Synthesis of Dimethyl Selenone and Studies on the Biomethylation Pt 1

Abstract Synthesis of Dimethyl Selenone and Studies on the Biomethylation Intermediates of Selenium Reduction and Methylation. Purpose This investigation was undertaken for the purpose of determin- ing whether or not dimethyl selenone could be an intermediate of biomethylation and transformation of inorganic selenium in bacterial cultures. The possible pathways for the synthesis of dimethyl selenone were also investigated. Method The reactions of the synthesis of dimethyl selenone were carried out in a methylene chloride solution of 3-chloroperoxybenzoic acid and dimethyl selenide. Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, elemental analysis and melting point analysis were used to characterize the dimethyl selenone formed. Pseudomonas fluorescens K27 was grown in tryptic soy broth containing 0.1% potassium nitrate, TSN medium. All test tubes and filters were autoclaved before using. Both medium and aqueous solutions of selenium containing salts were sterilized by using sterilized filters. A Hewlett Packard 5890 Series II gas chromatograph coupled with a Sievers Research Model 300 Sulfur Chemiluminescence Detector was used as the principal tool for this research. Findings 3-Chloroperoxybenzoic acid (m-CPBA) (65%) was found to readily oxidize dimethyl selenide (DMSe) at 200C in 2 hours. The optimum ratio of m-CPBA to DMSe was 3:1 mole equivalents. Pseudomonas fluorescens K27 grew well in sterilized TSN me- dium. Dimethyl selenone was tentatively identified as one of the intermediates of biomethylation of inorganic selenium because very large amount of volatile selenium compounds such as dimethyl selenide, dimethyl diselenide (DMDSe) and probably dimethyl selenenyl sulfide (DMSeS) were observed after the K27 cultures were dosed with dimethyl selenone. Gas phase concentrations of volatile DMSe and DMDSe formed above selenone dosed cultures were about 4000 times and 1000 times more (respectively) than those observed above sodium selenate dosed cultures poisoned with the same concentration. If dimethyl selenone is a biological intermediate of the reduction and methylation of selenium oxyanions, then the final reduction step to dimethyl selenide and dimethyl diselenide may occur by chemical means via reduction by dimethyl sulfide and dimethyl disulfide. Chapter I Introduction Selenium and its compounds have been of considerable biologi- cal interest for a long time because they are both required by and highly toxic to human beings and animals. From the public health standpoint, trace amounts of selenium are important but they are harmful to organisms in the environment at higher concentrations. Movement of toxic inorganic selenium through the geocycle and its biological methylation in the environment to volatile products have also attracted attention. Methylation often leads to a change in both mobility and toxicity of the element. The conversion of oxyanious compounds containing inorganic selenium such as selenate(VI) and selenite(IV) to volatile methylated species such as dimethyl selenide and dimethyl diselenide by plants, microorganisms and mammals has been well documented in the literature (Challenger and North, 1934; Lewis et al., 1966; Jiang et al., 1983). The biological methylation of inorganic selenium by microorgan- isms has been investigated since the early 1930s beginning with Challenger and co-workers. In 1934, Challenger and North reported that fungi in culture are able to methylate inorganic selenium (Challenger and North, 1934). In the 1970s, information on microorganisms' ability to methylate some heavy metals such as Hg (Wood, 1974), As (Braman and Foreback, 1973; Wood, 1974), and Pb (Wong et al., 1975) was published. For heavy metals such as Hg and Pb, microorganisms have the capacity to metabolize certain compounds containing these elements and the resulting transformations could thus yield more toxic products (Wood, 1974; Wong, et al. 1975). In 1974, Francis and co-workers noted that dimethyl selenide was evolved from samples of soil (Francis et al., 1974). Hence, methylation reactions may be of importance to the transformations of selenium in