Dual mode ratiometric recognition of zinc acetate: Nanomolar detection with in vitro tracking of endophytic bacteria in rice root tissue

Article abstract of DOI:10.1039/c5dt03431k

Several naphthalene-based aldazine derivatives were developed as efficient colorimetric and fluorescence probes for selective ratiometric recognition of traces of zinc acetate. The derivative structures were characterized by single-crystal X-ray diffraction. The probes were used for in vitro tracking of zinc acetate in endophytic bacteria within rice root tissue and to image zinc acetate in human breast cancer cells (MCF7) by normal and fluorescence microscopy. Density functional theoretical studies were in close agreement with the experimental findings.

Full text of DOI:10.1039/c5dt03431k

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S. Ta, M. Ghosh, S. Karmakar, A. Banik, T. K. Dangar and S. K. Mukhopadhyay, Dalton Trans., 2015, DOI:
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DOI: 10.1039/C5DT03431K
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ARTICLE TYPE
Dual mode ratiometric recognition of zinc acetate: nano-molar detection
with in-vitro tracking of endophytic bacteria in rice root tissue
a
a
a
b
c
Abhijit Ghosh , Sabyasachi Ta , Milan Ghosh , Subhajit Karmakar , Avishek Banik , Tushar Kanti
c
d
a
Dangar , Subhra Kanti Mukherjee and Debasis Das*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Single crystal X-ray structurally characterized several naphthalene based aldazine derivatives have been established
as efficient colorimetric and fluorescence probes for selective ratiometric recognition of traces zinc acetate. Moreover,
the probes are capable for in-vitro tracking of zinc acetate in endophytic bacteria within the rice root tissue. The
probes are also useful to image zinc acetate in human breast cancer cells (MCF7) with normal as well as fluorescence
microscopes. Density functional theoretical studies are in close agreement to the experimental findings.
1
0
1
Zinc deficiency is treated by administration of different
supplements like zinc gluconate (ZG), zinc gluconateꢀglycine
(
host. Detection of endophytic colonization and intracellular
tracking of bacteria, transformation of green fluorescent protein
(GFP) encoding plasmid is most popular for visualization.
ZGG), zinc gluconate citrate (ZGꢀC) and zinc acetate (ZA) etc.
Activity of zinc supplement depends on iZn (sum of all positive
1
6, 17
1
2
2
3
3
4
4
5
5
0
5
0
5
0
5
0
However, the method is very time consuming and expensive.
charged zinc species in solution) and the highest iZn value of ZA
55
2
(100%) makes it most promising.
Our new probes successfully and efficiently track in vitro
endophytic bacteria within six hours of infection in rice root
tissue.
Wilson's disease is treated with ZA that blocks the intestinal
3
copper absorption from diet. ZA lozenges are used to deal with
4
common cold, cough, nasal discharge, and muscle ache. It has
The probes are also useful to image ZA in human breastꢀcancer
mild antibacterial action and specific inhibitory effect against 60 cells (MCF7) under fluorescence microscope.
respiratory syncytial virus (RSV), the major cause of lower Three probes, viz. A4, A5 and A6 are prepared by
5
, 6
respiratory tract disease in children.
injection has positive impact to control prostate cancer growth.
Thus, sensitive and selective visual determination of traces ZA
Direct intraꢀtumoral ZA
condensation of hydrazone derivative of 2ꢀhydroxyꢀ1ꢀ
naphthaldehyde with different pꢀ substituted benzaldehyde in
methanol (Scheme 1). A model compound (A4a) has also been
7, 8
in human tumor and cancer cell lines after therapeutic treatment 65 prepared using 1ꢀnaphthaldehyde instead of 2ꢀhydroxyꢀ1ꢀ
is very essential in medicinal chemistry. In literature, there are
naphthaldehyde (Scheme S1). The1 compounds have been
9
,
10
13
two available ZA selective probes,
however none of them
characterized by ESIꢀTOF mass, HNMR, CNMR, FTIR
recognize ZA both colorimetric and ratiometric fluorescence
method.
Addressing the limitations, herein, we report several probes for 70 analysis (Fig. 1). Crystal packing and Hꢀbonding network of A4
selective determination of traces ZA through ratiometric
absorbance and fluorescence changes with improved detection
limits and binding affinities at physiological conditions.
spectra (Fig. S1–S19, ESI). Additionally, the structures of A4
and A6 have been confirmed by single crystal Xꢀray structure
and A6 are presented in Fig. S20–S21 (ESI). Crystal parameters,
refinement data, significant bond lengths and bond angles are
summarized in Tables S1ꢀS5 (ESI). Elemental analysis of the
compounds are provided as evidence of their purities (ESI).
