Nanomolar fluorogenic detection of Al(iii) by a series of Schiff bases in an aqueous system and their application in cell imaging

Article abstract of DOI:10.1039/c4ob00329b

Three positional isomers of a Schiff base containing -OH as end groups have been synthesized and evaluated for selective Al(iii) detection due to inhibition of ESIPT, PET and activation of CHEF in 70% aqueous medium. Devoid of any conventional fluorophore, these sensors have nanomolar detection limits with high quantum yields and naked eye sensing of Al(iii). Moreover, these probes have been demonstrated to enable the Al(iii) detection in live human HeLa cells and rat C6 glioma cells using a confocal microscope. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00329b

Organic &
Biomolecular Chemistry
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Nanomolar fluorogenic detection of Al(III) by a
series of Schiff bases in an aqueous system and
their application in cell imaging†
Cite this: Org. Biomol. Chem., 2014,
12, 4445
Sanyog Sharma,a Maninder Singh Hundal,*a Amandeep Walia,b Vanita Vanitab and
Geeta Hundal*a
Three positional isomers of a Schiff base containing –OH as end groups have been synthesized and eval-
uated for selective Al(III) detection due to inhibition of ESIPT, PET and activation of CHEF in 70% aqueous
medium. Devoid of any conventional fluorophore, these sensors have nanomolar detection limits with
high quantum yields and naked eye sensing of Al(III). Moreover, these probes have been demonstrated to
enable the Al(III) detection in live human HeLa cells and rat C6 glioma cells using a confocal microscope.
Received 13th February 2014,
Accepted 29th April 2014
DOI: 10.1039/c4ob00329b
www.rsc.org/obc
sensors for Al(III) have been reported based on various mecha-
nisms such as photoinduced charge transfer (PET), intramole-
Introduction
Aluminium is one of the most abundant metals in the earth’s cular charge transfer (ICT), aggregation induced emission
crust; it finds wide applications in our daily life such as in (AIE), excimer/exciplex formation, fluorescence resonance
food additives, packing materials, water treatment, paper energy transfer (FRET), CvN isomerization and chelation-
making, colours and in some medicines.1 The toxicity of Al(III) enhanced fluorescence (CHEF).6 From the literature,6 it is
causes diseases such as Alzheimer’s, Parkinson’s disease, clear that there are even fewer chemosensors which comply
bone softening, impaired lung function, fibrosis, chronic renal with all four desirable features in a chemical sensor at the
failure, etc.2 Moreover the concentration of aluminium has same time, i.e. working in aqueous systems, low detection
been found to be crucial for fish and also for agricultural pro- limit, high selectivity and applicability in living systems. We
duction as it increases the acidity of the soil.3 The FAO/WHO report here three probes which meet these criteria, namely
Joint Expert Committee on food additives recommends a daily they provide high selectivity with low detection limit in
intake of Al(III) ions for the human body of 3–10 mg and a aqueous medium and have good response in biological
weekly intake of 7 mg kg−1 body weight.4 Since there is a close systems.
association between Al(III) and human health, the detection of
Previously, our group reported sensors based on an aro-
Al(III) is crucial in controlling its concentration in the bio- matic platform which had imine, hydroxyl, urea/thiourea and
sphere. Among various sensing techniques, fluorescence sig- thiosemicarbazides as receptor-cum-transducers for sensing.7
nalling offers the advantages of high selectivity, sensitivity and As an extension of our work on imine/hydroxyl containing
rapid response.5 However, due to the poor coordination ability probes, we have now designed systems DBIH1–DBIH3 which
of aluminium, the development of its sensors is quite difficult contain a combination of –CONH, CvN and –OH groups
as compared to other biologically important cations such as (Scheme 1) to see their cooperative effect on sensing. Since Al(III)
Cu(II), Pb(II), Hg(II), Zn(II) etc. Recently, a few fluorescence is a hard acid, accordingly it prefers systems containing
hard base sites as O and N; hence, DBIH1–DBIH3 provide an
ideal co-ordination environment for selective and sensitive
detection of Al(III) in HEPES buffer (pH 7.4, containing 30%
DMSO as a co-solvent). Devoid of any conventional fluoro-
phore, these easily synthesized systems provide a highly selec-
aDepartment of Chemistry, UGC Center for Advance Studies, Guru Nanak Dev
University, Amritsar 143005, Punjab, India. E-mail: geetahundal@yahoo.com
bDepartment of Human Genetics, Guru Nanak Dev University, Amritsar 143005,
Punjab, India
tive detection of Al(III) ions with high quantum yield. The
detection limits achieved for DBIH1–DBIH3 are 8.91, 14.1 and
19.95 nM comparable to the concentration range of Al(III) ions
found in many chemical and biological systems. In addition,
DBIH1–DBIH3 have found practical application in the form of
‘dip-sticks’ which can provide instant detection of Al(III) and at
†Electronic supplementary information (ESI) available: Spectral characterization
of sensors, UV spectral studies, fluorescence titration, Benesi–Hildebrand plots,
Job’s plots, detection limit and free energy calculations, competitive selectivity,
spectral characterization and comparison of sensors with the literature and the
MTT assay of chemosensors DBIH1, DBIH2 and DBIH3. CCDC 995844. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4ob00329b
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Scheme 1 Synthesis of sensors.
the same time in the estimation of Al(III) in live HeLa cells and cular H-bonding (Fig. 1) between the imine nitrogens N2 and
C6 glioma cells using a confocal microscope.