natural ecosystems. With inorganic selenium, biomethylation processes by microor- ganisms can produce volatile organic metabolic compounds that are less toxic than the original forms (McConnell and Portman, 1952; Sigel and Sigel, 1993). As selenium is of interest as a potential environmental toxicant, attentions have been focused on the biodegradation and transformation processes of selenium in the environment. Some investigations have been done to develop a detailed under- standing of the mechanism of biomethylation and transformation of selenium. In 1945, Challenger suggested a mechanism (Scheme 1) that involves four steps in which the selenium atom is methylated and reduced to form volatile dimethyl selenide (Challenger, 1945). But this mechanism did not involve dimethyl diselenide. Many workers have observed dimethyl diselenide in the headspace above selenium resistant cultures (Reamer and Zoller, 1980; Chasteen et al., 1990; Chasteen, 1993; McCarty et al., 1993). None of the postulated, intermediate selenium compounds was detected in culture solutions; however, dimethyl selenone is the only postulated intermediate which may be stable enough to be isolated by normal means. Dimethyl selenone has never been synthesized and pur- posely introduced into bacterial cultures to probe the mechanism. HSeO3 - Methylation selenite Methylation Ionization & Reduction CH3 SeO3 H CH3 SeO2 - methane selenonic acid methaneselenic acid Reduction (CH3 )2SeO2 (CH3 ) 2 Se dimethyl selenone dimethyl selenide Scheme 1 More recently, Reamer and Zoller (1980) proposed an expanded mechanism (Scheme 2) based on their identification of dimethyl selenide, dimethyl diselenide, and dimethyl selenone as products from soil and sewage amended with selenite or elemental selenium. Recent evidence suggests, however that their detection of dimethyl selenone was probably in error and that they confused dimethyl selenone for dimethyl selenenyl sulfide instead (Chasteen, 1993). H2SeO3 or Na2SeO3 Hydrolysis selenite CH3SeO3H Methylation H+SeO3Hselenonic acid Reduction CH3SeO2 Reduction methane selenonic acid CH3SeH or CH3SeOH Methylation (CH3 ) 2S e dimethyl selenide (CH3 ) 2 SeO2 dimethyl selenone Scheme 2 CH3SeSeCH3 dimethyl diselenide Doran (1982) suggested that the methylation of inorganic Se may first involve reduction of selenite to the elemental Se and then re- duction to the selenide form, which is subsequently methylated to form dimethyl selenide (Scheme 3). Many workers have found el- emental selenium in bacterial cultures doped with oxidized selenium salts (Francis et al., 1974; Doran and Alexander, 1977; Steinberg and Oremland, 1990). Our bacterial cultures doped with oxyanions of selenium often yielded a red precipitate that is probably selenium. SeO3- Reduction selenite Methylation Reduction S e0 HSeX elemental Se CH3SeH Methylation methane selenol selenide (CH3)2S e dimethyl selenide Scheme 3 The present investigation was initiated to study one possible pathway of the biomethylation process of selenium salts by Pseudomonas fluorescens K27, a selenium resistant bacterial strain which was isolated from Kesterson Reservoir in the San Joaquin valley of central California (Burton et al., 1987). Our studies were focused on identifying whether or not dimethyl selenone may act as an intermediate of the biomethylation process. We have noticed that both of the mechanisms of Challenger and of Reamer and Zoller involved dimethyl selenone as an intermediate of the biomethylation process. This compound was never tested by Challenger but it was reported by Reamer & Zoller in soil and sew- age sludge samples inoculated with sodium selenite or elemental selenium. Because dimethyl selenone has a melting point at 1470C- 1480C (Paetzold and Bochmann, 1968), it is probably not possible to detect directly by gas chromatography as a volatile metabolic com- pound as proposed by Reamer & Zoller; we proposed another way to identify it as an intermediate. The method that we proposed is to synthesize dimethyl selenone, dose cultures with it and analyze the headspace released from the cultures by gas chromatography coupled with sulfur chemilumines- cence detection. If the methylation process