Name: preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation
Uploaded: 2018-03-09T13:00:00
Description: Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

This research was supported by Basic Science Research program (Project number: 2016R1A2B4013467) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT &#38; Future Planning 2016 and also supported by Korea Basic Science Institute Research Program (Project number: C36707).

1. Synthesis of DMMTAV
<ol>
	<li>Chemical preparation and molar ratio mixing of DMAV, Na2S, and H2SO4 
	NOTE: DMAV:Na2S:H2SO4 = 1:1.6:1.6
	<ol>
		<li>Dissolve 5.24 g of DMAV in 40 mL deionized and N2-purged (purged for at least 30 min) water in a 50 mL centrifuge tube.</li>
		<li>Prepare Na2S reagent by dissolving 14.41 g of Na2S&#183;9H2O in 50...

<ol>
	<li>Suzuki, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethylthioarsenicals+as+arsenic+metabolites+and+their+chemical+preparation.">Dimethylthioarsenicals as arsenic metabolites and their chemical preparation.</a> Chem. Res. Toxicol. 17, 914-921 (2004).</li><li>Kuroda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+metabolite+of+dimethylarsinic+acid+is+highly+toxic+and+genotoxic.">Microbial metabolite of dimethylarsinic acid is highly toxic and genotoxic.</a> Toxicol. Appl. Pharmacol. 198, 345-353 (2004).</li><li>Naranmandura, H., Iwata, K., Suzuki, K. T., Ogra, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+metabolism+of+four+different+dimethylated+arsenicals+in+hamsters.">Distribution and metabolism of four different dimethylated arsenicals in hamsters.</a> Toxicol. Appl. Pharmacol. 245, 67-75 (2010).</li><li>Naranmandura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+toxicity+of+arsenic+metabolites+in+human+bladder+cancer+EJ-1+cells.">Comparative toxicity of arsenic metabolites in human bladder cancer EJ-1 cells.</a> Chem. Res. Toxicol. 24, 1586-1596 (2011).</li><li>Wallschlager, D., London, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+methylated+arsenic-sulfur+compounds+in+groundwater.">Determination of methylated arsenic-sulfur compounds in groundwater.</a> Environ. Sci. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+dimethylthioarsenicals+in+red+blood+cells.">Formation of dimethylthioarsenicals in red blood cells.</a> Toxicol. Appl. Pharmacol. 227, 390-399 (2008).</li><li>Leffers, L., Ebert, F., Taleshi, S. M., Francesconi, A. K., Schwerdtle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+toxicological+characterization+of+two+arsenosugars+and+their+metabolites.">In vitro toxicological characterization of two arsenosugars and their metabolites.</a> Mol. Nutr. Food Res. 57, 1270-1282 (2013).</li><li>Wang, Q. Q., Thomas, J. D., Naranmandura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+of+being+thiomethylated:+Formation,+Fate+and+Effects+of+methylated+thioarsenicals.">Important of being thiomethylated: Formation, Fate and Effects of methylated thioarsenicals.</a> Chem. Res. Toxicol. 25, 281-289 (2015).</li><li>Kim, Y. T., Lee, H., Yoon, H. O., Woo, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+dimethylated+thioarsenicals+and+the+formation+of+highly+toxic+dimethylmonothioarsinic+acid+in+environment.">Kinetics of dimethylated thioarsenicals and the formation of highly toxic dimethylmonothioarsinic acid in environment.</a> Environ. Sci. Technol. 50, 11637-11645 (2016).</li><li>Cullen, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylated+and+thiolated+arsenic+species+for+environmental+and+health+research+-+A+review+on+synthesis+and+characterization.">Methylated and thiolated arsenic species for environmental and health research - A review on synthesis and characterization.</a> J. Environ. Sci. 49, 7-27 (2016).</li><li>Fricke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+separation+and+identification+of+products+form+the+reaction+of+dimethylarsinic+acid+with+hydrogen+sulfide.">Chromatographic separation and identification of products form the reaction of dimethylarsinic acid with hydrogen sulfide.</a> Chem. Res. Toxicol. 18, 1821-1829 (2005).</li><li>Fricke, M., Zeller, M., Cullen, W., Witkowski, M., Creed, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethylthioarsinic+anhydride:+a+standard+for+arsenic+speciation.">Dimethylthioarsinic anhydride: a standard for arsenic speciation.</a> Anal. Chim. Acta. 583, 78-83 (2007).</li><li>Suzuki, K. T., Iwata, K., Naranmandura, H., Suzuki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+differences+between+twon+dimethylthioarsenicals+in+rats.">Metabolic differences between twon dimethylthioarsenicals in rats.</a> Toxicol. Appl. Pharmacol. 218, 166-173 (2007).</li><li>Jeong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simultaneous+analytical+method+to+determine+arsenic+speciation+using+HPLC-ICP-MS:+Arsenate,+arsenite,+monomethylarsonic+acid,+dimethylarsinic+acid,+dimethyldithioarsinic+acid,+and+dimethylmonothioarsinic+acid.">Development of a simultaneous analytical method to determine arsenic speciation using HPLC-ICP-MS: Arsenate, arsenite, monomethylarsonic acid, dimethylarsinic acid, dimethyldithioarsinic acid, and dimethylmonothioarsinic acid.</a> Microchem. J. 134, 295-300 (2017).</li><li>Li, Y., Low, C. -. K., Scott, A. J., Amal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic+speciation+in+municipal+landfill+leachate.">Arsenic speciation in municipal landfill leachate.</a> Chemosphere. 79, 794-801 (2010).</li><li>Conklin, D. S., Fricke, W. M., Creed, A. P., Creed, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+pH+effects+on+the+formation+of+methylated+thio-arsenicals,+and+the+effects+of+pH+and+temperature+on+their+stability.">Investigation of the pH effects on the formation of methylated thio-arsenicals, and the effects of pH and temperature on their stability.</a> J. Anal. At. Spectrom. 23, 711-716 (2008).</li><li>Hansen, H. R., Raab, A., Jaspara, M., Milne, F. 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T., Anzai, K., Yamaguchi, K., Sei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SEC-HPLC-ICP-MS+hyphenated+technique+for+identification+of+sulfur-containing+arsenic+metabolites+in+biological+samples.">A SEC-HPLC-ICP-MS hyphenated technique for identification of sulfur-containing arsenic metabolites in biological samples.</a> J. Chromatogr. B. 874, 64-76 (2008).</li><li>Bartel, M., Ebert, F., Leffers, L., Karst, U., Schwerdtle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicological+characterization+of+the+inorganic+and+organic+arsenic+metabolite+thio-DMAV+in+cultured+human+lung+cells.">Toxicological characterization of the inorganic and organic arsenic metabolite thio-DMAV in cultured human lung cells.</a> J. 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The developed protocol has clarified critical steps that previous studies1,3,4,8,15 omitted or abbreviated, which may have led to difficulties with or failure during DMMTAV and DMDTAV synthesis. As DMMTAV is oxidation-sensitive1,5, chemical reagents for its synthesis we...

