catalase mimetics with cyclodestrins

Journal of Inorganic Biochemistry 103 (2009) 381–388

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New conjugates of superoxide dismutase/catalase mimetics with cyclodestrins Valeria Lanza, Graziella Vecchio * Dipartimento di Scienze Chimiche, Università di Catania, viale A. Doria 8, 95125 Catania, Italy

a r t i c l e

i n f o

Article history: Received 18 July 2008 Received in revised form 21 November 2008 Accepted 24 November 2008 Available online 7 December 2008 Keywords: Cyclodextrins Salen Manganese complexes SOD mimetics EUK 113 EUK 108

a b s t r a c t Mimetics of antioxidant enzymes such as superoxide dismutases (SOD) or catalases are reported as potential new drugs able to reduce oxidative stress damage. In particular, manganese(III) complexes of salen-type ligands have been studied as both SOD and catalase mimetics. In this paper, we report the synthesis of two novel conjugates of salen-type ligands with the b-cyclodextrin, the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N0 -bis(salicylidene))]-b-cyclodextrin and the 6-deoxy-6-[(S-cysteamidopropyl (1,2-diamino)N,N0 -bis(3-methoxysalicylidene))]-b-cyclodextrin, their spectroscopic characterization, and the synthesis and the characterization of their manganese(III) complexes. The SOD-like activity of the metal complexes was investigated by the indirect Fridovich method. The catalase like activity was tested using a Clark-type oxygen electrode. The peroxidase activity was tested using the ABTS (2,20 azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay. The glycoconjugation of salen–manganese(III) complexes yields compounds with enhanced SOD activity. These complexes also show catalase and peroxidase activities higher than the simple salen complexes (EUK 113 and EUK 108). Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Antioxidant compounds have been considered as therapeutic agents for a variety of disorders associated with the oxidative stress. In fact, oxidative stress processes have been implicated in a variety of detrimental health conditions including ischemiareperfusion injury [1], chronic inflammation [2], diabetes [3], neurodegenerative disease [4,5], cancer [6] and aging [7]. In this perspective, mimetics of antioxidant enzymes such as superoxide dismutases (SODs) or catalases are presented as potential new drugs to prevent oxidative stress damages [2,8,9]. Among these systems, metal complexes of manganese(II) or (III) seem to be among the most widely investigated molecules [10]. Particularly, the manganese(II) complexes of [15]aneN5 (1,4,7,10,13-pentaaza cyclopentadecane) type ligands [10–13] are reported in the literature as SOD mimetics and the manganese (III) complexes of porphyrins [14,15] or salen (H2salen = N,N0 bis(salicylidene) ethylenediamine) type ligands [9,16–18] have been reported as both SOD and catalase mimetics. The molecular mechanism by which the manganese complexes work is still not entirely understood, but a number of different hypotheses based on experiments in vivo have been made. The SOD and catalase activities of these molecules have been considered as the main reason for their tested pharmacological activity. Investigations have been carried out in order to understand the action mechanism [19].

* Corresponding author. Fax: +39 95580138. E-mail address: [email protected] (G. Vecchio). 0162-0134/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2008.11.017

As regards the salen–manganese(III) complexes, generally the SOD-like activity, investigated in vitro, is considered to be the operative mechanism in vivo [9,20,21]. It has been shown that salen complexes exert beneficial effects in several in vitro and in vivo animal models of human diseases which involve oxidative stress ranging from Alzheimer’s [22,23], Parkinson’s [24], Prion Disease [25] and motor neuron disease to multiple sclerosis and excitotoxic neuronal injury [26]. The Eukarion compounds have also been shown to afford protection from ischemia-reperfusion injury of the brain [20], heart [27] and kidney [28,29] in animal models. In the case of Mn(III) to salen derivatives, the mechanism for the dismutation of O 2 involves the reduction of Mn(III) to Mn(II) by O 2 , which is oxidized to O2. The Mn(II) is oxidized back to Mn(III) by another molecule of O 2 yielding H2O2 [10]:

MnðIIIÞ þ O 2 ¢ MnðIIÞ þ O2 þ MnðIIÞ þ O 2 þ 2H ¢ MnðIIIÞ þ H2 O2

The mechanism by which the Mn–salen acts as a catalase mimetic involves the oxidation of Mn(III) to oxomanganese by H2O2, releasing water [20]. The oxomanganese is then reduced to Mn(III) by another hydrogen peroxide molecule to form oxygen and water

MnðIIIÞ þ H2 O2 ¢ MnðVÞO þ H2 O MnðVÞO þ H2 O2 ¢ MnðIIIÞ þ H2 O þ O2 Starting from the most simple system [Mn(salen)X] (X = Cl or CH3COO, code name EUK 8 and EUK 108, respectively), other modified salen complexes have been investigated (Chart 1). The water solubility of this class of molecules is not very high, as has been reported by several authors [10].

