Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades

D‐Luciferin (D‐LH2), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D‐LH2 analogues with ≈51 % yield. The method can synthesize the novel 5′‐methyl‐D‐LH2 and 4′,5′‐dimethyl‐D‐LH2, which have never been synthesized or found in nature. 5′‐Methyl‐D‐LH2 emits brighter and longer wavelength light than the D‐LH2. Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment. Enzymatic reactions were developed to convert 19 phenolic derivatives containing halogen, nitro, and methyl substituents into eight D‐luciferin analogues, including the novel compounds 4′,5′‐dimethyl‐D‐luciferin and 5′‐methyl‐D‐luciferin, which emit brighter and longer‐wavelength light than the native D‐luciferin. These reactions enabled the further development of luminescence technology for the sensitive detection of organophosphate pesticides.