Robust performance of a live bacterial therapeutic chassis lacking the colibactin gene cluster.

E. coli Nissle (EcN) is a non-pathogenic probiotic bacterium of the Enterobacteriaceae family that has been used for over a century to promote general gut health. Despite the history of safe usage of EcN, concerns have been raised regarding the presence of the pks gene cluster, encoding the genotoxin colibactin, due to its association with colorectal cancer. Here, we sought to determine the effect of pks island removal on the in vitro and in vivo robustness and activity of EcN and EcN-derived strains. A deletion of the pks island (Δpks) was constructed in wild type and engineered strains of EcN using lambda red recombineering. Mass spectrometric measurement of N-myristoyl-D-asparagine, released during colibactin maturation, confirmed that the pks deletion abrogated colibactin production. Growth curves were comparable between Δpks strains and their isogenic parents, and wild type EcN displayed no competitive advantage to the Δpks strain in mixed culture. Deletion of pks also had no effect on the activity of strains engineered to degrade phenylalanine (SYNB1618 and SYNB1934) or oxalate (SYNB8802). Furthermore, 1:1 mixed dosing of wild type and Δpks EcN in preclinical mouse and nonhuman primate models demonstrated no competitive disadvantage for the Δpks strain with regards to transit time or colonization. Importantly, there was no significant difference on in vivo strain performance between the clinical-stage strain SYNB1934 and its isogenic Δpks variant with regards to recovery of the quantitative strain-specific biomarkers d5- trans-cinnamic acid, and d5-hippuric acid. Taken together, these data support that the pks island is dispensable for Synthetic Biotic fitness and activity in vivo and that its removal from engineered strains of EcN will not have a deleterious effect on strain efficacy.

Denitrative Hydroxylation of Unactivated Nitroarenes.

A one-step method for the conversion of nitroarenes into phenols under operationally simple, transition-metal-free conditions is described. This denitrative functionalization protocol provides a concise and economical alternative to conventional three-step synthetic sequences. Experimental and computational studies suggest that nitroarenes may be substituted by an electron-catalysed radical-nucleophilic substitution (SRN 1) chain mechanism.

An engineered bacterial therapeutic lowers urinary oxalate in preclinical models and in silico simulations of enteric hyperoxaluria.

Enteric hyperoxaluria (EH) is a metabolic disease caused by excessive absorption of dietary oxalate leading to the formation of chronic kidney stones and kidney failure. There are no approved pharmaceutical treatments for EH. SYNB8802 is an engineered bacterial therapeutic designed to consume oxalate in the gut and lower urinary oxalate as a potential treatment for EH. Oral administration of SYNB8802 leads to significantly decreased urinary oxalate excretion in healthy mice and non-human primates, demonstrating the strain's ability to consume oxalate in vivo. A mathematical modeling framework was constructed that combines in vitro and in vivo preclinical data to predict the effects of SYNB8802 administration on urinary oxalate excretion in humans. Simulations of SYNB8802 administration predict a clinically meaningful lowering of urinary oxalate excretion in healthy volunteers and EH patients. Together, these findings suggest that SYNB8802 is a promising treatment for EH.

Indole-ynones as Privileged Substrates for Radical Dearomatizing Spirocyclization Cascades.

Indole-ynones have been established as general substrates for radical dearomatizing spirocyclization cascade reactions. Five distinct and varied synthetic protocols have been developed─cyanomethylation, sulfonylation, trifluoromethylation, stannylation and borylation─using a variety of radical generation modes, ranging from photoredox catalysis to traditional AIBN methods. The simple and easily prepared indole-ynones can be used to rapidly generate diverse, densely functionalized spirocycles and have the potential to become routinely used to explore radical reactivity. Experimental and computational investigations support the proposed radical cascade mechanism and suggest that other new methods are now primed for development.

Dynamic Kinetic Sensitization of ß-Dicarbonyl Compounds-Access to Medium-Sized Rings by De†Mayo-Type Ring Expansion.

Herein, we present a photocatalyzed two-carbon ring expansion of ß-dicarbonyl compounds with unactivated olefins that provides facile access to medium-sized rings. Selective sensitization of the substoichiometric enol tautomer enables reactivity of substrates incompatible with the classical De Mayo reaction conditions. Key to success is the identification of the metal-based sensitizer fac-[Ir(CF3 -pmb)3 ], which can be excited using common near-visible LEDs, and possesses a high triplet excited state energy of 73.3 kcal mol-1 . This exactly falls in the range between the triplet energies of the enol and keto tautomer, thereby enabling a dynamic kinetic sensitization. Demonstrating the applicability of fac-[Ir(CF3 -pmb)3 ] as a photocatalyst in organic synthesis for the first time, we describe a two-step photocycloaddition-ring-opening cascade with ß-ketoesters, -diketones, and -ketoamides. The mechanism has been corroborated by time-resolved spectroscopy, as well as further experimental and computational studies.

Radical-anion coupling through reagent design: hydroxylation of aryl halides.

