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Vibrational spectroscopic investigations and biological activity of toxic material amitraz
Materials Today: Proceedings xxx (xxxx) xxx
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Vibrational spectroscopic investigations and biological activity of toxic material amitraz D.E. Nimmi a, Geethu Sudhi a, S.G. Praveen b, J. Binoy a,⇑ a b
Department of Physics, Govt. College for Women, University of Kerala, Thiruvananthapuram 695014, India School of Physics, Indian Institute of Science Education and Research, Thiruvananthapuram 695016, India
a r t i c l e
i n f o
Article history: Received 30 November 2019 Received in revised form 20 January 2020 Accepted 23 January 2020 Available online xxxx Keywords: Vibrational spectra DFT SERS Neurotoxic assay Molecular docking
a b s t r a c t Amitraz is a non-systemic acaricide and insecticide and has also been described as a scabicide which is slightly toxic to birds, fish and non-toxic to bees, whose structural features can be explained excellently using vibrational spectral investigations. In this work, the detailed molecular vibrational analysis of amitraz using FT-IR and FT-Raman spectral bands have been carried out with the aid of density functional theoretical (DFT) simulations, with B3LYP functional and 6-311++G(d, p) basis set and using potential energy distribution (PED) of vibrational modes. The conjugation of the lone pair of N with the psystem of C=N and the conjugation of the ring p-system with the p-system of C=N have been explored. Surface-enhanced Raman scattering (SERS) method was employed for the determination of trace amitraz, in silver-colloidal substrate. The normal Raman and SERS spectra of amitraz were analyzed, and the binding details of amitraz with silver nanocluster have been exploited. The molecular docking simulation of amitraz with AChE has been performed to find the mode of binding and the interactions with AChE has been investigated using UV–Visible spectroscopic method and fluorescence spectral studies to assess the strength of binding. Ó 2020 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on the Science and Technology of Advanced Materials.
1. Introduction The organophosphate compounds induce inhibitory attack on neurotransmitter enzyme acetylcholinesterase (AChE) and as a result of the high concentrations of AChE at nervous synapse, it has been observed to depress motor functions, respiratory depression and brain dysfunction, leading to death [1]. The organophosphate neurotoxic pesticides remain as substances largely applied in agriculture, accumulated in soil and water and affecting the animal and human health. The organophosphorous pesticides were used in substitution to organochlorine pesticides and it draws similar human health risks [2]. Amitraz(Fig. 1), a non-systemic acaricide and insecticide, has also been described as a scabicide [3] and is used as pesticide that shows a high activity and a low toxicity to bees [4], owing to its peculiar neurotoxic characteristics. Owing its specific properties, which are molecular structure dependent, the structure property correlation is of much research interest and can be effectively car⇑ Corresponding author. E-mail address: [email protected] (J. Binoy).
ried out using FT-IR and Raman spectral investigations, supported by the density functional theoretical (DFT) computation and the calculation of potential energy distribution (PED) of vibrational modes. The trace detection of pesticides such as amitraz is of highly relevant and SERS spectral explorations have been performed. The neurotoxicity has been investigated by examining the binding interaction between amitraz and AChE, using UV Visible and Fluorescence spectral assays and molecular docking.
2. Experimental 2.1. Chemicals and reagents Amitraz(C19H23N3 99%), Acetylcholinesterase (AChE, 99%), Silver nitrate(AgNO3, 99%), Sodium borohydride (NaBH499.98%) and Poly(N-vinyl-2-pyrrolidone) (PVP), all of AR grade, was obtained from Sigma Aldrich and used without further purification. Acetylcholinesterase (AChE), phosphate buffer and acetone were purchased from Sigma Aldrich and the phosphate buffer pH 8 has been used to prepare AChE solution of 0.1 M concentration. The
https://doi.org/10.1016/j.matpr.2020.01.404 2214-7853/Ó 2020 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on the Science and Technology of Advanced Materials.
Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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D.E. Nimmi et al. / Materials Today: Proceedings xxx (xxxx) xxx
Fig. 1. The optimized molecular structure of Amitraz calculated using DFT at the B3LYP/6-311++G(d,p) level.
title compound amitraz has been dissolved in acetone solution, for neurotoxic assay experiment. 2.2. Recording of spectrum The FT-IR spectrum (Fig. 2) was recorded on Shimadzu FTIR 8400S spectrophotometer by taking samples in KBr pellet, for the wavenumber range 4000–400 cm 1, with the spectral resolution of 2 cm 1. For recording Raman spectrum (Fig. 3), the compound is taken as such and is subjected to Ar laser excitation, with wavelength 632 nm. The spectrum has been recorded using Horiba JobinYvon FT Raman spectrometer in the region 4000–400 cm 1, with resolution 2 cm 1. 2.3. Preparation of SERS substrate and spectra recording Silver colloids were prepared by adding 0.01 M AgNO3 solution to excess of ice cold 0.002 M of NaBH4 under stirring, till the solution turns to light yellow [5]. For maintaining the stability, 0.005 g of (PVP) is added to yield the colloid. For the SERS spectral measurements (Fig. 4), 0.1 M of amitraz is added to the colloid [6] and the SERS spectra were obtained 10 min after the sample preparation to acquire the adsorption equilibrium of the analytes over
Fig. 3. Raman spectrum of Amitraz (a) experimental spectrum (b) Computed spectrum with scaled wavenumbers.
Fig. 4. SERS spectra of amitraz at 514 nm excitation.
Fig. 2. IR spectrum of Amitraz (a) experimental spectrum (b) Computed spectrum with scaled wavenumbers.
the metallic surfaces. SERS spectra were recorded using Horiba Lab Ram HR Raman Spectrometer with an excitation wavelength of 514 nm, 8 mW laser and the SERS spectra were obtained in the range from 3000 to 500 cm 1.
Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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2.4. Neurotoxic assay UV–Vis absorbance spectral measurements were performed on a UV-1800 Schimadzu UV spectrophotometer and the absorption spectra were recorded over the range of 220–400 nm. Amitraz concentration was kept constant at 0.1 mM with varying AChE concentrations of 0, 0.0109, 0.0218, 0.0437, 0.0875, 0.175 and 0.35 mM for the binding measurements. Fluorescence spectral measurements were performed using PerkinElmer fluorescence spectrometer LS 45. In fluorescence spectral experiments, 0.1 mM amitraz solution was allowed to react with different concentrations of AChE. The fluorescence spectra of the solution were obtained by exciting at 270 nm and measuring the emission spectra from 360 to 660 nm, using 2 nm slits.
Fig. 5. Conjugation with nitrogen lone pair with the p-system of C=N.
Table 2 The interaction of functional groups of amitraz with silver nano system.
2.5. Computational details DFT calculations, with B3LYP functional [7,8] and 6-311++ G(d, p) basis set [9],were carried out to optimize the molecular structure of Amitraz (Fig. 1), using Gaussian’09 software [10]. The calculated vibrational frequencies have been scaled, using standard scaling factor 0.9679 [11] and the molecular vibrational analysis has been performed using eigen vector distribution obtained from Gaussview 5.0 program [12] and vibrational potential energy distribution (PED) [13], computed using the software VEDA[14].The molecular docking simulation studies were performed using, Auto Dock 4.2 program [15] and the binding of the present compound Amitraz with Acetylcholinesterase (AChE) has been investigated and the output has been analyzed [16]. 3. Results and discussion 3.1. Structural and vibrational spectral analysis The structural analysis of amitraz reveals that the ring p-system has been found to conjugate with the p-system of C=N, reducing the bondlengths of C29-N28 and C4-N18 to 1.406 Å and 1.408 Å respectively, from the standard value of 1.47 Å [17]. The C29-N28 stretching vibrations were observed at 1593 cm 1 in FT-Raman spectra and the corresponding calculated frequencies were observed at 1592 cm 1 (Table. 1). Also, C4-N18 stretching modes are at 1491 cm 1 as medium band in IR spectrum (Fig. 2) and in Raman spectrum (Fig. 3), it can be observed at 1490 cm 1, supported by the computed band position at 1480 cm 1, higher than the normal C-N stretching frequency range of 1020–1250 cm 1
Table 1 Vibrational wavenumbers (cm
1
Relative Intensity
SERS Frequency
3043 2916 1663 1603 – 1490 1453 1243 1120 – 956 806 – 720 473 443
S S S S
– 2930 1698 – 1522 – 1427 1248 – 1062 – 796 – – – –
M M M S S
Assignment
Interacting functional group with Ag-nanosystem
2930 1698 1522 1427 1248 1062 796
m(C10H11 + C10H12 + C10H13) m(N28C26 + N18C19) + CH3 m(C35C32 + C29C30 + C31C34)
Me4 Ph1 + Me3 Ph1 Me3 Ph1 + Me1 + Me2 Me1 Ph2
ds(H24C22H23 + H25C22H24) dw(C29C30 + C38C31 + C42C35) dw(H45C42H44 + H45C42C35C32) c(H7C2C3C4 + H8C3C4C5)
[18]. The higher bond strength, concluded from the lower bondlength and higher stretching band position, can be attributed to the partial double bond character of C-N originated from the phenyl ring conjugation with the p-system of C=N. Another conjugation can be found between the lone pair of N21 with the p-system of C=N, stronger than the conjugation with the ring p-system (Fig. 5). The stronger conjugation is evident from the much higher reduction in C-N bondlength, whose values are 1.394 Å and 1.387 Å respectively for N21-C26 and N21-C19. The conjugation can be further confirmed by the lesser negative charge (-0.47e) acquired by N21, compared to neighbour nitrogen atoms N18 and N28 whose NBO charges are respectively equal to 0.51e and 0.49e. The conjugation can be further confirmed by the higher vibrational stretching wavenumbers and the stretching frequency of N21-C26 can be found in Raman at 1663 cm 1, where the computed band position is 1687 cm 1. Also, for N21-C19, the stretching vibrations were observed in FT-IR spectra at 1620 cm 1 and in FT-Raman spectra at 1603 cm 1, in correlation with the scaled computed frequency at 1628 cm 1. The higher shift in band positions from the normal frequency range for N21-C26 and N21-C19, compared to those of C29-N28 and C4-N18 further confirms
) and assignments of experimental Raman and SERS spectrum.