involves this compound, we should observe volatile selenium compounds as final products in headspace above the cultures. If dimethyl selenone were not in- volved, no volatile selenium compounds should be observed (assuming no other sink for reduced selenium exists). This process was carried out in an anaerobic and facultative bacterial culture. Little information on the synthesis of dimethyl selenone was available in the literature; however, Paetzold and Bochmann suc- cessfully synthesized dimethyl selenone by using ozone as oxidant in 1968 (Paetzold and Bochmann, 1968). More recently, workers reported the synthesis of dimethyl selenone by the oxidation of the corresponding dimethyl selenide by 3-chloroperoxybenzoic acid and potassium permanganate (Krief et al., 1985). Rebane reported the mass spectrum of this compound in 1974 (Rebane, 1974). As we did not have an ozone generator in our lab, we had to choose another appropriate oxidant for this synthesis. 3-Chloroperoxybenzoic acid was found to efficiently oxidize dimethyl selenide to the corresponding selenone, and the yield was higher than when using potassium permanganate as oxidant (Krief et al., 1985). So, the method that we followed was to use 3-chloroperoxybenzoic acid and dimethyl selenide as starting mate- rials and methylene chloride as solvent to perform this synthesis. In order to achieve purer dimethyl selenone, a modification of the Krief et al. method was used in our lab. Gas chromatography is the most widely used technique to analyze volatile compounds in all fields of science. A large number of detec- tors have been developed for gas chromatography. Most of them are specialized detectors which are selective for a certain kind of compounds. Three major sulfur/selenium selective detectors are discussed below. The flame photometric detector (FPD) is the most popular detec- tor for sulfur compounds (Brody and Chaney, 1966; Farwell and Barinaga, 1986), but is also used for volatile compounds containing selenium (Flinn and Aue, 1978). The most frequently cited impediments to its broader use is that the FPD produces an exponential response which is close to two for sulfur and selenium compounds. But it has been shown to have a number of inherent problems, such as non-linear response above two orders of magnitude (Farwell et al., 1981), variation in the response factor with molecular structure, and quenching by hydrocarbons and other species which are eluted together with the analyzed compounds (Driscoll and Berger, 1989). The detection limits of FPD for sulfur and selenium compounds are in the nanogram range which is not very satisfactory for trace analysis. So, more recently, two other sulfur and selenium selective detection methods which are more sensitive and selective and re- spond on a relatively constant mass basis, independent of the source of the sulfur or selenium, have become commercially available. Sulfur chemiluminescence detection (SCD) is specific for alkylated sulfur/selenium compounds; atomic emission spectrometric detec- tion (AED) is a muti-element method capable of detecting elements with atomic emission at vacuum-UV, UV, visible and near-IR por- tions of the electromagnetic spectrum. Both detectors have detection limits for sulfur or selenium in the low picogram range (Chasteen, 1990; Eckert-Tilotta et al., 1992). The linear dynamic range with AED and SCD are approximately five orders of magnitude and four orders of magnitude respectively. However, atomic emission detector instrumentation is much more expensive and requires more laboratory space than sulfur chemiluminescence detector instru- mentation. On account of its availability, high sensitivity, wide lin- earity, and good selectivity to alkylated selenium, the sulfur chemiluminescence detector was found well suited for our research. Chapter II Experimental Methods Part I. Synthesis of Dimethyl Selenone 1. Apparatus and Reagents All chemicals used in our synthesis were of analytical reagent grade and used without further purification. Dimethyl selenide was purchased from Strem Chemicals, Inc. (Newburyport, MA USA). 