A selective hydrolytic and restructuring approach through a Schiff base design on a coumarin platform for “turn-on” fluorogenic sensing of Zn2+

Article abstract of DOI:10.1039/c8dt03211d

A new Schiff base, CMD, designed based on a coumarin platform was synthesized and fully characterized through single crystal X-ray diffraction studies. CMD underwent selective Zn²⁺-triggered hydrolysis in ethanolic medium followed by restructur

Full text of DOI:10.1039/c8dt03211d

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A selective hydrolytic and restructuring approach
through a Schiff base design on a coumarin
platform for “turn-on” fluorogenic sensing
of Zn2+†
Cite this: DOI: 10.1039/c8dt03211d
Abha Pandey,a Sharad Kumar Asthana,a Anand Prakash,b Jagat Kumar Roy,b
a
Ida Tiwaria and K. K. Upadhyay
*
A new Schiff base, CMD, designed based on a coumarin platform was synthesized and fully characterized
through single crystal X-ray diffraction studies. CMD underwent selective Zn2+-triggered hydrolysis in
ethanolic medium followed by restructuring of its fragments, resulting in a “turn-on” green fluorogenic
response. This response was confirmed through various physico-chemical measurements along with
single crystal X-ray diffraction studies. This selective hydrolytic fluorogenic event was exploited for the
successful optical detection and live cell imaging of Zn2+ in SiHa cells. The above restructured products
were characterized as two new Schiff bases, viz. CM and NSA, of which NSA was highly fluorescent
(green). Hence, the formation of this green fluorogenic product accounted for the above fluorogenic
“turn-on” sensing of Zn2+ with a sub-nanomolar detection limit. Spectroscopic evidence along with mass
determinations indicated that the Zn-CMD ensemble took the form of CM-Zn-CM in solution, supporting
our above proposal of hydrolysis and restructuring. However, the X-ray diffraction studies of the Zn-CMD
ensemble further revealed it to consist of NSA and CM-Zn-CM’, where CM’ is yet another new Schiff base
formed in situ during the process of developing single crystals.
Received 6th August 2018,
Accepted 2nd January 2019
DOI: 10.1039/c8dt03211d
rsc.li/dalton
apoptosis.6 Consequently, the intracellular concentration of
Zn2+ is quite high, on the order of 10 to 100 mM.7 In view of
Introduction
Zinc is second only to iron among the most biologically rele- the great importance of Zn2+ in living systems, its recognition
vant d-metals found in human beings.1 The amounts of zinc is very important, particularly at low levels.8 A large number of
ranges from nM to mM in the human body.2 Most Zn2+ ions optical chemosensors for various types of analytes have been
are bound to proteins,3,1b while a small part remains in fluid. reported in the literature in the past few decades.9 Because it
Disruption in the mobility of Zn2+ leads to β-amyloid for- is highly relevant from the viewpoint of living systems, Zn2+
mation, which in turn is responsible for a number of diseases, has been targeted by chemists with a variety of chemo-
such as Alzheimer’s, ischemia, epilepsy, Parkinson’s, etc.4 On sensors.10 However, a considerable number of these chemo-
account of the unique stereochemical flexibility of Zn2+, it is sensors suffer from the problem of interference from one or
an essential cofactor in various enzymes and proteins.5 more metal ions, viz. Mg2+, Ca2+, Cd2+, etc.11 However, this
Additionally, it plays considerably important roles in
a
problem has been addressed to a large extent through chemo-
plethora of biological processes, such as gene transcription, dosimetric approaches.12 In this connection, the chemical lit-
regulation of metalloenzymes, neuronal transmission and erature incorporates a number of reports involving the quench-
ing of >CvN isomerization/hydrolysis, leading to remarkable
colour changes.13 A few mentionable reports in the literature
aDepartment of Chemistry, Institute of Science, Banaras Hindu University, Varanasi-
were also provided by our own research group.14 The design
221005, Uttar Pradesh, India. E-mail: drkaushalbhu@yahoo.co.in, kku@bhu.ac.in;
strategy of our probe (CMD) is simple; 7-hydroxy, 4-methyl cou-
Tel: +91542 670 2488
bDepartment of Zoology, Institute of Science, Banaras Hindu University,
Varanasi-221005, Uttar Pradesh, India
marin aldehyde was condensed with hydrazine hydrate in a
1 : 1 molar ratio, and the resulting product was further
condensed with 4-diethylaminosalicylaldehyde, again in a
1 : 1 molar ratio.