FT Raman Frequency
M M M M
SERS frequency m (cm 1)
Relative Intensity vvs m vw vw vw vw m
Computed and Scaled Frequency
Assignment
3043 2928 1687 1628 1549 1442 1408 1215 1102 1026 929 798 744 701 453 444
m (C19H20 + C22H25) m (C10H11 + C10H12 + C10H13) m (N28C26 + N18C19) + CH3 m (C30C32 + C31C34) m (C35C32 + C29 C30 + C31C34) ds (H13C10H12 + H15C14H16) ds (H24C22H23 + H25C22H24) dw(C29C30 + C38C31 + C42C35) dw (H36C32C35 + C29C30C32) dw (H45C42H44H43 + H37C32C35H36) c(H27C26N28C29 + N21C26N28C29) c (H7C2C3C4 + H8C3C4C5) c (H33C30C32C35) c (C29C30C32C35) c (C34C35C32) c(C38C29C34C31)
m = stretching; ds = scissors;dw = wagging; c = out of plane bend. Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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D.E. Nimmi et al. / Materials Today: Proceedings xxx (xxxx) xxx Table 3 Eigen vector distribution of vibrational modes of amitraz obtained from the DFT calculations.
Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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that the conjugation of nitrogen lone pair with p-system of C=N is higher than ring- (C=N) conjugation. 3.2. Experimental Raman and SERS spectra The detailed vibrational analysis of molecules and the vibrational modes observed in SERS (Fig. 4) provides insight to the binding characteristics of amitraz on silver surface. (Table. 2) [19]. The bands which show maximum enhancement in SERS spectrum can be observed at 2930 cm 1, 1698 cm 1 and 796 cm 1 and the corresponding Raman bands are 2916 cm 1, 1663 cm 1 and 806 cm 1. The band positions are in correlation with DFT computed spectrum and can be observed at 2928 cm 1, 1687 cm 1 and 798 cm 1 respectively. The maximum enhancement has been observed of mode involving methyl group and ring, where the CH out of plane bending of the ring has been enhanced. This favors the flat orientation of the ring over silver surface, whereas methyl groups are in cross proximity with silver surface (Table 3). 3.3. Neurotoxic assay 3.3.1. UV–Visible absorption studies UV absorption technique can be used to explore the structural changes of protein and to investigate protein–ligand complex formation. The UV–Vis absorption spectra of AChE-amitraz complex having different concentrations are shown in (Fig. 6). The interaction of amitraz-AChE at different concentrations (0, 0.0109, 0.0218, 0.175 and 0.35 (10 5mol/L) with amitraz (0.1 mM) was studied using UV–Vis spectroscopy. The spectrum was obtained by a strong peak at 324 nm, whose absorbance is increasing with increasing ratio of AChE/amitraz along with blue-shift, which stands for the binding in a more hydrophobic microenvironment [20].
Fig. 7. Fluorescence spectra of amitraz in the presence of AChE at different concentrations. CAmitraz = 0.1 mM, CAChE = 0, 0.0109, 0.0218, 0.0437, 0.0875, 0.175 and 0.35 (10 5mol/L) for curves 1–7, respectively.