On the other hand, plant–microbe interactions that promote plant
development and health has considerable research interest. Plants
constitute vast and diverse niches for endophytic organisms.
Among the microorganisms, endophytic bacteria occupy internal
tissues of apparently healthy plant without substantive harm or
benefit other than shelter, however provides beneficial effect.
Endophytic bacteria have agronomic interest as they may
enhance plant growth in nonleguminous crops and improve their
nutrition through nitrogen fixation, phosphate solubilisation or
iron chelation. The interaction between plant and endophytic
bacteria may have profound effect on plant health, growth,
development, yield as well as soil quality as it reduces the
75
Scheme 1
Results and discussion
As the probes contain pH sensitive hydroxyl donor site,
naturally their performance should be pH sensitive. Hence, the
consumption of chemicals, particularly for sustainable agriculture 80 emission characteristics of A4 in presence and absence of ZA are
1
1ꢀ15
and environmental protection.
So, in vitro intraꢀcellular
examined at different pH. Fig. S22 (ESI) indicates that A4
tracking of endophytic bacteria is one of the pioneering research
interests of biologist to study their niche and interaction with the
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Fig. 1 Single crystal Xꢀray structures for A4 and A6
detects ZA over a wide range of pH from 6.0 to 12.0. Naturally,
all experiments have been performed at pH 7.4, being closer to
physiological pH. All spectral studies have been performed in 0.1
M HEPES buffered aqueous DMSO media (DMSO/ water, 9/1,
v/v).
5
45
Fig. 3 Proposed Zn(OAc)
2
sensing mechanism
2
+
Fluorescence of the probes enhances when both Zn
−
2+
and OAc ions are present together. Zn salts of other counter
−
anions or OAc salts of other metal ions do not show any
50
fluorescence enhancement of A4 (Fig. 5 and 6). Common cations
(Fig. S31, ESI) and anions (Fig. S32, ESI) do not interfere. The
effect of other salts, including the acetate salts of different metal
ions and zinc salts of different anions) on the absorption spectra
of A4, A5 and A6 have also been studied. The results are
2
+
55
included in ESI and indicate that the presence of both Zn and
−
OAc ions is must to observe the desired absorbance changes
1
0
5
Fig. 2 Changes in the absorption spectra of A4 ((10 ꢁM, DMSO–water (9:
(Fig. S33, ESI).
1
, v/v, 0.1 M HEPES buffer, pH 7.4) upon gradual addition of ZA (0ꢀ400
ꢁ
M). Inset: naked eye colors of A4 before and after addition of ZA.
The binding and recognition events of the probes are studied
using different spectroscopic methods. UVꢀVis spectra of the
solution of A4 (Fig. 2) reveals two absorption peaks at 335 nm
1
(A5: 332 nm, A6: 333 nm; Fig. S23A) (π– π*) and 385 nm (A6:
3
81 nm, A6: 405 nm; Fig. S23B) (π– π*). Upon successive
addition of ZA, the bands at 335 nm and especially at 385 nm
gradually weakens with the appearance of a new red shifted band
at 446 nm (A5: 444 nm, A6: 465 nm) and an isobestic point at
20
25
30
35
40
4
10 nm (A5: 401 nm, A6: 433 nm), indicating the formation of a
new species (Fig. 3). The deprotonation of the naphthol group
decreases the probability of π–π* transition energy decreases
the probability of π– π* transition energy through improved
60
Fig. 4 Changes in the emission spectra of A4 (10 ꢁM, same media as
supra, pH 7.4, λex, 370 nm, λem, 487 nm) upon gradual addition of ZA (0ꢀ
400 ꢁM). Inset: UV light exposed colors of A4 in absence and presence of
ZA
18
delocalization to account for the new red shifted band ~446 nm
with the appearance of yellowꢀ green (A5: yellow, A6: orange)
coloration. The changes in the absorbance of the probes as a
function of added ZA are presented in Fig. S24ꢀS26 (ESI). The
corresponding linear regions are useful for colorimetric
determination of unknown ZA.
Fluorescence titrations of A4 (Fig. 4) and A5 (Fig. S27A, ESI)
with varying concentration of ZA show that very weak emissions
at 487 nm (for A4, λ , 370 nm) and 495 nm (for A5, λ , 430 nm)
ex
ex
gradually increase with increasing ZA concentration. While for
A6, the emission at 440 nm (λ , 337 nm) gradually decreases
ex
(Fig. S27B, ESI) with the appearance of a new band at 520 nm.
Emission intensities for A4, A5 and A6 increases with increasing
ZA concentration (Fig. S28A, S29A and S30A). The linear region
of those plots (Fig. S28B, S29B and S30B, ESI) are useful for
determination of unknown ZA by fluorescence methods using
A4, A5 or A6 respectively.
