Synthesis, crystal structure and bioactivity of manganese complexes with asymmetric chiral Schiff base

Accepted Manuscript Synthesis, crystal structure and bioactivity of manganese complexes with asymmetric chiral Schiff base Enfeng Zhang, Yi Wei, Fuping Huang, Qing Yu, Hedong Bian, Hong Liang, Fuhou Lei PII:

S0022-2860(17)31491-6

DOI:

10.1016/j.molstruc.2017.11.017

Reference:

MOLSTR 24503

To appear in:

Journal of Molecular Structure

Received Date: 31 August 2017 Revised Date:

5 November 2017

Accepted Date: 6 November 2017

Please cite this article as: E. Zhang, Y. Wei, F. Huang, Q. Yu, H. Bian, H. Liang, F. Lei, Synthesis, crystal structure and bioactivity of manganese complexes with asymmetric chiral Schiff base, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.11.017. 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.

ACCEPTED MANUSCRIPT

Synthesis, Crystal Structure and Bioactivity of Manganese Complexes with Asymmetric Chiral Schiff Base Enfeng Zhang, Yi Wei, Fuping Huang, Qing Yu, Hedong Bian, Hong Liang, Fuhou

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Lei

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A couple of chiral unsymmtrical Schiff base ligands, and their Mn (III) complexes had been synthesized and fully characterized. The interaction of the two Mn (III) complexes with bovine serum albumin (BSA) was investigated by spectroscopic techniques. SOD-like activity and ABTS free radical scavenging ability were also studied. The results showed that the microenvironment of BSA can enhance the activity of the complexes.

ACCEPTED MANUSCRIPT

Synthesis, Crystal Structure and Bioactivity of Manganese Complexes with Asymmetric Chiral Schiff Base Enfeng Zhanga,b, Yi Weib, Fuping Huang*b, Qing Yub, Hedong Bian*a,b, Hong Liangb, Fuhou Leia Guangxi Key Laboratory of Chemistry and Engineering of Forest Products, School of Chemistry and

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a

Chemical Engineering, Guangxi University for Nationalities, Nanning, Guangxi, 530008, PR China b

Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of

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Education of China), School of Chemistry and Pharmacy, Guangxi Normal University, Guilin, Guangxi, 541004, PR China

Abstract

(1R,2R)(-)chxn(salH)(naftalH)

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A couple of chiral unsymmtrical Schiff base ligands, and

(1S,2S)(-)chxn(salH)(naftalH)

had

been

synthesized by the condensation of salicylaldehyde and 2-hydroxy-1-naphthaldehyde with

two

isomers

of

(1R,2R)-trans-1,2-cyclohexanediamin

and

(1S,2S)-trans-1,2-cyclohexanediamin, respectively. At the same time, two manganese

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complexes have been synthesized and fully characterized by FT-IR spectrum, elemental analyses, single crystal X-ray diffraction. The interaction of the two Mn (III) complexes with bovine serum albumin (BSA) was investigated by spectroscopic

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techniques. The result reveals that the complexes can strongly quench the intrinsic fluorescence of BSA through a static quenching mechanism. The binding constant and binding mode has been determined. The secondary structure and the amino acid

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residues microenvironment of BSA change in the presence of these complexes. SOD-like activity and ABTS free radical scavenging ability were also studied. The antioxidant capacity of the compounds showed that the complexes and their corresponding BSA adducts showed some SOD activity. The results of ABTS free radical scavenging showed that the activity of the BSA adduct was more obvious than that of the complex.

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Key words

asymmetrical chiral Schiff base; BSA; SOD-like activity;

ABTS· + scavenging capacity

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1.Introduction Reactive oxygen species (ROS) is a naturally occurring, oxygen-containing chemical species, which is more reactive to a large number of substances than the ground state oxygen[1]. The ROS included superoxide anion (O2·-), hydroxyl radical

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(·OH), singlet oxygen (1O2), hydrogen peroxide (H2O2), peroxyl radical (ROO·), and hypochlorite radical (OHCl·)[2]. The intracellular ROS mainly from the mitochondrial

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respiratory chain, which generates a mass of superoxide radicals[3]. When introduced for the first time, ROS were considered highly toxic and associated with a variety of pathological conditions. From then on, a lot of researches have been issued connecting ROS with a variety of physiological processes as well[4, 5]. The balance between generation and scavenging of ROS is of crucial as ROS have been tied up

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with numerous disease conditions and biological processes[6]. Under normal circumstances, the metabolism of free radicals is in balance, like these conditions such as: aging or other factors. In cells, excessive ROS cause oxidative damage to cellular

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components for instence lipid, nucleic acid and protein[7]. However, ROS is also involved a variety of diseases states such as diabetes mellitus, cancer, cardiovascular

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diseases, and atherosclerosis to name few[8, 9]. Therefore, scavenging of superoxide ion free radicals timely is very important for the health of the organism. Superoxide dismutase (SOD) which catalyzes the dismutation of superoxide union

into molecular oxygen and hydrogen peroxide, is one of the most prominent antioxidant enzymes involved in lifespan extension[10]. The aerobic organisms have evolved various enzymatic and non-enzymatic antioxidant systems to eliminate the extra ROS and protect redox homeostasis[11]. A large number of studies have shown that SOD plays an important role in preventing cellular oxidative stress and suppress tumorigenicity[12]. It has long been recognized that SOD is essential in high levels of