N4 and the ortho-hydroxyl groups (O2–H2A⋯N2 2.678(4) and
O5–H5A⋯N4 2.717(4) Å), respectively. The meta-hydroxyl group
O3 at one end of the dipodal molecule is engaged in four inter-
molecular H-bonding interactions. It accepts one bond from
the imine nitrogen N3 and hydroxyl oxygen O5 whereas it
donates two H-bonds to imine N4 and carbonyl O4. The latter
three H-bonds help in forming a zigzag chain parallel to the
b-axis in a head to tail manner. The O6⋯O1 intermolecular
H-bonding interactions between the hydroxyl group and the
carbonyl oxygen (Table S2†) on the other end help in the
formation of another zigzag chain parallel to the previous one
(Fig. S1†), thus forming H-bonded undulating tapes, growing
the crystal structure in the bc plane. The N3⋯O3 interactions
result in similar but centrosymmetric tapes and the overall
crystal structure ends up as a double helical H-bonded struc-
ture as shown down the b-axis (Fig. S2†).
Results and discussion
DBIH1–DBIH3 were synthesized by Schiff base condensation
reaction (Scheme 1) and characterized by various spectroscopic
techniques (Fig. S3 and S14†) and X-ray analysis (for DBIH1).
X-ray crystal structure of DBIH1
The two arms of the DBIH1 are twisted with respect to each
and the central phenyl anchor, making dihedral angles of 16.9(1)
and 31.4(1)° with the anchor, respectively, and 46.5(1)°
between each other and they lie on opposite sides of the
central phenyl ring (inset, Fig. 1). There is strong intramole-
1H NMR of DBIH1–DBIH3
The chemical shifts of two –OH groups in the three isomers
follow a trend since using the same concentration (5 mM), the
α and β protons appear more downfield shifted in the meta
isomer DBIH2 (Fig. 2) which may be attributed to the -ortho,
-para directing effect of the –OH groups, which causes a shield-
ing effect on the C atoms bearing them. The aromatic protons
‘i’ of meta isomer are also much downfield shifted than ‘j’ and
‘l’ as they are shielded due to the presence of partial negative
charge on them, owing to the +R effect of –OH groups.
Colorimetric and chromogenic spectral response of sensors
(DBIH1–DBIH3)
The absorption spectrum of DBIH1 exhibits a maximum cen-
tered at 318 nm (ε = 3.90 × 104 M−1 cm−1) while for DBIH2 at
Fig. 1 ORTEP diagram of the DBIH1 at 50% probability showing the
labeling scheme. Inset: dihedral angle between two arms of DBIH1.
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Fig. 2 1H NMR of (A) DBIH1, (B) DBIH2 and (C) DBIH3 at 5 mM concentration of each sensor.
Fig. 3 Changes in absorption spectra of (a) DBIH1, (b) DBIH2 and (c) DBIH3 (10 µM) upon gradual addition of Al(III). Inset: color changes in sensor
solutions with Al(III).
340 nm (ε = 3.09 × 104 M−1 cm−1) in HEPES buffer (pH 7.4, rescence at λem 508 nm and λem 528 nm (excitation at 325 nm
containing 30% DMSO as a co-solvent). For DBIH3, the absorp- and 358 nm, respectively). Such a fluorescence is known to be
tion spectrum exhibit bands centered at 296 nm (ε = 2.19 × 104 due to the well-known ESIPT phenomenon in the case of
M−1 cm−1) and at 358 nm (ε = 1.37 × 104 M−1 cm−1) (Fig. S15– Schiff bases.9 Whereas DBIH2 shows a much weaker emission
S17†). The absorption bands, ranging from 318 to 358 nm for band at λem 455 nm (excitation at 340 nm) which may be due
the three sensors, have been designated as internal charge to the PET phenomenon owing to the availability of lone pairs
transfer (ICT) bands involving imine and hydroxyl groups.8
of N of the –CvN group and O of the OH group.10 This differ-
With Al(III), new bands are formed at λmax 403 nm for ence in their emission behaviour may again be attributed to
DBIH1, at 384 and 403 for DBIH2 and at 430 nm for DBIH3, the ortho–para directing capability of the –OH groups which
with isobestic points at 328 nm, 358 nm and at 310, 351 and places partial negative charges on the –OH bearing carbons in
387 nm, respectively (Fig. 3). These bathochromic shifts in all the case of ortho and para isomers, making them more suscep-
three cases are consistent with a color change from colorless tible to quinone forms, facilitating the proton transfer. These
to bright yellow (inset, Fig. 3). The presence of these isobestic effects are corroborated by the observed chemical shift values
points confirms the establishment of equilibrium between two of α- and β-OH and the aromatic protons (vide supra). All three
species and reflects the formation of 1 : 1 complexes.
isomers form a rigid chelated system with Al(III) due to coordi-
nation through OH and imine (CHvN) groups and conse-
quently exhibit a strong fluorescence enhancement due to the
Fluorogenic spectral response of sensors (DBIH1–DBIH3)
Fluorescence characteristics of DBIH1–DBIH3 (2.5 µM) were CHEF mechanism.
investigated in HEPES buffer (pH 7.4, containing 30% DMSO
DBIH1 exhibits strong fluorescence enhancement (Φ =
as a co-solvent). DBIH1 and DBIH3 exhibit very weak fluo- 0.63) upon addition of Al(III) centered at 508 nm (λex 325 nm)
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AlCl3 were used (Fig. S30, S32 and S34†). Consequently, due to
significant response of sensors towards Al(III), all the further
studies were carried out with Al(III) only.
