Directed Evolution of RebH for Site‐Selective Halogenation of Large Biologically Active Molecules

Abstract

We recently characterized the substrate scope of wild-type RebH and evolved variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. We have now used a substrate walking approach to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3-SS, which provides high conversion of tricyclic tryptoline derivatives; and 4-V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable functionalization of substrates not accepted by wild-type RebH and demonstrates the utility of RebH variants for site-selective halogenation of biologically active compounds.

Development of Halogenase Enzymes for Use in Synthesis

“…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). 225 Over a total of 4 generations of error-prone PCR, RebH was mutated to a quintuple mutant capable of halogenating the large C4-functionalised substrate 87 with complete regioselectivity. Together, these methods demonstrate the potential to develop a suite of biocatalysts for regiodivergent halogenation; either from a single parent enzyme or using halogenases of different natural regioselectivity.…”

“…[32][33][34]218,224,225 This is likely to be an artefact of these enzymes evolving as part of the biosynthetic pathways to non-essential secondary metabolites. Halogenases are not essential for the survival or growth of the native host, and hence there has been little evolutionary pressure for highly active halogenase enzymes.…”

“…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

“…56 In a subsequent study aimed at increasing activity and expanding substrate range, one of the stabilized mutants was used as a template for further mutagenesis, several cycles epPCR again being applied. 57 Following the screening of about 2200 transformants, two prominent variants were identified: 3-SS which is ideal for site-selective chlorination of tricyclic tryptoline derivatives, and 4-V which shows broad substrate range of indole and carbazole derivatives, likewise with high site-selectivity (Table 1). 57 All transformations are synthetically interesting, the meta-products such as 31 and 33 deserving special attention.…”