65
Fig. 5 Emission intensities of A4 (10 ꢁM, DMSO–water (9: 1, v/v, 0.1 M
HEPES buffer, pH 7.4, λex, 370 nm; λem, 487 nm) in presence of different
2+
combinations of Zn (400 ꢁM) and/or OAc− (400 ꢁM)
2
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downfield as one moves from free A4 (13.25 ppm) to A5 (13.40
ppm) to A6 (13.51 ppm). This is due to the stronger Hꢀbonding
provided by the more electron withdrawing group at the para
position of phenyl ring. However, upon gradual addition of ZA
to A4, the naphthol –OH weakens and eventually disappears at
55
1
.0 equiv. ZA affirming coordination of the naphthol oxygen to
ZA. Additionally, aldimine (–CH=N–) proton close to the
naphthyl ring experiences an upꢀfield shift from 9.72 ppm to 9.50
ppm. The –OH proton at the para position of the phenyl ring does
not shift or disappear even after addition of 5.0 equiv. of ZA
confirming its nonꢀinvolvement to coordinate ZA. However,
imine proton near to the phenyl ring and aromatic protons do not
show any significant change, only a small upꢀfield shift is
60
Fig. 6 Effect of different metal ions (400 ꢁM) on the emission intensities
of A4ꢀ OAc− (10 ꢁM & 400 ꢁM (DMSO– water, 9: 1, v/v, 0.1 M HEPES
buffer, pH 7.4, λex, 370 nm; λem, 487 nm) system
5
65 attributed to “through bond delocalization” or “diamagnetic
shielding” of the negative charge generated through
deprotonation of naphthol –OH. No further change in the
chemical shift values have been observed upon addition of ZA
over 1.0 equiv. indicating a 1 : 1 stoichiometry between A4 and
Very weak emission of the probes may be attributed to the
excited state intraꢀmolecular proton transfer (ESIPT) involving
the naphthol proton and the imine nitrogen. Upon interaction
1
9
1
1
2
2
3
3
4
4
5
0
5
0
5
0
5
0
5
0
with ZA, the chelationꢀenhanced fluorescence (CHEF) starts and 70 ZA which is further substantiated from Job's plot (Fig. S35, ESI)
ESIPT stops (Fig. 3). It is found that increasing water content of
the aqueousꢀ DMSO media decreases the emission intensity of
ZA adducts of A4, A5 and A6. Thus, the difference of emission
intensities of the adducts with free probes decreases significantly,
and mass spectra of their adducts with ZA (A4, Fig. S36; A5, Fig.
S37, and A6, Fig. S38, ESI) The FTIR spectra of the ZA
.
complexes of the receptors are shown in Fig. S39ꢀS41, (ESI†).
Inability of A4a to recognize ZA (Fig. S42, ESI) further
hindering the detection of ZA. Hence, we have used 10% 75 establishes the requirement and involvement of naphthol oxygen
aqueousꢀDMSO as optimum medium for spectral studies. This is
attributed to the preferential coordination of water with Zn over
acetate at high water content. This is illustrated in Fig. S34 (ESI).
donor for complex formation.
2
+
Introduction of electron withdrawing group at the para
position of phenyl ring assists delocalization of the negative
charge due to deprotonation of the naphthol proton.
Consequently, the extent of π– π* transition energy decreases as
reflected from the red shift in the emission as well as absorption
maxima in A6 than that of A4 and A5. This fact has also been
observed from the decrease of energy of both LUMOs (A4, –
1
.7064 eV; A5, – 2.0634 eV and A6, – 3.0316 eV) and
consequently the energy gap between frontier MOs of the probes
A4, 4.0466 eV; A5, 3.6180 eV and A6, 2.8534 eV) and their ZA
(
Fig. 7 Frontier molecular orbitals of A4 and [A4 + ZA] system
complexes (A4, 3.3067 eV; A5, 3.3021 eV and A6, 2.3587 eV)
80
upon changing ‘R’ from ‘OH’ to ‘NO ’ through ‘H’ from A4 to
2
A6 through A5 (Fig. 7ꢀ9). The decrease in energy of LUMO for
A6 is so pronounced to give a very small gap between the frontier
MOs resulting an intense emission at 440 nm for free A6. Thus,
explaining the ratiometric emission changes of A6 in presence of
Zn(OAc) . With the optimized structures of the complexes,
2
electronic transition energies were calculated by the TDDFT
method in the gas phase and in DMSO. For DMSO, the CPCM
formalism was imposed which considers the solvent as a
polarisable continuum and does not include discrete solvent
2
0
molecules for solvation of the solute. Calculated transition
energies for the prominent absorption bands are shown in Table
S6ꢀS8 (ESI†). The calculated absorption peaks agree well with
the experimentally observed peaks. Optimised structures for
Fig. 8 Frontier molecular orbitals of A5 and [A5 + ZA] system
The binding constants of A4, A5 and A6 for ZA, evaluated from
5
–
5
HOMOꢀ1, HOMOꢀ2 and HOMOꢀ3 of A6 are presented in Fig. 10 85 the respective fluorescence titrations are 3.9 × 10 M , 4.5 × 10
1
–1
5
–1
For deeper insight on the nature of host–guest interaction, H
M and 8.3 × 10 M (Fig. S43, ESI) respectively, indicating a
NMR titration of A4 with ZA has been performed in DMSOꢀd6
strong hostꢀguest interaction. The increasing binding constants
values from A4 to A6 are also in line with the aforesaid logic of
extra stability gained by higher electron delocalization. The
(
(
Fig. 11). Interestingly, appearance of a very downfield proton
for naphthol –OH, δ 13.25 ppm) in A4 is attributed to the
–
9
existence of intraꢀmolecular Hꢀbonding between imine nitrogen 90 lowest detection limits of A4, A5 and A6 for ZA are 3.0 × 10
–9
–9
and naphthol –OH. The naphthol –OH is found to move
M, 8.1 × 10 M and 4.9 × 10 M (Fig. S44, ESI) respectively.
