Tuning of azine derivatives for selective recognition of Ag+ with the in vitro tracking of endophytic bacteria in rice root tissue

Article abstract of DOI:10.1039/c6dt03117j

Several azine derivatives have been prepared and structurally characterized by spectroscopic and single-crystal X-ray diffraction analysis. Two of them, viz. naphthalene based (A10) and anthracene based (A11) show fluorescence enhancement in the presence of Ag⁺ in aqueous-methanol. Moreover, A11 efficiently tracks Ag⁺in vitro endophytic bacteria infected rice root tissue. Experimental results have been substantiated by theoretical DFT calculations.

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Trans., 2016, DOI: 10.1039/C6DT03117J.
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January 2016 Pages 1–398
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PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
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Tuning of azine derivatives for selective recognition
+
of Ag with in vitro tracking of endophytic bacteria in
Cite this: DOI: 10.1039/x0xx00000x
rice root tissue
Abhijit Ghosh, Sangita Adhikari, Sabyasachi Ta, Avishek Banik, Tushar Kanti
a
a
a
b
Received 00th January 2012,
Accepted 00th January 2012
b
c
d
e
Dangar, Subhra Kanti Mukherjee, Jesús Sanmartín Matalobos, * Paula Brandão,
e,f
a
Vítor Félix, * and Debasis Das *
DOI: 10.1039/x0xx00000x
www.rsc.org/
Several azine derivatives have been prepared and structurally
Bharadwaj group reported two azine derivatives, viz. LL (Fig. 1)
+
13
characterized by spectroscopic and single-crystal X-ray and HL to detect as low as 2 mM Ag in dry THF. To overcome
diffraction analysis. Two of them, viz. naphthalene based (A10) the limitations of sensing medium and poor sensitivity, systematic
+
and anthracene based (A11) show fluorescence enhancement in modification of probe architecture allows Ag detection at longer
+
presence of Ag in aqueous-methanol. Moreover, A11 efficiently emission wavelength.
+
tracks Ag in vitro endophytic bacteria infected rice root tissue.
Endophytic organisms associated with plants are variable and
Experimental results have been substantiated by theoretical complex but they occupy relatively privileged niche within plant
DFT calculations.
and usually contribute to uplift plant’s health, growth, development,
and improving soil quality. Besides, some endophytic
Silver ion (Ag ) plays key role in several industrial processes like microorganisms have been reported to be the mutualists that co-
electronics, photography, imaging, pharmacy and biology. Ag and evolved to protect plants against biotic stresses too. Furthermore, in
+
1
its compounds are widely used in consumer products such as the last two decades endophytes have been targeted as a valuable
2
detergents as well as for wound dressings. Silver stains are used to sources of new bioactive compounds through iron chelation,
enhance the visibility of cells and organelles through microscope. nitrogen fixation, phosphate solubilisation that allows least use of
Silver stains have been used to study the nervous system and Golgi chemicals for sustainable agriculture and environmental
3
+
14
apparatus since last fifteen decades. Ag belongs to the highest protection. Therefore, visualization and intra-cellular tracking of
4
+
toxicity class of heavy metal pollution. Intake of excessive Ag lead endophytic bacteria at their specific niche, remains one of the
to long-term accumulation of insoluble precipitates in skin and pioneering research interests of plant biologists. Detection of
eyes. Ag deactivates sulfhydryl enzymes and combine with amine, endophytic colonisation and intracellular tracking of bacteria,
imidazole, and carboxyl groups of various metabolites, causing transformation of green fluorescent protein (GFP) encoding plasmid
serious health problems. Recently, bioaccumulation and potential is very popular for visualisation. However, this method is very time
toxicity of Ag to benign bacteria, amphibian and fishes in water consuming and expensive. On the other hand, the biological
have been reported. Thus, positive impact of silver-related harmful activity of silver is very prominent at very low
technologies negatively impact to the environment due to ultimate concentration since it releases trace amount of Ag that strongly
release of silver containing products to sewage lines and rivers.
Accordingly, development of sensitive and selective method for successful to track down Ag challenged endophytic Azotobacter
1
b
+
1
b,5
+
15
6
+
1
6
binds anionic cellular macromolecules. The present probe, A11 is
+
+
+
detection of Ag is highly desirable. Traditional methods for Ag
chroococcum strain Avi2 inside the rice root tissue without
determination, such as inductively coupled plasma mass hampering it’s motility under fluorescence microscope.
spectrometry (ICP-MS), atomic absorption spectrometry or anodic
stripping voltammetry demand either expensive or time consuming
Results and discussion
7
procedures. Comparatively, optical detection, specially fluorescence
sensing is an interesting alternative for high selectivity, sensitivity,
cost effectiveness, rapidity, invasive nature and simplicity of
At first, the reported probe LL is prepared using a different
procedure that leads to a neutral molecule unlike ‘N’ protonated
cationic species. Subsequently two new probes, viz. A10 and A11
has been prepared (Fig.1) by condensation of hydrazone derivatives
of 1-naphthaldehyde and 9-anthracenecarboxaldehyde with 4-N, N-
dimethylamino benzaldehyde. A model compound (A10a) has also
been prepared replacing 1-naphthaldehyde of A10 by 2-hydroxy-1-
naphthaldehyde. The structures of LL, A10a, A11 and some of their
8
analysis.