The selection of N,N-diethyl salicylaldehyde as a constituent
was based on our previous experience and that of other
†Electronic supplementary information (ESI) available: 1H NMR, 13C NMR, IR,
mass spectrum of CMD, NSA, CM, CM-Zn-CM and CM-Zn-CM′ and crystal refine-
ment data of CMD, NSA and CM-Zn-CM′. CCDC 1588901, 1856059 and 1836451.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c8dt03211d
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Scheme 1 Synthetic scheme for CMD.
(∼100 mL) with stirring. The off-white precipitate thus formed
was filtered and was dried in a vacuum evaporator (Scheme 1).
Spectroscopic characterization data for 4-methyl-7-hydroxy-
coumarinol. Yield: 80%; M.p. 94–97 °C; IR/cm−1 of CMD:
3501, 3114, 2819, 1669, 1607, 1518, 1454, 1394, 1277, 1226,
1559, 1137, 989, 848, 807, 585 (Fig. S1, ESI†).
Fig. 1 Chemical structures of CMD, NSA, CM’, CM, CM-Zn-CM’ and
CM-Zn-CM.
Synthesis of ACM
The synthesis of ACM (7-hydroxy-4-methyl-2-oxo-2H-chromene-
8-carbaldehyde) is shown in Scheme 1. In a 100 mL round
bottom flask, glacial acetic acid (20.0 mL) along with 4-methyl-
7-hydroxycoumarin (5.0 mmol) was taken, and hexamethyl-
enetetramine (15.0 mmol) was added to the above reaction
mixture. The reaction mixture was boiled under reflux at 80 °C
to 85 °C for ∼20 h. Subsequently, 5 mL of water and 30 mL dil.
hydrochloric acid were added to this reaction mixture, and
refluxing was continued for ∼45 minutes. The reaction mixture
was cooled to r.t. and was further extracted with diethyl ether.
Upon evaporation of diethyl ether, a yellow solid was isolated
which was further recrystallized from ethanol to afford the
pure product.
researchers regarding its multiple uses on a wide scale in the
design and synthesis of chemosensors (Fig. 1).15
Zn2+ is a good Lewis acid in addition to being very biologi-
cally important; hence, we chose it as an analyte to examine
the possibility of successful optical fluorogenic recognition by
CMD. To our surprise, our strategy worked well in the sense
that one highly fluorescent restructured Schiff base, viz. NSA,
was selectively formed in due course of the interaction of CMD
with Zn2+. This provided an indirect but foolproof method to
detect the presence of Zn2+ in the system. This is the key step
of this sensing process. At the same time, yet another restruc-
tured Schiff base, CM, was formed and ligated successfully
with Zn2+ in a 2 : 1 molar ratio (CM-Zn-CM) with an arrange-
ment of N2O4 donor sets in an octahedral geometrical pattern.
Yet another structural rearrangement took place during the
course of single crystal development, where one more new
Schiff base, CM′, was synthesized through interaction with
dichloromethane, which was used for the layering. CM′ co-
ordinated with Zn2+ by replacing one CM in CM-Zn-CM, which
ultimately resulted in the isolation of CM-Zn-CM′. Here, we
observed N3O3 donor sets around Zn2+ in an octahedral geo-
metrical pattern. Although a number of chemodosimetric
approaches for analytes including Zn2+ have been reported in
the literature, the restructuring of the fragments and the
detailed characterization presented here are rare.
Spectroscopic characterization data for ACM. Yield: 24%. M.
p. 185.0–186.0 °C; IR/cm−1 of ACM: 3054, 2325, 1749, 1645,
1479, 1434, 1386, 1339, 1297, 1223, 1166, 1075, 927, 897, 750,
1
717, 650, 591, 473, 1455, 540, 428; H NMR (500 MHz, CDCl3,
TMS): δ ppm = 12.21 (s, –OH), 10.62 (s, –CHO), 7.74–7.72 (d,
1H), 6.92–6.90 (d, 1H), 6.20 (s, 1H) (Fig. S2 and S3, ESI†).
Synthesis of ACM-hydrazide (ACM-Hz)
(E)-8-(Hydrazonomethyl)-7-hydroxy-4-methyl-2H-chromen-2-one
was synthesized by adding NH2–NH2·2H2O (5.0 mmol, 160 μL)
to 20 mL of a mixture of ethanol and acetic acid (2 : 1) contain-
ing ACM (0.204 g, 1.0 mM). The reaction mixture was further
refluxed for ∼6 h at 75 °C. The product was isolated by filtering
over a sintered glass (G4) crucible. The product was washed
several times with distilled water and dried under vacuum over
anhydrous CaCl2 (Scheme 1).