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DOI: 10.1039/C5DT03431K
3
4
4
5
5
5
0
5
0
5
been used without further purification except when specified.
MiliꢀQ Milipore 18.2 Mꢂ cm–1 water has been used throughout
all the experiments.
A
Shimadzu Multi Spec 2450
spectrophotometer is used for recording UVꢀVis spectra. FTIR
spectra are recorded on a Shimadzu FTIR (model IR Prestige 21
CE) spectrophotometer. Mass spectra have been recorded using a
QTOF 60 Micro YA 263 mass spectrometer in ES positive mode.
1
H NMR titration of A4 with ZA in DMSOꢀd6 has been
1
3
performed using a Bruker Avance 500 (500 MHz). C NMR
spectrum of A4 and H NMR spectra of A5 and A6 are recorded
using a Bruker Avance 400 (400 MHz). While C NMR
spectrum of A5 and A6 and H NMR spectra of X1 and X2 are
1
1
3
1
recorded using a Bruker Avance 300 (300 MHz). Elemental
analyses are performed on a Perkin Elmer 2400 CHN analyzer.
The steady state emission and excitation spectra are recorded
with a Hitachi Fꢀ4500 spectrofluorimeter. Systronics digital pH
meter (model 335) is used for measurement of solution pH.
Dilute HCl or NaOH (50 ꢁM) are used for pH adjustment. The
imaging system is composed of an inverted fluorescence
microscope (Dewinter, Italy). Images are captured through an
attached CCD camera equipped with BioWizard 4.2 software.
The microscope is equipped with a 50 W mercury arc lamp. All
spectra are recorded at room temperature.
Fig. 9 Frontier molecular orbitals of A6 and [A6 + ZA] system
Fig. 10 HOMOꢀ1, HOMOꢀ2 and HOMOꢀ3 of A6
5
1
Fig. 11 H NMR titration of A4 with ZA in DMSOꢀd6
6
6
0
5
Fig. 12 Imaging of ZA in A. A. chroococcum cells infected within rice
seedlings under UVꢀ transluminator (UV A): (A) root, treated with
suspension of A. chroococcum in sterile distilled water, (B) root, treated
with ZA (100 ꢁM) incubated A. chroococcum, (C) A4 (10 ꢁM) treated
root, incubated only with A. chroococcum, (D) root incubated with ZA
(100 ꢁM) and A. chroococcum followed by treatment with A4 (10 ꢁM).
10
15
20
Rice seedling imaging
After 6h incubation, ZA (100 ꢁM) treated A. chroococcum
cells have been infected within rice seedlings followed by A4 (10
ꢁM) treatment. The lateral roots, especially at the zone of
elongation and differentiation appear green when placed on the
UVꢀ transluminator (UV A) due to colonization of the bacterium
(Fig. 12D). The phase contrast microscopy at 100 magnification
X
(UVꢀfilter) of transverse section of those selected green emitting
roots confirms the intra cellular localization of A. chroococcum
inside root of Oryza sativa var. Swarna (Fig. 13C and D). No
green emission has been observed from A. chroococcum used in
control (Fig. 12A, B, C and Fig. 13A, B).
Human breast cancer (MCF7) cell imaging
Fig. 14 reveals that A4 is cell membrane permeable and detects
intracellular ZA in human breast cancer cells (MCF7) under
fluorescence microscope without any harm as cells remain alive
even after several hours of exposure to 10 µM A4.
