Biomimetic enzymatic synthesis of polyphenolic bioactive molecules

Project leaders

Sergio Riva, Vladimir Kren

Agreement

REPUBBLICA CECA - CAS (ex AVCR) - Czech Academy of Sciences

Call

CNR-CAS (ex AVCR) 2016-2018

Department

Chemical sciences and materials technology

Thematic area

Chemical sciences and materials technology

Status of the project

New

Research proposal

Flavonolignans are plant natural products, composed of a flavonoid moiety and a lignan (phenylpropanoid) part. Current literature focuses on flavonolignans formed from taxifolin and coniferyl alcohol, as e.g. silybin and its congeners from fruit extract from the purple variety of the milk thistle (Silybum marianum) denoted as "silymarin". Further important flavonolignans are derived from apigenin, luteolin, tricin, chrysoeriol, naringenin and eriodictyol - as the flavonoid part. Flavonolignans have been shown to exhibit in vitro and/or in vivo anti-hepatotoxic, anti-oxidant, free radical scavenging, anti-inflammatory, anti-proliferative, anti-cancer, chemotherapy potentiating, anti-melanogenic, anti-bacterial, vasorelaxing, anti-platelet aggregation and/or hypotriglyceridemic activity. Many of these compounds inhibited Staphylococcus aureus multidrug resistance pump NorA and sensitized multidrug resistant cancer cell lines showing a potential as adjuvants.

The biomimetic synthesis of rare flavonolignans (silybin and isosilybin derivatives) using chemical promotors (e. g. silver oxide and other monoelectron oxidants) and/or enzymatic catalysis (peroxidases, laccases, tyrosinases) will be elaborated. By employing modified reactants it will be possible to isolate non-natural analogues of these compounds.
As it might be possible that, although controlled, the reactions will furnish complex products mixtures, including structural isomers and stereoisomers, advanced separation techniques will be exploited. If needed, analogously to our previous joint publications (a, S. RIVA, V. KREN, et al. (2010) "Large-scale separation of sylibin diastereoisomers using lipases" Process Biochem., 45, 1657-1663. b, S. RIVA, V. KREN et al. (2010) "Enzymatic kinetic resolution of sylibin diastereoisomers" J. Nat. Prod., 73, 613-619), the desired discrimination will be achieved by lipases-catalyzed reactions (acylations or alcoholysis) in organic solvents.
Up to know we have investigated the homocoupling of phenolic compounds catalyzed by oxidative enzymes (laccases). For instance, in joint publications we have recently described the coupling of a benzyl derivative of sylibin A and sylibin B (S. RIVA, V. KREN, et al. (2014) "Enzymatic oxidative dymerization of sylimarin flavolignans" J. Mol. Catal. B-Enzymatic, 109, 24-30) and of piceid, a natural glucoside of the well-known stilbenic derivative resveratrol ( V. KREN, S. RIVA, et al. (2015) "Laccase-catalyzed dimerization of piceid, a resveratrol glucoside, and its further enzymatic elaboration" Adv. Synth. Catal., 357, 1831-1839).
As a step further, an important part of this new project will be the investigation of heterocoupling reactions. Mimicking nature, in which - for instance - sylibin A and silybyn B are the products of the biocatalyzed heterocoupling of (+)-taxifolin and coniferyl alcohol, we will try to reproduce this condensation in vitro, and then, by using analogues both of (+)-taxifolin and of coniferyl alcohol, to isolate new derivatives of sylibin.
The same approach will be applied to quercetin and luteolin (flavonoid moiety) leading to respective 2,3-dehydroderivatives of silybin-type flavonolignans and to hydnocarpin-type of flavonolignans. Then on phenylpropanoid part we will investigate condensation of some other coniferyl alcohol derivatives (e.g. caffeyl alcohol, umbellyl alcohol and sinapyl alcohol).
From our previous experience and from what has been reported by other scientists active in this research area, it is well known that these reactions are not quite stereoselective: when new stereogenic carbons are formed in the coupling reactions, usually a racemic mixture is obtained (unless the coupling occurs in the presence of the so-called "dirigent proteins"). In order to overcome this limitation, we propose the concept of "chiral handle", e.g. we will try to control the selectivity by attaching bulky and chiral moieties, such as carbohydrates, to the starting substrates. Actually, in preliminary experiments we have observed that when coniferyl acohol is glycosylated, the resulting main dihydrobenzofuranon-type dimer obtained by laccase-catalyzed oxidation is enriched in one stereoisomer, whereas the same biocatalyzed homocoupling reaction performed on the "naked" alcohol gives a racemate (P. Gavezzotti, S. RIVA, et al. (2011) Synthesis of enantiomerically enriched dimers of vinylphenols by tandem action of laccases and lipases, Adv. Synth. Catal., 353, 2421-2430).
The new compounds will be tested for biological activities (in vitro and ex vivo) for SAR studies in close collaboration with international partners participating to a COST Action (CM1303, "System biocatalysis") as well as using our own facilities. As a fundamental infrastructure for this project allowing screening of various biological activities is available in the Prague Campus; it is a robotic high-throughput screening open-access facility CZ-Openscreen (www.openscreen.cz) allowing screeing of hundreds of compounds for numerous bio-activities (over 70), e.g. cytotoxicity and proapoptotic activity to cancer lines HL60, K562, HCT116, PC3; toxicity to the erythroid progenitor cells; effects upon nucler steroid receptors and many others.

Research goals

o To increase the knowledge in the area of the chemical and enzymatic oxidation of "sensitive" natural polyphenols.
o To develop new approaches to induce stereoselectivity in the coupling reactions, even in the absence of the so-called "dirigent proteins"
o To synthesize new derivatives of silybin, hydnocarpin and pinoresinol-type compounds
o To evaluate the biological activities of the synthesized products in terms of cytotoxicity, proapoptotic activity and inhibitory activity towards multidrug resistance protein (MDR inhibitors or P-gp inhibitors).
* To further increase the interaction between our two research groups and our two Institutions (SR and VK scientific collaboration started in 1998. The results have been reported in 19 joint publications (one more is going to be submitted) and 2 joint patents).

Last update: 03/08/2025