ENZYMATIC SYNTHESIS OF NEW HYBRID ANTIOXIDANTS BASED ON POLYPHENOLS AND VITAMINS

Project leaders

Sergio Riva, Vladimir Kren

Agreement

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

Call

CNR/AVCR 2013-2015

Department

Molecular Design

Thematic area

Chemical sciences and materials technology

Status of the project

New

Research proposal

ABSTRACT:
Multistep enzymatic processes for the modification of natural compounds showing antioxidant activity will be developed in order to obtain selectively functionalized products with improved properties. Specifically, chemo-enzymatically prepared hybrid dimers of silybin and resveratrol with antioxidant vitamins will be prepared and their antioxidant activity will be evaluated.
 
INTRODUCTION: Discovery of entirely new biologically active compounds is expensive and time-consuming, whereas modifications of known (lead) structures is often rewarding and moreover it can bring important SAR (structure-activity relationships) data. Biocatalysis has numerous advantages for modification of biologically active natural compounds mainly due to selectivity (stereoselectivity) and mild reaction conditions. This is highly important in complex multifunctional natural products.
ENZYMATIC MODIFICATION OF NATURAL COMPOUNDS : In recent years hydrolytic enzymes (e.g., esterases, lipases and proteases) have come into widespread use in organic synthesis due to their large availability, low cost, broad substrate specificity and easy handling (Carrea and Riva, 2000). For instance, flavonoid glycosides (i.e., rutin, hesperidin and naringin) have been successfully acylated at specific OH’s of their molecules by performing hydrolase-catalyzed esterifications in organic solvent and in the presence of activated esters.
OXIDATIVE STRESS AND ANTIOXIDANTS : Life requires oxygen. This runs the risk that, when oxygen leaks out from normal metabolism, reactive oxygen species (ROS) are formed, which - when too high - trigger disease. With the idea to overcome this, antioxidants are heavily marketed, yet without proof of their effectiveness. Rather, worrying evidence suggests adverse effects. This paradox is due to the fact that ROS are not only „bad“ but  in tightly regulated amounts  also act as essential signaling molecules. Unraveling the fine balance between ROS acting as a friend or a foe is fundamental to understand aerobic life. To advance this important area of biology and medicine, highly synergistic approaches combining diverse and scattered disciplines are needed.
ANTIOXIDATIVE POLYPHENOLS In our project we will concentrate to the modification of selected  natural polyphenols, specifically silybin and resveratrol, with antioxidant and radical scavenging activities, which we want to combine in a single molecule with antioxidant vitamins using selective and mild methods of enzymatic catalysis.
Flavonolignan SILYBIN (Figure 1) is one of the best pharmacologically documented natural product is an active constituent of phytopreparations used in the prevention and the treatment of various liver diseases (Saller et al., 2001) since ancient times. Although pharmacology of silybin has been largely studied (over 800 papers just in the last 12 years), there are still many chemical problems pending that are also reflected in numerous controversies in medical and pharmacological literature. E.g.: I. Silybin is a mixture (1:1) of two diastereomers (Fig. 1) that were separated and structurally characterized only quite recently. From practical reasons virtually all silybin chemistry is done with the stereomeric mixture that poses high demands on the spectral identification. II. Silybin carries five hydroxyl groups and their reactivity including  work on a chemistry of protection/deprotection strategy has not been systematically studied, yet. III. Silybin is very prone to oxidation and oxidative polymerization especially under alkaline conditions. IV. Mechanism(s) of antiradical and antioxidative action of the silybin molecule has not been systematically investigated and it is not quite clear.
In a previous Czech-Italian collaboration (sponsored by EU-COST) we have solved many of these problems, the most important being enzymatic separation of silybin diastereomers (D. Monti, R. Ga?ák, P. Marhol, D. Biedermann, K. Purchartová, M. Fedrigo, S. Riva, V. KYen, Enzymatic Kinetic Resolution of Silybin Diastereoisomers. J. Nat. Prod. 73(4), 613-619 , 2010. R. Ga?ák, P. Marhol, K. Purchartová, D. Monti, D. Biedermann, S. Riva, L. Cvak, V. KYen: Large-scale separation of silybin diastereoisomers using lipases. Proc. Biochem. 45, 1657-1663, 2010) as well as some problems of the antioxidant mechanisms (Ga?ák et al. 2009) of silybin and its congeners.
trans -RESVERATROL (3,5,4´-trihydroxystilbene, Figure 2) is a stilbenic phytoalexin produced by plants via a metabolic sequence induced in response to biotic or abiotic stress factors. This compound and its 3-O-beta-D-glucoderivative (piceid) are present in relatively high concentrations in grape juice and in red wine and it has been recently hypothesized that they could be responsible for the decrease in coronary heart disease observed among wine drinkers (French paradox). Moreover, growing evidences suggest that resveratrol plays a role in the prevention of carcinogenesis and its oligomers, the so-called `viniferins´, are reported to exhibit a wide-array of biological activities, such as antimicrobial, anti-HIV, anti-inflammatory; they are also reported to be potentially important cancer chemoprotective agents, being able to inhibit cellular events associated with carcinogenesis Thus, resveratrol may also play a role in cancer therapy and may be considered as a lead compound for preparation of analogues with enhanced activity. SAR studies on resveratrol derivatives provided only limited information on structural requirements of different biological activities.