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V. Lanza, G. Vecchio / Journal of Inorganic Biochemistry 103 (2009) 381–388

R

R O

X

containing cyclodextrins could be an interesting approach to carry an O 2 /H2O2 scavenger in the colon. In addition, cyclodextrins are able to react with the OH radicals and thus their appropriate metal complexes could act against a cocktail of radicals [31,36]. In this paper we report the synthesis of the 6-deoxy-6-[(S-cysteamand idopropyl(1,2-diamino)N,N0 -bis(salicylidene))]-b-cyclodextrin the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N0 -bis(3-methoxysalicylidene))]-b-cyclodextrin, their spectroscopic characterization, and the synthesis and the characterization of their manganese(III) complexes. The study includes the determination of superoxide and hydrogen peroxide scavenging properties of the new manganese(III) complexes. The SOD-like activity of the metal complexes was investigated by the indirect Fridovich method [37]. The catalase activity was tested using a Clark-type cell [20]. The peroxidase activity was tested using the ABTS assay [38].

O

Mn

N

N

R = H, X = Cl- EUK 8; X = CH3COO- EUK 108 R = OCH3, X = Cl- EUK 134; X = CH3COO- EUK 113 Chart 1.

We have synthesized some SOD mimetics based on copper(II) complexes, conjugating b-cyclodextrin (CD) with amine ligands and have found that the cyclodextrin cavity improves, in some case greatly, the SOD activity of the complexes [30,31]. On the basis of the wide interest in salen derivatives, we have also synthesized a Mn(III) complex based on functionalized cyclodextrin, similar to the salen ligand [32]. This compound shows a good water solubility and a SOD activity similar to the EUK 8, but is not very stable in water. In this paper we report the synthesis of two novel compounds, in which salen moieties have been conjugated to a b-cyclodextrin through a short chain. In particular, we have conjugated the N,N0 bis(salicylidene)ethylendiamine or N,N0 bis(3-methoxysalicylidene)ethylendiamine with the b-CD in order to obtain compounds analogous to EUK 108 and EUK 113, among the most studied Mn–salen type compounds (Chart 2). The presence of the cyclodextrin cavity, widely used in pharmaceutical preparations, could improve certain features of the salen moiety. Cyclodextrins, in fact, are used as complexing agents for drugs, and, in some cases, the CDs have been covalently conjugated to the drugs to enhance their stability, solubility and sitospecificity. Cyclodextrin-drug bioconjugates are reported in the literature as site specific carriers for the colon, being very promising as antiinflammatory pro-drugs for colon diseases [33]. Some results indi cate that ROS (reactive oxygen species) as O 2 , and OH have a role in mediating intestinal damage in inflammatory bowel diseases [34]. The protective effect of native SOD in pilot clinical trials in Crohn’s disease, related to the role of O 2 in this inflammatory injury, has been demonstrated [35]. The synthesis of SOD mimetics

2. Experimental Commercially available reagents were used directly, unless otherwise noted. TLC was carried out on silica gel plates (Merck 60-F254). CD derivatives were detected with UV light and with anisaldehyde reagent and CD amino derivatives with the ninhydrin test. EUK 108 and EUK 113 were synthesized as reported elsewhere [39]. The 6-tosyl-b-cyclodextrin was synthesized adding a solution of tosyl chloride (1.5 g) in anhydrous pyridine to a solution of dry b-cyclodextrin (5 g) in anhydrous pyridine at 5 °C After 5 h, the pyridine was distilled off in vacuo at 40 °C. The 6-tosyl-b-cyclodextrin was isolated by reversed phase Rp 8 (Lichroprep, 40–63 lm) chromatography (eluent H2O/DMF). Synthetic details are reported elsewhere [40]. 1 H NMR spectra were recorded at 25 °C with a Varian Inova 500 spectrometer at 499.883 MHz in CD3OD, without a reference compound. The 1H NMR spectra were acquired by using standard pulse programs from the Varian library. In all cases the length of the 90° pulse was ca. 7 ls. The 2D spectra (COSY, TOCSY, HSQC, T-ROESY) were acquired using 1 K data points, 256 increments and a relaxation delay of 1.5 s. T-ROESY spectra were obtained using a 300 ms spin-lock time. Electronic (UV-Visible(UV–Vis)) absorption spectra were recorded on a Beckman DU 650 spectrophotometer. The spectra were recorded at 25 °C, on freshly prepared aqueous solutions. R

R OH H2N

NH2

O

O

O HO

OH

HO

6

2

OH

O

Mn

N

N

CO

CO

HN

OH

S

Ac

O

N

R

HN OH

HO

N

CO

R

R

S

OH

O

O

O HO

HN OH O

O HO

OH 6

OH

O

S O

MnAc2 HO

O

O HO

OH 6

R= H 3a

R = H 4a

R = OCH3 3b

R = OCH3 4b

Chart 2.

