Synthesis of Novel Arsonolipids and Development of Novel Arsonoliposome Types.

Arsonolipids represent a class of arsenic-containing compounds with interesting biological properties either as monomers or as nanostructure forming components, such as arsonoliposomes that possess selective anticancer activity as proven by in vitro and in vivo studies. In this work, we describe, for the first time, the synthesis of novel arsono-containing lipids where the alkyl groups are connected through stable ether bonds. It is expected that this class of arsonolipids, compared with the corresponding ester linked, will have higher chemical stability. To accomplish this task, a new methodology of general application was developed, where a small arsono compound, 2-hydroxyethylarsonic acid, when protected with thiophenol, can be used in an efficient and simple way as a building block for the synthesis of arsono-containing lipids as well as other arsono-containing biomolecules. Thus, besides the above-mentioned arsonolipid, an arsono cholesterol derivative was also obtained. Both ether arsonolipid and arsono cholesterol were able to form liposomes having similar physicochemical properties and integrity to conventional arsonoliposomes. Furthermore, a preliminary in vitro anticancer potential assessment of the novel ether arsonolipid containing liposomes against human prostate cancer (PC-3) and Lewis lung carcinoma (LLC) cells showed significant activity (dose- and time-dependent), which was similar to that of the conventional arsonoliposomes (studied before). Given the fact that novel arsonolipids may be more stable compared to the ones used in conventional arsonoliposomes, the current results justify further exploitation of the novel compounds by in vitro and in vivo studies.

Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay.

A new fluorometric assay is presented for the ultrasensitive quantification of total protein carbonyls, and is based on their specific reaction with rhodamine B hydrazide (RBH), and the production of a protein carbonyl-RBH hydrazone the fluorescence of which (at ex/em 560/585 nm) is greatly enhanced by guanidine-HCl. Compared to the fluorescein-5-thiosemicarbazide (FTC)-based fluorometric assay, the RBH assay uses a 24-fold shorter reaction incubation time (1 h) and at least 1000-fold lower protein quantity (2.5 µg), and produces very reliable data that were verified by extensive standardization experiments. The protein carbonyl group detection sensitivity limit of the RBH assay, based on its standard curve, can be as low as 0.4 pmol, and even lower. Counting the very low protein limit of the RBH assay, its cumulative and functional sensitivity is 8500- and 800-fold higher than the corresponding ones for the FTC assay. Neither heme proteins hemoglobin and cytochrome c nor DNA interfere with the RBH assay.

Oxidation of structural cysteine residues in thioredoxin 1 by aromatic arsenicals enhances cancer cell cytotoxicity caused by the inhibition of thioredoxin reductase 1.

Thioredoxin systems, composed of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, play important roles in maintaining cellular redox homeostasis and redox signaling. Recently the cytosolic Trx1 system has been shown to be a cellular target of arsenic containing compounds. To elucidate the relationship of the structure of arsenic compounds with their ability of inhibiting TrxR1 and Trx1, and cytotoxicity, we have investigated the reaction of Trx1 system with seven arsenic trithiolates: As(Cys)3, As(GS)3, As(Penicillamine)3, As(Mercaptoethanesulfonate)3, As(Mercaptopurine)3, As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3. The cytotoxicity of these arsenicals was consistent with their ability to inhibit TrxR1 in vitro and in cells. Unlike other arsenicals, As(Mercaptopurine)3 which did not show inhibitory effects on TrxR1 had very weak cytotoxicity, indicating that TrxR1 is a reliable drug target for arsenicals. Moreover, the two aromatic compounds As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3 showed stronger cytotoxicity than the others. As(2-mercaptopyridine)3 which selectively oxidized two structural cysteines (Cys62 and Cys69) in Trx1 showed mild improvement in cytotoxicity. As(2-mercaptopyridine N-oxide)3 oxidized all the Cys residues in Trx1, exhibiting the strongest cytotoxicity. Oxidation of Trx1 by As(2-mercaptopyridine)3 and As(2-mercaptopyridine N-oxide)3 affected electron transfer from NADPH and TrxR1 to peroxiredoxin 1 (Prx1), which could result in the reactive oxygen species elevation and trigger cell death process. These results suggest that oxidation of structural cysteine residues in Trx1 by aromatic group in TrxR1-targeting drugs may sensitize tumor cells to cell death, providing a novel approach to regulate cellular redox signaling and also a basis for rational design of new anticancer agents.

