## 3 Results and Discussion
### Experimental Validation of the Computational Study  
We move to the experimental reaction design using an initial 1:1 molar ratio between _rac_-**1** and **2** in order to favor the formation of the above monoesters **3**, testing different amounts of biocatalyst and the effect of three different temperatures. The % conversion is calculated from the ratio between the esterified and free ibuprofen detected via 1H-MNR analysis (as shown in Figure 4a; the results of the reactions are shown in Figure 4b). Figure 4a demonstrates the application of NMR to calculate the conversion of **1** by using the shift of the CH\({}_{3}\) proton signal from the starting **1** (1.32 ppm) to the products **3** (1.38 ppm). L435 is known to be able to reach high working temperatures. For this reason, we tested three temperatures: 50 \({}^{\circ}\)C, 70 \({}^{\circ}\)C, and 90 \({}^{\circ}\)C, with a fixed reaction time of 24 h. Data shown in Figure 4b suggest that, in the presence of 2M2B as the solvent, at the lower enzyme amount, inactivation phenomena negatively affected the conversion at the higher temperatures. With an enzyme concentration of 20%, the conversion of the API decreased with the increase in temperature. Raising the enzyme concentration to  
Figure 4: (**a**) Proton NMR (region from 0.7 to 1.85 ppm) of **1** (**top**) and the raw reaction of esterification of **1** with **2** to attain **3** (**bottom**). (**b**) E20, E40, and E60 indicate the reaction with different \(w/w\) percentages of the enzyme with respect to ibuprofen (20, 40, and 60%, respectively). Reaction condition: 1:1 **1** to **3** molar ratio, 400 rpm, 24 h reaction time. 40% (E40), the amount of biocatalyst compensates for the inactivation phenomena, with a resulting increase in the conversion for the higher temperatures. This trend is even more evident by charging the reactor with 60% of the enzyme (E60). Corresponding blank experiments performed under the same conditions in the absence of the catalyst did not afford any products. Temperature also affects the solubilization of the substrates, thus implying that higher temperatures result in improved mass transfer. However, as the temperature increases, the active site may become more flexible, potentially leading to a reduction in enzyme selectivity and an increase in the formation of byproducts, such as polyacylated erythritols. Indeed, at 90 \({}^{\circ}\)C, with the enzyme concentration of 60%, the reaction afforded about 10% of the 1,4 diacylated derivative **4**. Both product **3** and the byproduct **4** were isolated via column chromatography and characterized via NMR analyses (\({}^{1}\)H-, \({}^{13}\)C-, DEPT-, COSY-, HSQC-, and HMBC-NMR; see Supplementary Materials), which confirmed that the monoester product is an equimolar mixture of diastereoisomers (**3a** and **3b**, Figure 2), as deductible by the double set of signals attributable to the protons of the esterified hydroxymethylene group of erythroid in the region between 4.3 and 3.8 ppm of the \({}^{1}\)H-NMR spectrum (Figure 5). The solubility of a compound in aqueous-based media is a crucial property. Solubility directly influences a compound's bioavailability, affecting its absorption, and serves to validate the in vitro assay data obtained. It is generally reported that compounds with solubilities greater than 60 ug/mL may have good absorption potential [33]. The water solubility of product **3** based on the turbidimetric method reported by [PERSON] et al. [34] was 463 ug/mL, which was 22 times better than the acid ibuprofen. In the attempt to maintain a high production of monoesters **3a** and **3b**, minimizing the diacylation of erythroid, we investigated the effect of different substrate molar ratios by raising the amount of polyol with respect to ibuprofen. The results are shown in Figure 6.  
In both the temperatures tested (50 and 90 \({}^{\circ}\)C: the lowest and the highest considered in this study), raising the alcohol molar concentration led to a decrease in the conversion.