Experimental
25
Materials and methods
30
Highꢀpurity HEPES, hydrazine hydrate, 2ꢀhydroxyꢀ1ꢀ
Fig. 13 Fluorescence microscope images of root (A) at 100x
magnification, infected with ZA (100 ꢁM) treated A. chroococcum; (B) at
naphthaldehyde,
1ꢀnaphthaldehyde,
benzaldehyde,
4ꢀ
nitrobenzaldehyde and 4ꢀhydroxybenzaldehyde are purchased 70 40x magnification, infected with A4 (10 ꢁM) treated A. chroococcum; (C)
at 40x magnification, infected with and (D) at 100x magnification,
infected with both ZA (100 ꢁM) and A4 (10 ꢁM) treated A. chroococcum
from Sigma Aldrich (India). Solvents used are of spectroscopic
grade. Other chemicals are of analytical reagent grade and have
4
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cells. Red arrow indicates localization of A. chroococcum cyst inside root
of Oryza sativa var. Swarna.
Synthesis
of
1-[(4-hydroxy-benzylidene)-
hydrazonomethyl]-naphthalen-2-ol (A4, Scheme 1):
A solution containing X1 (186 mg, ~1 mmol) and 4ꢀ
hydroxybenzaldehyde (122 mg, ~1 mmol) in methanol is refluxed
for 6h to get wine color solution which is kept in a test tube for
slow evaporation. After several days Xꢀray quality needle shaped
brown crystals appeared. Calculated yield: 70%. Anal. calcd (%):
5
6
6
7
5
0
5
0
C, 74.47; H, 4.86 and N, 9.65; found: C, 74.43; H, 4.89 and N,
5
Fig. 14 Fluorescence microscope images of MCF7 cells: (A) and (B) are
images of cells after 2 h incubation with ZA (100 ꢁM) and A4 (10 ꢁM)
respectively, (C) cells after 2 h incubation with 100 ꢁM ZA followed by
addition of 10 ꢁM A4 and (D) bright field image of the cells after
incubation with 10 µM A4 for 2h followed by addition of 100 µM ZA.
1
9.51. H NMR [400 MHz, CDCl
, TMS, J (Hz), δ (ppm)]: 13.25
3
(
(
(
1H, s), 9.72 (1H, s), 8.76 (1H, s), 6.86 (2H, d, J = 8.25), 7.24
1H, d, J = 7.8), 7.47 (2H, d, J = 8.20), 7.64 (1H, t, J = 8.0), 7.72
1H, d, J = 7.80), 7.80 (1H, m, J = 8.0), 7.97 (1H, t, J = 7.8), 8.00
1
0
1
3
(1H, d, J = 8.0). C NMR [400 MHz, CDCl , TMS, δ (ppm)]:
3
General method of UV-Vis and fluorescence titration:
1
1
61.12, 149.31, 147.21, 134.24, 132.84, 129.22, 128.25, 127.84,
26.35, 124.95, 123.68, 120.18, 119.29, 114.57, 108.77. ESIꢀ
P
ath length of the cells used for absorption and emission
studies is 1 cm. Stock solutions of A4, A5, A6 and ZA are
prepared in DMSO/ water (9/1, v/v). Working solutions of A4,
A5, A6 and ZA are prepared from their respective stock
solutions. Fluorescence measurements are performed using 2.5
nm x 2.5 nm slit width.
Determination of binding constant:
The binding constant of A4, A5, A6 for ZA is
MS(+) (m/z), calculated for C H N O : 291.11; found: 291.2,
1
8
14
2
2
+
[
M+H] .
1
5
Synthesis
naphthalen-2-ol (A5, Scheme 1):
A methanol solution of X1 (186 mg, ~1 mmol) is added to a
methanol solution of benzaldehyde (106 mg, ~1 mmol). The
resulting mixture is refluxed for 6h to obtain a yellow solution
which on slow evaporation gives bright yellow solid. The product
is recrystallized from DCM. Calculated yield: 87%. Anal. calcd
of
1-[(benzylidene)-hydrazonomethyl]-
2
0
determined using
equation :
a
modified Benesi–Hildebrand
7
5
21
ꢀ
ꢁꢂꢃ ꢄ ꢀ
ꢅ
ꢆ
ꢆ
=
1 + ( ) ( ꢋ); where Imax, I and I are emission
0 x
ꢇ ꢈꢉꢊ
ꢀ
ꢄ ꢀ
ꢅ
ꢃ
(%): C, 78.81; H, 5.14 and N, 10.21; found: C, 78.75; H, 5.07 and
N, 10.27. H NMR [400 MHz, CDCl , TMS, J (Hz), δ (ppm)]:
1
intensity values for A4, A5, A6 in the presence of ZA at
3
saturation, in the absence of ZA and at any intermediate 80 13.40 (1H, s), 9.75 (1H, s), 8.65 (1H, s), 8.15 (1H, d, J = 8.4),
ꢀ
ꢁꢂꢃ
ꢌ
ꢀ
ꢅ
ꢆ
7.89 (3H, m, J = 6.4), 7.77 (1H, d, J = 7.6), 7.56 (1H, m, J = 6.0),
2
5
ZA concentrations, respectively. A plot of
vs.
