Synthesis of Alkylseleno-Carbohydrates and Evaluation of their Antioxidant Properties

The chemistry and biology of simple small molecules containing selenium in their structures has received increased attention over the past decades. The first of such compounds to be developed was ebselen and after the discovery of its properties, a number of different small molecules containing selenium have been synthesized. The great deal of attention that has been paid to these compounds stems from the discovery of a selenium atom at the catalytic site of glutathione peroxidase (GPx). GPx is a key enzyme present in mammals with antioxidant activity and its mechanism of action involves the reduction of H2O2 at the expense of the oxidation of glutathione. A number of studies have been reported in the literature describing that small organic selenium compounds can effectively scavenge and eliminate reactive oxygen species (ROS) thus mimicking the activity of GPx enzyme. ROS are commonly generated in normal cellular oxygen metabolism playing important biological roles. However, an increase in the ROS production over cellular endogenous antioxidant system capacity of response could result in a condition characterized as oxidative stress (OS), which causes damage to cells leading to age related degenerative diseases, cancer and a wide range of different human diseases. A simple and effective way of preventing these conditions is by inhibition of the oxidative damage and, therefore, there is a great interest in the search for new organoselenides which could represent good pharmacological alternatives to counteract oxidative stress. In this context, our groups have been interested in the synthesis and biological properties of selenium-containing carbohydrates and as a result, we have developed a straightforward approach for the introduction of an organoselenium moiety at nonanomeric positions of sugars. Most of our efforts have been focused on the synthesis of arylselenium derivatives of carbohydrates and of diselenides, which were used as building blocks for the synthesis of more complex nonglycosidically linked selenium-linked pseudodisaccharides and neoglycopeptides. In addition, previous studies of our group demonstrated that arylseleno-furanosides and a carbohydrate-derived diselenide did not inhibited δ-aminolevulinate dehydratase (δ-ALA-D), demonstrating low toxicity and antioxidant effects. More recently, we demonstrated that arylseleno-furanoside therapy was effective in restoring δ-ALA-D activity from ovary that was inhibited by cadmium and arylselenoand aryltelluroxylofuranosides attenuate manganese-induced toxicity in Caenorhabditis elegans. In view of the above, herein we report additional studies on this area, more specifically on the synthesis of selenocarbohydrates possessing an alkylselenium moiety and the evaluation of their in vitro antioxidant profile.


Introduction
The chemistry and biology of simple small molecules containing selenium in their structures has received increased attention over the past decades.][5][6][7][8] The great deal of attention that has been paid to these compounds stems from the discovery of a selenium atom at the catalytic site of glutathione peroxidase (GPx). 9,10GPx is a key enzyme present in mammals with antioxidant activity and its mechanism of action involves the reduction of H 2 O 2 at the expense of the oxidation of glutathione. 11,124][15][16] ROS are commonly generated in normal cellular oxygen metabolism playing important biological roles. 17However, an increase in the ROS production over cellular endogenous antioxidant system capacity of response could result in a condition characterized as oxidative stress (OS), which causes damage to cells leading to age related degenerative diseases, cancer and a wide range of different human diseases. 18,19A simple and effective way of preventing these conditions is by inhibition of the oxidative damage and, therefore, there is a great interest in the search for new organoselenides which could represent good pharmacological alternatives to counteract oxidative stress.1][22] Most of our efforts have been focused on the synthesis of arylselenium derivatives of carbohydrates and of diselenides, which were used as building blocks for the synthesis of more complex nonglycosidically linked selenium-linked pseudodisaccharides and neoglycopeptides. 23In addition, previous studies of our group demonstrated that arylseleno-furanosides and a carbohydrate-derived diselenide did not inhibited δ-aminolevulinate dehydratase (δ-ALA-D), demonstrating low toxicity and antioxidant effects. 21More recently, we demonstrated that arylseleno-furanoside therapy was effective in restoring δ-ALA-D activity from ovary that was inhibited by cadmium 24 and arylseleno-and aryltelluroxylofuranosides attenuate manganese-induced toxicity in Caenorhabditis elegans. 25n view of the above, herein we report additional studies on this area, more specifically on the synthesis of selenocarbohydrates possessing an alkylselenium moiety and the evaluation of their in vitro antioxidant profile.

Chemistry
The synthesis of the selenocarbohydrates was straightforward, and we chose as sugar substrates two Vol.26, No. 4, 2015   furanoside derivatives 2 and 3, readily available from the parent carbohydrates D-xylose and D-ribose, respectively, and a pyranoside substrate, derived from D-galactose (Figure 1).
The tosylates 2-4 reacted with a selenium nucleophile, generated by reductive cleavage of the corresponding dialkyldiselenide with NaBH 4 , to yield seleno-carbohydrates 5-7 (Table 1).The reaction was performed using a 3:1 mixture of tetrahydrofurane (THF) and ethanol as solvents.The tosylate displacement reaction with furanoside derivatives 2 and 3 was easier than with the pyranoside derivative 4, which took longer reaction times to afford reasonable yields of the corresponding products.The reason for this behavior is the opposing influence of the fixed dipole caused by the presence of the axial C-O bond at the C-4, combined with the steric hindrance imposed by the acetonide protecting group that hinders the backside approach of the selenium nucleophile at C-6. 26,27 Deprotection of the acetonide, followed by acetal formation at the anomeric position with MeOH, under acidic conditions delivered the desired seleno-furanosides 8a-b, 9a-b, and 10a-b (Scheme 1).