Permeability of PEGylated immunoarsonoliposomes through in vitro blood brain barrier-medulloblastoma co-culture models for brain tumor therapy.

PURPOSE: Owing to restricted access of pharmacological agents into the brain due to blood brain barrier (BBB) there is a need: 1. to develop a more representative 3-D-co-culture model of tumor-BBB interaction to investigate drug and nanoparticle transport into the brain for diagnostic and therapeutic evaluation. 2. to address the lack of new alternative methods to animal testing according to replacement-reduction-refinement principles. In this work, in vitro BBB-medulloblastoma 3-D-co-culture models were established using immortalized human primary brain endothelial cells (hCMEC/D3). METHODS: hCMEC/D3 cells were cultured in presence and in absence of two human medulloblastoma cell lines on Transwell membranes. In vitro models were characterized for BBB formation, zonula occludens-1 expression and permeability to dextran. Transferrin receptors (Tfr) expressed on hCMEC/D3 were exploited to facilitate arsonoliposome (ARL) permeability through the BBB to the tumor by covalently attaching an antibody specific to human Tfr. The effect of anticancer ARLs on hCMEC/D3 was assessed. RESULTS: In vitro BBB and BBB-tumor co-culture models were established successfully. BBB permeability was affected by the presence of tumor aggregates as suggested by increased permeability of ARLs. There was a 6-fold and 8-fold increase in anti-Tfr-ARL uptake into VC312R and BBB-DAOY co-culture models, respectively, compared to plain ARLs. CONCLUSION: The three-dimensional models might be appropriate models to study the transport of various drugs and nanocarriers (liposomes and immunoarsonoliposomes) through the healthy and diseased BBB. The immunoarsonoliposomes can be potentially used as anticancer agents due to good tolerance of the in vitro BBB model to their toxic effect.

Arsonoliposomes: preparation and physicochemical characterization.

Arsonoliposomes (ARSL) which are liposomes that contain arsonolipids in their membranes have shown interesting anticancer and antiparasitic activity in vitro. Their lipid composition (the specific arsonolipids and/or phospholipids used for their preparation, and the relative amounts of each lipid type) highly influences their physicochemical properties as well as their in vivo kinetics and antiparasitic activity; however, their cytotoxicity towards cancer cells is minimally--if at all--modified. ARSL are prepared by a modification of the "one step" method followed or not by sonication (for formation of sonicated or non-sonicated ARSL, respectively). Arsonoliposomes may be composed only of arsonolipids (containing or not cholesterol) [plain ARSL], or they may contain mixtures of arsonolipids with phospholipids (with or without Chol) [mixed ARSL]. Herein, we describe in detail the preparation and physicochemical characterization of ARSL.

A high yield procedure for the preparation of arsonolipids (2,3-diacyloxypropylarsonic acids).

The crucial step in the preparation of the title arsonolipids starting from the dichloromethane-soluble dithioarsonite CH(2)(OH)CH(OH)CH(2)-As(SPh)(2) is to avoid an internal cyclization during the acylation which protects the primary -OH group from being acylated. This was to a large extent accomplished by using fatty acyl chloride in the presence of the weak base pyridine and controlling the temperature and rate of the acyl chloride addition, giving approximately 70% yields of arsonolipids. The presence of catalytic amounts of 4-dimethylaminopyridine boosted the yields to 82-85%. This yield is a great improvement over the yields (20-55%) previously achieved. The acylating systems (RCO)(2)O or RCOCl and BF(3).Et(2)O gave only moderate yields (25-60%) of arsonolipids.

Does the lipid membrane composition of arsonoliposomes affect their anticancer activity? A cell culture study.

