Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers.

Since the original work by Sweeley et al. (1) the silylation and subsequent gas-liquid chromatography of simple sugars has been well documented (2–5). The silylation of their biochemically important derivatives, notably sugar phosphates, has proven more difficult. Wells et al. (6) premethylated the phosphate with diazomethane in methanol before silylating the sugar group with the usual reagents [pyridine, hexamethyldisilazane (HMDS), and trimethylchlorosilane (TMCS)], Hashizume and Sasaki (7) refluxed the sugar phosphates in the reaction mixture for 1 hr to effect complete silylation, while Eisenberg and Bolden (8) achieved similar results by heating in a sealed vial at 100°C for 10 min. The latter researchers noted that glucose 1-phosphate tended to decompose under their conditions to give one major and two minor peaks. Horning et al. (9) and, later, Sherman et al. (10) used bis(trimethylsilyl)acetamide (BSA) to silylate sugar phosphates, but Pierce (11) has noted that BSA tends to give inconsistent silylation of simple sugars and we have also noted this in our work.In 1969, Ellis (12) reported on the use of a silylation system using dimethylsuifoxide (DMSO) or dimethylformamide (DMF) as the reaction solvent rather than pyridine. The silylated derivatives are not soluble in either of these solvents and they form a second, upper liquid layer (in 10 min in the case of DMSO, 18 hr for DMF). The silylated sample is thus concentrated and this avoids the necessity of removing the solvent [with its attendant problems (10)] after silylation.This paper reports a modification and extension of the silylation system described by Ellis which allows rapid, quantitative silylation of sugars and sugar phosphates at room temperature, i.e., the addition of cyclohexane to the silylation mixture to promote the silylation of sugar phosphates and other biologically important acids. Also reported is the first use of SE-52 stationary liquid phase for the glc of sugar phosphates.