**2.2 Reaction conditions**

The performance and regioselectivity of the enzyme-catalyzed flavonoid transformation is affected by several factors, including the type of enzyme, the nature of medium, reaction conditions, water content in the media, structure and concentration of substrates and their molar ratio. By varying these factors, significant changes in ester production and regioselectivity can be achieved.

### **2.2.1 Reaction media**

Reaction media play an important role in enzymatic transformations. Methodologies for enzymatic flavonoid acylation have focused on searching a reaction medium which allows appropriate solubility of polar acyl acceptor (flavonoid glycoside) and nonpolar acyl donor as well as the highest possible enzymatic activity. Moreover, the medium has often been required to be nontoxic and harmless to biocatalyst. In order to meet the above-mentioned requirements, several scientific teams have dealt with proper medium selection (Viskupicova et al., 2006; Mellou et al., 2005; Kontogianni et al., 2001, 2003; Gao et al., 2001; Nakajima et al., 1999; Danieli et al., 1997).

Non-aqueous biocatalysis has several advantages over conventional aqueous catalysis: the suppression of hydrolytic activity of the biocatalyst which is carried out in water (Fossati & Riva, 2006), the enhanced solubility of hydrophobic substrates, the improvement of enzyme enantioselectivity, the exclusion of unwanted side reactions, the easy removal of some products, the enhanced enzyme thermostability and the elimination of microbial contamination (Rubin-Pitel & Zhao, 2006; Torres & Castro, 2004). Laane (1987) pointed out that log P, as a solvent parameter, correlated best with enzyme activity. Zaks & Klibanov (1988) reported that the activity of lipases was higher in hydrophobic solvents than in hydrophilic ones. Narayan & Klibanov (1993) claimed that it was hydrophobicity and not polarity or water miscibility which was important, whereas the log P parameter could be called a measure of solvent hydrophobicity. Trodler & Pleiss (2008), using multiple molecular dynamics simulations, showed that the structure of CALB possessed a high stability in solvents. In contrast to structure, flexibility is solvent-dependent; a lower dielectric constant led to decreased protein flexibility. This reduced flexibility of CALB in

which is preferred due to its acceptance of a wide range of substrates, good catalytic activity and a high degree of regioselectivity (Viskupicova et al., 2010; Katsoura et al., 2006, 2007; Ghoul et al., 2006; Mellou et al., 2005, 2006; Stevenson et al., 2006; Ardhaoui et al., 2004a, 2004b, 2004c; Passicos et al., 2004; Moussou et al., 2004; Gayot et al., 2003; Ishihara & Nakajima, 2003; Ishihara et al., 2002; Kontogianni et al., 2001, 2003; Nakajima et al., 1999,

As for flavonoid aglycons, only two enzymes have been reported to be capable of acylating this skeleton – lipase from *Pseudomonas cepacia* and carboxyl esterase. Lambusta et al. (1993) investigated the use of *P. cepacia* lipase for catechin modification. They discovered that the acylation took place on the C5 and C7 hydroxyls. Sakai et al. (1994) observed that carboxyl esterase showed regioselectivity towards C3-OH of catechin. Sakai et al. (1994) explored the use of carboxyl esterase from *Streptomyces rochei* and *Aspergillus niger* for the 3-*O*-acylated

The performance and regioselectivity of the enzyme-catalyzed flavonoid transformation is affected by several factors, including the type of enzyme, the nature of medium, reaction conditions, water content in the media, structure and concentration of substrates and their molar ratio. By varying these factors, significant changes in ester production and

Reaction media play an important role in enzymatic transformations. Methodologies for enzymatic flavonoid acylation have focused on searching a reaction medium which allows appropriate solubility of polar acyl acceptor (flavonoid glycoside) and nonpolar acyl donor as well as the highest possible enzymatic activity. Moreover, the medium has often been required to be nontoxic and harmless to biocatalyst. In order to meet the above-mentioned requirements, several scientific teams have dealt with proper medium selection (Viskupicova et al., 2006; Mellou et al., 2005; Kontogianni et al., 2001, 2003; Gao et al., 2001;