Sonicated arsonoliposomes were prepared using arsonolipid with palmitic acid acyl chain (C16), mixed with phosphatidylcholine (PC)-based or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-based, and cholesterol (Chol) with C16/DSPC/Chol 8:12:10 molar ratio. PEG-lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) containing vesicles (PEGylated-arsonoliposomes; PC-based and DSPC-based) were also prepared. The cytotoxicity of these arsonoliposomes towards different cancer cells (human promyelocytic leukaemia NB4, Prostatic cancer PC3, human breast adenocarcinoma MDA-MB-468, human T-lymphocyte (MT-4) and also towards human umbilical vein endothelial cells (HUVECs) was evaluated by calculating the arsonoliposome-induced growth inhibition of the cells by the MTT assay. IC-50 values were interpolated from cell number/arsonoliposome concentration curves. The results reveal that all types of arsonoliposomes evaluated significantly inhibit the growth of most of the cancer cells studied (PC3, NB4, MT4) with the exception of the MDA-MB-468 breast cancer cells which were minimally affected by arsonoliposomes; in some cases even less than HUVEC. Nevertheless, for the same cell type the differences between the different types of arsonoliposomes were significant but not proportional to their stability, indicating that the formation of arsonoliposomes with very stable membranes is not a problem for their anticancer activity. Thereby it is concluded that arsonoliposome composition should be adjusted in accordance to their in vivo kinetics and the desired, for each specific application, biodistribution of As and/or encapsulated drug.

A reinvestigation of the synthesis of arsonolipids (2,3-diacyloxypropylarsonic acids).

A reinvestigation of the reactions leading to arsonolipids (2,3-diacyloxypropylarsonic acids) has been carried out in order to understand why the yields of their preparation were only moderate, although they are better than those reported for 2,3-diacyloxypropylphosphonic acid (phosphotidic acid). Thus, the reaction of glycidol and of 3-chloro-1,2-propanediol with alkaline sodium arsenite, "Na3AsO3", gives the desired product, 2,3-dihydroxypropylarsonic acid, and approximately 10% of an arsenic-containing glycerol dimer which is removed during the preparation of these arsonolipids. The step which is mainly responsible for the diminished yields is due to the reaction of the -As(SPh)2 or -AsO3H- precursor with the activated acid chlorides or carboxylic acid anhydrides to give an intermediate which cyclizes with the primary hydroxy group of the 2,3-dihydroxypropyl moiety. This cyclization does not allow the primary hydroxy group to be acylated. Such cyclization could not be avoided with RCOCl/py, (RCO)2O/DMAP, or RCOOH/DCC/DMAP acylating systems.

In vivo distribution of arsonoliposomes: effect of vesicle lipid composition.

Sonicated arsonoliposomes were prepared using arsonolipid with palmitic acid acyl chain (C16), mixed with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC-based), and cholesterol (Chol) with a molar ratio C16/DSPC/Chol 8:12:10. PEG-lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) containing vesicles (Pegylated-arsonoliposomes) were also prepared. DSPC-based and Pegylated-arsonoliposomes, were administered by intraperitoneal injection in balb/c mice (15 mg arsenic/kg) and the distribution of As in the organs was measured by atomic absorption spectroscopy. Results demonstrate that a high portion of the dose administered is rapidly excreted since 1 h post-injection only about 30-40% of the dose was detected cumulatively in animal tissues. After this, the whole body elimination of arsenic was a slow process with a half-life of 27.6 h for Pegylated-arsonoliposomes, and 83 h, for the DSPC-based ones. For both arsonoliposomes, arsenic distribution was greater in intestines, followed by liver, carcass+skin stomach, spleen, kidney, lung and heart. Different arsenic kinetics in blood between the two liposome types were observed. Compared to the results obtained previously with PC-based arsonoliposomes, both the DSPC-based and Pegylated-arsonoliposomes have better bioavailability. This proves that arsonoliposome lipid composition (and consequently their integrity) influences their pharmacokinetic profile. Thus, the proper arsonoliposome composition should be used according to the intended application.

Trypanocidal activity of arsonoliposomes: effect of vesicle lipid composition.

Sonicated arsonoliposomes were prepared using an arsonolipid with palmitic acid acyl chain (C16), mixed with phosphatidylcholine (PC-based) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC-based), and cholesterol (Chol) with a molar ratio C16 /PC or DSPC/ Chol 8:12:10. PEG-lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) containing vesicles (pegylated-arsonoliposomes) were also prepared. The in vitro and in vivo trypanocidal activity of the various types of arsonoliposomes was evaluated. Although PC-based arsonoliposomes exhibited in vivo activity on an acute trypanosomiasis animal model, no evidence of activity was demonstrated for DSPC-based or pegylated-arsonoliposomes on a chronic model. Despite the fact that DSPC-based and pegylated-arsonoliposomes have better bioavailability compared to PC-based ones, their in vitro activity is lower than that of PC-based arsonoliposomes, indicating the importance of arsonoliposome lipid composition on their trypanocidal activity and suggesting that further arsonoliposome structure design is required to overcome these disadvantages.

