Engineered RebH Halogenase Variants Demonstrating a Specificity Switch from Tryptophan towards Novel Indole Compounds

Abstract

Activating industrially important aromatic hydrocarbons by installing halogen atoms is extremely important in organic synthesis and often improves the pharmacological properties of drug molecules. To this end, tryptophan halogenase enzymes are potentially valuable tools for regioselective halogenation of arenes, including various industrially important indole derivatives and similar scaffolds. Although endogenous enzymes show reasonable substrate scope towards indole compounds, their efficacy can often be improved by engineering. Using a structure‐guided semi‐rational mutagenesis approach, we have developed two RebH variants with expanded biocatalytic repertoires that can efficiently halogenate several novel indole substrates and produce important pharmaceutical intermediates. Interestingly, the engineered enzymes are completely inactive towards their natural substrate tryptophan in spite of their high tolerance to various functional groups in the indole ring. Computational modelling and molecular dynamics simulations provide mechanistic insights into the role of gatekeeper residues in the substrate binding site and the dramatic switch in substrate specificity when these are mutated.

Indole and azaindole halogenation catalyzed by the RebH enzyme variant 3-LSR utilizing co-purified E. coli reductase

“…For indole halogenation, substituents at different positions on the benzenoid ring were well-tolerated by 3-LSR as monobrominated indoles were obtained in all cases as determined by LC-MS analysis. NMR analysis confirmed the formation of 3-bromoindoles (Supplementary Figure S2) (Sana et al, 2021). In analogy to the catalytic mechanism of tryptophan halogenases, the mechanism here is proposed to involve a Lys79-bromoamine intermediate active species which carries out electrophilic aromatic substitution at the most electron rich C3-position of the indole ring followed by deprotonation of the Wheland intermediate assisted by glutamate 346 oxygen to form 3bromoindoles (Figure 3) (Fraley et al, 2017;Karabencheva-Christova et al, 2017;Schnepel and Sewald, 2017;Ismail et al, 2019).…”

Extended Biocatalytic Halogenation Cascades Involving a Single‐Polypeptide Regeneration System for Diffusible FADH2

“…While activity and stability of wild type FDHs leave much to be desired, [4] enzyme engineering and directed evolution campaigns are underway to improve these shortcomings [5] . Tryptophan halogenases in particular have been thoroughly characterised and engineered regarding thermostability, [6] regioselectivity, [7] and their substrate spectrum [8,9] . Beyond protein engineering, another common approach to improve catalyst stability and even enable catalyst recyclability is enzyme immobilisation [10] …”

“…[5] Tryptophan halogenases in particular have been thoroughly characterised and engineered regarding thermostability, [6] regioselectivity, [7] and their substrate spectrum. [8,9] Beyond protein engineering, another common approach to improve catalyst stability and even enable catalyst recyclability is enzyme immobilisation. [10] A particularly convenient immobilisation strategy was first developed in Sheldon's laboratory.…”

One-Pot Synthesis of 1,2-Disubstituted Indoles from 2-Ethynylanilines and Benzaldehydes

“…Therefore, efficient protocols for the synthesis of substituted indoles are highly necessary. Since the development of the classical synthesis of indoles by Fischer, Batcho–Leimgruber, Fukuyama, Nenitzescu, Bartoli, etc., a number of synthetic methods to access indole derivatives have been investigated, − most of which start from 2-ethynylaniline derivatives. − Among these methods, the most frequently used reagents are the strong bases, − such as stoichiometric KH, n -BuLi, t -BuOK, or t -BuONa, and the reported methods mostly used palladium complexes as catalysts. − Although these methods are effective, there are still some disadvantages (Scheme ). (i) Usually, the reported methods require a strong base, , and some cases need a large excessive base (5 equiv), which is not friendly to sensitive functional groups (such as an ester or a carboxyl group).…”