ACCEPTED MANUSCRIPT hyperactive

oxygen

cancer,

diabetes,

inflammatory

diseases

and

various

cardiovascular diseases. According to different transition metal ions in the active site has been found that natural SOD can be divided into four categories: CuZn SOD, Mn-SOD, Fe-SOD and Ni-SOD[13]. In the mitochondrial matrix, manganese

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superoxide dismutase (MnSOD/SOD2) is responsible for the energy metabolism of O2·- cell protection[14, 15]. SOD2 activity is regulated and induced by the amount of ROS[16, 17]. In mitochondria, a change in SOD2 activity are likely to cause some interference related with dysfunction of the mechanisms for which mitochondria are

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responsible[18]. Artificial SOD mimic enzymes has been synthesized and it is superior to natural SOD enzyme in selectivity reactivity, membrane permeability, toxicity,

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stability, immunogenicity and cost[19].

ABTS method is the use of ABTS as a color reagent, which can determinate saliva, serum, plasma, urine and other body fluids, plants or herbal extracts, cells or tissues and other lysates or a variety of antioxidant solution total resistance oxidation capacity. ABTS method to determine the total antioxidant capacity of the principle is

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that ABTS in the role of oxidants prone to an electronic reaction, the formation of ABTS· + from the base was green, ABTS / ABTS· + redox potential of 0.68V

[20, 21]

.

When the presence of antioxidants, ABTS· + production will be inhibited, measured at

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734 nm or 405 nm ABTS absorbance can be measured and calculated the total antioxidant capacity of the sample. Since the native enzyme cannot penetrate across cell membranes and is not orally

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bioavailable, its therapeutic application is limited. Compared with native enzymes, metal based mimics have numerous advantages, including membrane permeability, low toxicity, stability and cheap cost[22]. The previous work focused on the ligands derived from Schiff base, prorphyrins, macrocyclic ligand, and so on[23-25]. Most of these mimics are unsoluble in water. More attention is needed to improve their solubility in water under physiological conditions Generally[26, 27], β-cyclodextrin or proteins are used as skeleton to improve water solubility. These conjugations not only are water soluble, but have higer activities[28-30].

ACCEPTED MANUSCRIPT In this paper, we report the synthesis, characterization and properties of a couple of Mn(III) complexes with chiral unsymmtrical Schiff base ligands. The antioxidant activity of the complexes and their bovine serum albumin (BSA)adducts have been

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determined.

2.Experimental

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2.1. Materials

All the starting materials and reagents were obtained from commercial sources and

synthesized

in

our

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used directly without further purification. The two ligands, H2L1 and H2L2 had been laboratory.

(1R,2R)-trans-1,2-cyclohexanediamin,

(1S,2S)-trans-1,2-cyclohexanediamin was purchased from J&K reagent co, LTD (Guangzhou). Bovine serum albumin (BSA) was purchased from Sigma.

Physical measurements

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2.2

IR spectra were recorded on a PE Spectrum One FT-IR spectrophotometer with KBr pellets in the 4000–400 cm-1 region. Elemental analyses for C, H and N were

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performed at a PE-2400-II apparatus. Diffraction data were recorded on a SuperNova single-crystal diffractometer. UV-Vis spectra were performed on a Cary 100

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UV-Visible Spectrophtometer ( Varian). Fluorescence measurements were performed on a RF-5301 Fluorescence spectrophotometer ( Japan Shimadzu Company). CD spectra were recorded with a Jasco-810 Circular Dichroism spectrometer ( Jasco, Japan).

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2.3. Synthetic procedures 2.3.1. General procedure for synthesis of ligands The anhydrous ethanol solution ( 100 mL) of 2-hydroxy-1-naphthaldehyde

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( 1.72g,10 mmol) was slowly added dropwise to the ethanol solution ( 200 mL) of (1R,2R)-trans-1,2-cyclohexanediamin ( 2.28 g, 20 mmol) with ice bath stirring. The solution gradually changed from colorless to yellow and was reacted at 0°C with

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stirring for 2 h. The solution of salicylaldehyde ( 1 mL) in ethanol solution ( 100 mL) was slowly added to the above yellow mixed solution used constant pressure funnel

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and was reacted at 0°C with stirring for 2 h. The yellow solution was suction filtered and washed with ethanol solution and then dried in vacuum. The bright yellow product of Schiff-base ligands was obtained. A schematic representation of ligand synthesis is given in Scheme 1.The synthetic procedures for H2L2 were very similar to the H2L1, except that (1R,2R)-trans-1,2-cyclohexanediamin is replaced by

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(1S,2S)-trans-1,2-cyclohexanediamin. And discussed together as follows: H2L1 yield: 38%.Anal. calc. ( %) for C24H24N2O2 (Mr = 372.18): C, 77.39; H, 6.49; N, 7.52. Found: C, 76.87; H, 7.12; N, 7.82. FT-IR ( KBr phase, cm-1) : 3433s, 3056w, 2932s, 2858s, 2664w, 1625s, 1534w, 1373w.

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H2L2 ligand: Yield: 41%. Anal. Calc. For C24H24N2O2 ( %): C, 77.39; H,6.49; N, 7.52. Found : C, 77.15; H, 7.23; N, 6.96. FT-IR ( KBr phase, cm-1) : 3681s, 3055w,

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2928s, 2852s, 2384w, 1625s, 1546w, 1373w.

