The Identification of Gamma-Butyrolactone in JUUL Liquids

Gamma-Butyrolactone (GBL), a commonly used industrial solvent, is used recreationally as a central nervous system (CNS) depressant and, therefore, a United States Drug Enforcement Agency (DEA) List 1 chemical of the Controlled Substances Act. GBL was identified presumptively in the liquid from JUUL Virginia Tobacco flavored pods during routine untargeted screening analysis of e-cigarette products by gas chromatography–mass spectrometry (GC–MS). Methods for the confirmation and quantitation of GBL were developed for GC–MS and liquid chromatography–tandem mass spectrometry (LC–MS-MS) in the liquids and the aerosol generated from the liquid. Three flavors of JUUL pods available at the time of analysis were obtained by direct purchase from the manufacturer, purchase from a local vape shop, and via submission from a 3rd party. The only liquid flavor to contain GBL was Virginia Tobacco, with an average of 0.37 mg/mL of GBL, and it was detected in the aerosol. Studies evaluating the pharmacological effects of inhaling GBL do not exist, however a case report of chronic oral GBL ingestion indicates acute lung injury. The identification of GBL in an e-cigarette product purportedly compliant with federal regulation continues to demonstrate public health and public safety concerns.

(Bio-)Synthesis of the Aquatic Phytotoxin Cyanobacterin – A Paradigm for Furanolide Core Structure Assembly

The gamma-butyrolactone structural motif is commonly found in many natural signaling molecules and other specialized metabolites. A prominent example is the potent aquatic phytotoxin cyanobacterin bearing a highly functionalized gamma-butyrolactone core structure. The enzymatic machinery assembling cyanobacterin and the many structurally related natural products – herein termed furanolides – has remained elusive over decades. Here we discover and characterize the underlying biosynthetic process of furanolide core structure assembly. The cyanobacterin biosynthetic gene cluster (<i>cyb</i>) is identified by targeted bioinformatic screening and validated by heterologous expression in <i>E. coli</i>. Functional evaluation of the recombinant key enzymes provides in-depth mechanistic insights into a streamlined <i>C</i>,<i>C</i>-bond-forming cascade that involves installation of compatible reactivity at seemingly unreactive C-alpha-positions of the amino acid precursors and facilitates development of a one-pot biocatalytic <i>in vitro</i> synthesis. Our work extends the biosynthetic and biocatalytic toolbox for gamma-butyrolactone formation. It thereby provides a general paradigm for the biosynthesis of furanolides and thus sets the stage for their targeted discovery, biosynthetic engineering and enzymatic synthesis.

(Bio-)Synthesis of the Aquatic Phytotoxin Cyanobacterin – A Paradigm for Furanolide Core Structure Assembly

The gamma-butyrolactone structural motif is commonly found in many natural signaling molecules and other specialized metabolites. A prominent example is the potent aquatic phytotoxin cyanobacterin bearing a highly functionalized gamma-butyrolactone core structure. The enzymatic machinery assembling cyanobacterin and the many structurally related natural products – herein termed furanolides – has remained elusive over decades. Here we discover and characterize the underlying biosynthetic process of furanolide core structure assembly. The cyanobacterin biosynthetic gene cluster (<i>cyb</i>) is identified by targeted bioinformatic screening and validated by heterologous expression in <i>E. coli</i>. Functional evaluation of the recombinant key enzymes provides in-depth mechanistic insights into a streamlined <i>C</i>,<i>C</i>-bond-forming cascade that involves installation of compatible reactivity at seemingly unreactive C-alpha-positions of the amino acid precursors and facilitates development of a one-pot biocatalytic <i>in vitro</i> synthesis. Our work extends the biosynthetic and biocatalytic toolbox for gamma-butyrolactone formation. It thereby provides a general paradigm for the biosynthesis of furanolides and thus sets the stage for their targeted discovery, biosynthetic engineering and enzymatic synthesis.