**2.3.1 Acyl donor**

270 Biochemistry

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

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

Water content in reaction media is a crucial parameter in lipase-catalyzed synthesis as it alters the thermodynamic equilibrium of the reaction towards hydrolysis or synthesis. Moreover, it is involved in noncovalent interactions which keep the right conformation of an enzyme catalytic site (Foresti et al., 2007). The amount of water required for the catalytic process depends on the enzyme, its form (native or immobilized), the enzyme support, and on the solvent nature (Arroyo et al., 1999; Zaks & Klibanov, 1988). The influence of water content in the reaction system on enzyme activity is variable with various enzymes (lipase from *Rhizomucor miehei*, *Rhizomucor niveus*, *Humicola lanuginosa*,

In general, the water amount which is considered to be optimal for esterifications in organic solvents is 0.2 – 3% (Rocha et al., 1999; Yadav & Piyush, 2003; Iso et al., 2001). The enzymatic esterification of flavonoids in non-aqueous media is greatly influenced by the water content of the reaction system (Ardhaoui et al., 2004b; Gayot et al., 2003; Kontogianni et al., 2003). Ardhaoui et al. (2004b) observed the best enzyme activity when water content was maintained at 200 ppm. Gayot et al. (2003) found that the optimal value of water in an organic reaction medium equaled 0.05% (v/v). Kontogianni et al. (2003) reported that highest flavonoid conversion was reached when initial water activity was 0.11 or less.

Temperature represents a significant physical factor in enzyme-catalyzed reactions. It affects viscosity of the reaction medium, enzyme stability, and substrate and product solubility.

Since lipase from *C. antarctica* belongs to thermostable enzymes, improved catalytic activity was observed at higher temperatures (Arroyo et al., 1999). To date, flavonoid transformation has been carried out in the temperature range 30 – 100°C (Ghoul et al., 2006; Katsoura et al., 2006; Stevenson et al., 2006; Mellou et al., 2005; Ardhaoui et al., 2004a, 2004b, 2004c; Moussou et al., 2004; Passicos et al., 2004; Enaud et al., 2004; Gayot et al., 2003; Kontogianni et al., 2003; Ishihara et al., 2002; Gao et al., 2001; Otto et al., 2001; Nakajima et al., 1999; Danieli et al., 1990). The choice of temperature depends on the enzyme and solvent used. The majority of authors performed flavonoid acylation at 60°C due to the best enzyme activity, good solubility of substrates and highest yields of resulting esters reached (Viskupicova et al., 2006, 2010; Ghoul et al., 2006; Katsoura et al., 2006; Stevenson et al., 2006;

challenging approach to conventional solvent catalysis.

the synthesized products (Chebil et al., 2006).

*Candida rugosa*, *Pseudomonas cepacia*).

**2.2.2 Water content** 

**2.2.3 Temperature** 

Since lipase-catalyzed acylation takes place through the formation of an acyl-enzyme intermediate, the nature of the acyl donor has a notable effect on reactivity. The ideal acyl donor should be inexpensive, fast acylating, and completely non-reactive in the absence of the enzyme (Ballesteros et al., 2006). Many acylating agents have been tested in flavonoid esterification, such as aromatic or aliphatic organic acids, substituted or not (Tab.3). Special attention was attributed to fatty acid ester production (Katsoura et al., 2006; Mellou et al., 2005, 2006; Ardhaoui et al., 2004a, 2004b, 2004c; Enaud et al., 2004; Gayot et al., 2003; Kontogianni et al., 2003). This approach enables to improve flavonoid solubility and stability in lipophilic systems. The proper acyl donor selection may significantly influence not only the physicochemical but also biological properties of the resulting esters.

A simple way to increase the reaction rate and conversion yield in acylation is to use an excess of acyl donor (Patti et al., 2000). Many authors have tried to determine the optimal molar ratio of flavonoid/acyl donor in order to achieve the highest possible yields. The molar ratios 1:1 to 1:15 (acyl acceptor/acyl donor) have been investigated, whereas the majority agreed on the ratio 1:5 to be the most suitable for the best reaction performance (Mellou et al., 2006; Gayot et al., 2003; Ishihara & Nakajima, 2003; Ishihara et al., 2002; Kontogianni et al., 2001). A better solution is offered by the use of special acyl donors which ensure a more or less irreversible reaction. This can be achieved by the introduction of electron-withdrawing substituents (esters), resulting in higher conversion yields and reaction rates. The use of vinyl esters allows a several times faster reaction progress than do other activated esters (Ballesteros et al., 2006). Enzymatic synthesis of flavonoid esters can be realized by two basic approaches, i.e. esterification and transesterification (Fig.3).


\*acyl donors found in anthocyanins

Table 3. Acyl donors found in flavonols, flavones (Williams, 2006) and anthocyanins (Andersen & Jordheim, 2006).

Fig. 3. Mechanism of isoquercitrin esterification and transesterification (Chebil et al., 2006).

Pleiss et al. (1998) studied the acyl binding site of CALB and found the enzyme to be selective for short and medium fatty acid chain length. This fact may be attributed to the structure of the lipase acyl binding pocket, which is an elliptical, narrow cleft of 9.5 × 4.5 Å. With increasing carbon number of a fatty acid or molecule size, the steric hindrance is involved resulting in low efficiency of the enzymatic reaction (Riva et al., 1988; Wang et al., 1988; Carrea et al., 1989). This fact was experimentally confirmed by Katsoura et al. (2006) and by Viskupicova & Ondrejovic (2007) whose results showed higher performance of the naringin and rutin esterification when fatty acids up to C10 were introduced. On the other hand, Ardhaoui et al. (2004b) and Kontogianni et al. (2003) reported that the fatty acid chain length had no significant effect on conversion yield when fatty acids of a medium and high chain length were used.

Thus, the effect of fatty acid chain length on flavonoid acylation still remains a matter of discussion. Our team conducted a series of experiments with both saturated and unsaturated fatty acids and found a correlation between log P of the acids tested and conversion yields (Viskupicova et al., 2010). It would be interesting to take this parameter into consideration when assessing the influence of an acyl donor on the reaction progress.

Only little progress has been achieved in flavonoid esterification with aromatic acids (Stevenson et al., 2006; Enaud et al., 2004; Gao et al., 2001; Nakajima et al., 2000). It has been observed that the performance of the process depends mainly on the nature of the substitutions, the position of the hydroxyls and the length of the spacers.