H

O

C

-H2O

OH

+ H2N

NH2

N

OH

NH2

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Scheme 1. Synthesis of the ligand H2L1

2.3.2. General procedure for synthesis of complexes

The anhydrous ethanol solution (15 mL) and triethylamine (5 drops) was added to

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the hydrothermal reactor with H2L1 (0.3 mmol) and MnCl2·4H2O (0.3 mmol) then sealed up, heated at 80 °C for 72 h and then cooled to 40 °C at a rate of 5 °C . The

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resulting brown-black massive crystals (1) was washed with anhydrous ethanol and dried in air. The synthetic procedures for complex (2) were very similar to the complex (1) except that H2L2 replaced H2L1 and discussed together as follows: Complex 1 yield: 61%. Anal. calc. (%) for C26H28ClMnN2O3 (Mr = 505.89): C, 61.73; H, 5.38; N, 5.54. Found: C, 60.93; H, 4.88; N, 5.57. FT-IR (KBr phase, cm-1):

925w, 765w, 583w.

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3435s, 3062w, 2857w, 1617s, 1538w, 1450m, 1341w, 1298w, 1188s, 1151s, 1033s,

Complex 2 yield: 63%. Anal. calc. (%) for C26H28ClMnN2O3 (Mr = 505.89): C,

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61.73; H, 5.38; N, 5.54. Found: C, 61.33; H, 5.78; N, 5.67. FT-IR (KBr phase, cm-1): 3427s, 3062w, 2946w, 2857s, 1617s, 1538m, 1437m, 1333m, 1313w, 1024w, 1196s,

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1114s, 1034s, 1029w, 824w, 758w, 633m, 583w.

2.4. X-ray crystallography Single-crystal X-ray diffraction data for the title complexes were obtained on a

SuperNova 1000 CCD diffractometer (graphite monochromized MoKα radiation, λ = 0.71073 Å) and collected by the Φ-ω scan technique at 298 (2) K in a certain θ range. The structure was solved using direct methods and refined by the full-matrix least-squares method on F2 data using SHELXTL-97. The non-hydrogen atoms were refined anisotropically. The final cycle of full-matrix least-squares refinement was

ACCEPTED MANUSCRIPT based on observed reflections and variable parameters. Further crystallographic data and structural refinement details are summarized in Table. 1. Table. 1 Crystal Data and Structure Refinement for 1 and 2

Goodness-of-fit on F2 Final R indices [I > 2σ (I)]

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R indices (all data)

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2 C26H27ClMnN2O3 505.89 Monoclinic 298(2) P21 7.2224 (13) 17.553 (3) 9.2288 (17) 90 92.672 (3) 90 1168.7 (4) 2 1.438 526 2.5 to 23.4 14534 5164 [R(int)=0.040] 1.02 R1 = 0.0399 ωR2= 0.0813 R1 = 0.0573 ωR2= 0.0881

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1 C26H28ClMnN2O3 506.89 Monoclinic 298(2) P21 7.2025 (9) 17.486 (8) 9.2096 (10) 90 92.553 (14) 90 1158.7 (5) 2 1.453 528 3.1 to 28.5 6244 4165 [R(int)= 0.050] 0.99 R1 = 0.0666 ωR2= 0.1237 R1 = 0.1115 ωR2= 0.1461

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Compound formula Formula weight Crystal system Temperature(K) Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) V/(Å3) Z Dc (g cm-3) F (000) θ range for data collection (°) Reflections collected Independent reflections

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2.5. BSA binding experiments The concentration of 1.0×10-3 mol·L-1 BSA stock solution and the concentration of

1.0×10-3 mol·L-1 complex solution were prepared with pH = 8.0 Tris-HCl buffer solution. The interaction between metal complexes and BSA was analyzed by UV -Vis spectroscopy and fluorescence titration. Quenching measurements were taken in 3.0 mL of a solution containing 1.0 ×10

-6

mol·L-1 BSA, which was titrated by

successive additions of the metal complex solution. The fluorescence intensity was recorded at an excitation at 280 nm using slit widths of 5 / 5 nm. Measurement of UV

ACCEPTED MANUSCRIPT and circular dichroism spectra is similar to the method of measuring fluorescence quenching.

2.6.1 SOD activity experiment

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2.6 Antioxidant activity measurements

SOD-like activity of the ligands and their complexes was measured according to essay[31], which is, it was determined as the ability to inhibit the reduction of blue

(NBT)

by

superoxide

ions

generated

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nitrotetrazolium

by

xanthine-xanthine-oxidase system ( X-XO). The NBT reduction was observed by

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measuring the absorbance at 550 nm on a spectrophotometer for inhibited and uninhibited systems and presented as relative inhibition rate. The reaction mixture was composed of NBT 0.5 µM, xanthine 0.1 µM in phosphate buffer 17 µM, at pH 7.4. An appropriate amount of xanthine oxidase was added to 2.0 mL reaction mixture to produce a ∆A550 nm min

-1

of 0.024. The NBT reduction rate was measured in the

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presence and absence of the investigated complex or complex-BSA adduct. All measurements were conducted at 25 ºC using 1.0 cm constant temperature cuvettes in which solutions were magnetically stirred. The metal complex-BSA adduct was

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synthesized by incubating equimolar amounts of BSA and the metal complex for 10 minutes in the buffer solution at room temperature. Each sample was measured in six

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concentrations while each of which was subjected to three parallel experiments, and the average was calculated. The SOD activity was quantified via the IC50 value, i.e. as the concentration of the SOD mimic that induces a 50% inhibition of the reduction of NBT

in

the

X-XO

system.

