- Email: [email protected]
Fluoride selective colorimetric sensor based on cefetamet pivoxil drug
Accepted Manuscript Title: Fluoride selective colorimetric sensor based on cefetamet pivoxil drug Author: Saravana S. Kumar Shilpa Bothra Suban K. Sahoo Ashok S.K. Kumar PII: DOI: Reference:
S0022-1139(14)00117-1 http://dx.doi.org/doi:10.1016/j.jfluchem.2014.04.015 FLUOR 8311
To appear in:
FLUOR
Received date: Revised date: Accepted date:
26-2-2014 25-4-2014 29-4-2014
Please cite this article as: S.S. Kumar, S. Bothra, S.K. Sahoo, A.S.K. Kumar, Fluoride selective colorimetric sensor based on cefetamet pivoxil drug, Journal of Fluorine Chemistry (2014), http://dx.doi.org/10.1016/j.jfluchem.2014.04.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Graphical Abstract - Pictogram
A simple fluoride ion selective chemosensor was developed using cefetamet pivoxil (L) drug. In the presence of F- ions, the drug L selectively portrayed a naked-eye detectable color change from colorless to red with the appearance of a new charge transfer band at 500 nm. No significant color change was observed with other anions such as Cl−, Br−, I−, AcO−, HSO4− and
3.0 0.8
absorbance
2.0
1.5
us
2.5
0.4
0.2
0.0
0
M
1.0
0.0 300
400
2
500
4
6
8
10
12
Fluoride, M
600
700
wavelength, nm
Ac ce p
te
200
d
0.5
79M
an
absorbance at 500nm
Lowest detection limit = 0.6
cr
ip t
H2PO4−.
Page 1 of 30
*Graphical Abstract - Synopsis
Graphical Abstract A simple fluoride ion selective chemosensor was developed using cefetamet pivoxil (L) drug. In the presence of F- ions, the drug L selectively portrayed a naked-eye detectable color change from colorless to red with the appearance of a new charge transfer band at 500 nm. No significant color change was
0.8
absorbance
2.0
1.5
0.4
0.2
0.0
0
M
1.0
0.0
te
300
400
2
500
4
6
8
10
12
Fluoride, M
600
700
wavelength, nm
Ac ce p
200
d
0.5
79M
us
2.5
0.6
an
absorbance at 500nm
Lowest detection limit =
cr
3.0
ip t
observed with other anions such as Cl−, Br−, I−, AcO−, HSO4− and H2PO4−.
Page 2 of 30
*Highlights (for review)
Highlights We have developed a simple fluoride ion selective colorimetric sensor by using the Cefetamet Pivoxil drug. The drug showed a naked-eye detectable color change upon addition of fluoride ion
ip t
with the appearance of a new charge transfer band at 500 nm, without any interfering effects of
Ac ce p
te
d
M
an
us
cr
other tested anions. The detection limit was found to be 79 µM under competitive environment.
Page 3 of 30
*Manuscript
Fluoride selective colorimetric sensor based on cefetamet pivoxil drug Saravana Kumar Sa, Shilpa Bothrab, Suban K Sahoob and Ashok Kumar S.Kc* a
Department of Applied Chemistry, SV National Institute of Technology (SVNIT), Surat-
ip t
b
Research and Development, Orchid Pharma, Chennai-600119, India
c
cr
395007, Gujarat, India. E-mail: [email protected]
Materials Chemistry Division, School of Advanced Sciences, VIT University, Vellore-632014,
an
us
India. India. E-mail: [email protected]
Abstract
M
A simple fluoride ion selective chemosensor was developed using cefetamet pivoxil (L) drug. In the presence of F- ions, the drug L selectively portrayed a naked-eye detectable color change
d
from colorless to red with the appearance of a new charge transfer band at 500 nm. No
te
significant color change was observed with other tested anions such as Cl−, Br−, I−, AcO−, HSO4− and H2PO4−. Further, the antimicrobial activity of L was screened in the absence and presence of
Ac ce p
F- by agar well diffusion method.
Keywords: Cefetamet pivoxil drug, colorimetric, fluoride, DFT, antimicrobial activity.