However, fluorescence sensing of heavy transition metal ions
(
HTM) is challenging as they quench fluorescence through spin–
9
orbit coupling or energy/ electron transfer processes. Therefore,
most of the reported fluorescence sensors for Ag are based on
quenching mechanism while fluorescence turn-on probes for Ag
are scarce. Moreover, most of the Ag sensors have limitations like
+
1
0
+
+
Ag complexes are confirmed by single crystal X-ray diffraction
1
1
+
analysis. Crystal data together with selected refinement details are
summarised in Tables S1-S2 (ESI).
1
2
poor water solubility, sensitivity and selectivity.
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As A10 and A11 contain pH sensitive imine donor sites, their
performance is pH dependent. The emission characteristics of A10
+
and A11, in presence and absence of Ag are examined at different
+
pH. Fig. S1 (ESI†) indicates that A10 and A11 sense Ag over a
wide range of pH from 3.0 to 8.0, allowing to perform experiments
at pH 7.4, close to physiological pH. All spectral studies have been
performed in 0.1 M HEPES buffered aqueous-methanol (methanol/
water, 4/1, v/v).
Free probes show absorption peaks centred at 350 nm and
+
3
55 nm respectively for A10 and A11. Addition of Ag ion red-
shifts the bands to 396 nm and 403 nm respectively, associated
with well-defined isobestic points at 372 nm and 377 nm
respectively (Fig.4). A10 and A11 show weak emissions at 470
nm and 507 nm respectively. Among different metal ions, only
+
Ag remarkably enhances the emission intensity as shown in
Fig. 5. The emission intensities of A10 and A11 gradually
+
increases with increasing Ag concentration along with red shift
of emission maxima to 508 nm and 553 nm respectively (Fig.
Fig.1 Chemdraw structures of the probes.
6).
The molecular structures of three probes, presented in Fig. 2,
show the azine central core adopting a trans spatial disposition
characterised by a -C=N–N=C- torsion angle of 180, -179.8(2) and
1
73.1(2)º for LL, A10a and A11, respectively. In addition, A10a
exhibits an O-H···N intermolecular hydrogen between the naphthol
and imine groups (O···N = 2.548(3) Å and ∠ O-H···N = 152(2) º).
LL has a centro-symmetric structure with the middle point of the N–
N bond positioned at a crystallographic inversion centre of
symmetry and accordingly the non-hydrogen atoms are co-planar.
A10a also displays a planar shape with a dihedral angle between the
naphthalene moiety and phenyl ring of 8.0(1)º. In contrast, in A11
the anthracene entity is rotated relatively to the remaining molecular
backbone with a dihedral angle between the polyaromatic and
phenyl rings of 38.16(5)º. This structural feature is consistent with
the Ag(I) assisted red shifts observed for these three ligands (vide
infra).
Overall, the three molecules have bond lengths and bond angles
within the expected values, being consistent with the existence of an
azine fragment in the ligands LL, A10a and A11.The crystal packing
analysis of the three molecules shows non-existence of relevant π–π
stacking interactions.
Fig. 2 Molecular structures of LL (top), A11 (middle) and A10a
bottom), determined by single crystal X-ray diffraction. Nitrogen,
carbon, hydrogen and oxygen atoms are drawn in blue, grey, white and
red, respectively, with thermal ellipsoids drawn at 50% probability
level. The O-H···N intermolecular hydrogen bond in A10a is drawn as
purple dashed lines.
(
+
Single crystal X-ray structures of Ag complexes, presented in
Fig. 3, exhibit Ag(I) centre(s) coordinated by a nitrate anion in a
chelating fashion. Moreover, moving across the series, ligand
binding pattern changes from LL to A10 to A11. Whereas A10
Fluorophores having electron-donating and electron-withdrawing
groups in conjugation, normally susceptible to visible light
+
produces a monomeric Ag complex coordinated by two A10
ligands, consistent with the molecular formula [Ag(A10)
produces a dinuclear Ag complex with two AgNO fragments
2
(NO
3
3
)]; LL
excitation and consequently, intra-molecular charge transfer from
+
17, 18
donor to the acceptor occurs.
