Generation of High-Affinity Aptamers for Indazole Synthetic Cannabinoids.

Synthetic cannabinoids are a widely abused class of dangerous psychoactive substances, especially among youths and young adults. Dozens of such drugs have been identified to date, and new ones continue to emerge. The ability to detect these drugs is important for interdiction efforts and the diagnosis of drug overdose, but existing analytical methods lack broad cross-reactivity to diverse members of this drug family. Here, we have utilized library-immobilized SELEX to generate DNA aptamers that can broadly recognize various members of the indazole-3-carboxamide synthetic cannabinoid family. Using two representatives of this family, AB-FUBINACA and 5F-AMB, we identify two aptamers FUB4 and AMB2F with respective dissociation constants (KDs) of 138 ± 15 and 411 ± 20 nM for their targets. These aptamers can recognize many indazole-based synthetic cannabinoids with high affinity and excellent specificity against natural cannabinoids as well as other structurally similar interferents like serotonin and tryptophan. We use these two aptamers to develop fluorescence strand-displacement sensors that successfully detect these synthetic cannabinoids at concentrations as low as 50 nM in human serum. The sensors can also detect up to 14 different drugs from this family─a major improvement over the six recognized by an existing commercial immunoassay.

Decomposition of 2,4-dihalophenols by dehaloperoxidase activity and spontaneous reaction with hydrogen peroxide.

The enzyme dehaloperoxidase (DHP) found in the marine worm Amphitrite ornata is capable of enzymatic peroxidation of 2,4-dichlorophenol (DCP) and 2,4-dibromophenol (DBP). There is also at least one parallel oxidative pathway and the major products 2-chloro-1,4-benzoquinone (2-ClQ) and 2-bromo-1,4-benzoquinone (2-BrQ) undergo aspontaneous secondary hydroxylation reaction. The oxidation and hydroxylation reactions have been monitored by UV-visible spectroscopy, High Performance Liquid Chromatography (HPLC), and mass spectrometry. Evidence from time-resolved UV-visible spectroscopy suggests that the hydroxylations of 2-ClQ and 2-BrQ in the presence of hydrogen peroxide (H2O2) are non-enzymatic spontaneous processes approximately ∼10 and âˆ¼ 5 times slower, respectively, than the enzymatic oxidation of DCP or DBP by DHP in identical solvent conditions. The products 2-ClQ and 2-BrQ have λmaxat 255 nm and 260 nm, respectively. Both substrates, DCP and DBP, react to form a parallel product peaked at 240 nm on the same time scale as the formation of 2-ClQ and 2-BrQ. The 240 nm band is not associated with the hydroxylation process, nor is it attributable to the catechol 3,5-dihalobenzene-1,3-diol observed by mass spectrometry. One possible explanation is that muconic acid is formed as a decomposition product, which could follow decomposition either the catechol or hydroxyquinone. These reactions give a more complete understanding of the biodegradation of xenobiotics by the multi-functional hemoglobin, DHP, in Amphitrite ornata. SYNOPSIS: The decomposition of 2,4-dihalophenols catalyzed by dehaloperoxidase was studied by UV-visible spectroscopy, High Performance Liquid Chromatography and Liquid Chromatography-Mass Spectrometry. Spectroscopic evidence suggests two major products, which we propose are 2-halo-1,4-benzoquinone and 2-halomuconic acid. These complementary techniques give a high-level view of the degradation of xenobiotics in marine ecosystems.