Dual responsive salen-type Schiff bases for the effective detection of l-arginine: Via a static quenching mechanism

Article abstract of DOI:10.1039/c8nj02657b

l-Arginine is one of the most important amino acids and plays a key role in diagnostic studies. Herein, we report four salen-type Schiff base fluorimetric/colorimetric sensors for the rapid detection of l-arginine. Substituents play a major role in the se

Full text of DOI:10.1039/c8nj02657b

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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DOI: 10.1039/C8NJ02657B
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Dual Responsive Salen-type Schiff Bases for the Effective
Detection of L-Arginine via Static Quenching Mechanism.
Nithya Mohan,a S. S. Sreejith,a P. M. Sabura Begum a and M. R. Prathapachandra Kurup*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
L-Arginine is one of the most important amino acid and plays a key role in diagnostic studies. Herein we report four Salen-
type Schiff base fluorimetric/colorimetric sensors for the rapid detection of L-arginine. Substituents play a major role in the
sensing mechanism of the probes with the nitro-substituent bearing probe showing the highest efficiency. Evidence of a 1:2
complexation between all the probes and the analyte was evidenced from the absorption titrations; and the fluorescence
studies revealed quenching phenomenon occurring during complex formation. From the life time measurements, the nature
of quenching was found to be static and the formation of a ground state complex was confirmed. DFT calculations done at
B3LYP/TZVP level of theory corroborated the experimental findings and further established the proton transfer happening
from probe to the analyte during the complex formation.
DOI: 10.1039/x0xx00000x
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hyperammonemia disorders and astrocytes of aggregating
neural cell culture.8
Introduction
Biological functions of Arginine include secretion of certain
L-Arginine is the most propitious, naturally occurring amino
acid among the non-thiol containing amino acids. The most
inimitable property of Arginine among the rest of the natural
amino acids is the high basicity of one of its terminal position
i.e. guanidine part.1 Its pKa value is found to be 13.8 and is a
conditionally essential amino acid.2 It is also found in cerebral
system in 14μM concentration and has the highest isoelectric
point3. Studies especially sensing appertaining to the detection
of L-Arginine(L-Ar) has inordinate importance in the scientific
community as well as in the medical and biological fields4.
Arginine is one of the most important diagnostic indicator for
some of the human diseases like hypertension, renal damage
and also the varying concentration of Arginine or its derivatives
hormones such as insulin growth hormone, glucagon and
prolactin.9 It also has a vital role in cell division, healing of
wounds etc. Nitric oxide which has many biological functions is
also produced from the conversion of L-Arginine into
Citrulline.10 Besides these, some cell-cell, signalling molecules
are also generated from Arginine in the presence of oxygen.
Owing to its ability to attain different charge states, Arginine is
involved in a large number of crucial biochemical process which
includes some steps of metabolic pathways, trans-membrane
transport etc. Also, Arginine is essential for the quantum
chemical study of the interactions involved in proton transfer
and other selective binding processes.1, 2
Extensive methods are available in the literature for the
selective detection of Arginine. Apart from the voluminous data
of detecting Arginine using methods like HPLC, mass
spectrometry etc. Reports are there in the literature for the
detection of thiol-containing amino acids using fluorescent
probes. Fluorescent sensors for the detection and sensing of
amino acids other than thiol-based ones are limited in number
and still limited when it comes to colorimetric as well as
fluorescent sensors.11-13
6
cause many health issues that can even lead to death.5,
Administration of L-Ar orally on patients with preeclampsia was
found to accelerate foetal gain and improve biophysical profile.7
Arginine also plays a pivotal role in the pathophysiology of
a.Department of Applied chemistry
Cochin University of Science and Technology
Kochi, Kerala-682022, India
E-mail: mrpcusat@gmail.com.
Among the reported sensors colorimetric detection are
mainly reported in the field of gold based nano particles, which
again has a limitation i.e. incorporation of costly Au particles.9,
b.Department of chemistry, School of Physical Sciences
Central University of Kerala
Riverside Transit campus, Neeleshwar, Kerala-671314, India.