 ANTIOXIDANT VITAMINES Some vitamins (Figure 3) have notable antioxidant and radical-scavenger activity – typically ASCORBIC ACID, TOCOFEROL and RETINOL (plus carotenoids). Besides their specific roles in the organism they exert antioxidant activity, which is stronger in the combination with other plant derived antioxidants, typically polyphenols and flavonoids. This is why the consumption of natural plant derived fresh food is so beneficial for health as the complexes act often in a synergic way.
VITAMIN A is a generic term for a large number of related compounds. Retinol and retinal  are often referred to as preformed vitamin A. Retinal can be converted by the body to retinoic acid, the form of vitamin A known to affect genetranscription. Retinol, retinal, retinoic acid, and related compounds are known as retinoids. Beta-carotene and other carotenoids that can be converted by the body into retinol are referred to as provitamin A carotenoids. Hundreds of different carotenoids are synthesized by plants, but only about 10% of them are provitamin A carotenoids.
VITAMIN C, also known as ascorbic acid, is a water-soluble vitamin. Unlike most mammals and other animals, humans do not have the ability to make their own vitamin C, therefore, we must obtain vitamin C through our diet. Vitamin C is required for the synthesis of collagen, an important structural component of blood vessels, tendons, ligaments, and bone. Vitamin C also plays an important role in the synthesis of the neurotransmitter, norepinephrine. Moreover, research also suggests that vitamin C is involved in the metabolism of cholesterol to bile acids, which may have implications for blood cholesterol levels and the incidence of gallstones. Vitamin C is also a highly effective antioxidant. Even in small amounts vitamin C can protect indispensable molecules in the body, such as proteins, lipids (fats), carbohydrates, and nucleic acids (DNA and RNA), from damage by free radicals and reactive oxygen species that can be generated during normal metabolism as well as through exposure to toxins and pollutants. Ascorbyl palmitate is actually a vitamin C ester (i.e., vitamin C that has been esterified to a fatty acid). In this case, vitamin C is esterified to the saturated fatty acid, palmitic acid, resulting in a fat-soluble form of vitamin C. Ascorbyl palmitate has been added to a number of skin creams due to interest in its antioxidant properties as well as its importance in collagen synthesis.
The term Vitamin E describes a family of eight antioxidants: four tocopherols (alfa-, beta-, gamma-, and delta-) and four tocotrienols (alfa-, beta-, gamma-, and delta-). alfa-Tocopherol is the only form of vitamin E that is actively maintained in the human body; therefore, it is the form of vitamin E found in the largest quantities in blood and tissuesand appears to have the greatest nutritional significance. The main function of the fat-soluble alfa-tocopherol in humans appears to be that of an antioxidant, being uniquely suited to intercept free radicals and thus prevent a chain reaction of lipid destruction. When a molecule of alpha-tocopherol neutralizes a free radical, it is altered in such a way that its antioxidant capacity is lost. However, other antioxidants, such as vitamin C, are capable of regenerating the antioxidant capacity of alpha-tocopherol.
SYNTHESIS OF HYBRID MOLECULES
Combinations of antioxidants with different mechanisms of action in one formulation produce often an increased efficency. More sophisticated approach to this problem is a combination of various antioxidants in one molecule, i.e., hybrid molecules. This methodology was already used, e.g., for synthesis of hybrid molecule of tocoferol and procain (Koufak et al., 2003) providing substance with antioxidant and antiarrhytmic properties or a hybrid radical scavenger/iron chelator based on combination of 3-hydroxy-2-methyl-4(1H)-pyridinone (deferiprone) and di-t-butyl-hydroxytoluene (BHT) (Bebbington et al., 2002) and many others.
In this project two approaches will be studied for the synthesis of the hybrid antioxidants : a) enzyme-catalyzed formation of diesters using divinyl ester of dicarboxylic acids or b) diether linkage using α,w-dibromo-linkers with appropriate base – we used this concept e.g. in the coupling of ergot alkaloids (Kren, V., et al: Free Rad. Biol. Med. 46, 745-758 (2009).
In previous works (P. Magrone, S. Riva, et al. “Exploiting enzymatic regioselectivity: a facile methodology for the synthesis of polyhydroxylated hybrid compounds”, Org. Biol. Chem. 8, 5583 – 5590, 2010; C. Dolle, S. Riva, et al., "Symmetric and asymmeteric bolaamphiphiles from ascorbic acid", J. Phys. Chem. B., 115, 11638-11649, 2011), we have shown that lipases regioselectivity can be efficiently exploited to link together any kind of natural molecule (including vitamins) carrying a nucleophilic group (obviously accessible to the enzyme active site) via diesters bridges of different length and nature. Moreover, it has been shown that also activated esters of dithio dicarboxylic acids are well-accepted acyl donors, thus offering a simple and efficient two-step synthetic approach to the so-called “dynamic libraries” of bioactive compounds.
The syntheses of the target new hybrid compounds will be performed by exploiting the regioselectivity of lipases, mainly of Novozym-435, thus avoiding cumbersome protective and deprotective steps.
 

