The late-stage site-selective derivatisation of peptides has many potential applications in structure-activity relationship studies and postsynthetic modification or conjugation of bioactive compounds. The development of orthogonal methods for C-H functionalisation is crucial for such peptide derivatisation. Among them, biocatalytic methods are increasingly attracting attention. Tryptophan halogenases emerged as valuable catalysts to functionalise tryptophan (Trp), while direct enzyme-catalysed halogenation of synthetic peptides is yet unprecedented. Here, it is reported that the Trp 6-halogenase Thal accepts a wide range of amides and peptides containing a Trp moiety. Increasing the sequence length and reaction optimisation made bromination of pentapeptides feasible with good turnovers and a broad sequence scope, while regioselectivity turned out to be sequence dependent. Comparison of X-ray single crystal structures of Thal in complex with d-Trp and a dipeptide revealed a significantly altered binding mode for the peptide. The viability of this bioorthogonal approach was exemplified by halogenation of a cyclic RGD peptide.

Halogenating enzymes are able to introduce halogen substituents under ambient conditions using non‐hazardous reagents with intriguing selectivity, which is highly desired in green chemistry. Although C−H functionalization such as halogenation is a well‐known transformation in synthetic chemistry, the selective incorporation of halogens using conventional chemical approaches often remains challenging. Therefore, enzyme‐based strategies have been emerging as valuable alternatives in recent years. Inspired by manifold developments of enzymatic halogenation, this review focuses on advances of halogenating enzymes and their application with particular emphasis on FAD‐dependent halogenases (FDHs). Catalytic strategies, application scope and engineering of FDHs are outlined pointing to the increasing utility of halogenases as promising biocatalysts. Current limitations as well as potential future developments of their synthetic utility are being discussed.

Halogenases are valuable biocatalysts for selective C-H activation, but despite recent efforts to broaden their application scope by means of protein engineering, improvement of thermostability and catalytic efficiency is still desired. A directed evolution campaign aimed at generating a thermostable flavin-dependent tryptophan 6-halogenase with reasonable activity suitable for chemoenzymatic purposes. These characteristics were tackled by combining successive rounds of epPCR along with semi-rational mutagenesis leading to a triple mutant (Thal-GLV) with substantially increased thermostability (T-M=23.5 K) and higher activity at 25 degrees C than the wild type enzyme. Moreover, an active-site mutation has a striking impact on thermostability but also on enantioselectivity. Our data contribute to a detailed understanding of biohalogenation and provide a profound basis for future engineering strategies to facilitate chemoenzymatic application of these attractive biocatalysts.

Flavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH2, oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate trypto-phan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 Å resolution. The structure revealed that the C6 of tryptophan is positioned next to the ϵ-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of l-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases.

Halogenated compounds, like 7-chloro-l-tryptophan, are important intermediates or components of bioactive substances relevant for the pharmaceutical, chemical and agrochemical industries. About 20% of all pharmaceutical small molecule drugs and around 30% of all active compounds in agrochemistry are halogenated. Chemical halogenation procedures usually are characterized by the use of hazardous or even highly toxic chemicals. Recently, a biocatalytic process for l-tryptophan halogenation at the gram-scale using FAD-dependent halogenase and NADH-dependent flavin reductase enzymes has been described. Many proteinogenic amino acids are produced by fermentation using Corynebacterium glutamicum. The fermentative production of l-glutamate and l-lysine, for example, is operated at the million-ton scale. However, fermentative production of halogenated amino acids has not yet been described. In this study, fermentative production of the halogenated amino acid 7-chloro-l-tryptophan from sugars, ammonium and chloride salts was achieved. This required metabolic engineering of an l-tryptophan producing C. glutamicum strain for expression of the genes coding for FAD-dependent halogenase RebH and NADH-dependent flavin reductase RebF from Lechevalieria aerocolonigenes. Chlorination of l-tryptophan to 7-chloro-l-tryptophan by recombinant C. glutamicum was improved by optimizing the RBS of rebH. Metabolic engineering enabled production of 7-chloro-l-tryptophan and l-tryptophan from the alternative carbon sources arabinose, glucosamine and xylose.