E-mail: mrp@cukerala.ac.in.
† Footnotes relating to the title and/or authors should appear here.
14, 15
Works including fluorescent, electrochemical and surface
plasmon resonance detection of Arginine were reported but the
sensor or probe used for this purpose are mainly of macrocyclic
type like crown ether and quantum dots (QDs).3, 16-18 These
methods also bear some unavoidable limitations such as poor
solubility, advanced experimental set up, sophisticated
characterization methods and the use of costly metals.
Recently colorimetric as well as fluorescent probes were
Electronic Supplementary Information (ESI) available:
IR, UV-VIS 1H-NMR spectra, GC-MS data, crystal data refinement table and,
hydrogen bonding interactions, Jobs plots, life-time data, KSV value and Stern-
Volmer plot, fluorimetric response times. CCDC 1845374 contain the supplementary
crystallographic data for this paper. These data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 IEZ, UK; fax: (+44) 1223-336-033; or e-
mail: deposit@ccdc.cam.ac.uk.] [details of any supplementary information available
should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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developed for the selective detection of Arginine, but none of and ORTEP-3 for windows30.All the C bound HVaietwoAmrtisclewOnelirnee
DOI: 10.1039/C8NJ02657B
them were able to propose the mechanism behind the derived from Fourier difference maps and were placed
sensing.19, 20 These also fail in explaining the effect/presence of geometrically (C–H = 0.98-0.99 Å) and refined in the riding
Arginine in the excited state life time of the probe. Also, the model approximation with U(H) set to 1.2 U (C) (1.5 U for CH3).
reason behind the quenching of fluorescent intensity of the Anisotropic displacement parameters of C6 and C7 were
probe is still unknown.
restrained using enhanced rigid bond restrain for the 1,2
Through this work, we propose
a
Salen-type distances using RIGU command and some reflections were
bicompartmental Schiff base systems for the effective sensing omitted owing to disagreement.
of the amino acid L-Arginine. To date, to the best of our
knowledge, this is the first reported Salen-type system for the Synthesis and characterization of the compounds
colorimetric detection of L-Arginine even though there are
In order to study the structural effect of Salen type Schiff
reports on the use of Schiff bases and its metal complexes for base compounds, we have synthesized four different types of
the sensing of cations and anions etc.21-25 But the number is still compounds by changing the substitution at the salicylaldehyde
limited in salen like systems.26 The proposed detection moiety as well as in the spacer groups. Compounds
1 to 4 were
technique is rapid, facile, cost effective and also has high synthesized according to the route shown in Scheme 1.
selectivity.
Experimental
Materials
2-Hydroxy-3-methoxybenzaldehyde, 5-Bromo-2-hydroxy-3-
methoxybenzaldehyde, 5-Nitro-2-hydroxy-3-
.
methoxybenzaldehyde, Ethylene diamine and L- Arginine were
purchased from Sigma-Aldrich Chemical Co. Pvt. Ltd.; 2,2’-
dimethyl ethylene diamine was purchased from Nice Chemicals
Pvt. Ltd. and used as received. Fluorescein and Quinine sulphate
were bought from LobaChemie Private Ltd., The stock solution
of L-Arginine was prepared in distilled water. All other solvents
and chemicals used were of analytical grade.
Scheme 1 Synthetic route for the formation of Schiff bases.
Physical Measurements
Compound
1 was synthesized by the addition of a
Elemental analyses and GC-MS data were collected from a
Vario EL III CHNS elemental analyzer and using Trace 1300 GC
and ISQ QD single quadruple GC-MS instrument respectively. IR
spectra of the compounds were recorded on a JASCO FT-IR-
5300 Spectrometer in the range 4000-400 cm-1 using KBr
pellets. Electronic spectra of the compounds were taken on a
Thermo scientific Evolution 220 spectrophotometer in the 260-
700 nm range. Emission and lifetime measurements were taken
using 530 nm laser source in Fluorolog and Flurolog TCSPC,
Horiba Scientific respectively. 1H NMR spectra of all the ligands
except compound 2 and 4 were recorded from Bruker AVANCE
II 500 NMR Spectrometer using CDCl3 and compound 2 and 4 in
d6-DMSO.
