The stereochemistry of alpha-oxidation of fatty acids in plants: the configuration of biosynthetic long-chain 2-hydroxy acids.

All natural long-chain 2-hydroxy acids that have been isolated and tested by polarimetry are optically active and have the D-configuration at C-2; in particular, lipids of pea (Pi8um sativum) leaves contain a small proportion ofD-2-hydroxypalmitate (Hitchcock, Morris & James, 1968b). This evidently accumulates after biosynthesis by a-hydroxylation of palmitate (Hitchcock & James, 1966; Morris & Hitchcock, 1968), though ac-oxidation of palmitate apparently involves preferential formation and oxidation of L-2-hydroxypalmitate (Hitchcock, Morris & James, 1968a). When [1-14C]palmitic acid is incubated with pea leaves or solutions of their acetone-dried powders, 2-hydroxy[1-14C]palmitate accumulates; the configuration of this metabolite cannot be determined directly, but appears to be the D-hydroxy acid by crystallization of the isolated hydroxy esters with authentic enantiomers (Hitchcock et al. 1968b). This finding has now been confirmed by converting the hydroxy esters into their L-acetylmandelates and resolving the derivatives by t.l. c. (Morris & Crouchman, 1971). Methods. (a) Preparation of L-acetylmandelyl chloride. L-Mandelic acid (1 g) and acetyl chloride (1.5g) were allowed to react at room temperature until a clear mixture was obtained (15min). The excess of acetyl chloride was removed in vacuo at 600C for 30min, and the residue was taken up in 25ml of benzene. Oxalyl chloride (2g) was added; after 2h at room temperature followed by refluxing for 1h, the solvent was removed in vacuo. The crude yellow oil was used without purification. (b) Preparation of methyl DL-2-(L-acetylmandelyloxy)palmitate. Methyl DL-2-hydroxypalmitate (20mg) was dissolved in 2ml of pyridine, and 40mg of crude L-acetylmandelyl chloride was slowly added. After 16h at room temperature, the reaction mixture was diluted with diethyl ether, and washed successively with aqueous hydrochloric acid until acid, aqueous sodium hydrogen carbonate until alkaline and water. The ethereal solution was dried (over anhydrous sodium sulphate) and evaporated; the required mandelates were isolatedl from the residue, and their subsequent purity was checked, by t.l. c. They were separated on t.l. c. plates of silica gel G by two developments with 3% (v/v) diethyl ether in benzene; under these conditions the R, values of the methyl esters are approximately as follows: 2-hydroxypalmitate, 0.42; L-2-(L-acetylmandelyloxy)palmitate, 0.56; D-2-(L-acetylmandelyloxy)palmitate, 0.72; palmitate, 0.85. (c) Biosynthesis of 2-hydroxy[1-_4C]palmitate. [1-14C]Palmitic acid was incubated with either freshly sliced pea leaves or acetone-dried powders therefrom, and the biosynthesized 2-hydroxy[1-14C]palmitate was isolated as the methyl ester as described by Hitchcock et al. (1968b). After dilution with unlabelled DL-2-hydroxypalmitate, the mandelates were prepared and separated as described above with excess of L-acetylmandelyl chloride. They were located by comparing with extemal markers and by scanning for radioactivity; the radioactivity of appropriate areas was determined by scraping off the adsorbent, eluting the esters with ether and measuring the radioactivity