As a result of this excited state
assembled by a LL bridging ligand. In addition, the coordination
sphere of each Ag(I) centre is completed with an imine nitrogen
from another LL ligand, leading to the molecular formula
charge transfer, the donor site becomes positively charged and
subsequently, dipole moment changes to cause a Stokes shift that
19
depends on the microenvironment of the fluorophore. Thus, cations
[
Ag
and Ag is disordered, being composed of two polymeric one-
dimensional non-ending polymeric structures [Ag (A11) (NO
and [Ag (A11) (NO (H O) ], with the refined occupations of x and
-x being 0.727(7) and 0.273(7), respectively. These two covalent
chains differ only in the silver coordination sphere. The first one
2 3 3 2
(LL) (NO ) ] (vide infra). The crystal structure between A11
interacting to donor or acceptor unit will influence the photo-
+
physical properties of the fluorophore and affect the charge transfer
n
n
3
)
n
]
20-23
(CT) process.
When cation interacts with the donor moiety, its
n
n
3
)
n
2
n
electron-donation ability decreases. This diminished conjugation
results blue shift of the absorption band with lesser extinction
coefficient. On the other hand, cation interacting with the acceptor
moiety enhances its electron-withdrawing character and causes a
red-shift of the absorption band with higher molar extinction
1
–
3
contains a chelating NO anion (Ag-O = 2.564(2) and 2.460(3) Å),
while the second one has a nitrate anion and a water molecule, both
+
coordinated to Ag in a monodentate fashion with Ag-O distances of
24
coefficient. The emission band shifts in the same direction.
2
.564(2) and 2.547(12) Å, respectively. Henceforth, this component,
Obviously, these photo-physical processes depend on the charge and
with minor occupancy, will be not considered.
+
size of the cation, therefore cation selectivity, e.g., Ag in present
In summary, the three complexes have four coordinate geometry
with comparable Ag–O and Ag–N bond lengths (Table S3).
Moreover, the former ones are longer than the second ones.
25
case is observed.
2
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increased electron-withdrawing conjugation character of the ligands.
A10 and A11 are asymmetrically substituted azines with enhanced
formal electron transfer between electron acceptor (naphthalene or
anthracene) and electron donor (dimethyl amino phenyl) through
2
9
azine spacer, which is not feasible for symmetric LL.
Dipole moment of CT states is higher than the ordinary exciplex.
So, to impose stability to CT states followed by CT fluorescence,
3
0
solvation in polar solvent is essential. Consequently, to get CT
emission from symmetrical azine like LL, highly polar solvent, viz.
+
DMF is required. Our objective is to determine Ag in a medium
compatible to biological system. Both A10 and A11 being good
donor-acceptor conjugated unsymmetrical azine, are inherently CT
active, and hence excludes the requirement of highly polar solvent
for stabilization of CT states. Thus, methanol behaves sufficiently
polar to enhance the emission intensities of either A10 or A11 in
+
presence of Ag (Fig. S2, ESI†). Consequently, strategic
modification of the probe structure allows us to use less polar
+
aqueous-methanol medium for Ag detection at longer wavelengths.
Single crystal X-ray structure of [Ag (A11) (NO ) ] reveals that Ag
n n 3 n
+
is bonded either of N-donors of the azine function, while only one
+
N-donor (closer to donor di-methyl amino group) is involved in Ag
2 3 3 2 2 3
coordination in [Ag (LL) (NO ) ] and [Ag(A10) (NO )] (Fig. 3). This
+
feature leads to higher PCT for the Ag complex of A11 than that of
A10. Hence, highest red-shift is observed in the emission band of
+
Ag complex of A11. However, while a 2:1 stoichiometry (mole
+
ratio) between A10 and Ag is supported from the Job’s plot using
fluorescence method, a 1:2 stoichiometry (mole ratio) is predicted
+
between A11 and Ag (Fig. S3, ESI†). These findings are also in
+
accordance with the ESI-MS spectra of the corresponding Ag
complexes of A10 (Fig. S4, ESI†) and A11 (Fig. S5, ESI†). While
+
the molecular ion peak at m/z, 772.60 is observed for the Ag
+
complex of A10, it is observed at m/z, 703.81 for the Ag complex
+
of A11 in methanol. In solution, the polymeric structure of the Ag
3
1
complex of A11 (observed from SCXRD) is not retained rather the
+
solvent molecules coordinate to Ag centre. Moreover, presence of
-
+
+
NO
3
ion is not essential for Ag recognition as other soluble Ag
also enhances emission intensities of A10 and
salts like AgClO
4
A11 (Fig. S6, ESI†). However, A10a having almost similar structure
+
Fig.3 Single crystal X-ray structures of [Ag
2
(LL)
3
(NO
3
)
2
] (top, with to A10 is non-responsive to Ag (Fig. S7, ESI†), probably due to
a
a
stands for 1-x,1-y,-z), [Ag(A10)
2 3
(NO )] (middle, with stands for 1-x, preferential binding of naphthol oxygen and nearby imine nitrogen
a
+
y, 1/2-z) and [Ab g
n
(A11) (NO
x,5/2-y, -1/2+z, and for x,5/2-y,1/2+z).
