## 3 Results and Discussion  
### Screening of Free and Immobilized Lipases for the Synthesis of Ibuprofen (IBU)-Erytritol Ester  
The methodology developed in this study focuses on the functionalization of ibuprofen (indicated as IBU) to obtain hydrophilic prodrugs, embracing the principles of green chemistry. The process reported in this study deviates from the esterification of ibuprofen previously reported (for instance, the _solventless_ reaction with glycerol or those in biphasic systems involving the ester of ibuprofen with sorbitol). Herein, both the polyalcohol and ibuprofen acid were in solid form and studied in a monophasic reaction medium. Thus, it was necessary to use a solvent able to solubilize both substrates, characterized as different in polarity and, thus, hydrophilic/hydrophobic profile (the logP of ibuprofen is 3.97, while that of erythroid is \(-\)2.29). This work demonstrates that erythroid has a detrimental effect on the esterification capacity of the exploited lipases, and that the effect of the solvent on the enzymatic activity is not negligible. The work here reported provides an important integration into the already vast literature concerning processes biocatalyzed by lipases, extending the knowledge even beyond the widespread _solventless_ reactions (which see the use of an alcohol as both solvent and reagent) and the intrinsically limited biphasic reactions(which, due to the presence of water, cannot achieve high yields). The limitations of this approach are underlined, and for each one, we provide experimental evidence to support the hypotheses. For this purpose, bioreactors were employed, harnessing biocatalysts with molecular mechanisms capable of producing the desired prodrugs under environmentally sustainable conditions. The herein studied approach can, in principle, be extended to a variety of poorly water-soluble APIs. Scheme 1 shows the esterification of ibuprofen with erythroid for the synthesis of the desired prodrug (IBU-erythnitol ester). The lipase could, in principle, produce a mixture of four possible stereoisomers of the monoester **3** (**3a-3d**, Figure 1). In this study, the stereoisomeric outcome was predicted through a computational approach. In the rational design of the prodrug **3**, the polyol moiety represents both a hydrophilicity enhancer (due to its high water solubility) as well as a potential functional improving agent (for its antioxidant properties) for a dualistic IBU derivative. To promote this esterification reaction, four lipases were tested as biocatalysts: _Candida antarctica_ lipase **B**, _Rhizomucor mielei_ lipase, _Candida rugosa_ lipase, and _Apergillus oryzae_ lipase, starting from the commercially available immobilized form of _Candida antarctica_ lipase type B Lipozyme(r) 435 (hereafter indicated as L435). Although water/organic solvent systems can be exploited for biocatalyzed reactions, in this case, the presence of water needed to be avoided to shift the esterification-hydrolysis equilibrium [14]. As we previously demonstrated, \(t\)-amyl alcohol 2-methylbutan-2-ol (2M2B) was a suitable solvent able to solubilize both the substrates of this study, and it was used to create a homogeneous reaction mixture [2]. It is important to highlight that, to obtain the IBU-erythnitol ester **3**, the biocatalyzed route needs to agree with the green chemistry principles, as it allows for the selective esterification of the primary hydroxyl groups of erythroid [28] without the need for protection-deprotection strategies, which strongly decrease the process atom and step economies. Furthermore, this reaction design, which employs the direct esterification of IBU as acid instead of its ester derivatives, produces water, avoiding the formation of organic coproducts (alcohols, such as methanol). Due to its two primary hydroxyl groups, erythnitol can be potentially mono- or di-esterified by the biocatalyst.