The design and development of an oxime-based hydroxylation reagent, which can chemoselectively convert aryl halides (X = F, Cl, Br, I) into phenols under operationally simple, transition-metal-free conditions is described. Key to the success of this approach was the identification of a reducing oxime anion which can interact and couple with open-shell aryl radicals. Experimental and computational studies support the proposed radical-nucleophilic substitution chain mechanism.

Metabolic modulation of tumours with engineered bacteria for immunotherapy.

The availability of L-arginine in tumours is a key determinant of an efficient anti-tumour T cell response1-4. Consequently, increases of typically low L-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies5. However, currently no means are available to locally increase intratumoural L-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours6, to L-arginine. Colonization of tumours with these bacteria increased intratumoural L-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by L-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies.

A Thiol-Mediated Three-Step Ring Expansion Cascade for the Conversion of Indoles into Functionalized Quinolines.

An operationally simple, high yielding three-step cascade process is described for the direct conversion of indole-tethered ynones into functionalized quinolines. A single "multitasking" thiol reagent is used to promote a three-step dearomatizing spirocyclization, nucleophilic substitution, and one-atom ring expansion reaction cascade under remarkably mild conditions. In addition, a novel route to thio-oxindoles is described, which was discovered by serendipity.

Evaluation of the Structure-Activity Relationship of Microtubule-Targeting 1,2,4-Triazolo[1,5-a]pyrimidines Identifies New Candidates for Neurodegenerative Tauopathies.

Studies in tau and Aß plaque transgenic mouse models demonstrated that brain-penetrant microtubule (MT)-stabilizing compounds, including the 1,2,4-triazolo[1,5-a]pyrimidines, hold promise as candidate treatments for Alzheimer's disease and related neurodegenerative tauopathies. Triazolopyrimidines have already been investigated as anticancer agents; however, the antimitotic activity of these compounds does not always correlate with stabilization of MTs in cells. Indeed, previous studies from our laboratories identified a critical role for the fragment linked at C6 in determining whether triazolopyrimidines promote MT stabilization or, conversely, disrupt MT integrity in cells. To further elucidate the structure-activity relationship (SAR) and to identify potentially improved MT-stabilizing candidates for neurodegenerative disease, a comprehensive set of 68 triazolopyrimidine congeners bearing structural modifications at C6 and/or C7 was designed, synthesized, and evaluated. These studies expand upon prior understanding of triazolopyrimidine SAR and enabled the identification of novel analogues that, relative to the existing lead, exhibit improved physicochemical properties, MT-stabilizing activity, and pharmacokinetics.

Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity.

Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine.

Characterization of novel conformation-selective α-synuclein antibodies as potential immunotherapeutic agents for Parkinson's disease.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are progressive neurodegenerative diseases for which there is no disease-modifying treatment. PD and DLB are characterized by aggregation of the synaptic protein α-synuclein, and there is compelling evidence to suggest that progression of these diseases is associated with the trans-cellular spread of pathogenic α-synuclein through the brains of afflicted individuals. Therapies targeting extracellular, pathogenic α-synuclein may therefore hold promise for slowing or halting disease progression. In this regard, it has been suggested that highly-selective antibodies can be administered as therapeutic agents targeting pathogenic proteins. In the current study, we screened a series of antibodies using multiple selection criterion to identify those that selectively bind pathogenic α-synuclein and show potent inhibition of pathology seeding in a neuronal model of α-synucleinopathy. A lead antibody was tested in a mouse model of PD, and it was able to reduce the spread of α-synuclein pathology in the brain and attenuate dopamine reductions in the striatum. This study highlights the therapeutic potential of α-synuclein immunotherapy for the treatment of PD and DLB, and provides a framework for screening of α-synuclein antibodies to identify those with preferred properties.

Visible-Light-Mediated Charge Transfer Enables C-C Bond Formation with Traceless Acceptor Groups.

The development and application of traceless acceptor groups in photochemical C-C bond formation is described. This strategy was enabled by the photoexcitation of electron donor-acceptor (EDA) complexes with visible light. The traceless acceptors, which were readily prepared from amino acid and peptide feedstocks, could be used to alkylate a wide range of heteroarene and enamine donors under metal- and peroxide-free conditions. The crucial role of the EDA complexes in the mechanism of these reactions was explored through combined experimental and computational studies.

Visible-light-induced intramolecular charge transfer in the radical spirocyclisation of indole-tethered ynones.

Indole-tethered ynones form an intramolecular electron donor-acceptor complex that can undergo visible-light-induced charge transfer to promote thiyl radical generation from thiols. This initiates a novel radical chain sequence, based on dearomatising spirocyclisation with concomitant C-S bond formation. Sulfur-containing spirocycles are formed in high yields using this simple and mild synthetic protocol, in which neither transition metal catalysts nor photocatalysts are required. The proposed mechanism is supported by various mechanistic studies, and the unusual radical initiation mode represents only the second report of the use of an intramolecular electron donor-acceptor complex in synthesis.