Experimental section
Synthesis of 4-methyl-7-hydroxy-coumarinol
Spectroscopic characterization data for ACM-Hz. Yield: 70%;
First, 20.0 mmol (2.202 g) resorcinol was dissolved in 20 mL decomposed 241–246 °C; IR/cm−1 of ACM-Hz: 3379, 3215,
dil. sulfuric acid in a 100 mL round bottom flask; then, 1916, 1717, 1611, 2972, 2928, 1736, 1633, 1615, 1514, 1387,
25.0 mmol (∼3.2 mL) ethyl acetoacetate was added to the 1356, 1293, 1240, 1190, 1165, 1134, 1074, 1052, 936, 897, 844,
1
solution with constant stirring at 0 °C to 5 °C for ∼6 hours. 773, 705, 651, 595, 458; H NMR (500 MHz, DMSO-d6, TMS): δ
Finally, the reaction mixture was poured into ice cold water ppm = 12.76 (s, –OH), 8.37 (s, –CvN), 7.53–7.51 (d, 1H), 7.34
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(s, 2H), 6.84–6.82 (d, 1H), 6.18 (s, 1H), 2.36 (s, 3H) (Fig. S4 and
S5, ESI†).
To verify the in situ synthesized products, we freshly syn-
thesized and characterized NSA, CM and CM-Zn-CM.
Synthesis of NSA
Synthesis of CMD
NSA was synthesized by the reaction of an equimolar mixture
The synthesis of CMD, 8-((E)-((E)-(4-(diethylamino)-2-hydroxy-
benzylidene)hydrazono)methyl)-7-hydroxy-4-methyl-2H-chromen-
2-one, is shown in Scheme 1. A 2.0 mmol absolute ethanolic
solution of 4-(diethylamino)-2-hydroxybenzaldehyde was
added to a separate equimolar absolute ethanolic solution of
(E)-8-(hydrazonomethyl)-7-hydroxy-4-methyl-2H-chromen-2-
one at room temperature for ∼5 hours. A dark yellow solid was
finally filtered and was dried under vacuum over anhydrous
CaCl2. CMD was characterized through various spectroscopic
of
4-(diethylamino)-2-hydroxybenzaldehyde
and
NH2–
NH2·2H2O according to a method previously reported by our
research group.15a
Spectroscopic characterization data for NSA. IR/cm−1: 2972,
2929, 1628, 1593, 1557, 1515, 1485, 1453, 1414, 1375,
1352, 1300, 1228, 1132, 1080, 1014, 787, 761, 709, 650, 560;
1H NMR (300 MHz, DMSO-d6, TMS): δ ppm = 1H NMR of
NSA (500 MHz, DMSO-d6, TMS): δ ppm = 11.45 (s, –OH),
8.56 (s, –CvN), 7.24–7.22 (d, 1H), 6.27–6.25 (d, 1H), 6.07
(s, 1H), 3.00–3.00 (q, 4H), 1.08–1.05 (t, 6H). 13C NMR
1
techniques such as IR, H and 13C NMR, and ESI-mass spec-
tral studies along with single crystal XRD analysis (Fig. S6–S9,
ESI†).
(125 MHz, DMSO-d6)
δ ppm = 160.66, 160.64, 150.93,
133.03, 106.40, 104.06, 97.05, 43.87, 12.54. ESI-MS: m/z calcu-
lated for [C22H30N4O2] = 382.24, found = 383.15 (Fig. S13–S16,
ESI†).
Spectroscopic characterization data for receptor CMD. Yield:
70%; M.p. 224–228 °C; IR/cm−1 of CMD: 3441, 2972, 2928,
1736, 1633, 1615, 1514, 1387, 1356, 1293, 1240, 1190, 1165,
1134, 1074, 1052, 936, 897, 844, 773, 705, 651, 595, 458; 1H
NMR (500 MHz, CDCl3, TMS): δ ppm = 12.89 (s, –OH), 11.42 (s,
–OH), 9.28 (s, –CH), 8.52 (s, –CH), 7.55–7.53 (d, –1H),
7.12–7.10 (d, –1H), 6.95–6.93 (d, –1H), 6.28–6.26 (d, –1H), 6.24
(s, 1H), 6.15 (s, –H), 3.43–3.38 (q, –4H), 2.42 (s, 3H), 1.23 (t,
6H); 13C NMR (125 MHz, CDCl3) δ ppm = 164.32, 162.95,
162.21, 160.25, 155.01, 153.55, 153.00, 152.23, 134.18, 127.87,
113.79, 112.07, 111.39, 106.32, 106.23, 104.41, 97.82, 44.66,
18.90, 12.67; ESI-MS: m/z calculated for [C22H23N3O4] = 393.17,
found = 394.3.
