Petr Klán’s research while affiliated with Masaryk University and other places

Bilirubin (BR) is a water-insoluble product of heme catabolism in mammals. Elevated blood concentrations of BR, especially in the neonatal period, are treated with blue-green light phototherapy. The major mechanism of BR elimination during phototherapy is photoisomerization, while a minor, less studied mechanism of degradation is oxidation. In this work, we studied the oxidation of the bilirubin model tetramethyl-dipyrrinone (Z-13) by singlet oxygen in methanol using UV–vis and ESI-MS spectroscopy, resulting in propentdyopents as the main oxidation products. We also identified two additional intermediates that were formed during the reaction (hydroperoxide 21a and imine 17). The structure of the hydroperoxide was confirmed by helium-tagging IR spectroscopy. Such reaction intermediates formed during the oxidation of BR or bilirubin models have not been described so far. We believe that this work can be used as a first step in studying the complex oxidation mechanism of BR during phototherapy.

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.

The engineering of efficient enzymes for large-scale production of industrially relevant compounds is a challenging task. Utilizing rational protein design, which relies on a comprehensive understanding of mechanistic information, holds significant promise for achieving success in this endeavor. Pre-steady-state kinetic measurements, obtained either through fast-mixing techniques or photoswitchable substrates, provide crucial mechanistic insights. The latter approach not only furnishes mechanistic clarity but also affords real-time structural elucidation of reaction intermediates via time-resolved femtosecond crystallography. Unfortunately, only a limited number of such valuable mechanistic probes are available. To address this gap, we applied a multidisciplinary approach, including computational analysis, chemical synthesis, physicochemical property screening, and enzyme kinetics to identify promising candidates for photoswitchable probes. We demonstrate the approach by designing an azobenzene-based photoswitchable substrate tailored for haloalkane dehalogenases, a prototypic class of enzymes pivotal in developing computational tools for rational protein design. The probe was subjected to steady-state and pre-steady-state kinetic analysis, which revealed new insights about the catalytic behavior of the model biocatalysts. We employed laser-triggered Z-to-E azobenzene photoswitching to generate the productive isomer in situ, opening avenues for advanced mechanistic studies using time-resolved femtosecond crystallography. Our results not only pave the way for the mechanistic understanding of this model enzyme family, incorporating both kinetic and structural dimensions, but also propose a systematic approach to the rational design of photoswitchable enzymatic substrates.

Cyanine dyes are a class of organic, usually cationic molecules containing two nitrogen centers linked through conjugated polymethine chains. The synthesis and reactivity of cyanine derivatives have been extensively investigated for decades. Unlike the recently described phototruncation process, the thermal truncation (chain shortening) reaction is a phenomenon that has rarely been reported for these important fluorophores. Here, we present a systematic investigation of the truncation of heptamethine cyanines (Cy7) to pentamethine (Cy5) and trimethine (Cy3) cyanines via homogeneous, acid–base-catalyzed nucleophilic exchange reactions. We demonstrate how different substituents at the C3′ and C4′ positions of the chain and different heterocyclic end groups, the presence of bases, nucleophiles, and oxygen, solvent properties, and temperature affect the truncation process. The mechanism of chain shortening, studied by various analytical and spectroscopic techniques, was verified by extensive ab initio calculation, implying the necessity to model catalytic reactions by highly correlated wave function-based methods. In this study, we provide critical insight into the reactivity of cyanine polyene chains and elucidate the truncation mechanism and methods to mitigate side processes that can occur during the synthesis of cyanine derivatives. In addition, we offer alternative routes to the preparation of symmetrical and unsymmetrical meso-substituted Cy5 derivatives.

