Triphenylphosphine

All reagents were used as received, without further purification. Hydrogen tetrachloroaurate(III) tetrahydrate (HAuCl₄·4H₂O, 99.0%) was purchased from Kanto Chemical Co., Inc. Silver(I) nitrate (AgNO₃, >99.9%) was purchased from Kojima Chemicals Co., Ltd. Tetrabutyl ammonium bromide {TBABr, [N(C₄H₉)₄]Br, >98.0%}, triethylamine [N(C₂H₅)₃, >99.0%], tetrakis(triphenylphosphine)palladium(0) [Pd(PPh₃)₄, >97.0%], and tetrakis(triphenylphosphine)platinum(0) [Pt(PPh₃)₄, >97.0%] were purchased from Tokyo Chemical Industry Co., Ltd. Sodium borohydride (NaBH₄, 95.0%), triphenylphosphine (PPh₃, 97.0%), 12 molybdo(VI) phosphoric acid n-hydrate [H₃(PMo₁₂O₄₀)·nH₂O, >95.0%], disodium molybdate(VI) dihydrate (Na₂MoO₄·2H₂O, >99.0%), disodium tungstate(VI) dihydrate (Na₂WO₄·2H₂O, 99.0%–100.5%), hydrochloric acid (HCl, 35.0%–37.0%), and acetic anhydride [(CH₃CO)₂O, >97.0%] were purchased from Wako Pure Chemical Industry.

13(S)-HPOs of linoleic and linolenic acid were prepared with partially purified soybean seed LOX-1 as reported previously^{39 (link)}. The HPOs were purified to remove fatty acids with silica gel chromatography (Wakogel C-300, Wako Pure Chemicals, Osaka, Japan) with the solvent system of hexane/ether. Reduction of the HPOs to corresponding hydroxides was carried out by the addition of a large excess of triphenylphosphine (Wako Pure Chemicals). Conversion of the HPOs was monitored by following absorption at 234 nm with a spectrophotometer. In a typical assay, 2 µL of 10 mM 13S-HPOT in ethanol was added into 1 mL of 50 mM sodium borate buffer (pH 9.0) containing an appropriate amount of enzyme solution. Formation of the corresponding oxo-fatty acid was monitored by reading absorption at 280 nm. After the reaction, the mixture was acidified to pH 4.0 with perchloric acid, and the products were extracted with ether. The ether extract was dried with nitrogen gas. The resulting residue was dissolved in 100 µL of hexane/2-propanol/acetic acid (98:2:0.05 by volume) for HPLC analysis as follows.