OH

O

V. Lanza, G. Vecchio / Journal of Inorganic Biochemistry 103 (2009) 381–388

2.1. Superoxide dismutase assay The SOD-like activity was determined by the indirect Fridovich method [37]. The superoxide anion was enzymatically generated by the xanthine–xanthine oxidase system and spectrophotometrically detected by monitoring the reduction of cytochrome c at 550 nm or of NBT (nitro blue tetrazolium salt) at 560 nm. The reaction mixture was composed by cytochrome c (50 lM) or NBT (250 lM), xanthine (50 lM), catalase (30 lg/ml) in phosphate buffer (10 mM) at pH 7.4. An appropriate amount of xanthine oxidase was added to 2 mL reaction mixture to produce a DA550 nm min1 of 0.024. This corresponded to O2 production rate of 1.1 lM min1. The detector molecule reduction rate was measured in the presence and in the absence of the investigated complex for 600 s. All measurements were carried out at 25.0 ± 0.2 °C using 1  1 cm thermostatted cuvettes in which solutions were magnetically stirred. In separate experiments, urate production by xanthine oxidase was spectrophotometrically monitored at 295 nm, ruling out any inhibition of xanthine oxidase activity. The I50 (the concentration which causes the 50% inhibition of cytochrome c or NBT reduction) values of metal complexes at pH 7.4 were determined. 2.2. Catalase activity assay The catalase activity was assayed by monitoring the conversion of hydrogen peroxide to oxygen polarographically, using a Clarktype oxygen electrode (Amel, Model 360). In a typical experiment, the H2O2 solution was thermostatted and stirred and, when the baseline was steady, the manganese complex was added. The oxygen concentration versus time data were collected on a PC using the Data-List program purchased from Amel. The initial rate method was used to determine the rate constants. Initial rates were calculated by linear regression using data from the first 5 s of the reaction. Manganese–salen complexes were tested in a range of concentrations 10–50 lM, in reaction mixtures consisting of hydrogen peroxide (10 mM) in phosphate buffer (50 mM) at pH 7.4 and at 25.0 ± 0.1 °C. In other experiments, the initial rates were measured at constant catalyst concentration for different concentrations of hydrogen peroxide (1–50 mM). Oxygen concentration was monitored at 1 s intervals. Stock solutions of complexes were prepared in water in the case of cyclodextrin derivatives and in water/ethanol for the eukarion compounds. The final concentration of ethanol did not exceed 5%, and did not affect the measurement.

383

Sephadex C-25 column (15  130 mm, NHþ 4 form). Water and then a linear gradient 0–0.15 M of NH4HCO3 solution (400 mL) were used as eluents. The product 1 was isolated in yield 68%. Rf = 0.42 (PrOH-H2O-AcOEt-NH3, 5:2:2:1). 1 H NMR (500 MHz, MeOD) d (ppm) 5.03 (1H, d, H-1A) 4.96 (6H, d, other H-1 of CD), 3.90 (1H, m, H-5A), 3.87–3.75 (25H, m, H-3, H5, H-6), 3.58–3.45 (14H, m, H-2, H-4), 3.02 (1H, dd, H-6aA), 2.89 (2H, t, H del CH2 in a to the NH2), 2.82 (1H, dd, H-6bA), 2.71 (2H, t, CH2 in b to NH2). (d = doublet, t = triplet, m = multiplet, dd = doublet of doublet). ESI-MS m/z = 1194 (1+H)+ Calcd. Elemental analysis 1, C44H75NO34S  4H2O: C, 41.7; H, 6.6; N, 1.1; S, 2.5. Found: C, 42.0; H 6.7; N, 1.2; S, 2.4. 2.3.2. Synthesis of 6-deoxy-6-[S-cysteamidopropyl(1,2-diamino)]-bcyclodextrin 2 The Na,Nb-Di-Boc-L-2,3-diaminopropionic acid (dicyclohexylamine salt) (DAP-Boc, 0.250 g, 0.82 mmol), the (O-(benzotriazole1-yl)-N,N,N0 ,N0 -bis(tetramethylene) uronium hexa fluorophosphate (0.315 g, 0.82 mmol) and 1-hydroxybenzotriazole (0.111 g, 0.82 mmol) were solubilized in anhydrous DMF (30 ml). The reaction mixture was stirred under nitrogen at room temperature. After 15 min 1 was added (0.979 g, 0.82 mmol) and the reaction was checked by TLC (PrOH-H2O-AcOEt-NH3, 5:2:1:1) and was stopped when the reagents disappeared in the mixture. The solvent was evaporated and the remaining solid was purified by chromatography, using a reversed phase C-8 column and a linear gradient H2O/MeOH (0?50%). The product isolated was deprotected in CF3COOH, under stirring for 1 h. The CF3COOH was evaporated and then the solid was purified by CM-Sephadex C-25 (NHþ 4 form), eluting with water and then with a linear gradient of a NH4HCO3 solution 0?0.25 M (total volume 2 l). Appropriate fractions, containing 2, were collected, Rf = 0.23 (PrOH-H2OAcOEt-NH3, 5:2:2:2) and the solvent was evaporated. The final product (315 mg, 0.246 mmol; yield 40%) was characterized by 1 H NMR and ESI-MS. 1 H NMR (500 MHz, D2O) d (ppm) 5.03 (1H, d, H-1A of CD), 4.96 (5H, m, H-1 of the CD), 4.94 (1H, d, H-1 of CD), 3.92 (1H, t, H-5A), 3.87–3.75 (25H, m, H-3, H-5, H-6 of CD), 3.58–3.45 (14H, m, H-2, H-4 of CD), 3.36 (2H, t, 3.34 CH2 in b to S), 3.33 (1H, t, CH of DAP), 3.04 (1H, dd, H-6aA CD), 2.81 (2H, m, H-6bA of CD, CH2 of DAP, Ha del CH2 of DAP), 2.70 (3H, m, Hb of CH2 of DAP, CH2 in a to S). ESI-MS: m/z = 1280.1 (2+H)+, m/z = 1302.3 (2+Na)+ Calcd. Elemental analysis 2, C47H81N3O35S  4H2O: C, 41.7; H, 6.6; N, 3.1; S, 2.4. Found: C, 41.2; H 6.7; N, 2.9; S, 2.3.