Carbon monoxide (CO) is notorious for its toxic effects but is also recognized as a gasotransmitter with considerable therapeutic potential. Due to the inherent challenges in its delivery, the utilization of organic CO photoreleasing molecules (photoCORMs) represents an interesting alternative to CO administration characterized by high spatial and temporal precision of release. This paper focused on the design, synthesis, and photophysical and photochemical studies of 20 3-hydroxyflavone (flavonol) and 3-hydroxyflavothione derivatives as photoCORMs. Newly synthesized compounds bearing various electron-donating and electron-withdrawing groups show bathochromically shifted absorption maxima and considerably enhanced CO release yields compared to the parent unsubstituted flavonol, exceeding 0.8 equiv of released CO in derivatives exhibiting excited states with a charge-transfer character. Until now, such outcomes have been limited to flavonol derivatives possessing a π-extended aromatic system. In addition, thione analogs of flavonols, 3-hydroxyflavothiones, show substantial bathochromic shifts of their absorption maxima and enhanced photosensitivity but provide lower yields of CO formation. Our study elucidates in detail the mechanism of CO photorelease from flavonols and flavothiones, utilizing steady-state and time-resolved spectroscopies and photoproduct analyses, with a particular emphasis on unraveling the structure–photoreactivity relationship and understanding competing side processes.

Cyanine dyes are a class of organic, usually cationic molecules containing two nitrogen centers linked through conjugated polymethine chains. Unlike phototruncation, the thermal truncation (chain-shortening) reaction is a phenomenon that has rarely been described for these important fluorophores. Here, we present a systematic investigation of the truncation of heptamethine cyanines (Cy7) to pentamethine (Cy5) and trimethine (Cy3) cyanines via homogeneous, acid-base catalyzed nucleophilic exchange reactions. We demonstrate how different substituents at the C3′ and C4′ positions of the chain and dif-ferent heterocyclic end groups, the presence of different bases, nucleophiles and oxygen, solvent properties, and tempera-ture affect the truncation process. The mechanism of chain shortening, studied by various analytical and spectroscopic techniques, was verified by extensive ab initio calculation, demonstrating the need to model catalytic reactions by highly correlated wavefunction-based methods. We show that entropic effects control the course of this process. The study provides a critical insight into the reactivity of the polyene chains of cyanines and offers new approaches to the synthesis of meso-substituted symmetrical and unsymmetrical pentamethine cyanines from Cy7 derivatives.

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.

Small‐molecule dyes are generally designed based on well‐understood electronic effects. However, steric hindrance can promote excited‐state geometric relaxation, increasing the difference between the positions of absorption and emission bands (the Stokes shift). Accordingly, we hypothesized that sterically induced central ring puckering in xanthene dyes could be used to systematically increase their Stokes shift. Through a combined experimental/quantum‐chemical approach, we screened a group of (9‐acylimino)‐pyronin dyes with a perturbed central ring geometry. Our results showed that an atom with sp3 hybridization in position 10 of (9‐acylimino)‐pyronins induces central ring puckering and facilitates excited‐state geometric relaxation, thereby markedly enhancing their Stokes shifts (by up to ~2000 cm−1). Thus, we prepared fluorescent (9‐acylimino)‐pyronin pH sensors, which showed a Stokes shift disparity between acid and base forms of up to ~8700 cm−1. Moreover, the concept of ring puckering‐enhanced Stokes shift can be applied to a wide range of xanthene analogues found in the literature. Therefore, central ring puckering may be reliably used as a strategy for enhancing Stokes shifts in the rational design of dyes.

Bilirubin is the principal product of heme catabolism. High concentrations of the pigment are neurotoxic, yet slightly elevated levels are beneficial. Being a potent antioxidant, oxidative transformations of bilirubin occur in vivo and lead to various oxidized fragments. The mechanisms of their formation, intrinsic biological activities, and potential roles in human pathophysiology are poorly understood. Degradation methods have been used to obtain samples of bilirubin oxidation products for research. Here, we report a complementary, fully synthetic method of preparation. Our strategy leverages repeating substitution patterns in the parent tetracyclic pigment. Functionalized ready-to-couple γ-lactone, γ-lactam, and pyrrole monocyclic building blocks were designed and efficiently synthesized. Subsequent modular combinations, supported by metal-catalyzed borylation and cross-coupling chemistries, translated into the concise assembly of the structurally diverse bilirubin oxidation products (BOXes, propentdyopents, and biopyrrins). The discovery of a new photoisomer of biopyrrin A named lumipyrrin is reported. Synthetic bilirubin oxidation products made available in sufficient purity and quantity will support future in vitro and in vivo investigations.