ꢋ
ꢀ
ꢄ ꢀ
ꢅ
ꢈꢉꢊ
7.48 (3H, d, J = 6.0), 7.38 (1H, m, J = 6.8), 7.24 (1H, t, J = 5.2).
ꢃ
13
C NMR [300 MHz, CDCl , TMS, δ (ppm)]: 171.79, 161.58,
3
(
here n = 1.0) yields the binding constant values from the
1
34.58, 133.71, 132.79, 131.50, 130.18, 129.37, 128.89, 127.85,
slopes.
85
123.65, 120.07, 119.20, 108.23. ESIꢀMS(+), m/z: calculated for
Determination of detection limit
:
+
+
C H N O: 275.11. Found: 275.12, [M+H] ; 297.08, [M+Na] .
18
14
2
From the fluorescence titration experiments, emission
intensities at λem = 487 nm, 495 nm and 520 nm (for A4, A5 and
A6 respectively) are normalized between the minimum and
3
0
Synthesis
of
1-[(4-nitro-benzylidene)-
hydrazonomethyl]-naphthalen-2-ol (A6, Scheme 1):
Condensation between X1 (186 mg, ~1 mmol) and 4ꢀ
nitrobenzaldehyde (151 mg, ~1 mmol) as described above gives
A4 as orange solid (Scheme 1) which is recrystallized from DCM
to get Xꢀray quality needle shaped deep orange color crystals.
Yield: 85%. Anal. calcd (%): C, 67.71; H, 4.10 and N, 13.16;
ꢃ ꢅ
ꢀ ꢄ ꢀ
maximum values ꢀꢁ
ꢅ; where Imax, I and I are emission
90
0
x
ꢄ ꢀ
ꢂꢃ
intensity values for A4, A5, A6 in the presence of ZA at
saturation, in the absence of ZA and at any intermediate ZA
concentrations in the low concentration regions, respectively.
Linear regression curves are then fitted to the normalized data,
and the points at which this line crosses the ordinate axes have
3
5
1
95
found: C, 67.61; H, 3.96 and N, 13.24. H NMR [400 MHz,
CDCl , TMS, J (Hz), δ (ppm)]: 13.51 (1H, s), 9.76 (1H, s), 8.69
3
2
2
been considered as the detection limits. Detail calculations
related to the determination of detection limits are listed in the
ESI.
(1H, s), 8.19 (1H, d, J = 8.4), 7.99 (2H, t, J = 7.8), 7.93 (1H, d, J
=
9.2), 7.84 (1H, d, J = 8.0), 7.79 (2H, d, J = 8.4), 7.63 (1H, m, J
4
0
13
=
7.2), 7.45 (1H, t, J =7.6), 7.29 (1H, t, J = 2.0). C NMR [300
Synthesis of 1-hydrazonomethyl-naphthalen-2-ol (X1, 100 MHz, CDCl , TMS, δ (ppm)]: 163.47, 161.76, 158.57, 139.56,
3
Scheme 1):
135.47, 132.77, 129.26, 129.14, 128.15, 124.07, 123.92, 119.94,
119.16. ESIꢀMS(+), m/z, Calculated for C H N O : 320.10;
found: 320.19 [M+H] .
1
0 mL (~ 200 mmol) hydrazine hydrate is taken in a 50 mL
18
13
3
3
+
round bottom flask and placed over a magnetic stirrer. Another
solution containing 2ꢀhydroxyꢀ1ꢀnaphthaldehyde (3.0 g, ~17.44
4
5
mmol) dissolved in minimum methanol is added drop wise with 105 Synthesis of Naphthalen-1-ylmethylene-hydrazine (X2,
continuous stirring. After stirring for 6h, the solution is filtered
off. The filtrate is then poured in 50 mL water whereby a
Scheme S1):
precipitate appeared which is collected by filtration and dried in
X2 is prepared following the same procedure described above
air. ESIꢀMS(+) m/z, Calculated for C H N O: 187.08; found:
5
0
1
1
10
2
+
for X1 where naphthaldehyde (3.0 g, ~19.23 mmol) is used in
10 place of 2ꢀhydroxyꢀ1ꢀnaphthaldehyde and reacted with10 mL (~
1
87.02 [M+H] .
