Amide-imine conjugate involving gallic acid and naphthalene for nano-molar detection, enrichment and cancer cell imaging of La3+: Studies on the catalytic activity of the La3+complex

Article abstract of DOI:10.1039/d0nj02415e

A single crystal X-ray structurally characterized amide-imine conjugate (GAN) derived from gallic acid and naphthalene selectively recognizes La3+ ions via TURN ON fluorescence through ESIPT and CHEF mechanisms. GAN can detect as low as 23.93 × 10-9 M La3+ ions and image intracellular La3+ ions in live HeLa and SiHa cells under a fluorescence microscope in a time- and concentration dependent manner. The corresponding [La(iii)-GAN] complex is established as an efficient catalyst for the synthesis of benzimidazole derivatives from o-phenylenediamine and substituted benzaldehydes. Moreover, GAN is very useful for enrichment of La3+ in ethyl acetate medium. This journal is

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Amide-imine conjugate involving gallic acid and
naphthalene for nano-molar detection, enrichment and
cancer cell imaging of La3+: Studies on catalytic activity of
La3+ complex
4Received 00th January
20xx,
Accepted 00th January
20xx
Ahad Shaikh,a Milan Ghosh,a Pallabi Mukherjeea, Avijit Ghosh,b Rostam Ali Molla,c Sabyasachi Ta*a and Debasis Das*a
DOI: 10.1039/x0xx00000x
aDepartment of Chemistry, The University of Burdwan, Burdwan, 713104, W.B., India
bCentre for Research in Nanoscience & Nanotechnology, (CRNN), University of Calcutta, Technology Campus, Sector-III,
Block-JD 2, Salt Lake, Kolkata, 700098, West Bengal, India.
www.rsc.org/
cDepartment of Science and Humanities, S. N. Bose Govt. Polytechnic College, Ratua, Malda, 73213, India
Abstract
Single crystal X-ray structurally characterized amide-imine conjugate (GAN), derived from gallic acid and naphthalene
selectively recognizes La3+ ion via TURN ON fluorescence through ESIPT and CHEF mechanism. The GAN can detect as
low as 23.93×10−9 M La3+ ion and image intracellular La3+ ion in live HeLa and SiHa cells under fluorescence microscope in
a time- and concentration dependent manner. The corresponding [La(III)-GAN] complex is established as an efficient
catalyst for the synthesis of benzimidazole derivative from o-phenylenediamine and substituted benzaldehyde. Moreover,
GAN is very useful for enrichment of La3+ in ethyl acetate medium.
Introduction
antiviral and anti-HIV.24-25 One-pot synthesis of 2-substituted
Lanthanum (La), a rare earth element is extensively used in benzimidazole derivatives from o-phenylenediamine and different
industries as a mischmetal alloy in “flints”, anode in nickel hydride aldehydes requires lanthanum chloride as catalyst.26 However,
batteries, to manufacture special optical glasses, carbon lighting in several issues like solvent, temperature and product yield still remain
studio, catalyst in petroleum refining etc. In medicinal field, La is a major concern.
used as biological tracer of Ca2+. The radioactive La is used in Optical detection and separation of La3+ from other Ln3+ ions is an
cancer treatment.1 Lanthanum carbonate, Fosrenol depresses high attractive research area. Notably, luminescent La3+ complexes are
blood phosphate levels, known as hyperphosphatemia in human to relatively rare.27 The electronic configuration is probably responsible
reduce heart disease and strokes. It is also useful during dialysis of for non-luminous nature of La3+ ion that makes it different from
kidney disease.2-3 Thus, determination of trace amount La is other Ln3+ ions. Several fluorescent ligands have been employed to
demanding. Large ionic radii, Lewis acidity, unoccupied 5d and 6s recognize La3+ ion. For example, Zhao et. al. reported a pyridine
orbitals and high coordination number make La3+ ion an a useful quinoxaline derivative (HPDQ) for detection of La3+ through red
catalyst for several organic trasformation.4
shifted fluorescence enhancement.28 Ahmed et. al reported
On the other hand, gallic acid (G), an important antioxidant5-6 albendazole (ABZ) based La3+ sensor where emission intensity is
inhibits pancreatic cholesterol esterase.7-9 The G, extracted from tea measured as a function of drug concentration.29 Sayed and his co-
leaf, oak bark, grapes etc.7-8 is also used in tanning and ink dye. The workers reported 5-fluorouracil (5-FU) based La3+ sensor30 having
G and several other polyphenols selectively damage cancer cell, binding constant, 0.364 x 104 M-1.
