A FRET operated sensor for intracellular pH mapping: Strategically improved efficiency on moving from an anthracene to a naphthalene derivative

Article abstract of DOI:10.1039/c3ra41591k

Two novel fluorescent probes based on anthracene (AAC) and naphthalene (ANC) have been synthesised and characterised. Both the AAC and ANC derivative show green fluorescence at basic pH, but at acidic pH AAC is non-fluorescent, while ANC emits red light. This is attributed primarily to the FRET on-off processes. ANC has better pH discrimination abilities than AAC and can be used to distinguish between different pH environments inside a living cell for the first time. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra41591k

RSC Advances
View Article Online
View Journal | View Issue
PAPER
A FRET operated sensor for intracellular pH mapping:
strategically improved efficiency on moving from an
anthracene to a naphthalene derivative†
Cite this: RSC Advances, 2013, 3,
14397
Arnab Banerjee,a Animesh Sahana,a Sisir Lohar,a Bidisha Sarkar,b
b
a
*
Subhra Kanti Mukhopadhyay and Debasis Das
Two novel fluorescent probes based on anthracene (AAC) and naphthalene (ANC) have been synthesised
and characterised. Both the AAC and ANC derivative show green fluorescence at basic pH, but at acidic pH
AAC is non-fluorescent, while ANC emits red light. This is attributed primarily to the FRET on–off processes.
ANC has better pH discrimination abilities than AAC and can be used to distinguish between different pH
environments inside a living cell for the first time.
Received 3rd April 2013
Accepted 31st May 2013
DOI: 10.1039/c3ra41591k
www.rsc.org/advances
best strategies for making an efficient FRET based pH sensor is
that the wavelength of absorption of the acceptor varies with
1. Introduction
Intracellular pH plays an important role1,2 in different physi-
ological and pathological processes viz. cell growth and
apoptosis,3,4 the proliferation of multidrug resistance (MDR),5
ion transport,6,7 calcium regulation and cell adhesion,8 endo-
cytosis,4 along with homeostasis and muscle contraction.9 An
unusual intracellular pH indicates irregular cell function,
growth and division, and is commonly observed in cancers10
and Alzheimer’s disease.11 The acidic environment in lyso-
somes (pH 4.5–5.5)12,13 facilitates the degradation of proteins in
cellular metabolism. Intracellular pH becomes alkaline
($8.0)14 at the early stage of pollen grain germination. There-
fore, pH responsive uorescent probes with different colours at
acidic and basic pH are in high demand for biological appli-
cations. Most of the reported uorescent pH sensors function
either in the acidic or basic range and have not been used for
intracellular studies.15 Most importantly, they are not sensitive
in the vicinity of neutral pH, and hence, not useful for moni-
toring intercellular pH. Noticeably, most previously reported
pH sensors are polymers or large molecules that requires
tedious and expensive synthetic protocols. Presently, we are
working to develop an inexpensive small molecule/analyte for
use as a selective uorescent sensor.16
pH. The absorption wavelength of 2,6-diformyl-4-methylphenol
changes with pH and its Schiff base contains an extended
conjugation system making the p-cresol unit a good acceptor.
Initially, we have chosen anthracene as a donor unit because
its emission energy is comparable to the absorption energy of
the p-cresol unit. On moving from the anthracene to a naph-
thalene derivative, the pH dependent FRET process can be
tuned signicantly resulting in a pH sensor that can operate at
both acidic and basic pH.
The probe based on AAC contains three units, viz. 2-ami-
nothiophenol, an anthracene compound, and p-cresol,
whereas in the ANC probe, the anthracene is replaced by a
naphthalene compound. Scheme 1 shows an outline of their
synthesis.
2. Experimental
2.1 General procedures
2-Aminothiophenol, 9-chloromethyl anthracene and 2-bromo-
methyl naphthalene were purchased from Sigma-Aldrich (India).
p-Cresol, p-formaldehyde and hexamethylenetetramine were
purchased from Merck (India). Spectroscopic grade solvents were
used. Other analytical reagent grade chemicals were used without
further purication. Mili-Q 18.2 M U cmꢀ1 water was used
throughout all of the experiments. A JASCO (model V-570) UV-Vis
spectrophotometer was used for measuring the absorption
spectra. FTIR spectra were recorded on a JASCO FTIR spectrom-
eter (model: FTIR-H20). Mass spectra were obtained using a
QTOF Micro YA 263 mass spectrometer in ES positive mode. 1H
NMR spectra were recorded using Bruker Advance 300 (300
MHz), 400 (400 MHz) and 500 (500 MHz) instruments in CDCl3.
13C NMR spectra have been recorded using Bruker Advance 400
(100 MHz) and 500 (125 MHz) instruments in CDCl3 or DMSO-d6.
Selective determination of an analyte with a FRET based
probe minimizes the effects of aggregation, photo bleaching or
interference from accompanying biomolecules.17 One of the
aDepartment of Chemistry, The University of Burdwan, Burdwan-713104, West Bengal,
India
bDepartment of Microbiology, The University of Burdwan, Burdwan-713104, West
Bengal, India. E-mail: ddas100in@yahoo.com; Fax: +91-342-2530452; Tel: +91-342-
2533913
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ra41591k
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 14397–14405 | 14397

View Article Online
RSC Advances
Paper
and methanol was used as the solvent. The conductor-like
polarizable continuum model (CPCM) considers the dielectric
effect of the solvent.
2.2 Imaging system
The imaging system was composed of an inverted uorescence
microscope (Leica DM 1000 LED), a digital compact camera
(Leica DFC 420C), and an image processor (Leica Application
Suite v3.3.0). The microscope is equipped with a 50 W mercury
arc lamp and a UV lter.