3.3.2. Fluorescence spectral studies The fluorescence spectrum at the excitation wavelength of 270 nm was used to explore the interaction between amitraz (0.1 mM) and AChE at of different concentrations of AChE (0, 0.0109, 0.0218, 0.0437, 0.0875, 0.175 and 0.35 (10 5 mol/L)) and the results are shown (Fig. 7). This revealed that the fluorescence intensity of amitraz increase with the increase in AChE concentrations, and the maximum emission wavelength of the fluorescence is found shifted to the blue, slightly. This indicates that the hydrophobic interaction occurred between amitraz and
Molecular docking is a useful method for studying the interaction between small molecules and proteins. It can also be used for predicting and simulating the binding mode and to quantify the affinity of drugs or toxic material to protein. The molecular docking of amitraz with AChE (PDB: 5DTI) were performed [22]. The docking conformations of amitraz-AChE, were obtained and the docked conformer with highest binding has been chosen for the analysis (Table 4). Molecular docking reveals that Amitraz is found to bind with AchE, with binding energy equal to 5.56 kcal/mol (Fig. 8). The three dimensional structure of AChE consist of a catalytic triad which include an active site (A-site), a peripheral anionic site (PAS) and a long narrow hydrophobic gorge which connects the Asite and the PAS. The amino acid residues TYR 72, TRP 286, PHE 297, TYR 341 and PHE 338 belonging to PAS of AChE make noncovalent interactions with amitraz, leading to AchE-Amitraz complex formation (Table 4). It was observed that molecule preferred to interact with the Tyrosine72 residue (TYR 72) of AchE strongly, via O N interaction, the O N distance being 2.97 Å. The interaction has been favored by the partial positive charge acquired by N21 originated from lone pair-p conjugation which has been confirmed by DFT based vibrational spectral analysis and NBO charges.
AChE, leading to the microenvironment change of the amino acid residues of AChE [21]. These results indicated that molecular interaction between amitraz and AChE is strong.
3.4. Molecular docking
Table 4 The interactions of amitraz with AChE.
Fig. 6. UV absorption spectra of AChE with amitraz. The concentrations of AChE are 0, 0.0109, 0.0218, 0.175 and 0.35 (10 5 mol/L) and that of amitraz is 0.1 mM.
Interacting Residue
Type of Bonding
Distance of interaction (Å)
Tyrosine72 Tryptophan 286 Tyrosine 341 Phenylalanine 338 Tryptophan 286
N . . .. . .O interaction Pi-sigma interaction Amide-Pi stacked interaction Pi-Alkyl interaction Pi-Pi stacked interaction
2.97 3.64 5.31 4.51 5.01
Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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Fig. 8. Molecular docking of amitraz with AChE.
4. Conclusion
References
In the present study, we have carried out the experimental and theoretical spectroscopic analysis of amitraz using FTIR and FT Raman, aided by density functional theoretical simulation. The DFT study shows that stronger conjugation of the lone pair of N21 with the p-system of C=N, in addition to the conjugation of the ring p-system with the p-system of C=N. The stronger conjugation is evident from the much higher reduction in C-N bondlength and the higher vibrational stretching wavenumbers of C-N bond. The conjugation can be further confirmed by the lesser negative charge acquired by N21, compared to neighbour nitrogen atoms N18 and N28. The SERS spectral investigations reveal that maximum enhancement has been observed of mode involving methyl group and ring. The CH out of plane bending of the ring has been enhanced which favors the flat orientation of the ring over silver surface. The spectral assay using UV–Vis and fluorescence spectroscopy shows hyperchromism with blue shift, which indicated the hydrophobic interaction of amitaz with AChE. The molecular docking interaction of AChE with amitraz, the binding energy is equal to 5.56 kcal/mol and the results favor the Amitraz- AChE complex formation. The amitraz molecule preferred to interact with AchE via Tyrosine72 residue (TYR 72),through O N interaction, favoured by the loss of negative charge on nitrogen arising due to lone pair-p interactions, as confirmed by geometric and vibrational spectral analysis.
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CRediT authorship contribution statement D.E. Nimmi: Conceptualization, Methodology, Software. Geethu Sudhi: Data curation, Writing - original draft. S.G. Praveen: Visualization, Investigation. J. Binoy: Supervision, Writing - review & editing.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements The authors Nimmi.D.E. and Geethu Sudhi are thankful for the financial support from the University of Kerala. Author, J Binoy, is thankful to DST FIST scheme, department of physics, Govt College for Women, Thiruvananthapuram for instrumental support.
Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
Contents lists available at ScienceDirect
Materials Today: Proceedings journal homepage: www.elsevier.com/locate/matpr
Vibrational spectroscopic investigations and biological activity of toxic material amitraz D.E. Nimmi a, Geethu Sudhi a, S.G. Praveen b, J. Binoy a,⇑ a b
Department of Physics, Govt. College for Women, University of Kerala, Thiruvananthapuram 695014, India School of Physics, Indian Institute of Science Education and Research, Thiruvananthapuram 695016, India
a r t i c l e
i n f o
Article history: Received 30 November 2019 Received in revised form 20 January 2020 Accepted 23 January 2020 Available online xxxx Keywords: Vibrational spectra DFT SERS Neurotoxic assay Molecular docking
a b s t r a c t Amitraz is a non-systemic acaricide and insecticide and has also been described as a scabicide which is slightly toxic to birds, fish and non-toxic to bees, whose structural features can be explained excellently using vibrational spectral investigations. In this work, the detailed molecular vibrational analysis of amitraz using FT-IR and FT-Raman spectral bands have been carried out with the aid of density functional theoretical (DFT) simulations, with B3LYP functional and 6-311++G(d, p) basis set and using potential energy distribution (PED) of vibrational modes. The conjugation of the lone pair of N with the psystem of C=N and the conjugation of the ring p-system with the p-system of C=N have been explored. Surface-enhanced Raman scattering (SERS) method was employed for the determination of trace amitraz, in silver-colloidal substrate. The normal Raman and SERS spectra of amitraz were analyzed, and the binding details of amitraz with silver nanocluster have been exploited. The molecular docking simulation of amitraz with AChE has been performed to find the mode of binding and the interactions with AChE has been investigated using UV–Visible spectroscopic method and fluorescence spectral studies to assess the strength of binding. Ó 2020 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on the Science and Technology of Advanced Materials.
1. Introduction The organophosphate compounds induce inhibitory attack on neurotransmitter enzyme acetylcholinesterase (AChE) and as a result of the high concentrations of AChE at nervous synapse, it has been observed to depress motor functions, respiratory depression and brain dysfunction, leading to death [1]. The organophosphate neurotoxic pesticides remain as substances largely applied in agriculture, accumulated in soil and water and affecting the animal and human health. The organophosphorous pesticides were used in substitution to organochlorine pesticides and it draws similar human health risks [2]. Amitraz(Fig. 1), a non-systemic acaricide and insecticide, has also been described as a scabicide [3] and is used as pesticide that shows a high activity and a low toxicity to bees [4], owing to its peculiar neurotoxic characteristics. Owing its specific properties, which are molecular structure dependent, the structure property correlation is of much research interest and can be effectively car⇑ Corresponding author. E-mail address: [email protected] (J. Binoy).
ried out using FT-IR and Raman spectral investigations, supported by the density functional theoretical (DFT) computation and the calculation of potential energy distribution (PED) of vibrational modes. The trace detection of pesticides such as amitraz is of highly relevant and SERS spectral explorations have been performed. The neurotoxicity has been investigated by examining the binding interaction between amitraz and AChE, using UV Visible and Fluorescence spectral assays and molecular docking.
2. Experimental 2.1. Chemicals and reagents Amitraz(C19H23N3 99%), Acetylcholinesterase (AChE, 99%), Silver nitrate(AgNO3, 99%), Sodium borohydride (NaBH499.98%) and Poly(N-vinyl-2-pyrrolidone) (PVP), all of AR grade, was obtained from Sigma Aldrich and used without further purification. Acetylcholinesterase (AChE), phosphate buffer and acetone were purchased from Sigma Aldrich and the phosphate buffer pH 8 has been used to prepare AChE solution of 0.1 M concentration. The
https://doi.org/10.1016/j.matpr.2020.01.404 2214-7853/Ó 2020 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on the Science and Technology of Advanced Materials.
Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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Fig. 1. The optimized molecular structure of Amitraz calculated using DFT at the B3LYP/6-311++G(d,p) level.
title compound amitraz has been dissolved in acetone solution, for neurotoxic assay experiment. 2.2. Recording of spectrum The FT-IR spectrum (Fig. 2) was recorded on Shimadzu FTIR 8400S spectrophotometer by taking samples in KBr pellet, for the wavenumber range 4000–400 cm 1, with the spectral resolution of 2 cm 1. For recording Raman spectrum (Fig. 3), the compound is taken as such and is subjected to Ar laser excitation, with wavelength 632 nm. The spectrum has been recorded using Horiba JobinYvon FT Raman spectrometer in the region 4000–400 cm 1, with resolution 2 cm 1. 2.3. Preparation of SERS substrate and spectra recording Silver colloids were prepared by adding 0.01 M AgNO3 solution to excess of ice cold 0.002 M of NaBH4 under stirring, till the solution turns to light yellow [5]. For maintaining the stability, 0.005 g of (PVP) is added to yield the colloid. For the SERS spectral measurements (Fig. 4), 0.1 M of amitraz is added to the colloid [6] and the SERS spectra were obtained 10 min after the sample preparation to acquire the adsorption equilibrium of the analytes over
Fig. 3. Raman spectrum of Amitraz (a) experimental spectrum (b) Computed spectrum with scaled wavenumbers.
Fig. 4. SERS spectra of amitraz at 514 nm excitation.
Fig. 2. IR spectrum of Amitraz (a) experimental spectrum (b) Computed spectrum with scaled wavenumbers.
the metallic surfaces. SERS spectra were recorded using Horiba Lab Ram HR Raman Spectrometer with an excitation wavelength of 514 nm, 8 mW laser and the SERS spectra were obtained in the range from 3000 to 500 cm 1.
Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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2.4. Neurotoxic assay UV–Vis absorbance spectral measurements were performed on a UV-1800 Schimadzu UV spectrophotometer and the absorption spectra were recorded over the range of 220–400 nm. Amitraz concentration was kept constant at 0.1 mM with varying AChE concentrations of 0, 0.0109, 0.0218, 0.0437, 0.0875, 0.175 and 0.35 mM for the binding measurements. Fluorescence spectral measurements were performed using PerkinElmer fluorescence spectrometer LS 45. In fluorescence spectral experiments, 0.1 mM amitraz solution was allowed to react with different concentrations of AChE. The fluorescence spectra of the solution were obtained by exciting at 270 nm and measuring the emission spectra from 360 to 660 nm, using 2 nm slits.
Fig. 5. Conjugation with nitrogen lone pair with the p-system of C=N.
Table 2 The interaction of functional groups of amitraz with silver nano system.
2.5. Computational details DFT calculations, with B3LYP functional [7,8] and 6-311++ G(d, p) basis set [9],were carried out to optimize the molecular structure of Amitraz (Fig. 1), using Gaussian’09 software [10]. The calculated vibrational frequencies have been scaled, using standard scaling factor 0.9679 [11] and the molecular vibrational analysis has been performed using eigen vector distribution obtained from Gaussview 5.0 program [12] and vibrational potential energy distribution (PED) [13], computed using the software VEDA[14].The molecular docking simulation studies were performed using, Auto Dock 4.2 program [15] and the binding of the present compound Amitraz with Acetylcholinesterase (AChE) has been investigated and the output has been analyzed [16]. 3. Results and discussion 3.1. Structural and vibrational spectral analysis The structural analysis of amitraz reveals that the ring p-system has been found to conjugate with the p-system of C=N, reducing the bondlengths of C29-N28 and C4-N18 to 1.406 Å and 1.408 Å respectively, from the standard value of 1.47 Å [17]. The C29-N28 stretching vibrations were observed at 1593 cm 1 in FT-Raman spectra and the corresponding calculated frequencies were observed at 1592 cm 1 (Table. 1). Also, C4-N18 stretching modes are at 1491 cm 1 as medium band in IR spectrum (Fig. 2) and in Raman spectrum (Fig. 3), it can be observed at 1490 cm 1, supported by the computed band position at 1480 cm 1, higher than the normal C-N stretching frequency range of 1020–1250 cm 1
Table 1 Vibrational wavenumbers (cm
1
Relative Intensity
SERS Frequency
3043 2916 1663 1603 – 1490 1453 1243 1120 – 956 806 – 720 473 443
S S S S
– 2930 1698 – 1522 – 1427 1248 – 1062 – 796 – – – –
M M M S S
Assignment
Interacting functional group with Ag-nanosystem
2930 1698 1522 1427 1248 1062 796
m(C10H11 + C10H12 + C10H13) m(N28C26 + N18C19) + CH3 m(C35C32 + C29C30 + C31C34)
Me4 Ph1 + Me3 Ph1 Me3 Ph1 + Me1 + Me2 Me1 Ph2
ds(H24C22H23 + H25C22H24) dw(C29C30 + C38C31 + C42C35) dw(H45C42H44 + H45C42C35C32) c(H7C2C3C4 + H8C3C4C5)
[18]. The higher bond strength, concluded from the lower bondlength and higher stretching band position, can be attributed to the partial double bond character of C-N originated from the phenyl ring conjugation with the p-system of C=N. Another conjugation can be found between the lone pair of N21 with the p-system of C=N, stronger than the conjugation with the ring p-system (Fig. 5). The stronger conjugation is evident from the much higher reduction in C-N bondlength, whose values are 1.394 Å and 1.387 Å respectively for N21-C26 and N21-C19. The conjugation can be further confirmed by the lesser negative charge (-0.47e) acquired by N21, compared to neighbour nitrogen atoms N18 and N28 whose NBO charges are respectively equal to 0.51e and 0.49e. The conjugation can be further confirmed by the higher vibrational stretching wavenumbers and the stretching frequency of N21-C26 can be found in Raman at 1663 cm 1, where the computed band position is 1687 cm 1. Also, for N21-C19, the stretching vibrations were observed in FT-IR spectra at 1620 cm 1 and in FT-Raman spectra at 1603 cm 1, in correlation with the scaled computed frequency at 1628 cm 1. The higher shift in band positions from the normal frequency range for N21-C26 and N21-C19, compared to those of C29-N28 and C4-N18 further confirms
) and assignments of experimental Raman and SERS spectrum.