Job’s plot13 obtained from emission data showed 1 : 1
stoichiometry for all the Al(III) complexes of DBIH1–DBIH3
(Fig. S21–S23†). This complex formation was further supported
by ESI/MS, peaks at m/z 459.0798 [(M − 2H) + Al(III)]+ (calc.
459.0885), 495.0797 [(M − 2H) + Al(III) + 2H2O]+ (calc.
495.1097), 537.0963 [((M − 2H) + Al(III) + NO3 + H2O) − 2]+
−
−
(calc. 537.0713), 615.1080 [((M − 2H) + Al(III) + NO3 + H2O +
DMSO) − 2)]+ (calc. 615.0852) for DBIH1. Similarly, peaks at
m/z 459.0888, 537.1035, 615.1165 for DBIH2 and at m/z
459.0457, 537.1008, 615.0988 for DBIH3 were observed
(Fig. S37, S39 and S41†). The occurrence of a peak at 537.07 in
all three cases indicates the formation of the proposed
[(C22H18AlN5O10) − 2] complex (Fig. 6).
Titration of DBIH1–DBIH3 with Al(III) (Fig. S24, S26 and
S28†) was followed by fluorescence to determine binding con-
stants, 5.57 × 105
M ,
−1, 2.5 × 106 M−1 and 7.0 × 104 M−1
Fig. 4 Changes in fluorescence spectra of DBIH1 (2.5 µM) upon the
addition of 10 equiv. of metal nitrates. Inset: color changes in sensor
solution with Al(III) under a UV lamp.
respectively, employing the Benesi–Hildebrand plot14 (Fig. S25(a),
S27(a) and S29(a)†) [comparable to the absorbance data
(Fig. S18, S19 and S20†)]. The metal complexation is confirmed
(Fig. 4) accompanied by a green fluorescence (inset, Fig. 4). by the free energy values of the complexation processes such
Similarly, upon Al(III) addition to DBIH2 a remarkable fluo- as −32.75 kJ mol−1, −36.50 kJ mol−1 and −27.64 kJ mol−1 for
rescence enhancement (Fig. 5a) at λem 455 nm (λex 340 nm) DBIH1–DBIH3, respectively, obtained using the equation ΔG =
was observed with quantum yield11 (Φ = 0.7) giving blue fluo- −2.303 RT log Ka (S20). Respective detection limits of these
rescence (inset, Fig. 5a). In DBIH3, when excited at λex 358 nm sensors for Al(III) calculated according to the literature15 were
after Al(III) addition, fluorescence enhancement (Fig. 5b) was found to be 8.91 × 10−9 M, 14.1 × 10−9 M and 19.95 × 10−9 M,
observed at 528 nm with quantum yield (Φ = 0.77) yielding which are quite low to detect the submicromolar concentration
green fluorescence (inset, Fig. 5b). For all the sensors, the of Al(III) (Fig. S25(b), S27(b) and S29(b)†).
addition of Li(I), Na(I), K(I), Ag(I), Mg(II), Ca(II), Cd(II), Ba(II),
Competitive selectivity of DBIH1–DBIH3 for Al(III) (Fig. S31,
and Sr(II) showed no significant change, whereas Bi(III) and S33 and S35†) was determined by fluorescence titration with
Zn(II) caused very little enhancement and Co(II), Cr(III), Cu(II), Al(III) in the presence of other metal ions under study, which
Ni(II), Hg(II), Pb(II), and Fe(III) showed quenching to different revealed that Al(III) can be detected in the presence of other
extents.10d,12 Selectivity of sensors DBIH1–DBIH3 showed no competitive metal ions. For this experiment, DBIH1–DBIH3
change when other aluminium salts such as Al2(SO4)3 and were treated with 10 equivalents of Al(III) in the presence of 50
Fig. 5 Changes in fluorescence spectra of (a) DBIH2 and (b) DBIH3 (2.5 µM) upon the addition of 10 equiv. of metal nitrates under study. Inset:
color changes in sensor solutions with Al(III) under a UV lamp.
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Al(NO3)3·9H2O in ethanol and characterizing them using
NMR, IR and mass spectroscopy (Fig. S36–S41†).
In order to check the practical applicability of DBIH1–
DBIH3, we prepared dip sticks by coating paper strips with
DMSO–H2O solution of sensors. After drying, these strips were
dipped in the solution (5 µM) of Al(NO3)3 in distilled water,
dried and observed under a UV lamp. Similar color changes
were observed in the solid state (Fig. 8) as those found earlier
in the solution state. The different fluorescent colors obtained
not only detect the presence of Al(III) but also distinguish
between three positional isomers.
