Hypersensitive quantification of major astringency markers in food and wine by substoichiometric quenching of silicon-rhodamine conjugates.

Tannins are chemically diverse polyphenols in plant-derived products that not only show diverse biological activities but also play a crucial role in determining the sensory attributes of food and beverages. Therefore, their accurate and cost-effective quantification is essential. Here, we identified a novel fluorescence quenching mechanism of different synthetic rhodamine fluorophores, with a high selectivity towards tannic acid (TA) and catechin-3-gallate (C3G) compared to a structurally diverse panel of tannins and polyphenols. Specific chemical conjugates of silicon-rhodamine with alkyl linkers attached to bulky apolar moieties had a limit of detection near 500 pM and a linear range spanning 5-100 nM for TA. We validated the assay on 18 distinct red wine samples, which showed high linearity (R2 = 0.92) with methylcellulose precipitation with no interference from anthocyanins. In conclusion, a novel assay was developed and validated that allows the sensitive and selective quantification of major astringency markers abundant in food and beverages.

Silicon-Rhodamine Functionalized Evocalcet Probes Potently and Selectively Label Calcium Sensing Receptors In Vitro, In Vivo, and Ex Vivo.

The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled receptor (GPCR) that regulates extracellular calcium signals via the parathyroid glands. CaSR has recently also been implicated in noncalcitropic pathophysiologies like asthma, gut inflammation, and cancer. To date, molecular tools that enable the bioimaging of CaSR in tissues are lacking. Based on in silico analyses of available structure-activity relationship data on CaSR ligands, we designed and prepared silicon-rhodamine (SiR) conjugates of the clinically approved drug evocalcet. The new probes EvoSiR4 and EvoSiR6, with differing linker lengths at the evocalcet carboxyl end, both showed a 6-fold and 3-fold increase in potency toward CaSR (EC50 < 45 nM) compared to evocalcet and the evocalcet-linker conjugate, respectively, in an FLIPR-based cellular functional assay. The specificity of the EvoSiR probes toward CaSR binding and the impact of albumin was evaluated in live cell experiments. Both probes showed strong albumin binding, which facilitated the clearance of nonspecific binding interactions. Accordingly, in zebrafish embryos, EvoSiR4 specifically labeled the high CaSR expressing neuromasts of the lateral line in vivo. EvoSiR4 was also assessed in human parathyroid tissues ex vivo, showing a specific absolute CaSR-associated fluorescence compared to that of parathyroid autofluorescence. In summary, functionalization of evocalcet by SiR led to the preparation of potent and specific fluorescent CaSR probes. EvoSiR4 is a versatile small-molecular probe that can be employed in CaSR-related biomedical analyses where antibodies are not applicable.