Coligand driven efficiency of catecholase activity and proteins binding study of redox active copper complexes

Journal Pre-proofs Research paper Coligand driven efficiency of catecholase activity and proteins binding study of redox active copper complexes Vaishali Chhabra, Bidyut Kumar Kundu, Rishi Ranjan, Pragti, Shaikh M Mobin, Suman Mukhopadhyay PII: DOI: Reference:

S0020-1693(19)30919-3 https://doi.org/10.1016/j.ica.2019.119389 ICA 119389

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Inorganica Chimica Acta

Received Date: Revised Date: Accepted Date:

25 June 2019 18 October 2019 12 December 2019

Please cite this article as: V. Chhabra, B. Kumar Kundu, R. Ranjan, Pragti, S.M. Mobin, S. Mukhopadhyay, Coligand driven efficiency of catecholase activity and proteins binding study of redox active copper complexes, Inorganica Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.119389

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Coligand driven efficiency of catecholase activity and proteins binding study of redox active copper complexes Vaishali Chhabra,a Bidyut Kumar Kundu,a Rishi Ranjan,a Pragti,a Shaikh M Mobin,a Suman Mukhopadhyaya,b* Department of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore, Khandwa Road, Simrol, Indore 453552, India. Tel: +91 731 2438 735 Fax: +91 731 2361 482 E-mail: [email protected] a

bCentre

for Biosciences and Biomedical Engineering, School of Basic Sciences, Indian Institute of Technology Indore, Khandwa Road, Simrol, Indore 453552, India.

Keywords Copper Schiff base complexes Single crystal XRD Catecholase like activity Michaelis-Menten kinetics Ligand field effect Abstract Two new copper complexes [Cu(HL)SCN] (1) and [Cu(HL)Cl(CH3OH)] (2) with tri-dentate Schiff-base ligand with alcoholic arm[H2L = 3-[(2-hydroxy-propylimino)-methyl]naphthalen-2-ol] have been synthesized and characterized through several spectroscopic techniques and single crystal X-ray crystallography. The field strength of co-ligand has been found to be the key to dictate the geometry surrounding the metal ion. Where a moderate field strength of isothiocyanate ion stabilizes the complex in square-planar geometry, a weaker co-ligand like chloride induces a square-pyramidal geometry with one more additional ligand viz. methanol. Both the complexes have shown excellent catecholase like activity where compound 2 has been found to be more active through faster binding of substrate molecule through quicker dissociation of weaker co-ligands. In addition to their potential use as a bio-mimic catalyst in catechol oxidation, the interaction of these complexes with protein (BSA) was also studied to establish their potent role as metal-drug system. 1. Introduction Among all the transition metal ions, copper plays an important role in diverse biological activities including transportation of oxygen molecules, redox reactions, electron transport, 1

and prevention of build-up of harmful entity like superoxide in body etc.[1, 2] Various biological processes rely heavily on the catalytic prospects of transition metals for their specific activity[3]. Among them, many copper catalyzed reactions are well known and many model complexes in terms of structural and functional analogue have been designed and explored[4-6]. Catechol oxidase is a type 3 dicopper enzyme which catalyzes oxidation of catechol to ortho-quinone along with subsequent reduction of molecular oxygen into water[79]. 1/2 O2

H2O O

OH OH

Catechol Oxidase

O

Quinones used to get polymerize to form melanin, which can protect the wound from oxidative atmosphere by formation of insoluble barrier. Various di- and mono-nuclear model copper complexes have been derived over the years and their ability to perform similar catalytic action have been studied[10-14]. The active center of the enzyme has been found to be a hydroxo-bridged copper system along with histidine imidazole residues, which completes the trigonal pyramidal geometry surrounding the metal ion. There are certain reports where hydroxo-bridged complexes have been developed as functional model of catechol oxidase where the other coordination positions are fulfilled by Schiff base, reduced Schiff base or other polydentate ligands[3, 12, 15, 16]. However, apart from bridging dimeric copper complexes a fair number of mononuclear copper systems many times with Schiff bases are also known to mimic the same enzymatic catalytic reaction[3, 16, 17].. Schiff bases with multi-donor atoms are generally found to be very popular platform to design biomimetic transition metal based active center as it can provide the stability of the metal complexes keeping few coordination position open through which the metal can get bonded to the substrate molecules[12, 18].Some labile ligands are found to be attached to the metal center in the catalyst, which are very important as very often these may dissociate to allow the substrate molecules to bind to the metal center. To test this hypothesis, we have designed a simple tri-dentate Schiff base ligand (Scheme 1) with an alcoholic arm, which may or may not act as a bridge between copper centres. The number of donating site has been purposefully kept limited so that the other donating position can be taken up by relatively labile monodentate ligand.

