6-d-glucopyranosyl Fatty Acid Esters from Brassica Napus Pollen

6-D-Glucopyranosyl esters of palmitic, oleic, linoleic and linolenic acids were identified in Brassica napus (rape) pollen. These esters are inactive as plant growth promoters in the bean second-internode bioassay. acids as oleic. linoleic and linolenic acids. The identitv of o-glucose in hydrolysates of esters subsequently isolated by PLC was established by TLC, GLC and colour reaction. Individual glucosyl esters were separated by PLC on AgN0 3-Si gel. Although the layers exhibited gray streaks indicating reduction of Ag ion, the reaction was only partial. Because reduction of Ag ion was not observed with synthetic 1and 2-substituted glucosyl esters, an alternate site of fatty acid attachment in the natural material was indicated. Three bands were removed which afforded the following major glucosyl esters in order of decreasing R (: palmitate, linoleate and linolenate. Alternatively. glucosyl esters could be separated by reversed phase HPLC on ,u-Bondapak C 18 , Esters were eluted in the order: linolenate, linoleate. palmitate and oleate (contaminated with palmitate). HPLC ofa biologically active fraction that also contained glucosyl esters resulted in elution of material that exhibited brassin growth-promoting activity well before the elution of glucosyllinolenate. GC-MS of TMSi derivatives of natural glucosyl esters (see Experimental) gave ions for M + minus Me and/or one and two TMSiOH groups. In addition An iso-PrOH extract of H,O washed Brassica napus to acylium ions (RCO+) indicative of the fatty acid, (rape) pollen was fractionated by countercurrent distribuions corresponding to rearranged RCO,DMSi + were tion (CCD) and column chromatography on Si gel. also observed. The derivatized esters exhibited two The bean second-internode bioassay was used to follow GLC peaks of variable intensity representing the fractionation. Rechromatography of those fractions exanomeric mixture; the first peak due to the fl-anomer hibiting significant brassin-type activity afforded an (see below) was usually less intense. The MS of the first inactive major fraction which was eluted just prior G LC peak exhibited strong ions at m/e 217 (100 %) and to the growth-promoting material. The IR (Nujol) 204 (ca 50 %) while the second peak showed these spectrum of the inactive fraction showed ester absorption ions with the intensities reversed [m/e 217 (50 %), at 1730 cm 1. Transesterification with NaGMe gave 204 (100 %)J. These ions represent threeand twoMe esters which were analyzed by GLC on the basis carbon fragments from the carbohydrate ring [5]. of equivalent chain length. The following fatty acid In comparison, MS of synthetic TMSi-I-glucopyranosyl Me esters were characterized (percent composition palmitates showed the ion at m/e 217 three times stronger given in parentheses): 14: 0(0.6), 16: 0(39), 18: 0(0.8), than the 204 ion. 18: 1(3), 18:2(11.8), 18:3(44.8). Assignments were conThe 90 MHz PMR spectrum (CsDsN) of natural firmed by GC-MS which showed the appropriate glucosyl palmitate showed it to be an anomeric mixture M + for each ester. Double bond positions in unsaturated of 75 % C( (<5 5.88, J = 3.5 Hz): 25 % fl (<55.32, J = 7.0 Hz). esters were established by GC-MS of TMSi derivatives In contrast, anomeric protons for synthetic 1and 2of the alcohols obtained by per-hydroxylation with glucopyranosyl palmitates [6. 7J were observed as OsO4 [4]. These procedures identified the unsaturated follows: I-C(, <5 6.92 (J = 3.5 Hz): I-fl, <56.41 (J = 6.7 Hz):