Single-crystal X-ray diffraction measurement were
performed on a Bruker SMART APEX diffractometer, equipped
with a graphite crystal incident-beam monochromator, and a
methanolic solution of 2,2’–dimethyl-1,3-propane diamine into
a round bottom flask containing 5-bromo-3-methoxy-2-hydroxy
salicylaldehyde in methanol.32 A fluorescent yellow colored
solution was formed immediately after the addition of diamine.
The reaction mixture was refluxed using simple water
condenser at a temperature of 50 °C for 2 hours.33 Yellow
colored product formed was filtered, washed with methanol
and recrystallized from DMSO. Purification method adopted
here is recrystallization instead of chromatography since
degradation of Schiff base happens during the purification step
through hydrolysis.26 Same method was adopted for the
syntheses of other compounds:
crystals were obtained for
precipitate from DMSO. Immediate precipitation was observed
in the case of and respectively. The precipitate was collected
2
to
4. Diffraction quality single
2
after recrystallization of the
3
4
and washed with methanol and recrystallized in DMSO solvent.
fine focus sealed tube with Mo K ( = 0.71073 Å) as the X-ray
source. The unit cell dimensions were measured and data
collection was performed at 293(2) K. Data acquisition and
integration were done using Bruker SMART and Bruker SAINT
software27. Absorption corrections were carried out using
SADABS based on Laue symmetry using equivalent reflections28.
The structure was solved by direct methods and refined by full-
matrix least-squares calculations with the SHELXL-2016/1
software29in the WINGX software package30. The structures
were plotted using the graphics tools DIAMOND version 3.2 k31
1
Compound
1
: Yield (73%); H NMR (500 MHz, CDCl3,TMS): δ
14.84 (s, –OH, 2H), δ 8.24 (s, 2H), δ 6.90 – 7.01 (m, Aromatic
Protons 4H), δ 3.91 (s, –OCH3, 6H), δ 3.49 (d, 4H), δ 1.07 (s, 6H),
(Fig S1, ESI†). FTIR/cm-1 (KBr): 3481 (–OH), 2982 (CHst), 1613
(C=N), 1472(Ar C=C), 1229 (C-O), (Fig. S2, ESI†). GC-MS: 528.94
(M+2, 100%) (Fig. S3, ESI†). Anal.calc. (%) for C21H24Br2N2O4: C,
47.75; H, 4.58;N, 5.30. Found (%) C, 47.35;H,4.61; N, 5.31.
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1
Compound
2 : Yield, (70%); H NMR (500 MHz, DMSO,TMS): δ Job’s plot Analysis.
View Article Online
14.09 (s, –OH, 2H), δ 8.47 (s, 2H), δ 6.9 – 7.01 (m, Aromatic
Job’s Plot analysis was done by addiDnOgI:510t.o103590/Cμ8LNJo0f26L5-A7Br
Protons 4H), δ 3.74 (s, –OCH3, 6H), δ 3.45 (d, 4H), δ 0.95 (s, 6H), solution to a quartz cuvette containing 100 μL of the sensor and
(Fig S1, ESI†). FTIR/cm-1 (KBr): 3441 (–OH), 2982 (CHst), 1633 the total volume was made to 2000 μL. The UV spectra of the
(C=N), 1465(Ar C=C), 1249 (C-O), 736(C-Br), (Fig. S2, ESI†). GC- sensor–L-Ar mixture were recorded separately and the Job’s
MS: 371.23 (M+1, 100%) (Fig. S3, ESI†). Anal.calc. (%) for Plot were drawn to confirm the composition of adduct formed.
C21H26N2O4: C, 68.09; H, 7.07;N, 7.56. Found (%) C, 68.23;H,7.09;
N, 7.27.
