Esterases and lipases can process amphiphilic esters used as drugs and prodrugs and impact their pharmacokinetics and biodistribution. These hydrolases can also process ester components of drug delivery systems (DDSs), thus triggering DDSs destabilization with premature cargo release. In this study we tested and optimized assays that allowed us to quantify and compare individual esterase contributions to the degradation of substrates of increased lipophilicity and to establish limitations in terms of substrates that can be processed by a specific esterase/lipase. We have studied the impact of carbonic anhydrase; phospholipases A1, A2, C and D; lipoprotein lipase; and standard lipase on the hydrolysis of 4-nitrophenyl acetate, 4-nitrophenyl palmitate, DGGR and POPC liposomes, drawing structure–property relationships. We found that the enzymatic activity of these proteins was highly dependent on the lipophilicity of the substrate used to assess them, as expected. The activity observed for classical esterases was diminished when lipophilicity of the substrate increased, while activity observed for lipases generally increased, following the interfacial activation model, and was highly dependent on the type of lipase and its structure. The assays developed allowed us to determine the most sensitive methods for quantifying enzymatic activity against substrates of particular types and lipophilicity.

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ChemInform Abstract Commercially available pig liver esterase (PLE) is separated in 6 different main isozyme fractions and it is shown that their individual stereospecifities are essentially the same in the hydrolysis of representative monocyclic and acyclic diester substrates (I), (III) (→ (II), (IV)). This unexpected discovery removes previous considerations that different isozymal compositions of PLE, a widely used catalyst for chiral synthon production, could result in an inconsistent behavior when applied for asym. synthetic purposes. The results establish that commercial PLE can be exploited synthetically as a chiral catalyst with confidence that it will behave as if it were a single protein.

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