Fig. 6 Proposed structure of Al(III) complexes of DBIH1–DBIH3.
Bioimaging of Al(III) in live cells
For the investigation of biological applications of the sensors
DBIH1–DBIH3, cell imaging studies have been performed with
both the human cervical cancer cell line (HeLa cells) (Fig. 9)
and glial cells of the rat brain (C6 glioma cells) (Fig. 10). Both
the HeLa and C6 glioma cells, themselves and after incubation
with Al(III) (10 µM and 50 µM), did not exhibit any fluorescence
(Fig. 9(a)–9(c) and 10(a)–10(c), respectively). Further, HeLa and
C6 glioma cells were incubated with 10 µM of DBIH1, DBIH2
and DBIH3 for 30 min at 37 °C. When imaged after incu-
equivalents of other metal ions under study. There is no sig-
nificant interference for the detection of Al(III) in the presence
of other metal ions except in the case of Cu(II) and Fe(III) which
showed some quenching of fluorescence but enhancement is
still very prominent.
1H NMR titration experiments of sensors (DBIH1–DBIH3)
In order to investigate the mode of the binding sensor to Al(III) bation, HeLa as well as C6 glioma cells showed no fluo-
ions, NMR titrations were carried out in DMSO-d6 (Fig. 7). In rescence for DBIH1 (Fig. 9(d), 10(d)) and DBIH2 (Fig. 9(g),
all the cases, signals due to α-OH protons disappear as a result 10(g)) but faint green fluorescence for DBIH3 (Fig. 9( j) and
of deprotonation and complexation but those of β-OH, imine 10( j)) was observed in both HeLa and C6 glioma cells. The
and –NH groups sustain. The chemical shift values of β-OH excitation laser used for DBIH1, DBIH2 and DBIH3 was
protons show low frequency shifts in DBIH1 and DBIH2 (Δδ 405 nm. Once the treated cells (HeLa and C6 glioma) were
0.042 and 0.712) and a high frequency shift in DBIH3 (Δδ incubated with Al(III) (10 µM) for another 30 min at 37 °C,
0.267), which may be due to different kinds and extents of bright green fluorescence was observed in both the types of
H-bonding interactions. The complexation was confirmed by cells for DBIH1 (Fig. 9(e) and 10(e)) which increased signifi-
isolating the solid complexes by reacting DBIH1–DBIH3 with cantly with 50 µM Al(III) (Fig. 9(f) and 10(f)). Similarly, green
Fig. 7 Changes in partial 1H NMR of DBIH1–DBIH3 (5 mM) upon the addition of Al(III) in DMSO-d6.
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Fig. 8 Fluorescent color changes with dip sticks formed from DBIH1–
DBIH3 (2.5 µM) in DMSO–H2O upon treatment with 5 µM of Al(III). Left –
before and right – after Al(III) treatment.
Fig. 10 Images of C6 glioma cells: (a) brightfield image of C6 glioma
cells, (b) fluorescence image of C6 glioma cells incubated with Al(III)
(10 µM) for 30 min, (c) fluorescence image of C6 glioma cells incubated
with Al(III) (50 µM) for 30 min, (d) fluorescence image of C6 glioma cells
incubated with DBIH1 (10 µM) for 30 min and further incubation with (e)
10 µM Al(III) and (f) 50 µM Al(III), (g) fluorescence image of C6 glioma
cells incubated with DBIH2 (10 µM) for 30 min and further incubation
with (h) 10 µM Al(III) and (i) 50 µM Al(III), ( j) fluorescence image of
C6 glioma cells incubated with DBIH3 (10 µM) for 30 min and further
incubation with (k), 10 µM Al(III) and (l) 50 µM Al(III).
To investigate the cytotoxicity of DBIH1, DBIH2 and DBIH3,
the MTT assay with HeLa cells as well as C6 glioma cells was
performed. No significant differences in the proliferation of
the HeLa and C6 glioma cells were observed in the absence or
presence of 10 µM of chemosensors DBIH1 and DBIH3
Fig. 9 Images of HeLa cells: (a) brightfield image of HeLa cells, (b) flu-
orescence image of HeLa cells incubated with Al(III) (10 µM) for 30 min,
(c) fluorescence image of HeLa cells incubated with Al(III) (50 µM) for
30 min, (d) fluorescence image of HeLa cells incubated with DBIH1 (10
µM) for 30 min and further incubation with (e) 10 µM Al(III) and (f) 50 µM (80–90% cell viability with the chemosensor DBIH1 and
Al(III), (g) fluorescence image of HeLa cells incubated with DBIH2
(10 µM) for 30 min and further incubation with (h) 10 µM Al(III) and (i)
50 µM Al(III), ( j) fluorescence image of HeLa cells incubated with DBIH3
(10 µM) for 30 min and further incubation with (k) 10 µM Al(III) and (l)
90–94% cell viability with DBIH3) for both the tested cell lines
(Fig. S42†). However, with the chemosensor DBIH2 HeLa cells
showed nearly 27% cell survival while C6 glioma cells had
more than 50% (53%) cell viability, indicating the chemo-
sensor DBIH2 to be toxic for the cells. With Al(III) (50 µM) alone
there was not much effect on cell viability (90% cell viability)
with both the tested cell lines. Again, addition of 10 µM or
50 µM of Al(III) in the presence of chemosensors DBIH1 and
DBIH3 (10 µM) did not show any significant effect but DBIH2
showed a considerable effect on the cell viability of both the
cell types. These data show that chemosensors DBIH1 and
DBIH3 have very low cytotoxicity while DBIH2 is substantially
cytotoxic.