leaving normal cell unaffected.10-17 The G is also a potential anti- Efficient separation of La3+ from mixture of lanthanides (Ln3+) is
inflammatory drug.11-12 Ester form of G has higher bioavailability challenging and demanding.31-33 Literature suggest that Tian, et. al.
over G.13 The
G inhibits the rancidity, produced by lipid used N,N-di-alkyl-diglycolamic acid (HDMDGA) for efficient
peroxidation for use in cosmetics and food packaging materials. The extraction of lanthanides from concentrated nitric acid medium.34
G is chosen because it’s three –OH groups may stabilize the Binnemans and co-workers developed methane sulphonic acid and
designed La3+ complex in solution through several hydrogen bonds di-(2-ethylhexyl)phosphoric acid based organic extractant that
leading to enhanced rigidity of the system, with strong fluorescence.
separate La3+ from terbium-rich lamp phosphor waste.35 Yin et. al.
As already mentioned, La3+ compounds are useful catalyst for used a low cost chelator, H3cit with P204 for separation of La3+
several organic transformations18-19 like direct amidation of esters,20 from other Ln3+ ions.36 Bieke et.al. have reported extraction of La3+
highly enantio-selective conjugate addition of thioglycolate to ion using ammonium and phosphonium nitrate system.37
chalconescan,21 trans-esterification of carboxylic esters,22 These facts inspired us to develop a new probe for trace level
asymmetric Mannich-type reaction etc.23 The benzimidazole selective optical recognition and determination of La3+ ion. Herein,
derivative being structurally similar to purine bases and binding single crystal X-ray structurally characterized amide-imine conjugate
constituent of several natural products like vitamin B12 are used as (GAN), derived from gallic acid and 2-hydroxy napthaldehyde is
potential drug for antitumor/ anticancer, antibacterial, antifungal, explored for trace level selective recognition of La3+ ion in live cells.
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Besides, the new [La(III)-GAN] complex is exploited as an efficient The ortep view of GAN is presented in Fig. 1. Intermolecular
View Article Online
catalyst for synthesis of benzimidazole derivative by coupling o- hydrogen bond contributes to the packing oDfOGI:A10N.103u9n/iDts0NviJa02–4O15HE
phenylenediamine and substituted benzaldehyde at room group having P21/n space group. The crystallographic data and
temperature. In addition, GAN is useful for enrichment of La3+ from refinement details are collected in Table S1 [ESI]. Selected bond
6
mixture of lanthanides (Ln).
lengths and angles are listed in Table S2 [ ESI].
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Experimental
Synthesis (Scheme 1)
3, 4, 5 –Trihydroxybenzohydrazide (GA)
Excess thionyl chloride is added to the 3, 4, 5-trihydroxybenzoic
acid (133 mg, ~1 mmol) under stirring condition and resulting
solution is refluxed for 6h. The solid obtained after removal of
solvent is purified by solvent extraction using ethyl acetate. The
solid isolated after removal of ethyl acetate is refluxed with excess
hydrazine for 5h. Finally, removal of solvent yielded solid GA.
