 4735.
(b) D. Jacquemin, B. Mennucci and C. Adamo, Phys. Chem.
Chem. Phys., 2011, 13, 16987; (c) A. Pedone and V. Barone,
Phys. Chem. Chem. Phys., 2010, 12, 2722; (d) D. Jacquemin,
A. Planchat, C. Adamo and B. Mennucci, J. Chem. Theory
Comput., 2012, 8, 2359; (e) R. Improta, G. Scalmani,
M. J. Frisch and V. Barone, J. Chem. Phys., 2007, 127,
074504; (f) A. Pedone, J. Chem. Theory Comput., 2013, 9,
4087.
13 (a) M. Kleerekoper, Endocrinol. Metab. Clin. North Am.,
1998, 27, 441; (b) G. M. Whitford, J. Dent. Res., 1987, 66,
1056.
14 R. Hu, J. Feng, D. Hu, S. Wang, S. Li, Y. Li and G. Yang,
Angew. Chem., Int. Ed., 2010, 49, 4915.
19 (a) J. Bloino, M. Biczysko, F. Santoro and V. Barone,
J. Chem. Theory Comput., 2010, 6, 1256; (b) A. Pedone,
J. Bloino and V. Barone, J. Phys. Chem. C, 2012, 116, 17807;
(c) C. Adamo and D. Jacquemin, Chem. Soc. Rev., 2013, 42,
845; (d) M. Biczysko, J. Bloino, G. Brancato, I. Cacelli,
C. Cappelli, A. Ferretti, A. Lami, S. Monti, A. Pedone,
G. Prampolini, C. Puzzarini, F. Santoro, F. Trani and
G. Villani, Theor. Chem. Acc., 2012, 131, 1201; (e) V. Barone,
J. Bloino, S. Monti, A. Pedone and G. Prampolini, Phys.
Chem. Chem. Phys., 2010, 12, 10550; (f) R. Improta,
V. Barone and F. Santoro, Angew. Chem., Int. Ed., 2007, 46,
405.
15 J. F. Zhang, C. S. Lim, S. Bhuniya, B. R. Cho and J. S. Kim,
Org. Lett., 2011, 13, 1190.
16 (a) M. Chiba, T. Tsuneda and K. Hirao, J. Chem. Phys.,
2007, 126, 034504; (b) P. Wiggins, J. A. G. Williams and
D. J. Tozer, J. Chem. Phys., 2009, 131, 091101;
(c) C. A. Guido, B. Mennucci, D. Jacquemin and C. Adamo,
Phys. Chem. Chem. Phys., 2010, 12, 8016.
17 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, 20 M. Boiocchi, L. Del Boca, D. Esteban-Gomez,
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng,
J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda,
L. Fabbrizzi, M. Licchelli and E. Monzani, Chem.–Eur. J.,
2005, 11, 3097.
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, 21 K. L. Weiss, G. Alshafie, J. S. Chapman, S. M. Mershon,
H. Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta,
F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers,
H. Abou-Issa, M. Clagett-Dame and R. W. Curley, Bioorg.
Med. Chem. Lett., 2001, 11, 1583.
K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, 22 F. Qian, C. Zhang, Y. Zhang, W. He, X. Gao, P. Hu and
K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, Z. Guo, J. Am. Chem. Soc., 2009, 131, 1460.
J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, 23 D. Kand, A. M. Kalle and P. Talukdar, Org. Biomol. Chem.,
J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo,
2013, 11, 1691.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 2143–2149 | 2149