1
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2
00 mmol) hydrazine hydrate. ESIꢀMS(+) m/z, Calculated for
bacteria within the rice root tissue and intracellular imaging of
60 ZA in human breast cancer cells (MCF7) has been performed
with fluorescence microscope. Theoretical results are in full
agreement to the experimental findings.
+
C H N : 171.08; found: 171.11 [M+H] .
1
1
10
2
Synthesis
of
4-(naphthalen-1-ylmethylene-
5
0
5
hydrazonomethyl)-phenol (A4a, Scheme S1):
A methanol solution containing X2 (170 mg, ~1 mmol) and 4ꢀ
hydroxybenzaldehyde (122 mg, ~1 mmol) is refluxed for 6h. The 65 AG is grateful to BU for fellowship. ST and MG are grateful to
Acknowledgements
resulting straw color solution is kept for slow evaporation to get a
light yellow solid that is recrystallized from acetonitrile. The
yield is 80%. Anal. calcd (%): C, 78.81; H, 5.14 and N, 10.21;
UGCꢀDAEꢀCSRꢀKolkata and State DST (Govt. of W.B.) for
funding. We thank Dr A Banerjee, Department of Chemistry &
Biochemistry, University of Texas at Austin, USA for routine cell
imaging studies.
1
1
found: C, 78.53; H, 5.23 and N, 9.98. H NMR [400 MHz,
CDCl , TMS, J (Hz), δ (ppm)]: 9.76 (1H, s), 9.12 (1H, s), 8.18
3
70 Notes and references
(
(
1H, d, J = 7.6), 7.75 (4H, t, J = 8.4), 7.40 (1H, d, J = 6.8), 7.30
1H, t, J = 8.0), 7.26 (2H, q, J = 3.2), 6.97 (3H, m, J = 4.4), 5.70
CCDC of A4 and A6 are 1018133 and 1415557.
a
Department of Chemistry, The University of Burdwan, Burdwan, West
Bengal, India. Eꢀmail: ddas100in@yahoo.com; Fax: +91ꢀ342ꢀ2530452;
1
(1H, s). ESIꢀMS (+), m/z, calculated for C H N O: 275.11.
1
8
14
2
+
Tel: +91ꢀ342ꢀ2533913
found: 275.10 [M+H] .
b
7
8
5
0
University Science Instrumentation Center, The University of Burdwan,
Burdwan, West Bengal, India.
Synthesis of ZA adducts of the probes:
c
To a magnetically stirred solution of A4 in acetonitrile,
methanol solution of ZA (A4: ZA = 1: 1, mole ratio) is added
dropꢀwise such that total solvent composition remains
acetonitrile: methanol, 9:1 (v/v). After 10ꢀ15 min stirring, the
solution is filtered and kept for slow evaporation while white
solid appeared after several days.
Microbiology Laboratory, Crop production division, ICARꢀ Central Rice
Research Institute. Cuttack, Odisha, India.
d
Department of Microbiology, The University of Burdwan, Burdwan,
20
West Bengal, India.
Electronic Supplementary Information (ESI) available: [Schemes, tables,
figures, and some spectra], See DOI: ######.
The ZA complexes of A5 and A6 are prepared following the
same procedure.
Rice seedling imaging
ZA (100 ꢁM) is incubated with Azotobacter chroococcum (16S
rRNA gene NCBI acc. no. KP099933) cells for 6h and infected
within rice seedlings. Then, the system is treated with A4 (10
ꢁM) and observed over UVꢀ transluminator (UV A).
Corresponding systems either without ZA or A4 are used as
control.
1
2
R. M. RoatꢀMalone. Bioinorganic Chemistry: A Short Course.
Hoboken: John Wiley & Sons, 2002.
(a). G. A. Eby, Bioscience Reports, 2004, 24 (1), 23ꢀ39; (b). A. S.
Prasad, F. W. J. Beck, B. Bao, D. Snell and J. T. Fitzgerald, J. Infect.
Dis., 2008, 197, 795ꢀ802.
G. J. Brewer, Expert Opin. PharmOActher., 2001, 2 (9), 1473ꢀ1477.
A. S. Prasad, J. T. Fitzgerald, B. Bao, F. W. J. Beck and P. H.
Chandrasekar, Ann. Intern Med., 2000, 133, 249.
A. Osol, (ed.). Remington's Pharmaceutical Sciences. 16th ed.
Easton, Pennsylvania: Mack Publishing Co., 1980., p. 718.
R..O. Suara, J. E. Jr. Crowe, Antimicrob. Agents Chemother., 2004,
48 (3), 783ꢀ790.