The

IC50

values

were

determined

from

concentration-dependent plots[32].

2.6.2 ABTS· + scavenging ability experiment ABTS free radical scavenging assay was done using the method by Roberta Re. et al[33] with modifications. ABTS was dissolved in water to a 5 mM concentration.

ACCEPTED MANUSCRIPT ABTS radical cation ( ABTS· +) was produced by reacting ABTS stock solution with 5 mM potassium persulfate at avolume ratio of and allowing the mixture to stand in the dark at room temperature for 12~16 h before use. The ABTS· + solution was diluted with PBS buffer at a concentration of 0.01 and pH = 7.4, to an absorbance of 0.70 ±

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0.02 at 734 nm and equilibrated at 30 ºC. Each sample was measured in five

the average was calculated.

3 Results and discussion

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3.1. Description of crystal structure

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concentrations while each of which was subjected to three parallel experiments, and

1 (Fig. 1) and 2, a pair of enantiomers, have the similar crystal structures. Therefore, the crystal structure of 1 is discussed in detail here. 1 crystallizes in monoclinic system with space group P21. The bond length and angle of the complexes are shown

(2)

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(1)

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in Table 2.

Fig . 1 X-ray crystal structure of 1 (left) and 2 (right). Hydrogen atoms are omitted for clarity

The central metal ion Mn1 coordinates with two nitrogen atoms (N1, N2) and two oxygen atoms (O1, O2) from the ligand H2L1, one oxygen atom of ethanol molecule, and one chlorine atom (Cl1) to form an octahedral configuration of MnN2O3Cl. The

ACCEPTED MANUSCRIPT oxygen atoms and nitrogen atoms on the ligand are located at the equatorial position of octahedron. The average bond lengths of Mn-O and Mn-N are 2.1615 Å and 1.9725 Å, respectively. The chlorine atom and the alcoholic hydroxyl group occupy the axial position. The bond lengths of Mn-Cl and Mn-O on the axial position are

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2.556 (2) Å and 2.466 (2) Å, respectively, which are longer than the bond length in the equatorial direction.

2.556 (2) 2.443 (5) 1.880 (5) 169.99 (14) 98.18 (17) 90.3 (2) 169.7 (2) 92.8 (2) 90.5 (2) 90.37 (17) 80.7 (2)

2 Mn1—Cl1 Mn1—O3 Mn1—N1 O3—Mn1—Cl1 N1—Mn1—O3 N1—Mn1—N2 O2—Mn1—Cl1 O2—Mn1—O3 O2—Mn1—N1 O2—Mn1—N2 O2—Mn1—O1

2.5660 (9) 2.464 (2) 1.979 (2) 169.82 (6) 80.65 (8) 83.32 (10) 97.96 (7) 90.45 (8) 169.66 (9) 90.25 (9) 92.95 (9)

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1 Mn1—Cl1 Mn1—O3 Mn1—O1 O3—Mn1—Cl1 O1—Mn1—Cl1 O1—Mn1—O3 O1—Mn1—N2 O1—Mn1—O2 O1—Mn1—N1 N2—Mn1—Cl1 N2—Mn1—O3

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Selected bond lengths (Å) and angles (º) for the complexes 1 and 2.

Mn1—N2 Mn1—O2 Mn1—N1 N2—Mn1—N1 O2—Mn1—Cl1 O2—Mn1—O3 O2—Mn1—N2 O2—Mn1—N1 N1—Mn1—Cl1 N1—Mn1—O3

1.969 (5) 1.881 (6) 1.977 (6) 83.4 (3) 96.38 (19) 88.5 (3) 91.9 (2) 170.2 (3) 92.27 (19) 82.3 (3)

Mn1—O2 Mn1—N2 Mn1—O1 N1—Mn1—Cl1 N2—Mn1—Cl1 N2—Mn1—O3 O1—Mn1—Cl1 O1—Mn1—O3 O1—Mn1—N1 O1—Mn1—N2

1.8767 (18) 1.991 (2) 1.877 (2) 90.41 (7) 92.34 (7) 81.85 (11) 96.74 (7) 88.50 (11) 92.07 (10) 169.85 (10)

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Tab. 2

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3.2. BSA Binding Properties 3.2.1. UV–Vis absorption spectra UV-vis absorption spectroscopy is a simple technique to explore the structural

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changes of molecules and to recognize the complex formation between different chemical entities[34]. Fig. S1 is UV-vis absorption spectroscopy of interaction between the complex and BSA. As shown in Fig. S1, there are two characteristic absorption

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peaks in BSA molecules. Where the characteristic absorption peak near 203 nm belongs to the π-π * transition of C=O in the peptide bond, with the protein skeleton

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changes related. The characteristic absorption peak at about 279 nm is caused by the π-π * and n-π * transitions of the benzene ring in the phenylalanine, tyrosine and tryptophan residues[35-37]. As shown in Fig. S1, theabsorption band at 203 nm exhibits a hypochromic effect accompanied by a small red-shift in the presence of manganese(III) complexes. The results reveal a definite interaction of the

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manganese(III) complexes with BSA protein.

