Enantioselective synthesis of chiral α,α-dialkyl indoles and related azoles by cobalt-catalyzed hydroalkylation and regioselectivity switch.

General, catalytic and enantioselective construction of chiral α,α-dialkyl indoles represents an important yet challenging objective to be developed. Herein we describe a cobalt catalyzed enantioselective anti-Markovnikov alkene hydroalkylation via the remote stereocontrol for the synthesis of α,α-dialkyl indoles and other N-heterocycles. This asymmetric C(sp3)-C(sp3) coupling features high flexibility in introducing a diverse set of alkyl groups at the α-position of chiral N-heterocycles. The utility of this methodology has been demonstrated by late-stage functionalization of drug molecules, asymmetric synthesis of bioactive molecules, natural products and functional materials, and identification of a class of molecules exhibiting anti-apoptosis activities in UVB-irradiated HaCaT cells. Ligands play a vital role in controlling the reaction regioselectivity. Changing the ligand from bi-dentate L6 to tridentate L12 enables CoH-catalyzed Markovnikov hydroalkylation. Mechanistic studies disclose that the anti-Markovnikov hydroalkylation involves a migratory insertion process while the Markovnikov hydroalkylation involves a MHAT process.

The global nitrogen regulator GlnR is a direct transcriptional repressor of the key gluconeogenic gene pckA in actinomycetes.

In most actinomycetes, GlnR governs both nitrogen and non-nitrogen metabolisms (e.g., carbon, phosphate, and secondary metabolisms). Although GlnR has been recognized as a global regulator, its regulatory role in central carbon metabolism [e.g., glycolysis, gluconeogenesis, and the tricarboxylic acid (TCA) cycle] is largely unknown. In this study, we characterized GlnR as a direct transcriptional repressor of the pckA gene that encodes phosphoenolpyruvate carboxykinase, catalyzing the conversion of the TCA cycle intermediate oxaloacetate to phosphoenolpyruvate, a key step in gluconeogenesis. Through the transcriptomic and quantitative real-time PCR analyses, we first showed that the pckA transcription was upregulated in the glnR null mutant of Amycolatopsis mediterranei. Next, we proved that the pckA gene was essential for A. mediterranei gluconeogenesis when the TCA cycle intermediate was used as a sole carbon source. Furthermore, with the employment of the electrophoretic mobility shift assay and DNase I footprinting assay, we revealed that GlnR was able to specifically bind to the pckA promoter region from both A. mediterranei and two other representative actinomycetes (Streptomyces coelicolor and Mycobacterium smegmatis). Therefore, our data suggest that GlnR may repress pckA transcription in actinomycetes, which highlights the global regulatory role of GlnR in both nitrogen and central carbon metabolisms in response to environmental nutrient stresses. IMPORTANCE: The GlnR regulator of actinomycetes controls nitrogen metabolism genes and many other genes involved in carbon, phosphate, and secondary metabolisms. Currently, the known GlnR-regulated genes in carbon metabolism are involved in the transport of carbon sources, the assimilation of short-chain fatty acid, and the 2-methylcitrate cycle, although little is known about the relationship between GlnR and the TCA cycle and gluconeogenesis. Here, based on the biochemical and genetic results, we identified GlnR as a direct transcriptional repressor of pckA, the gene that encodes phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, thus highlighting that GlnR plays a central and complex role for dynamic orchestration of cellular carbon, nitrogen, and phosphate fluxes and bioactive secondary metabolites in actinomycetes to adapt to changing surroundings.

Synthesis of Thiophene-Substituted Ketones via Manganese-Catalyzed Dehydrogenative Coupling Reaction.

This study reports an efficient and green one-step method for synthesizing thiophene-substituted ketones from 2-thiophenemethanol and ketones via dehydrogenative coupling using manganese complexes as catalysts. The manganese complex demonstrated a broad applicability under mild conditions and extended the range of usable substrates. Utilizing this strategy, we carried out an efficient and diverse reaction of ketones with 2-thiophenemethanol, and successfully synthesized a series of thiophene-substituted saturated ketones and α, ß-unsaturated ketones in good isolated yields.

Chemodivergence in Pd-catalyzed desymmetrization of allenes: enantioselective [4+3] cycloaddition, desymmetric allenylic substitution and enynylation.

