Within our enzyme design work package, we successfully expressed a protein host harboring the uAA para-aminophenylalanine (pAF), following established protocols. However, in our hands, this designer protein did not demonstrate the expected nucleophilic activation via enamine formation. To overcome this obstacle, we embarked in the design and genetic incorporation development of novel uAA’s bearing cyclic secondary amines, which are known to promote enamine chemistry. However, this task proved technically challenging, and no successful candidates were obtained
In parallel, within our cofactor development work package, we synthesize a small library of transition metal complexes with phenanthroline derivatives, including various cobalt-containing species. These were evaluated with a panel of allylic electrophilic substrates under different conditions. Yet, the desired allylic functionalization was not observed.
At this stage, as our initial objectives faced substantial challenges to be achieved, likely due to the incompatibility of the targeted transformation with aqueous media, we reassessed our proposed plan and decided to pivot towards late-transition metal catalysis, whose reaction conditions were more compatible with our biocatalytic platform proposal. With the aim of achieving allylic or benzylic functionalization, we adjusted our objective: developing a gold-mediated benzylic alcohol formation., a structural motif present in drug candidates with, i.e. immunosuppressant activity. Moreover, we set our attention to developing an asymmetric version of this transformation by taking advantage of the chiral environment given by our protein host. Inspired by in-house preliminary work, we explored the coordination of gold via canonical and non-canonical amino acid residues. Upon reaction optimization, these systems successfully gave chiral secondary alcohols in a wide range of yield and enantioselectivities. One of these systems was further refined under a directed evolution campaign to improve its catalytic performance in terms of both activity and selectivity.
The outcomes of this project were presented in six international congresses. The work on uAA contributed to the publication of two open-access articles in internationally recognized high impact journals.