Synthesis of CM
The compound CM [((8,8′-((1E,1′E)-hydrazine-1,2-diylidenebis
(methanylylidene))bis(7-hydroxy-4-methyl-2H chromen-2-one))]
was synthesized by the reaction of an equimolar mixture of
previously synthesized ACM-HZ and ACM (8-formyl-7-hydroxy-
4-methyl-coumarin) in ethanol at reflux for six hours
according to a previously reported method.16 CM was syn-
thesized as a yellow precipitate, which was filtered and dried
under vacuum.
Spectroscopic characterization data for CM. Yield: 82%; IR/
cm−1 of ACM-Hz: 3417, 3084, 2960, 1732, 1633, 1620, 1585,
1488, 1436, 1390, 1293, 1220, 1185, 1074, 936, 844, 807, 773,
457; 1H NMR (500 MHz, DMSO-d6, TMS): δ ppm = 12.72 (s,
–OH), 8.40 (s, –CvN), 7.54–7.52 (d, 1H), 7.26 (s, 1H), 6.85–6.83
(d, 1H), 6.17 (s, 1H), 2.35 (s, 3H). 13C NMR (125 MHz,
DMSO-d6) δ ppm = 160.81, 160.02, 154.39, 151.39, 135.76,
125.70, 113.39, 112.21, 110.90, 107.37, 18.76; ESI-MS: m/z cal-
culated for [C22H16N2O6] = 404.10, found = 405.10 (Fig. S17–
S20, ESI†).
Synthesis of single crystals of the complex
Suitable crystals for X-ray diffraction studies of the complex
were developed by slow evaporation of a saturated solution of
CMD and ZnOAc prepared in 2.0 mL of a dimethylsulfoxide :
ethanolic (1 : 1) mixture followed by layering with 1.0 mL di-
chloromethane at room temperature over a period of a few
days. To our surprise, the X-ray diffraction pattern revealed the
existence of two different molecular entities in the crystals.
The first was identified as a new Schiff base in the form of
NSA, while the second was identified as a zinc complex
(CM-Zn-CM′) of yet another in situ synthesized Schiff base,
Synthesis of CM-Zn-CM
CM, by Zn2+-triggered hydrolysis of CMD as well as one of its To a 10 mL ethanolic solution of CM, (0.404 g, 1 mmol),
reaction products with dichloromethane (CM′). This in situ Zn(OAc)2·2H2O (0.676 g, 3 mmol) in ethanol (10 mL) was
restructuring also proves the hydrolysis of CMD in solution added. The yellow solution was then stirred overnight and a
(Scheme S1, ESI†).
yellow precipitate was obtained; this was filtered and washed
Spectroscopic characterization data for CM-Zn-CM′ + NSA. with a cold ethanol–water mixture (1 : 1, v/v) and dried under
IR/cm−1: 3430, 2971, 2929, 1771, 1734, 1630, 1596, 1515, 1454, vacuum.
1411, 1386, 1352, 1300, 1233, 1192, 1132, 1079, 828, 788, 710,
Spectroscopic characterization data for complex CM-Zn-CM.
457; 1H NMR (500 MHz, DMSO-d6, TMS): δ ppm = 11.44 (s, IR/cm−1 of CM-Zn-CM: 3407, 2923, 1732, 1714, 1620, 1584,
–OH), 9.57 (s, –CvN), 8.53 (s, –CvN, 2H), 7.66 (s, 1H), 1526, 1489, 1403, 1330, 1292, 1222, 1187, 1166, 1075, 937, 842,
7.22–7.21 (d, 2H), 6.25–6.23 (d, 2H), 6.048 (s, 1H), 5.96 (s, 1H), 816, 774, 458. 1H NMR (500 MHz, DMSO-d6, TMS): δ ppm =
5.65–5.63 (d, 2H), 4.58 (s, 1H), 3.33–3.30 (t, 4H), 2.46 (s, 3H), 8.46 (s, 1H), 7.49–7.47 (d, 1H), 6.59–6.57 (d, –1H), 5.83 (s, –
1.08–1.05 (q, 6H); ESI-MS: m/z calculated for [C22H30N4O2] = 1H), 1.76 (s, –3H); 13C NMR (125 MHz, DMSO-d6) δ ppm
382.24, found = 383.24. [C22H14N2O6Zn] = 466.01, found = 174.33, 160.04, 156.55, 130.34, 120.45, 117.15, 114.44, 107.14,
467.18 (Fig. S10–S12, ESI†).