2.3 Preparation of the cells
Pollen grains were obtained from the freshly collected mature
buds of Tecoma stans, a common ornamental plant with a bright
yellow ower, by crushing the stamens on a sterile Petri plate, then
suspending them in normal saline solution. Debris was removed
by ltration through a thin layer of non-absorbent cotton. The
suspended pollen was collected by centrifugation at 5000 rpm for
5 min. The pollen pellet was then washed twice in normal saline
solution and incubated in a DMSO-buffer solution containing the
probes (50 mM) for 1 h at room temperature. Aer incubation, the
products were washed again with normal saline solution and
observed under a orescence microscope using a UV lter.
2.4 Cell viability studies
At rst, the pollen grains were treated with 10% HgCl2 solution to
sterilize their surfaces. The treated pollen was washed three times
with a pH 7.0 buffer. One set of pollen was incubated in a pollen
germination medium (PGM: 0.01% H3BO3, 0.07% CaCl2$2H2O,
3.0% PEG-4000 and 20% sucrose)3 and acted as a control. Another
set of pollen was incubated in the same medium, but also con-
taining the probes (AAC or ANC, at pH 7.0). This set of pollen was
then incubated for up to three days at 30–35 ꢁC.
The pollen germination was monitored from time to time
under a light microscope. The Tecoma stans pollen treated with
the probes exhibited more than 90% viability, in terms of
germination, compared to the control set.
Scheme 1
Elemental analysis was performed using a Perkin Elmer CHN
analyser with a rst 2000 analysis kit. Steady-state uorescence
experiments were performed using a Hitachi F-4500 spectrou-
orimeter. Time-resolved uorescence life time measurements
were carried out using a picosecond pulsed diode laser-based
time-correlated single photon counting (TCSPC) spectrometer
from IBH (UK) at 375 nm (lex) with a MCP-PMT detector. The
emission from the sample was collected at a right angle to the
direction of the excitation beam maintaining a magic angle
polarization of 54.71. The full width at half maximum (FWHM) of
the instrument response function is 250 ps and the resolution is
28 ps per channel. The data were tted to multi-exponential
functions aer deconvolution of the instrument response func-
tion using an iterative reconvolution technique with IBH DAS 6.2
data analysis soware in which reduced w2 and weighted resid-
uals serve as parameters of best t.
pH measurements were carried out using a Systronics digital
pH meter (model 335, India). All spectra were recorded at room
temperature. The structures of the probes at acidic, basic and
neutral pH were optimized using DFT/TDDFT techniques.
Theoretical calculations were performed at the DFT/B3LYP level
with a 6-31G basis set using the Gaussian 03 soware package.18
The optimized geometry of the probes in three different forms
were performed in the gas phase as this represents the solid
state structure. However, the theoretical UV-Vis spectra were
generated by a TDDFT method using the CPCM formalism,19
2.5 Synthesis of 2-((anthracen-9-yl)methylthio)benzenamine
(AA) (Scheme 1)
276.0 mg (12.0 mmol) of Na (a little more than a stoichiometric
amount) was added pinch-wise to dry ethanol (20 mL, 0 ꢁC)
under a nitrogen atmosphere with constant stirring, until
complete dissolution occurred.
A solution of 2-amino-
thiophenol (1 g, 7.98 mmol in 5 mL dry EtOH) was added to the
above solution, while stirring, over 30 min followed by the
pinch-wise addition of 2.51 g (7.98 mmol) of 9-cholor-
omethylanthracene. Stirring was continued for another 4 h at 70
ꢁC. Aer removing the solvent using a rotary evaporator, the
crude product was subjected to column chromatography
(hexanes : EtOAC ¼ 95 : 5, v/v). Yield, 90%. 1H NMR (300 MHz,
CDCl3) (Fig. S1, ESI†): d (ppm): 4.3 (2H, s); 4.9 (2H, s); 6.6 (1H, m,
J ¼ 7.5 Hz); 6.7(1H, m, J ¼ 6.8 Hz); 7.1 (1H, m, J ¼ 7.9 Hz); 7.2
(1H, s); 7.3 (2H, m, J ¼ 6.18 Hz); 7.5 (3H, m, J ¼ 3.3 Hz); 8.0 (2H,
m, J ¼ 4.4 Hz); 8.2 (2H, m, J ¼ 7.3 Hz); 8.3(1H, m, J ¼ 5.1 Hz). 13
C
14398 | RSC Adv., 2013, 3, 14397–14405
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
RSC Advances
NMR (125 MHz, CDCl3) (Fig. S2 and S3, ESI†): d (ppm): 148.6, reuxed for 4 h. Aer removing the solvent using a rotary
136.8, 133.2, 132.4, 130.1, 128.1, 127.7, 127.6, 127.4, 127.1, evaporator, the crude product was puried by column chro-
126.0, 125.7, 118.5, 114.9, 40.01. QTOF-MS ES+: [M + H+]+ matography (hexanes : EtOAC ¼ 82 : 18, v/v). Yield, 75%. M.
(Fig. S4, ESI†) ¼ 316.4 (100%). Elemental analysis data calcu- P. ¼ 130 ꢁC (ꢂ1 ꢁC). 1H NMR (300 MHz, CDCl3) (Fig. S12, ESI†): d
lated for C17H15NS (%): C, 76.94; H, 5.70 and N 5.28. Found (%): (ppm): 2.3 (3H, s); 4.2 (4H, s); 7.0 (4H, m, J ¼ 7.2 Hz); 7.2 (6H, m,
C, 77.43; H, 5.96 and N, 4.89.