1 Page 4 of 30
1. Introduction Fluorine is the thirteenth most abundant element in the earth’s crust and is the lightest member of the halogens. It is the most electronegative and reactive of all the elements and as a
ip t
result, elemental fluorine does not occur in nature but is found as fluoride mineral complexes [1]. Fluoride is present in soil and rock formations: fluorapatite [Ca5(PO4)3F], fluorspar (CaF2),
cr
amphiboles [Na(CaNa)Mg5 Si8O22F2], micas [K(Fe,Mg)3AlSi3O10(F,OH)2] [2-5]. The fluoride
us
present in these soil/rock/minerals is substituted by hydroxide ions resulting in the release of fluoride ions to the circulating water [5]. Hence, the presence of high fluoride content in drinking
an
water is a serious health hazard and found to cause arthritis, osteoporosis, hip fractures, cancer, infertility, Alzheimer’s disease and brain damage [6,7]. Human body is also exposed to fluoride
M
mainly through consumption of water and other edible products; for example, fluoride contents
d
in drinking water is generally in the range of 0.5-1.5 mg/L, tea leaves containing 4-138 µg of
te
fluoride per gram, toothpaste containing 53-338 µg of fluoride per gram and 16-306 µg per gram for pan masala with tobacco [8]. Besides its biological role, fluoride is also used as a potential
Ac ce p
catalyst in a number of inorganic and organic syntheses [9,10]. The importance of accurate determination of fluoride has been increased with the growth of the practice of fluoridation of water supplies as a public health measure. Taking into account of the importance of fluoride, an attempt has been made to design and develop a low cost sensor. Fluoride ions mainly recognized through hydrogen-bonding interactions [11], electrostatic interactions [12] and coordination with metal ions [13]. Among various noncovalent interactions, hydrogen-bonding is particularly useful and effective in this respect [14]. Among the different types of chemosensors, the sensors based on colorimetric have many advantages due to the simplicity and high sensitivity. However, the development of anion sensor is challenging in compared to cations because anions are larger (lower charge to radius ratio), pH 2 Page 5 of 30
sensitive, highly solvated and they come in a wide range of different geometries: spherical, linear, trigonal, tetrahedral, octahedral, etc., [15,16]. Recently, numerous charged/neutral receptors containing binding groups such as imidazoles [17,18], pyrroles [19], calixpyrroles [20],
[29,30] have been studied for the selective sensing of target anions.
cr
ip t
amides [21,22], cabamides [23], phenols [24], ureas [25,26], thioureas [27,28] and amidoureas
us
Cephalosporins are β-lactam antibiotics that differ from the penicillin's in that the β-ring is 6-membered dihydrothiazine ring (Fig. 1) [31]. Variations among the cephalosporins are made
an
on either the acyl side chain at 7-position to change antibacterial activity or at the 3-position to alter the pharmaokinetic profile. Cephalosporin C was first isolated in 1948 by Dr. Abraham
M
from a fungus, Cephalosporium acremonium, collected in seawater near a seage outlet in
d
Sardinia by Professor Guiseppe Brotzu in 1945. Moreover, Cefetamet is classified as a third
te
generation cephalosporin with excellent activity against many aerobic gram positive and gram negative organisms [32,33]. In this paper, we have investigated the colorimetric sensing ability
Ac ce p
of cefetamet pivoxil (L) drug towards different anions such as F−, Cl−, Br−, I-, AcO¯, HSO4− and H2PO4− by various experimental (naked-eye, UV-Vis, 1H NMR and FT-IR) and theoretical (B3LYP/6-31G(d,p)) methods. Also, the antimicrobial activity of L was screened in the absence and presence of F- by agar well diffusion method.
