Studies of nitrile oxide cycloadditions, and the phenolic oxidative coupling of vanillin aldoxime by Geobacillus sp. DDS012 from Italian rye grass silage

Abstract

During studies directed towards the discovery of nitrile hydrolysing enzymes from thermophiles, vanillin aldoxime was incubated with the thermophilic organism, Geobacillus sp. DDS012 isolated from Italian rye grass (Lolium multiflorum) silage. The predominant product was a dihydro-dimer, which could only be characterised by LC-MS. This was initially imagined to be the product of cycloaddition of vanillin aldoxime with the corresponding nitrile oxide, but preparation of the supposed adduct and model studies excluded this possibility. The rate constant for the second order dimerisation of 4-O-acetyl vanillin nitrile oxide was measured (1.21 × 10⁻⁴ M⁻¹ s⁻¹, 0.413 M, 25 °C) and the ¹³C-NMR signal for the nitrile oxide carbon was observed (δ_C 34.4, br. t ¹J¹³C,¹⁴N circa 50 Hz). Treatment of vanillin aldoxime with potassium persulfate and iron sulfate gave material with the same LC-MS properties as the natural product, which is therefore identified as 5,5′-dehydro-di-(vanillin aldoxime) 1d formed by phenolic oxidative coupling.