FT Raman Frequency
M M M M
SERS frequency m (cm 1)
Relative Intensity vvs m vw vw vw vw m
Computed and Scaled Frequency
Assignment
3043 2928 1687 1628 1549 1442 1408 1215 1102 1026 929 798 744 701 453 444
m (C19H20 + C22H25) m (C10H11 + C10H12 + C10H13) m (N28C26 + N18C19) + CH3 m (C30C32 + C31C34) m (C35C32 + C29 C30 + C31C34) ds (H13C10H12 + H15C14H16) ds (H24C22H23 + H25C22H24) dw(C29C30 + C38C31 + C42C35) dw (H36C32C35 + C29C30C32) dw (H45C42H44H43 + H37C32C35H36) c(H27C26N28C29 + N21C26N28C29) c (H7C2C3C4 + H8C3C4C5) c (H33C30C32C35) c (C29C30C32C35) c (C34C35C32) c(C38C29C34C31)
m = stretching; ds = scissors;dw = wagging; c = out of plane bend. Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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D.E. Nimmi et al. / Materials Today: Proceedings xxx (xxxx) xxx Table 3 Eigen vector distribution of vibrational modes of amitraz obtained from the DFT calculations.
Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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that the conjugation of nitrogen lone pair with p-system of C=N is higher than ring- (C=N) conjugation. 3.2. Experimental Raman and SERS spectra The detailed vibrational analysis of molecules and the vibrational modes observed in SERS (Fig. 4) provides insight to the binding characteristics of amitraz on silver surface. (Table. 2) [19]. The bands which show maximum enhancement in SERS spectrum can be observed at 2930 cm 1, 1698 cm 1 and 796 cm 1 and the corresponding Raman bands are 2916 cm 1, 1663 cm 1 and 806 cm 1. The band positions are in correlation with DFT computed spectrum and can be observed at 2928 cm 1, 1687 cm 1 and 798 cm 1 respectively. The maximum enhancement has been observed of mode involving methyl group and ring, where the CH out of plane bending of the ring has been enhanced. This favors the flat orientation of the ring over silver surface, whereas methyl groups are in cross proximity with silver surface (Table 3). 3.3. Neurotoxic assay 3.3.1. UV–Visible absorption studies UV absorption technique can be used to explore the structural changes of protein and to investigate protein–ligand complex formation. The UV–Vis absorption spectra of AChE-amitraz complex having different concentrations are shown in (Fig. 6). The interaction of amitraz-AChE at different concentrations (0, 0.0109, 0.0218, 0.175 and 0.35 (10 5mol/L) with amitraz (0.1 mM) was studied using UV–Vis spectroscopy. The spectrum was obtained by a strong peak at 324 nm, whose absorbance is increasing with increasing ratio of AChE/amitraz along with blue-shift, which stands for the binding in a more hydrophobic microenvironment [20].
Fig. 7. Fluorescence spectra of amitraz in the presence of AChE at different concentrations. CAmitraz = 0.1 mM, CAChE = 0, 0.0109, 0.0218, 0.0437, 0.0875, 0.175 and 0.35 (10 5mol/L) for curves 1–7, respectively.