n
3
)
n
]
(bottom, with stands for to Ag inhibits the charge transfer process. The charge transfer may
also be hindered due to presence of electron donating hydroxyl
group in the naphthalene moiety. Changes in the emission intensities
Here, Ag interacts with azine acceptor and the excited state of A10 and A11 with added Ag are shown in Figs. S8 and S9
dipole moment becomes higher than that of ground state due to (ESI†), the linear regions of which are useful for determination of
+
+
+
+
-6
accelerated PCT from amino group. Gradual addition of Ag
enhances PCT emission along with red shift of both the absorption 10 M Ag respectively (Fig. S10, ESI†). The binding constants of
unknown Ag . Their detection limits are 1.07 × 10 M and 4.14 ×
-
6
+
2
6
+
32
6
-1/2
and emission maxima. This is attributed to the higher stabilisation A10 and A11 for Ag , obtained from Hill plots are 7.62 × 10 M
and 4.89 × 10 M (Fig. S11, ESI†) respectively. The effect of
To unfold this mechanism, the molecular interaction between A10 competing metal ions on the fluorescence detection of Ag is
2
7
6
-2
of the excited state than the ground state.
+
+
and Ag was studied by density functional theory (DFT). These insignificant (Fig. S12, ESI†).
calculations on the probe A10 and complex [Ag(A10)
2 3
(NO )] were
carried out with B3LYP functional and 6-31G and Lanl2dZ basis set
+
28
(
Fig. 7) for non-metal atoms and Ag respectively . It is observed
+
that the energy of both HOMO and LUMO decreases in Ag bound
state, however the extent of stabilization of the later is higher than
that of the former orbital causing an overall decrease of the gap from
3
.2700 eV to 3.0265 eV. The figure also reveals that the charge
density at HOMO of A10 is distributed throughout the molecule.
Upon excitation, the charge density is slightly moved to the azine
+
and naphthalene part. After binding to Ag , the charge transfer
Fig.4 Changes in the absorption spectra of (A) A10 and (B) A11 (20
μM, in methanol/ water, 4/1, v/v, 0.1 M HEPES buffer, pH 7.4) upon
gradual addition of Ag (0-600 μM)
becomes more prominent at the vicinity of acceptor azine bound to
+
Ag , observed from LUMO.
+
On moving from LL to A11 via A10, emission maxima gradually
red shifted from 425 nm to 553 nm via 508 nm. This is due to
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Rice seedling imaging
Fluorescence microscope images (Fig. 10C and D) (UV filter)
suggest that A11 (10 µM) successfully penetrated the rice root
+
tissues and bind to Ag treated Azotobacter chroococcum cells to
produce a pink fluorescence from the root hairs, especially in the
root elongation and differentiation zones. Moreover, when the rice
+
seedlings challenged with Ag treated Azotobacter chroococcum,
followed by incubation in A11 (10 µM), the roots turn pink
indicating the zone of bacterial colonization upon exposure to UV
transluminator (UV-A) (Fig. 10D). However no emission is
Fig.5 Effect of different metal ions (600 μM) on the emission observed from fluorescence microscope image of root at 100x
+
intensities of (A) A10 (λex, 410 nm, λem, 508) and (B) A11 (λex, 440 magnification, colonised with Ag (100 µM) treated Azotobacter
nm, λem, 553) (20 μM, methanol– water, 4: 1, v/v, 0.1 M HEPES buffer,
pH 7.4
chroococcum (without A11- control) (Fig. 10A) and sterile distilled
water treated Azotobacter chroococcum colonised root, incubated in
A11 (10 µM) at 100x magnification (without Ag+ - control) (Fig.
1
0B).
Human breast cancer (MCF7) cell imaging
Fig. 11 reveals that A11 is cell permeable to image intracellular
Ag+ in human breast cancer cells (MCF7) by fluorescence
microscope. The cells remain alive after several hours of exposure to
1
0 μM of A11.
Fig.6 Changes in the emission spectra of (A) A10 (λex, 410 nm, λem
,
5
08) and (B) A11 (λex, 440 nm, λem, 553) (20 μM, media and pH are
+
same as supra) upon gradual addition of Ag (0-600 μM)
+
1
To unfold solution state interactions between A11 and Ag , H
NMR titration have been performed in CD OD–D O media (Fig.8).
