2-Methoxybenzoic acid → Salicylic Acid

Participants

Reaction Conditions

Reaction Steps

Salicylic acid was extracted and analyzed by HPLC using a modification of the methods described in Meuwly et al., 1993. For salicylic acid analysis, 0.3 to 0.5 gFW leaf tissue from E. orontii-infected leaves (7 dpi) was frozen in glass tubes with liquid nitrogen and either used directly or stored at −80° C. The frozen tissue was ground in liquid nitrogen to a fine powder using a chilled glass rod. Three ml of 90% methanol and 250 ng o-anisic acid (internal standard) were added to each sample. Samples were vortexed, sonicated for 20 minutes, and centrifuged for 20 minutes at 3000 rpm in a table-top centrifuge. The supernatant was transferred to a new tube, and the pellet was re-extracted with 2 ml of 90% methanol. The two supernatants were combined, divided into two portions of equal volumes (for total SA and free SA measurements), vacuum dried, and frozen at −80° C. For total SA, 500 μl β-glucosidase (80 U/ml in 100 mM sodium acetate, pH 5.2; Sigma, St. Louis, Mo.) was added to each sample. The samples were sonicated for 5 minutes, vortexed, covered with foil, and incubated for 90 minutes at 37° C. For both total and free SA samples, 2.5 ml 5% trichloroacetic acid was added, and the samples were vortexed, sonicated for 5 minutes, and centrifuged at 3000 rpm for 15 minutes. The supernatant was extracted twice with 2.5 ml of a 1:1 (v/v) mixture of ethyl acetate:cyclopentane. The organic phases were combined, vacuum dried, and frozen at −80° C. Just prior to loading samples on the HPLC, each was resuspended in 250 μl of 20% methanol, vortexed, sonicated for 5 minutes, and filtered through a 0.22 μm nylon filter.

Provenance & Evidence