3.2.2. Fluorescence quenching spectra

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Fluorescence quenching refers to any process that decreases the fluorescence intensity from a fluorophore induced by a variety of molecular interactions including

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excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation and collisional quenching[38]. BSA contains some residues such as phenylalanine, tyrosine and tryptophan, which can emit strong endogenous fluorescence. Actually, the intrinsic fluorescence of BSA is almost contributed by tryptophan alone[39], since phenylalanine has a very low quantum yield and the fluorescence of tyrosine is almost totally quenched if it is ionized or near an amino, a carboxyl, or a tryptophan. In other words, the change of endogenous fluorescence intension of BSA is that of fluorescence intension of the tryptophan residue when small molecules combine with proteins[40, 41]. There are two tryptophan residues in the

ACCEPTED MANUSCRIPT BSA molecule (located at positions 134 and 212 respectively). When λex = 280 nm, a strong fluorescence emission peak is generated in the BSA molecule[42]. When the small molecule is added to the protein molecule, it will cause the fluorescence of Trp-212 to quench. At the same time, it can not be excluded that the drug molecule

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interacts with the other adjacent amino acid residues, resulting in a change in the secondary structure of the protein[43]. The interaction of the manganese(III) complexes with BSA have been studied by fluorescent quenching experiment at three temperature (293K, 299K, 306K). As shown in Fig. 2, at 293K, when λex = 280 nm,

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BSA has a strong fluorescence emission near 343 nm. Keeping the peak shape unchanged, the increase of complex concentration leaded the BSA fluorescence

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intensity gradually decresed in the case of constant BSA concentration, which indicates that the interaction between the complex and BSA.

700000

(C)BSA-Mn-(RR) 293K

a

1000

600000

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500000

Fluorescence Intensity

Fluorescence intensity

j

400000 300000 200000

0 300

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100000

350

400

BSA-2 293K

j 600

400

200

0

450

300

400

wavelength(nm)

Wavelength (nm)

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a

800

Fig. 2 Emission spectra of BSA in the absence and the presence of complexes. T = 293 K,

λex= 280 nm,

λem= 343 nm, pH = 7.43 and BSA concentration was 1.0 × 10 –6 mol·L–1. From

a to j, the concentration of complex (1 and 2), were varied from 0 to 9×10 -6 mol·L -1, as a step of 1×10 -6 mol·L -1.

3.2.3 Fluorescence quenching mechanism The fluorescence quenching can be better explained by the Stern-Volmer relation[34]:

ACCEPTED MANUSCRIPT F0/F = 1 + KSV [Q] = 1 + kqτ0[Q]

(3.1)

where F0 and F are the fluorescence intensities of BSA in the absence and presence of the quencher, respectively. KSV is the Stern-Volmer dynamic quenching constant, [Q] is the concentration of quencher, kq is the bimolecular quenching rate constant, τ0 is

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the average fluorescence lifetime of the biomolecule in the absence of quencher ( about 10 -8 s).

1.7

1.5

1.6

293K 299K 306K

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1.7

293K 299K 306K

1.6

1.5

F0/F

1.3

1.2

1.4

1.3

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F0/F

1.4

1.2

1.1

1.1

1.0

1.0

2

4

6

8

-6

10

2

4

6

8

10

-6

[Mn-SS]10 mol/L

[Mn-RR]10 mol/L

Fig. 3 The Stern-Volmer plots for the binding of the complexes with BSA.

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According to the equation Stern-Volmer, the interaction curve between the complexes and the BSA can be obtained by mapping of [Q]. And the values of Ksv and kq are calculated according to the slope of the curve. The interaction curves are

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shown in the Fig. 3 and the values of Ksv and kq are listed in Table 3. The quenching rate constant kq of the interaction between the complex and BSA is much larger than

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that of the maximum scattering collision quenching constant (2 × 10 10 L·mol -1 s -1)[44]. These illustrate that the quenching mechanism is static quenching and is due to the formation of new complexes[45].

Tab. 3 The Stern−Volmer quenching constants of complexes with BSA.

Complexes

T (K)

KSV (×10 4 L·mol −1)

Kq (×10 12 L·mol−1·s−1)

Ra

S.D. b

293

7.1

7.1

0.99352

0.00204

299

3.4

3.4

0.99368

0.00096

BSA-1

ACCEPTED MANUSCRIPT 3.2

3.2

0.99395

0.00093

293

7.4

7.4

0.9987

0.00094

299

5.2

5.2

0.98923

0.0019

306

3.7

3.7

0.99349

0.00109

a

R is the correlation coefficient

b

S.D. is the standard deviation for the KSV values

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BSA- 2

306

3.2.4. The binding constant and binding site of the complex with

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BSA

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For the static quenching process, the binding constant (K) and the number of binding sites (n) can be determined by logarithmic Eq. (3.2) [46]. lg( F0 − F ) / F = lg K + n lg[Q ]

(3.2)

where K is the binding constant, and n is the number of binding sites. Using the Eq.(3.2) to plot, the double logarithmic curve of the interaction between the complex

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and BSA is obtained, and the values of K and n are calculated according to the slope of the curve Fig.4. The values are listed in Table 4. -0.2

293K 299K 306K

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-0.4

-1.0

-0.6 -0.8 -1.0

-1.4

-1.2

-1.6

-4.8

-4.6

-0.2 -0.4

-1.2

-5.0

293K 299K 306K

lg[(F0-F)/F]

-0.8

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lg[(F0-F)/F]

-0.6

0.0

-1.4

-4.4

lg[Mn-SS]

-4.2

-4.0

-5.0

-4.8

-4.6

-4.4

-4.2

-4.0

lg[Mn-SS]

Fig. 4 The Logarithmic plots for the binding of complex with BSA.