3
2
1
The HNMR spectrum of A11 reveals that -CH=N proton close to
anthracene appears at downfield (9.79 ppm) compared to other
-CH=N proton (8.73 ppm). The reason is the presence of electron
donating dimethylamino phenyl group closer to that -CH=N proton.
+
However, upon addition of Ag , both imine protons gradually
downfield shifted to 9.95 ppm and 8.90 ppm respectively (upon
+
addition of maximum 5.0 equiv. Ag ). Other protons, including
1
+
anthracene ring protons remain unaffected. This indicates that both Fig.8 H NMR titration of A11 with Ag (A. 0.0, B. 0.5, C. 1.0, D. 2.0,
+
3 2
E. 5.0 equiv.) in CD OD-D O
-
CH=N nitrogen coordinates Ag , also corroborated from X-ray
1
structure of polymeric chain [Ag
n
(A11)
n
(NO )
3 n
] vide supra. H NMR
titration nullifies any stacking of anthracene moieties in presence of
Ag , otherwise, anthracene protons would have been shifted to
higher field due to diamagnetic shielding.
+
Experimental
Materials and methods
High-purity
HEPES,
hydrazine
hydrate,
9-
anthracenecarboxaldehyde,
1-naphthaldehyde,
2-hydroxy-1-
naphthaldehyde and 4-dimethylaminobenzaldehyde were purchased
from Sigma-Aldrich (USA). Solvents used were of spectroscopic
grade. Other chemicals were analytical reagent grade and used
−1
without further purification. Milli-Q Millipore 18.2 MΩ cm water
was used throughout the experiments. A Shimadzu Multi Spec 2450
spectrophotometer was used for recording UV-Vis spectra. FTIR
spectra were recorded on a Shimadzu FTIR spectrophotometer (IR
Prestige 21 CE). Mass spectra were recorded with a QTOF 60 Micro
1
YA 263 mass spectrometer in ES positive mode. H NMR titration
+
of A11 with Ag in MeOD-D
2
O was performed with a Bruker
1
3
Advance 600 (600 MHz) NMR spectrometer. The C NMR
spectrum of A10, A11, A10a and H NMR spectra of A10 in CDCl
1
3
were recorded with a Bruker Advance 400 (400 MHz) NMR
spectrometer. Elemental analyses were performed on a Perkin-Elmer
2
400 CHN analyzer. The steady state emission and excitation spectra
were recorded with a Hitachi F-4500 spectrofluorimeter.
Systronics digital pH meter (model 335) was used for measuring the
A
Fig.7 Frontier molecular orbitals of the probe, A10 and complex,
[
Ag(A10)
2
(NO
3
)]
solution pH. Dilute HCl or NaOH (50 μM) are used to adjust pH.
4
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The imaging system was an inverted fluorescence microscope
Dewinter, Italy). Images were captured by a CCD camera with
(
BioWizard 4.2 software. The microscope was equipped with a 50 W
mercury arc lamp. All spectra were recorded at room temperature.
General method of UV-Vis and fluorescence titration
The path length of the cells used for absorption and emission
studies was 1 cm. Stock solutions of A10, A11 and Ag+ were
prepared in methanol/water (4/1, v/v). Working solutions of A10,
+
A11 and Ag were prepared from their respective stock solutions.
Fluorescence measurements were performed using 2.5 × 2.5 nm slit
width.
Determination of detection limit
From fluorescence titration, emission intensities at λem = 508 nm
and 553 nm (for A10 and A11 respectively) were normalised
ꢀ
ꢁ − ꢀ0
between the minimum and maximum values ꢀ
; where Imax, I
0
ꢂꢃꢁ − ꢀ0
+
and I
x
were emission intensities for A10, A11 in presence of Ag at
+
+
saturation, in absence of Ag and at any intermediate Ag
concentrations at low concentration region, respectively (Fig. S9,
ESI†). Linear regression curves were then fitted to the normalised
data, and the point at which the line crosses the ordinate axes was
33
considered as the detection limit.