In conclusion, we have reported a series of three chromo-
fluorogenic sensors for Al(III) which can detect Al(III) up to the
nanomolar level, with high quantum yields in aqueous
medium. The detection mechanism involved is CHEF acti-
vation due to the formation of rigid aluminium complexes.
50 µM Al(III).
fluorescence of DBIH3 got enhanced when incubated with
Al(III) (10 µM) (Fig. 9(k) and 10(k)) and fluorescence got further
enhanced significantly with 50 µM Al(III) (Fig. 9(l) and 10(l)).
For DBIH2, blue fluorescence was observed with 10 µM Al(III)
treatment (Fig. 9(h) and 10(h)) which also increased consider-
ably with 50 µM Al(III) (Fig. 9(i) and 10(i)) in both the tested
cell lines. The fluorescence was observed in the perinuclear
region as well as the cytosol; hence, it indicates that chemo-
sensors DBIH1, DBIH2 and DBIH3 are permeable to HeLa and
C6 glioma cells and can be used for detecting Al(III) in the live
cells.
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Additionally, on complexation these positional isomers can be UV-vis and fluorescence studies
distinguished visually under UV light illumination, as well as
Molecular interactions of DBIH1, DBIH2 and DBIH3 with 19
different metal nitrates under study were investigated by UV-
vis spectroscopy at 10−5 M and fluorescence spectroscopy at
2.5 × 10−6 M in HEPES buffer (pH 7.4, containing 30% DMSO
as a co-solvent). Stock solutions of DBIH1, DBIH2 and DBIH3
(10−3 M) and of metal nitrates (10−1–10−3 M) were prepared in
DMSO and distilled water respectively. Selectivity tests were
performed by 2.5 × 10−6 M of all the sensors and 10 equiv. of
Al(III) in the presence of 50 equiv. of other interfering metal
ions. The binding stoichiometries of Al(III) complexes of
DBIH1–DBIH3 were determined by the method of continuous
by dip stick experiment, even at 5 µM of Al(III) without the aid
of any sophisticated instrument. Moreover, cell imaging experi-
ments with HeLa cells and C6 glioma cells establish the utility
of these sensors for tracking Al(III) in live cells.
Experimental
General procedures
All the commercially available chemicals were purchased from variation (Job’s plot). Ten solutions were prepared by varying
Aldrich and used without further purification. All solvents the L/M ratio and keeping the total concentration of all the
were dried by standard methods. Dimethyl isophthalate (DIP) sensors and the cationic guest constant (2.5 × 10−5 M) with
and isophthalohydrazide (IPH) were prepared according to lit- continuous variation of mole fraction of DBIH1–DBIH3. The
erature methods.16 TLC was carried out on glass sheets pre- results indicate the formation of complexes with a stoichio-
coated with silica gel. Elemental analyses (C, H, N) were per- metric ratio of 1 : 1. The stability constant was determined
1
formed on a Perkin-Elmer model 2400 CHN analyzer. The H using a Benesi–Hildebrand plot in each case.
and 13C NMR spectra were carried out in DMSO-d6 with TMS
Calculation of quantum yield
as an internal reference on a JEOL-FT NMR-300 MHz spectro-
photometer. The infrared spectra (KBr pellet) were recorded
using a Perkin-Elmer FT-IR C92035 spectrophotometer in the
range 400–4000 cm−1. The electronic absorption spectra were
recorded on a Shimadzu Pharmaspec UV-1700 UV-vis spectro-
photometer with a quartz cuvette (path length, 1 cm). The
absorption spectra have been recorded between 1100 and
200 nm. The cell holder of the spectrophotometer was thermo-
stated at 25 °C for consistency in the recordings. Fluorescence
spectra were recorded on a Varian fluorospectrophotometer.
HRMS spectra were recorded on a Bruker’s microTOF-QII
spectrophotometer.
The fluorescence quantum yield Φf for DBIH1–DBIH3 was
determined at room temperature using optically matching
solutions of 9,10-diphenylanthracene (Φf = 0.90) in ethanol as
the standard at an excitation wavelength of 325, 340 and
358 nm respectively. The quantum yield was calculated using
eqn (1), in which Φfs is the radiative quantum yield of the
sample, Φfr is the radiative quantum yield of the reference, As
and Ar are the absorbances of the sample and the reference,
respectively, Ds
1 ꢁ 10ꢁA L
Ns
Nr
Ds
Dr
2
s
s
r
Φfs ¼ Φfr ꢀ
ꢀ
2 ꢀ
ð1Þ
1 ꢁ 10ꢁA L
r
and Dr are the areas of emission for the sample and the refer-
ence, Ls and Lr are the lengths of the absorption cells, and Ns
and Nr are the refractive indices of the sample and reference
solutions (pure solvents were assumed).
