Directed evolution of RebH for catalyst-controlled halogenation of indole C–H bonds

Abstract

RebH variants capable of chlorinating substituted indoles ortho-, meta-, and para- to the indole nitrogen were evolved by directly screening for altered selectivity on deuterium-substituted probe substrates using mass spectrometry. This systematic approach allowed for rapid accumulation of beneficial mutations using simple adaptive walks and should prove generally useful for altering and optimizing the selectivity of C-H functionalization catalysts. Analysis of the beneficial mutations showed that structure-guided selection of active site residues for targeted mutagenesis can be complicated either by activity/selectivity tradeoffs that reduce the possibility of detecting such mutations or by epistatic effects that actually eliminate the benefits of a mutation in certain contexts. As a corollary to this finding, the precise manner in which the beneficial mutations identified led to the observed changes in RebH selectivity is not clear. Docking simulations suggest that tryptamine binds to these variants as tryptophan does to native halogenases, but structural studies will be required to confirm these models and shed light on how particular mutations impact tryptamine binding. Similar directed evolution efforts on other enzymes or artificial metalloenzymes could enable a wide range of C-H functionalization reactions.

Development of Halogenase Enzymes for Use in Synthesis

“…Starting from a RebH mutant previously found to be more thermostable 224 and using a total of 6 rounds of screening, two RebH mutants were found which halogenated the 6-and 5-positions of an indolic substrate (84) with good selectivity and reasonable conversion. 223 This method does not require structural information about the enzyme and can be used to modulate regioselectivity in a semi high-throughput manner, in contrast to most high-throughput screening (HTPS) methods which focus on improving stability or activity of an enzyme. Random mutagenesis has also been used in a "substrate walking" approach to allow the late-stage halogenation of a number of large bioactive substrates such as 85 to 88 (Figure 22).…”

“…As the monooxygenase screening methods reported so far are dependent upon detection of a specific reaction product or sequence of reactions occurring, they are not appropriate for application to the flavin-dependent halogenases, although methods which could be applied to their directed evolution are slowly emerging and have been demonstrated feasible. 223,240,241 In combination with what is known about the modification of their substrate scope, regioselectivity and stability through mutagenesis, 150,155,[222][223][224][225] in addition to the discovery of naturally-occurring thermostable Fl-Hals, 145 a number of reliable starting points for further engineering efforts are known.…”

“…Additionally, work on adapting the Fl-Hals to accept larger, bioactive, substrates 223,225 may mean that ultimately they find application as a means of late-stage C-H activation.…”

Enzymatic site-selectivity enabled by structure-guided directed evolution

“…Indeed, this was demonstrated in a subsequent study in which tryptamine (40) was used as the model substrate (Scheme 14). 58 At the outset of the project, the goal was defined as the search for three different RebH mutants leading to the ortho-, meta-and para-products 41, 42 and 43, respectively. In the case of chlorination product 41, epPCR sufficed, but in order to evolve meta-and para-selectivity, it was more productive to apply saturation mutagenesis and iterative saturation mutagenesis at rationally chosen sites lining the binding pocket (CAST/ISM).…”

“…In the case of chlorination product 41, epPCR sufficed, but in order to evolve meta-and para-selectivity, it was more productive to apply saturation mutagenesis and iterative saturation mutagenesis at rationally chosen sites lining the binding pocket (CAST/ISM). 58 Directed evolution of flavin-dependent regioselective halogenases is a relatively new research area from which more advances can be expected. It is likely that structure-guided saturation mutagenesis will play the dominant role in future studies.…”