Synthesis and spectroscopic characterization of double chained and sulfurated derivatives of L-ascorbic acid

Lipophilic saturated and unsaturated L-ascorbyl 5,6-O-diesters from fatty acids are prepared and fully characterized through NMR and MS spectra. Derivatives with different sulfurated moieties are obtained as well through thio-Michael addition of thiols on ascorbyl acrylates. The new amphiphilic structures exhibit very high antioxidant activity using the DPPH assay.


Introduction
Antioxidants are widely used in different fields, for example the food, cosmetic and pharmaceutical industries.Our interest in the chemistry of sulfurated and selenated compounds led us to develop an access to organochalcogen derivatives, such as selenides, diselenides and heterocyclic compounds, [1][2][3][4][5][6] which evidenced interesting antioxidant activity.The chemistry of organosulfur, and more recently of organoselenium compounds has in fact experienced a strong development. 7,8Such derivatives are used as reagents in organic synthesis and, due to their interest from the biological point of view, in the preparation of molecules with biological activity.Recently we were involved in the study of modifications of natural products, with the aim to introduce on the same molecular skeleton sulfurated or selelenated moieties, which could possess a synergistic effect together with the natural core.We found a convenient method for the synthesis of selenium-derivatives of resveratrol as antioxidants and free radical scavengers. 9These structures evidenced antioxidant activity with Trolox-like capacity.
In this context considerable interest has also been devoted to the study of liposoluble ester derivatives of L-ascorbic acid (L-ASC), which is a well known, potent and versatile antioxidant.Their physico-chemical properties have been extensively investigated [10][11][12][13][14][15][16][17] in particular their phase behavior in the solid state and in aqueous dispersions.The amphiphilic derivatives of ascorbic acid fully retain the antioxidant power of the parent molecule, and produce nanostructured self-assembled systems both in aqueous and non aqueous media, and therefore they can act as carriers for important hydrophobic molecules such as drugs and nutraceutics.
9][20][21][22] Generally 6-O-ascorbyl esters are obtained via different methods, by reacting vitamin C and fatty acids under catalysis of lipases 20 or in concentrated sulfuric acid. 23ere we report the preparation and full characterization of lipophilic saturated and unsaturated 5,6-Odiesters of L-ASC, as well as of sulphur-containing 6-O-ascorbyl alkanoates.Preliminary evaluation of their antioxidant properties will also be described.

