Conjugation of Aromatic Compounds in Some Invertebrates

Abstract

Insects from a variety of orders were surveyed for phosphate and glucoside phosphate conjugates after p-nitrophenol was administered. It was observed that out of the ten species of insects investigated, nine produced phosphate conjugates of p-nitrophenol. Glucoside phosphate conjugation was less common and detected in five insect species. In all the insects investigated, p-nitrophenyl-β-glucoside was the major metabolite and was easily detected. p-Nitrophenyl sulphate was also detected in five of the insect species investigated. From the results of this survey e mealworms Tenebrio molitor, were used to produce large amounts of metabolites. Crystals of the biosynthetic p-nitrophenyl-β-glucoside and p-nitrophenyl phosphate were successfully obtained. The nature and structure of these compounds were determined and confirmed by comparison with authentic reference compounds using spectroscopic methods such as infrared and nuclear magnetic resonance analysis. Large amounts of the biosynthetic glucoside phosphate of p-nitrophenol were obtained but attempts to obtain this compound in crystalline form as the cyclohexylamine salt were unsuccessful. The synthesis of the reference p-nitrophenyl glucoside-6-phosphate was also attempted using the method as described by Heenan and Smith (1974). It was observed that repeats of this method produced two different glucoside phosphates. Nuclear magnetic resonance spectral analysis of the compounds obtained has revealed that one compound was the glucoside-6-phosphate while the other was the glucoside-4-phosphate of p-nitrophenol. In addition to the glucoside, phosphate and glucoside phosphate obtained, a new metabolite (metabolite X) was also isolated. The metabolite was identified by ionophoresis, paper chromatography, acid and alkali hydrolysis. The results obtained suggested that metabolite X was the β-glucoside sulphate conjugate of p-nitrophenol. The metabolite was not obtained in crystalline form. Attempts at the synthesis of the reference glucoside sulphate by the method of Dodgson and spencer (1954) produced a mixture of compounds. These were tentatively identified as the p-nitrophenyl-β-glucoside-6-sulphate and a disulphate. The formation of phosphate conjugates was not limited to p-nitrophenol. Phosphate conjugation was also present when other phenols were used. These included α-naphthol, 8-hydroxyquinoline and 4-methylumbelliferone. Detection of the glucoside phosphate and the new metabolite X of these other phenols was more difficult. The presence of these strong acidic conjugates (phosphate, glucoside phosphate, metabolite X) was also examined when 14[C] benzoic acid was administered to these insects. It was observed that these strong acidic conjugates were completely absent in these insects. In the case of mealworms, blowflies and houseflies, only weakly acid metabolites were obtained. Hippuric acid was found in this fraction together with other unidentified metabolites. In the other insects, a neutral metabolite fraction was also isolated in addition to the weakly acid metabolites. However, no attempts were made to identify the metabolites in this neutral fraction.