## 3 Results and Discussion
### Computational Analysis for the Evaluation of the CalB Stereoselectivity  
Although the broad substrate specificity and the stereoselectivity of lipases are some of the characteristics that decide the synthetic success of these catalysts, the enantioselectivity of L435 can restrict its activity towards a preferred enantiomer of ibuprofen racemate [30]. The enantioselectivity of this enzyme can be described by using computational analyses (docking) able to assess the most stable poses of the two enantiomers of ibuprofen in the active site of the enzyme, highlighting the most favored for the catalysis. The (R)- and (S)-enantiomers of ibuprofen were docked into the catalytic site of L435 using AutoDock Vina, based on a Monte Carlo search and rapid gradient-optimization conformational search coupled with a simple scoring function for free-energy estimation [31]. The Near Attack Conformation (NAC) [32] is the assumption to identify the \"productive\" pose, namely, that which is able to produce the attack of the catalytic serine (Ser105) to the carbon atom of the acyl group of the ligands (the (R)- and (S)-ibuprofen, in this case). The results of the docking experiments are reported in Table 1.  
The lower the energy (E), the more stable the pose of the enantiomer, and the more favored the catalysis. To be considered as a productive pose, the acyl carbon of ibuprofen must have a maximum distance (_d_) to the serin oxygen (Ser105 of CalB) of 3.2 A. Furthermore, the orientation of the acyl carbon of the ligands towards the serine oxygen (\(\emptyset\)) must be such so as not to exceed a 90\({}^{\circ}\) angle. Based on these assumptions, the data shown in Table 1 display how the (R)-enantiomer of ibuprofen is the energetically favored substrate for the esterification with L435.  
Figure 3 shows the best poses of the two enantiomers of ibuprofen in the active site of the enzyme. Based on these data, molecules **3a** and **3b** of Figure 1 are the expected products.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Lipase** & **Ligand** & **E (kcal/mol)** & \(d\) (Å)** & \(\emptyset\) (\({}^{\circ}\)) \\ \hline \multirow{2}{*}{CalB} & (R)-ibuprofen & \(-6.7\) & 3.21 & 67.7 \\  & (S)-ibuprofen & \(-6.2\) & 3.97 & 101.8 \\ \hline \hline \end{tabular}
\end{table}
Table 1: AutoDock Vina data relative to the docking free energies (E) computed only for the poses corresponding to the lowest energy values. The distances (_d_) and angles (\(\emptyset\)) were calculated by taking as a reference the C atom of the ibuprofen carbonyl group.  
Figure 2: Undesired byproduct **4** resulting from the 1,3-diacylation of erythroid catalyzed by lipases (the three possible configurations of product **4** are schematically represented in the Supplementary Material on page 25, Figure S24).  
Figure 3: AutoDock Vina representation of the best poses of (\(R\))-ibuprofen (**left**, purple) and (\(S\))-ibuprofen (**right**, blue) in the active site of CalB (PDB ID: 1 TCA; catalytic triad: Ser105, His224, and Asp187). (**a**,**b**) \(d\) values computed for the distance between the acyl carbon of ibuprofen and serin oxygen of CalB. (**c**,**d**) Residues in the active site involved in binding interactions with ibuprofen. (**e**,**f**) Kyle–Doolittle hydrophobicity scale representation of the active site of CalB with docked ibuprofen (blue, hydrophobic regions; red, hydrophilic regions).