Preparation of DL-2,3,4-trihydroxybutylarsonic acid and dl-2,3-dihydroxybutane-1,4-bis(arsonic acid): starting compounds for novel arsonolipids.

The reaction of DL-1,3-butadiene diepoxide and of DL-1,4-dibromo-2,3-butanediol with aqueous alkaline sodium arsenite, "Na(3)AsO(3)", gave mixtures of the title arsonic acids which can be separated by anion exchange resin. Characterization of by-products leads to a better understanding of these reactions. These compounds are valuable intermediates for the preparation of novel arsonic acids and bis(arsonic acids).

Arsonoliposomes: novel nanosized arsenic-containing vesicles for drug delivery.

Natural and synthetic arsenolipids, have been discovered, synthesized, and evaluated for their biological activity. Arsonolipids, are analogs of phosphonolipids, in which P has been replaced by As. The synthesis of arsonolipids has been explored and a simple one-pot method with high yield is currently available for their preparation. However, although arsonolipids posses interesting biophysical and biochemical properties their anticancer or antiparasitic activity is not considered adequate for therapeutic applications. But when arsonolipids are incorporated in liposomes, the vesicles formulated have interesting possibilities, as seen in a number of studies. In cell culture studies, nanosized arsonolipid-containing liposomes or else arsonoliposomes, showed increased toxicity against cancer cells (compared to that of arsenic trioxide) but at the same time were less toxic than arsenic trioxide for normal cells. Furthermore, arsonoliposomes also demonstrate antiparasitic activity in vitro. Nevertheless, As is rapidly cleared from blood after in vivo administration of arsonoliposomes, and this will highly limit possible therapeutic applications. In addition, the fact that arsonoliposomes were observed to aggregate and subsequently fuse into larger particles in presence of cations, may also be considered as a problem. Thereby, methods to modulate the stability of arsonoliposomes and, perhaps, their in vivo distribution (as surface property modification) are currently being investigated. In very recent experiments it has been shown that arsonoliposome pegylation results in the formation of liposomes with very high membrane integrity. In addition, pegylation results in increased physical stability of arsonoliposomes and abolishment of cation-induced aggregation and fusion. Nevertheless, further in vivo studies are required in order to prove if pegylation alters arsonoliposome in vivo kinetics in a positive way, without affecting their activity. From studies performed thus far it is concluded that arsonoliposomes are nanosized-vesicles with interesting properties that justify further exploitation towards the development of therapeutic systems for cancer or parasitic diseases.

Arsonoliposome interaction with thiols: effect of pegylation and arsonolipid content of arsonoliposomes on their integrity during incubation in glutathione.

Increased toxicity of arsonoliposomes towards cancer cells may be attributed to interaction between arsonolipids and cellular thiols which, would result in reduction of As(V) to the more toxic As(Ill). Cancer cells with high thiol contents may thus be more sensitive to arsonoliposomes, providing that the arsonolipid molecules that are incorporated in the liposome membrane can interact with thiol-containing compounds. For examination of this possibility we investigate the effect of incubating various compositions of arsonoliposomes with glutathione, on their integrity. If glutathione does interact with the As(V) of the arsonolipid headgroup, this should result in an alteration of the arsonoliposome membrane stability. We followed arsonoliposome integrity by measuring the release of vesicle-encapsulated calcein from arsonoliposomes with different lipid compositions, during incubation in glutathione. The results of this study show that the effect of glutathione on arsonoliposome integrity is higher (arsonoliposomes are less stable) when the arsonolipid content of their membranes increases. This indicates that arsonolipid molecules interact with glutathione, and in some cases, depending on the rigidity of their membranes; this interaction leads to a (higher or lower) destabilization of arsonoliposomes. The destabilizing effect of glutathione was higher for arsonoliposomes that were previously found to be less stable during incubation in serum proteins or, in other words, have lower membrane rigidity. In the case of pegylated-arsonoliposomes membrane destabilization was minimal and this may be related to the high stability demonstrated previously for these specific arsonoliposomes, or, it may indicate that pegylation results in prevention (total or partial) of arsonolipid-As interaction with thiols (perhaps because of steric repulsion).

Thermal behavior of novel non-sonicated arsonolipid-containing liposomes.