2

Apart from the oxidation activity, the metal complexes have also been found to play an important role as drug carriers. The drugs interact with macromolecules and proteins are the most abundant macromolecules in cells and are crucial to maintaining normal cell functions. Hence it is important to investigate the potential drug–protein interactions. Bovine serum albumin (BSA) has been one of the most extensively studied proteins, especially because of its structural homology with human serum albumin. So to test the potential role of complexes as drug carriers, their binding to serum albumin in vitro is considered as a model in protein chemistry. The changes in fluorescence intensities of BSA-metal complex adduct could give considerable information regarding the binding characteristics of the metal complex with proteins. In this work we are going to report synthesis and characterization of two new redox active copper complexes viz [Cu(HL)SCN] (1) and [Cu(HL)Cl(CH3OH)] (2)[H2L = 3-[(2-hydroxypropylimino)-methyl]-naphthalen-2-ol]and compare their catalytic activity towards oxidation of 3,5-di-tert-butylcatechol. The probable mechanistic pathway of one of the complexes has been also investigated through various analytical tools. Moreover, binding ability of both the complexes with proteins have been studied using BSA as a model protein. 2. Experimental 2.1. Materials and Methods All of the chemicals were of analytical grade and used as received without further purification. These chemicals included 2-hydroxynaphthaldehyde (Sigma Aldrich), 1-amino2- propanol (Sigma Aldrich, assay 93%), copper chloride dihydrate (CuCl2·2H2O) (Merck assay 99%), trimethylamine (Avantor, assay 99.5%), sodium thiocyanate (Fluka, assay 98%). Infrared spectra (4000 to 500 cm-1) were recorded with a BRUKER TENSOR 27 instrument in KBr pellets. NMR spectra were recorded on an AVANCE III 400 Ascend Bruker BioSpin machine at ambient temperature. Mass spectrometric analyses were done on BrukerDaltonics, microTOF-Q II mass spectrometer. Spectrophotometric measurements were performed on a Varian UV-Vis spectrophotometer (Model: Cary 100) (for absorption) and a Fluoromax-4p spectrofluorometer from Horiba JobinYvon (Model: FM-100) (for emission) using a quartz cuvette with path length of 1 cm. 2.2. X-ray crystallography The X-ray structural studies were performed on a CCD Agilent Technologies (Oxford Diffraction) SUPER NOVA diffractometer. Data for all the complexes were collected at 3

