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Palladium-catalyzed cross-coupling reactions have emerged as one of the most versatile tools in organic chemistry. Extensive efforts were made to adapt these reactions to aqueous media, not only for the purpose of environmental conservation but also to expand the scope, increase the efficiency and implement bio-compatible protocols. Among different palladium cross-coupling reactions, the Heck reaction turned out to be the most challenging in an aqueous environment. This led to various original developments in catalyst design. This review summarizes the different approaches pursued to perform Heck reactions in neat water as well as aqueous mixtures. Both, homogeneous and immobilized catalysts, including nanoparticles are presented herein. Graphical Abstract

The palladium-catalyzed Suzuki–Miyaura cross-coupling reaction of organic halides with boronic acids is one of the most versatile methods for the synthesis of biaryls. Green chemistry is a rapidly developing new field that provides us a proactive avenue for the sustainable development of future science and technologies. When designed properly, clean chemical technology can be developed in water as a reaction medium. The technologies generated from such green chemistry endeavours may often be cheaper and more profitable. This review covers the literature on palladium-catalysed the Suzuki–Miyaura cross-coupling reaction in water. Graphical Abstract

For life to emerge, the confinement of catalytic reactions within protocellular environments has been proposed to be a decisive aspect to regulate chemical activity in space1. Today, cells and organisms adapt to signals2–6 by processing them through reaction networks that ultimately provide downstream functional responses and structural morphogenesis7,8. Re-enacting such signal processing in de novo-designed protocells is a profound challenge, but of high importance for understanding the design of adaptive systems with life-like traits. We report on engineered all-DNA protocells9 harbouring an artificial metalloenzyme10 whose olefin metathesis activity leads to downstream morphogenetic protocellular responses with varying levels of complexity. The artificial metalloenzyme catalyses the uncaging of a pro-fluorescent signal molecule that generates a self-reporting fluorescent metabolite designed to weaken DNA duplex interactions. This leads to pronounced growth, intraparticular functional adaptation in the presence of a fluorescent DNA mechanosensor11 or interparticle protocell fusion. Such processes mimic chemically transduced processes found in cell adaptation and cell-to-cell adhesion. Our concept showcases new opportunities to study life-like behaviour via abiotic bioorthogonal chemical and mechanical transformations in synthetic protocells. Furthermore, it reveals a strategy for inducing complex behaviour in adaptive and communicating soft-matter microsystems, and it illustrates how dynamic properties can be upregulated and sustained in micro-compartmentalized media.Genetically improved artificial metalloenzymes in DNA protocells convert signalling molecules into DNA-interacting metabolites that induce downstream growth, functional adaptation and fusion processes inside protocells and between protocells.

An artificial metalloenzyme is compartmentalized and evolved in vivo for olefin metathesis—an archetypal organometallic reaction without equivalent in nature; the evolved metathase reveals broad substrate scope and compares favourably with commercial catalysts. Directed evolution of an artificial metalloenzyme Artificial metalloenzymes—made by incorporating an abiotic cofactor within a protein scaffold—have the potential to engineer non-natural in vivo reactions. To be of practical use, such catalysts must maintain their activity in a cellular environment, which means overcoming the tendency of introduced metal cofactors to be inhibited by cellular components. This paper demonstrates that directed evolution can overcome this difficulty. Markus Jeschek et al . report on the in vivo evolution of a ruthenium–protein complex that can catalyse olefin metathesis—an archetypal organometallic reaction with no equivalent in nature—in the periplasm of Escherichia coli . The evolved metathase compares favorably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the protocol. The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions 1 . Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions 2 , 3 . For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist 3 : repurposing natural metalloenzymes for abiotic transformations 2 , 4 ; in silico metalloenzyme (re-)design 5 , 6 , 7 ; and incorporation of abiotic cofactors into proteins 8 , 9 , 10 , 11 . The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein 12 , 13 , 14 , 15 . This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction 16 , 17 , 18 , 19 , 20 , 21 , 22 without equivalent in nature. Building on previous work 6 on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor 15 . This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction in vivo , with potential applications in, for example, non-natural metabolism.