3-Chloroperoxybenzoic acid (65%) was acquired from the Spectrum Chemical Mfg. Corp. (Gardena, CA USA). Methylene chloride was obtained from the Aldrich Chemical Company, Inc. (St. Louis, MO). 2. Methods Synthesis of dimethyl selenone Dimethyl selenone was synthesized by oxidizing dimethyl selenide with an excess of 3-chloroperoxybenzoic acid in methylene chloride solution. One mL dimethyl selenide (0.013 moles) was dissolved in 5 mL methylene chloride. Three mol equiv. of 3-chloroperoxybenzoic acid (65%, 10.35 g), dissolved in 25 mL methylene chloride solution, was added to the selenide solution dropwise. The reaction was stirred for two hours at 200C and the white cloudy solution was dried using an evaporator and extracted three times with 20 mL ethyl ether. The by-product, 3-chlorobenzoic acid, which dissolved in ether solution, was separated from the crude solid dimethyl selenone by vacuum filtration. The crude selenone was recrystallized with methanol two times to give white odorless crystals. Elemental analysis of dimethyl selenone, (CH 3 ) 2 S e O2 Elemental analysis of dimethyl selenone was performed by Galbraith Laboratories, Inc. Knoxville, TN USA. A fifty-five mg sample was sent to this lab and the results returned by mail. Fourier transfer infrared spectroscopy of (CH 3 ) 2 S e O2 A Bomem DA3 fourier transfer infrared spectrometer was used to identify dimethyl selenone. The spectrometer was controlled by a DEC PDP/11 computer. The data were exported to a 486sx personal computer for storage and display. The sample preparation was performed by Mr. D. Ellinwood by mixing 2 mg dimethyl selenone and 200 mg potassium bromide in a small sample cell. The sample was pressed at 10,000 psi pressure for 2 minutes to achieve a thin transparent sample sheet in order for infrared radiation to pass through or be absorbed by the sample. The sample cell was kept in a desiccator after pellet preparation until FTIR analysis was performed. Nuclear magnetic resonance measurement of (CH 3 ) 2 S e O2 The proton NMR spectrum of dimethyl selenone was carried out on a Varian EM 360 NMR spectrometer. This instrument was a 60 MHz system that provided an accurate and non-destructive method of determining structure in liquids and soluble chemical compounds. Deuterium oxide was the solvent for dimethyl selenone in this NMR measurement. It was obtained from Aldrich Chemical Com- pany, Inc. (St. Louis, MO USA) and NMR test tubes were purchased from Alltech (Deerfield, IL). About 10 mg of sample was dissolved in 6 drops of solvent and the spectrum was recorded at room temperature. The stability of dimethyl selenone was checked by NMR; the spectrum was taken one month after dimethyl selenone was synthesized. Melting point analysis of dimethyl selenone The measurement of melting point of dimethyl selenone was performed on a Fisher-John melting point apparatus in our lab. The thermometer for this measurement was not calibrated. Part. II Studies on the Biomethylation Intermediates o f Selenium Reduction and Methylation 1. Apparatus and Reagents Dimethyl sulfide, dimethyl disulfide, and dimethyl diselenide were obtained as pure solutions from Aldrich Chemical Company, Inc. (St. Louis, MO USA). Dimethyl selenide and sodium selenate were purchased from Strem Chemicals, Inc. (Newburyport, MA USA). Tryptic soy broth was obtained from Difco Laboratories (Detroit, MI USA). One mL gas syringes were acquired from Alltech (Deerfield, IL USA), and sterilized filters were purchased from MSI (Westboro, MA USA). 2. Gas Chromatography-Chemiluminescence Detection A gas chromatograph-sulfur chemiluminescence detector system was used for all the headspace experiments. The chromatograph was a Hewlett Packard Model 5890 Series II. The detector was a Sievers Research Model 300. In this instrument, organo-sulfur, -selenium and -tellurium compounds react with molecular fluorine (F2) which is produced in-line by a high frequency electrical discharge of a stream of SF6 (99.99%). This reaction is carried out in a steel cell in which the pressure is below one torr, maintained by a vacuum pump; the temperature of the cell is room temperature. A red sensitive photomultiplier tube using photon counting electronics is used to detect chemiluminescence which is produced by the reaction