Martina Hurtová

Glucuronidation is a major phase II biotransformation of (poly)­phenols leading to potentially bioactive metabolites. Due to the limited availability of authentic standards, in this work, we have focused on the glucuronidation of a series of mono- and dihydroxyphenolic acids. Their reactivity with two glucuronidation reagents, 2,3,4-triaceto-1-bromo-α-d-glucuronic acid methyl ester and Schmidt imidate, was investigated. The use of Schmidt imidate led to the successful synthesis of six target glucuronides in moderate to excellent yields. Subsequent deprotection of these compounds afforded the final glucuronides of 2-hydroxyphenylacetic, 3-hydroxyphenylacetic, 4-hydroxyphenylacetic, 3,4-dihydroxyphenylacetic, 3-(4-hydroxyphenyl)­propionic, and 3-(3,4-dihydroxyphenyl)­propionic acid. These compounds, which are plausible polyphenolic metabolites, were fully characterized and used in a pilot metabolic study in rats after administration of a hawthorn berry extract (475 mg/kg). UHPLC-HRMS analysis identified two of the glucuronides, namely 4-hydroxyphenylacetic acid glucuronide and 3-hydroxyphenylacetic acid glucuronide in rat plasma, confirming their in vivo formation.

Flavonoids are naturally occurring compounds found in fruits, vegetables, and other plant-based foods, and they are known for their health benefits, such as UV protection, antioxidant, anti-inflammatory, and antiproliferative properties. This study investigates whether flavonoids, such as quercetin and 2,3-dehydrosilybin, can act as photoactivatable carbon monoxide (CO)-releasing molecules under physiological conditions. CO has been recently recognized as an important signaling molecule. Here, we show that upon direct irradiation, CO was released from both flavonoids in PBS with chemical yields of up to 0.23 equiv, which increased to almost unity by sensitized photooxygenation involving singlet oxygen. Photoreleased CO reduced cellular toxicity caused by high flavonol concentrations, partially restored mitochondrial respiration, reduced superoxide production induced by rotenone and high flavonol levels, and influenced the G0/G1 and G2/M phases of the cell cycle, showing antiproliferative effects. The findings highlight the potential of quercetin and 2,3-dehydrosilybin as CO-photoreleasing molecules with chemopreventive and therapeutic implications in human pathology and suggest their possible roles in plant biology.

In this work, we report the application of Buchwald-Hartwig amination for the preparation of new derivatives of quercetin and luteolin. Our investigation delves into the impact of aniline moiety on antioxidant, and anti-inflammatory activity, cytotoxicity, and the ability of flavonoids to modulate drug-resistance mechanisms in bacteria. The anti-inflammatory activity disappeared after the introduction of aniline into the flavonoids and the cytotoxicity remained low. Although the ability of quercetin and luteolin to modulate bacterial resistance to antibiotics has already been published, this is the first report on the molecular mechanism of this process. Both flavonoids attenuate erythromycin resistance by suppressing the ribosomal methyltransferase encoded by the ermA gene in Staphylococcus aureus. Notably, 4-(trifluoromethyl)anilino quercetin emerged as a potent ErmA inhibitor, likely by interacting with the RNA-binding pocket of ErmA. Additionally, both 4-fluoroanilino derivatives effectively impended the staphylococcal efflux system. All the prepared derivatives exhibited superior activity in modulating gentamicin resistance in S. aureus compared to the parent compounds. Overall, the incorporation of substituted anilines into the flavonoid core significantly enhanced its ability to combat multidrug resistance in bacteria.

Photooxygenation of flavonoids leads to the release of carbon monoxide (CO). Our structure-photoreactivity study, employing several structurally different flavonoids, including their ¹³C-labeled analogs, revealed that CO can be produced via two completely orthogonal pathways, depending on their hydroxy group substitution pattern and the reaction conditions. While photooxygenation of the enol 3-OH group has previously been established as the CO liberation channel, we show that the catechol-type hydroxy groups of ring B can predominantly participate in photodecarbonylation.

A library of previously unknown halogenated derivatives of flavonolignans (silybins A and B, 2,3-dehydrosilybin, silychristin A, and 2,3-dehydrosilychristin A) was prepared. The effect of halogenation on the biological activity of flavonolignans was investigated. Halogenated derivatives had a significant effect on bacteria. All prepared derivatives inhibited the AI-2 type of bacterial communication (quorum sensing) at concentrations below 10 µM. All prepared compounds also inhibited the adhesion of bacteria (Staphyloccocus aureus and Pseudomonas aeruginosa) to the surface, preventing biofilm formation. These two effects indicate that the halogenated derivatives are promising antibacterial agents. Moreover, these derivatives acted synergistically with antibiotics and reduced the viability of antibiotic-resistant S. aureus. Some flavonolignans were able to reverse the resistant phenotype to a sensitive one, implying that they modulate antibiotic resistance.

Herein, we report the use of the Suzuki–Miyaura cross-coupling reaction for the preparation of a library of synthetic derivatives of flavonoids for biological activity assays. We have investigated the reactivity of halogenated flavonoids with aryl boronates and with boronyl flavonoids. This reaction was used to prepare new synthetic derivatives of flavonoids substituted at C-8 with aryl, heteroaryl, alkyl, and boronate substituents. The formation of flavonoid boronate enabled a cross-coupling reaction with halogenated flavones yielding biflavonoids connected at C-8. This method was used for the preparation of natural compounds including C-8 prenylated compounds, such as sinoflavonoid NB. Flavonoid boronates were used for the preparation of rare C-8 hydroxyflavonoids (natural flavonoids gossypetin and hypolaetin). A series of previously unknown derivatives of quercetin and luteolin were prepared and fully characterized.

The natural diastereomeric mixture of silybins A and B is often used (and considered) as a single flavonolignan isolated from the fruit extract of milk thistle (Silybum marianum), silymarin. However, optically pure silybin diastereomers are required for the evaluation of their biological activity. The separation of silybin diastereomers by standard chromatographic methods is not trivial. Preparative chemoenzymatic resolution of silybin diastereomers has been published, but its optimization and scale-up are needed. Here we present a continuous flow reactor for the chemoenzymatic kinetic resolution of silybin diastereomers catalyzed by Candida antarctica lipase B (CALB) immobilized on acrylic resin beads (Novozym^® 435). Temperature, flow rate, and starting material concentration were varied to determine optimal reaction conditions. The variables observed were conversion and diastereomeric ratio. Optimal conditions were chosen to allow kilogram-scale reactions and were determined to be −5 °C, 8 g/L silybin, and a flow rate of 16 mL/min. No significant carrier degradation was observed after approximately 30 cycles (30 days). Under optimal conditions and using a 1000 × 15 mm column, 20 g of silybin per day can be easily processed, yielding 6.7 and 5.6 g of silybin A and silybin B, respectively. Further scale-up depends only on the size of the reactor.