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Fig. 1 ORTEP view of GAN (50% thermal ellipsoid probability)
(E)-3,
4,
5-Trihydroxy-N'-((2-hydroxynaphthalen-1-
Result and discussion
Spectroscopic studies
yl)methylene)benzohydrazide (GAN)
The mixture of GA (133 mg, 1 mmol) and 2-hydroxynapthaldehyde
(172 mg, 1 mmol) in methanol is refluxed for 6h to obtain wine color
solution which upon slow evaporation produces needle shape red
crystals, suitable for single crystal X-ray diffraction (SC-XRD)
analysis. The yield is 70%. Anal. calcd. (%): C, 63.90; H, 4.17 and
N, 8.28; found: C, 63.40; H, 4.82 and N, 8.95. ESI-MS(+), m/z,
calcd. for C18H14N2O5, 338.09; found, 339.09 [GAN+H]+ [Fig. S1,
ESI]. 1HNMR [400 MHz, CDCl3, TMS, J (Hz), δ (ppm)]: 11.41 (1H,
s), 9.691 (1H, s), 9.249 (1H, s), 9.03 (1H, s), 6.94- 8.24 (aromatic
region), 7.518(1H, s) [Fig. S2, ESI].
As GAN have pH-sensitive hydroxyl group, the effect of pH on its
emission characteristics in presence and absence of La3+ is
thoroughly investigated and presented in Fig. S7 [ESI]. Thus, GAN
and La3+ are mixed in different sets at different pH from pH 3.0 -
12.0 and their emission profile is monitored. Fig. S7 [ESI] the
optimum performance of GAN is observed near physiological pH,
7.4 that allow to carry out the entire studies at pH 7.4 using HEPES
buffered aqueous DMSO (DMSO/ H2O, 4/1, v/v, 10 mM HEPES
buffer) media. Interestingly, optimum performance is observed in
20% aqueous DMSO (v/v) media.
13CNMR [400 MHz, CDCl3, TMS, J (Hz), δ (ppm)]: 167.85, 161.81,
145.86, 139.11, 135.20, 134.75, 131.83, 129.31, 127.79, 127.56,
123.31, 122.32, 106.83, 106.17, 105.86 and 104.77 [Fig. S3, ESI].
FTIR (cm-1), 3446 (NH), 3335.66 (OH), 2981.74 (imine CH), 1608
(C=O) and 1446 (C=N) [Fig. S4, ESI].
The interaction of the GAN with La3+ is monitored by different
spectroscopic techniques. The steady state emission of GAN is
perturbed by La3+ at nano-molar level while other tested common
cations viz. Ce4+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+,
Er3+, Yb3+, Lu3+, Fe3+, Fe2+, VO2+, Cr3+, Mn2+, Co2+, Ni2+, Cu2+,
Zn2+, Hg2+, Pb2+ and Cd2+ remain reluctant (λex, 336 nm)[Fig. S8,
ESI]. The effect of tested cations on the UV light irradiated bare eye
view of GAN indicates that La3+ turns colourless GAN to bright
green [Fig. S9, ESI] while other cations remain spectator.
The competitive experiment in presence of common cations
indicates no significant interference during GAN assisted La3+
sensing (Fig. S10, ESI).
The weak emission of GAN at 396 nm (λex = 336 nm) experiences
red shift in presence of La3+ to 472 nm resulting green emission [Fig.
S11, ESI]. The intensity of green emission gradually increases upon
gradual addition of La3+ (Fig. 2a) to maximum 97 fold with an
isobestic point at 435 nm. Moreover, the plot of emission intensity
vs. La3+ concentration follows a sigmoidal pattern [Fig. S12, ESI],
the linear region of which is useful for determination of unknown
La3+ concentration [Fig. S13, ESI]. It is noteworthy that addition of
75 equiv. or more La3+ quenches the fluorescence probably due to
static quenching. While mathematically calculated lowest detection
limit for La3+ is 23.93×10-9 M,38 the practical detection limit is
5.0×10-9M [Fig. S14, ESI]. The quantum yields of GAN and its La3+
complex are 0.016 and 0.321 respectively.
Scheme 1 Synthesis of GAN
[La(III)-GAN] complex
To a magnetically stirred acetonitrile solution of GAN, methanol
solution of La(NO3)6.6H2O is added drop-wise. After stirring for 40-
45 min, the solution is filtered. The filtrate is kept for slow
evaporation whereby yellow solid is collected after 5 days. The ESI-
MS(+), m/z calcd. for C18H13LaN5O4, [La(III)-GAN+H]+ 663.23.