2.3. Peroxidase activity assay The peroxidase activity of 4a, 4b, EUK 108 and EUK 113 compounds was assayed by monitoring the hydrogen peroxide-dependent oxidation of ABTS (2,20 -azino-bis-(3-ethylbenzothiazoline-6sulfonic acid)) spectrophotometrically [38]. The assay mixture consisted of ABTS (0.2 mM), the manganese(III) complex (1 lM), H2O2 (0.5 mM) in phosphate buffer (50 mM) at pH 7.4. Assays were conducted at 25 °C. The ABTS oxidation was mon4 itored at 734 nm (eþ ABTS ¼ 1; 5  10 ) to eliminate interferences from the absorption of metal complexes. No ABTS oxidation with the complexes or the hydrogen peroxide was observed in the condition of the assay (pH 7.4, T = 25 °C). 2.3.1. Synthesis of 6-deoxy-6-(S-cysteamine)-b-cyclodextrin 1 Cysteamine (1.0 g) in water (2 ml) in the presence of an equivalent of NaOH, was added to a solution of 6-tosyl-b-cyclodextrin [40] (1.70 g) in DMF. The reaction was carried out at 60 °C, under stirring and under nitrogen. After 7 h, the solvent was evaporated under vacuo and the crude product was purified using a CM-

2.3.3. Synthesis of 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N0 bis(salicylidene))]-b-cyclodextrin 3 The salen derivatives were synthesized as reported in the literature [41]. Salicylaldehyde (12.5 ll) was added to 2 (0.200 g) in absolute ethanol (about 10 ml), under stirring at room temperature. A yellow solid was formed during the reaction. After 3 h, the solvent was evaporated in vacuo and the solid obtained was washed with acetone. Yield 88%. 1 H NMR: (MeOD, 500 MHz) d (ppm) 8.60 (1H, s, iminic proton in alfa to the amidic bound), 8.49 (1H, s, other iminic proton), 7.45– 7.32 (4H, m, Hs in ortho to the OH and Hs in ortho to the iminic group), 7.00–6.89 (4H, m, Hs in para and in meta to the OH), 4.98 (1H, d, J1,2 = 3.57 Hz, H-1 of CD), 4.96 (3H, m, H-1 of CD), 4.94 (1H, d, J1,2 = 3.57 Hz, H-1 of CD), 4.92 (1H, d, J1,2 = 3.57 Hz, H-1 of CD), 4.90 (1H, d, J1,2 = 3.48 Hz H-1A of b-CD), 4.55 (1H, t, J = 5.77 Hz, H in a to CO), 4.11 (2H, m, Hs in b to CO), 3.92–3.65 (26H, m, H-3, H-6, H-5), 3.58–3.40 (14 H, m, H-2, H-4 of CD), 3.07 (1H, dd, H-6aA of CD), 2.72 (3H, m, H-6bA of CD, CH2 in a to S). ESI-MS: m/z = 1488.1 (3a+H)+ and m/z = 1510.2 (3a+Na)+.