The thermal properties of novel arsonolipid-containing liposomes in PBS pH 7.4 and in water in absence and presence of Ca(2+) ions are reported. Liposomes composed of arsonolipids with different acyl chains (C(12), C(16) and C(18)) were prepared by the one step method. Microcalorimetry results showed that (i) the thermotropic transitions of arsonoliposomes (in PBS, pH 7.4, and in water) increase as a function of arsonolipid fatty acyl chain length, (ii) arsonoliposomes of long fatty acyl chain arsonolipids (C(16) and C(18)) showed higher enthalpy and transition temperature in the buffer compared to those observed in water (for arsonoliposomes of C(12)-fatty acyl chain arsonolipid, the order was reversed which might be attributed to their different structure), and (iii) the presence of 2 mM CaCl(2) has more pronounced effects on the thermal properties of arsonoliposomes in distilled water than in buffer, which suggests that the ionic strength of the dispersion medium plays an important role in determining the thermal properties of arsonoliposomes.

Incorporation of PEG-lipids in arsonoliposomes results in formation of highly stable arsenic-containing vesicles.

We investigated the effect of pegylation on the physical stability, morphology and membrane integrity of arsonoliposomes. Arsonoliposomes composed of distearoylglycerophosphocholine (DSPC), cholesterol (Chol) and the palmitoyl side chain arsonolipid (with concentrations ranging from 0 mol% [DSPC/Chol vesicles] to 53 mol% of total lipid) containing either 4 or 8 mol% DPPE-PEG2000 or DSPE-PEG2000, were prepared by sonication. Arsonoliposome membrane integrity was evaluated by measuring the retention of encapsulated calcein in vesicles (during incubation in buffer or fetal calf serum [FCS]) while physical stability was evaluated by measuring vesicle dispersion turbidity (during incubation in water or CaCl(2)). Vesicle morphology was studied by cryo-electron microscopy. Experimental results show that: (i) PEG-lipids are incorporated in arsonoliposomes (as confirmed by the vesicle zeta potential modulation), (ii) pegylation of arsonoliposomes prevents their aggregation and fusion in the presence of calcium ions and (iii) when 8 mol% of PEG-DSPE is incorporated in arsonoliposomes based on their arsonolipid content, two groups of pegylated vesicles are formed: low content arsonoliposomes (<20 mol% arsonolipid) which are highly leaky and high content arsonoliposomes (>27 mol% arsonolipid) which are highly stable (70% calcein retention after 24h incubation in fetal calf serum [FCS]). In addition to high membrane integrity, the high content pegylated arsonoliposomes are morphologically perfect round-shaped vesicles without the sharp edges typically observed with non-pegylated DSPC-containing arsonoliposomes.

Stability and aggregation studies of non-sonicated arsonolipid-containing vesicles.

In this study, we investigated non-sonicated arsonolipid-containing liposomes (arsonoliposomes) in terms of the influence of lipid composition on their stability, assessed as membrane integrity and physical stability [size]. Vesicles consisting of plain arsonolipids or mixtures of arsonolipids with cholesterol [Chol] or with distearoyl-phospatidylcholine [DSPC] were studied. Membrane integrity was evaluated by measuring the retention of incorporated 5-(6)carboxyfluorescein [CF] during incubation of the vesicles in Tris buffer, pH 7.4. Photon correlation spectroscopy was used to investigate the time-dependent aggregation of arsonoliposomes in the absence and presence of Ca(2+)ions. Vesicles composed of plain C18 (acyl fatty chain) arsonolipids were found to be unstable, with only 15% of the initially incorporated CF remaining in the vesicles after 24 hours. The addition of Chol to the membrane (1:1 mol/mol) significantly increased the stability of arsonoliposomes, while the addition of DSPC to the lipid bilayer (1:1 mol/mol) increased vesicle stability to a lower extent. The results of particle size analysis showed that non-sonicated arsonoliposomes consisting of plain arsonolipid Ars/Stearic are highly and rapidly aggregated, while calcium-induced aggregation is also significant, but slower. Aggregation could not be always explained on the basis of zeta potential changes, indicating that the process is complex.

Preparation of dehydro-L-ascorbic acid dimer by air oxidation of L-ascorbic acid in the presence of catalytic amounts of copper(II) acetate and pyridine.

The catalytic system Cu(AcO)2-pyridine 1:4 mol% in methanol, slowly catalyses the air oxidation of ascorbic acid to the 2-methyl hemi-ketal of dehydroascorbic acid 5, and hydrogen peroxide. However, with Cu(AcO)2-pyridine 3:4 mol% the air oxidation is quite fast and no hydrogen peroxide is present at the end of the reaction. Removal of the catalyst and refluxing the foamy 5 in MeCN gives the oxidized, dimeric, dehydroascorbic acid in very good yields (approximately 70%) contaminated by approximately 1-2% MeCN.