Found : 663.45; [La(III)-GAN+Na+H2O]+, 703.23; found: 702.70;
The absorption spectrum of GAN exhibits peaks at 325 nm (ε =
7.5×104 M−1 cm−1) and 361 nm (ε =5.5×103 M−1 cm−1), assigned to
π–π* and n-π* electronic transitions respectively. Gradual addition
of La3+ weakens both the peaks gradually along with the appearance
of a new band at 412 nm through an isobestic point at 387 nm (Fig.
2b).
[La(III)-GAN+2H2O+C2H5OH]+,
744.33,
found:
743.72;
[GA+2LiCl+H]+, 269.94; found: 269.93 [Fig. S5, ESI]. FTIR (cm-1),
3426 (OH), 1636 (C=O), 1457 (C=N) [Fig.S6, ESI].
[La(III)-GAN] complex catalyzed synthesis of benzimidazole
derivative
The Job’s plot39 derived from fluorescence titration data indicates
1:1 (mole ratio) interaction between GAN and La3+ [Fig. S15, ESI],
also corroborated from ESI-MS mass spectrum of the resulting
complex [Fig. S6, ESI]. The association constant of GAN for La3+,
determined from Benesi-Hildebrand plot using fluorescence titration
data is 1.1×105 M [Fig. S16, ESI].
In general, 1.0 mmol of o-phenylenediamine is added to the ethanol
solution of the substituted aldehyde (1.0 mmol, 5.0 mL) in presence
of 10 mg catalyst. The reaction mixture is stirred at room
temperature while the progress is monitored by thin layer
chromatography (TLC). After 45 min, the solid product isolated by
filtration is purified by crystallization from ethanol.
The weak emission of the GAN is attributed to the excited state
intramolecular proton transfer (ESIPT) involving naphthol proton
Single crystal X-ray diffraction studies
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and imine nitrogen. Interaction of GAN with La3+ inhibits the ESIPT
process leading to chelation-enhanced fluorescence (CHEF)
(Scheme 2).
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DOI: 10.1039/D0NJ02415E
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Fig. 3. 1H NMR titration of GAN with La3+ ion in CDCl3 at 15 min interval
Catalytic activity
Literature on use of La3+ complex as catalyst for condensation
reaction inspired us to study the catalytic activity of the [La(III)-
GAN] complex for synthesis of benzamidazole derivative by
condensing aromatic aldehyde with o-phenylenediamine in ethanol
(Scheme 3).
CHO
NH2
N
La-Complex
+
ethanol
NH2
N
H
R
room temperature
R
Scheme 3 Synthesis of benzimidazole derivative using [La(III)-GAN]
complex as catalyst.
Fig. 2. Changes in the spectra of GAN (20 µM) upon addition La3+ in
DMSO/ H2O (4 /1, v/v, 10 mM HEPES buffer, pH 7.4; λex, 336 nm) [La3+] =
0.001, 0.01, 0.1, 1, 10, 50, 100, 200, 300, 400, 600, 800, 1000, 1200, 1400,
1500, 1800, 2000, 2400 and 3000 µM: (a) emission and (b) absorption
Different parameters for this catalytic condensation reaction have
been optimized.
Although, 22% yield of the product is observed after 24h reaction in
ethanol without catalyst, the reaction time significantly reduces in
presence of the catalyst (Table S4, ESI). At 10 mg catalyst load, the
product yield reaches maximum. The effect of solvent on product
yield indicates that polar protic solvent like ethanol and methanol are
preferable over polar aprotic solvent like acetonitrile. In fact, the
product yield becomes highest in ethanol. Effect of reaction time on
the yield of the product indicates a maximum 95% yield after 45 min
under optimized reaction conditions.
At optimized reaction conditions, the condensation of o-
phenylenediamine with different substituted aldehydes lead
reasonably good yield of the product at short time (Table S5, ESI).