106.87, 105.03, 22.75, 18.908, 18.79 (Fig. S21–S23, ESI†).
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X-ray diffraction studies of the CMD-Zn ensemble
Results and discussion
Both obtained crystals, NSA and CM-Zn-CM′, crystalized as
monoclinic systems with the space group P21/n (Table S1,
ESI†). The asymmetric unit of CM-Zn-CM′ is shown in Fig. 4. A
probable mechanistic description showing the genesis of NSA,
CM, CM′ and CM-Zn-CM′ is presented in diagrammatic form
in Scheme S1, ESI.†
The X-ray crystal structure of NSA (Fig. S26, ESI†) along
with its detailed lattice parameters is given in (Table S1, ESI†);
these are highly similar to the crystal details previously pub-
lished by us elsewhere.15b
In the crystal structure of CM-Zn-CM′, a number of dis-
torted solvents were found to be present; however, these did
not affect the overall structure of the complex. Most of these
were subsequently removed by the PLATON/SQUEEZE tool.17
The few remaining distorted solvents were removed using
Mercury software. The CM-Zn-CM′ ensemble crystallized in the
form of a binuclear complex where the interseparation dis-
tance between two zinc atoms was equal to 3.919 Å. Each Zn2+
has a N3O3 coordination atmosphere with distorted octahedral
positioning. Of the two Zn, Zn(1) suffers slightly more distor-
tion than Zn(2), which is also evident from relevant bond
angle measurements: O(1)WZn(1)O(3) = 99.9(1), N(1)Zn(1)N(3)
= 73.9(1), O(1)WZn(1)N(6) = 157.8(1), O(3)Zn(1)N(3) = 159.1(1),
O(10) Zn(1)N(3) = 104.3(1), O(3)Zn(1)N(6) = 100.9(1), and O(9)
Zn(2)N(2) = 95.4(1). The Zn(2) center has angles O(2)WZn(2)N(5)
= 169.8(1), O(4)Zn(2)O(9) = 104.7(1), and O(4)Zn(2)N(2) =
158.9(1). Furthermore, the crystal structure also indicates con-
siderable uplifting of both zinc atoms from the crystal plane,
by 0.196 Å [Zn(1)] and 0.176 Å [Zn(2)]. This observation further
substantiates the distortion in the crystal structure as dis-
cussed above.
X-ray diffraction studies of CMD
CMD was fully characterized through single-crystal X-ray diffr-
action studies. Crystals of CMD (Fig. 2) with suitable shapes
and sizes were developed through slow evaporation of its satu-
rated solution in dimethyl formamide over a period of 3 to 4
days. CMD crystalized as a monoclinic system with the space
group P2(1)/n (Table S1, ESI†).
The strongly fluorescent nature (orange yellow) of CMD in
the solid state (Fig. S24, ESI†) can be attributed to a variety of
non-classical intermolecular hydrogen bonds, viz. N(2)⋯H–C(1),
O(1)⋯H–C(3) and O(1)⋯H–C(21A), with distances of separ-
ation ranging from 2.734 Å to 2.878 Å. In solution, the weakly
fluorescent nature of CMD can be understood in terms of
rupture of the above intermolecular hydrogen bonds while pre-
serving the intramolecular hydrogen bonds between N(1)⋯H–
O(3) and N(2)⋯H–O(4) with distances of separation of 1.849 Å
and 1.909 Å, respectively (Fig. 3 and Fig. S25a, ESI†). These
hydrogen bonds affect the availability of lone pairs over the
nitrogen atoms in CMD for photoinduced electron transfer
(PET) processes.
The torsion angle between the phenyl ring and coumarin
ring of CMD was found to be ∼4°, indicating only slight
non-planarity in the system (Fig. S25b†). Molecules of CMD
underwent intermolecular π⋯π stacking involving
phenyl rings in a head to tail fashion with an intersepara-
tion distance of 3.472 Å along the a-axis (Fig. S25c and
S25d, ESI†).