Fig.10 (A) Fluorescence microscope image of root at 100x
+
magnification, colonised with Ag (100 µM) treated Azotobacter
chroococcum Avi2 (without A11
- control); (B) fluorescence
microscope image of sterile distilled water treated Azotobacter
chroococcum Avi2 colonised root incubated in A11 (10 µM) at 100x
magnification (without Ag+ - control); (C) fluorescence microscope
+
image of root at 40x magnification, treated with Ag (100 µM)
Azotobacter chroococcum Avi2, followed by incubation in A11 (10
µM);
(D) fluorescence microscope image of root at 100x
+
magnification, treated with Ag (100 µM) Azotobacter chroococcum
Fig.9 Different treatments of rice seedlings under ultraviolet A, to Avi2, followed by incubation in A11 (10 µM). The arrows represent in
screen endophytic colonisation (a) root, incubated with suspension of vitro colonization of Azotobacter chroococcum Avi2, inside rice roots
+
Azotobacter chroococcum Avi2 in sterile distilled water (without Ag
+
and A11) (b) root, challenged with Ag (100 µM) treated Azotobacter
chroococcum Avi2 (without A11). (c) A11 (10 µM) incubated root,
incubated with sterile distilled water treated Azotobacter chroococcum
+
+
Avi2 (without Ag ). (d) root challenged with Ag (100 µM) treated
Azotobacter chroococcum Avi2, followed by incubation in A11 (10
+
µM) (Ag + A11). The arrow represents the intracellular localization of
+
Ag treated Azotobacter chroococcum Avi2, incubated in ligand A11
inside rice root seedlings
Synthesis
Fig.11 Fluorescence microscope images of MCF7 cells: (A) and (B) are
4
, 4'-(Hydrazine-1, 2-diylidenebis-(methanylylidene))
+
images of cells after 2 h incubation with Ag (100 μM) and A11 (10
bis(N,N-dimethylaniline) (LL)
+
μM) respectively, (C) cells after 2 h incubation with 100 μM Ag
1
0 mL (~ 200 mmol) hydrazine hydrate was added to 4- followed by addition of 10 μM A11
dimethylamino)benzaldehyde (2.0 g, ~13.41 mmol) in methanol and
(
stirred for 4h.The solution was neutralised with dilute sulphuric acid
to obtain yellow precipitate. It was recrystallized from methylene
chloride whereby yellow crystals suitable for X-ray diffraction
analysis found. Anal. calcd (%): C, 73.44; H, 7.53 and N, 19.03;
Naphthalen-1-ylmethylene-hydrazone (X2, Scheme
S1†)
X2 was prepared following the literature procedure by controlled
condensation of 1-naphthaldehyde and hydrazine hydrate in
found: C, 73.40; H, 7.59 and N, 19.01. ESI-MS(+): m/z, calcd. for
3
4
+
methanol.
C
18
H
22
N
4
: 295.18; found: 295.30, [M+H] (Fig. S13, ESI†). FTIR
spectrum is presented in Fig. S14 (ESI†).
Anthracen-9-ylmethylene-hydrazone (X3, Scheme
S1†)
1
-Hydrazonomethyl-naphthalen-2-ol (X1, Scheme
1
0 mL (~ 200 mmol in methanol) hydrazine hydrate was added
S1†)
drop-wise to methanol solution of 9-anthracene carboxaldehyde (3.0
g, ~14.55 mmol) under stirring condition. The precipitate appeared
X1 was prepared following the literature procedure by controlled
condensation of 2-hydroxy-1-naphthaldehyde and hydrazine hydrate
3
4
12 2
was collected and dried in air. ESI-MS(+): m/z, calcd. for C15H N :
in methanol.
+
2
21.10; found: 221.20 [M+H] (Fig. S15, ESI†).
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The procedure was same as above, here, we used A11 (0.1 g,
0.28 mmol) and AgNO (0.035 g, ~0.20 mmol). Slow evaporation
of the solvent generated plate shape crystals suitable for X-ray
diffraction. FTIR (Fig. S28, ESI†).
Dimethyl-[4-(naphthalen-1-ylmethylene-
hydrazonomethyl)-phenyl]-amine (A10, Scheme S1)
Methanol solution of X2 (170 mg, ~1 mmol) and 4-
~
3
(
6
dimethylamino)benzaldehyde (149 mg, ~1 mmol) was refluxed for
h. Upon slow evaporation of the resulting yellow solution, a pale
Rice seedling imaging
Oryzasativa var. Swarna seedlings were incubated for 6 h with
Ag+ (100 µM) treated rice root endophytic Azotobacter
chroococcum strain Avi2 (16S rRNA gene National Center for
Biotechnology Information Gen Bank Accession KP099933) cells.
Bacteria treated seedlings were washed five times with sterile
HEPES buffer (0.1 M) to remove loosely associated bacteria from
the root surface. Subsequently, the seedlings were incubated with
A11(10 µM) for 1 h at room temperature and washed five times with
sterile HEPES buffer (0.1 M) and photographed under fluorescence
yellow solid found that was recrystallized from acetonitrile. The
yield was 75%. Anal. calcd. (%): C, 79.70; H, 6.35 and N, 13.94;
1
found: C, 80.23; H, 6.20 and N, 13.44. H NMR [400 MHz, CDCl
3
,
TMS, J (Hz), δ (ppm)]: 9.34 (1H, s), 8.98 (1H,d, J = 8.4), 8.57 (1H,
s), 8.14 (1H, d, J = 7.2), 7.95 (1H, d, J = 8.0), 7.91 (1H, t, J = 8.4),
7
3
.69 (3H, m, J = 7.2), 7.65 (2H, m, J = 7.2), 6.73 (2H, q, J = 6.8),
.06 (6H, m, J = 3.6), (Fig. S16, ESI†). C NMR [100 MHz, CDCl ,
3
1
3
TMS, J (Hz), δ (ppm)]: 190.67, 163.34, 162.37, 161.10, 160.06,
1
1
52.44, 134.27, 132.32, 131.82, 130.64, 130.19, 129.55, 128.82,
27.73, 126.62, 125.71, 124.94, 112.06, 111.34, 40.51 (Fig. S17,
+
microscope. Corresponding systems either without Ag or A11 were
used as control.