Fluorescence studies
Fluorometric Titration
1
Compound
3
: Yield, (86%); H NMR (500 MHz, DMSO,TMS): δ
In order to study the fluorescence spectral response of the
13.57 (s, –OH, 2H), δ 8.23 (s, 2H), δ 6.9 – 7.2(m, Aromatic synthesized sensors towards L-Ar, fluorescence titration was
Protons 4H), δ 3.88 (s, –OCH3, 6H), δ 3.96 (t, 4H), (Fig S1, ESI†). performed with the probe and the analyte in a concentration
FTIR/cm-1 (KBr): 3420 (–OH), 2928 (CHst), 1617 (C=N), 1459(Ar same as above. But the addition of the analyte was little bit
C=C), 1235 (C-O), 689(C-Br), (Fig. S2, ESI†). GC-MS: 485.92 different i.e 10 to 150 μL since this method is much more
(M+2, 100%) (Fig. S3, ESI†). Anal.calc. (%) for C18H18Br2N2O4: C, sensitive than the absorption titration.
44.47; H, 3.73; N, 5.76. Found (%) C, 44.62; H, 3.76; N, 5.33.
Lifetime Measurement
Compound
4
: Yield (82%); mp, 212 °C. 1H NMR (500 MHz,
The Lifetime measurement was carried out to study the
DMSO,TMS): δ 14.2 (s, –OH, 2H), δ 8.58 (s, 2H), δ 7.27 – 7.94 (m, mechanism behind the sensing of L-Ar by the synthesized
Aromatic Protons 4H), δ 3.76 (s, –OCH3, 6H), δ 3.63 (t, 4H), δ 1.1 sensors using a 530 nm laser source. The study was conducted
(s, 6H) (Fig S1, ESI†). FTIR/cm-1 (KBr): 3420 (–OH), 2955 (CHst), with and without the analyte L-Ar and the plot was constructed
1654 (C=N), 1532 (Assy. NO2st), 1317 (Sym. NO2st), 1438( C-N), with counts against time of decay. Stern-Volmer plots were
1256 (C-O), (Fig. S2, ESI†). GC-MS: 485.92 (M+2, 100%) (Fig. S3, analyzed to confirm the mechanistic route for the detection of
ESI†). Anal.calc. (%) for C21H24Br2N4O8: C,54.78; H, 5.25; N, analyte.
12.17. Found (%) C, 54.44; H, 5.28; N, 12.31.
NMR Titration Experiment
Colorimetric sensing of L-Arginine
UV-Vis titration measurements
Stock solution of compound
prepared in 10-3 M (0.0052 g in 10 mL) concentration in DMSO probes
solvent. It is then diluted to obtain a concentration of 10-5 M. interacting part of the probe. To carry out the experiment,
The analyte L-Ar was prepared in distilled water having a were dissolved in d6-DMSO and the analyte was dissolved in
1H NMR titration with single addition of L-Ar was carried out
.
in probes
) was individually and 2 equivalents of the analyte was added to the
and in order to study the change as well as the
and
2 and 4. NMR spectrum of each probe was recorded
1
(sensor/probe 1
2
4
2
4
concentration of 10-3 M (0.0017 g in 10mL). From this a volume D2O solvent. The solvent d6-DMSO was chosen for the
of 2500 μL was pipetted out into a 25 mL std. flask and made up experiment in order to avoid the possibility of solvent exchange
to the mark in order to obtain a concentration of 10-4 M. of OH protons with the added D2O solvent.
Colorimetric response was studied by the addition of 100 μL of
analyte into a cuvette containing 100 μL of the sensor, it is then Theoretical Calculations
diluted to a total volume of 2 mL. An immediate color change
DFT calculations were done employing ORCA 3.0.3 software
35
from colorless to light yellow was observed. The UV spectra package34,
using the Becke’s three-parameter hybrid
were then recorded at room temperature. The titration exchange functional including the Lee–Yang–Parr nonlocal
experiment was conducted with the addition of L-Ar in the correlation functional (B3LYP)36, 37 and the triple zeta valence
volume range of 20 – 200 μL.