The N3O3 coordination of each Zn comprises two imine
nitrogens (one from CM and another from CM′) and one
amine nitrogen [N(3) or N(2)] (Scheme S1, ESI†). On the other
hand, of the three coordinating oxygens, two are in the form of
phenolates, while the third originates from water, i.e. an aqua
ligand. Thus, there are two aqua ligands (one per Zn2+ in the
CM-Zn-CM′ complexes). The Zn–O bond lengths ranged from
Fig. 2 ORTEP view of a single crystal of CMD with 50% probability.
Fig. 3 Crystal structure of compound CMD showing stair-like packing
Fig. 4 ORTEP view of a single crystal of zinc complex CM-Zn-CM’ with
due to non-classical hydrogen bonding.
displacement ellipsoids at 60% probability.
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2.021(3) to 2.129(3) Å and were found to be consistent with chromic shifting along with red shifting of 5 nm. An isosbestic
other zinc-phenolate complexes.18,15b On the other hand, the point at 377 nm indicated a chemical interaction between
Zn–N distances for the three nitrogen donors lie in the range CMD and Zn2+ (Fig. S27a and S27b, ESI†). Separate additions
of 2.104(3) to 2.520(3) Å, which are well within the range for of 10 equiv. of a number of cations, viz. Na+, K+, Mg2+, Ba2+,
analogous zinc complexes.19 Among the nitrogens, there are Al3+, Ca2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Cd2+, Hg2+ and
two types of donors, viz. imine and the amine nitrogens. The Pb2+, as their chloride salts did not perturb the visible colour
bond length with the imine nitrogen is considerably shorter or the UV-visible spectral pattern of CMD to a significant
than that with the amine nitrogen. Detailed crystallographic extent (Fig. S28a and S28b, ESI†). This is somewhat surprising
data are given in Table S2, ESI.†
for Cd2+ and Hg2+ in the sense that both belong to the same
In addition to the above routine bond formations, supra- group as zinc. The reason for this may be understood in terms
molecular interactions in terms of hydrogen bonding also of the variations of the physical and chemical behaviour of 3d
appear to play an important role in the final architecture of elements compared to 4d and 5d elements. It is well reported
the CM-Zn-CM′ ensemble. The hydrogen atoms of the co- in chemistry textbooks that the 4d elements resemble their 5d
ordinated water molecules W(A) and W(B) undergo inter- counterparts more closely (due to lanthanide contraction) than
molecular hydrogen bonding with oxygen atoms of the next their 3d congeners. Moreover, the chemosensing action
complex moiety, i.e. CM-Zn-CM′. This ultimately results in a reported here is chemolytic in nature; Zn2+ (ionic radius = 74
helical chain architecture. Thus, the two asymmetric units of pm) is more suitable for this due to its smaller size compared
CM-Zn-CM′ are interconnected through the O(3)–H(2)WB and to Cd2+ and Hg2+ (ionic radii = 95 pm and 102 pm,
O(10)–H(2)WA bonds with separation distances of 1.903 Å and respectively).
1.947 Å, respectively (Fig. 5a and b).
To validate the fluorescence turn-on nature of its sensing,
the photophysical properties of the probe CMD were investi-
gated in ethanol. Free CMD upon excitation at 420 nm showed
a weak fluorescence signal in the form of a broad band cen-
tered at ∼560 nm. Upon individual addition of a wide range of
metal ions, viz. Li+, Na+, K+, Ba2+, Mg2+, Al3+, Ca2+, Cr3+, Mn2+,
Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+ and Pb2+, as their chlo-
ride salts, only Zn2+ caused a remarkable fluorescence enhance-
ment at 535 nm with efficient ‘turn-on’ behavior (Fig. 6 and 7).
However, a competition experiment with a number of the
Photophysical studies of CMD
In the UV-visible absorption spectrum of a 10 μM ethanolic
solution of CMD, two broad absorption bands centered at
∼350 and ∼420 nm were assigned to the π–π* and n–π* tran-
sitions, respectively. Upon the addition of Zn2+ (0.0 to 17.5
equiv.) as its acetate salt, the former band underwent gradual
hyperchromic shifting while the latter band underwent hypo-
Fig. 6 Relative fluorescence changes of CMD (1.0 μM) showing
responses to relevant competitive metal ions (10 equiv.) (λex = 420 nm,
λem = 535 nm) with inset showing the fluorescence spectra.
Fig. 5 (a) Weaker intermolecular interactions stabilizing the supramole-
cular framework and side view of a 1D single-stranded right-handed
helical chain along the c axis formed due to extension of the inter-
molecular hydrogen bonding. (b) Upper view of the supramolecular
architectures along the c* axis.