Engineered enzyme cascades offer powerful tools to convert renewable resources into value-added products. Man-made catalysts give access to new-to-nature reactivities that may complement the enzyme’s repertoire. Their mutual incompatibility, however, challenges their integration into concurrent chemo-enzymatic cascades. Herein we show that compartmentalization of complex enzyme cascades within E. coli whole cells enables the simultaneous use of a metathesis catalyst, thus allowing the sustainable one-pot production of cycloalkenes from oleic acid. Cycloheptene is produced from oleic acid via a concurrent enzymatic oxidative decarboxylation and ring-closing metathesis. Cyclohexene and cyclopentene are produced from oleic acid via either a six- or eight-step enzyme cascade involving hydration, oxidation, hydrolysis and decarboxylation, followed by ring-closing metathesis. Integration of an upstream hydrolase enables the usage of olive oil as the substrate for the production of cycloalkenes. This work highlights the potential of integrating organometallic catalysis with whole-cell enzyme cascades of high complexity to enable sustainable chemistry. Cycloalkenes are bulk petrochemicals that are currently obtained from fossil fuels. Here, the authors developed multi enzyme pathways in combination with a Ru-catalyzed metathesis reaction for the one-pot production of cyclopentene, cyclohexene, and cycloheptene from olive oil-derived intermediates.

Olefin metathesis is one of the most powerful C–C double-bond-forming reactions. Metathesis reactions have had a tremendous impact in organic synthesis, enabling a variety of applications in polymer chemistry, drug discovery and chemical biology. Although challenging, the possibility to perform aqueous metatheses has become an attractive alternative, not only because water is a more sustainable medium, but also to exploit biocompatible conditions. This review focuses on the progress made in aqueous olefin metatheses and their applications in chemical biology.

Enzymes provide an exquisitely tailored chiral environment to foster high catalytic activities and selectivities, but their native structures are optimized for very specific biochemical transformations. Designing a protein to accommodate a non-native transition metal complex can broaden the scope of enzymatic transformations while raising the activity and selectivity of small-molecule catalysis. Here, we report the creation of a bifunctional artificial metalloenzyme in which a glutamic acid or aspartic acid residue engineered into streptavidin acts in concert with a docked biotinylated rhodium(III) complex to enable catalytic asymmetric carbon-hydrogen (C-H) activation. The coupling of benzamides and alkenes to access dihydroisoquinolones proceeds with up to nearly a 100-fold rate acceleration compared with the activity of the isolated rhodium complex and enantiomeric ratios as high as 93:7.

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin–streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells. Artificial enzymes can be used to elicit reactions in cells. Here, the authors developed such an artificial catalyst combined with a genetic switch, and showed that it was readily taken up by human cells and able to kick off a reaction cascade resulting in the biosynthesis of the desired product.

The bacterial envelope plays a critical role in maintaining essential cellular functions by selectively regulating import and export. The selectivity of this envelope can restrict the utilisation of externally provided compounds, thereby restricting the functional space of cellular engineering. This study systematically investigates the potential of large pore outer membrane proteins (OMPs) to enhance outer membrane permeability for diverse challenging compounds. We focus on the general porin OmpF, which facilitates the diffusion of water and small molecules, and specific OMP transporters FhuA and FepA, which mediate the translocation of small hydrophilic compounds. Through comprehensive characterisation, we evaluate the effects of recombinant expression of OMPs and engineered variants for small and hydrophilic compounds, aromatic molecules and bulky molecules and apply our findings to address two critical contemporary transport challenges: the uptake of large metal‐containing cofactors for artificial metalloenzymes and non‐permeant fluorescent Halo‐ligands for in vivo protein labelling. Notably, we demonstrate significant improvements in ArM‐catalysis and labelling. This study provides a practical guide for designing experiments that include outer‐membrane‐transport‐limiting steps. This study highlights the potential of engineered OMPs to overcome the limitations imposed by the cell envelope, enabling the incorporation of complex molecules and expanding the frontiers of cellular engineering. Access to synthetic compounds in the bacterial cytoplasm is hindered by the selective cell envelope, particularly in Gram‐negative bacteria like Escherichia coli. This study systematically compares three outer membrane proteins (OMPs) and their engineered variants, highlighting the potential of OMP engineering for the permeabilisation of the outer membrane.

We report herein the synthesis of N -substituted indoles resulting from the ring-closing metathesis of indole precursors bearing N -terminal alkenes. The aqueous metathesis of the indole precursors gave good yields of N -substituted indoles (up to 72%) with commercial metathesis catalysts and with artificial metalloenzymes based on the biotin-streptavidin technology. Strikingly, the yield of the N -acetylindole increases in presence of a second metathesis substrate. Graphic Abstract