293(2) K using graphite-monochromated CuKα radiation (λα = 1.54184 Å). The strategy for the data collection was evaluated by using the CrysAlisPro CCD software. The data were collected by the standard ‘phi-omega’ scan techniques and were scaled and reduced using CrysAlis- Pro RED software. The structures were solved by direct methods using SHELXS97 and refined by full matrix least squares with SHELXL-97, refining on F2. The positions of all the atoms were obtained by direct methods. All non-hydrogen atoms were refined anisotropically. The remaining hydrogen atoms were placed in geometrically constrained positions and refined with isotropic temperature factors, generally 1.2Ueq of their parent atoms. Crystal data and structural parameters are provided in Table 1. 2.3. Synthesis of Schiff-base ligand (H2L) The ligand H2L has been prepared by a reported procedure[19] by mixing 5 mmol of 1amino-2-propanol (0.37g) with 5 mmol of 2-hydroxynaphthalene-1-carbaldehyde (0.86 g) in 20 mL of ethanol. The reaction mixture was stirred under reflux condition for 4 h at 80˚C. Yield: 83%. The clear bright yellow solution obtained was dried off completely and crude was collected, characterized and used further . 2.4. Synthesis of [Cu(HL)(SCN)] (1) The mononuclear complex 1was prepared by refluxing the reaction mixture containing 0.11 g (0.5 mmol) of H2L, 0.05 g (0.5 mmol) of triethylammine, 0.18 g (0.5 mmol) of Cu(ClO4)2.6H2O followed by addition of 0.04 g (0.5 mmol) of sodium thiocyanate in 30 mL of methanol (Scheme 1). A green precipitate appears after 4 hrs, reaction was stopped and filtered. The precipitate was recrystallized from methanol. Yield: 76%. Formula: [C15H14CuN2O2S], Elemental analysis: Calculated- C, 51.49; H, 4.03; N, 8.01; Found- C, 51.28; H, 4.10; N, 8.11. (m/z) calculated – 372.0 (M+Na) (m/z); obtained –372.0 (M+Na) (m/z). Selected IR on KBr (m/cm-1): 2084 cm-1(-N=C=S), 1623 cm-1(-C=N).[20, 21] 2.5. Synthesis of [Cu(HL)Cl(MeOH)] (2) Complex 2 was also formed in a similar method by utilization of 0.11 g (0.5 mmol) of H2L, 0.05 g (0.5 mmol) of triethylammine followed by addition of 0.08 g (0.5 mmol) of CuCl2.2H2O (Scheme 1). Similar green precipitate was resulted which has been recrystallized from methanol ether mixture. Yield: 78%. Formula: [C15H18ClCuNO3]; Elemental analysis: Calculated- C, 29.38; H, 2.96; N, 2.28; Found- C, 29.36; H, 2.95; N, 2.23. (m/z) calculated –

4

291.0 (Cu(HL))+ (m/z); obtained – 291.0(Cu(HL))+(m/z). Selected IR on KBr (m/cm-1): 1621 cm-1(-C=N). 2.6. Protein Binding study The protein binding studies of these complexes were investigated using bovine serum albumin (BSA) by means of fluorescence spectroscopy recording excitation at 295 nm and the corresponding emission at 340 nm. The excitation and emission slit widths and scan rates were kept constant throughout the experiment.A 10 μM stock solution of BSA was prepared using Tris-HCl buffer (pH ∼ 7.4) solution and stored at 4°C for further use. Stock solutions of complexes 1 and 2(1 mM in strength) were also prepared in Tris-HCl buffer and 5% DMSO. Fluorescence intensity of 2 mL stock solution of BSA was measured and recorded as blank. Thereafter it was titrated by successive additions of 10 μL of the respective stock solution of complexes (upto 200 μL). The fluorescence data was further analyzed by the Stern–Volmer equation. 2.7. Catecholase activity study The catalytic experiments were conducted in aerobic condition at room temperature with 3,5di-tert-butylcatechol (3,5-DTBC) in methanol as model substrate. For this purpose, 10-4 M methanolic solution of the complexes was added to 100 equivalents of 3,5-DTBC separately and the time course of the reaction was monitored spectrophotometrically. As the reaction progresses, a gradual increase in the absorbance band at 400 nm owing to the increase in concentration of 3,5-DTBQ was observed. The initial rate method was applied to determine the rate of reaction and Michaelis-Menten approach was used to calculate TOF (Turn Over Frequency). 2.8. Detection of hydrogen peroxide in the catalytic reaction of catechol oxidase activity The formation of H2O2 during the catalytic reaction of catechol oxidation by the complexes 1 and 2 was detected iodometrically by assaying I3− that was formed from the reaction of KI with the reaction mixture. For this purpose, the reaction mixtures were prepared as in the kinetics experiments and after 1 h of reaction, formed quinone was extracted using dichloromethane and water. Quinone gets separated with dichloromethane while the aqueous layer was acidified with H2SO4 to pH ≈ 2 to stop further oxidation, and 1 mL of 10% solution of KI was added to it followed by addition of catalytic amount of ammonium molybdate (3 drops of 3% solution) to accelerate the formation of I3−. In the presence of hydrogen 5