J ¼ 7.5 Hz); 7.4 (8H, m, J ¼ 5.1 Hz); 7.7 (8H, m, J ¼ 7.5 Hz); 13
C
NMR (125 MHz, CDCl3) (Fig. S13 and S14, ESI†) 159.6; 134.6;
133.3; 132.3; 132.5; 131.8; 128.2; 127.7; 127.6; 127.5; 127.3;
127.1; 127.0; 126.1; 125.7; 118.1; 117.9; 40.0; 20.3. QTOF-MS ES+
(Fig. S15 in ESI†): [M + H]+ ¼ 659.23 (100%). Elemental analysis
data calculated for C43H34N2OS2 (%): C, 78.39; H, 5.20 and N,
4.25. Found (%): C, 78.54; H, 4.89 and N, 4.52. The light yellow
solid monomer was also formed in an 8% yield.
2.6 Synthesis of 2,6-bis(-(2-((anthracen-9-yl)methylthio)-
phenylimino)methyl)-4-methylphenol (AAC) (Scheme 1)
2,6-Diformyl-4-methylphenol was synthesized starting from p-
cresol following a published procedure.20 To a solution of 2,6-
diformyl-4-methylphenol (0.328 g, 2 mmol in 20 mL dry meth-
anol), AA (1.26 g, 4 mmol in 20 mL dry methanol) was added
drop-wise while stirring. The reaction mixture was reuxed for 4
h. Aer removing the solvent using a rotary evaporator, the
crude product was puried by column chromatography
2.9 Synthesis of 2-((pyridin-2-yl)methylthio)benzenamine
(AP) (Scheme 1)
(hexanes : EtOAC ¼ 82 : 18, v/v). Yield, 75%. M. P. ¼ 130 ꢁC (ꢂ1 276.0 mg (12.0 mmol) of Na was added pinch-wise to 20 mL of
1
ꢁ
ꢁC). H NMR (300 MHz, CDCl3) (Fig. S5, ESI†): d (ppm): 2.1 (3H, dry EtOH under a nitrogen atmosphere (at 0 C) with constant
s); 4.2 (4H, m, J ¼ 7.8 Hz); 7.4–7.1 (8H, m, J ¼ 8.1 Hz); 7.8 (2H, t, stirring, until complete dissolution occurred. A 5 mL solution of
J ¼ 13.5 Hz); 8.2–8.0 (14H, m, J ¼ 8.7 Hz); 8.5 (2H, d, J ¼ 8.2 Hz); 2-aminothiophenol (1 g, 7.98 mmol in dry EtOH) was then
8.7 (2H, d, J ¼ 9.4 Hz); 9.3 (2H, s); 13.7 (1H, d, J ¼ 8.2 Hz); 13C added, while stirring, over 30 min followed by the addition of
NMR (125 MHz, CDCl3) (Fig. S6 and S7, ESI†): d (ppm): 163.1; 2.02 g (7.98 mmol) of 2-bromomethylpyridine hydrobromide.
143.3; 137.3; 135.8; 134.0; 131.8; 131.5; 131.2; 130.1; 129.2; Stirring was continued for another 2 h. Aer removing the
129.1; 127.9; 127.5; 126.7; 125.9; 125.5; 125.23; 125.0; 124.6; solvent using a rotary evaporator, the crude product was sub-
59.9; 40.1. QTOF-MS ES+: [M + H+]+ (Fig. S8, ESI†) ¼ 759.25 jected to column chromatography (hexanes : EtOAC ¼ 95 : 5, v/
(100%). Elemental analysis data calculated for C43H34N2OS2(%): v). Yield, 80%. 1H NMR (400 MHz, CDCl3) (Fig. S16, ESI†): d
C, 78.39; H, 5.20; N, 4.25. Found (%): C, 78.84; H, 4.79; N, 4.82. (ppm): 3.9 (2H, s); 3.9 (2H, s); 6.5 (1H, t, J ¼ 7.6 Hz); 6.6 (1H, d,
No monomer has been found.
J ¼ 8 Hz); 6.9 (1H, d, J ¼ 7.6 Hz); 7.0 (2H, m, J ¼ 5.2 Hz); 7.1 (1H,
d, J ¼ 8 Hz); 7.4 (1H, m, J ¼ 7.6 Hz); 8.4 (1H, d, J ¼ 4.4 Hz). QTOF-
MS ES+: [M + H]+ (Fig. S17, ESI†) ¼ 217.17 (100%). Elemental
analysis data calculated for C12H12N2S (%): C, 66.63; H, 5.59 and
N, 12.95. Found (%): C, 66.46; H, 5.63 and N, 13.04.