3.3.2. Fluorescence spectral studies The fluorescence spectrum at the excitation wavelength of 270 nm was used to explore the interaction between amitraz (0.1 mM) and AChE at of different concentrations of AChE (0, 0.0109, 0.0218, 0.0437, 0.0875, 0.175 and 0.35 (10 5 mol/L)) and the results are shown (Fig. 7). This revealed that the fluorescence intensity of amitraz increase with the increase in AChE concentrations, and the maximum emission wavelength of the fluorescence is found shifted to the blue, slightly. This indicates that the hydrophobic interaction occurred between amitraz and
Molecular docking is a useful method for studying the interaction between small molecules and proteins. It can also be used for predicting and simulating the binding mode and to quantify the affinity of drugs or toxic material to protein. The molecular docking of amitraz with AChE (PDB: 5DTI) were performed [22]. The docking conformations of amitraz-AChE, were obtained and the docked conformer with highest binding has been chosen for the analysis (Table 4). Molecular docking reveals that Amitraz is found to bind with AchE, with binding energy equal to 5.56 kcal/mol (Fig. 8). The three dimensional structure of AChE consist of a catalytic triad which include an active site (A-site), a peripheral anionic site (PAS) and a long narrow hydrophobic gorge which connects the Asite and the PAS. The amino acid residues TYR 72, TRP 286, PHE 297, TYR 341 and PHE 338 belonging to PAS of AChE make noncovalent interactions with amitraz, leading to AchE-Amitraz complex formation (Table 4). It was observed that molecule preferred to interact with the Tyrosine72 residue (TYR 72) of AchE strongly, via O N interaction, the O N distance being 2.97 Å. The interaction has been favored by the partial positive charge acquired by N21 originated from lone pair-p conjugation which has been confirmed by DFT based vibrational spectral analysis and NBO charges.
AChE, leading to the microenvironment change of the amino acid residues of AChE [21]. These results indicated that molecular interaction between amitraz and AChE is strong.
3.4. Molecular docking
Table 4 The interactions of amitraz with AChE.
Fig. 6. UV absorption spectra of AChE with amitraz. The concentrations of AChE are 0, 0.0109, 0.0218, 0.175 and 0.35 (10 5 mol/L) and that of amitraz is 0.1 mM.
Interacting Residue
Type of Bonding
Distance of interaction (Å)
Tyrosine72 Tryptophan 286 Tyrosine 341 Phenylalanine 338 Tryptophan 286
N . . .. . .O interaction Pi-sigma interaction Amide-Pi stacked interaction Pi-Alkyl interaction Pi-Pi stacked interaction
2.97 3.64 5.31 4.51 5.01
Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404
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Fig. 8. Molecular docking of amitraz with AChE.
4. Conclusion
References
In the present study, we have carried out the experimental and theoretical spectroscopic analysis of amitraz using FTIR and FT Raman, aided by density functional theoretical simulation. The DFT study shows that stronger conjugation of the lone pair of N21 with the p-system of C=N, in addition to the conjugation of the ring p-system with the p-system of C=N. The stronger conjugation is evident from the much higher reduction in C-N bondlength and the higher vibrational stretching wavenumbers of C-N bond. The conjugation can be further confirmed by the lesser negative charge acquired by N21, compared to neighbour nitrogen atoms N18 and N28. The SERS spectral investigations reveal that maximum enhancement has been observed of mode involving methyl group and ring. The CH out of plane bending of the ring has been enhanced which favors the flat orientation of the ring over silver surface. The spectral assay using UV–Vis and fluorescence spectroscopy shows hyperchromism with blue shift, which indicated the hydrophobic interaction of amitaz with AChE. The molecular docking interaction of AChE with amitraz, the binding energy is equal to 5.56 kcal/mol and the results favor the Amitraz- AChE complex formation. The amitraz molecule preferred to interact with AchE via Tyrosine72 residue (TYR 72),through O N interaction, favoured by the loss of negative charge on nitrogen arising due to lone pair-p interactions, as confirmed by geometric and vibrational spectral analysis.
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CRediT authorship contribution statement D.E. Nimmi: Conceptualization, Methodology, Software. Geethu Sudhi: Data curation, Writing - original draft. S.G. Praveen: Visualization, Investigation. J. Binoy: Supervision, Writing - review & editing.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements The authors Nimmi.D.E. and Geethu Sudhi are thankful for the financial support from the University of Kerala. Author, J Binoy, is thankful to DST FIST scheme, department of physics, Govt College for Women, Thiruvananthapuram for instrumental support.
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Please cite this article as: D. E. Nimmi, G. Sudhi, S. G. Praveen et al., Vibrational spectroscopic investigations and biological activity of toxic material amitraz, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.404