Biocatalysis is an effective tool to access chiral molecules that are otherwise hard to synthesize or purify. Time-efficient processes are needed to develop enzymes that adequately perform the desired chemistry. We evaluated machine-directed evolution as an enzyme engineering strategy using a moderately stereoselective imine reductase as the model system. We compared machine-directed evolution approaches to deep mutational scanning (DMS) and error-prone PCR. Within one cycle, it was found that machine-directed evolution yielded a library of high-activity mutants with a dramatically shifted activity distribution compared to that of traditional directed evolution. Structure-guided analysis revealed that linear additivity might provide a simple explanation for the effectiveness of machine-directed evolution. The most active and selective enzyme mutant, which was identified through DMS and error-prone PCR, was used for the gram-scale synthesis of the H4 receptor antagonist ZPL389 with full conversion, > 99% ee (R), and a 72% yield.

The immunodominant central portion of the circumsporozoite (CS) surface protein of the malaria parasite Plasmodium falciparum contains a tetrapeptide motif, Asn-Pro-Asn-Ala (NPNA), tandemly repeated almost 40 times. The three-dimensional structure of the CS protein, including the central repeat region, is presently unknown. We have investigated an approach to stabilize β-turns in a single NPNA motif, by its incorporation into a template-bound cyclic peptide comprising the sequence ANPNAA. The template was designed to stabilize β-turns in the peptide loop and to allow its conjugation to T-cell epitopes in a multiple-antigen-peptide. NMR studies and MD simulations with time-averaged NOE-derived upper distance restraints support the formation of a stable β-I turn conformation in the NPNA motif of this template-bound antigen. Balb/c mice immunized with a multiple-antigen-peptide containing four copies of the template-bound loop conjugated to a single universal T-cell epitope produced antibodies that bound P. falciparum sporozoites in immunofluorescence assays. These results provide further support for the immunological relevance of a type-I β-turn conformation based on the NPNA cadence in the repeat region of the CS protein and illustrate the use of a novel template for the evaluation of conformationally constrained peptide immunogens.

Cladosporin, a natural product known for decades, has recently been discovered to display potent and selective antiplasmodial activity by inhibition of lysyl‐tRNA synthetase. It was subjected to a panel of oxidative biotransformations with one fungal and two actinomycetes strains, as well as a triple mutant bacterial CYP102A1, yielding eight, mostly hydroxylated, derivatives. These new compounds covered a wide chemical space and contained two pairs of epimers in the tetrahydropyran ring. Although less potent than the parent compound, all analogues showed activity in a cell‐based synthetase assay, thus demonstrating uptake and on‐target activity in living cells with varying degrees of selectivity for the enzyme lysyl‐tRNA synthetase from Plasmodium falciparum and highlighting sites suitable for synthesis of future cladosporin analogues. Compounds with adjacent hydroxy functions showed different MS/MS fragmentation that can be explained in terms of an, in some cases, regioselective loss of water followed by a retro‐Diels–Alder reaction.

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