2.7 Synthesis of 2-((naphthalen-6-yl)methylthio)-
benzenamine (AN) (Scheme 1)
276.0 mg (12.0 mmol) of Na was added pinch-wise to 20 mL of dry
EtOH under a nitrogen atmosphere (at 0 C) with constant stir-
ring, until complete dissolution occurred. A 5 mL solution of 2-
aminothiophenol (1 g, 7.98 mmol in dry EtOH) was then added,
ꢁ
2.10 Synthesis of 2,6-bis((Z)-(2-((pyridin-2-yl)methylthio)-
phenylimino)methyl)-4-methylphenol (APC) (Scheme 1)
while stirring, over 30 min followed by the addition of 1.76 g (7.98 To a solution of 2,6-diformyl-4-methylphenol (0.328 g, 2 mmol
mmol) of 2-bromomethylnaphthalene. Stirring was continued for in 30 mL methanol), AP (0.864 g, 2 mmol in 20 mL methanol)
another 2 h. Aer removing the solvent using a rotary evaporator, was added drop-wise while stirring. The reaction mixture was
the crude product was subjected to column chromatography reuxed for 4 h. Aer removing the solvent using a rotary
1
(hexanes : EtOAC ¼ 95 : 5, v/v). Yield, 85%. H NMR (300 MHz, evaporator, the crude product was puried by column chro-
CDCl3) (Fig. S9, ESI†): d (ppm): 4.0 (2H, s); 4.2 (2H, s); 6.5 (1H, t, matography (hexanes : EtOAC ¼ 82 : 18, v/v). Yield, 65%. 1H
J ¼ 7.2 Hz); 6.6 (1H, d, J ¼ 7.5 Hz); 7.1 (1H, t, J ¼ 7.8 Hz); 7.2 (1H, NMR (400 MHz, CDCl3) (Fig. S18, ESI†): d (ppm): 2.3 (3H, s); 3.9
m, J ¼ 0.9 Hz); 7.3 (1H, m, J ¼ 1.5 Hz); 7.4 (3H, m, J ¼ 3.3 Hz); 7.7 (4H, s); 6.5 (3H, m, J ¼ 7.6 Hz); 7.2 (10H, m, J ¼ 6.8 Hz); 7.4 (5H,
(1H, m, J ¼ 3.3 Hz); 7.9 (2H, m, J ¼ 4.5 Hz); 13C NMR (125 MHz, m, J ¼ 7.6 Hz); 8.5 (2H, m, J ¼ 4.4 Hz); 8.8 (2H, s); 10.4 (1H, s);
CDCl3) (Fig. S10, ESI†) 148.7; 136.6; 131.5; 130.3; 130.0; 129.0; 13C NMR (100 MHz, CDCl3) (Fig. S19, ESI†) 163.5; 158.9; 157.2;
127.4; 125.9; 125.0; 124.1; 118.6; 114.8; 32.5. QTOF-MS ES+: [M + 155.5; 149.0; 136.8; 132.5; 129.4; 128.03; 127.4; 127.1; 126.6;
H]+ (Fig. S11, ESI†) ¼ 266.01 (100%). Elemental analysis data 126.1; 123.3; 122.2; 118.1; 114.3; 37.1; 20.0. QTOF-MS ES+
calculated for C17H15NS (%): C, 76.94; H, 5.70 and N, 5.28. Found (Fig. S20, ESI†): [M + H]+ ¼ 561.1 (100%). Elemental analysis
(%): C, 77.23; H, 5.86 and N, 4.99.
data calculated for C33H28N4OS2 (%): C, 70.68; H, 5.03 and N,
9.99. Found (%): C, 70.54; H, 4.99 and N, 10.07. No monomer
was detected.
2.8 Synthesis of 2-(-(2-((naphthalen-2-yl)methylthio)-
phenylimino)methyl)-6-(2-((naphthalen-3-yl)methylthio)-
phenylimino)methyl)-4-methylphenol (ANC) (Scheme 1)
2.11 Synthesis of (Z)-2-((anthracen-9-yl)methylthio)-N-
((pyridin-2-yl)methylene)benzenamine (AAP) (Scheme 2)
To a solution of 2,6-diformyl-4-methylphenol (0.328 g, 2 mmol
in 30 mL methanol), AN (1.06 g, 4 mmol in 20 mL methanol) To a solution of pyridine-2-carboxaldehyde (0.328 g, 2 mmol in
was added drop-wise while stirring. The reaction mixture was 30 mL methanol), AA (0.864 g, 2 mmol in 20 mL methanol) was
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 14397–14405 | 14399

View Article Online
RSC Advances
Paper
395.0 nm, respectively. This new peak at 439.0 nm is attributed
to the phenolate ion, which is also supported by the theoretical
TDDFT results (vide infra).
In the case of ANC, an increase of pH (above pH 7.0) causes a
decrease in the absorbance at 365 nm, with a concomitant
increase at 437 nm through an isobestic point at 398 nm
(Fig. S26, ESI†). In acidic pH, the peak at 365 nm undergoes a 25
nm blue shi to 340 nm and the absorbance of this peak
increases with decreasing pH. Moreover, at acidic pH, a weak
intra-molecular charge transfer (ICT) from the naphthalene
moiety to the AC unit (which is supported TDDFT studies vide
infra) was observed at 525 nm. Interestingly, such a CT band is
absent in the AAC example, probably due to the greater steric
crowding of the anthracene moiety compared to naphthalene.
Thus, AAC is intense green at basic pH, faint yellow at
neutral pH and colorless at acidic pH (inset, Fig. S24, ESI†). On
the other hand, ANC is light green at basic pH, colorless at
neutral pH and red at acidic pH (inset, Fig. S26, ESI†).
Scheme 2
added drop-wise while stirring. The reaction mixture was
reuxed for 4 h. Aer removing the solvent using a rotary
evaporator, the crude product was puried by column chro-
matography (hexanes : EtOAC ¼ 82 : 18, v/v). Yield, 65%. 1H
NMR (500 MHz, CDCl3) (Fig. S21, ESI†): d (ppm): 5.1 (2H, s); 7.1
(3H, m, J ¼ 10.0 Hz); 7.3 (2H, m, J ¼ 2.5 Hz); 7.4 (5H, m, J ¼ 4.5
Hz); 7.9 (2H, m, J ¼ 6.5 Hz); 8.1 (1H, m, J ¼ 7.5 Hz); 8.3 (2H, d,
J ¼ 9.0 Hz); 8.3 (1H, s); 8.4 (1H, s); 8.5 (1H, d, J ¼ 4.0 Hz); 8.6 (1H,
d, J ¼ 4.0 Hz). QTOF-MS ES+ (Fig. S22, ESI†): [M + Na]+ ¼ 427.04
(100%). Elemental analysis data calculated for C27H20N2S(%):
C, 80.17; H, 4.98 and N, 6.92. Found (%): C, 80.34; H, 4.99 and
N, 6.77.