M. R. Shah, C. L. Kriedt, N. H. Lents, M. K. Hoyer, N. Jamaluddin,
C. Klein and J. Baldassare, J. Exp. Clin. Canc. Res., 2009, 28, 84.
P. E. Spiess, Prostate Cancer ꢀ From Bench to Bedside, InTech, 2011
D. Karak, S. Das, S. Lohar, A. Banerjee, A. Sahana, I. Hauli, S. K.
Mukhopadhyay, D. A. Safin, M. G. Babashkina, M. Bolte, Y. Garcia
and D. Das, Dalton Trans., 2013, 42, 6708
2
5
85
3
4
9
9
0
5
30
5
6
7
Human breast cancer (MCF7) cell imaging
Human breast cancer cell line MCF7 are grown in DMEM
35
40
45
50
(Sigma, St. Louis, USA) supplemented with 10% fetal bovine
ꢀ
1
serum (Sigma, St. Louis, USA), 2 mM glutamine, 100 U mL
8
9
penicillinꢀstreptomycin solution (Gibco, Invitrogen, USA) in
1
00
presence of 5% CO at 37°C. For in vitro imaging studies, the
2
cells are seeded in 6 well culture plate with a seeding density of
1
0
H. J. Zo, J. Y. Song, J. J. Lee, S. Velmathi and J. S. Park, Talanta,
5
10 cells per well. After reaching 60–70% confluence, the
2
013, 112, 80ꢀ84.
previous media is replaced with serum free media, supplemented
ZA and A4 at a concentration of 100 and 10 ꢁM, and incubated 105
for 2h to facilitate their uptake by cells. The cells are then
observed under an inverted microscope at different
magnifications to examine any adverse effect on cellular
11 G. Welbaum, A.V. Sturz, Z. Dong and J. Nowak, Crit. Rev. Plant.
Sci., 2004, 23, 175–193.
1
1
1
2
3
4
D. Wilson, Oikos, 1995, 73(2), 274–276.
M. Rosenblueth and E. M. Romero, MPMI, 2006 (8), 827–837.
S. Compant, H. Kaplan, A. Sessitsch, J. Nowak, E. A. Barka and C.
Clement, Microbiol Ecol., 2008, 63, 84–93.
morphology. A4 treated cells are then incubated with ZA alone 110 15 G. Brader, S. Compant, B. Mitter, F. Trognitz and A. Sessitsch, Curr.
for 15–30 min and observed under fluorescence microscope at
different magnifications using blue filter. Images are captured
through an attached CCD camera equipped with BioWizard 4.2
software. Control experiment is performed using medium devoid
of ZA or A4.
Opin. Biotechnol., 2014, 27, 30–37.
R. Anand and C.P. Chanway, Biol.Fert. of soils, 2013, 49(1), 111ꢀ
1
1
1
1
6
7
8
9
1
18.
A. Banik, S. K. Mukhopadhyay, A. Sahana, D. Das and T.K. Dangar,
(2015). Biol. Fert. Soils, DOI: 10.1007/s00374ꢀ015ꢀ1064ꢀ6.
H. Qin, Y. He, C. Hu, Z. Chen and L. Hu, Tetrahedron: Asymmetry,
1
1
1
15
2
007, 18, 1769–1774.
M. Royzen, A. Durandin, V. G. Young, N. E. Geacintov and J. W.
Canary, J. Am. Chem. Soc., 2006, 128, 3854–3855.
Conclusions
For the first time, we report a dual mode ratiometric approach
for colorimetric and fluorescence recognition of ZA using a
single crystal Xꢀray structurally characterized naphthalene based
aldazine probe. The CHEF assisted ESIPT inhibition is
responsible for the sensing of ZA in aqueous solution with the
best detection limit ever. In-vitro tracking of ZA in endophytic
20 20 (a) S. Miertus, E. Scrocco and J. Tomasi, Chem. Phys., 1981, 55,
17; (b) M. Cossi, V. arone, R. Cammi and J. Tomasi, Chem. Phys.
1
55
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.
25 21 H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, 71,
2
703ꢀ2707.
6
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2
2 (a) M. Shortreed, R. Kopelman, M. Kuhn and B. Hoyland, Anal.
Chem., 1996, 68, 1414; (b) A. Caballero, R. Martinez, V. Lloveras, I.
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Sahana, A. Banerjee, S. Lohar, D. A. Safin, M. G. Babashkina, M.
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5
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Graphical abstracts
Structurally characterized naphthalene based aldazine derivatives efficiently detect traces
zinc acetate (ZA) in a ratiometric manner both by fluorescence and naked eye. In-vitro
tracking of ZA in endophytic bacteria within the rice root tissue and intracellular imaging of
ZA in human breast cancer cells (MCF7) have been performed with fluorescence microscope.
Density functional theoretical studies fully support the experimental findings.