2. Experimental 2.1. Materials and methods All chemicals of AR grade were purchased from Sigma-Aldrich, Alfa Aesar or Spectrochem based on their availability and used without further purification. All the solvents were procured from SD Fine, India of HPLC grade and used without further purification. The 3 Page 6 of 30
anions were added in the form of tetra-n-butyl ammonium (TBA) salts and were obtained commercially in the purest form. UV-Vis spectra were recorded on a VARIAN CARY 50 spectrophotometer in the wavelength range of 250-700 nm with a quartz cuvette of 1 cm path
ip t
length. All spectroscopic experiments were carried out at room temperature. Stock solutions of drug (1.0 ×10-3 M) and different anions (1.0 × 10-3 M) were prepared in CH3CN and stored in the
cr
dark chamber. 1H NMR spectra were recorded on a Bruker AM 400 spectrometer with
us
tetramethylsilane (TMS) as internal reference and DMSO-d6 as solvent. The infrared spectrum (KBr pellet) was recorded using a Perkin-Elmer IR spectrophotometer in the range of 400-4000
an
cm-1. The drug was collected from Orchid Pharma, Chennai, India and characterized by 1H NMR (DMSO-d6, δ, ppm: 1.16 (s, 9H, CH3), 2.03 (s, 3H, CH3), 3.42 & 3.63 (two doublets, 2H, CH2),
M
3.93 (s, 3H, CH3), 5.16 (d, 1H, CH), 5.73 (dd, aH, CH), 5.78 & 5.89 (two doublets, 2H, CH2),
d
6.92 (s, 1H, CH), 8.68 (sb, 2H, NH2) and 9.77 (d. 1H, amide-NH).
te
2.2. Determination of antimicrobial activity
The materials Muller Hinton Agar plates, Haemophilus test medium plates, sterile saline,
Ac ce p
densi-la-meter, cotton swabs, cefepime (30 µg) discs were used for the antimicrobial test. The cultures E. Coli ATCC 25922 and H. Influenzae ATCC 33533 were used. Muller Hinton Agar plate and Haemophilus medium plate were inoculated with overnight grown cultures, previously adjusted to 0.5 McFarland standard turbidity and diluted to 1:10 using sterile saline solution, following the CLSI recommendations. Wells were made in the plates with a well puncture rod. Then, 10µL of the drug (1mM), fluoride and 20µL (1mM) of the mixture of drug and fluoride were added to the wells. Similarly, 30µL of standard drug was added as a control. All plated were incubated overnight at 37º C, and the zone of inhibition was measured to determine the antimicrobial activity. 2.3. Computational methods 4 Page 7 of 30
The theoretical calculations were carried out with the Gaussian 09W computer code using the density functional theory (DFT) method [34]. The structural optimization of receptor L and the fluoride-L complex was performed without symmetry constraints by applying B3LYP/6-
ip t
31G(d,p) method in the gas phase. Then, the harmonic vibrational frequency calculations were carried out to ascertain the presence of a local minimum.
cr
3. Results and discussion
us
3.1. Anion sensing ability of L
The selective colorimetric sensing ability of L (5 × 10-4 M) towards different anions (5 ×
an
10-4 M) were monitored by naked-eye observation and UV-Vis absorption spectra in CH3CN. The naked-eye test inferred that the colorless solution of L changed into red on selective
M
interaction with one equivalent of F- (Fig. 2). However, the addition of other anions (Cl¯, Br¯,
d
I¯, AcO¯, H2PO4¯ and HSO4¯) did not result in obvious visual responses even in abundance.
te
Simultaneously, the absorption spectra of L were recorded in the absence and presence of one equivalent of different anions to investigate the qualitative anion binding ability of the drug in
Ac ce p
CH3CN (Fig. 2). The colorless drug showed an absorption band at 275 nm can be assigned due to π→π* transition. With the addition of F- ions to the drug solution resulted in the appearance of a new broad peak at ~500 nm which indicates the possible interaction of drug (L) with fluoride anion through intermolecular hydrogen bonding and/or deprotonation process. The new broad red-shifted band can be attributed to the internal charge transfer (ICT). The addition of other anions Cl-, Br-, I-, AcO¯, H2PO4¯ and HSO4¯ did not result in obvious spectral changes even in abundance. Further experiments (Fig. 3) inferred that the lower concentration (3 × 10-4 M) of L and F- at equivalent amount required for the detectable color changes.
S0022-1139(14)00117-1 http://dx.doi.org/doi:10.1016/j.jfluchem.2014.04.015 FLUOR 8311
To appear in:
FLUOR
Received date: Revised date: Accepted date:
26-2-2014 25-4-2014 29-4-2014
Please cite this article as: S.S. Kumar, S. Bothra, S.K. Sahoo, A.S.K. Kumar, Fluoride selective colorimetric sensor based on cefetamet pivoxil drug, Journal of Fluorine Chemistry (2014), http://dx.doi.org/10.1016/j.jfluchem.2014.04.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Graphical Abstract - Pictogram
A simple fluoride ion selective chemosensor was developed using cefetamet pivoxil (L) drug. In the presence of F- ions, the drug L selectively portrayed a naked-eye detectable color change from colorless to red with the appearance of a new charge transfer band at 500 nm. No significant color change was observed with other anions such as Cl−, Br−, I−, AcO−, HSO4− and
3.0 0.8
absorbance
2.0
1.5
us
2.5
0.4
0.2
0.0
0
M
1.0
0.0 300
400
2
500
4
6
8
10
12
Fluoride, M
600
700
wavelength, nm
Ac ce p
te
200
d
0.5
79M
an
absorbance at 500nm
Lowest detection limit = 0.6
cr
ip t
H2PO4−.