3.2 Emission studies
Fig. S27 in the ESI† shows that the excitation of AAC at 380 nm
(in a DMSO–Britton–Robinson buffer, 1 : 9, v/v, pH 7.0) results
in a very weak emission band at 460 nm (the donor emission of
an AA unit) along with a strong emission band at 537 nm (the
acceptor emission). At pH 5.0, the weak emission at 460 nm is
enhanced, while the acceptor emission at 537 nm is suppressed
(FRET OFF). The reverse phenomenon was observed at basic pH
(pH 11.0, FRET ON). This is well supported by the uorescence
life time data. The emission intensity of AAC at 537 nm (in a
DMSO–Britton–Robinson buffer, 1 : 9, v/v, lex ¼ 450 nm)
increases from neutral (quantum yield ¼ 0.31) to basic pH,
whereas it decreases at acidic pH (pH 5.0, quantum yield ¼
0.14). Over pH 11.0 (quantum yield ¼ 0.75) the emission
intensity remains unaltered (Fig. 1(i)). The absorption peak of
the C unit in AAC undergoes a blue shi at acidic pH, whereas
the peak position remains the same both at neutral and basic
pH. Thus, at basic and neutral pH, the emission spectrum of the
AA unit overlaps with the absorption spectrum of the C unit
resulting in FRET ON (Fig. 2a). However, FRET alone is not
responsible for the uorescence enhancement of AAC at basic
pH, the strong extended delocalization of the lone pair of the
phenoxide ion (Fig. 3a)21 (which is greatly reduced at neutral
pH) also has a signicant contribution. In contrast, at acidic
pH, both of the imine nitrogens get protonated, thus inhibiting
the extended delocalization.
2.12 Synthesis of (Z)-2-(naphthalen-2-ylmethylthio)-N-
(pyridin-2-ylmethylene)aniline (ANP) (Scheme 2)
To a solution of pyridine-2-carboxaldehyde (0.328 g, 2 mmol in
30 mL methanol), AN (0.864 g, 2 mmol in 20 mL methanol) was
added drop-wise with stirring. The reaction mixture was
reuxed for 4 h. Aer removing the solvent using a rotary
evaporator, the crude product was puried by column chro-
matography (hexanes : EtOAC ¼ 82 : 18, v/v). Yield, 65%. M.
P. ¼ 125 ꢁC (ꢂ1 ꢁC). 1H NMR (500 MHz, CDCl3) (Fig. S23, ESI†): d
(ppm): 4.3 (2H, s); 7.0 (1H, m, J ¼ 7.6 Hz); 7.2 (2H, m, J ¼ 7.0 Hz);
7.3(1H, m, J ¼ 4.5 Hz); 7.4 (2H, m, J ¼ 5.0 Hz); 7.5 (1H, m, J ¼ 8.5
Hz); 7.8 (6H, m, J ¼ 6.5 Hz); 8.3 (1H, d, J ¼ 8.0 Hz); 8.5 (1H, s); 8.6
(1H, d, J ¼ 4.5 Hz). QTOF-MS ES+ (Fig. S24, ESI†): [M + Na]+ ¼
377.01 (100%). Elemental analysis data calculated for
C
33H28N4OS2 (%): C, 77.93; H, 5.12 and N, 7.90. Found (%): C,
77.84; H, 4.99 and N, 8.07.
3. Results and discussion
3.1 Absorption studies
Replacement of the anthracene moieties by naphthalene
The effect of pH on the UV-Vis spectra of AAC and ANC (in units (ANC) causes dramatic changes in the uorescence
methanol–water, 7 : 3, v/v) is presented in Fig. S25 and S26 properties (Fig. 1(ii)). The emission intensity increases on
(ESI†), respectively. Methanol is chosen instead of a buffer as moving from neutral to basic pH (lEm ¼ 535 nm, lEx ¼ 365 nm,
the solvent in order to compare the results with the theoretical quantum yield ¼ 0.01 at pH 7.0). Above pH 11.0, the emission
TDDFT studies. At low pH, AAC shows three peaks at 355.0, intensity remains unaltered (quantum yield ¼ 0.39 at pH 11.0).
375.0 and 395.0 nm, which can be attributed to the anthracene Interestingly, the emission intensity also increases with a
unit (Fig. S25, ESI†). With a gradual increase in the pH, a new decrease in pH (from pH 6.5) (lEm ¼ 600 nm, quantum yield ¼
peak at 439.0 nm appears (at pH 7.0), the absorbance of which 0.48 at pH 2.0). Thus, the probe can recognize all three condi-
gradually increases with increasing pH, which is coupled with a tions, viz. acidic, basic and neutral pH. Fig. 2b demonstrates
concomitant decrease in the absorbance at 355.0, 375.0 and that the emission spectrum of the AN unit overlaps with the
14400 | RSC Adv., 2013, 3, 14397–14405
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
RSC Advances
Fig. 1 Fluorescence spectra at different pH values: (i) AAC (10 mM) at pH (from bottom to top) 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0 and 12.0. (ii)
ANC (10 mM). Red lines (from bottom to top) indicate pH 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5 and 2.0, whereas green lines (from bottom to top) indicate pH 7.0, 7.5,
8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5 and 12.0. Inset: a, b and c show the colours at basic, neutral and acidic pH, respectively. (Solvent: DMSO–Britton–Robinson buffer,
1 : 9, v/v, lex ¼ 450 nm).
Fig. 2 Overlap between the donor emission and acceptor absorbance for the FRET process of (a) AAC and (b) ANC. The red and blue lines show the normalized
absorption of C at acidic and basic pH, respectively. The light green line shows the normalized emission spectra of (a) AA and (b) AN.
absorption spectrum of the C unit at acidic pH to a greater
extent than at basic or neutral pH. Hence, a stronger “FRET ON”
is observed at acidic pH. Moreover, protonation of both of the
imine nitrogens causes a loss of planarity and brings the
naphthalene moieties closer to the C unit (this is also supported
by DFT studies). In addition, an accumulation of positive charge
at the nitrogen centers makes intra-molecular charge transfer
(ICT) easier, illustrated by the appearance of a new charge
transfer absorption band at 525 nm and a substantial red shi
of the emission band from 535 nm to 600 nm. Furthermore, an
overlap of the emission spectrum of the AN unit with the
absorption band of the C unit at acidic pH makes the FRET
process feasible and, therefore, an enhancement of the emis-
sion intensity at 600 nm is observed. Thus, the observed
enhancement of emission intensity at 600 nm in the acidic pH
range is due to the ICT-assisted FRET process.