X-Ray measurements and structure determination
The crystals of the compound DBIH1 were grown by slow evap-
oration from
a mixture of N,N-dimethylformamide and
ethanol. X-Ray data were collected on a Bruker’s Apex-II CCD
diffractometer using Mo Kα (λ = 0.71069 Å) at room tempera-
Cell imaging studies
ture. The data were corrected for Lorentz and polarization Both the tested cell lines (HeLa and C6 glioma) were grown in
effects and empirical absorption corrections were applied Dulbecco’s modified Eagle’s medium (DMEM) supplemented
using SADABS from Bruker. A total of 15 046 reflections were with 10% fetal bovine serum (FBS) and 100 IU ml−1 penicillin,
measured out of which 3788 were independent and 1850 were 100 µg ml−1 streptomycin and 100 µg ml−1 gentamycin. The
observed [I > 2σ(I)] for theta 26°. The structures were solved by cells were maintained in a humidified incubator at 37 °C with
direct methods using SIR-92 and refined by full-matrix least 5% CO2. On the day before treatment, a total of 2 × 105 cells
squares refinement methods based on F2 using SHELX-97. The were seeded on 11 mm glass coverslips into each well of a
hydrogens of the –OH and –NH groups were located from the 24-well plate and these were grown for 24 hours (till 60–70%
difference Fourier synthesis and were refined isotropically with confluence) and treatment was carried out in triplicates in FBS
Uiso values 1.2 times that of their carrier oxygen atoms, with and antibiotic free media. HeLa and C6 glioma cells were incu-
restraints on the bond distances. All non-hydrogen atoms were bated with three different ligands, i.e., DBIH1, DBIH2 and
refined anisotropically. All other hydrogen atoms were fixed DBIH3 (10 µM) (each in triplicates), at 37 °C with 5% CO2 for
geometrically with their Uiso values 1.2 times those of the 30 min followed by three times washing with 1× phosphate
phenylene carbons. All calculations were performed using the buffered saline (PBS) (pH = 7.4) and treatment with Al(III)
Wingx [3] package. The important crystal and refinement para- (10 µM and 50 µM conc. each in three replicates) for another
meters are given in Table S1.†
30 min by incubating the cells under the same conditions. The
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cells were then washed three times with 1× PBS, fixed in ice were filtered, washed with chilled methanol and dried. Yield
cold 4% paraformaldehyde, washed again three times with 50%. Yellow solid. Mp = 300–302 °C; 1H NMR (300 MHz,
1× PBS and mounted on glass slides. To investigate the cyto- DMSO-d6) (Fig. S9,† δ): 6.75 (4H, s, Ar), 7.01 (2H, s, Ar), 7.71
toxicity of DBIH1, DBIH2 and DBIH3, the MTT [3-(4,5- (1H, t, Ar, J = 7.8 Hz), 8.13 (2H, d, Ar, J = 8.1 Hz), 8.50 (1H, s,
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay Ar), 8.61 (2H, s, –CHvN), 8.96 (2H, s, –OH), 10.31 (2H, s,
with HeLa cell lines as well as C6 glioma cells was performed –OH), 12.13 (2H, s, –NH); 13C NMR (75 MHz, DMSO-d6)
to determine the effect of DBIH1, DBIH2 and DBIH3 on cell (Fig. S10,† δ): 113.6 (Ar), 117.2 (Ar), 119.1 (Ar), 127.0 (Ar), 128.9
proliferation. Confocal microscopy imaging was performed on (Ar), 130.9 (Ar), 133.4 (Ar), 147.8 (CHvN), 149.9 (–C–OH),
a NIKON A1R confocal laser scanning microscope using diode 150.3 (–C–OH), 162.3 (–CvO); IR (KBr, cm−1) (Fig. S11†): 3385
laser excitation at 405 nm. Imaging was performed using Plan (OH), 3204 (NH), 1625 (CvO), 1580 (CvN); elemental analysis
Apo 60× oil immersion objective lens.
calculated for C22H18N4O6: C, 60.83; H, 4.18; N, 12.90%.
Found: C, 60.38; H, 4.19; N, 12.11%; HRMS m/z (Fig. S12†):
435.1300 [M + 1]+ (calc. 435.1299).