Page 1 of 30
*Graphical Abstract - Synopsis
Graphical Abstract A simple fluoride ion selective chemosensor was developed using cefetamet pivoxil (L) drug. In the presence of F- ions, the drug L selectively portrayed a naked-eye detectable color change from colorless to red with the appearance of a new charge transfer band at 500 nm. No significant color change was
0.8
absorbance
2.0
1.5
0.4
0.2
0.0
0
M
1.0
0.0
te
300
400
2
500
4
6
8
10
12
Fluoride, M
600
700
wavelength, nm
Ac ce p
200
d
0.5
79M
us
2.5
0.6
an
absorbance at 500nm
Lowest detection limit =
cr
3.0
ip t
observed with other anions such as Cl−, Br−, I−, AcO−, HSO4− and H2PO4−.
Page 2 of 30
*Highlights (for review)
Highlights We have developed a simple fluoride ion selective colorimetric sensor by using the Cefetamet Pivoxil drug. The drug showed a naked-eye detectable color change upon addition of fluoride ion
ip t
with the appearance of a new charge transfer band at 500 nm, without any interfering effects of
Ac ce p
te
d
M
an
us
cr
other tested anions. The detection limit was found to be 79 µM under competitive environment.
Page 3 of 30
*Manuscript
Fluoride selective colorimetric sensor based on cefetamet pivoxil drug Saravana Kumar Sa, Shilpa Bothrab, Suban K Sahoob and Ashok Kumar S.Kc* a
Department of Applied Chemistry, SV National Institute of Technology (SVNIT), Surat-
ip t
b
Research and Development, Orchid Pharma, Chennai-600119, India
c
cr
395007, Gujarat, India. E-mail: [email protected]
Materials Chemistry Division, School of Advanced Sciences, VIT University, Vellore-632014,
an
us
India. India. E-mail: [email protected]
Abstract
M
A simple fluoride ion selective chemosensor was developed using cefetamet pivoxil (L) drug. In the presence of F- ions, the drug L selectively portrayed a naked-eye detectable color change
d
from colorless to red with the appearance of a new charge transfer band at 500 nm. No
te
significant color change was observed with other tested anions such as Cl−, Br−, I−, AcO−, HSO4− and H2PO4−. Further, the antimicrobial activity of L was screened in the absence and presence of
Ac ce p
F- by agar well diffusion method.
Keywords: Cefetamet pivoxil drug, colorimetric, fluoride, DFT, antimicrobial activity.