Arsonoliposomes: effect of lipid composition on their stability and morphology.

The influence of the lipid composition of arsonoliposomes on their membrane integrity was investigated to evaluate whether it is possible to combine their action with drugs that can be encapsulated in their aqueous interior. This was investigated by measuring the retention of vesicle-encapsulated calcein (100 mM) during incubation, in the absence and presence of serum proteins. Liposomes containing various concentrations of arsonolipid (with the palmitoyl side chain) as well as egg-lecithin (phosphatidylcholine, PC) and cholesterol (lipid/chol 2:1 mol:mol) were prepared. In some experiments, PC was replaced by the synthetic phospholipid DSPC. All PC/arsonoliposomes tested are stable after 24 h of incubation in buffer at 37 degrees C. After incubation in the presence of serum proteins, arsonoliposomes that contain low amounts of arsonolipid (up to 5 mol% of the lipid content without cholesterol) are stable, whereas increased release of calcein is observed when vesicle arsonolipid concentration is raised (from 5 to 15 mol%). Further increase of arsonolipid content results in immediate decrease of calcein latency while the remaining calcein is rapidly released during incubation. DSPC/arsonoliposomes are comparably more stable, and membrane integrity is independent of the vesicle arsonolipid content, in the range investigated (15-40 mol% of the lipid content without cholesterol). Thereby, we conclude that more stable arsonoliposomes that incorporate high arsonolipid concentrations may be produced when PC is replaced by DSPC. The latter arsonoliposomes provide a system that may be used for combining arsonolipid activity with the activity of other drugs.

Preparation of dehydro-l-(+)-ascorbic acid dimer by oxidation of ascorbic acid with arsenic acid/iodine and formation of complexes between arsenious acid and ascorbic acid.

Ascorbic acid in the presence of a catalytic amount of iodine reduces arsenic acid in methanol giving the arsenious acid bound to the 2-methyl hemi-ketal of dehydroascorbic acid, 5, in 1:1 and in a more stable 2:1 5/As(III) molar ratio. Removal of the As(III) and treating the 2-methyl hemi-ketal of dehydroascorbic acid with refluxing acetonitrile affords the pure, crystalline dehydroascorbic acid dimer in good yields. Ascorbic acid also binds to As(III) of H(3)AsO(3) in a 1:1 and 2:1 ascorbic acid/As(III) molar ratio. The 1:1 complex is not stable and by expulsion of H(3)AsO(3) is transformed to the more stable 2:1 complex. The data do not permit distinguishing the 2:1 complexes between [AsL(2)(H(2)O)](-)H(+) or AsL(LH)(H(2)O) where L is the bis deprotonated and LH is the mono deprotonated 2-methyl hemi-ketal of dehydroascorbic acid or ascorbic acid. The 2:1 ascorbic acid/As(III) complex is oxidized by dioxygen, in a solvent-dependent manner, to dehydroascorbic acid implying dioxygen activation by the bound As(III). With thiophenol the same complex gives quantitatively triphenyl trithioarsenite, As(SPh)(3).

The reaction of allyl and benzylarsonic acids with thiols: mechanistic aspects and implications for dioxygen activation by trivalent arsenic compounds.

The reaction of allyl and benzylarsonic acids with thiophenol gives not only the expected diphenyl alkyldithioarsonites and diphenyl disulfide but also various other compounds arising from the decomposition at the arsenic(V) oxidation level (the arsonic acids) by thiophenol and at the arsenic(III) oxidation level (mainly the alkyldithioarsonites) by thiophenol and by dissolved dioxygen. The reaction of these arsonic acids with 4-nitrothiophenol, which is not oxidized by dioxygen, revealed that the arsenic(III) of these alkyldithioarsonites is the active atom towards dioxygen. However, the reaction of allyl, benzyl, and 2-picolylarsonic acids with DL-penicillamine gives the expected products with no or very small oxidative decomposition. The decomposition pathways of allyl and benzylarsonic acids were elucidated. The results are briefly discussed in the contexts of the use of arsonic acids in chemotherapy and the ability of arsenic(III) compounds to generate reactive oxygen species.