Enzymatic Late‐Stage Modifications: Better Late Than Never

From Wiley via Jisc Publications RouterHistory: received 2020-11-08, rev-recd 2021-01-15, pub-electronic 2021-03-08, pub-print 2021-07-26Article version: VoRPublication status: PublishedFunder: EPSRC; Grant(s): EP/S005226/1Funder: BBSRC; Grant(s): EP/S005226/1Funder: AstraZeneca plc; Id: http://dx.doi.org/10.13039/100004325; Grant(s): EP/S005226/1Funder: European Research Council; Id: http://dx.doi.org/10.13039/100010663; Grant(s): 742987-BIO-H-BORROW-ERC-2016-ADG, 788231-ProgrES-ERC-2017-ADGAbstract: Enzyme catalysis is gaining increasing importance in synthetic chemistry. Nowadays, the growing number of biocatalysts accessible by means of bioinformatics and enzyme engineering opens up an immense variety of selective reactions. Biocatalysis especially provides excellent opportunities for late‐stage modification often superior to conventional de novo synthesis. Enzymes have proven to be useful for direct introduction of functional groups into complex scaffolds, as well as for rapid diversification of compound libraries. Particularly important and highly topical are enzyme‐catalysed oxyfunctionalisations, halogenations, methylations, reductions, and amide bond formations due to the high prevalence of these motifs in pharmaceuticals. This Review gives an overview of the strengths and limitations of enzymatic late‐stage modifications using native and engineered enzymes in synthesis while focusing on important examples in drug development

Enzymkatalysierte späte Modifizierungen: Besser spät als nie

From Wiley via Jisc Publications RouterHistory: received 2020-11-08, rev-recd 2021-01-15, pub-electronic 2021-03-08, pub-print 2021-07-26Article version: VoRPublication status: PublishedFunder: EPSRC; Grant(s): EP/S005226/1Funder: BBSRC; Grant(s): EP/S005226/1Funder: AstraZeneca plc; Id: http://dx.doi.org/10.13039/100004325; Grant(s): EP/S005226/1Funder: European Research Council; Id: http://dx.doi.org/10.13039/100010663; Grant(s): 742987-BIO-H-BORROW-ERC-2016-ADG, 788231-ProgrES-ERC-2017-AD

Biocatalytic Aryl Functionalization Using Flavin-Dependent Halogenases. Enzyme Engineering and Chemoenzymatic Cascades

Schnepel C. Biocatalytic Aryl Functionalization Using Flavin-Dependent Halogenases. Enzyme Engineering and Chemoenzymatic Cascades. Bielefeld: Universität Bielefeld; 2019

Late-Stage Halogenation of Peptides Using Engineered FAD-Dependent Halogenases

Montua N, Schnepel C, Sewald N. Late-Stage Halogenation of Peptides Using Engineered FAD-Dependent Halogenases. In: Journal of Peptide Science. Vol 28. Hoboken: Wiley; 2022

One-Pot Synthesis of D-Halotryptophans by Dynamic Stereoinversion Using a Specific L-Amino Acid Oxidase

Schnepel C, Kemker I, Sewald N. One-Pot Synthesis of D-Halotryptophans by Dynamic Stereoinversion Using a Specific L-Amino Acid Oxidase. ACS Catalysis. 2019;9(2):1149-1158.Tryptophan (Trp) derivatives constitute important building blocks found in many natural products, peptides, and drugs. Accordingly, there is a high demand for suitable biocatalytic pathways providing selective derivatization of Trp like C-H functionalization or C-alpha-oxidation. The specific l-amino acid oxidase RebO from the actinomycete L. aerocolonigenes was harnessed for chemoenzymatic oxidation of substituted Trp derivatives. An array of potential substrates was tested, and several Trp derivatives are being accepted with reasonable turnover. The highest selectivity was observed for 7-halotryptophan being converted about 35-fold faster than nonsubstituted Trp. This selectivity is also useful for establishing a colorimetric halogenase high-throughput assay. RebO was also employed in dynamic stereoinversion in the presence of an ammonia-borane complex to provide access to non-native D-configured Trp analogues. Optimized reaction conditions yielded similar to 70% D-amino acid with >98% ee for halotryptophans on an analytical scale. Dynamic stereoinversion preceded by enzymatic halogenation in a sequential one-pot reaction cascade provided D-configured 5- or 7-bromotryptophan with improved conversion of approximately 90%, >92% ee, and good isolated yields

Novel Arylindigoids by Late-Stage Derivatization of Biocatalytically Synthesized Dibromoindigo.

Schnepel C, Dodero VI, Sewald N. Novel Arylindigoids by Late-Stage Derivatization of Biocatalytically Synthesized Dibromoindigo. Chemistry - A European Journal . 2021;27(17):5404-5411.Indigoids represent natural product-based compounds applicable as organic semiconductors and photoresponsive materials. Yet modified indigo derivatives are difficult to access by chemical synthesis. Thus a biocatalytic approach applying several consecutive selective C-H functionalizations was developed that selectively provides access to various indigoids: Enzymatic halogenation of l-tryptophan followed by indole generation with tryptophanase yields 5-, 6- and 7-bromoindoles. Subsequent hydroxylation using a flavin monooxygenase furnishes dibromoindigo that is derivatized by acylation. This four-step one-pot cascade gives Boc-protected dibromoindigo in good isolated yields. The halogen substituent allows for late-stage diversification by cross-coupling directly performed in the crude mixture, thus enabling synthesis of 5,5'- and 6,6'-diarylindigo derivatives. This chemoenzymatic approach provides a modular platform towards novel indigoids with attractive spectral properties. © 2021 Wiley-VCH GmbH

A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases

Schnepel C, Minges H, Frese M, Sewald N. A High-Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases. Angewandte Chemie International Edition. 2016;55(45):14159-14163.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

Ein Hochdurchsatz-Fluoreszenz-Assay zur Bestimmung der Aktivität von Tryptophan-Halogenasen

Schnepel C, Minges H, Frese M, Sewald N. Ein Hochdurchsatz-Fluoreszenz-Assay zur Bestimmung der Aktivität von Tryptophan-Halogenasen. Angewandte Chemie. 2016;128(45):14365-14369

PEPTIDE STAPLING BY SUZUKI- MIYAURA CROSS-COUPLING

Gruß H, Frese M, Schnepel C, Sewald N. PEPTIDE STAPLING BY SUZUKI- MIYAURA CROSS-COUPLING. In: JOURNAL OF PEPTIDE SCIENCE. Vol 22. Wiley; 2016: S124