Since dimethylarsinic acid (DMAV) has been demonstrated to exhibit both acute toxicity and genotoxicity due to undergoing methylation and thiolation upon ingestion1,2, the metabolic pathway of arsenic thiolation has been intensively studied both in vitro and in vivo3,4 as well as in environmental media (e.g., landfill leachate)5,6. Previous studies have found both reduced and thiolated analogs of DMAV in living cells, for example, dimethylarsinous acid (DMAIII), dimethylmonothioarsinic acid (DMMTAV), and dimethyldithioarsinic acid (DMDTAV)7,8,9, with dimethylated thioarsenicals such as DMMTAV exhibiting greater toxicity than other known inorganic or organic arsenicals10. The abundance of highly toxic thioarsenicals has serious environmental implications, since they may pose a risk to humans and the environment under highly sulfidic conditions11. However, the mechanisms of DMMTAV and DMDTAV (trans)formation and their fates in environmental media still require further study. Thus, the quantitative analysis of thioarsenicals is required to improve understanding of the environmental effects of DMMTAV and DMDTAV.
Although standard chemicals are the key requirement for quantitative analysis, the standards of DMMTAV and DMDTAV are difficult to obtain by replicating previous studies, owing to the high risk of species transformation into other species and unstandardized synthesis procedures12. Moreover, the methods referenced have limitations that may lead to practical difficulties in synthesizing the standard chemicals and performing quantitative analysis. DMMTAV and DMDTAV are commonly prepared by mixing DMAV, Na2S, and H2SO4 in a certain molar ratio1 or bubbling H2S gas through a solution of DMAV 13,14. The bubbling method features substitution of oxygen by sulfur using a direct supply of H2S gas, which, is highly toxic and difficult to control for an inexperienced user. Conversely, the above mixing method1, widely used for the qualitative analysis of DMMTAV and DMDTAV in environmental sudies5,6,12, features the thiolation of DMAV with H2S generated by mixing Na2S and H2SO4 and produces DMMTAV and DMDTAV, allowing easier stoichiometric control to produce target chemicals, as compared to the direct use of H2S gas.
The reference mixing method procedures1,3,4,8,15 mentioned in this study exhibit limitations in some of their critical experimental steps, which might lead to experimental failure. For example, the details of specific solvent (i.e., deionized water) preparation and the extraction and crystallization of the synthesized arsenicals are over-abbreviated or not described in sufficient detail. Such dispersed and limited information on procedural steps might lead to the inconsistent formation of thioarsenicals and unreliable quantification analysis. Therefore, the modified protocol developed herein describes the synthesis of DMMTAV and DMDTAV stock solutions with quantitative species separation analysis.

