**2.3.2 Acyl acceptor**

272 Biochemistry

**Aliphatic acids Aromatic acids**  Acetic\* Caffeic\* Malic\* p-Coumaric\* Malonic\* Ferulic\* Succinic\* Gallic\* Tartaric\* p-Hydroxybenzoic\* Butyric Sinapic\* Crotonic Benzoic n-Butanoic Cinnamic Isobutyric Isoferulic Isovaleric Methylsinapic

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

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

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.

Lactic 3-methylbutyric Quinic Vinylpropionic Tiglic

\*acyl donors found in anthocyanins

(Andersen & Jordheim, 2006).

chain length were used.

The structure of acyl acceptor (flavonoid), especially stereochemistry of glycosidic bonds, plays an important role in flavonoid acylation. The structural differences, such as the number and position of hydroxyl groups, the nature of saccharidic moiety, as well as the position of glycosidic bonds, influence the flavonoid solubility, and thus affect the overall conversion yield.

Available studies are concerned mainly with acylation on flavonoid glycosides. Among polyphenolic compounds, naringin and rutin are the most widely used substrates. For the naringin molecule, which possesses a primary hydroxyl group on glucose, the acylation takes place on the 6''-OH (Katsoura et al., 2006; Konntogianni et al., 2001, 2003; Ishihara et al., 2002; Gao et al., 2001; Otto et al., 2001; Danieli et al., 1990) since the primary hydroxyl is favored by CALB (Fig.4). However, in rutin, which has no primary hydroxyl available, either the 3''-OH of glucose (Ishihara et al., 2002; Danieli & Riva, 1994) or the 4'''-OH of rhamnose (Fig.4) (Viskupicova et al., 2010; Mellou et al., 2006; Ardhaoui et al., 2004a, 2004b, 2004c) can be acylated. Danieli et al. (1997) observed the rutin-3'',4'''-*O*-diester formation. When subtilisin was used as biocatalyst, naringin-3''-*O*-ester and rutin-3''-*O*-ester were synthesized (Danieli et al., 1990).

Fig. 4. Acylation sites of naringin (left) and rutin (right) molecule.

The concentration of the flavonoid also affects the performance of the acylation reaction. The conversion yield and the initial rate rise with increasing flavonoid concentration. However, the amount of flavonoid is limited by its solubility in a reaction medium (Chebil et al., 2006, 2007).