of the analytes with F2. The resulting analog signal from the detector is recorded by a Hewlett Packard Model 3396 Series II integrator. There is a heated nickel transfer line between the gas chromatograph and the detector. The capillary column from the GC is in- serted through this transfer line into the reaction cell of the detector and the temperature of this transfer line is thermostatically maintained at approximately 2130C. The gas chromatograph was equipped with a cryogenic oven which was used 1) to trap samples at the head of the column upon injection at the very beginning of temperature program and 2) to cool down the column in a short time when the temperature program ran out. A thirty meters long capillary column (Alltech, Deerfield, IL USA) was used. The chromatographic phase of the column was SE-54 (5% phenyl methyl silicone), which was an almost completely non-polar phase so that the elution order of analytes were generally based on the boiling point of compounds. Helium was used as the carrier gas at a flow rate of about 1 mL/min in our experiments. 3. Microbial Incubations Bacterial strains used in this study were Pseudomonas fluorescens K27, isolated from Kesterson Reservoir in central California (Burton et al., 1987). The K27 cultures were supplied by Ray Fall at University of Colorado, Boulder. This selenium resistant bacterial strain was grown at 300C in TSN medium containing 10 g/L tryptic soy broth and 1 g/L potassium nitrate as the electron sink (terminal electron acceptor). The medium was filter sterilized before inoculation and the inoculated cultures were incubated at 300C overnight in a thermostatic water bath. After 24 hours of growth, the cultures were diluted 1:1 with fresh sterilized medium and dosed with a desired amount of poison (selenate or selenone) using the stock solution described below. The cultures were then incubated for 24 hours at 300C before headspace sampling. The TSN medium is a complex medium, for which the exact chemical composition can not be defined. It is made of nutrients such as digests of proteins from soy broth so that the energy, car- bon, nitrogen, and sulfur requirements of the growing microorgan- isms are met largely by protein. In order to avoid complex medium, two chemically defined media in which the exact chemical composition are known were also attempted for the growth of K27. The minimal succinate medium consisted of 40 mM Na2C4H4O4, 20 mM NH4Cl, 2.5 mM K2HPO4, 2.5 mM KH2PO4, 0.8 mM MgSO4.7H2O in 100 mL distilled water with 0.1% (vol/vol) trace mineral solution (250 mg of EDTA, 500 mg of FeSO4.7H2O, 154 mg of MnSO4.H2O, 10 mg of CuSO4.5H2O, 24.5 mg of Co(NO3)2.6H2O, 17.7 mg of Na2B4O7.10H2O, water to 100 mL), and adjust to pH 6.8 (Bryan et al ,1985). Another minimal medium consisted of 0.544 g KH2PO4, pH 7.4 and 0.6 mL of a salt solution consisting of (L-1) 10 g MgSO4.7H2O, 1.0 g MnCl2.4H2O, 0.4 g FeSO4.7H2O and 0.1 g CaCl2.2H2O in 100 mL distilled water with the addition of 0.2 g glu- cose and 0.38 g NH4Cl (McEldowney and Fletcher, 1986). Both mini- mal media did not grow the K27 bacteria under the same incubation conditions that was successful using TSN medium. So, the TSN me- dium was the only medium used for growing the K27 bacteria in this research. 4. Sample Preparation Sterilization was accomplished by autoclaving all test tubes, caps and septa before preparing samples. Solutions were sterilized via sterile filtering. One mole/L aqueous solutions of sodium selenate, sodium selenite and dimethyl selenone were prepared as stock solutions. The dimethyl selenone was recrystallized the same day that the stock solution was prepared. The diluted solutions were pre- pared by taking different amount of filter sterilized stock solutions, diluting into 2 mL and 10 mL cultures to get either 1 mM or 10 mM final concentration in the media. 