Research goals

a) Coupling of various antioxidants into one molecule
Examples of some of the target hybrid molecules that we will attempt to synthesize are shown in the FIGURES 4-8. (N.B. all the Figures have been e-mailed as an attached file, as it is not possible to draw chemical structures in these forms).
 
b) Antioxidant tests of the new molecules
Radical scavenging and antioxidant activity i) Antioxidant capacity of the new hybrids will be estimated by observing hydrogen transfer from a target compound to 2,2-diphenyl-1-(2,4,6-trinitro-phenyl)hydrazyl (DPPH) radical spectrophotometrically. The stoichiometry and the rate constant of the transfer reaction will be calculated. ii) The deoxyribose degradation assay and the xanthine/xanthine oxidase method will be used to assess hydroxyl radical and superoxide anion scavenging, respectively. iii) Inhibition of lipid peroxidation induced by Fe(III)/ADP/NADPH in in microsomal membranes will be assayed by thiobarbituric acid method. iv) 5-Lipoxygenase inhibiting activity will be determined by measuring the arachidonic acid metabolites 5-HETE and LTB4 in polymorphonuclear leukocytes using HPLC.
 
c) Cooperation      
This joint project is planned for 3 years. It combines materials and skills available in the Istituto di Chimica del Riconoscimento Molecolare, CNR, Milano, e.g., a large set of enzymes from different classes for biotransformations (hydrolases, glycosyltransferases and oxidoreductases); facilities and skills for the purification and characterization of proteins (HPLC, slab-gel and capillary electrophoresis, centrifuges etc.), for the enzymatic acylation and glycosylation reactions, for the characterization of the products) and the material and skills available in the Laboratory of Biotransformation, Inst. of Microbiology, Czech Academy of Sciences, Prague (e.g., materials: large fungal collection for search of new enzymes, large collection of glycosidases (ca 200 enzymes), testing facilities for antioxidant activities (free radical scavenging assay, inhibition of lipid peroxidation), facilities for structure determination (NMR, MS, MALDI-TOF)).
A main benefit for both participating partners will be the combination of sophisticated techniques and unique materials available in collaborating laboratories.
It deserves to be mentioned here that the two groups have already a longstanding tradition of cooperation, mainly sponsored by EU-COST, the results being reported in 16 joint papers and 2 joint patents

Last update: 02/08/2025