In vitro live cell imaging
Human cervical cancer HeLa and SiHa cells are cultured in
Dulbecco’s modified eagle medium (DMEM) supplemented with
10% heat-inactivated fetal bovine serum (FBS) and 1%
penicillin/streptomycin at 37°C and 5% CO2. For in vitro imaging
studies, the cells are seeded in 12-well tissue culture plates with a
seeding density of 105 cells per well. After reaching 60%–70%
confluence, the previous DMEM medium is replaced with serum-
free DMEM medium (colorless), supplemented with 20 μM GAN
and incubated for 2h to facilitate the probe uptake by cells. Then
cells are washed three times with PBS buffer to remove any
extracellular GAN. Then La3+ (50 µM) is added to the medium and
further incubated for 1h. The control experiments have been
performed by incubating the cell only with GAN and La3+
separately. After washing with PBS buffer, images of live cells (Fig
4-5) are captured using an EVOS® FL cell imaging system, Life
Technologies, USA).
Scheme 2. Probable sensing mechanism
NMR titration
The mode of binding of GAN with La3+ in solution is established by
1HNMR titration. After addition of 1.0 equiv. La3+ to GAN, among
three types of OH protons (two types from gallic acid and one type
from 2-hydroxy-1-napthaldehyde), the most downfield peak at 11.39
ppm (-OH of 2-hydroxy-1-napthaldehyde) disappear along with
minor shift of other two type protons (from 9.25 to 9.32 and 9.06 to
9.12). This fact clearly indicates the involvement of phenol-O to the
binding of La3+. In addition, imine proton of GAN downfield shifted
from 9.69 to 9.72 ppm, indicating coordination of imine-N to La3+
leading to inhibition of PET process.
No fluorescence is observed in cells that are not previously exposed
either to La(NO3)3 (Fig. 4e, 5e) or GAN (Fig. 4b, 5b). Cells that are
exposed to La3+ display strong fluorescence in green channel (Fig.
4h, 4k and 5h, 5k) and no fluorescence in red channel. Thus, the
probe efficiently detects intra-cellular La3+ ion in live cell.
The comparison table (Table S3, ESI) shows the efficiency of GAN
for La3+ sensing over other pioneering La3+ sensors.
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image of the cells after incubating with different concentration of La3+
Time and concentration dependent cell imaging studies
For fluorescence visualization of intracellular La3+, HeLa and SiHa
cells are incubated with varying concentration of GAN (20-100 μM )
and La3+ (20-100 μM ) at 370C and 5% CO2 in culture medium. After
washing with PBS thrice, the fluorescence images of the cells are
captured (Figs. 6-7).
ranging from 20, 30, 40, 50 and 100 μM respectively and (a’’V-eie’w’)Aarrtieclme Oerngliende
DOI: 10.1039/D0NJ02415E
images (bright field and fluorescence) of cells (scale bar 200 μm).
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Fig. 7. GAN concentration dependent imaging of HeLa cells: (a-d) bright
field image of cells after incubating with La3+ (50 μM); (a’-d’) fluorescence
image of the cells after incubating with different concentration of GAN
ranging from 20, 30, 40, 50 and 100 μM respectively; (a’’-d’’) merge images
(bright field and fluorescence) of cells (scale bar 200 μm).
Moreover, it is observed that with increasing incubation time, the
fluorescence intensity in green channel increases. Therefore, a time
dependent fluorescence imaging is performed with live SiHa cells
and presented in Fig. 8.
Fig. 4. Imaging of La3+ in HeLa cells: (a) bright field image of cells after
incubating with GAN (20 μM); (b) fluorescence image of the same cells of
set (a); (c) overlay images of (a) and (b); (d) bright field image of cells after
incubating with La3+ (50 μM) only; (e) fluorescence image of the same cells
Thus, cell permeable and stable GAN effectively bind intracellular
of set (d); (f) merge images of (d) and (e); (g) and (j) are bright field images La3+ that allow its intracellular imaging. The cytotoxicity of the
GAN on Hela cells have been tested by MTT assay (Fig.S17, ESI).