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3b was synthesized as reported for 3a, using the 3-methoxy salicylaldehyde instead of the salicylaldehyde. Yield: 90%. 1 H NMR: (MeOD, 500 MHz) d (ppm) 8.58 (1H, s, iminic proton in a to the amidic bound), 8.45 (1H, s, other iminic proton), 7.05 (2H, m, H-4, H-6 of benzene ring bound to the 2-imino group), 7.00 (1H, d, H-4 of benzene ring bound to 3-imino group), 6.95 (1H, d, H-6 of benzene ring bound to 3-imino group), 6.87 (1H, t, H-5 of benzene ring bound to 2-imino group), 6.80 (1H, t, H-5 of benzene ring bound to 3-imino group), 4.99–4.88 (7H, m, H-1 of CD), 4.55 (1H, t, J = 5.77 Hz, H in a to CO), 4.11 (2H, m, Hs in b to CO), 3.92– 3.65 (32H, m, H-3, H-6, H-5, OCH3), 3.58–3.35 (14H, m, H-2, H-4 of CD), 3.05 (1H, d, J = 13.92 Hz, H-6aA of CD), 2.81 (2H, dd, J6b,5 = 6.00 Hz, J6a,6b = 13.92 Hz, H-6bA of CD), 2.74 (2H, m, CH2 in a to S). ESI-MS: m/z = 1548.5 (3b+H)+; m/z = 1570.5 (3b+Na)+. 2.3.4. Synthesis of manganese(III) complexes of 3a and 3b 3a or 3b (0.115 g, 0.086 mmol) was dissolved in absolute ethanol (5 ml) and two equivalents of NaOH (ethanol solution) were added. The mixture was stirred and MnAc2 (0.086 mmol) was added. The yellow solution turned to brown immediately and was refluxed for 3 h. The mixture was left to reach room temperature, the precipitated solid was filtered off and washed with acetone. Yield: 90%. The purity of the products was checked by HPLC (column: Econosphere ODS, eluent: H2O-CH3CN, 0?70%, k = 220 nm and 400 nm). 4a: ESI-MS: m/z = 1540.4 [4a-Ac]+; m/z = 781.5 [4a-Ac+Na]2+. UV–Vis kmax/nm (e/M1 cm1) (H2O-MeOH 9/1): 214 (69,800) (sh, ligand transition), 236 (66,000) (phenyl ring p–p*), 284 (30000) (phenyl ring p–p*), 314 (sh) (p–p* azomethine), 406 (br. 8740) (CT). IR (KBr): m/cm1: 3394; 2922; 2358; 1610; 1558;1540; 1408; 1290; 694; 574. Calcd. Elemental analysis 4a, C63H90N3O39SMn  6H2O: C, 44.1; H 6.5; N, 2.4; S, 1.9. Found: C, 43.6; H 6.4; N, 2.6; S, 2.1. 4b: ESI-MS: m/z = 1600.40 [4b-Ac]+; m/z = 811.5 [4b-Ac+Na]2+. UV–Vis kmax/nm (e/M1 cm1) (H2O-MeOH 9/1): 228 (65240) (phenyl ring p–p*), 275 (sh) (phenyl ring p–p*), 284 (6200), 326 (sh) (p–p* C'N), 413 (6775) (CT). IR (KBr): m/cm1: 3394; 2910; 2360; 1616; 1554; 1540; 1292; 1052; 668; 576. Calcd. Elemental analysis 4b, C65H94N3O41SMn  6H2O: C, 44.1; H 6.0; N, 2.4; S, 1.8. Found: C, 44.5; H 6.0; N, 2.2; S, 1.9. 3. Results and discussion 3.1. Ligands An appropriately functionalized b-CD was synthesized to build various salen type ligand conjugates. The synthesis of these glycoconjugates was carried out starting from the new derivative of bcyclodextrin, 6-deoxy-6-[S-cysteamidopropyl(1,2-diamino)]-bcyclodextrin 2. This derivative was synthesized from the 6deoxy-6-(S-cysteamine)-b-cyclodextrin 1, obtained in a good yield, from cyclodextrin 6-tosylate, as reported for the cysteine derivatives [42]. In particular, 2 was synthesized from 1 by condensation of the 2,3 amino propionic acid, protected by boc (t-butyloxycarbonyl), in the presence of activating and condensating agents, typically used for condensation reactions [43]. This reaction was carried out at room temperature and the product was easily isolated and characterized by ESI-MS and NMR spectroscopy. The boc groups were hydrolyzed in CF3COOH. The final product 2 was fully characterized. NMR spectra of the intermediate 2 confirm the identity of the compound: on the 1H NMR, in addition to the signals due to the cyclodextrin protons, the signals of the chain are evident. The protons of the ethylenic chain of the cysteamine