Catalytic anti-Markovnikov oxidation of alkene feedstocks could simplify synthetic routes to many important molecules and solve a long-standing challenge in chemistry. Here we report the engineering of a cytochrome P450 enzyme by directed evolution to catalyze metal-oxo-mediated anti-Markovnikov oxidation of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidation over the kinetically favored alkene epoxidation by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions.

Bei der gerichteten Evolution von Halogenasen ist die Identifizierung verbesserter Mutanten aus einer umfangreichen Bibliothek vergleichbar mit einer Suche nach der Nadel im Heuhaufen. In der Zuschrift auf S. 14365 ff. nutzen N. Sewald et al. eine Pd-katalysierte Kreuzkupplung beim Hochdurchsatz-Screening von Halogenasen. Dank der fluorogenen Kreuzkupplung und der Bildung eines fluoreszierenden Aryltryptophans fanden die Autoren eine thermostabile Tryptophan-6-Halogenase.

AbstractDie Anwendung von Tryptophan‐Halogenasen für die enzymatische Halogenierung hat noch gravierende Limitierungen. Diese könnten mittels gerichteter Evolution überwunden werden, jedoch bedarf die Durchmusterung großer Mutantenbibliotheken eines robusten Hochdurchsatz‐fähigen Testsystems. Im Hinblick darauf wurde eine Suzuki‐Miyaura‐Kreuzkupplung als quantitativer Halogenase‐Assay auf Basis der Bildung eines fluoreszierenden Aryltryptophans entwickelt. Die Methode wurde für die Anwendung im E.‐coli‐Lysat ohne intermediäre Aufarbeitung optimiert und ermöglicht es, halogenierte Tryptophanderivate mit hoher Spezifität in der Mikrotiterplatte quantitativ nachzuweisen. Unter Verwendung dieses Verfahrens wurde eine thermostabile Tryptophan‐6‐Halogenase gefunden, indem mittels fehlerbehafteter PCR eine Mutantenbibliothek erzeugt wurde, die mithilfe der fluorogenen Kreuzkupplung auf erhöhte Thermostabilität durchmustert wurde. Diese Methode lieferte eine verbesserte Enzymvariante mit deutlich gesteigerter Stabilität sowie 2.5‐fach verbesserter Aktivität.

Biocatalytic halogenation with tryptophan halogenases is hampered by severe limitations such as low activity and stability. These drawbacks can be overcome by directed evolution, but for screening large mutant libraries, a facile high-throughput method is required. Therefore, we developed a quantitative halogenase assay based on a Suzuki-Miyaura cross-coupling towards the formation of a fluorescent aryltryptophan. The technique was optimized for application in crude E.coli lysate without intermediary purification steps, and was used for quantitatively monitoring the formation of halogenated tryptophans with high specificity by facile fluorescence screening in microtiter plates. This novel screening approach was exploited to engineer a thermostable tryptophan 6-halogenase. Libraries were constructed by error-prone PCR and selected for improved thermal resistance simply by fluorogenic cross-coupling. Our method led to an enzyme variant with substantially increased thermal stability and 2.5-fold improved activity.

The combination of the biocatalytic halogenation of l‐tryptophan with subsequent chemocatalytic Suzuki–Miyaura cross‐coupling reactions leads to the modular synthesis of an array of C5, C6, or C7 aryl‐substituted tryptophan derivatives. In a three‐step one‐pot reaction, the bromo substituent is initially incorporated regioselectively by immobilized tryptophan 5‐, 6‐, or 7‐halogenases, respectively, with concomitant cofactor regeneration. The halogenation proceeds in aqueous media at room temperature in the presence of NaBr and O2. After the separation of the biocatalyst by filtration, a Pd catalyst, base, and boronic acid are added to the aryl halide formed in situ to effect direct Suzuki–Miyaura cross‐coupling reactions followed by tert‐butoxycarbonyl (Boc) protection. After a single purification step, different Boc‐protected aryl tryptophan derivatives are obtained that can, for example, be used for peptide or peptidomimetic synthesis.