peroxide, I− is oxidised to I2, and with an excess of iodide ions, the tri-iodide ion is formed. The formation of I3− was monitored by UV-Vis spectroscopy due to the development of the characteristic I3− band at 353 nm for complex 1 and 350 nm for complex 2. 3. Results and discussion 3.1. Synthesis and characterization The synthesis of ligand, H2L prepared by a reported procedure[19] and its two metal complexes have been depicted in Scheme 1. All the characterization data of the ligand has been included in the supporting information (Fig. S1a-S1d). The IR spectrum of free ligand reveals a prominent band at 1636 cm-1 which can be assigned as C=N imine stretching band. However, in compounds 1 and 2 the band shifts to lower frequency (1623 and 1621 cm-1 respectively) suggesting coordination to the metal ion through imine nitrogen atom.[20-22] Furthermore compound 1 has shown a strong broad band at 2084 cm-1 which can be attributed to stretching frequency of isothiocyanate group (Fig. S2a-b).[23]The electrospray ionization mass (ESI-MS positive) spectra of the metal complexes provide reliable evidence for the formation of compounds with the suggested molecular formula. Complex 1 showed molecular ion peak at 372 corresponding to [M + Na]+ (Fig.S3), whereas compound 2 has shown peak at 291 corresponding to monocationic moiety with formula [Cu(HL)]+ (Fig.S4). O OH

MeOH, 4 hrs Reflux

+

H2N

OH

OH N OH

[H2L] Cu(ClO4)2.6H2O NaSCN MeOH, 4 hrs Reflux

O N

[1]

CuCl2.2H2O MeOH, 4 hrs Reflux

H O N

Cu SCN O

H

Cl Cu O O H

[2]

6

Scheme 1. Reaction scheme for the synthesis of ligand and its complexes. 3.2. Crystal structure of complex 1 and 2 A blue block like specimen of complex 1 and dark green needle like specimen of complex 2 have been used for X-ray crystallographic analysis. The structures elucidation indicates that both the complexes are monomeric in nature. Complex 1 has been found to posses square planar geometry (Fig. 1) where the central metal is surrounded by three donor centers of ligand where the alcoholic group remains protonated making the ligand effectively uninegative in nature. Even increase of amount of organic base during synthetic procedure does not lead to deprotonation of alcoholic group. The fourth coordination position is taken up by N-donor center of isothiocyanate. The bond distances and angles are found to be similar to those of reported earlier[24, 25]. Complex 2 which crystallizes in monoclinic crystal system and P21/n space group has shown five coordinated geometry (Fig. 2). The deviation from square-planar to square-pyramidal geometry is because of the weaker field produced by labile chlorido ligand with respect isothiocyanate. The configuration sorrounding the metal ion is measured by means of τ value [ τ = (β-α)/60 where α and β are two largest coordination angles][26] which was found to be 0.058 indicating slight distortion from ideal square pyramidal geometry. Here also the alcoholic protons remains attached to the ligand. Two adjacent molecules are found to be attached each other through Cl(1)...H101-O(4) and O(1)...H(2)-O(2) making a complicated three dimensional network structure (Fig. S5-7). All the crystal parameters and bond length and bond angles are given in Table S1 and Table 1, respectively.

7

(a)

(b)

(c)

(d)

Fig. 1(a) Crystal structure (b) Crystal close packing (c) 1D polymeric form (d) 2D polymeric framework of complex 1via H-bonding interaction.