Antioxidant activity
9][30] With this in mind, we screened selected selenocarbohydrates for their in vitro antioxidant activity.We first screened the new alkylseleno carbohydrate in the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferric ion reducing antioxidant power (FRAP) assays.As can be seen in Table 2, xylo-pyranoside 8a presented ABTS and DPPH radical scavenging at concentration of 50 and 500 µM, respectively.Compound 8a possessed potent inhibitory activity against ABTS radical with IC 50 value of 210 µM.In addition, compound 8a exhibited ferricreducing ability, and the reducing power was improved by increasing its concentration.Comparing the values of the DPPH, ABTS and FRAP assays, we can suggest that the mechanism of antioxidant action of compound 8a is most likely based on single electron transfer.
On the other hand, compound 8b, possessing the longer C-10 alkyl chain and displaying a ribo-furanoside backbone, exhibited ferric-reducing ability (Table 3 The galactose-derived compounds 10a-b were also evaluated.Galacto-pyranoside 10a presented ABTS scavenging at concentration of 500 µM (Table 4) but didn't show significant result in both assays DPPH and FRAP (data not shown).On the other hand, results presented in Table 5 indicated that galacto-pyranoside derivative 10b presented ABTS scavenging at concentrations 50-500 µM and exhibited ferric-reducing ability (at 500 µM).However, it didn't exhibit any protection against DPPH radical at any concentration tested (data not shown).
Compounds 5b and 9b were also evaluated against ABTS, DPPH and FRAP assays but didn't show any significant antioxidant effects.In addition to the ABTS, DPPH and FRAP studies, compounds 5b, 8a, 8b, 9b, 10a and 10b had their superoxide dismutase (SOD)-like, nitric oxide 31 and hydroxyl radical 32 scavenging activities evaluated (data not shown) and none of them displayed significant activity, therefore ruling out these mechanisms as being responsible for their antioxidant profile.
Inspection of all the results gathered revealed that seleno-xylofuranoside 8a, containing an alkyl group with eight carbon atoms, showed the highest antioxidant activity when compared with the closely related compound 8b (with an alkyl group with ten carbon atoms) and selenogalactopyranoside 10b, which possesses a C-8 alkyl chain.Direct comparison between 8a and the acetonide-protected compounds 5a-b revealed that the presence of the acetonide moiety has a deleterious effect for the antioxidant activity, since 5 did not exhibit antioxidant activity, suggesting that the increased water-solubility of 8a when compared to 5a might play a role on its antioxidant profile.

Conclusions
Herein we have reported a straightforward synthesis of simple alkylseleno carbohydrates with different sugar scaffolds.Screening of selected compounds for antioxidant activity revealed that the presence of an alkyl group with eight carbon atoms has an important influence in the free radical scavenging activity of these compounds and therefore they can potentially prevent damage of protein and lipids.

Experimental
General procedure for the synthesis of 5-7 Under an argon atmosphere, sodium borohydride (2.5 equiv) was added to a solution of the dialkyldiselenide (1.0 mmol) in THF (7.5 mL).Ethanol (2.5 mL) was then added dropwise and the clear solution formed was stirred at room temperature for 10 min.After this time, a solution of the appropriate tosylate (1.5 mmol in 1 mL THF) was added dropwise.After stirring under reflux for the time indicated in Table 1, the reaction mixture was quenched with aqueous saturated NH 4 Cl (10 mL) and extracted with CH 2 Cl 2 (3 × 25 mL).The combined organic layers were dried with MgSO 4 , filtered and concentrated.The crude product was purified by flash chromatography, first eluting with hexanes and then with a mixture of hexanes/ethyl acetate.
Analytical data for compound 5a: yield 65%; white solid; purified using hexane:EtOAc 80:    In a round bottomed flask, the appropriate selenocarbohydrate (0.5 mmol) was stirred in an aqueous solution of trifluoracetic acid (50% v/v, 10 mL) for 1 h at room temperature.After this time, the reaction mixture was concentrated in vacuum, co-evaporated with toluene (3 × 10 mL) and the residue dissolved in MeOH (10 mL), in the presence of a catalytic amount of sulfuric acid, and stirred for additional 24 h, at room temperature.Following this time, the mixture was neutralized by the addition of solid sodium bicarbonate.The mixture was filtered and the solvents evaporated to afford the product.

Table 1 .
Synthesis of alkyl-Se-carbohydrates a Isolated yields.

Table 5 .
Antioxidant activity of compound 10b on ABTS and FRAP assays a

Table 4 .
Antioxidant activity of compound 10a on ABTS assay a a Denote p < 0.05; b p < 0.01 as compared to the respective control sample (one way ANOVA/Newman-Keuls).