From the Fig.4 and Table 4, it can be seen that the number of binding sites of each complex with BSA was about 1, indicating that each complex has about one binding

ACCEPTED MANUSCRIPT site in BSA, that is, the complex binds BSA to 1:1 in a certain concentration range. This determines the subsequent determination of SOD-like activity by 1:1 mixing. Tab. 4 The static binding constants K and the number of binding sites n of complexes with BSA at three temperatures.

BSA-2

n

(×10 4 L·mol−1)

( kJ·mol-1)

292K

1.98

1.109

-256.99

299K

0.17

0.932

-253.43

306K

0.019

0.767

-253.34

292K

0.57

0.945

-113.28

299K

0.31

0.933

-111.90

306K

0.075

0.841

-111.87

∆S0

( kJ · mol -1)

( J·mol -1·K -1)

-256.86

-11.49

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BSA-1

T(K)

∆H0

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Complexes

∆G0

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K

-113.26

-4.53

3.2.5 Combination type determination

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The main forces between small molecule drugs and biological macromolecules are: hydrogen bond, van der Waals force, electrostatic attraction and hydrophobic interaction. Its determination is based on entropy change and enthalpy change of

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thermodynamic parameters. The enthalpy change ∆H0 and the entropy change ∆S0 of the reaction can be calculated from the Van't -Hoff equation. Van't- Hoff equation is: lnK = -∆H0/RT+ R∆S0

(3.3)

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where K is the binding constant for the complex binding to BSA , R is the gas molecular constant ( R = 8.3151 J·mol-1·K-1). Using the Eq. (3.3) to map, the curve is obtained. The values of ∆H0 and ∆S0 are calculated according to the slope of the curve Fig.5 and are listed in Table 4.

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V-1 V-2 Mn-(RR) Mn-(SS)

11

LnK

10 9 8

6 5 0.00325

0.00330

0.00335

1/T

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7

0.00340

buffer of pH 7.43.

∆G0 = ∆H0 - T∆S0

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Fig. 5 Van’t Hoff plot for the binding of Complex with BSA in 0.05 mol·L-1 phosphate

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(3.4)

The Gibbs free energy ∆G0 of the reaction can be obtained by the Eq.(3.4). As can be seen from Table 4, ∆G0 at three experimental temperatures is negative, indicating that the binding reaction between the complexes and BSA can be spontaneous. In addition, ∆H0 < 0, ∆S0 < 0, indicate that the main force between the complexes and BSA is a

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typical hydrophobic force.

3.2.6 Circular dichroism spectra

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The CD spectra have been utilized as a powerful tool for exploring the chiral aspect of complexes and to provide valuable information about the chiral complexes[47]. The

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technique of CD spectroscopy can give useful information on changes in BSA morphology, since CD signals are quite sensitive to the mode of BSA interactions with small molecules[48]. It is also an excellent method for rapidly evaluating the secondary structure, folding and binding properties of proteins[49,

50]

. The CD

spectrum in the near UV region (250~320 nm) reflects the tertiary structure of the protein. In the far UV region (185~250 nm), which corresponds to peptide bond absorption, the CD spectrum can be analyzed to give the content of regular secondary structural features such as α-helix and β-sheet[51]. There are two negative peaks in the CD spectra of BSA, which are characteristic of the α-helix structure of the protein at

ACCEPTED MANUSCRIPT 208 nm and 222 nm, respectively[50]. Fig.6 shows the CD spectra for the different concentrations of the complexes with BSA. It can be seen from the figure that the peak shape of BSA does not change with the addition of the complex, but the intensity of the peak decreases with the increase of the complex concentration. This suggests

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that the complex binds to BSA and causes a change in the secondary structure of the protein. The α-helix structure in BSA is reduced, but the α-helix structure remains the main conformation in BSA [52].

The structural content of α-helix in the protein molecule is calculated by Eq.(3.5) .

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[53]

MRE = θ obs (m deg)/ (10 × n × l × Cp )

(3.5)

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where MRE is the molar ellipticity, θobs is ellipticity, Cp is the molar concentration of human protein, n is the number of amino acid residues (BSA 582), and l is the thickness of the cell (1 cm).

The α-helix structure of BSA was calculated by the ellipticity of 208 nm at CD spectra, such as Eq.(3.6)[54].

a %helix = [( − MRE 208 − 4000)/(33000 − 4000)] × 100

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(3.6)

where MRE208 is the ordinate value corresponding to the molar ellipticity at 208 nm in the CD spectrum. The content of α-helix of pure BSA and complex-BSA system can

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be calculated by Fig. 6, Eqs.(3.5) and (3.6). The results are shown in Table 5.

20

0

-20

-20

-40

-40

Elipti city[mdeg]

Elipticity[medg]

20

BSA-(2)

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BSA-(1)

0

-60 -80

c

-100

a

-120 -140 -160 200

-60

c

-80 -100

a -120 -140

210

220

230

Wavelength(nm)

240

250

-160 200

210

220

230

240

250

Wavelength(nm)

Fig. 6 The CD spectra of complex-BSA system. [BSA] = 1.0 × 10 –6 mol·L –1. From a to c, the ratio of [BSA] : [complex] were 1:0, 1:1and 1:2.

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Tab. 5 The content of α−helix of BSA in the absence of and presence of complexes.