1 Page 4 of 30
1. Introduction Fluorine is the thirteenth most abundant element in the earth’s crust and is the lightest member of the halogens. It is the most electronegative and reactive of all the elements and as a
ip t
result, elemental fluorine does not occur in nature but is found as fluoride mineral complexes [1]. Fluoride is present in soil and rock formations: fluorapatite [Ca5(PO4)3F], fluorspar (CaF2),
cr
amphiboles [Na(CaNa)Mg5 Si8O22F2], micas [K(Fe,Mg)3AlSi3O10(F,OH)2] [2-5]. The fluoride
us
present in these soil/rock/minerals is substituted by hydroxide ions resulting in the release of fluoride ions to the circulating water [5]. Hence, the presence of high fluoride content in drinking
an
water is a serious health hazard and found to cause arthritis, osteoporosis, hip fractures, cancer, infertility, Alzheimer’s disease and brain damage [6,7]. Human body is also exposed to fluoride
M
mainly through consumption of water and other edible products; for example, fluoride contents
d
in drinking water is generally in the range of 0.5-1.5 mg/L, tea leaves containing 4-138 µg of
te
fluoride per gram, toothpaste containing 53-338 µg of fluoride per gram and 16-306 µg per gram for pan masala with tobacco [8]. Besides its biological role, fluoride is also used as a potential
Ac ce p
catalyst in a number of inorganic and organic syntheses [9,10]. The importance of accurate determination of fluoride has been increased with the growth of the practice of fluoridation of water supplies as a public health measure. Taking into account of the importance of fluoride, an attempt has been made to design and develop a low cost sensor. Fluoride ions mainly recognized through hydrogen-bonding interactions [11], electrostatic interactions [12] and coordination with metal ions [13]. Among various noncovalent interactions, hydrogen-bonding is particularly useful and effective in this respect [14]. Among the different types of chemosensors, the sensors based on colorimetric have many advantages due to the simplicity and high sensitivity. However, the development of anion sensor is challenging in compared to cations because anions are larger (lower charge to radius ratio), pH 2 Page 5 of 30
sensitive, highly solvated and they come in a wide range of different geometries: spherical, linear, trigonal, tetrahedral, octahedral, etc., [15,16]. Recently, numerous charged/neutral receptors containing binding groups such as imidazoles [17,18], pyrroles [19], calixpyrroles [20],
[29,30] have been studied for the selective sensing of target anions.
cr
ip t
amides [21,22], cabamides [23], phenols [24], ureas [25,26], thioureas [27,28] and amidoureas
us
Cephalosporins are β-lactam antibiotics that differ from the penicillin's in that the β-ring is 6-membered dihydrothiazine ring (Fig. 1) [31]. Variations among the cephalosporins are made
an
on either the acyl side chain at 7-position to change antibacterial activity or at the 3-position to alter the pharmaokinetic profile. Cephalosporin C was first isolated in 1948 by Dr. Abraham
M
from a fungus, Cephalosporium acremonium, collected in seawater near a seage outlet in
d
Sardinia by Professor Guiseppe Brotzu in 1945. Moreover, Cefetamet is classified as a third
te
generation cephalosporin with excellent activity against many aerobic gram positive and gram negative organisms [32,33]. In this paper, we have investigated the colorimetric sensing ability
Ac ce p
of cefetamet pivoxil (L) drug towards different anions such as F−, Cl−, Br−, I-, AcO¯, HSO4− and H2PO4− by various experimental (naked-eye, UV-Vis, 1H NMR and FT-IR) and theoretical (B3LYP/6-31G(d,p)) methods. Also, the antimicrobial activity of L was screened in the absence and presence of F- by agar well diffusion method.
2. Experimental 2.1. Materials and methods All chemicals of AR grade were purchased from Sigma-Aldrich, Alfa Aesar or Spectrochem based on their availability and used without further purification. All the solvents were procured from SD Fine, India of HPLC grade and used without further purification. The 3 Page 6 of 30
anions were added in the form of tetra-n-butyl ammonium (TBA) salts and were obtained commercially in the purest form. UV-Vis spectra were recorded on a VARIAN CARY 50 spectrophotometer in the wavelength range of 250-700 nm with a quartz cuvette of 1 cm path
ip t
length. All spectroscopic experiments were carried out at room temperature. Stock solutions of drug (1.0 ×10-3 M) and different anions (1.0 × 10-3 M) were prepared in CH3CN and stored in the
cr
dark chamber. 1H NMR spectra were recorded on a Bruker AM 400 spectrometer with
us
tetramethylsilane (TMS) as internal reference and DMSO-d6 as solvent. The infrared spectrum (KBr pellet) was recorded using a Perkin-Elmer IR spectrophotometer in the range of 400-4000
an
cm-1. The drug was collected from Orchid Pharma, Chennai, India and characterized by 1H NMR (DMSO-d6, δ, ppm: 1.16 (s, 9H, CH3), 2.03 (s, 3H, CH3), 3.42 & 3.63 (two doublets, 2H, CH2),
M
3.93 (s, 3H, CH3), 5.16 (d, 1H, CH), 5.73 (dd, aH, CH), 5.78 & 5.89 (two doublets, 2H, CH2),
d
6.92 (s, 1H, CH), 8.68 (sb, 2H, NH2) and 9.77 (d. 1H, amide-NH).