The same procedure was adopted for the colorimetric accelerate the calculations, the resolution of identity (RI)
sensing of L-Ar using compounds to (Fig. 2). The approximation was utilized with the decontracted auxiliary
concentration of compound (sensor def2–TZV/J Coulomb fitting basis sets and the chain–of–spheres
) was reduced to 10-6
basis set with one set of polarization function (TZVP)38. To
1
3
4
4
M
since it shows a higher optical density than the other ones. This (RIJCOSX) approximation to exact exchange as implemented in
is an indication of its higher sensitivity towards our interest ie. ORCA.39 Initial geometries used for the optimization process
L-Ar.
were taken either from crystal structure or from NMR results.
The optimized geometries were then confirmed to be global
energy minima by performing frequency calculations which
Selectivity test
Prior to the above absorption titration measurements, returned no imaginary frequency. TZVP basis set was used since
competition tests were performed with 5 equivalents of it reduces the basis set superposition error (BSSE) to negligible
fourteen amino acids (Gly, Ala, Lys, Trp, Arg, Val, His, Leu, Ph ala, in the calculation of systems with noncovalent interactions.
Ser, Asp, Cys, Tyr and Glu (Fig. S4)).
Avogadro software was used for the visualization of orbitals.40
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observation, it was very interesting to observe theVcieowloArrticclheaOnnlginee
of the probe from colorless to light yellow which makes it a
colorimetric naked eye sensor. It is worth mentioning that the
naked eye detection was viable only at relatively high L-Ar
concentration.
Results and Discussion
Crystal structure description of 2
DOI: 10.1039/C8NJ02657B
The asymmetric unit of the compound consists of a single
molecule having the molecular formula C21H26N2O4. It
crystalizes into monoclinic P21/n space group (Table S1). The
torsional angle between C1–C7–N4–O8 was found to be
178.74(2)° which suggests an E conformation of the molecule
w.r.t. the azomethine nitrogen atom. The two terminal moieties
including the benzene ring of the aldehydic parts lie nearly in a
perpendicular fashion as evident from the angle measured
between the two planes which returned a value of 124°. The
two intramolecular hydrogen bonding interactions (O(2)–H(2)···
N(1) and O(3)–H(3)··· N(2)) present in the crystal system results
in the formation of S(6) ring motifs and also adds to the stability
of the solid structure of the compound (Fig 1b, Table S2).41, 42
One of the azomethine nitrogen atom N(2) acts as an acceptor
in the intramolecular nonclassical hydrogen bonding interaction
(C(11)–H(11)··· N(2)) which results in the formation of S(5) ring
motif. Thus N(2) forms a bifurcated hydrogen bond with two
different donor atoms i.e. O(3) and C (11) (Fig1b). The ORTEP
The sensors reported here (1 to 4) show a similar pattern in
the absorption titration curves but the position of the newly
formed peak and isosbestic points were shifted accordingly with
the substituents on the aldehydic as well as the diimine spacer
part (Fig 2). Apart from competitive experiments, analogous
investigations were carried out using absorption titration
method with alanine but the response was negligible and could
be ignored.
image including the numbering scheme of
1a.
2 is shown in Figure
Figure 2 Absorption titration curves of sensors 1-4 with incremental addition
(20-200 μL) of L-Arginine.
In order to probe into the stoichiometry of the complex
formed, Jobs plot analysis were done and the ratio was found
to be 1:2 (sensor: L-Ar) for all the four sensors. The mass data
also support the result from the jobs plot (Fig S5). Initially the
m/z peak observed for sensor
When 2 equivalents of L-Ar was added to the sensor, new m/z
peak was found in consonance with that of ( + 2 L-Ar) (Fig S6).
The sensors are then subjected to fluorescent titration
measurements and quenching of fluorescence was observed
when L-arginine was added to the fluorescent probe (Fig 3).
