Bo Duan and Dr. Merritt Andrus, Chemistry and Biochemistry

6–methylsulfinylhexyl isothiocyanate (MS-ITC) is the active component of wasabi, Japanese horseradish. Wasabi plant (Wasabia Japonica), a member of the Cruciferae family, is native to Japan and is traditionally found growing in or by cold mountain streams. The root and stem of this plant has a fiery hot flavor that quickly dissipates in the mouth, leaving a lingering sweet taste.

Much attention was given to MS-ITC, the major spicy wasabi flavor compound, because of its extensive biological and cancer-fighting activities. Experiments proved that MS-ITC has antibacterial activities against Escherichia coli and Staphylococcus aurous, induces apoptotic cell death, and induce phase II enzyme and suppresses phase I enzymes. MS-ITC is also reported as a potential inhibitor of human platelet aggregation, inhibitor cancer metastasis of and inhibitor of skin tumor mutation. Research reports have recommended that everyone eat two servings of wasabi each week to dramatically lower risk of all types of cancer.

The naturally occurring MS-ITC has the R configuration as shown in Figure 1

Since the MS-ITC used for biological evaluation was the organic solvent extracts from natural plants, only R configuration MS-ITC has been tested. My main task was through organic synthesis route to synthesis both R and S configuration forms of MS-ITC.

To synthesize MS-ITC, I followed five major steps showing below:

Step1: With solvent N, N–dimethylformamide (DMF), 1, 6-dibromohexane reacted with 1.2 molar equivalent of potassium phthalimide overnight at room temperature to obtain 6-bromohexyl phthalimide with yield 91% for the most yield attempt. Column chromatography was employed to purify the product. In fact, column chromatography was employ for step 1 to step 3 as the main purification process; Thin Layer Chromatography (TLC) was used to track reaction progress; and Nuclear Magnetic Resonance spectroscopy NMR was employed to detect and confirm the aimed product.

Step2: 6–bromohexyl phthalimide reacted with 1.1 molar equivalent of sodium monohydrate to obtain 6-methylthiohexyl phthalimide with yield 92% at room temperature overnight and with one day cool down in refrigerator.

Step3: 6–methylthiohexyl phthalimide reacted with 1.5 molar equivalent of 30% peroxide at room temperature with TLC monitored the reaction progress to obtain 6-methylsulfinlyhexyl phthalimide and with overnight cool down and the yield was only 45%.

Step4: This step presented major significant difficulty for this project. I failed twice at this step and spent more than 8 weeks time to overcome this step. 6–methylthiohexyl phthalimide reacted with 1.2 molar equivalent hydrazine monohydrate at 0 °C overnight to obtain 6–methylsulfinylhexyl amine. Since, 6–methylsulfinylhexyl amine doesn’t have aromatic group, it could not be detected by TLC method instead we used NMR to roughly detect the aimed product. Also, since this product doesn’t have aromatic group, the usual column chromatography couldn’t be used. Through research, I found reduced pressure distillation was the ideal solution for this step’s purification. By distillation, I had purified 6–methylsulfinylhexyl amine yield 19%.

Step5: The racemic mixture of 6–methylsulfinylhexyl amine reacted with Thiophosgene (CSCl2), would give final product 6-methylsulfinylhexyl amine. However, the experiment condition was improperly controlled, thus I didn’t have successful result from this step yet. This step requires highly hydrophobic or anhydrous condition. Some sodium hydroxide solution was been misused and caused no yield for this step’s experiment. The expected result was the MS-ITC light yellowish liquid product would be obtained, and when treated with steric acid such as D-(-) tartaric acid, it will bind the (+) configuration of 6–methylsulfinylhexyl amine and precipitate it out. Alternatively, it treat the racemic mixture with L-(+)tartaric acid, the (-)6–methylsulfinylhexyl amine will be precipitated; thus, separation of R and S configuration of MS-ITC can result.

Further study can include the structural effect of the two different configurations of 6–methylsulfinzlhexyl isothiocyanate on its biological activities. In addition, future work can then be proposed to test their antibacterial, antioxidative and antiplatelet aggregation properties according to the known assays. This will help to know is the S configuration has the same, more or less biological activity as its R configuration of MS-ITC. Also, some alternative synthetic route can be researched to improve the yield for the step 4 reaction.

I attempted total three times during this fall semester to synthesize MS-ITC. For the first attempt I didn’t start with big enough reactant scale, and then all the product was lost when reactions were carried to later steps. For the second attempt, the optimal condition for the reduced pressure distillation was not known; thus, almost all the step 3 product was spent to just find the optimal condition for the distillation such as to find the temperature and pressure.

Overall, this was an invaluable learning experience for me. I have never done any project like this before and this project enhanced my BYU education greatly. Because of this project, I learned how to gather research information and apply laboratory skills and knowledge to carry out experiment reactions. To see all these chemical reaction phenomena and learn the principle behind those reactions is extremely helpful for gaining insights of chemical reaction. Also, because of this project, I had the chance to learn how to use some analytic equipment such as NMR and Mass Spectrometer. I am graduating in the summer and applying for graduate school, and the experience gain from this project will give me great advantage for preparing me to go to graduate school, and I thanks ORCA for funding me this project and giving this wonderful opportunity to gain experience that will certainly benefit me in my career.