Synthesis of compounds
Synthesis of DBIH1. 200 mg (1.02 mmol) of IPH was dis-
Synthesis of the DBIH1–Al(III) complex. To a 5 ml suspen-
solved in 10 ml of ethanol to which was added 284 mg sion of DBIH1 (0.05 g, 1.15 mmol) in ethanol, a 10 ml solution
(2.05 mmol) of 2,3-dihydroxybenzaldehyde in 10 ml of ethanol of Al(NO3)3·9H2O (0.086 g, 2.30 mmol) in distilled water was
along with 2–3 mg of zinc perchlorate. The color of the solu- added dropwise over 15 minutes and then stirred for half an
tion changed immediately to turbid yellow and precipitates hour. After stirring, the reaction mixture was concentrated and
were separated out within 10 minutes. These precipitates were placed in an ice bath. The dark brown precipitates were col-
filtered, washed with methanol and dried under vacuum for lected on a Buchner funnel. Mp = 300–302 °C. 1H NMR
1
24 hours. Yield 90%. Light yellow solid. Mp = 238–240 °C; H (300 MHz, DMSO-d6) (Fig. S28(a),† δ): 6.74 (t, 2H, Ar, J = 7.5
NMR (300 MHz, DMSO-d6) (Fig. S1, ESI,† δ): 6.78 (t, 2H, Ar, J = Hz), 6.86 (d, 2H, Ar, J = 7.5 Hz), 6.98 (d, 2H, Ar, J = 7.8 Hz),
7.8 Hz), 6.89 (d, 2H, Ar, J = 6.6 Hz), 7.03 (d, 2H, Ar, J = 6.6 Hz), 7.72 (t, 1H, Ar, J = 7.5 Hz), 8.14 (d, 2H, Ar, J = 7.8 Hz), 8.51 (s,
7.76 (t, 1H, Ar, J = 7.5 Hz), 8.19 (d, 2H, Ar, J = 7.8 Hz), 8.56 (s, 1H, Ar), 8.63 (s, 2H, –CHvN), 9.27 (s, 2H, –OH), 12.30 (s, 2H,
1H, Ar), 8.67 (s, 2H, –CHvN), 9.31 (s, 2H, –OH); 11.09 (s, 2H, –NH); IR (KBr, cm−1) (Fig. S28(b)†): 3242 (OH), 3064 (NH),
–OH); 12.32 (s, 2H, –NH); 13C NMR (75 MHz, DMSO-d6) 1660 (CvO), 1614 (CvN), 1385 (–NO3); HRMS m/z (Fig. S29†):
(Fig. S2,† δ): 117.8 (Ar), 119.2 (Ar), 119.6 (Ar), 120.3 (Ar), 127.4 459.0798 [(M − 2H) + Al(III)]+ (calc. 459.0885), 495.0797 [(M −
(Ar), 129.4 (Ar), 131.4 (Ar), 133.6 (Ar), 146.0 (CHvN), 146.5 2H) + Al(III) + 2H2O]+ (calc. 495.1097), 537.0963 [((M − 2H) + Al(III)
−
(–C–OH), 149.6 (–C–OH), 162.6 (–CvO); FTIR (KBr, cm−1
)
+ NO3 + H2O) − 2]+ (calc. 537.0713), 615.1080 [((M − 2H) +
(Fig. S3†): 3299 (OH), 3090 (NH), 1647 (CvO), 1609 (CvN); Al(III) + NO3− + H2O + DMSO) − 2)]+ (calc. 615.0852).
elemental analysis calculated for C22H18N4O6: C, 60.83; H, Synthesis of the DBIH2–Al(III) complex. To a 5 ml solution
4.18; N, 12.90%. Found: C, 61.34; H, 4.20; N, 11.32%; HRMS of DBIH2 (0.05 g, 1.15 mmol) in ethanol, a 10 ml solution of
m/z (Fig. S4†): 457.1046 [M + Na]+ ion (calc. 457.1119).
Al(NO3)3·9H2O (0.086 g, 2.30 mmol) in distilled water was
Synthesis of DBIH2. The same procedure as that for DBIH1 added dropwise over 15 minutes and then stirred for half an
was used except that 2,4-dihydroxybenzaldehyde was used in hour. After stirring, the reaction mixture was concentrated and
place of 2,3-dihydroxybenzaldehyde. The color of the solution placed in an ice bath. The dark brown precipitates were col-
changed immediately to light orange and precipitates were lected on a Buchner funnel. Mp = 300–302 °C; 1H NMR
separated out within half an hour. These precipitates were fil- (300 MHz, DMSO-d6) (Fig. S30(a),† δ): 6.33 (s, 2H, Ar), 6.35 (d,
tered, washed with chilled methanol and dried under vacuum 2H, Ar, J = 8.4 Hz), 7.33 (d, 2H, Ar, J = 8.9 Hz), 7.69 (t, 1H, Ar,
for 24 hours. Yield 80%. Orange solid. Mp = 298–300 °C; 1H J = 8.1 Hz), 8.10 (d, 2H, Ar, J = 8.1 Hz), 8.45 (s, 1H, Ar), 8.54 (s,
NMR (300 MHz, DMSO-d6) (Fig. S5,† δ): 6.33 (s, 2H, Ar), 6.36 2H, –CHvN), 9.91 (s, 2H, –OH), 12.05 (s, 2H, –NH); IR (KBr,
(d, 2H, Ar, J = 8.4 Hz), 7.33 (d, 2H, Ar, J = 8.1 Hz), 7.69 (t, 1H, cm−1) (Fig. S30(b)†): 3391 (OH), 3206 (NH), 1639 (CvO), 1618
Ar, J = 8.1 Hz), 8.12 (d, 2H, Ar, J = 7.8 Hz), 8.47 (s, 1H, Ar), 8.54 (CvN), 1385 (–NO3); HRMS m/z (Fig. S31†): 459.0888 [(M −
(s, 2H, –CHvN), 9.95 (s, 2H, –OH), 11.40 (s, 2H, –OH), 12.05 2H) + Al(III)]+ (calc. 459.0885), 537.1035 [((M − 2H) + Al(III) +
−
(s, 2H, –NH); 13C NMR (75 MHz, DMSO-d6) (Fig. S6,† δ): 102.6 NO3 + H2O) − 2]+ (calc. 537.0713), 615.1165 [((M − 2H) +
(Ar), 107.7 (Ar), 110.5 (Ar), 126.7 (Ar), 128.8 (Ar), 130.6 (Ar), Al(III) + NO3− + H2O + DMSO) − 2)]+ (calc. 615.0852).