8

(a)

(b)

(c)

(d)

Fig. 2(a) Crystal structure (b) Crystal close packing (c) 1D polymeric form (d) 2D polymeric framework of complex 2

9

Table 1. Selected bond lengths (Ȧ) and bond angles (˚) of complex 1 and 2 Complex 1

Complex 2

Cu(1)-N(1)

1.9015(18)

Cu(1)-O(1)

1.915(5)

Cu(1)-O(1)

1.9129(15)

Cu(1)-N(1)

1.928(5)

Cu(1)-N(2)

1.919(2)

Cu(1)-O(2)

2.028(5)

Cu(1)-O(2)

2.0265(17)

Cu(1)-Cl(1)

2.2392(19)

N(1)-Cu(1)-O(1)

94.05(7)

Cu(1)-O(4)

2.331(6)

N(1)-Cu(1)-N(2)

171.35(8)

O(1)-Cu(1)-N(1)

92.9(2)

O(1)-Cu(1)-N(2)

93.60(8)

O(1)-Cu(1)-O(2)

168.0(2)

N(1)-Cu(1)-O(2)

82.96(7)

N(1)-Cu(1)-O(2)

82.1(2)

O(1)-Cu(1)-O(2)

160.41(8)

O(1)-Cu(1)-Cl(1)

91.52(15)

N(2)-Cu(1)-O(2)

91.14(8)

N(1)-Cu(1)-Cl(1)

171.47(18)

C(1)-O(1)-Cu(1)

125.85(13)

O(2)-Cu(1)-Cl(1)

92.22(14)

C(13)-O(2)-Cu(1)

112.08(13)

O(1)-Cu(1)-O(4)

98.9(2)

Cu(1)-O(2)-H(101)

106(2)

N(1)-Cu(1)-O(4)

86.1(2)

C(11)-N(1)-Cu(1)

125.63(15)

O(2)-Cu(1)-O(4)

91.6(2)

C(12)-N(1)-Cu(1)

113.92(14)

Cl(1)-Cu(1)-O(4)

100.43(16)

C(15)-N(2)-Cu(1)

170.4(2)

C(1)-O(1)-Cu(1)

125.1(4)

C(13)-O(2)-Cu(1)

110.9(4)

Cu(1)-O(2)-H(2)

124.5

C(15)-O(4)-Cu(1)

119.5(6)

Cu(1)-O(4)-H(101)

109(7)

C(11)-N(1)-Cu(1)

125.6(4)

C(12)-N(1)-Cu(1)

113.9(4)

3.3. Catecholase activity study It was interesting to check the catecholase like activity of both the complexes as it was expected with a labile chlorido and a methanol ligand compound 2 should be more pro-active to catalyze oxidation of 3,5-di-tert-butyl catechol (DTBC) in presence of oxygen. The reaction has been monitored through UV-Vis spectrophotometry by monitoring the increase in absorbance at 400 nm as a function of time due to increase in concentration of 3,5-di-tertbutylquinone (DTBQ). Methanol solution of compound 1 and 2 (10-4 M) were taken separately with 10-2M solution of 3,5-DTBC in open atmosphere. Initial rate method has been employed to understand the kinetic aspects of the reaction. The observed rate vs substrate concentration was analyzed based on Michaelis-Menten approach (Fig.3a and Fig. 4a)[3, 10

27].Linearization using Lineweaver-Burk plot (Fig.3b and Fig. 4b) furnished MichaelisMenten constant (KM) and maximum initial rate (Vmax). The turnover number (Kcat) was calculated from dividing maximum initial rate with concentration of the corresponding complexes. The obtained values indicate both the complexes are quite active in oxidizing 3,5DTBC (Table S2 shows the efficiency of copper complexes to oxidise catechol reported in literature). Compound 2 following our expectation shows greater activity as compared to compound 1. The details of all the enzyme kinetic parameters are tabulated in Table 2. The high reactivity of both the complexes may be attributed to the fact that the coordination number of surrounding the metal is less and co-ligand is easily dissociable which can help the substrate to get attached to the metal center in a relatively easier way.

Fig. 3(a) UV-Vis spectra of oxidation of 3,5-DTBC to 3,5-DTBQ with time using complex 1;(b) Plot of rate vs. substrate concentration for complex 1, inset shows the Lineweaver–Burk plot.

