Synthesis of glutathione ester 【University of Florida】

This procedure for preparation of monoethyl GSH-ester is based on that described by Anderson and Meister, 1989 (Anal. Biochem. 183:16-20).

1. Add 100 ml anhydrous ethanol to a 1 1 round-bottomed flask and then add 2.74 ml of sulfuric acid.

2. Add 10 g of reduced GSH (Sigma; G4251; MW=307.3) to flask. Place stopper on flask and shake in a warm water bath (31 to 34 C) for at least 9 h.

3. Cool mixture on ice and treat with 700 ml ice-cold anhydrous diethyl ether. Allow the precipitate that immediately forms to settle by placing the flask at 4 C overnight (4-16h).

4. Remove the supernatant and wash precipitate with ether. Partially dry the ether by gently blowing nitrogen over the flask. Dry further by placing flask in a vacuum desiccator containing P₂0₅.

5. Dissolve the dried product in 15 ml water and add 25 ml ethanol. Crystallization will begin at this time. Allow crystallization to continue for 18-36 h at 4 C.

6. Filter crystals through a sintered glass funnel. Wash filter with cold ethanol followed by ether.

7. Place solution in a vacuum desiccator over P₂0₅.

8. Store monoethyl GSH ester in a desiccator at room temperature. The resulting ester (GSH monoethyl ester 1/2 H₂S0₄) has a MW of 402.3.