Biomed Res Trace Elements 17(4):423-426, 2006 423 Antioxidant Activities of Synthesized Selenocompounds without Selenol Groups Yoshihiro Saito, Daisuke Umemoto, Ai Matsunaga, Takaji Sato and Masahiko Chikuma Department of Bio-analytical Chemistry, Osaka University of Pharmaceutical Sciences, Osaka 569-1094, Japan Abstract Low-molecular-weight selenocompounds without selenol groups, N-methyl,N-phenyl-(2-selenomethyl)benzoylamide, N-phenyl-(2-selenoallyl)benzoylamide, N-phenyl-(2-selenopropyl)benzoylamide, and bis[1-methyl-2-(N-phenylcarboxa- mido)ethyl] diselenide, dibenzyl diselenide, were synthesized and their antioxidant activities were evaluated. Among the synthesized selenocompounds, N-phenyl-(2-selenoallyl)benzoylamide had the highest glutathione peroxidase(GPx)-like activity, and the activity increased depending on the concentration. The activity of N-phenyl-(2-selenoallyl)benzoylamide was several times higher than that of selenomethionine, though it was about 1/10 of ebselen. These results suggest that se- lenocompounds which don’t have selenol groups can also behave as a GPx-like reductant, where a different reaction mechanism from that for selenocompounds with selenol groups is present. Spectrophotometric studies also supported this suggestion. It may be possible that selenium atom located between two carbon atoms in the molecule is oxidized to se- lenoxide by an oxidant and then selenoxide is reduced to selenium by reduced form of gluta-thione, thus GPx-like reduc- tion of hydrogen peroxide by selenocompounds without selenol groups occurs. N-phenyl-(2-selenoallyl)benzoylamide had superoxide anion scavenging activity, although it was comparatively low. These results indicated that selenocom- pounds without selenol groups such as N-phenyl-(2-selenoallyl)benzoylamide can be reductants against peroxide or other oxidant species possibly by forming selenoxide, though their antioxidant activity may not be high. Keywords :selenium, selenocompounds, antioxidant activity, glutathione peroxidase-like activity, superoxide anion scavenging activity Introduction tants even in the absence of glutathione [1]. A synthetic Selenol group in glutathione peroxidase (GPx) plays a selenocompound ebselen has high GPx-like activity [2]. central role in its catalytic reaction in the presence of re- Ebselen has a covalent Se-N bond in the molecule, and a duced form of glutathione. Selenocompounds with se- selenol group is readily produced in the aqueous reaction lenol groups may also act as GPx, and they can be reduc- mixture. Therefore, it seems that selenol group is an es- ――――――――――――――――――――――― sential chemical form for selenocompounds to become Address correspondence to : reducing agents. Yoshihiro Saito Recently, Silva et al. measured the redox potentials of Department of Bio-analytical Chemistry, phenylaminoethyl selenoxides, which are selenocom- Osaka University of Pharmaceutical Sciences, pounds without selenol groups, and showed the possibil- 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, ity of non-selenol selenocompounds to become GPx-like Japan reductants in the presence of glutathione [3]. On the Tel & Fax : +81-72-690-1061 other hand, oxidation of selenomethionine and reduction E-mail : [email protected] of selenomethionine selenoxide has been studied for ――――――――――――――――――――――― years, though selenomethionine does not have a selenol Received : 27 September 2006 group [4-6]. Thus it is possible that selenocompounds Accepted : 20 October 2006 without selenol groups may be antioxidants as well as 424 Biomed Res Trace Elements 17(4):423-426, 2006 those with selenol groups. of NADPH, then for 200 seconds after the addition of In this study, we have synthesized several selenocom- hydrogen peroxide (substrate) to evaluate enzyme-like pounds without selenol groups, and investigated their activity. GPx-like activity and superoxide anion scavenging activ- ity. We have also studied the oxidation and subsequent 3. Superoxide anion scavenging activity reduction of selenomethionine by spectrophotometry and Superoxide anion scavenging activity of the com- HPLC to clarify the reaction. pounds was measured by chemiluminescent method. Su- peroxide anion was produced by hypoxanthine-xanthine Materials and Methods oxidase system, and the chemiluminescence derived from 1. Materials superoxide anion was measured with 2-methyl-6-p- N-methyl, N-phenyl-(2-selenomethyl)benzoylamide, methoxyphenylethinylimidazopyrazinone (MPEC), a N-phenyl-(2-selenoallyl)benzoylamide, N-phenyl-(2-se- chemiluminescence probe. The reaction mixture con- lenopropyl)benzoylamide, bis[1-methyl-2-(N-phenyl- sisted of the following solutions : 1) 10μLofMPECso- carboxamido)ethyl] diselenide, and dibenzyl diselenide lution (300mM), 2) 10μLofsamplesolution, 3) 