Like AAC, at basic pH, ANC becomes more planar with an
extended delocalization of the lone pair of the phenoxide ion,
Fig. 3 A plausible mechanism for the emission properties of (a) AAC and (b)
which enhances the emission intensity at 535 nm (Fig. 3b).
Although this extended delocalization is absent at acidic pH, a
ANC.
larger emission–absorption overlap and
a shorter inter-
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 14397–14405 | 14401

View Article Online
RSC Advances
Paper
Fig. 4 Fluorescence lifetime decay study of (a) AAC, lEm ¼ 460 nm; (b) AAC, lEm ¼ 537 nm; (c) ANC, lEm ¼ 400 nm; and (d) ANC, lEm ¼ 600 nm at pH 2.0 and lEm ¼ 535
nm at pH 11.0.
chromophore distance22 make the FRET process stronger three model compounds (APC, AAP and ANP) at acidic pH
(Fig. 2b and 3b). The weak emission intensity of ANC at neutral strongly suggests the absence of a FRET processes in these
pH may be attributed to the weak extended delocalization of the monochromophoric compounds.
lone pair of the phenolic OH group and the relatively high inter-
Fluorescence lifetime decay and DFT studies (see ESI†)
chromophore distance compared to that seen at an acidic pH. further support the presence of a FRET process in AAC and ANC.
In order to characterize the FRET mechanism, the donor and The pKa values of AAC and ANC have been calculated from
acceptor have been investigated individually by studying the uorescence experiments and are discussed in detail in the
monochromophoric model compounds; AAP, ANP and APC). By ESI.† The two pKa values for AAC are 6.2 ꢂ 0.1 (for pKa1 at acidic
replacing the acceptor C unit with a pyridine moiety, two new pH, Fig. S31, ESI†) and 8.2 ꢂ 0.1 (for pKa2 at basic pH, Fig. S32,
model compounds, AAP and ANP, were prepared (Scheme 2). ESI†). While for ANC, the values are 5.8 ꢂ 0.1 (for pKa1 at acidic
Meanwhile, replacing the donor (either anthracene or naph- pH, Fig. S33, ESI†) and 8.3 ꢂ 0.1 (for pKa2 at basic pH, Fig. S34,
thalene) unit with the same pyridine moiety, another model ESI†). These pKa values indicate that the color change occurs
compound, APC was also prepared (Scheme 1). AAP and ANP do within the vicinity of neutral pH, which is useful for intra-
not show any signicant uorescence changes with variation of cellular studies. The pKa values of AAC and ANC were calculated
pH (Fig. S28 and S29, ESI†). However, APC shows a uorescence from the uorescence experiments.
enhancement at basic pH due to extended delocalization
(Fig. S30, ESI†). However, the extent of the uorescence
enhancement is much less than that observed for either AAC or
ANC. Negligible changes in the emission intensity for all the
3.3 Fluorescence life-time decay studies
The average donor lifetime in AAC (lEm ¼ 460 nm) is 0.06 ns
(Fig. 4a) at pH 12.0, which increases as the pH changes from
Table 1
Donor lifetime (AA or AN)
Acceptor (C unit) lifetime
AAC
ANC
pH 4.0 (lem ¼ 460 nm)
pH 7.0 (lem ¼ 460 nm)
pH 12.0 (lem ¼ 460 nm)
pH 2.0 (lem ¼ 400 nm)
pH 7.0 (lem ¼ 400 nm)
pH 12.0 (lem ¼ 400 nm)
4.23 ns
2.45 ns
0.06 ns
0.64 ns
6.57 ns
1.23 ns
pH 4.0 (lem ¼ 537 nm)
pH 7.0 (lem ¼ 537 nm)
pH 12.0 (lem ¼ 537 nm)
pH 2.0 (lem ¼ 600 nm)
pH 7.0 (lem ¼ 535 nm)
pH 12.0 (lem ¼ 535 nm)
0.05 ns
0.32 ns
1.35 ns
0.20 ns
0.03 ns
0.07 ns
14402 | RSC Adv., 2013, 3, 14397–14405
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
RSC Advances
Fig. 5 The optimized geometry of AAC in its protonated (a), neutral (b) and
deprotonated (c) forms along with the corresponding donor–acceptor distances.
neutral (pH 7.0) to acidic (pH 4.0), having values of 2.45 ns and
4.23 ns, respectively. In contrast, average acceptor lifetime in
AAC (lEm ¼ 537 nm) at pH 12.0 is 1.35 ns, signicantly longer
than its value (0.05 ns) at pH 4.0 (Fig. 4b). Likewise, the average
donor lifetime in ANC (lEm ¼ 400 nm) at neutral pH is 6.57 ns,
which is reduced (by almost 5 times) at pH ¼ 12.0 to 1.23 ns and
is further reduced (by almost 10 times) at pH 2.0 to 0.64 ns
(Fig. 4c). The average acceptor lifetime in ANC at acidic pH
(lEm ¼ 600 nm) is 0.20 ns, whereas it is 0.07 ns in basic pH
(lEm ¼ 535 nm, Fig. 4d) (vide supra). All of the results are pre-
sented in Table 1.
3.4 Computational studies
Fig. 7 Charge density distribution of different HOMOs and LUMOs, along with
the respective energy gaps for AAC at acidic, neutral and basic pH.