It is observed that 90% of the cells upon treatment with 20 μM of
GAN for 12h remain alive. Hence, the concentration of the GAN has
been fixed to 20 µM in all cell line experiments.
of cells after incubating with GAN (20 μM); (h) and (k) are fluorescence
image of the cells in (g) and (j) upon incubation with 50 μM La3+; (i) and (l)
are merge images of (g), (h) and (j), (k) respectively (scale bar 200 μm).
Fig. 8. Incubation time dependent fluorescence images of SiHa cells exposed
to 50 µM La3+ and 20 µM GAN: (a) bright field image of cells after 30 min;
(b) fluorescence image of the same set of cells; (c) overlay images of (a) and
(b); (d) bright field image of cells after 60 min; (e) fluorescence image of the
same set of cells; (f) merge images of (d) and (e); (g) bright field image of
cells after 90 min, (h) fluorescence image of the same set cells; (i) overlay
images of (g) and (h); (j) bright field images of cells after 120 min; (k) are
fluorescence image of the same set cells; ((l) merge images of (j), (k)
respectively (scale bar 200 um).
Fig. 5 Imaging of La3+ in SiHa cells: (a) bright field image of cells after
incubating with GAN (20 μM); (b) fluorescence image of the same set of
cells; (c) overlay images of (a) and (b); (d) bright field image of cells after
incubating with La3+ (50 μM) only; (e) fluorescence image of the same set of
cells; (f) merge images of (d) and (e); (g) and (j) are bright field images of
cells after incubating with GAN (20 μM); (h) and (k) are fluorescence image
of the cells in (g) and (j) upon incubation with 50 μM La3+; (i) and (l) merge
images of (g), (h) and (j), (k) respectively (scale bar 200 μm).
La3+ enrichment studies
The selective and quantitative extraction of La3+ from aqueous
solution to organic solvent is tested with GAN. Among different
solvents tested for improved efficiency, namely CH3COOC2H5 (E%,
98.95), CHCl3 (E%, 98.89), CCl4 (E%, 98.80) and DCM (E%,
98.69), the extraction efficiency is highest with CH3COOC2H5. The
extraction efficiency is measured using present method through
monitoring of emission intensity of [La(III)-GAN] complex in
organic phase at 472 nm (Fig. 9-10). It is found that the efficiency of
La3+ extraction using GAN is higher than that of gallic acid, ethyl
gallate and gallic acid hydrazide (Table S6, Fig. S18, ESI). The
La3+ extraction efficiency of GAN is compared with other
pioneering extractant available in the literature (Table S7, ESI).
Fig. 6. La3+ concentration dependent imaging of HeLa cells: (a-e) bright field
image of cells after incubating with GAN (20 μM); (a’-e’) fluorescence
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Fig. 9 Solvent extraction of La3+ using GAN
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Conclusion
An amide-imine conjugate (GAN) derived from gallic acid and
naphthalene is structurally characterised by single crystal X-ray
diffraction analysis. The GAN selectively recognizes La3+ ion as low
as 23.93×10−9M. La3+ triggered CHEF assisted inhibition of ESIPT
process is responsible for TURN ON fluorescence of the GAN. The
GAN can image intracellular La3+ ion in live HeLa and SiHa cells
under fluorescence microscope in
a time- and concentration
dependent manner. In addition, the [La(III)-GAN] complex
efficiently catalyses the synthesis of benzimidazole derivative from
o-phenylenediamine and substituted benzaldehyde. Finally, GAN is
very useful for enrichment of La3+ from aqueous to ethyl acetate
medium.
Conflicts of interest
There are no conflicts of interest
Acknowledgement
A. Shaikh acknowledge CSIR, New Delhi for financial assistance. P.
Mukherjee acknowledges DST (New Delhi) for Inspire fellowship.
We acknowledge the assistance rendered by CAS(II) (UGC) to
Department of Chemistry, The University of Burdwan.
Notes and references
CCDC No. of GAN is 1889365. These data can be obtained free of
charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html or from
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
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