Recent reports on Salen Schiff bases discusses anion or amino
acid sensing via fluorescence enhancement.43, 44 Similarly a new
1 at 528.94 corresponds to m+2.
Figure 1 (a) ORTEP diagram of 2 with atom numbering scheme (for clarity
hydrogen atoms are not labelled); (b) Intramolecular hydrogen bonding
interactions generating two types of ring motifs.
1
Sensors
1 to 4 were synthesized, initially characterized and
were subjected to absorption titrations to study their sensing
ability towards L-Arginine (L-Ar). The absorption peak around
331 nm in sensor 1 was found to significantly decrease with the
type of emission enhancement called aggregation-induced
46
emission (AIE) is also reported.45,
The two enhancement
incremental addition of the analyte and a concomitant increase
in absorbance at 418 nm was also found. Absorbance @418 nm
is the newly formed band and was absent in the sensor which
also gave the initial experimental proof for (sensor + L-Ar)
complex formation. As the phenolic OH groups in the sensor are
acidic in nature, it gets deprotonated in presence of a strongly
basic analyte L-Ar. Hence this band may be attributed to the
deprotonation of the probe in presence of the strong base L-
Ar.19 In addition to the new band, a clear isosbestic point was
noted at 315 nm which also gave the evidence for the existence
of the complex/adduct formation. In addition to the above
phenomenon are entirely different, as in the case of
anion/amino acid sensing the fluorescence enhancement is due
to the uniform distribution of π-function Frontier molecular
orbitals in the molecule skeleton. While in AIE it is originated
from aggregated molecules in its solid state or in H2O due to
weak intermolecular face–face π⸱⸱⸱π interactions, multiple
hydrogen bonds and other strong non-covalent intermolecular
interactions between adjacent moieties. In contrast, in this
work, the quenching may be due to the intermolecular proton
transfer between the sensor and the L-Ar.47 To explore this
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quenching phenomena, life time measurement was done in thus no changes in the absorption spectra after the addition. On
View Article Online
each sensor and also with its analyte (see later).
the other hand, static quenching maDyOIo: c10c.u10r39d/uCe8NJt0o26t5h7eB
formation of a complex between the fluorophore and quencher.
When this complex absorbs light it immediately returns to the
ground state without emission of a photon and the observed
fluorescence is from the non-complexed fluorophores. In this
case, the life time of the sensor will remain unchanged even
after the addition of an analyte and 휏0/휏 = 1, where 휏0and 휏
are life time of the probe and mixture respectively.51
To investigate the quenching behaviour and thereby the
underlying mechanism of recognition, lifetime measurements
of the probe was done with and without the addition of analyte
L-Ar. The average life time was calculated and it was observed
that the life time remains unchanged even after the addition of
L-Ar (Table S3, Fig 4). The above discussed spectroscopic results
reveal the existence of a static type of quenching and the
formation of a non-emissive ground state complex. To get
further insight into the type of quenching, we analysed the UV-
Vis spectra of the probe and the mixture (Probe + L-Ar) carefully.
The obtained UV-Vis spectra are in good agreement with the
static quenching mechanism for our proposed sensors since the
UV-Vis spectra for the mixtures obtained through incremental
addition of arginine consists of a new peak and the presence of
isosbestic points confirms the formation of a ground state
complex.52
Figure 3 Quenching of emission intensity in compounds 1-4.
The most important facets of a sensor are its ability for the
rapid detection of an analyte and its stability. This was
confirmed by measuring the fluorescence intensity at different
time intervals after the initial scan and it was noticed that the
intensity remains constant after 12-14 seconds (Fig S7). This
immediate time response towards the analyte may increase its
practical utility. Among the four sensors, sensor
stable after 12 seconds even though the concentration of
lower than that of the rest (Fig S7). Further sensitivity of probes
4
became
4
is
was measured and 4 exhibited the lowest detection limit of 1 ×
10-6molL-1
.
Mechanism of Sensing
Complex or adduct formation of the sensor with the analyte was
confirmed from the UV-Vis absorption titration studies. Further
to explore the reason behind sensing/recognition of analyte and
quenching of fluorescence, life time measurements were done.