<table width="712">
	<tbody>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Cacodylic acid</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:179px;">20835-10G-F</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Sodium sulfide nonahydrate</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:179px;">S2006-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Sulfuric acid 96%</td>
			<td style="width:179px;">J.T.Baker</td>
			<td style="width:179px;">0000011478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Ammonium acetate</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:179px;">A7262-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Formic acid 98%</td>
			<td style="width:179px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:179px;">066-00461</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Diethyl ether (Extra Pure)</td>
			<td style="width:179px;">Junsei Chemical</td>
			<td style="width:179px;">33475-0380</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Adapter cap for 60 mL Bond Elut catridges</td>
			<td style="width:179px;">Agilent Technologies</td>
			<td style="width:179px;">12131004</td>
			<td>Syringe type of SPE</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">Bond Elut C18 cartridge</td>
			<td style="width:179px;">Agilent Technologies</td>
			<td style="width:179px;">14256031</td>
			<td>Syringe type of SPE</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:179px;height:21px;">HyPURITY C-18</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:179px;">22105-254630</td>
			<td>5 um, 125 x 4.6 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Glovebox</td>
			<td style="width:179px;">Chungae-chun, Rep. of Korea</td>
			<td style="width:179px;"></td>
			<td>Customized&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Agilent 1260 Infinity Bio-inert LC</td>
			<td style="width:179px;">Agilent Technologies</td>
			<td style="width:179px;">DEAB600252, DEACH00245</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:179px;height:42px;">Agilent Technologies 7700 Series ICP-MS</td>
			<td style="width:179px;">Agilent Technologies</td>
			<td style="width:179px;">JP12031510</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:179px;height:63px;">Finnigan LCQ Deca XP MAX Mass Spectrometer System</td>
			<td style="width:179px;">Thermo Electron Corporation</td>
			<td style="width:179px;">LDM10627</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dmmtav and dmdtav using dmav for environmental applications: synthesis, purification, and confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Since DMMTAV has been mistakenly prepared by the DMAIII synthesis method19, verification of synthesized DMMTAV and DMDTAV is a critical step for synthesis and extraction and determining the ideal standard chemical materials. Synthesized chemicals can be verified by the peak of DMMTAV (MW 154 g&#183;mol-1) and DMDTAV (MW 170 g&#183;mol-1) mass-to-charge ratio (m/z) using...

