Engineering of RuMb: Toward a Green Catalyst for Carbene Insertion Reactions

The small, stable heme protein myoglobin (Mb) was modified through cofactor substitution and mutagenesis to develop a new catalyst for carbene transfer reactions. The native heme was removed from wild-type Mb and several Mb His64 mutants (H64D, H64A, H64V), and the resulting apoproteins were reconstituted with ruthenium mesoporphy­rin IX (RuMpIX). The reconstituted proteins (RuMb) were characterized by UV–vis and circular dichroism spectroscopy and were used as catalysts for the N–H insertion of aniline derivatives and the cyclopropanation of styrene derivatives. The best catalysts for each reaction were able to achieve turnover numbers (TON) up to 520 for the N–H insertion of aniline, and 350 TON for the cyclopropanation of vinyl anisole. Our results show that RuMb is an effective catalyst for N–H insertion, with the potential to further increase the activity and stereoselectivity of the catalyst in future studies. Compared to native Mb (“FeMb”), RuMb is a more active catalyst for carbene transfer reactions, which leads to both heme and protein modification and degradation and, hence, to an overall much-reduced lifetime of the catalyst. This leads to lower TONs for RuMb compared to the iron-containing analogues. Strategies to overcome this limitation are discussed. Finally, comparison is also made to FeH64DMb and FeH64AMb, which have not been previously investigated for carbene transfer reactions.