There are reports explaining the sensing mechanism of analyte
by life time study. But to the best of our knowledge, this is the
first reported salen system which senses arginine and also the
method employed here (life time study) is hitherto unexplored
to investigate mechanism in these class of systems.
Fluorescence life time method is not only the most reliable one
but is an accurate method to get an insight into the mechanistic
pathway of a reaction.48, 49 The quenching may be due to the
excited-state reactions, molecular rearrangements, ground-
Figure 4 Life time plots of sensors 1-4 and their respective mixture with
L-arginine.
The static fluorescent quenching was further analysed with
the Stern-Volmer equation
state
complex
formation,
collisional
quenching,
energy/electron transfer and emission group destruction etc.
There are two important types of fluorescent quenching, first is
the collisional/ dynamic quenching and second is the static
quenching. For either static or dynamic quenching to occur, the
fluoro probe and the quencher must be in contact with each
other.50 Dynamic quenching may be attributed to the
nonradiative electron transfer from the excited state of the
fluoroprobe to the analyte molecule. Due to its diffusion-
controlled collision, change in life time may be observed and
also it affects only the excited states of the fluorophores, and
I0/I = KSV[Q] + 1
Where,
I0 = fluorescent intensity of the sensor before the addition of the
analyte
I = fluorescent intensity after the addition of the analyte
KSV = Stern-Volmer constant
[Q] = molar concentration of the analyte (M-1).
The Stern-Volmer (SV) plots show good agreement with the
static type of quenching and ground state interaction of the
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probe and the analyte with
S8, Table S4 in ESI).
4
having the highest KSV value (Fig.
Since the guanidine part in L-Ar is more VbieawsAircti,clemOnolirnee
probable interacting part with the acidic DpOarI:t1o0.f10th39e/Cse8NnsJ0o2r6w57iBll
As all the sensors have two acidic moieties (two phenolic be the guanidine terminal. The hypothesis of guanidine part
OH) and the analyte is a strong base, there is a high chance of interacting in the complex/adduct formation was verified by
complex/adduct formation through proton transfer.47 1H-NMR performing absorption titration. For confirming this, the
experiments were performed for its verification. To elucidate absorption titration was performed with guanidine
the deprotonation, 1 eqv. of L-Ar (D2O) was added to the sensor hydrochloride and the spectra obtained was exactly similar to
and the 1HNMR spectra of the sensor
2 and 4 were then that of the Sensor-L-Ar spectra (Fig S9).
recorded in d6-DMSO solvent before and after the addition.
DMSO solvent was selected so as to avoid the D2O exchange.
The OH peak was found to be absent in the NMR spectrum of
the mixture containing (sensor
2 + L-Ar) confirming the proton
transfer and involvement of OH group in the complex formation
(Fig. 5). From the literature survey also, it was clear that the
amino acid L-arginine is a strong base having a pKa value of 13.8
and is strong enough to deprotonate these OH groups.
It can be proposed that the sensing mechanism involves the
complex/adduct formation between the acidic part of the
sensors and the basic part of the arginine. Since there is no
major difference in the structure between the sensors, we can
Scheme 2 Formation of 1:2 adduct via proton transfer from probe to the
analyte.
Experimentally obtained results and the proposed
mechanism were further verified by performing DFT
calculations of the compounds at the B3LYP/def2-TZVP level of
theory. To find out the plausible stable complex, the frontier
orbitals of the optimized geometries of all the Salen Schiff bases
explain the mechanism by taking the example of sensor 1. The
complex formation is initiated by the proton transfer of the
acidic phenolic OH by the basic NH group of the guanidine part
of the arginine (Scheme 2). This results in the formation of
slightly negative charge at the proximity of the phenolic oxygen
which in turn attracts the guanidine part of the arginine where
now there is a slight positive charge formation due to the
aforementioned proton transfer. This is also evidenced from
1HNMR where the OCH3 protons due to their position near to
the negative charge of the adduct are downfield shifted (Fig 5).
