Alcohol dehydrogenase-catalyzed in vitro oxidation of anandamide to N-arachidonoyl glycine, a lipid mediator: synthesis of N-acyl glycinals

Abstract

N-Arachidonoyl ethanolamide or anandamide is an endocannabinoid found in most tissues where it acts as an important signaling mediator in a number of physiological and pathophysiological processes. Consequently, intense effort has been focused on understanding all its biosynthetic and metabolic pathways. Herein we report human alcohol dehydrogenase-catalyzed sequential oxidation of anandamide to N-arachidonoyl glycine, a prototypical member of the class of long chain fatty acyl glycines, a new group of lipid mediators with a wide array of physiological effects. We also present a straightforward synthesis for a series of N-acyl glycinals including N-arachidonoyl glycinal, an intermediate in the alcohol dehydrogenase-catalyzed oxidation of anandamide.

Figure 1

Chromatographic and mass spectral fragmentation analysis of standard and ADH7-synthesized N-arachidonoyl glycinal. LC/MS/MS MRM chromatograms of standard and ADH7-synthesized N-arachidonoyl glycinal (A and B). MS/MS spectra of standard and ADH7-synthesized N-arachidonoyl glycinal (C and D).

Figure 2

Chromatographic and mass spectral fragmentation analysis of standard and ADH7-synthesized N-arachidonoyl glycine. LC/MS/MS MRM chromatograms of standard and ADH7-synthesized N-arachidonoyl glycine (A and B). MS/MS spectra of standard and ADH7-synthesized N-arachidonoyl glycine (C and D).

Figure 3

Time course for the formation of N-arachidonoyl glycinal (A) and N-arachidonoyl glycine (B) by human ADH7. Anandamide (8 μM) and NAD⁺ (2.4 mM) were incubated with human ADH7 (56 nM) in 100 mM sodium phosphate buffer, pH 7.4 (3 ml) at 37 °C for the times indicated. Aliquots (100 μL) were withdrawn, diluted with acetonitrile (100 μl) and analyzed by LC/MS/MS using MRM methods.

Scheme 1. Synthesis of N-acyl-(1,2-dihydroxypropyl)amides (1a-1d) and N-Acyl glycinals (2a-2d)