[ML]:[BSA]

[ML]:[BSA]

[ML]:[BSA]

0:1

1:1

2:1

BSA-1(%)

52.76

41.29

BSA-2 (%)

52.76

40.35

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Complexes

37.69

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39.20

As can be seen from Table 5, the content of α-helix of the BSA molecule decreases

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as the concentration of the complex increases. The complexes bind to the amino acid residues in the BSA molecule and destroy the networked hydrogen bond backbone formed between the amino acid residues[55]. So that the secondary structure of BSA changes, thereby reducing the α-helix structure of the content[56]. The order of the

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effect of the complex on BSA secondary structure is 1> 2.

3.3. Superoxide dismutase activity

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3.3. 1 SOD activity

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There are many direct and indirect methods reported for measuring the SOD activity. In this essay, we have used the indirect method. Superoxide scavenger activities of the Schiff base metal complexes and their BSA adduct was assayed by measuring the competition kinetics of nitro blue tetrazolium (NBT) and it was an indicator[57]. At room temperature, in the xanthine-xanthine oxidase method to generate O2·-, O2·- react with NBT and generated formazan which have the maximum absorption value at 550nm. Thus, we could use the absorption value (A) at 550 nm to plot against time (t) and get a straight line with a slope that can represent the rate of the generation.

ACCEPTED MANUSCRIPT Fig.S2 shows the change in the NBT reduction O2· - reaction rate for metal complexes and their protein adducts under different concentrations. The experimental results showed that in the same concentration the degree of decrease for the protein adduct is much greater than that of the corresponding complex. It suggested that the

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protein adduct catalyzed the decomposition of O2· - was much faster than that of the complex, that is, the protein adduct was much more active than the complex. According to the following Eq. (3.7) to determine their SOD activity.

V0 k cat -1 = [cat ] Vcat k det ector [detector ]

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(3.7)

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where V0 is the reaction rate at which no inhibitor is added; Vcat is the rate at which the inhibitor is added; kdetector is a reaction constant for monitoring molecules with superoxide anion radicals, kcat is the apparent rate constant of the inhibitor. In this paper, kdetector is reaction constant of NBT with O2· - and its value is 5.88×10 4 M-1·s-1 [58]

; [detector] is the concentration of NBT; [cat] is the concentration of the complex

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or complex-BSA adduct.

According to Eq.(3.7), the complexes and it’s BSA adduct as abscissa, and (V0 / Vcat) -1 is plotted as ordinate. The results are shown in Fig. 7. 0.7 0.6

BSA-Mn-(3)

BSA-Mn-(4) 1.2 1.0

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V0/VC-1

0.4

0.8

0.3

0.6

0.2 0.1

0.4

0.0

0.2

0.5

1.0

1.5

Mn-(4)

V0/VC-1

0.5

1.6 1.4

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Mn-(3)

2.0

concentration(10-6M)

2.5

3.0

0.5

1.0

1.5

2.0

2.5

3.0

concentration(10-6M)

Fig. 7 The SOD-like activity of complex (■) and complex-BSA adduct (●) in the Fridovich assay ( NBT=250 µM). V0 is the reduction rate of the NBT and Vcat is the rate of reduction of the NBT in the presence of the complex.

ACCEPTED MANUSCRIPT When ( V0 / Vcat) -1 = 1, the IC50 value ( concentration for 50% inhibition) can be determined. According to the following Eq.(3.8), the IC50 and kcat values could be obtained and listed in Table 6[59-62].

Tab.6

(3.8)

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kcat = kdetector [detector] / IC50

IC50 and kcat values of the complexes and the corresponding BSA adducts

IC50(×10 -6 mol·L−1)

kcat(×10 7 L·mol-1·s−1)

Native SOD

0.0158

-

1

5.01

2.95

BSA-1

4.76

2

3.23

BSA-2

2.08

R

0.9942

0.9736

3.09

0.9783

4.56

0.9829

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substance

0.9961

7.06

Under the experimental conditions, the IC50 value of the natural SOD enzyme was 0.0158 µmol·L-1, which was very close to the reported value of 0.0147 µmol · L-1[63],

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so the experimental method was reliable. In the results of SOD activity determination, it can be found that when the protein is present, for the complex 1, the IC50 value decreased slightly, and its kcat value increased slightly. Indicating that complex 1 with BSA did not significantly increase its SOD activity. For the complex 2, the IC50 value

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was significantly decreased, and the kcat value was significantly increased when the protein is present. The results showed that the complex activity of complex 2 and

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BSA resulted in a significant increase in SOD activity. It can be concluded that the activity sequence of these two SOD mimics is in order BSA-2 > 2 > BSA-1 > 1. The results indicate that the SOD activity is higher for S,S ligand than the R,R ligand, which suggests that the configuration of the ligand had a certain effect on SOD activity. In the previous work, most Mn(II) complexes with Schiff base present medium SOD activity with kcat of 10 5 ̶ 10 6 M -1 s -1, such as slen complexes of EUK -8, EUK-134, and EUK-189[23]. SOD activity higher than 107 M -1 s -1was found at the manganese

complexes

with

(2-((2-phenyl-2-(pyridin-2-yl)hydazono)methyl)phenol,

tridentate

ligands

ACCEPTED MANUSCRIPT 2-((2-phenyl-2-(pyridin-2-yl)hydrazono)methyl)napthalen-1-ol,

and

2-(1-(2-phenyl-2-(pyridine-2-yl)hydrazono)ethyl)phenol)2-(1-phenyhydrazinyl)pyridi ne)[64]. Compared with the complexes in the literature, 1 and 2 present higther SOD activities. It also can be seen that BSA can promote SOD activities of the complexes.

