Synthesis of GSH-MDI Conjugate

Reduced glutathione, GSH (CAS # 70-18-8), and 4,4’-methylenebis(phenyl isocyanate) or MDI (CAS # 101-68-8) were from Sigma-Aldrich (St. Louis, MO) and were of ≥ 98.0% purity. GSH was reacted with MDI as previously described (Wisnewski, Liu et al. 2013 (link)). Briefly, 50 µl of 10% (w/v) MDI in acetone from JT Baker (Phillipsburg, NJ) was added dropwise with stirring to 25 ml of 10 mM GSH in 200 mM sodium phosphate, pH 7.4 (final acetone concentration 0.2% v/v). The reaction mixture was rotated end-over-end for 2 hours at 37°C, and then centrifuged at 10,000 g, and 0.2 µm filtered before use.