A Modular Library of Small Molecule Signals Regulates Social Behaviors in Caenorhabditis elegans

Comparative metabolomics reveals a modular library of small molecule signals that function as aggregation pheromones in the nematode C. elegans.

Result: there are 18 icas#3 molecules contained in one worm volume of agar at an icas#3 concentration of 10 fM (femtomolar).

Synthesis of indole ascarosides
2.1 Synthesis of ascr#9. (R)-hex-5-en-2-ol 2 (32 mg, 0.32 mmol, prepared as described [2]) was coupled to trichloroacetimide 1 (150 mg, 0.3 mmol, [2]) using the conditions described for the synthesis of ascr#6. The resulting glycoside 3 (92 mg, 0.21 mmol) was dissolved in acetone (2 ml) and treated with 2 ml of a 1 M solution of potassium permanganate. After 30 min, the reaction mixture was poured into a mixture of ice-cold saturated aqueous sodium chloride solution (5 ml), acetic acid (0.1 ml), and dichloromethane (5 ml). The organic phase was separated and the aqueous phase extracted with two 5 ml-portions of dichloromethane. The combined organic extracts were dried over sodium sulfate, evaporated to dryness and re-dissolved in a mixture of 0.5 M aqueous lithium hydroxide (2 ml) and dioxane (6 ml). The mixture was stirred for 3 h at 70 ºC, then cooled to 23 ºC and acidified with 0.2 M aqueous hydrochloric acid. The mixture was evaporated to dryness and purified via Combiflash column chromatography using a methanol-dichloromethane solvent gradient, yielding 15.6 mg (0.063 mmol) of pure ascr#9 as a viscous oil.
Spectroscopic data for ascr#9. 1 3; 71.5, 71.4, 69.9, 68.4, 36.0, 33.5, 31,3, 19.1, 18 A stirred solution of ascr#3 (3.2 mg, 10.6 µmol) [2] in 10 ml of ethanol was hydrogenated using palladium on activated carbon (10% Pd, 1 atm H 2 pressure) at 23 ºC for 18 h. After completion, the reaction was evaporated to dryness, and the residue filtered over a short pad of silica using a 1:8 (v/v) mixture of methanol and dichloromethane yielding 3.0 mg (9.9 µmol) of pure ascr#10. Conversion of ascr#1 into the corresponding methyl ester. Ascr#1 (10 mg, 0.036 mmol), prepared using previously described methods [2], was dissolved in a mixture of toluene (1 mL) and methanol (1 mL Subsequently, the above solution of indole-3-carboxylic acid chloride was added drop wise over a period of 10 min with vigorous stirring. The well-stirred reaction was gradually warmed to -7 ºC at which temperature ice-cold saturated aqueous sodium bicarbonate solution (2 ml) was added. The biphasic mixture was allowed to warm to 20 ºC and extracted three times with ethyl acetate. The combined ethyl acetate extracts were evaporated in vacuo and subjected to column chromatography on silica gel using 0-10% methanol in dichloromethane.
Fractions containing the bis-2,4-O-(-indole-3-carbonyl)-derivative of the ascr#1 methyl ester were combined, evaporated to dryness and treated with a mixture of 3 ml aqueous 0.5 M lithium hydroxide solution and 7 ml dioxane at 67 ºC for 3 h. Subsequently, the reaction mixture was cooled to 23 ºC, neutralized by addition of 0.2 M aqueous hydrochloric acid and evaporated in vacuo. The residue was purified by HPLC, using the Agilent 1100 Series HPLC system equipped with an Agilent Eclipse XDB C-18 column (25 cm x 9.4 mm, 5 µm particle diameter). Acetonitrile and 0.1% aqueous acetic acid were used as solvents, increasing the percentage of acetonitrile from 15% at 0 min to 85% at 30 min. Icas#1-containing fractions were evaporated yielding 5.8 mg (0.014 mmol)) of the target compound as a wax-like white solid.
Coupling constants are given in Hertz [Hz].

Synthesis of icas#9.
O Icas#9 was obtained from ascr#9 as described above for the preparation of icas#1 from ascr#1. NMR-spectroscopic data are in agreement with published data [3].