ESI†).ESI-MS (+), m/z, calcd. for C20
H
19
N
3
: 302.16. found: 302.36
+
[
M+H] (Fig. S18, ESI†). FTIR (Fig. S19, ESI†).
Human breast cancer (MCF7) cell imaging
Human breast cancer cell line MCF7 were grown in DMEM
[
4-(Anthracen-9-ylmethylene-hydrazonomethyl)-
(
(
Sigma, St. Louis, USA) supplemented with 10% fetal bovine serum
Sigma, St. Louis, USA), 2 mM glutamine, 100 U mL penicillin-
phenyl]-dimethyl-amine (A11, Scheme S1)
Methanol solution of X3 (220 mg, ~1 mmol) and 4-
-
1
streptomycin solution (Gibco, Invitrogen, USA) in presence of 5%
CO at 37°C. For in vitro imaging studies, the cells were seeded in 6
(
6
dimethylamino)benzaldehyde (149 mg, ~1 mmol) was refluxed for
h. The resulting wine colour solution was kept for slow evaporation
2
well culture plate with a seeding density of 105 cells per well. After
reaching 60–70% confluence, the previous media was replaced with
whereby brown crystal suitable for single crystal X-ray diffraction
was found. The yield was 85%. Anal. calcd (%): C, 82.02; H, 6.02
+
serum free media, supplemented Ag and A11 at a concentration of
1
and N, 11.96; found: C, 81.53; H, 5.87 and N, 12.60. H NMR [400
1
00 and 10 μM, and incubated for 2h to facilitate their uptake by
MHz, CDCl
3
, TMS, J (Hz), δ (ppm)]: 9.86 (1H, s), 8.79 (1H, s), 8.74
cells. The cells were then observed under an inverted microscope at
different magnifications to examine any adverse effect on cellular
(2H, d, J = 11.6), 8.51 (1H, s), 8.03 (2H, d, J = 10.8), 7.82 (2H, d, J
=
9.2), 7.56 (4H, m, J = 8.8), 6.76 (2H, d, J = 9.2), 3.05 (6H, t, J =
+
morphology. A11 treated cells were then incubated with Ag alone
3
2
.4), (Fig. S20, ESI†). 1 C NMR [100 MHz, CDCl
3
, TMS, J (Hz), δ
ppm)]: 163.48, 159.28, 152.55, 131.40, 130.58, 130.47, 129.95,
28.91, 126.85, 126.06, 125.45, 125.37, 121.52, 111.73, 40.17, (Fig.
S21, ESI†). ESI-MS (+), m/z, calcd. for C24 : 352.17. found:
52.40 [M+H] (Fig. S22, ESI†). FTIR (Fig. S23, ESI†).
for 15–30 min and observed under fluorescence microscope at
different magnification using blue filter. Images were captured
through an attached CCD camera equipped with BioWizard 4.2
software. Control experiments were performed using medium devoid
(
1
21 3
H N
+
3
+
of Ag or A11.
1
-[(4-Dimrthylamino-benzylidene)-
hydrazonomethyl]-naphthalen-2-ol (A10a, Scheme 1) Single crystal X-ray diffraction analysis
Methanol solution of X1 (186 mg, ∼1 mmol) and 4-
Single crystal X-ray diffraction data were collected with on a
(
dimethylamino)benzaldehyde (149 mg, ~1 mmol) was refluxed for Bruker Kappa Apex II CCD diffractometer using graphite-
6
h while a dark yellow solution obtained. The solution was allowed monochromated Mo-Kα radiation (λ = 0.71073Å) at 100 K for A11-
to evaporate slowly for several days whereby X-ray diffraction Ag complex, at 150 K for A10-Ag complexes and at 296 K for free
quality rectangular orange crystals appear. Yield: 80%. Anal. calcd. LL, A10a and A11 ligands. The data collection of LL-Ag was
(
%): C, 75.69; H, 6.03 and N, 3.24; found: C, 76.01; H, 5.93 and N, carried out at low temperature (T = 149 K), also using an area
1
1
(
3.19. H NMR [400 MHz, CDCl
1H, s), 9.78 (1H, s), 8.56 (1H, s), 8.22 (1H, t, J = 8.0), 7.83 (4H, m, diffractometer. Data were processed and corrected for Lorentz and
J = 8.0), 7.58 (1H, m, J = 4.0), 7.41 (1H, q, J = 4.4), 7.28 (1H, m, J = polarisation effects. Multi-scan absorption corrections were
3
, TMS, J (Hz), δ (ppm)]: 13.4 detector and Mo-Kα radiation, on a Bruker D8 QUEST CCD
3
5
1
.6), 6.78 (2H, t, J = 1.6), (Fig. S24, ESI†). ESI-MS(+) m/z, calcd. performed using SADABS, as implemented in SAINT plus
+
36
19 3
for C20H N
O: 318.15; found: 318.15, [M+H] , (Fig. S25, ESI†). software suite. The structures were solved by direct methods with
3
7
FTIR (Fig. S26, ESI†).