te
2.2. Determination of antimicrobial activity
The materials Muller Hinton Agar plates, Haemophilus test medium plates, sterile saline,
Ac ce p
densi-la-meter, cotton swabs, cefepime (30 µg) discs were used for the antimicrobial test. The cultures E. Coli ATCC 25922 and H. Influenzae ATCC 33533 were used. Muller Hinton Agar plate and Haemophilus medium plate were inoculated with overnight grown cultures, previously adjusted to 0.5 McFarland standard turbidity and diluted to 1:10 using sterile saline solution, following the CLSI recommendations. Wells were made in the plates with a well puncture rod. Then, 10µL of the drug (1mM), fluoride and 20µL (1mM) of the mixture of drug and fluoride were added to the wells. Similarly, 30µL of standard drug was added as a control. All plated were incubated overnight at 37º C, and the zone of inhibition was measured to determine the antimicrobial activity. 2.3. Computational methods 4 Page 7 of 30
The theoretical calculations were carried out with the Gaussian 09W computer code using the density functional theory (DFT) method [34]. The structural optimization of receptor L and the fluoride-L complex was performed without symmetry constraints by applying B3LYP/6-
ip t
31G(d,p) method in the gas phase. Then, the harmonic vibrational frequency calculations were carried out to ascertain the presence of a local minimum.
cr
3. Results and discussion
us
3.1. Anion sensing ability of L
The selective colorimetric sensing ability of L (5 × 10-4 M) towards different anions (5 ×
an
10-4 M) were monitored by naked-eye observation and UV-Vis absorption spectra in CH3CN. The naked-eye test inferred that the colorless solution of L changed into red on selective
M
interaction with one equivalent of F- (Fig. 2). However, the addition of other anions (Cl¯, Br¯,
d
I¯, AcO¯, H2PO4¯ and HSO4¯) did not result in obvious visual responses even in abundance.
te
Simultaneously, the absorption spectra of L were recorded in the absence and presence of one equivalent of different anions to investigate the qualitative anion binding ability of the drug in
Ac ce p
CH3CN (Fig. 2). The colorless drug showed an absorption band at 275 nm can be assigned due to π→π* transition. With the addition of F- ions to the drug solution resulted in the appearance of a new broad peak at ~500 nm which indicates the possible interaction of drug (L) with fluoride anion through intermolecular hydrogen bonding and/or deprotonation process. The new broad red-shifted band can be attributed to the internal charge transfer (ICT). The addition of other anions Cl-, Br-, I-, AcO¯, H2PO4¯ and HSO4¯ did not result in obvious spectral changes even in abundance. Further experiments (Fig. 3) inferred that the lower concentration (3 × 10-4 M) of L and F- at equivalent amount required for the detectable color changes.
5 Page 8 of 30
The UV-Visible absorption titration of L was performed with the successive addition of F- to determine the linearity range and the detection limit by applying the equation 3/slope [6]. As shown in Fig. 4, the drug showed an excellent linearity for the detection of F- ions between
ip t
79-810 µM with the detection limit down to 79 µM. Then, the competitive experiments were
cr
performed to examine the specificity of the drug L for detecting fluoride anion in the presence of other interfering anions. As shown in Fig. 5, the fluoride ions detection ability of L was not
us
significantly influenced in the presence of other interfering anions. Also, the absorption titrations of L with F- ions were performed in the presence of other interfering anions (Fig. 6), which
an
resulted spectral changes as observed only with fluoride (Fig. 4).
M
d
te
The fluoride detection ability of the drug L was also investigated in mixed CH3CN/H2O medium (Fig. 7). The higher tendency of fluoride to form its hydrated ions can altered the
Ac ce p
hydrogen bonding interactions between the drug L and the fluoride ions in the presence of protic solvents such as H2O, which consequently resulted a reversal of the visual color and the spectral changes [35]. In case of L, the charge transfer band assigned for the L(F-) complex and also the red color was observed up to the 15% water content in CH3CN. Therefore, this drug can be applied for the detection of inorganic fluoride present in a water medium.
The UV-Visible absorption titration of L was performed with the successive addition of F- to determine the linearity range and the detection limit by applying the equation 3/slope [6]. As shown in Fig. 4, the drug showed an excellent linearity for the detection of F- ions between
ip t
79-810 µM with the detection limit down to 79 µM. Then, the competitive experiments were
cr
performed to examine the specificity of the drug L for detecting fluoride anion in the presence of other interfering anions. As shown in Fig. 5, the fluoride ions detection ability of L was not
us
significantly influenced in the presence of other interfering anions. Also, the absorption titrations of L with F- ions were performed in the presence of other interfering anions (Fig. 6), which
an
resulted spectral changes as observed only with fluoride (Fig. 4).