Fig. 7 Visual color changes observed upon addition of different metal
ions (10 equiv.) to CMD (10 μM) as seen under UV light (λ = 365 nm).
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The effects of counter anions were also studied here, and
faster kinetics was observed for acetate compared to chloride
as the counter anion (Fig. S30, ESI†).
Time-dependent intensity measurements of the fluorescence
behaviour of CMD (1.0 µM) with respect to Zn2+ (10 equiv.)
addition reveled that within only 30 seconds, a visible fluo-
rescence change was observed (Fig. S31, ESI†). The detection
limit (LOD)21 calculated from fluorescence titration data of
CMD for Zn2+ was found to be 3.03 × 10−8 M (R2 = 0.998) with a
linearity range of 4.00 × 10−6 to 1.80 × 10−5 M (Fig. S32, ESI†).
Fig. 8 Effects of most competitive cations on the emission spectrum of
CMD + Zn2+ at 535 nm.
Interaction mechanism of CMD with Zn2+
To obtain a deeper understanding of the interaction patterns
of CMD with Zn2+, we performed a number of studies, viz. ESI-
mass spectral studies, 1H NMR spectroscopy, photophysical
studies and single crystal X-ray diffraction studies. The ESI-
mass spectrum of CMD in ethanolic medium showed a promi-
nent molecular ion peak m/z at 394.3, corresponding to [CMD
+ H]+. However, upon adding 4 equiv. of Zn2+ (with acetate as
the counter anion), a number of peaks at m/z = 380.5 (NSA),
m/z = 403.5 (CM), m/z = 217.4 (ACM), 203.0 (coumarinol)
(Fig. S33, ESI†), m/z = 1138.4 (CM-Zn-CM ensemble), etc.
(Fig. S34, ESI†) were obtained in negative mode. Thus, the
mass spectral analysis further supported the dogma of Zn2+-
triggered hydrolysis of CMD, as we have described above
through single crystal X-ray diffraction studies.
abovementioned cations was also performed, and it was found
that four ions, viz. Fe3+, Co2+, Ni2+, and Cu2+, did show inter-
ference to a considerable extent (Fig. 8).
Fluorescence titration studies were carried out with
concomitant additions of Zn2+ (0.0 to 44.0 equiv.) to 1.0 μM
probe solution. A broad peak appeared around ∼535 nm and
became fully grown, exhibiting a blue shift of ∼20 to 25 nm com-
pared to CMD with ∼9 fold enhancement of its fluorescence
intensity. At this stage, green fluorescence was observed (Fig. 9).
The weakly fluorescent nature of CMD (quantum yield = 0.105)
can be understood in terms of intermolecular π–π interactions
as well as quenching of the PET phenomenon due to intra-
molecular hydrogen bonding between the imine nitrogen and
phenol (Fig. S29, ESI†). The CMD-Zn2+ coordination results in
two different effects: (i) hampering the intermolecular π–π
interactions between CMD molecules, which may result in a
blue shift; (ii) coordination of CMD through its nitrogen
donors, which is likely to decrease the chance of PET and may
be responsible for the hyperchromic shift, i.e. intensity magni-
fication, along with enhancement of the quantum yield
(0.514). Here, it is worth mentioning that of the number of
ratiometric probes reported to date in the literature, only a few
of them show Zn2+-triggered hypsochromic shifting of the
emission wavelength of the receptor.20
1H NMR studies
In order to substantiate the above findings, we further carried
1
out H NMR titration experiments between CMD and Zn2+ in
DMSO-d6. A number of new peaks and shifting in the routine
peaks of CMD clearly indicated the fragmentation of CMD trig-
gered by Zn2+. CMD shows peaks at 12.69 δ ppm and 11.12 δ
ppm for its –OH protons and for its aldimine protons at 9.10 δ
ppm and 8.81 ppm. Upon addition of Zn(OAc)2·2H2O (0.0–5.0
equiv. in DMSO-d6) to CMD, the –OH proton signals shifted
upfield and gradually disappeared; the aldimine proton
signals of CMD at 9.11 and 8.82 δ ppm were shifted upfield to
8.92 and 8.45 ppm. The peak at 8.45 δ ppm was assigned to
NSA and the peak at 8.92 δ ppm was assigned to the aldimine
proton of ACM-Hz. A new peak at 9.03 δ ppm appeared and
was assigned to the aldimine proton of CM. The peak of the
amine group of ACM-Hz appeared at 6.93 δ ppm. Finally, the
solution became strongly fluorescent green (Fig. 10). We also
carried out 1H NMR time-dependent experiments and com-
pared the resulting spectra with those of our synthesized com-
pounds. The intermediate ACM-Hz signal appeared at 5 equiv.
