Simplified Flow Photosynthesis of Deuterium-Labeled Pyocyanin.

Abstract

Deuterium-labeled pyocyanin was prepared from deuterium-labeled phenazine methosulfate in gram scale by a simplified flow photosynthesis in water. The main product was the protonated red form of pyocyanin-d₃ (Pyo-d₃-H⁺) in 85 % yield. Quantum chemical calculations of NMR support that nitrogen-10 is protonated. The by-products of the photolysis and the stability of the photolysis mixture were carefully characterized by LC-MS and NMR. Four by-products were identified: An isomer of pyocyanin-d₃ (9%), 8-hydroxypyocyanin-d₃ (4%), 1-hydroxyphenazine (0.4%), and phenazine (1%). The Pyo-d₃-H⁺ product was stable in the photolysis solution after storage at 8°C for 2.5 years. Pure blue pyocyanin-d₃ powder was isolated from the red photolysis solution by the Surrey method in 94 % yield. The addition of the red photolysis solution of Pyo-d₃-H⁺ (100 μM) and commercial pyocyanin (100 μM) to Pseudomonas aeruginosa cultures showed the same growth curves demonstrating that the minor impurities in the photolysis solution do not affect the growth behavior of the bacteria. The protonated deuterium-labeled pyocyanin may be used directly in biological experiments, which make the methodology extremely simple and useful for biologists.