(b)

(a)

Catechol Oxidation

11

Fig. 4(a) UV-Vis spectra of oxidation of 3,5-DTBC to 3,5-DTBQ with time using complex 2;(b) Plot of rate vs. substrate concentration for complex 2, inset shows the Lineweaver–Burk plot. Table 2. Different enzyme kinetic parameter for complex1 and complex2 in MeOH Catalyst

Complex

Vmax

Std. Error

Km (M)

Kcat/T.O.F

Conc. (M)

(M min-1)

Complex 1

0.0001

0.01357

4.1992 x 10-4

0.00418

0.8142 x 104

Complex 2

0.0001

0.02006

5.9415 x 10-4

0.00262

1.2036 x 104

(h-1)

3.4. Mechanistic study of the catecholase activity To find out a probable mechanism of catecholase activity of complex 2 (as it is more active), we have investigated the probable complex-substrate intermediate through ESI-MS and a qualitative detection of the I3− band (at ∼353 nm.) in UV-Vis spectrum. The ESI-MS spectrum of complex 2 recorded after 10 min of mixing exhibited two peaks of considerable intensity at m/z = 243 and 463 owing to the species [(3,5-DTBQ) + Na]+ and [(3,5-DTBQ)2 + Na]+, respectively (Fig. 5). A peak corresponding to the complex-substrate aggregate "C" (Scheme 2) has been observed at 511.2 which corresponds to the simulated pattern (Fig. 5), and similar with some earlier reports[28]. Based on the assignable peaks a plausible mechanistic pathway has been proposed (Scheme 2), which indicates the formation of hydrogen peroxide as the by-product of reduction of aerial oxygen. To investigate it further formation of hydrogen peroxide has been monitored through UV-Vis spectroscopy.

12

t

Bu

HO t

HO

Bu

- HCl

N II O H Cu Cl O O H

- MeOH

([M] = 358.03)

N II O H Cu t Bu O O 1e HO

- H+

[A]

t

([M] = 512.19)

1/2 H2O2

t

Bu

[B]

HO HO

Bu

t

Bu 1/2 O2 + H+

- H+

1e

t

Bu

N I O H Cu O t O Bu O

O t

O

Bu

1e 1/2 O2 + H+

1/2 H2O2

([M] = 511.18)

t

Bu

[C] t

Bu

O O

t

Bu

Scheme 2. Probable mechanism of catechol oxidation by 2.

Fig. 5 ESI-MS spectrum of the reaction mixture obtained 10 min after the addition of substrate 3,5-DTBC, showing the peak of product as well as intermediate. 13

Quantitative analysis of H2O2 indicates that 0.8 mol (≈1 mol) of H2O2 gets produced per mol of 3,5-DTBC along with the formation of 1 mol 3,5-DTBQ, which strongly supports the mechanism of reaction involving a two electron reduction process of areal oxygen, as indicated in our previous reports.[29] Modified iodometric method was used to detect H2O2 formed during the catalytic reaction. For this purpose, the reaction mixtures were prepared as in the kinetic experiments and after 1 h of reaction, formed quinone was extracted using dichloromethane and water. Quinone gets separated with dichloromethane while the aqueous layer was acidified with H2SO4 to pH ≈ 2 to stop further oxidation, and 1 mL of a 10% solution of KI and three drops of 3% solution of ammonium molybdate were added. In the presence of hydrogen peroxide I− is oxidised to I2, and with an excess of iodide ions, the triiodide ion is formed. The reactions are given as: H2O2 + 2I− + 2H+ → 2H2O + I2 and I2 (aq.) + I− → I3The formation of I3-was monitored by UV-Vis spectroscopy due to the development of the characteristic I3-band at 350 nm for complex 2 and 353 nm for complex 1 (Figure 6).

Fig. 6 Characterized peak for I3−at 353 nm for qualitative detection of H2O2 in complex 2 (on left) and complex 1 (on right) during catalytic oxidation process. 3.5. Protein binding studies The protein binding studies of these complexes were accomplished by investigating their interaction with bovine serum albumin (BSA) by means of fluorescence spectroscopy (Fig. 7). 2 mL of 10-6 M BSA solution prepared in Tris-buffer was titrated against 1mM stock 14

solution of complexes 1 and 2 (prepared in tris-buffer and 5% DMSO) distinctly. With each 10 µL addition of the stock solution of the complex, a decrease in the intrinsic fluorescence emission band of protein was observed for both the complexes. The fluorescence quenching data was further analysed by the Stern–Volmer equation, according to which: F0 = 1 + kqτ0 [Q] = 1 + KSV [Q] F where F0 and F are the fluorescence intensities in the absence and the presence of a quencher, kq is the bimolecular quenching rate constant, τ0 is the average lifetime of fluorophore in absence of a quencher and [Q] is the concentration of a quencher (metal complexes).

