β-(3-Thienyl)-D-β-homoglycine

Mutants of Spirulina platensis resistant to 5-fluorotryptophan, beta-3-thienyl-alanine, ethionine, p-fluorophenylalanine, or azetidine-2-carboxylic acid were isolated. Some of these mutants appeared to be resistant to more than one analog and to overproduce the corresponding amino acids. A second group was composed of mutants that were resistant to one analog only. Of the latter mutants, one resistant to azetidine-2-carboxylic acid was found to overproduce proline only, whereas one resistant to fluorotryptophan and one resistant to ethionine did not overproduce any of the tested amino acids.

Engineered myoglobins were recently shown to be effective catalysts for abiological carbene and nitrene transfer reactions. Here, we investigated the impact of substituting the conserved heme-coordinating histidine residue with both proteinogenic (Cys, Ser, Tyr, Asp) and non-proteinogenic Lewis basic amino acids (3-(3'-pyridyl)-alanine, p-aminophenylalanine, and β-(3-thienyl)-alanine), on the reactivity of this metalloprotein toward these abiotic transformations. These studies showed that mutation of the proximal histidine residue with both natural and non-natural amino acids result in stable myoglobin variants that can function as both carbene and nitrene transferases. In addition, substitution of the proximal histidine with an aspartate residue led to a myoglobin-based catalyst capable of promoting stereoselective olefin cyclopropanation under nonreducing conditions. Overall, these studies demonstrate that proximal ligand substitution provides a promising strategy to tune the reactivity of myoglobin-based carbene and nitrene transfer catalysts and provide a first, proof-of-principle demonstration of the viability of pyridine-, thiophene-, and aniline-based unnatural amino acids for metalloprotein engineering.