Fig. 5 clearly shows that the distance between anthracene and C
unit in AAC is less at acidic pH (a) compared to its other forms
(b and c). A similar observation is found in ANC (Fig. 6). The
theoretical UV-Vis spectrum of the protonated AAC shows
predominant transitions (due to the AA unit) at 394, 414 and
435 nm, which are assigned to HOMO ꢀ 5 / LUMO + 1, HOMO
ꢀ 1 / LUMO + 3 and HOMO ꢀ 6 / LUMO, respectively. On the
other hand, the deprotonated form of AAC shows a dominant
transition (due to the C unit) at 504 nm, which is attributed to
HOMO / LUMO + 2 (Fig. 7).
electron density of the LUMO is greater on the AC unit, indi-
cating an extended delocalization.
3.5 Cell imaging studies
Pollen grains are single cells with different pH environments. In
the presence of AAC, the pollen grain turns green indicating a
Moreover, two theoretical peaks (from the TDDFT studies)
of protonated ANC at 532 nm (HOMO ꢀ 1 / LUMO + 1) and
527 nm (HOMO / LUMO + 1) are in close agreement with the
experimental intra-molecular CT band at 525 nm (Fig. S8,
ESI†). As indicated in Fig. 8, the electron densities of the
HOMO and HOMO ꢀ 1 reside on the naphthalene unit,
whereas the electron density of LUMO + 1 resides on the AC
unit. The deprotonated form of ANC shows two theoretical
peaks at 365 nm and 437 nm, which are close to the experi-
mental peaks at 355 nm and 406 nm assigned to HOMO ꢀ 4
/ LUMO and HOMO / LUMO transitions, respectively. In
both cases, the charge densities of the HOMO ꢀ 4 and HOMO
are localized predominantly on the C unit, whereas the
Fig. 6 The optimized geometry of ANC in its protonated (a), neutral (b) and
deprotonated (c) forms along with the corresponding donor–acceptor distances.
Fig. 8 Charge density distribution in different HOMOs and LUMOs along with
the respective energy gaps for ANC at (a) acidic, (b) neutral and (c) basic pH.
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 14397–14405 | 14403

View Article Online
RSC Advances
Paper
8 (a) H. Zhao, X. Xu, J. Diaz and S. Muallem, J. Biol. Chem.,
1995, 270, 19599; (b) I. Yuliand and A. Oplatka, Science,
1987, 235, 340; (c) A. J. Janecki, M. H. Montrose,
P. Zimniak, A. Zweibaum, C. M. Tse, S. Khurana and
M. Donowitz, J. Biol. Chem., 1998, 273, 8790.
Fig. 9 Cell imaging studies showing (a) blank cell, (b) cell treated with AAC, (c)
enlarged view of the cell in (b), (d) cell treated with ANC and (e) enlarged view of
the cell in (d). The white scale bar is 10 mM.
9 A. J. Bullock, R. A. Duquette, N. Buttell and S. Wray, Puegers
Arch., 1998, 435, 575.
10 H. Izumi, T. Torigoe, H. Ishiguchi, H. Uramoto, Y. Yoshida,
M. Tanabe, T. Ise, T. Murakami, T. Yoshida, M. Nomoto and
K. Kohno, Cancer Treat. Rev., 2003, 29, 541.
11 T. A. Davies, R. E. Fine, R. J. Johnson, C. A. Levesque,
W. H. Rathbun, K. F. Seetoo, S. J. Smith, G. Strohmeier
and L. Volicer, et al. , Biochem. Biophys. Res. Commun.,
1993, 194, 537.
basic pH (Fig. 9b and 9c). Interestingly, ANC can be used to
illuminate different parts of the cell showing red and green
colors, which indicates that there are different pH environ-
ments inside the living cell (Fig. 9d and 9e). Cell viability studies
(Fig. S35, ESI†) indicate that 90% of the cells remain alive in the
presence of the probe even aer three days.
12 M. Schindler, S. Grabski, E. Hoff and S. Simon, Biochemistry,
1996, 35, 2811.
13 Y. Mathieu, J. Guern, A. Kurkdjian, P. Manigault,
J. Manigault, T. Zielinska, B. Gillet, J. C. Beloeil and
J. Y. Lallemand, Plant Physiol., 1988, 89, 19.
14 S. Ohkuma and B. Poole, Proc. Natl. Acad. Sci. U. S. A., 1978,
75, 3327.
4. Conclusion
The facile synthesis of two novel uorescent probes, AAC and
ANC, has been reported. AAC emits green light at basic pH, but
is colorless at acidic pH. On the other hand, ANC emits red light
at acidic pH, green light at basic pH, but is colorless at neutral
pH. The color changes of the sensors can be attributed to the
combined effect of extended delocalization and a FRET process.
The different behaviour of AAC and ANC at acidic pH has been
rationalized on the basis of an intra-molecular charge transfer
and enhanced FRET efficiency. ANC can be used to distinguish
different pH environments allowing the monitoring of pH
within a living cell for the rst time.