Proper orientation of –OH and –OCH3 plays a significant role in
the recognition of L-Ar. The deprotonated ArO− form is
stabilised by the presence of electron donating –OCH3 which is
critical in the formation of the adduct/complex. It does not
mean that these functionalities are mandatory for the sensing
of an amino acid since in some other systems where there is no
–OCH3 group, sensing happens but the interaction is somewhat
different.43, 47
(1
-
4
) and the arginine analyte were compared (Table 1, Fig 6).
It was found that among the probes, has the most stabilized
4
LUMO (-2.339 eV) which means that it is the strongest acid53
and the LUMO is concentrated on the phenol moieties on both
the sides (Fig 6) (For details about orbital composition see ESI).
This electrophilic nature of
transfer to the analyte retaining its negative charge. This results
in the formation of a -L-Ar (1:2) ground state complex (Scheme
4 helps in the effective proton
4
2). This is obvious since the presence of electron withdrawing
nitro group in the para position to the phenolic OH on both
aldehydic moieties in
4 helps in the deprotonation thereby
attaining a negative charge in the complex/adduct (Scheme 2).
From the table 1, it can be seen that the second most
stabilized LUMO is that of
2 (-2.274 eV) but its HOMO is having
the highest energy (-4.056 eV) among the selected probes
which results in a strong basic nature. For the two bromo
substituted compounds
comparable with each other (-1.513 and -1.484 eV) and higher
than that of and . But the low energy for HOMO in both and
(-5.858 and -5.713 eV) when compared to that of results in
low nucleophilicity and a better overall acidic character than
that of . Thus, the theoretical calculations substantiate the
experimental observations whereby the effective proton
transfer to the analyte follows the order
1 and 3 the LUMO values are
4
2
1
3
2
2
4>1>3>2.
Table 1 HOMO-LUMO energies calculated for the probes and L-arginine at
B3LYP/TZVP level of theory
Compounds HOMO(eV)
LUMO(eV)
0.130
Band Gap(eV)
-6.236
Arginine
-6.106
-5.858
-4.056
-5.713
-5.995
1
2
3
4
-1.513
-2.274
-1.484
-2.339
-4.345
-1.782
-4.229
-3.656
Figure 5 NMR plots of 2 alone and after the addition of 1 equivalent of L-
arginine.
6 | J. Name., 2012, 00, 1-3
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New Journal of Chemistry
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Journal Name
ARTICLE
Conclusions
Conflicts of interest
View Article Online
DOI: 10.1039/C8NJ02657B
There are no conflicts to declare.
Herein we report novel Salen-type Schiff bases for the
recognition and sensing of biologically important amino acid L-
Arginine. These are the first reported Salen-type systems for
both fluorimetric and colorimetric sensing of L-Arginine in
aqueous solution. Among the tested probes the one with nitro
substituent in the aldehydic moiety showed the highest sensing
character which shows the effect of substituents on the sensing
ability. Studies carried out using absorption titrations confirmed
the formation of 1:2 complex between respective probes and L-
arginine. Further fluorescence quenching measurements and
life time studies revealed static quenching and the formation of
Acknowledgements
NM is thankful to Cochin University of Science and Technology
and UGC-SAP for the financial assistance. NM acknowledges
Prof. S. Prathapan and Dr. N. Manoj for their timely help. The
authors are thankful to SAIF-CUSAT, NIIST-Trivandrum for
various analyses.
a
ground state complex. The experimental results are
Notes and references
substantiated with DFT calculations involving frontier orbitals
performed at B3LYP/TZVP level of theory which shows the
possibility of proton transfer during complex formation. The
facile synthetic procedure and appreciable detection limit of the
synthesized probes make them cheap and practical sensors for
L-arginine. We believe that this work will serve as a foundation
for further investigations on the use of these class of
compounds as biologically important sensors.
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Page 9 of 9
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ02657B
Effect of substituents of Salen Schiff bases on the detection of L-arginine via static quenching
mechanism is discussed.