(a)

(b)

Fig. 7(a) Fluorescence quenching of BSA by complex 1and inset shows the corresponding Stern-Volmer plot, (b) Scatchard plot for complex 1. (c) Fluorescence quenching of BSA by complex 2 and inset shows the corresponding Stern-Volmer plot, and (d) Scatchard plot for complex 2.

15

The calculated values of Ksv and kq for the interaction of the complexes with the BSA are given in Table 3 indicate a good BSA binding tendency of the complexes with complex 2 showing greater binding ability (Kb). Table 3. Table for Stern–Volmer quenching constant, binding constant and binding site. Catalyst

KSV (M-1)

kq (M-1 s-1)

Kb (M-1)

n

Complex 1

2.2895 x 104

3.8158 x 1012

0.3010 x 106

1.26

Complex 2

1.8521 x 104

3.0868 x 1012

2.7918 x 106

1.52

4. Conclusion In a nutshell two new monomeric copper complexes based on tri-dentate Schiff-base alcoholic armed ligand have been synthesized and characterized successfully. The alcoholic arms remained protonated in the final structure. The ligand field exerted by the coligand dictates the geometry surrounding the metal ion as evident from the square-planar geometry with isothiocyanate ligand and square-pyramidal geometry with relatively weak chlorido ligand. As expected the detailed catecholase activity reveals the higher activity of complex 2 which could be due to the presence of more labile ligands ensuring higher binding tendency of the complex with the substrate. This was further confirmed with the BSA protein binding experiment in which it was observed that binding constant (Kb) has more value in case of complex 2 as compared to that of complex 1..A further detailed study with coligands of different field strength is currently underway. Acknowledgements We thank SIC IIT Indore for analytical support and structural elucidation. Appendix A: Supplementary data Crystallographic data for compound 1 and 2 have been deposited with CCDC (1544785 and 1544784). Supplementary data associated with this article can be found in the online version. References:

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The authors have no Conflict of Interest

Highlights

Coligand driven efficiency of catecholase activity and proteins binding study of redox active copper complexes Vaishali Chhabra,a Bidyut Kumar Kundu,a Shaikh M Mobin,a Suman Mukhopadhyaya,b* Department of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore, Khandwa Road, Simrol, Indore 453552, India. Tel: +91 731 2438 735 Fax: +91 731 2361 482 E-mail: [email protected] a

Centre for Biosciences and Biomedical Engineering, School of Basic Sciences, Indian Institute of Technology Indore, Khandwa Road, Simrol, Indore 453552, India. b



Two Cu(II) complexes have been synthesised using tridentate Schiff base ligand with alcoholic arm.



Crystal structure of complexes were determined and the effect of ligand field on the geometry of complexes was studied.

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Both the complexes show excellent catecholase like activity and actively interact with protein (BSA).

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Through mechanistic investigation of catechol oxidation, it has been observed that copper participates electron transfer redox sensitive reaction.

Graphical Abstract

Coligand driven efficiency of catecholase activity and proteins binding study of redox active copper complexes Vaishali Chhabra,a Bidyut Kumar Kundu,a Shaikh M Mobin,a Suman Mukhopadhyaya,b* Department of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore, Khandwa Road, Simrol, Indore 453552, India. Tel: +91 731 2438 735 Fax: +91 731 2361 482 E-mail: [email protected] a

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Centre for Biosciences and Biomedical Engineering, School of Basic Sciences, Indian Institute of Technology Indore, Khandwa Road, Simrol, Indore 453552, India. b

Representation of natural catecholase activity in a piece of apple.

N

t

O

H

Cl Cu O O H

HO HO

t

Bu

Bu t

Bu

O2

O O

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t

Bu