moiety appear at 3.34 ppm and at 2.70 ppm, while the protons of the ABX system of the propionyl chain resonate at 3.33 ppm (X), at 2.81and 2.70 ppm (A,B). In this region the signals due to the 6Hs of the functionalized glucose ring of cyclodextrin are also present. 3a and 3b ligands were synthesized from 2, following the literature procedure [41]. 3a ligand was characterized in methanol and the 1H NMR spectrum is reported in Fig. 1a. On the 1H NMR spectra, in addition to the signals of the cyclodextrin in the 3.2–4.0 region, the signals due to the chain protons and the signals of the modified glucose ring are clearly evident. The imino protons appear at 8.60 and at 8.49 ppm, particularly the signal at 8.60 ppm is due to the imino group in a to the amido group. The other aromatic protons are also different from each other for the two rings. The H-1s of cyclodextrin moiety are divided in six groups as often observed in the case of other derivatives [44,45]. The signals due to the ABX system of the diamino propionyl chain are downfield shifted for the Schiff base formation at 4.36 (CH) and 4.11 ppm (CH2). The up-field signals due to the 6-Hs of the functionalized glucose ring are present together with the signals due to the methylene of the cysteamine in a to the sulphur atom. 3b ligand was also characterized in methanol and the 1H NMR spectrum is reported in Fig. 1b. The spectrum, in the main features, is very similar to the spectrum of 3a. The methyl group of the aromatic ring is identified by ROESY spectra at about 3.8 ppm. In the aromatic region, the two aromatic rings are different, due to the presence of the functionalizing moiety. The imino protons appear at 8.56 and at 8.45 ppm, the first signal due to the imino group in a to the amido group. 3.2. Manganese(III) complexes Manganese complexes, 4a and 4b, were synthesized as reported in the literature [39] for the salen complexes, at room temperature, and were isolated by adding acetone to the reaction mixture. The UV–Vis spectra are not significantly different from those of simple corresponding salen complexes EUK 108 and EUK 113, in agreement with the presence of the same coordination environment (Fig. 2). In the ESI mass spectra, two main peaks are present: the peak at 1540 m/z corresponding to complex species MnL+ (L = 3a) and at 781.5 m/z corresponding to the double charged sodiated species. Analogously when L = 3b, the peak due to the ML+ species is present at 1600 m/z and the peak due to the double charged sodiated species is at 811.5 m/z. On the basis of the data reported in the literature for similar ligands [46], the coordination of the amido group at neutral pH with the manganese could be excluded. Thus the same coordination environment of Eukarion complexes can be proposed for 4a and 4b. 3.3. SOD activity Rate constants for the reaction of 4a and 4b with the superoxide anion, were determined by competition kinetics using cytochrome c or NBT as the detector molecule, as reported elsewhere [47]. Possible interferences with the xanthine/xanthine oxidase reaction of the test compounds were examined by following the rate of urate production at 295 nm in the absence of the target molecule. The complexes did not interfere with the reaction of the xanthine/xanthine oxidase system. Inhibition of cyt c (50 lM) or of NBT (250 lM) reduction by the complex, when plotted as Vo/Vcat -1 against the [complex], yielded a straight line with a slope kcat/ kdetector[detector]. Vo is the uninhibited reduction rate of the detector molecule and Vcat is the reduction rate of the detector molecule inhibited by the complex (Fig. 3). Kdetector is the constant for the reaction between the O 2 and the detector molecule and it is

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a

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

ppm

4.5

4.0

3.5

3.0

b

8.5

8.0

7.5

7.0

6.5

6.0

5.5

ppm

5.0

4.5

4.0

3.5

3.0

2.5

Fig. 1. 1H NMR spectra (MeOD, 500 MHz) of 3a (a) and 3b (b).

1.2

EUK-113 4b 4a EUK-108

1.0 0.8

A

0.6 0.4 0.2 0.0 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

λ nm

Fig. 2. UV–Vis spectra of 4a, EUK 108, 4b and EUK 113 (2.0  105 M) in H2O-MeOH 9:1.

2.6  105 M1s1 when the detector is cyt c and 5.88  104 M1s1 when the detector is NBT. The slope of the line, obtained by the best fit of the experimental data, allowed the calculation of the kcat value [32,48]. When cyt c was used as the detector molecule, the CD conjugates showed an atypical behaviour. When the concentration of complex increased, the activity appeared to decrease. The experimental values of Vo/Vcat 1 were not fitted by a linear function but by an exponential function. This behaviour was not observed in the case of the simple EUK 113 and EUK 108. The interaction of the cyclodextrin derivatives and the cyt c was supposed, on the basis of the recent literature, reporting on the interaction of some cyclodextrin derivatives and cyt c [49]. When NBT was used as the detector, a linear correlation between the Vo/Vcat 1 and complex concentration of the CD conjugates was shown, as expected. In the light of this data, we could hypothesize the occurrence of an interaction of 4a and 4b with the cytochrome c, interfering with the assay. For this reason we have determined the I50

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3.4. Catalase activity

V0/Vcat-1

5 4 3 2 I 50

1 0 0.0

2.0x10

-7

4.0x10

-7

6.0x10

-7

8.0x10

-7

1.0x10

-6

1.2x10

-6

1.4x10

-6

4b (M) Fig. 3. The SOD-like activity of 4b in the Fridovich assay (NBT = 250 lM). Vo is the reduction rate of the NBT and Vcat is the rate of reduction of the NBT in the presence of the complex.

values using the NBT assay. The I50 and kcat values are reported in Table 1. For comparison we have also determined the SOD-like activity of EUK 108 and EUK 113, in our experimental conditions. The results are very similar to those in the literature [9]. The I50 values were maintained also in the presence of albumin, while the activity was lost when the assay was carried out in the presence of EDTA. This behaviour is reported in the literature for the manganese salen complexes [50]. EDTA is able to complex the manganese(II) ion, formed during the first step of the dismutation reaction, destroying the system. 4a and 4b show good I50 values, about 10 times lower than the values for corresponding salens without cyclodextrins (Table 1). In the literature a number of manganese–salen complexes have been investigated as SOD mimetics [20] and their activities are very similar to that of EUK 108. For this class of compounds the best molecule, the manganese complex of Phenol-2,20 -[(1R,2R)-1,2cyclohexanediylbis[(E)-nitrilomethylidyne]]bis[6-(1,1-dimethylethyl)-4-methoxy, has a I50 value of 0.32 lM [51] in the SOD assay in very similar experimental conditions to those of the present study, but this complex is very poorly soluble in water. 4a and 4b are among the most efficient salen-based SOD mimetics. On the other hand the improvement of the SOD activity as a consequence of the conjugation of the cyclodextrin has been already observed in copper(II) complexes of functionalized cyclodextrins [31,32,51]. In the present case, the improvement of SOD activity is better than that observed in other studies. On the basis of the data in the literature [31,32,52] and of the present results we can hypothesize that the cyclodextrin cavity could, in general, improve the catalytic activity for the presence, in proximity to the catalytic moiety, of an hydrophobic environment able to encapsulate radical species [53,54] and favour their reaction with the catalytic centre. In any case, the additional role in the catalytic mechanism of a side chain containing the amido group and of the OHs of CD could not be excluded.