5. Standard solutions preparation The volatile sulfur and selenium compounds released from our samples including methanethiol, dimethyl sulfide, dimethyl se- lenide, dimethyl disulfide and dimethyl diselenide were determined by comparing their retention times with those of standard solutions prepared by serial dilutions. The standard solutions of DMS, DMSe, DMDS, DMDSe were prepared by diluting 50 µ L pure standards into 1 mL acetonitrile solution in a 2 mL glass vial, then diluting 50 µ L solutions from the first vial into 1 mL acetonitrile in another 2 mL glass vial. This process was repeated five to six times to get different concentration diluted standard solutions. The standard solution of methanethiol was prepared in our lab by mixing 25 µ L dimethyl disulfide and 0.2 gram powder zinc with 1 mL concentrated HCl into 2 mL water in a 6 mL glass vial sealed with a Teflon® lined septum (Chasteen, 1990; Chasteen, 1993). Methanethiol was produced after the mixture was allowed to react for 15 min at room temperature. One mL gas of headspace above these standard solutions were removed from the vials by a gas syringe and analyzed by GC. The retention times of these standards were applied to identify headspace species in our samples. 6. Experimental Procedure In our experiments, two media blanks and two culture blanks (with microorganisms) were prepared as controls. Also, we grew several 2 mL and 10 mL cultures in 15 mL glass vials sealed with Teflon lined septa and poisoned them with different concentrations of sodium selenate and dimethyl selenone. On the other hand, we poisoned three media blanks (without microorganisms) with 10 mM sodium selenate, sodium selenite and dimethyl selenone respec- tively, with the addition of DMS and DMDS to see if the reduction of dimethyl selenone was a chemical reaction only. Known volumes of headspace gas above samples were taken from the vials by a gas tight syringe for GC analysis after 24 hours incubation at 300C. A splitless injection technique was used for sodium selenate or selenite samples and a split injection technique was used for dimethyl sele- none samples to avoid column overloading. The split ratio was 1/65, which means only 1/65 of the sample introduced into the injector was injected into the column and the other was thrown away to waste. The peak areas that we reported were normalized to a one mL gas sample, 10 mL culture volume, and splitless injection. For example, a 0.5 mL gas sample taken from above a 2 mL culture sample and injected in the split injection mode had the chromatogram’s peak areas multiplied by 2 (to normalize to 1 mL gas samples), by 5 (to normalize to 10 mL culture samples) and by 65 to normalize to a splitless injection. In order to get high resolution chromatograms, we set up a two- step temperature program. In the first ramp, the headspace samples were cryogenically trapped in the capillary column at -200C for one minute, then the temperature was increased at 80C/min until the temperature arrived at 200C. In the second ramp which began immediately, the temperature was increased at 150C/min to a final temperature of 2000C for three minutes. We used the same temperature program for all the standards and headspace samples analysis. Media blanks and culture blanks (sample vials inoculated with the K27 bacteria without selenate salts or dimethyl selenone) were run along with poisoned samples as controls. By this method, we routinely identified which gases detected in the headspace were the result of the addition of the selenate salts or dimethyl selenone and which were not. A syringe cleaning device was used to clean the gas syringes be- fore each headspace analysis; one mL of lab air in the gas tight sy- ringe was analyzed by GC to make sure the syringe was clean before each sample run. The stability of sodium selenate and sodium selenite containing TSN solution with regard to added DMS and DMDS was also mea- sured by GC-SCD. Sodium selenate and selenite amended TSN solutions with DMS and DMDS were prepared by Mr. Steven L. McCarty in our research group. First, sodium selenate and sodium selenite were dissolved in two 100 mL TSN media blanks at a final concentration of 10 mM. Nanogram range concentrations of DMS and DMDS solutions were prepared by serial dilutions of concentrated DMS and DMDS solutions. Then 1 ng DMS and 1 ng DMDS in water solutions were pipetted into the TSN media with selenate and selenite salts by Eppendorf pipet. The final solutions were sterilized by sterilized filters before sampling.