Results and Discussion
To the best of our knowledge, few examples of synthesis of 5,6-diesters of L-ASC have been reported.Nevertheless, a very large number of examples of esterification at C-6 with palmitoyl chloride were described in a patent, bearing different substituents and protecting groups on selected hydroxyls. 24Chemical modifications of vitamin C are often limited by its intrinsic instability.Typically, together with the methods cited above, the chemical synthesis of ascorbyl esters is carried out by selective protection and deprotection of functional groups.Several steps are required to obtain the target compounds.Usually the first step involves the protection of hydroxyl groups on C-6 and C-5 as an acetonide, followed by treatment of the enol hydroxyls with benzyl bromide. 21,25Nevertheless, on the basis of results recently reported 26 and of our own results in separate experiments, we observed a high, selective reactivity of the ene-diol portion with benzyl bromide by comparison with the alcohols at the 5 and 6 positions.This allowed us to treat L-ASC 1 with BnBr and K 2 CO 3 in THF/DMSO (Scheme 1) directly, thus avoiding preliminary protection as 5,6-O-isopropylidene derivative and the following deprotection step.The dibenzyl ether 2a was then treated with saturated fatty acid derivatives.Thus acyl chlorides of octanoic (caprylic, C 8 ), decanoic (capric, C 10 ) and dodecanoic (lauric, C 12 ) acid were reacted in the presence of DMAP/DCC in acetonitrile (Scheme 1).L-Ascorbyl 5,6-O-dialkanoates 3a-c were obtained in high yields (Table 1, entries 1-3).Cleavage with H 2 , Pd/C led to the formation in quantitave yield of the previously unreported 2,3-(OH) 2 -5,6-diesters 4a-c, which were fully characterized by means of NMR and MS spectra.In order to synthesize derivatives with different acyl chain lengths, for evaluating the influence on physicochemical properties, compound 3d (Table 1, entry 4) was prepared in two steps from lauroyl-and acetylchlorides (see experimental).The final deprotection afforded the mixed diester 4d (Scheme 1) .All the diesters were obtained with high selectivity (>99%), no trace of monoesters being observed.
Our interest also focused in the preparation of ascorbyl oleates.These compounds bear, on the same molecular skeleton, both oleic and ascorbic acid residues, that each possess antioxidant properties.However, the synthesis was not straightforward, the products being rather sensitive compounds.This makes ascorbyl oleate difficult to produce with traditional chemical methods.][30][31][32][33][34] The search for alternative methods is therefore a challenge to access these molecules.
Following a similar approach, oleoyl chloride was reacted with 2a leading to the formation of 2,3-Odibenzyl-5,6-O-ascorbyl dioleate 3e in good yield (Scheme 1 and Table 1, entry 5).For the cleavage of benzylic ethers, the incompatibility of H 2 , Pd/C system with the double bonds prompted us to seek alternative, mild methods.Unfortunately, treatment with CSI (chlorosulfonyl isocyanate) 35 did not afford the desired compound, but a complex mixture of products was formed.Thus, a different protecting group -pmethoxybenzyl (PMB) -was chosen.PMB can be more easily cleaved with numerous reagents, avoiding strong conditions. 36,37PMB-protected ascorbic acid 2b was prepared under the same conditions by direct treatment of L-ASC with PMBBr/K 2 CO 3 (Scheme 1).Subsequent reaction with oleyl chloride afforded 2,3-O-pmethoxybenzyl-5,6-O-ascorbyl dioleate 3f (Scheme 1 and Table 1, entry 6) or 2,3-O-p-methoxybenzyl-6-Oascorbyl oleate 5a (Table 1, entry 8), depending on the reaction conditions (treatment with DMAP/DCC for diester, with Et 3 N for monoester).Efficient cleavage of 3f with SnCl 4 /thiophenol 37 at -78 °C led to the isolation of the 2,3-deprotected ascorbyl dioleate 4e, while reaction on 5a allowed cleavage of only one PMBO-group (most likely on C-2).Further experiments are under investigation to obtain complete deprotection at both positions.Nonetheless, the result obtained is rather interesting, providing new ascorbyl oleates through a convenient, alternative access.
As a further step, we decided to evaluate the esterification on the secondary OH at position 5, retaining a free hydroxyl at C-6.To achieve this, a preliminary protection of 6-OH with trityl chloride was carried out on 2a and 2b following literature procedures (Scheme 2). 25  After purification, the so obtained products 6a,b were reacted respectively with lauroyl chloride and oleoyl chloride in the presence of DMAP/DCC, leading to 7a and 7b in good yields (Scheme 2).Deprotection of 6-OTrt under acidic conditions 38 was performed by treatment of 7a with BF 3 etherate, leading to 8 (Scheme 2, via A).When reductive cleavage was carried out to obtain complete deprotection on C-2/C-3, a very complex mixture was observed, and the desired product 10 was present in only very low yield (<10%).
In order to seek better conditions, we firstly considered deprotection of benzyl groups in 7a with H 2 /Pd/C (Scheme 2, via B).Compound 9 was isolated, and then treated under acidic conditions (BF 3 ) to remove the trityl group.Unfortunately, through this sequence, compound 10 was also formed in comparable yields (ca.10%), and optimization of this step is surely required.Nevertheless, compound 9 is interesting, in so far as the enediol moiety, responsible of the antioxidant activity of vitamin C, is still present in this molecule.
Finally, to introduce a sulfurated or selenated group on the skeleton of vitamin C, we prepared ascorbyl 5,6-O-diacrylate 3g and 6-O-acrylates 5b, 24 5c (Scheme 1 and Table 1, entries 3, 9 and 10).Then 5b, 5c and 3g were reacted respectively with one or two equivalents of a sulfur nucleophile (PhSH) in the presence of Al 2 O 3 39 (Scheme 3).Under these conditions compounds 11a, 11b and 12 were isolated, arising from a thio-Michael addition to the enones.
Even more interesting was the reaction of 5b with 1,2-ethanedithiol, providing the synthesis of the diascorbyl derivative 13, containing a sulfurated linker between the two vitamin C moieties (Scheme 3).It will be of interest to investigate the characteristics of such derivatives, including chains of different length, to compare their properties with the behaviour of bolaform surfactants studied by some of us. 12Search for conditions to deprotect 2,3-positions is currently under study in our laboratory.
Finally, in order to preliminary evaluate whether the antioxidant capacity was maintained for these new amphiphilic structures, they were tested through the DPPH assay, 40 which provides an easy and rapid way to assess antioxidant potency.Substances 4a-e and 9 exhibited a very high ability to act as free radical scavengers, in fact they instantaneously reacted with DPPH.

Conclusions
Different saturated and unsaturated ascorbyl derivatives, including novel sulfurated compounds, were prepared from fatty acids, and characterized by mono-and bidimensional NMR experiments.Their antioxidant activity was determined with the DPPH assay.

Experimental Section
General.NMR spectra were recorded in CDCl 3 with Varian Gemini 200, Mercury 400, Inova 400 and Bruker 400 spectrometers operating at 200 and 400 MHz ( 1 H), 50 and 100 MHz ( 13 C).NMR signals were referenced to non deuterated residual solvent signals of deuterochloroform (7.26 ppm for 1 H, 77.0 ppm for 13 C).Mass spectra (MS) were obtained by ESI.IR spectra were recorded on a Perkin Elmer Instrument (FT-IR).Only selected absorptions are reported, in wavenumbers (cm -1 ).Solvents were dried using a solvent purification system (Pure-Solv TM ).Flash column chromatography was performed using silica gel (230-400 mesh).Where not specified, products were commercially available or obtained through reported procedures.4-Methoxybenzyl bromide (PMBBr) was synthesised from (4-methoxyphenyl)methanol (PMBOH) upon treatment with phosphorus tribromide. 41DPPH assay was performed according to the literature, 40 by measuring the adsorbance decreasing of a DPPH methanolic solution (10 -4 M) treated with an equimolar amount of ascorbic acid derivatives (4 a-e, 9).

Table 1 .
Synthesis of saturated and unsaturated L-ASC esters a Isolated yields.b Global yield over two steps.c 10% of diester was also formed.