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2‐methyl‐5,6,7,8‐tetrahydro‐2H‐chromen‐4(3H)‐one (called 6‐oxo) is presented as a new AI‐1 quorum sensing inhibitor for Vibrio harveyi. The development of a chemical process to afford traceable materials for new biological assays demands the development of analytical methods to ensure their purity and quality. This work describes the use of quantitative 1H nuclear magnetic resonance (NMR) spectroscopy (qNMR) to assess the purity of a sample of 6‐oxo (99.88%) and a sample of its major process impurity (E)‐1‐(2‐hydroxycyclohex‐2‐en‐1‐yl)but‐2‐en‐1‐one (called HCB; 98.28%). To explore the scope of the use of qNMR to quantify the amount of low‐content components in samples related to the chemical process for 6‐oxo synthesis, this work also determined the amount of 6‐oxo in two HCB samples: (a) the high‐purity HCB sample described above and (b) a crude HCB sample collected during the chemical process. Despite the complexity of the crude sample, the amount of 6‐oxo was readily assessed and could help to estimate the extent to which 6‐oxo was already formed during the HCB synthesis. This information can help the understanding of how the process parameters can be modified to improve the performance of the whole process, by controlling the reaction mechanisms working at each step of this chemical process. In this context, our results reinforce qNMR as a complementary analytical tool for the quantification of the main component found in a sample, contributing to the standardization of reference materials and thus allowing the development of analytical methods for process control and traceability of the samples used for biological assays. [ABSTRACT FROM AUTHOR]

[Display omitted] • Novel modulators of Quorum Sensing (QS) phenotypes in CV026. • Readily prepared from inexpensive starting chemicals. • Mode of action validated by competition assay and RT-qPCR. • Blind docking calculations found them at the autoinducer binding domain. • QS modulation at the transcriptional level in CV026. This work presents Morita-Baylis-Hillman adducts (MBHA) as new modulators of the violacein biosynthesis in the biosensor strain Chromobacterium CV026. Seven adducts displayed violacein reduction higher than 50±1% when tested at concentrations lower than 625 μM. The most active MBHA inhibited hydrolysis of chitin concomitantly with inhibition of violacein production in CV026, suggesting the disruption of its QS machinery. One MBHA induced the violacein biosynthesis in CV026 in the absence of the cognate AI, also supporting its role as a QS modulator of this QS system. Further, RT-qPCR and competition experiments characterize these two molecules as transcriptional modulators of the QS-regulated operon vioABCDE. Blind docking studies for MBHA showed a strong preference for adducts positioned within the CviR autoinducer-binding domain (AIBD). Our results show MBHA as a promising chemical scaffold for the development of novel QS inhibitors affecting LuxR/LuxI-systems. [ABSTRACT FROM AUTHOR]