Non-aqueous biocatalysis has several advantages over conventional aqueous catalysis: the suppression of hydrolytic activity of the biocatalyst which is carried out in water (Fossati & Riva, 2006), the enhanced solubility of hydrophobic substrates, the improvement of enzyme enantioselectivity, the exclusion of unwanted side reactions, the easy removal of some products, the enhanced enzyme thermostability and the elimination of microbial contamination (Rubin-Pitel & Zhao, 2006; Torres & Castro, 2004). Laane (1987) pointed out that log P, as a solvent parameter, correlated best with enzyme activity. Zaks & Klibanov (1988) reported that the activity of lipases was higher in hydrophobic solvents than in hydrophilic ones. Narayan & Klibanov (1993) claimed that it was hydrophobicity and not polarity or water miscibility which was important, whereas the log P parameter could be called a measure of solvent hydrophobicity. Trodler & Pleiss (2008), using multiple molecular dynamics simulations, showed that the structure of CALB possessed a high stability in solvents. In contrast to structure, flexibility is solvent-dependent; a lower dielectric constant led to decreased protein flexibility. This reduced flexibility of CALB in

2003; Gao et al., 2001; Otto et al., 2001; Danieli et al., 1997).

catechin production.

**2.2 Reaction conditions** 

**2.2.1 Reaction media** 

regioselectivity can be achieved.

Nakajima et al., 1999; Danieli et al., 1997).

non-polar solvents is not only a consequence of the interaction between organic solvent molecules and the protein, but it is also due to the interaction with the enzyme-bound water and its exchange on the surface (Trodler & Pleiss, 2008). In organic solvents, the surface area has been suggested to be reduced, leading to improved packing and increased stability of the enzyme (Toba & Merz, 1997).

Polar aprotic solvents such as dimethyl sulfoxid (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF) and pyridine were first investigated (Nakajima et al., 1999; Danieli et al., 1997). However, it was observed that enzyme activity was readily deactivated in these solvents. To date enzymatic acylation of flavonoids has been successfully carried out in various organic solvents (Tab.2), while the most frequently used are 2-methylbutan-2-ol and acetone because of their low toxicity, their polarity allowing proper solubilization of substrates and high conversion yields.


Table 2. Organic solvents used in flavonoid acylation.

The effect of the solvent on conversion yield depends on the nature of both the acyl donor and the flavonoid (Chebil et al., 2006). Although much has been done in this area, it is quite difficult to deduce any general conclusion on solvent choice because the available data are controversial and sometimes even contrary.

Recently, ionic liquids have received growing attention as an alternative to organic solvents used for the enzymatic transformation of various compounds (Katsoura et al., 2006; Kragl et al., 2006; Jain et al., 2005; Lozano et al., 2004; Reetz et al., 2003; Van Rantwick et al., 2003). The potential of these "green solvents" lies in their unique physicochemical properties, such as non-volatility, nonflammability, thermal stability and good solubility for many polar and less polar organic compounds (Jain et al., 2005; Wilkes, 2004; Itoh et al., 2003; Van Rantwick et al., 2003). Probably the most promising advantage of the use of ionic liquids is their potential application in food, pharmaceutical and cosmetic preparations due to their reduced toxicity (Jarstoff et al., 2003). Due to the many above-mentioned advantages of ionic liquids for enzyme-mediated transformations, several flavonoid esters have been recently prepared in such media (Katsoura et al., 2006, 2007; Kragl et al., 2006). The biocatalytic process showed significantly higher reaction rates, regioselectivity and yield conversions compared to those achieved in organic solvents. Thus ionic liquid use seems to be a challenging approach to conventional solvent catalysis.

The solvent-free approach for elimination of the co-solvent of the reaction has been recently introduced as an alternative for conventional solvents (Enaud et al., 2004; Kontogianni et al., 2001, 2003). It is based on the use of one reactant in the role of the solvent. The authors reported rapid reaction rates; however, the conversion yields were slightly decreased. In spite of the attractiveness, the use of solvent-free systems is characterized by a serious drawback due to the necessity to eliminate the excess of the acyl donor for the recovery of the synthesized products (Chebil et al., 2006).