131.3 (Ar), 133.3 (Ar), 149.5 (CHvN), 159.4 (–C–OH), 160.8
Synthesis of the DBIH3–Al(III) complex. To a 5 ml solution
(–C–OH) 161.9 (–CvO); FTIR (KBr, cm−1) (Fig. S7†): 3349 (OH), of DBIH3 (0.05 g, 1.15 mmol) in ethanol, a 10 ml solution of
3128 (NH), 1660 (CvO), 1607 (CvN); elemental analysis calcu- Al(NO3)3·9H2O (0.086 g, 2.30 mmol) in distilled water was
lated for C22H18N4O6: C, 60.83; H, 4.18; N, 12.90%. Found: added dropwise over 15 minutes and then stirred for half an
C, 60.86; H, 4.16; N, 12.87%; HRMS m/z (Fig. S8†): 435.1283 hour. After stirring, the reaction mixture was concentrated and
[M + 1]+ (calc. 435.1299).
placed in an ice bath. The dark brown precipitates were col-
Synthesis of DBIH3. In this case 2,5-dihydroxybenzaldehyde lected on a Buchner funnel. Mp = 300–302 °C; 1H NMR
was used in place of 2,3-dihydroxybenzaldehyde. The color of (300 MHz, DMSO-d6) (Fig. S32(a),† δ): 6.75 (s, 4H, Ar); 7.00 (s,
the solution changed immediately to brownish orange and pre- 2H, Ar); 7.71 (t, 1H, Ar, J = 7.8 Hz); 8.13 (d, 2H, Ar, J = 8.1 Hz);
cipitates were separated out within an hour. These precipitates 8.49 (s, 1H, Ar); 8.61 (s, 2H, –CHvN); 10.17 (s, 2H, –OH); 12.13
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(s, 2H, –NH); IR (KBr, cm−1) (Fig. S32(b)†): 3408 (OH), 3250
(NH), 1666 (CvO), 1590 (CvN), 1385 (–NO3); HRMS m/z
(Fig. S33†): HRMS m/z: 459.0457 [(M − 2H) + Al(III)]+ (calc.
Analyst, 2012, 137, 3975; (c) S. Sinha, R. R. Koner,
S. Kumar, J. Mathew, P. V. Monisha, I. Kazi and S. Ghosh,
RSC Adv., 2013, 3, 345; (d) C.-H. Chen, D.-J. Liao, C.-F. Wan
and A.-T. Wu, Analyst, 2013, 138, 2527; (e) W.-H. Ding,
W. Cao, X.-J. Zheng, D.-C. Fang, W.-T. Wong and L.-P. Jin,
Inorg. Chem., 2013, 52, 7320; (f) X. Shi, H. Wang, T. Han,
X. Feng, B. Tong, J. Shi, J. Zhi and Y. Dong, J. Mater. Chem.,
2012, 22, 19296; (g) S. Samanta, B. Nath and J. B. Baruah,
Inorg. Chem. Commun., 2012, 22, 98; (h) Q. Meng, H. Liu,
S. Cheng, C. Cao and J. Ren, Talanta, 2012, 99, 464;
(i) A. Sahana, A. Banerjee, S. Lohar, B. Sarkar,
S. K. Mukhopadhyay and D. Das, Inorg. Chem., 2013, 52,
3627; ( j) H. M. Park, B. N. Oh, J. H. Kim, W. Qiong,
I. H. Hwang, K.-D. Jung, C. Kim and J. Kim, Tetrahedron
Lett., 2011, 52, 5581; (k) D. Maity and T. Govindaraju,
Chem. Commun., 2012, 48, 1039; (l) K. K. Upadhyay and
A. Kumar, Org. Biomol. Chem., 2010, 8, 4892; (m) A. Sahana,
A. Banerjee, S. Lohar, A. Banik, S. K. Mukhopadhyay,
D. A. Safin, M. G. Babashkina, M. Bolte, Y. Garcia and
D. Das, Dalton Trans., 2013, 42, 13311.
−
459.0885), 537.1008 [((M − 2H) + Al(III) + NO3 + H2O) − 2]+
−
(calc. 537.0713), 615.0988 [((M − 2H) + Al(III) + NO3 + H2O +
DMSO) − 2)]+ (calc. 615.0852).
Acknowledgements
MSH and GH are thankful to CSIR (India) for research grant
no. 01(2406)/10/EMR-II. SS is thankful to UGC for a senior
research fellowship. Live cell imaging was supported by DBT,
India (BT/IN/German/13/VK/2010) and BMBF (IND 10/036) to
VV under the framework of the Indo-German bilateral
cooperation for research.
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Org. Biomol. Chem., 2014, 12, 4445–4453 | 4453