A Leader-Guided Substrate Tolerant RiPP Brominase Allows Suzuki–Miyaura Cross-Coupling Reactions for Peptides and Proteins

Abstract

Bioorthogonal derivatization of peptides and proteins enables investigations into their biological function and allows for exploitation of their therapeutic potential, among other varied deliverables. Herein, we describe a marine halogenating enzyme-assisted bioconjugation strategy in which an N-terminal leader peptide guides bromination of a C-terminal Trp residue in genetically encoded peptides and proteins, setting up further Trp arylation by Suzuki–Miyaura reactions. The bromination and subsequent cross-coupling reactions are residue-specific and regiospecific for the indole-6 position, occur under mild aqueous conditions, and do not require any modification of other Trp residues in the substrate peptide and/or protein. Workflows described herein demonstrate the applicability of halogenating enzymes in bioorthogonal conjugation chemistry.

Enzymatic Peptide and Protein Bromination: The BromoTrp Tag

“…It is worth noting, that similar results for palladium-catalyzed cross-coupling were reported very recently for bromotryptophan functionalized SrpE-leader peptides by the Agarwal group, who also relied on enzymatic digest of crude mixtures containing their protein followed by LC-MS for analysis. [20] Crucially, while their halogenation assays were limited to microscale reactions and required purified halogenase and cofactor regeneration enzymes, our approach to protein bromination decisively simplifies the procedure. It does not rely on a long 78-residue leader peptide that also requires maturation and readily enables preparative-scale synthesis of brominated proteins in a single-step that can subsequently undergo cross-coupling.…”

“…were able to demonstrate on an analytical‐scale the leader‐peptide dependent bromination of ribosomally encoded sequences by the RiPP brominase SrpI. This approach was also successful with maltose‐binding protein that was C‐ terminally modified with the SrpE leader peptide [20] . They were also able to demonstrate cross‐coupling of various aryl boronic acids to these peptides using the same Pd/ADHP catalyst described by the Davis group.…”

“…This approach was also successful with maltose-binding protein that was C-terminally modified with the SrpE leader peptide. [20] They were also able to demonstrate cross-coupling of various aryl boronic acids to these peptides using the same Pd/ADHP catalyst described by the Davis group. However, the dependence on a long 78-residue leader peptide that also requires maturation compromises the elegance and the synthetic utility of this approach.…”

Enzymatische Peptid‐ und Proteinbromierung: Der BromoTrp Tag

“…Es sollte hier angemerkt werden, dass ähnliche Ergebnisse vor Kurzem für Bromtryptophan-funktionalisierte SrpE-Leader-Peptide von der Agarwal-Gruppe veröffentlicht wurden, deren Analytik ebenfalls auf der LC-MS-Analyse eines enzymatischen Verdaus ihrer proteinhaltigen rohen Reaktionsmischungen beruhte. [20] Ein entscheidender Unterschied zwischen diesen Ergebnissen und unseren ist, dass SrpI-katalysierte Bromierung auf Assays im Microlitermaßstab beschränkt waren, unter Verwendung von aufgereinigter Halogenase und Cofaktorregenerierungs-Enzymen. Unser Vorgehen hingegen stellt eine entscheidende Vereinfachung der Proteinbromierung dar.…”

“…Diese Strategie erwies sich ebenfalls bei einem Maltosebindenden Protein, das C-terminal mit dem SrpE Leader-Peptid modifiziert wurde, als erfolgreich. [20] Darüber hinaus konnten sie verschiedene Arylboronsäuren unter Einsatz desselben Pd/ADHP-Katalysators, den die Davis-Gruppe nutzte, kreuzkuppeln. Jedoch schränkt hier die Abhängigkeit von einem 78 Aminosäuren langen Leader-Peptid, welches auch erst zusätzliche Maturierungsschritte durchlaufen muss, die Eleganz und den synthesechemischen Nutzen dieses Ansatzes ein.…”