Cystamine hydrochloride was added to dioxane, and triethylamine was added to prepare a mixed solution. Maleic anhydride was dissolved in the mixed solution under stirring for 23 hours, and then the solvent was distilled off under reduced pressure. The residue was dissolved in a 60% sodium carbonate solution, and the resulting solution was washed three times with diethyl ether and once with ethyl acetate. Water was added under ice cooling to adjust the pH to 2, and after cooling for 1 hour, a precipitate was formed. The material was dried under vacuum, and then acetic anhydride was dissolved. Sodium acetate was added to the obtained solution, and the mixture was heated and stirred at 100°C for 2 hours under a nitrogen atmosphere. After returning to room temperature, ice water was added, and the mixture was stirred for 2 hours while cooling. Then, excess water and diethyl ether were added to extract the reaction product into the ether layer. After washing the ether layer with sodium carbonate solution, it was dried with anhydrous sodium sulfate, and the ether was distilled off under reduced pressure to give the product oil. Purification was performed by silica gel chromatography (using Merck Kiesel gel 60, eluting with benzene-ethyl acetate at a ratio of 5:1 to 1:1), and the solvent was evaporated from the solvent at a considerable rate of 7 under reduced pressure to obtain DITHIO-BIS-MALEIMIDOETHANE.
DTME is a homobifunctional, maleimide crosslinker for conjugation between sulfhydryl groups (-SH). Such bismaleimide crosslinkers are commonly used to explore and characterize protein structure (i.e., oligomerization) or protein interactions. DTME is similar in length to BMH but differs in containing a disulfide bond in its spacer arm, allowing crosslinks with DTME to be cleaved with reducing agent such as dithiothreitol (DTT).