A brain-penetrant triazolopyrimidine enhances microtubule-stability, reduces axonal dysfunction and decreases tau pathology in a mouse tauopathy model.

BACKGROUND: Alzheimer's disease (AD) and related tauopathies are neurodegenerative diseases that are characterized by the presence of insoluble inclusions of the protein tau within brain neurons and often glia. Tau is normally found associated with axonal microtubules (MTs) in the brain, and in tauopathies this MT binding is diminished due to tau hyperphosphorylation. As MTs play a critical role in the movement of cellular constituents within neurons via axonal transport, it is likely that the dissociation of tau from MTs alters MT structure and axonal transport, and there is evidence of this in tauopathy mouse models as well as in AD brain. We previously demonstrated that different natural products which stabilize MTs by interacting with ß-tubulin at the taxane binding site provide significant benefit in transgenic mouse models of tauopathy. More recently, we have reported on a series of MT-stabilizing triazolopyrimidines (TPDs), which interact with ß-tubulin at the vinblastine binding site, that exhibit favorable properties including brain penetration and oral bioavailability. Here, we have examined a prototype TPD example, CNDR-51657, in a secondary prevention study utilizing aged tau transgenic mice. METHODS: 9-Month old female PS19 mice with a low amount of existing tau pathology received twice-weekly administration of vehicle, or 3 or 10 mg/kg of CNDR-51657, for 3 months. Mice were examined in the Barnes maze at the end of the dosing period, and brain tissue and optic nerves were examined immunohistochemically or biochemically for changes in MT density, axonal dystrophy, and tau pathology. Mice were also assessed for changes in organ weights and blood cell numbers. RESULTS: CNDR-51657 caused a significant amelioration of the MT deficit and axonal dystrophy observed in vehicle-treated aged PS19 mice. Moreover, PS19 mice receiving CNDR-51657 had significantly lower tau pathology, with a trend toward improved Barnes maze performance. Importantly, no adverse effects were observed in the compound-treated mice, including no change in white blood cell counts as is often observed in cancer patients receiving high doses of MT-stabilizing drugs. CONCLUSIONS: A brain-penetrant MT-stabilizing TPD can safely correct MT and axonal deficits in an established mouse model of tauopathy, resulting in reduced tau pathology.

Deaminative Borylation of Aliphatic Amines Enabled by Visible Light Excitation of an Electron Donor-Acceptor Complex.

A deaminative strategy for the borylation of aliphatic primary amines is described. Alkyl radicals derived from the single-electron reduction of redox-active pyridinium salts, which can be isolated or generated in situ, were borylated in a visible light-mediated reaction with bis(catecholato)diboron. No catalyst or further additives were required. The key electron donor-acceptor complex was characterized in detail by both experimental and computational investigations. The synthetic potential of this mild protocol was demonstrated through the late-stage functionalization of natural products and drug molecules.

Energy transfer catalysis mediated by visible light: principles, applications, directions.

Harnessing visible light to access excited (triplet) states of organic compounds can enable impressive reactivity modes. This tutorial review covers the photophysical fundamentals and most significant advances in the field of visible-light-mediated energy transfer catalysis within the last decade. Methods to determine excited triplet state energies and to characterize the underlying Dexter energy transfer are discussed. Synthetic applications of this field, divided into four main categories (cyclization reactions, double bond isomerizations, bond dissociations and sensitization of metal complexes), are also examined.

Dearomative Cascade Photocatalysis: Divergent Synthesis through Catalyst Selective Energy Transfer.

The discovery and application of dearomative cascade photocatalysis as a strategy in complex molecule synthesis is described. Visible-light-absorbing photosensitizers were used to (sequentially) activate a 1-naphthol derived arene precursor to divergently form two different polycyclic molecular scaffolds through catalyst selective energy transfer.

Ag(I)-Catalyzed Synthesis of Azabicyclic Alkaloid Frameworks from Ketimine-Tethered Ynones: Total Synthesis of Indolizidine 209D.

An efficient Ag(I)-catalyzed π-acid activation method for the cyclization of cyclic ketimine-tethered ynones is reported. Various nitrogen-containing scaffolds commonly found in bioactive alkaloids can be prepared in high yields, and the utility of the method is demonstrated by a formal synthesis of (±)-lasubine II and in a short total synthesis of (±)-indolizidine 209D.

Increasing Catalyst Efficiency in C-H Activation Catalysis.

C-H activation reactions with high catalyst turnover numbers are still very rare in the literature and 10 mol % is a common catalyst loading in this field. We offer a representative overview of efficient C-H activation catalysis to highlight this neglected aspect of catalysis development and inspire future effort towards more efficient C-H activation. Examples ranging from palladium catalysis, Cp*RhIII - and Cp*CoIII -catalysis, the C-H borylation and silylation to methane C-H activation are presented. In these reactions, up to tens of thousands of catalyst turnovers have been observed.