Thomas R. Ward

Selective drug activation in living systems remains a central challenge in targeted cancer therapy. Bioorthogonal catalysis addresses this by using abiotic transition-metal catalysts to unmask prodrugs at the tumor site, but the repertoire of suitable reactions remains limited. Here, we exploit the Buchner ring-expansion as a new uncaging strategy, in which a metal-carbene catalyzes a regioselective intramolecular ring-expansion followed by spontaneous 1,6-elimination to release a caged payload─a cascade with no biological equivalent. The iridium porphyrin complex [Ir(mPIX)Me] catalyzes this reaction at nanomolar concentrations, and the platform can accommodate structurally diverse payloads. As a proof of concept, functionalization with a bivalent human carbonic anhydrase IX (hCA IX) inhibitor enabled tumor-selective release of epirubicin (Epi) in a cancer cell model. This work thus delineates a new reaction class to the growing toolkit of transition-metal-mediated bioorthogonal chemistry.

Photoenzymes have emerged as powerful biocatalysts for light-driven transformations, yet their engineering has been constrained by low-to-medium-throughput screening methods, and their catalytic efficiency for non-native reactions/substrates typically remains moderate. Here, we report the first ultrahigh-throughput screening (uHTS) platform for the directed evolution of a photoenzyme, achieved by engineering transcription factor-based biosensors coupled with fluorescence-activated cell sorting. Using the fatty acid photodecarboxylase CvFAP as a model, we performed three iterative rounds of uHTS combining multi-site saturation and random mutagenesis, and successfully obtained evolved variants (M9T and M9P) that exhibited >200-fold improvements in productivity towards two non-native aromatic substrates, with turnover numbers reaching up to 3,700. Molecular dynamics simulations revealed that the accumulated mutations synergistically optimized substrate access, pocket hydrophobicity, and catalytic distances. When integrated into enzyme cascades from ʟ-phenylalanine and ʟ-tyrosine, these CvFAP variants enabled photobiocatalytic production of toluene (67.9 mM) and p cresol (198 mM). Furthermore, by coupling with high cell density fermentation, we achieved direct conversion of glucose derived crude amino acids to 4.3 g l-1 toluene and 21.4 g l-1 p-cresol without intermediate purification. Collectively, this work establishes uHTS as a powerful platform for reprogramming photoenzymes towards non native substrates, and provides a sustainable route from renewable biomass to industrially important aromatic chemicals.

Olefin metathesis is a powerful transformation for carbon–carbon bond formation, yet its application in biological settings is challenged by the instability of most ruthenium catalysts in aqueous media. Herein, we report a Ru(CAAC‐5) olefin metathesis catalyst bearing a functional handle for bioconjugation. The resulting hybrid catalyst catalyzes ring‐closing metathesis (RCM) and displays extended lifetimes in aqueous media. Functionalized CAAC‐5 ligands bearing a protected aniline linker were prepared via enamine alkylation and coordinated to ruthenium to afford the corresponding Ru(CAAC‐5) complex. In a benchmark RCM reaction under biologically relevant conditions, the ruthenium complex exhibits enhanced catalytic robustness and reaches turnover numbers of up to 600. Deprotection of an amine‐functionalized handle enables the bioconjugation to proteins and peptides. Using this platform, the catalyst was anchored to biomolecules through supramolecular anchoring to human carbonic anhydrase II (hCA II), click conjugation to peptides (SS01), and covalent attachment to ubiquitin (Ubi). To the best of our knowledge, this aniline‐functionalized Ru(CAAC‐5) complex is the first CAAC‐bearing metathesis catalyst amenable to bioconjugation, while retaining the prolonged catalytic activity characteristic of CAAC‐based systems in aqueous media.

ZUSAMMENFASSUNG: Die Olefinmetathese ist ein leistungsstarker Umwandlungsprozess zur Bildung von Kohlenstoff‐Kohlenstoff‐Bindungen, doch ihre Anwendung in biologischen Systemen wird durch die Instabilität der meisten Rutheniumkatalysatoren in wässrigen Medien erschwert. Hier berichten wir über einen Ru(CAAC‐5)‐Olefinmetathese‐Katalysator, der eine funktionelle Gruppe für die Biokonjugation aufweist. Der resultierende Hybridkatalysator katalysiert die ringschließende Metathese (RCM) und weist in wässrigen Medien eine verlängerte Lebensdauer auf. Funktionalisierte CAAC‐5‐Liganden mit einem geschützten Anilin‐Linker wurden durch Enamin‐Alkylierung hergestellt und an Ruthenium koordiniert, um den entsprechenden Ru(CAAC‐5)‐Komplex zu erhalten. In einer Benchmark‐RCM‐Reaktion unter biologisch relevanten Bedingungen zeigt der Rutheniumkomplex eine verbesserte katalytische Robustheit und erreicht eine Umsatzzahl von bis zu 600. Die Entschützung einer Anilin‐Gruppe ermöglicht die Biokonjugation an Proteine und Peptide. Mithilfe dieser Plattform wurde der Katalysator durch supramolekulare Verankerung an Carboanhydrase II (hCA II), Click‐Konjugation an Peptide (SS01) und kovalente Bindung an Ubiquitin (Ubi) an Biomoleküle gebunden. Nach unserem Kenntnisstand ist dieser anilinfunktionalisierte Ru(CAAC‐5)‐Komplex der erste CAAC‐haltige Metathese‐Katalysator, der sich für die Biokonjugation eignet und dabei die für CAAC‐Systeme charakteristische katalytische Aktivität in wässrigen Medien beibehält.

Asymmetric alkene hydration provides direct access to enantiopure alcohols and has been extensively developed through ionic mechanisms. Radical pathways remain largely unexplored, despite their potential for late-stage functionalization and functional-group tolerance. In this context, the Drago-Mukaiyama hydration—which proceeds via a metal–hydride hydrogen atom transfer (MHAT) mechanism—has found numerous applications in total synthesis, yet achieving high stereocontrol remains challenging. Here we show that cytochrome P450BM3 can be repurposed as a radical hydratase that catalyzes the asymmetric radical hydration of both terminal and internal alkenes under aerobic conditions, affording a range of enantioenriched alcohols with up to 99% enantiomeric excess. Mechanistic investigations reveal a pathway initiated by metal–hydride hydrogen atom transfer (MHAT) and asymmetric intermolecular radical addition of dioxygen. A subsequent kinetic resolution of the hydroperoxide intermediate further upgrades the enantioselectivity of the alcohol product. This study highlights that radical alkene hydration can be catalyzed by haemoproteins, thus expanding the scope of challenging asymmetric reactions accessible to haemoproteins.

Artificial metalloenzymes (ArMs) offer a versatile and evolvable platform to extend the biocatalytic repertoire. Here we report the assembly of an ArM resulting from supramolecular anchoring an N-heterocyclic carbene Au(I) complex into a de novo designed tandem repeat protein (TRP). We identified a variant that, compared to the free cofactor, led to a higher catalytic activity for the Au-catalyzed hydroamination of 2-ethynylaniline. Structure-guided mutagenesis of this variant improved the activity, resulting in a double mutant displaying up to fourfold higher catalytic rates than the original TRP. Biophysical and crystallographic analysis revealed distinct cofactor binding poses, with single mutations reshaping the active site and correlating with improved catalytic performance. Importantly, the TRP scaffold imparted robustness, preserving catalytic activity under acidic conditions, in the presence of organic cosolvent, and at elevated temperatures, where the free cofactor was deactivated. This work highlights the potential of de novo designed proteins to harbor non-natural metal cofactors and points to design principles for stabilizing sensitive catalysts under chaotropic conditions.