## 3 Results and Discussion
### Lipozyme RM IM-Catalyzed Synthesis of (S)-IBU-Glycerol Ester  
Due to the herein reported limitations imposed by 2M2B and erythiritol for the esterification of ibuprofen, we decided to move towards _solventless_ strategies involving glycerol as a hydrophilizer for the preparation of an (\(S\))-ibuprofen prodrug with increased hydrophilicity compared to the lead compound (Scheme 2). Similar to what was previously reported for the reaction between _rac_-**1** and **2** (Scheme 1), the reaction in Scheme 2 can also yield two different ester configurations between (S)-**1** and **5**, resulting in **6a**/**6b** (Figure 8). Glycerol showed the ability to completely dissolve ibuprofen without the need for cosolvents. The reaction with free _C. rugosa_ lipase was unsuccessful, leading to no ester production after 24 h. The _C. rugosa_ enzyme is an interfacial enzyme reported as strongly influenced by the presence of an interfacial area for its shift to an active conformation [40]. In this case, as the _solventless_ reaction medium was a monophasic system with limited interfacial surface between substrates, probably the enzyme did not expose the active site via lid movement, not reaching its catalytically active form. The lipase from _Aspergillus oryzae_ showed moderate activity in the esterification of **1** with **5**, reaching a conversion of 23% after 24 h. Instead, the reaction catalyzed by RM IM achieved a conversion of 83 \(\pm\) 5% after 24 h, without traces of the diglyceride, confirming glycerol as a great reagent for the _solventless_ esterification of (\(S\))-ibuprofen. Additional advantages of using glycerol lie in its safety, liquid nature at room temperature, viscosity, high boiling point, and low vapor pressure. Glycerol is a coproduct of the biodiesel industry, and its availability has grown enormously in recent years, lowering its price. By virtue of this, its uses have multiplied, extending beyond its uses in the food, cosmetic, and pharmaceutical fields, such as the enzymatic synthesis of esters and polyesters [41]. Compared to the above-reported esterification with erythroid, where the use of a reaction solvent was mandatory, in this _solventless_ synthesis, glycerol acts not only as a substrate but also as a protein stabilizer, shifting the native structure of the catalyst to a more compact state [42]. This helps prevent the loss of enzymatic activity, enhances the thermal unfolding temperature, and prevents irreversible protein aggregation. Consequently, the utilization of this highly versatile short polyalcohol provides greater flexibility in the experimental procedures of enzymatic syntheses. To verify the role of 2M2B in the failure of the RM IM-catalyzed esterification of ibuprofen with erythroid, we repeated the reaction of (\(S\))-ibuprofen with glycerol by adding 2M2B in the same amount of glycerol (glycerol was miscible to 2M2B). Under this condition, the conversion dropped to 31 \(\pm\) 5%, thereby testifying to the negative effect of 2M2B on RM IM activity. Figure 9 shows the diagnostic signals in the \({}^{1}\)H-NMR spectrum of compound 6**a**/**6**b**, which is composed of an equimolar mixture of diastereoisomers because of the prochiral nature of glycerol (the complete NMR characterization is reported in the Supplementary Materials). Figure 8: Stereochemistries of the two possible reaction products 6**a** and 6**b**. Figure 9: Diagnostic region of the \({}^{1}\)H-NMR spectrum of the equimolar diastereomeric mixture of compound 6**a**/**6**b**. The docking of **1** to the active site of the _Rhizomucor miehei_ lipase confirmed the preference of the enzyme towards the (\(S\))-enantiomer of **1** (Figure 10). Indeed, the distance between the acyl carbon of 1 and the serin144 of the catalytic triad of RM lipase is 6.07 A for (\(R\))-**1**, compared to the 3.22 A for (\(S\))-**1** (energy (kcal/mol): -6.5 for (\(R\))-1 and -6.7 for (\(S\))-**1**).