M
d
te
The fluoride detection ability of the drug L was also investigated in mixed CH3CN/H2O medium (Fig. 7). The higher tendency of fluoride to form its hydrated ions can altered the
Ac ce p
hydrogen bonding interactions between the drug L and the fluoride ions in the presence of protic solvents such as H2O, which consequently resulted a reversal of the visual color and the spectral changes [35]. In case of L, the charge transfer band assigned for the L(F-) complex and also the red color was observed up to the 15% water content in CH3CN. Therefore, this drug can be applied for the detection of inorganic fluoride present in a water medium.
To investigate the practical application of drug L, colorimetric paper-made test strips were prepared by immersing the filter papers into a CH3CN/H2O (9:1, v/v) solution of the drug L (0.001 M) and then dried (Fig. 8a). The test strips containing L were utilized to sense different anions. Upon addition of several drops of different anions (1.0×10-3 M) to test strips, the obvious 6 Page 9 of 30
color change from colorless to dark brown was observed only with the F- anion solution (Fig. 8b). However, no significant color change was observed with other tested anions. Therefore, the test strip would have potential application for the colorimetric detection of F- anion easily and
ip t
rapidly.
cr
3.2. Sensing mechanism
us
The host-guest interaction between L and fluoride anion was investigated by the 1H NMR and 19F NMR measurements of L in the absence and presence of different equivalents of TBAF
an
in DMSO-d6. The free drug L showed a signal at ~9.77 ppm due to amide-NH proton (Fig. 9). Upon addition of different equivalents of F- ions resulted in the disappearance of the drug peak
M
due to amide-NH proton and a new broad peak at ~16 ppm was appeared (Fig. 9b) due to the
d
formation of bi-fluoride ion (FHF-) [36]. The broad peak at 16 ppm due to FHF- dimmer clearly
te
delineated the deprotonation of the drug L. Simultaneously, no remarkable variations were observed with other signals which inferred that the drug L is recognizing fluoride anion through
Ac ce p
N-H…F hydrogen bonds followed by a deprotonation process without any apparent conformational changes. Further, on addition of D2O, the FHF- dimmer broad peak at 16 ppm was disappeared (Fig. 9c) and the peaks due to drug (L) were restored. Moreover, the deprotonation mechanism was confirmed from 19F NMR experiments (Fig. 10). The peak due to free fluoride of TBAF was appeared at ~ -99 ppm. Upon addition of different equivalents of TBAF to L solution in DMSO-d6, the red color solution gave an intense peak at ~ -145 ppm due to the formation of FHF- dimmer.
7 Page 10 of 30
The participation of amide-NH in fluoride recognition was also supported by FTIR analysis of L and its L(F-) complex (Fig. 11). Fig. 11 stated that the δNH of the free drug appeared at 3262 cm-1 was disappeared in the presence of fluoride. The results clearly
ip t
demonstrate that the fluoride anion was functioning here as a base, giving rise to the deprotonation upon interaction with the receptor L.
cr
us
The structural optimization of L and its host-guest complex with fluoride anion was performed at B3LYP/6-31G(d,p) level by focusing on the proposed 1:1 binding mode to
an
complement the experimental evidences. The optimized global minimum structure of L and its L (F-) complex (Fig. 12) indicates that the amide-NH bond length of L increases from 1.012 Å to
M
1.551 Å on interaction with fluoride through hydrogen bonding followed by deprotonation
d
process. Further analysis of HOMO and LUMO diagrams (Fig. 12) of L and L(F-) indicate the
te
→* electronic transition and the internal charge transfer occurred during the anion recognition process. The HOMO and LUMO electron density of L was distributed mainly over thiazol unit.
Ac ce p
However, on interaction with fluoride ion, the charge delocalization occurred by deprotonation process of amide-NH, and the HOMO and LUMO electron density was shifted to dihydrothiazine unit. Also, the HOMO-LUMO band gap of L was decreased on interaction with fluoride, which supports the absorbance at longer wavelength and red coloration.