SIR2008 for complex A11-Ag and with SHELXS (version 2013/1)
3
8
for the remaining compounds. All structures were refined by full
[
Ag
2
(LL)
3
(NO
3
)
2
].CH
2
Cl (LL-Ag) complex
2
2
matrix-least squares on F using the SHELXL program (version
Yellow crystals of LL (0.1 g, ~0.34 mmol) were dissolved in 1:1
3
9
2
016/4). The unit cell of LL-Ag complex is composed of one
Ag (LL) (NO ] molecule, with a centro-symmetric structure, and
one methylene chloride molecule of crystallization, affording the
molecular formula [Ag (LL) (NO ].CH Cl . The mother liquor
(
(
v/v) mixture of DMF and methylene chloride. To this, AgNO
0.035 g, ~0.20 mmol) dissolved in minimum volume of DMF was
3
[
2
2
3 2
)
added drop-wise at room temperature. The resulting red solution
yielded X-ray quality plate-shape crystals after 2 days.
2
3
3
)
2
2
2
molecule was found disordered over an inversion centre and was
refined with an occupancy factor of 0.5. All non-hydrogen atoms
were refined with anisotropic thermal displacements. The hydrogen
atoms, apart the one from the naphthol group of A10a, were included
in the structure refinement in geometrically idealised positions with
isotropic thermal parameters equivalent to 1.2 times of those of the
atom to which they were attached (1.5Ueq when methyl groups).
The position of the hydroxyl hydrogen atom of A10a was obtained
from the difference Fourier map and was refined with an individual
[
Ag(A10)
2
(NO )] (A10-Ag) complex
3
The procedure was same as described above, however in present
case, we used A10 (0.1 g, ~0.33 mmol) and AgNO (0.035 g, ~0.20
3
mmol). Slow evaporation of the solvent generated block shape red
crystals suitable for X-ray diffraction. FTIR (Fig. S27, ESI†).
[
(
Ag
n
(A11)
n
(NO
3
)
n
] and [Ag
n
(A11)
n
(H
2
O)
n
(NO
3
n
) ]
A11-Ag) complex
6
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Journal Name
Dalton Transactions
ARTICLE
isotropic thermal displacement. The crystal structure diagrams were
drawn with Mercury software.
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4
0
8
9
Conclusion
Highly selective azine based probe for Ag+ is reported. The
sensing is attributed to inhibition of PCT process. The probes and
+
their Ag complexes are characterised by single crystal X-ray
+
diffraction studies. In vitro tracking of Ag in endophytic bacteria
+
within rice root tissue and intracellular imaging of Ag in human
breast cancer cells (MCF7) has been performed with fluorescence
microscope.
Acknowledgement
AG was grateful to BU for fellowship. We thank Dr A Banerjee,
Department of Chemistry & Biochemistry, University of Texas at
Austin, USA for routine cell imaging studies. We sincerely
acknowledge USIC, BU for XRD facility to some ligands. The
single crystal X-ray structure determinations were also carried out at
the University of Aveiro, supported by CT/MEC via financial
support to CICECO–Aveiro Institute of Materials (FCT
UID/CTM/50011/2013) and iBiMED-Institute of Biomedicine (FCT
UID/ BIM/ 04501/2013) through national funds and, when
applicable, co-financed by FEDER, within the PT2020 Partnership
Agreement.
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DOI: 10.1039/C6DT03117J
Graphical Abstract
+
Single crystal X-ray structurally characterized azine derivatives show Ag selective turn-on
+
fluorescence, attributed to inhibition of PCT process. The probe efficiently tracks Ag in vitro
+
endophytic bacteria infected rice root tissue. Intracellular Ag imaging in human breast
cancer cells (MCF7) has also been achieved.