-5

1.2x10

-5

1.0x10

-6

8.0x10

-6

6.0x10

-6

4.0x10

-6

2.0x10

0.0 0.00

0.01

0.02

0.03

0.04

0.05

[H2O2] (M) Fig. 4. Initial rate of H2O2 decomposition vs. [H2O2] at constant concentration (3.0  105 M) of 4a (j) and 4b (d).

Table 2 Kinetic parameters for the catalase activity of 4a and 4b at pH 7.4.

Table 1 SOD-like activity of 4a and 4b at pH 7.4. Complex

I50 (lM)

kcat (M1 s1)

4a 4b EUK 108 EUK 113

0.22 0.18 2.05 1.85

6.7  107 8.2  107 7.2  106 7.9  106

(±0.03) (±0.02) (±0.03) (±0.03)

The catalase activity was tested using a Clark-type oxygen electrode, in similar conditions to those in the literature [20]. 4a and 4b reacted with the H2O2 forming oxygen. The initial rates of oxygen production for the disproportion reactions were measured under pseudo first order conditions in excess of H2O2. Unlike the Eukarion complexes, the cyclodextrin derivatives exhibit substrate saturation kinetics in the presence of an excess of hydrogen peroxide. Fig. 4 shows the plots of replicate data sets of the initial rate of H2O2 decomposition versus the substrate concentration at constant concentration of 4a and 4b. The data were fitted to the Michaelis–Menten equation using the Origin 7.0 program and KM and kcat were determined (Table 2). These manganese complexes are among the few examples [55] of mononuclear complexes of manganese(III) with salen type ligands that show Michaelis–Menten behaviour. Substrate saturation behaviour implies a rapid equilibrium between unbound substrate and a catalyst–substrate complex. In these glyconjugates, the cyclodextrin cavity could bond with the hydrogen peroxide forming a complex substrate–catalyst by hydrogen bonding or simple inclusion in the cavity [54]. In the literature, manganese salen type complexes with a functionalizing ureido group have been investigated recently [56]. These complexes exhibit saturation kinetics in catalyzing the H2O2 decomposition and the KM values are higher than those of our complexes (about three times higher). These results could confirm the hypothesis that the oligosaccharide residue could act as binding site. At non-saturating levels of hydrogen peroxide, the reaction order was determined by initial rate method. At low substrate concentration, the reaction is first order in peroxide concentration. The molecularity of the reaction with respect to the metal complex concentration was measured at a constant hydrogen peroxide concentration (5 mM). The linear dependence of the rate confirms the

-1

6

v (M s )

7

(±5  106) (±9  106) (±9  105) (±9  105)

Complex

k (M1 s1)

Initial rate (lM O2/mina)

kcat (s1)

KM (mM)

4a 4b EUK108 EUK 113

33 49 20 27

198 288 120 156

0.21 (±0.03) 0.42 (±0.02) – –

5.1 (±0.2) 6.5 (±0.1) – –

a

(±2) (±3) (±1) (±2)

(±10) (±12) (±7) (±10)

Complex concentration is 10 lM.

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V. Lanza, G. Vecchio / Journal of Inorganic Biochemistry 103 (2009) 381–388 -5

1.5x10

-6

6.0x10

-6

5.0x10 -5

-6

4.0x10

.+

-1

v (M s )

[ABTS ] (M)

1.0x10

-6

5.0x10

-6

3.0x10

-6

2.0x10

-6

1.0x10

0.0 0.0

-5

1.0x10

-5

2.0x10

-5

3.0x10

-5

4.0x10

-5

5.0x10

-5

6.0x10

0.0 0

[4a] or [4b] (M)

10

15

20

25

30

time (s)

Fig. 5. Initial rate of H2O2 decomposition vs. catalyst, 4a (j) and 4b (d), at low concentration of substrate (5 mM).

