Alborn et al. 10.1073/pnas.0705947104. |
Fig. 5. Negative ion electrospray analyses of AF1 (caeliferin A16:1) and AF2 (caeliferin A16:0) with repeated daughter ion analyses.
Fig. 6. Isobutane CI GC/MS chemical analyses of AF1 (caeliferin A16:1) (A) and AF2 (caeliferin A16:0) (B) after methanolysis. Analyses of the corresponding acetylated methylester of AF1 (C) and AF2 (D).
Fig. 7. Electron impact GC/MS analyses of AF1 (caeliferin A16:1) (A) and AF2 (caeliferin A16:0) (B) after methanolysis.
Fig. 8. Schematic synthesis of 2,16-dihydroxy-E 6-hexadecenoic acid.
Fig. 9. Schematic synthesis of 2,16-dihydroxy-hexadecanoic acid.
SI Text
Analysis 1.
Negative ion electrospray analyses of AF1 (caeliferin A16:1) with (M-1)- ions at m/z 445 (Fig. 5A, and AF2 (caeliferin A16:0) with (M-1)- ions at m/z 447 (Fig. 5B). Daughter ions of both parent ions (MS2) gave for both compounds a neutral loss of 80 amu yielding strong ions at m/z 365 and 367 respectively. The neutral loss of 80 amu was repeated with MS3 analyses indicating that both compounds lost 2 x S03 resulting in 2,16-dihydroxypalmitoleic acid with (M-1)- ions at m/z 285 and m/z 287 for 1,16-dihydroxypalmitic acid respectively.
Analysis 2.
Isobutane CI GC/MS chemical analyses of AF1 (caeliferin A16:1), and B: AF2 (caeliferin A16:0) after methanolysis (and subsequent loss of the sulfooxy esters) (Fig. 6 A and B) gave strong (M + 1)+ ions at m/z 301 and 303 respectively. The different combinations of a consecutive neutral loss of 60 amu (-CH3OCOH), and 2 ´ 18 amu (-H2O), indicating alpha cleaving of an 2-hydroxy acid methylester, and the subsequent loss of 2 alcohols. Analyses of the corresponding acetylated methylesters (Fig. 6 C and D) shows (M + 1)+ ions at m/z 385 and 387 respectively which corresponded to the expected addition of 2 acetates (+2 ´ 42 amu). The fragmentation pattern shows a consequent loss of 3 ´ 60 amu (-CH3OCOH and -2x CH3COOH) but also the, for a normal carboxylic acid methyl ester, loss of 32amu (-CH3OH) is prominent.
Analysis 3.
Electron impact GC/MS analyses of AF1 (Caeliferin A16:1) (Fig. 7A) and AF2 (Caeliferin A16:0) (Fig. 7B) after methanolysis (and subsequent loss of the sulfooxy esters). Note the neutral losses of 59 amu for both compounds ( m/z 300-241 and m/z 302-243) indicative of a 2-hydroxy carboxylic acid methylesters. For AF2 there is also a clear neutral loss of 30 amu (m/z 302-272) indicating a loss of CH2O after long range proton transfer to the carbonyl and consequently the second hydroxyl was determined to be at the w position. The lack of a similar fragment for AF1 is probably due to the presence of the trans double bond.
Analysis 4.
The synthesis of 2,16-dihydroxy-E 6-hexadecenoic acid (SI Fig. 8) started with the ethoxyethylether derivative of 1-undecyne-11-ol 1. The protected undecynol was treated sequentially with n-butyl lithium, 1.2 equivalents of oxetane and boron trifluoride etherate according to the literature (1) to provide the alkynol 2 as the major product. The acetylene bond was reduced to the trans double bond using Red-Al in hot diglyme, and the resulting alcohol was oxidized to the unsaturated aldehyde 3 with pyridinium dichromate. Treatment of 3 with isopropyl dichloroacetate in the presence of potassium isopropoxide, and subsequent reduction with sodium cyanoborohydride in the manner described (2) provided the protected 2-hydroxy ester 4. The protected product was base hydrolyzed, and acidified to give 2,16-dihydroxy-E 6-hexadecenoic acid 5. It was desalted on a C18 sample prep column and then dried completely under N2 before sulfating as described below.
The synthesis of 2,16-dihydroxy-hexadecanoic acid (SI Fig. 9) was based on the methanolysis of commercially available w-pentadecalactone (Sigma Aldrich) 6 under acidic conditions to provide 15-hydroxy-methylpentadecanoate 7. The TBDS protected hydroxy aldehyde 8 was obtained by sequentially protecting the alcohol with tert-butyldimethylsilyl chloride (TBDMSCl) (3), carefully hydrolyzing the ester, reducing the resulting acid with borane methysulfide, followed by Swern oxidation to provide 15-OTBDMS pentadecanal. Treatment of 8 with trimethylsilyl cyanide (TMS-CN) (4, 5) provided the cyanohydrin trimethylsilyl ether which was hydrolyzed to an intermediate 2, 16-hydroxy amide (6). The amide was steam distilled under strong basic conditions to afford the 2,16-dihydroxy hexadecanoic acid 9.
Production of the sulfate esters was accomplished using chlorosulfonic acid (7), using dry acetonitrile as the solvent and less reagent than necessary for a complete reaction.
Dry di-hydroxy acids from the two syntheses above were partially dissolved in dry acetonitrile. While stirring, 3 molar equivalents of chlorosulfonic acid were added, and the solution stirred until clear which normally took less than 1 min. A large volume of water (10´ the original volume) was added and the solution adjusted to pH 8 by slow drop wise adding of a 1M sodium hydroxide solution. The solution was concentrated to approximately half the volume and desalted on a C18 sample prep column as described above. The intentional use of an insufficient amount of chlorosulfonic acid left an intact double bond for caeliferin A16:1 but also resulted in mixtures of mono- and di-sulfate esters that were separated using C18 reverse phase LC/ MS with a solvent split that allowed 90% of the injected material to be collected for bioassaying simultaneously with MS detection.
Analysis 5.
Glass chambers (30 cm long ´ 4 cm i.d.) were used for volatile collection. The chambers were placed under the same type and intensity of light as used in the rearing. A push/pull volatile collection system was used. Humidified and purified air (570 ml/min) was passed over the plants through the glass chamber and drawn by vacuum through an adsorption filter containing 25 mg of a polymeric adsorbent, Super Q 80/100 (catalog no. 2735 Alltech Associates, Deerfield, IL) positioned at the downwind end of the chamber. Volatiles were collected for 2 h for the cut stem assays and 1 h for the intact plant assays. The filters were then removed and extracted with 170 ml methylene chloride. Five ml of an 80 ng/ml solution of n-nonyl-acetate was added as an internal standard and the samples injected on a gas chromatograph (HP 6890, Hewlett Packard Palo Alto, CA) by splitless injection at 220°C. The methyl silicone column, (HP1, 15 m ´ 0.25 mm I.D. ´ 0.1 mm film thickness, Hewlett-Packard) was kept at 40°C for 0.5 min and then temperature programmed 12°C /min. to 180°C. The He carrier gas flow rate was 40 cm/sec. (constant flow), and the detector temperature was 250°C.
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