## 3 Results and Discussion
### Experimental Validation of the Computational Study
Although a protective effect on the biocatalyst has been reported for certain polyalcohols, such as glycerol (mainly attributed to its viscosity) [35], reactions with polyols solid at room temperature, such as erythritol, can be challenging. In fact, it was reported that some polyhydric alcohols can reduce the lipase activity [36], and erythroid seems to be no exception. Polyhydric alcohols can interfere with the water necessary for maintaining  
Figure 5: Proton NMR of the equimolar mixture of diastereoisomers **3a**/**3b**. the catalytically active conformation of the enzyme. The scientific literature discusses how three key characteristics of polyalcohols, the molecular size, hydrophobicity, and solubility, impact the enzyme's efficiency. The binding energy, which is influenced by the size of the polyhydroxylated molecule, plays a crucial role in activating the enzyme when the substrate binds to the active site. A low binding energy results in a suboptimal three-dimensional conformation of the lipase, leading to a slower reaction rate. Additionally, the enzyme's exposure to alcoholic functions increases with the solubility of the polyhydroxylated substrate in water, thereby promoting denaturation and subsequent inactivation. Furthermore, the influence of the reaction solvent on the distribution of water, understood as the quantity of water in the aqueous layer surrounding the catalyst, must be highlighted. Indeed, it is generally reported that solvents with logP values lower than 2 are less suitable for biocatalytic purposes [37]. In our case, the use of high volumes of 2M2B for the solubilization of the erythroid excess was detrimental to the enzyme activity. For these reasons, we moved to test reactions performed with a higher amount of racemic ibuprofen with respect to erythroid (molar ratio: 3:1). The reaction course was monitored for 6 days. Data are shown in Figure 7.  
Figure 6: Conversion (%) for the reactions conducted with acid/alcohol molar ratios of 1:1, 1:3.5, and 1:6; and 20, 40, and 60% of the enzyme for 24 h at temperatures of 50 \({}^{\circ}\)C (**a**) and 90 \({}^{\circ}\)C (**b**). Figure 7: Conversion (%) of erythroid to an equimolar mixture of mono-acylated IBU–erythitol ester **3a**-**3b** (purple) and 1,3 di-acylated ester **4** (blue) as a function of the reaction time. Reaction condition: 20% (\(w/w\) with respect to **1**) L435, **1/2** molar ratio of 3:1, 50 \({}^{\circ}\)C, 400 RPM. It was evident that the ibuprofen excess afforded high erythroid conversion. The low temperature of 50 \({}^{\circ}\)C was chosen in order to increase the selectivity, avoiding the formation of the undesired 1,4-bis ester **4**[10,35]. Taking into account the preference of L435 for the (\(R\))-enantiomer of ibuprofen, the 3:1 molar ratio provided enough substrate for the complete conversion of erythiritol into the derivatives **3a** and **3b**, limiting at the same time the bis-acylation of erythiritol, which, however, increased at longer reaction times. After the first 24 h, a progressive lowering of the conversion was also observed, probably due to the increasing amount of water in the reaction medium. Tests conducted at higher enzyme concentrations led to increased diester concentrations (data not shown), which were not favorable for the objectives of our study. With the enzyme amount used for the reaction reported in Figure 7, we achieved significant amounts of the monoester in comparison to the diester, aided by the lower temperature. Furthermore, due to the excess of ibuprofen over erythroid, the adverse effects of the polyol on the enzyme were mitigated, enabling high conversions at low enzyme concentrations. The reaction performed under the same conditions on the pure (5)-enantiomer of ibuprofen reached a 12% conversion after 6 days, confirming _C. antarctica_ lipase B (L435 in this case) as an (\(R\))-selective enzyme towards ibuprofen. In virtue of the above results, we moved to investigate the esterification of the (\(S\))-enantiomer of ibuprofen by testing biocatalysts alternative to the _C.