60μLof were synthesized in our laboratory. Ebselen was a gift xanthine oxidase solution (~0.1U/mL), 4) 170μLof from Daiichi Seiyaku Co., Ltd.. Glutathione (reduced phosphate buffer solution-0.05mM EDTA (0.1M, pH7.5), form), glutathione reductase, and NADPH were pur- and 5) 50μLofhypoxanthine solution (0.72mM). Hy- chased from Nacalai Tesque Inc.. A chemiluminescence poxanthine solution was added by an internal pump and probe, 2-methyl-6-p-methoxyphenylethinylimidazopyra- the chemiluminescence counts were accumulated for 60 zinone (MPEC) was purchased from Atto Co.. Other seconds after the addition of hypoxanthine solution. Su- chemicals were reagent grade. Chemiluminescence was peroxide anion scavenging activity of the compounds measured by an AB-2200 Luminescencer-PSN (Atto was represented by EC50,theconcentration reducing the Co.). chemiluminescence to 50% of the control. Se Results and Discussion R 1 Synthesized selenocompounds showed low GPx-like O activity though ebselen and diphenyl diselenide, which R Se 3 can be converted to selenol compounds in the reaction N Se R3 R2 mixture, had higher activity. The activity at 20μMofN- phenyl-(2-selenoallyl)benzoylamide, which was about Fig. 1 Structures of synthesized selenocompounds. 1/10 of ebselen, was the highest among the synthesized compounds, and the activity increased depending on the R1 =CH3-, R2 =CH3-: N-methyl, N-phenyl- ( 2- selenomethyl) benzoylamide ; R1=CH2CHCH2-, R2=H- : concentration. The activity of N-phenyl-(2-selenoallyl)- N-phenyl-(2-selenoallyl)benzoylamide ; R1=CH3CH2CH2-, benzoylamide was several times higher than that of se- R2=H-: N-phenyl-(2-selenopropyl)benzoylamide ; R3= lenomethionine (Fig.2). C6H5CH2-: dibenzyl diselenide ; R3=C6H5NHCOCH2CH (CH3)- :bis[1-methyl-2-(N-phenylcarboxamido)ethyl] 15 diselenide. 2. Glutathione peroxidase(GPx)-like activity 10 Selenocompounds were dissolved in dimethyl sulfox- ide, and diluted with water or assay buffer to the concen- 5 ke activity (mU/ml) ke activity trations studied. Glutathione peroxidase(GPx)-like activ- ity of these compounds was measured according to the GPx-li 0 method of Müller et al. [2]. The assay mixture consisted 04080120 160 of 50mM potassium phosphate buffer (pH7.0), 1mM Concentration (µM) EDTA, 1mM glutathione (reduced form), 1mM sodium Fig. 2 Glutathione peroxidase-like activity of N-phenyl- azide, 0.25mM NADPH, 1U/ml glutathione reductase, (2-selenoallyl)benzoylamide(●), selenome- and samples. Absorbance at 340nm was recorded for 100 thionine(○), and methionine (△). Data are seconds to estimate the nonenzymatic consumption rate means of three determinations. Antioxidant Activities of Selenocompounds 425 Methionine did not show the activity even at 160µM, zoyamide, ebselen, ascorbic acid, chlorogenic acid, and indicating that selenium is important for the activity. Trolox were 4.6×10-2,0.14, 1.7×10-3,7.5×10-3,6.4×10-2, These results suggest that selenocompounds which don’t and 0.1(mM), respectively. The activity of N-phenyl-(2- have selenol groups can also behave as a GPx-like reduc- selenoallyl) benzoylamide was about 1/6 of ascorbic acid. tant and another reaction mechanism other than that for the compounds with selenol group is present. Spectro- 100 photometric studies also supported this suggestion (Fig.3). It is reported that selenomethionine selenoxide or 80 selenomethionine dihydroxy selenide is formed by the 60 reaction of selenomethionine with hydrogen peroxide 40 [4,5], and methionine selenoxide can be reduced by glu- 20 tathione [6]. In our HPLC study, it was shown that se- 0 lenomethionine peak disappears by the reaction with hy- 0.0001 0.001 0.01 0.1 1 drogen peroxide and appears again after the addition of Reductionof luminescence (%) Concentration (mM) glutathioine (data not shown). The changes in ultraviolet Fig. 4 Superxoide anion scavenging activity of N- absorption spectra observed in Fig.4 are supposed to be phenyl-(2-selenoallyl)benzoylamide and some corresponding to these processes, though the reaction of reference compounds. Compounds studied were N-phenyl-(2-selenoallyl)benzoylamide may not com- N-methyl-N-phenyl(2-selenomethyl) benzoyla- pletely be the same as that of selenomethionine. It is pos- mide(●), ebselen(▲), N-phenyl(2-selenoallyl) benzoylamide(■), Trolox(○), chlorogenic acid sible that selenium atom located between two carbon at- (◇), and ascorbic acid(□). oms in the molecule is oxidized to selenoxide and the se- Data are means±SD (n=3). lenoxide can be reduced to selenium by reduced form of glutathione, thus GPx-like reduction of hydrogen perox- These results indicated that selenocompounds without ide by non-selenol compounds occurs (Fig.5). selenol groups such as N-phenyl-(2-selenoallyl)benzoyl- amide can be reductants against peroxide or other