15 A. Abiko and S. Masamune, Tetrahedron Lett., 1996, 37, 1081.
16 A. E. Ashford and R. B. Knox, Cell Sri., 1980, 44, 1.
17 N. P. Matveeva, D. S. Andreyuk, O. O. Voitsekh and
I. P. Ermakov, Russ. J. Plant Physiol., 2003, 50, 318.
18 (a) J. Han and K. Burgess, Chem. Rev., 2010, 110, 2709; (b)
R. Wang, C. Yu, F. Yu and L. Chen, TrAC, Trends Anal.
Chem., 2010, 29, 1004; (c) J. Han, A. Loudet, R. Barhoumi,
R. C. Burghardt and K. Burgess, J. Am. Chem. Soc., 2009,
131, 1642; (d) S. Chen, J. Liu, Y. Liu, H. Su, Y. Hong,
C. K. W. Jim, R. T. K. Kwok, N. Zhao, W. Qin, J. W. Y. Lam,
K. S. Wong and B. Z. Tang, Chem. Sci., 2012, 3, 1804.
19 (a) A. Banerjee, A. Sahana, S. Guha, S. Lohar, I. Hauli,
S. K. Mukhopadhyay, J. S. Matalobos and D. Das, Inorg.
Chem., 2012, 51, 5699; (b) A. Banerjee, A. Sahana, S. Das,
S. Lohar, S. Guha, B. Sarkar, S. K. Mukhopadhyay,
A. K. Mukherjee and D. Das, Analyst, 2012, 137, 2166; (c)
A. Banerjee, D. Karak, A. Sahana, S. Guha, S. Lohar and
D. Das, J. Hazard. Mater., 2011, 186, 738; (d) A. Sahana,
A. Banerjee, S. Das, S. Lohar, D. Karak, B. Sarkar,
S. K. Mukhopadhyay, A. K. Mukherjee and D. Das, Org.
Biomol. Chem., 2011, 9, 5523; (e) A. Sahana, A. Banerjee,
S. Guha, S. Lohar, A. Chattopadhyay, S. K. Mukhopadhyay
and D. Das, Analyst, 2012, 137, 1544; (f) S. Das, S. Guha,
A. Banerjee, S. Lohar, A. Sahana and D. Das, Org. Biomol.
Chem., 2011, 9, 7097; (g) S. Guha, S. Lohar, A. Banerjee,
A. Sahana, A. Chaterjee, S. K. Mukherjee, J. S. Matalobos
and D. Das, Talanta, 2012, 91, 18; (h) A. Sahana, A. Banerjee,
S. Lohar, S. Guha, S. Das, S. K. Mukhopadhyay and D. Das,
Analyst, 2012, 137, 3910; (i) A. Banerjee, A. Sahana, S. Lohar,
I. Hauli, S. K. Mukhopadhyay, D. A. San,
M. G. Babashkina, M. Bolte, Y. Garcia and D. Das, Chem.
Commun., 2013, 49, 2527–2529; (j) S. Lohar, A. Sahana,
A. Banerjee, A. Banik, S. K. Mukhopadhyay, J. S. Matalobos
and D. Das, Anal. Chem., 2013, 85, 1778; (k) A. Sahana,
A. Banerjee, S. Lohar, B. Sarkar, S. K. Mukhopadhyay and
D. Das, Inorg. Chem., 2013, 52, 3627.
Acknowledgements
Arnab Banerjee, Animesh Sahana and Sisir Lohar are grateful to
CSIR, New Delhi for a fellowship. Sincere thanks to the Indian
Institute of Chemical Biology (IICB), Kolkata for extending NMR
and mass spectrometer facilities. Special thanks to the Univer-
sity Science Instrumentation Center (USIC), Burdwan Univer-
sity, for providing the uorescence microscope facility. We are
grateful to Prof. Asok Kumar Mukherjee for his helpful discus-
sion and inspiration. The authors sincerely thank Prof. Samita
Basu and Mr Ajay Das, Chemical Science Division, SINP, Kol-
kata for extending TCSPC instrument facility.
Notes and references
1 A. Roos and W. F. Boron, Physiol. Rev., 1981, 61, 296.
2 A. Kotyk and J. Slavik, Intracellular pH and its Measurement,
CRC Press, Boca Raton, FL, 1989
3 D. Perez-Sala, D. Collado-Escobar and F. Mollinedo, J. Biol.
Chem., 1995, 270, 6235.
4 R. A. Gottlieb, J. Nordberg, E. Skowronski and B. M. Babior,
Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 654.
5 S. Simon, D. Roy and M. Schindler, Proc. Natl. Acad. Sci. U. S.
A., 1994, 91, 1128.
6 A. Varadiand and G. A. Rutter, Endocrinology, 2004, 145, 4540.
7 E. Liang, P. Liu and S. Dinh, Int. J. Pharm., 2007, 338, 104.
14404 | RSC Adv., 2013, 3, 14397–14405
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
RSC Advances
20 (a) M. Royzen, Z. Dai and J. W. Canary, J. Am. Chem. Soc., 22 (a) J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd
2005, 127, 1612; (b) A. Ajayaghosh, P. Carol and S. Sreejith,
J. Am. Chem. Soc., 2005, 127, 14962; (c) K. Kiyose,
edn, 1999, Ch. 13; (b) A. Albini, E. Fasani and D. Faiardi, J.
Org. Chem., 1987, 52, 155.
H. Kojima, Y. Urano and T. Nagano, J. Am. Chem. Soc., 23 (a) S. Miertus, E. Scrocco and J. Tomasi, Chem. Phys., 1981,
2006, 128, 6548; (d) S. Banthia and A. Samanta, J. Phys.
Chem. B, 2006, 110, 6437; (e) J. R. Lakowicz, Principles of
Fluorescence Spectroscopy, 3rd edn, Springer, New York, 2008.
21 (a) M. Mukhopadhyay, D. Banerjee and S. Mukherjee, J. Phys.
Chem. A, 2006, 110, 12743; (b) S. Mitra and S. Mukherjee, J.
Lumin., 2006, 118, 1.
55, 117; (b) M. Cossi, V. Barone, R. Cammi and J. Tomasi,
Chem. Phys. Lett., 1996, 255, 327; (c) M. Cossi, V. Barone
and M. A. J. Robb, Chem. Phys., 1999, 111, 5295; (d)
M. Cossi, G. Scalmani, N. Rega and V. J. Barone, Chem.
Phys., 2002, 117, 43.
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 14397–14405 | 14405