but disappeared at 10 equiv., probably due to the faster reac-
tion rate at a higher equiv (10 equiv.) of Zn2+. These results
indicate that the peaks of NSA (formed in situ) matched quite
well with those of synthesized NSA. However, slight variations
in the aldimine protons (8.407 to 9.044 δ ppm) as well as in
other protons were observed in the case of CM, probably as a
consequence of its binding interactions with Zn2+ (available in
the reaction medium); the corresponding spectrum is given in
ESI, Fig. S35.†
Fig. 9 Fluorescence intensity changes of CMD (1.0 μM) in the presence
of Zn2+ (0.0 to 44.0 equiv.).
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Scheme 2 Chemodosimetric and supramolecular strategy: hydrolysis
of CMD in the presence of Zn2+
.
Fig. 10 Partial 1H NMR spectra showing the gradual changes in the
NMR signals upon addition of up to 5 equiv. of Zn2+ to CMD.
Fluorescence studies were also performed for aldehyde
(ACM) and amine (ACM-Hz), i.e. constituents of CMD. At the
same time, NSA and CM were also synthesized from their con-
stituents and subjected to physicochemical studies under
identical conditions (Fig. S36a, S36b and S37, ESI†). The
results were similar to those of the CM and NSA formed in situ
during the interaction of CMD with Zn2+. Systematic fluo-
rescence studies were also performed with separate additions
of Zn2+ to all the above molecules under identical experimental
conditions. Here, it is worthwhile to mention that the fluo-
rescence characteristics of NSA (515 nm) were similar to those
of CMD solution upon addition of Zn2+. This further fortifies
our dogma of scission of CMD into NSA and other products,
as mentioned above.
Fig. 11 Chromo-fluorogenic response of test paper strips of CMD in
the presence of Zn2+
.
layer coatings of CMD. These strips were prepared by placing
filter paper strips (Whatman) in an ethanolic solution of CMD
(50 micromolar); the strips were dried and used to sense Zn2+.
As shown in Fig. 11, when Zn2+ was added to the test kits,
obvious color changes were observed with Zn2+ solution under
a 365 nm UV lamp.
Thus, comparison of the 1H NMR titration experiments
(Fig. 10 and Fig. S35†) as well as the fluorescence (S36a and
S36b, ESI†) and UV-visible (Fig. S37, ESI†) studies also sup-
ported the above mass spectral and X-ray diffraction studies.
The Zn2+-triggered hydrolysis of CMD as well as the genesis of
various recombination products discussed above are con-
veniently shown in Scheme 2.
A comparison of the scanning electron micrographs (SEM)
of CMD and its reaction products with Zn2+ is shown in
Fig. S38, ESI.† The corresponding morphologies of CMD prior
to (a) and after (b) the addition of Zn2+ are shown in Fig. S38,
ESI,† which indicate a categorical change and finally suggest
an interaction between CMD and Zn2+.
Cell culture and fluorescence imaging
Treatment of SiHa cells with 10 µM CMD without Zn2+ showed
very little or no fluorescent signals in the green channel;
however, after treatment with Zn2+ (5 times higher than the
CMD concentration), stronger florescent signals appeared in
the green channel. Similarly, 20 µM CMD without Zn2+ showed
very little green fluorescent signals; however, treatment with
Zn2+ (5 times higher than the CMD concentration) produced
very intense green florescent signals. In the presence of Zn2+,
cells treated with 20 µM CMD produced a more intense green
Practical utility of CMD
Detection of Zn2+ using a fluorescence spectrophotometer is a signal in comparison to cells treated with 10 µM CMD. To
cumbersome approach for onsite detection. Hence, to over- identify the nucleus, the cells were treated with DAPI, which
come this drawback, we prepared filter paper strips with thin can visualize nuclei with a blue florescent signal (Fig. 12). Cell
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Conclusions
We present here a simple, cost-effective and easy-to-use che-
modosimeter for Zn2+ in the form of CMD synthesized
through Schiff base condensation of aldehydes and hydra-
zine. The very quick response time of CMD (only 30 seconds)
and its sub-nanomolar limit of detection are additional
benefits. The successful imaging of Zn2+ in live cells indicate
further promise for CMD. The various single crystal X-ray
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Conflicts of interest
There is no conflict of interest among the authors of this
paper.
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