3.3. Determination of antimicrobial activity of L The antimicrobial activity was screened to understand whether L(F-) complex will preserve the originality of drug L or not. When TBAF contacted with selected microganisms (E.coli ATCC 25922 and H. Influenzae ATCC33533), there was no zone formation. However, when pure drug (L) and complexed form of drug L(F-) contacted with selected microrganisms 8 Page 11 of 30
showed a clear inhibition zone formation (Table 1). Moreover, inhibition zones exhibited by pure drug and L(F-) complex were comparable with the standard drug. This is because beta lactam moiety of the drug remain unbroken on interaction with fluoride.
ip t
4. Conclusion
color change from colorless to dark brown was observed only with the F- anion solution (Fig. 8b). However, no significant color change was observed with other tested anions. Therefore, the test strip would have potential application for the colorimetric detection of F- anion easily and
ip t
rapidly.
cr
3.2. Sensing mechanism
us
The host-guest interaction between L and fluoride anion was investigated by the 1H NMR and 19F NMR measurements of L in the absence and presence of different equivalents of TBAF
an
in DMSO-d6. The free drug L showed a signal at ~9.77 ppm due to amide-NH proton (Fig. 9). Upon addition of different equivalents of F- ions resulted in the disappearance of the drug peak
M
due to amide-NH proton and a new broad peak at ~16 ppm was appeared (Fig. 9b) due to the
d
formation of bi-fluoride ion (FHF-) [36]. The broad peak at 16 ppm due to FHF- dimmer clearly
te
delineated the deprotonation of the drug L. Simultaneously, no remarkable variations were observed with other signals which inferred that the drug L is recognizing fluoride anion through
Ac ce p
N-H…F hydrogen bonds followed by a deprotonation process without any apparent conformational changes. Further, on addition of D2O, the FHF- dimmer broad peak at 16 ppm was disappeared (Fig. 9c) and the peaks due to drug (L) were restored. Moreover, the deprotonation mechanism was confirmed from 19F NMR experiments (Fig. 10). The peak due to free fluoride of TBAF was appeared at ~ -99 ppm. Upon addition of different equivalents of TBAF to L solution in DMSO-d6, the red color solution gave an intense peak at ~ -145 ppm due to the formation of FHF- dimmer.
7 Page 10 of 30
The participation of amide-NH in fluoride recognition was also supported by FTIR analysis of L and its L(F-) complex (Fig. 11). Fig. 11 stated that the δNH of the free drug appeared at 3262 cm-1 was disappeared in the presence of fluoride. The results clearly
ip t
demonstrate that the fluoride anion was functioning here as a base, giving rise to the deprotonation upon interaction with the receptor L.
cr
us
The structural optimization of L and its host-guest complex with fluoride anion was performed at B3LYP/6-31G(d,p) level by focusing on the proposed 1:1 binding mode to
an
complement the experimental evidences. The optimized global minimum structure of L and its L (F-) complex (Fig. 12) indicates that the amide-NH bond length of L increases from 1.012 Å to
M
1.551 Å on interaction with fluoride through hydrogen bonding followed by deprotonation
d
process. Further analysis of HOMO and LUMO diagrams (Fig. 12) of L and L(F-) indicate the
te
→* electronic transition and the internal charge transfer occurred during the anion recognition process. The HOMO and LUMO electron density of L was distributed mainly over thiazol unit.
Ac ce p
However, on interaction with fluoride ion, the charge delocalization occurred by deprotonation process of amide-NH, and the HOMO and LUMO electron density was shifted to dihydrothiazine unit. Also, the HOMO-LUMO band gap of L was decreased on interaction with fluoride, which supports the absorbance at longer wavelength and red coloration.
3.3. Determination of antimicrobial activity of L The antimicrobial activity was screened to understand whether L(F-) complex will preserve the originality of drug L or not. When TBAF contacted with selected microganisms (E.coli ATCC 25922 and H. Influenzae ATCC33533), there was no zone formation. However, when pure drug (L) and complexed form of drug L(F-) contacted with selected microrganisms 8 Page 11 of 30
showed a clear inhibition zone formation (Table 1). Moreover, inhibition zones exhibited by pure drug and L(F-) complex were comparable with the standard drug. This is because beta lactam moiety of the drug remain unbroken on interaction with fluoride.
ip t