Watch this Scientific Journal Video about Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation at JoVE.com

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

The overall goal of this procedure is to synthesize, purify and isolate stable dimethyl monothiol arsenic acid and dimethyl dithioarsenic acid for use as standards in quantitative speciation analysis of thioarsenicals. This experimental guideline can help address key issues in quantitative thioarsenical analysis such as unreliability caused by errors in critical steps of the synthesis of thioarsenical standards. The main advantage of this modified technique is that it produces stable chemical standards that can be used to quantify thioarsenical content in samples.To begin the DMMTA synthesis in a fume hood prepare solutions of dimethylmonothioarsinic acid, sodium sulfide and sulfuric acid in deionized water purged for 30 minutes with nitrogen gas. Add the DMA solution to the sodium sulfide solution. Rinse the DMA solution container with 10 milliliters of N2 purged water and add the rinse to the mixture.Note the time at which the solutions were combined. Connect a three hole rubber stopper fitted with glass tubes to a nitrogen gas line. Stopper the flask and immediately begin flowing nitrogen gas through the flask ensuring that the solution does not splash under the flow then use acid resistant tubing to connect a 50 milliliter syringe containing the sulfuric acid solution to another of the glass tubes.Slowly add the sulfuric acid solution, four to five milliliters at a time drop wise to obtain a cloudy white solution. Once the sulfuric acid has been added and one hour has elapsed since the DMA and sodium sulfide solutions were initially combined transfer the reaction mixture to separatory funnel containing 200 milliliters of diethyl ether. Shake the funnel for five to 10 minutes while regularly venting the funnel.Collect the aqueous layer in a beaker and then collect the organic layer containing the DMMTA in a separate bottle. Extract the DMMTA from the aqueous layer with 200 milliliter portions of diethyl ether three more times then wash the combined ether layers with 100 milliliters of N2 purge deionized water. Collect the washed ethyl layer in a glass crystallizing dish and transfer the solution to a nitrogen filled glovebox.Allow the solvent to evaporate under a flowing nitrogen atmosphere to obtain DMMTA as a white crystalline precipitate. Measure and record the mass of the crystallized DMMTA then dissolve the solid in 50 milliliters of N2 purged deionized water. Filter the solution through a 0.2 micron syringe filter and store the solution at four degrees celsius in the dark.To begin the DMDTA synthesis prepare solutions of DMA, sodium sulfide and sulfuric acid in deionized water. Combine the the DMA and sodium sulfide solutions in a 250 milliliter flask. Rinse the container of DMA solution with 10 milliliters of deionized water and add the rinse to the flask.Slowly add the sulfuric acid solution to the mixture four to five milliliters at a time drop wise. Monitor the appearance of the mixture as it becomes white to yellow and cloudy. Allow the mixture to sit uncovered overnight.Then, pass the reaction mixture through C18 silicon based solid phase extraction cartridge to trap the DMDTA on the resin. Elute the DMDTA into a glass crystallizing dish with about one liter of a 10 millimolar pH 6.3 ammonium acetate solution. Transfer the eluate to a nitrogen filled glovebox.Allow the solvent to evaporate under a flowing nitrogen atmosphere to obtain crystallized DMDTA as a white precipitate. Measure and record the mass of the crystallized DMDTA. Dissolve the solid in 50 milliliters of deionized water and filter the solution through a 0.2 micron syringe filter.Store the solution at four degrees celsius in the absence of light. Electrospray ionization mass spectrometry was used to verify the successful synthesis of DMMTA and DMDTA from DMA and to confirm that dimethylarsinic acid was not the major product. HPLCICPMS also showed that DMA starting material had been consumed and that DMMTA and DMDTA had been produced.After being stored at four degrees Celsius in the absence of light for 13 weeks, the DMMTA and DMDTA solutions showed only a 2.2 and a 5.8 percent change in the species distributions respectively. Before attempting this procedure remember to read the reference material and safety data. It is important to understand the mechanism of DMMTA and DMDTA synthesis when preforming this experiment.

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Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to ...