Abstract
Enzyme-specific activation and the substrate mimetics strategy are effective ways to circumvent the limited substrate recognition often encountered in protease-catalyzed peptide synthesis. A key structural element in both approaches is the guanidinophenyl (OGp) ester, which enables important interactions for affinity and recognition by the enzyme—at least, this is usually the explanation given for its successful application. In this study we show that leaving group ability is of equal or even greater importance. To this end we used both experimental and computational methods: 1) synthesis of close analogues of OGp, and their evaluation in a dipeptide synthesis assay with trypsin, 2) molecular docking studies to provide insights into the binding mode, and 3) ab initio calculations to evaluate their electronic properties.
Highlights
These days enzymes are commonly used in organic synthesis.[1]
The guanidinophenyl (OGp) ester, which in essence resembles the naturally recognized side chain of arginine, is claimed to serve as a recognition moiety for trypsin, thereby making recognition independent of the side chain of the amino acid and broadening substrate scope. This approach is typically applied under aqueous conditions; but because other proteases.[3] The guanidinophenyl (OGp) functions as a leaving group, the commonly occurring secondary hydrolysis is prevented, as the product formed becomes unrecognizable for the enzyme
To distinguish between the effects of affinity for the enzyme and leaving group ability, we evaluated a set of close analogues of OGp that differed slightly in both properties
Summary
These days enzymes are commonly used in organic synthesis.[1]. The benefits of enzymatic reactions, including their generally excellent regio- and enantioselectivity, are widely recognized. A prerequisite for this enzymatic activity, irrespective of whether aqueous media or organic solvents are used, is that specific amino acids are recognized.[2] This problem of recognition can be circumvented by applying the “substrate mimetics” strategy as previously described for trypsin and other proteases.[3] The guanidinophenyl (OGp) ester, which in essence resembles the naturally recognized side chain of arginine, is claimed to serve as a recognition moiety for trypsin, thereby making recognition independent of the side chain of the amino acid and broadening substrate scope This approach is typically applied under aqueous conditions; but because OGp functions as a leaving group, the commonly occurring secondary hydrolysis is prevented, as the product formed becomes unrecognizable for the enzyme. An ab initio study provided insight into the electronic properties of the analogues under investigation
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