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The combination of protein is an effective way to improve the antioxidant activity.

3.3.2 ABTS· + scavenging ability

ABTS· + testing is usually used to assess the antioxidant capacity of biological

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fluids and many pure compounds. The inhibition rate is calculated by Eq. (3.9). Using inhibition rate and sample concentration to plot Fig. 8, the antioxidant concentration

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can be determined.

Activity = [ ( A0 -A) / A ] × 100%

(3.9)

where A0 is the absorbance of the non-suppressing agent; A is the absorbance after adding the inhibitor. When the inhibition rate is 50%, the corresponding sample concentration is IC50 value and listed in Table 7.

60 50 40

Scavenging activity(%)

VC 1 BSA-1 BSA

70

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Scavenging activity(%)

80

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90

30 20 10

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0

10

20

30

40

90 80

VC 2 BSA-2 BSA

70 60 50 40 30 20 10 0 10

50

20

30

40

50

Concentration(uM)

Concentration(uM)

Fig. 8 Trends in the inhibition of ABTS radicals by complexes with standard ascorbic acid

Tab.7

at various concentrations.

The radical scavenging activity of the compounds and ascorbic acid.

Compounds

VC

BSA

1

BSA- 1

2

BSA- 2

IC50 values (µM)

5.02

40.25

>100

21.66

>100

27.34

ACCEPTED MANUSCRIPT The protein contains amino acid residues (tyrosine, tryptophan, cysteine) capable of scavenging ABTS· +[65], so that the BSA adducts of the complexes have the ability to remove ABTS free radicals. As can be seen from Fig. 8, with the increase in concentration of VC on the ability to clear ABTS· + little effect. This shows that it

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reacts quickly with ABTS· + and quickly reaches equilibrium[66]. The ability of metal complexes scavenging ABTS· + is weak and does not change much as the concentration increase. The scavenging ability of BSA and its adduct with metal complexes increased with the increasing concentration. After the formation of hybrid

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protein, the IC50 value of the complexes decreased significantly. It is not difficult to see that the IC50 value of complex 1 is obviously smaller than that of complex 2. This

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illustrate that under the condition of R,R configuration ligand L1, the ability of scavenging ABTS radical was stronger than that of S,S configuration ligand L2. The

Fig. 8 and the IC50 values in the Table 7 suggested that the activity of the complexes and BSA adduct corresponding to the complex is more pronounced than that of the

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complex. Thus, the order of activity is VC > BSA-1 > BSA-2.

4.Conclusion

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In the article, a new asymmetric chiral Schiff base complexes were synthesized and well characterized by IR, X-ray and elemental analyses. At the same time, we

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synthesized and characterized the SOD activity of a pair of chiral Schiff base metal complexes and their BSA adducts, and the SOD and ABTS activities were tested. We have obtained the Single-crystal X-ray diffraction data and Crystal Structure of the two complexes and determine their chiral relationship. The interaction between the complex and BSA was studied by fluorescence spectroscopy, UV spectroscopy and circular dichroism. The results show that the complex interacts with BSA. The results of fluorescence analysis showed that the complexes could lead to endogenous fluorescence quenching of BSA, which was mainly static quenching. BSA binding experiment showed that the complex binds BSA to 1: 1 in a certain concentration

ACCEPTED MANUSCRIPT range. The results of CD spectroscopy showed that the complexes interacted with BSA to decrease the α-helix structure in BSA and change the secondary structure. Antioxidant activity studies have shown that the conformation of complexes has some effect on its properties. The results indicate that the SOD activity is higher for S,S

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ligand than the R,R ligand, which suggests that the configuration of the ligand had a certain effect on SOD activity. ABTS free radical scavenging experiments illustrate that under the condition of R,R configuration ligand L1, the ability of scavenging ABTS radical was stronger than that of S,S configuration ligand L2. BSA has a

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catalytic effect on the activity of the complex, it will be more applications in the future. At the same time, Schiff base complex in all aspects of the application

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prospects will be more broad.

Acknowledgements

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We gratefully acknowledge the National Nature Science Foundation of China (No. 21361003 and 21461003), Guangxi Natural Science Foundation of China (2016GXNSFDA380005, 2016GXNSFFA380010), the Foundation of Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Education of China (CMEMR2016-A11), New Century Ten, Hundred, Thousand Talents Project in Guangxi andInnovative Research Team in University of Ministry of Education of China (IRT_16R15). the Guangxi University for Nationalities Graduate Scientific Research Innovation Project (gxun-chxzs2016116).

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Appendix A. Supplementary material CCDC 1561194 and 1561195 contains the supplementary crystallographic data for C26H28ClMnN2O3(1) and C26H28ClMnN2O3(2). These data can be obtained free of

charge

from

The

Cambridge

Crystallographic

http://www.ccdc.cam.ac.uk/[email protected]

Data

Centre

via

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ACCEPTED MANUSCRIPT Highlights:

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A couple of chiral ligands and their Mn(III) complexes has been synthesized and characterized experimentally. The interaction between BSA and the complexes has been studied systematically. The superoxide and ABTS free radical scavenging ability of the complexes and their BSA adducts have been studied. BSA can promote antioxidant activities of the complexes.