first order rate constant for the catalyst (see Fig. 5). The rate equation at low hydrogen peroxide concentration is v = k[cat][H2O2]. These data have been determined also for EUK 108 and EUK 113 in our experimental conditions for comparison and are reported in Table 3. In order to compare these systems with other salen complexes in the literature [20,56], we also report the initial rate measured at a concentration of H2O2 of 10 mM and of catalyst of 10 lM. Both the cyclodextrin derivatives show a twofold increase in the kinetic constants, in comparison to the Eukarion compounds. The CD functionalized with EUK 113 moiety is a more efficient system than that with EUK 108. In the literature a number of salen complexes have been tested as catalase mimetics [20]. In contrast to the SOD activities, the catalase activities are profoundly influenced by the ligand structure and a wide range of values are reported. The initial rate of oxygen production, at a concentration of catalysts of 10 lM, ranges from 36 to 1073 lM/min [20,55]. The values for 4a and 4b are within this data range. The functionalization with the oligosaccharide moiety improves the catalase activity, even if not very markedly, in comparison to the corresponding salen complexes, in agreement with the presence of the same structure of the salen moiety. 3.5. Peroxidase activity Peroxidase activities of 4a, 4b and Eukarion compounds chosen as references, were investigated. The following mechanism is reported in the literature [20] for the peroxidase activity in the case of salen complexes, where A is a typical substrate

MnðIIIÞ þ H2 O2 ¢ MnðVÞ ¼ O þ H2 O MnðVÞ ¼ O þ AH2 ¢ MnðIVÞ  OH þ AHþ MnðIVÞ  OH þ AH2 ¢ MnðIIIÞ þ H2 O þ AHþ In order to quantify the peroxidase activity of salen cyclodextrin conjugates, ABTS was used as the substrate [56–58]. The reaction of ABTS with H2O2 in the presence of 4a or 4b generates ABTS+:

Table 3 Peroxidase activity of 4a, 4b, EUK 108 and EUK 113. Complex 4a 4b EUK108 EUK 113

5

(lM 23.3 30.8 18.3 26.2

ABTS/min) (±0.8) (±0.9) (±0.7) (±0.8)

Fig. 6. Plot of ABTS+ formation in the presence of 4a (j) or 4b (d) vs. time [4a] = [4b] = 1.0  105 M.

2ABTS þ H2 O2 þ 2Hþ ¢ 2ABTSþ þ 2H2 O The reduced form of ABTS is colourless, while the oxidized ABTS, the ABTS+ radical cation, is green and its Vis spectra show several characteristic absorption bands at 415, 650, 735 and 815 nm. In the absence of the complex, a solution of ABTS and H2O2 is stable for several hours at 25 °C, in the dark, without showing any formation of ABTS+. In order to compare our results with the data in the literature, the amount of ABTS+ formed per minute was determined at the concentration of the complex of 1.0  105 M. In Fig. 6, the ABTS+ formation in the presence of 4a or 4b is reported vs. time. The peroxidase activities of the cyclodextrin derivatives are slightly higher than those of the Eukarion compounds, as is the case of the catalase activities. The same trend observed for the Eukarion complexes is observed for the cyclodextrin conjugates and 4b shows a higher activity than 4a. 4. Conclusion On the basis of the increasing interest in Eukarion complexes as potential drugs against diseases related to oxidative stress processes, EUK 108 and EUK 113 were conjugated with the b-cyclodextrin. As expected, the conjugation with the cyclodextrin gives the new manganese(III) complexes, 4a and 4b, a marked enhancement of water solubilities compared to EUK 108 and EUK 113. 4a and 4b are very promising systems in the field of SOD mimetics based on salens. Their SOD-like activities are about 10 times higher, in the Fridovich assay, than the activities of EUK 108, EUK 113, and thus they are among the most highly active SOD mimetics reported in the literature, based on salens. Compared to our previous salen complex conjugate with cyclodextrin [32], these new conjugates show a catalase activity about twice that of simple complexes. Furthermore 4a and 4b show a saturation kinetic behaviour, unlike the Eukarion compounds. The peroxidase activities of these conjugates are also slightly higher than the corresponding Eukarion compounds. In addition, the presence of the cyclodextrin cavity, which constitutes a typical scavenger of the OH radicals as reported in the literature [31,36,53], could cooperate with the other antioxidant activities shown by these complexes, as reported for similar cyclodextrin derivatives [59], rendering these bioconjugates potentially able to react with a cocktail of ROS species. Furthermore, because it is known that cyclodextrins are site specific delivery agents, the bioconjugation of a salen type manga-

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nese(III) complex with a cyclodextrin could be an interesting approach in order to carry antioxidant species into various organs, for example the colon, because inflammatory bowel diseases are known to be mediated by ROS species. 5. Abbreviations SOD superoxide dismutase CD cyclodextrin ABTS 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) [15]aneN5 1,4,7,10,13-pentaazacyclopentadecane ROS reactive oxygen species ac acetate Cyt c cytochrome c NBT nitro blue tetrazolium H2salen N,N0 bis(salicylidene)ethylendiamine EUK eukarion boc t-butyloxycarbonyl DMF dimethylformamide COSY correlated spectroscopy TOCSY total correlation spectroscopy HSQC heteronuclear single quantum coherence T-ROESY transverse rotating-frame overhauser enhancement spectroscopy ESI-MS electrospray ionization mass spectrometry

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