A class of prochiral allenylic di-electrophiles have been introduced for the first time as three-atom synthons in cycloadditions, and a new type of [4+3] cycloaddition involving transition metal-catalyzed enantioselective sequential allenylic substitution has been successfully developed, enabling challenging seven-membered exocyclic axially chiral allenes to be accessed in good yields with good enantioselectivity. Through the addition of a catalytic amount of ortho-aminoanilines or ortho-aminophenols, the racemization of the [4+3] cycloaddition products is effectively suppressed. Mechanistic studies reveal that elusive Pd-catalyzed enantioselective intramolecular allenylic substitution rather than intermolecular allenylic substitution is the enantio-determining step in this cycloaddition. By tuning the ligands, a Pd-catalyzed enantioselective desymmetric allenylic substitution leading to linear axially chiral tri-substituted allenes or a Pd-catalyzed tandem desymmetric allenylic substitution/ß-vinylic hydrogen elimination (formal enynylation) leading to multi-functionalized 1,3-enynes is achieved chemodivergently.

Synthesis of Tetrahydro-ß-carbolines by a Manganese-Catalysed Oxidative Pictet-Spengler Reaction.

Herein, an efficient and green procedure for the synthesis of tetrahydro-ß-carbolines via dehydrogenative coupling of alcohols with tryptamines is reported. The reaction was carried out under mild conditions in the presence of a catalytic amount of the iPr PNP-Mn catalyst and a weak base (Na2 CO3 ). This method tolerated a variety of benzylic and aliphatic alcohol substrates with different functional groups and afforded diverse products in good to excellent isolated yields using tryptamines. Using this strategy, we successfully synthesised pharmaceutical molecules harman, harmaline, and harmine in a concise manner.

Direct Catalytic Asymmetric and Regiodivergent N1- and C3-Allenylic Alkylation of Indoles.

Herein we report a Pd-catalyzed asymmetric allenylic alkylation strategy for the direct functionalization of 1H-indoles by employing P-chiral BIBOP-type ligands. The regioselectivity (N1/C3) of this process can be switched efficiently. Using Cs2 CO3 at elevated temperatures in MeCN, N1-alkylated indoles bearing axial chirality with a stereocenter non-adjacent (ß) to the nitrogen are produced in good yields with high enantioselectivity and complete N1-regioselectivity regardless of the electronic properties and substitution patterns of diverse indoles. Using K2 CO3 at room temperature in CH2 Cl2 , chiral C3-alkylated indoles can also be obtained. Notably, we introduce a new class of tri-substituted allenylic electrophiles that proceeded through different pathways from di-substituted allenylic electrophiles.

Transcriptional Self-Regulation of the Master Nitrogen Regulator GlnR in Mycobacteria.

As a master nitrogen regulator in most actinomycetes, GlnR both governs central nitrogen metabolism and regulates many carbon, phosphate, and secondary metabolic pathways. To date, most studies have been focused on the GlnR regulon, while little is known about the transcriptional regulator for glnR itself. It has been observed that glnR transcription can be upregulated in Mycobacterium smegmatis under nitrogen-limited growth conditions; however, the detailed regulatory mechanism is still unclear. Here, we demonstrate that the glnR gene in M. smegmatis is transcriptionally activated by its product GlnR in response to nitrogen limitation. The precise GlnR binding site was successfully characterized in its promoter region using the electrophoretic mobility shift assay and the DNase I footprinting assay. Site mutagenesis and genetic analyses confirmed that the binding site was essential for in vivo self-activation of glnR transcription. Moreover, based on bioinformatic analyses, we discovered that most of the mycobacterial glnR promoter regions (144 out of 147) contain potential GlnR binding sites, and we subsequently proved that the purified M. smegmatis GlnR protein could specifically bind 16 promoter regions that represent 119 mycobacterial species, including Mycobacterium tuberculosis. Together, our findings not only elucidate the transcriptional self-regulation mechanism of glnR transcription in M. smegmatis but also indicate the ubiquity of the mechanism in other mycobacterial species. IMPORTANCE In actinomycetes, the nitrogen metabolism not only is essential for the construction of life macromolecules but also affects the biosynthesis of secondary metabolites and even virulence (e.g., Mycobacterium tuberculosis). The transcriptional regulation of genes involved in nitrogen metabolism has been thoroughly studied and involves the master nitrogen regulator GlnR. However, the transcriptional regulation of glnR itself remains elusive. Here, we demonstrated that GlnR functions as a transcriptional self-activator in response to nitrogen starvation in the fast-growing model Mycobacterium species Mycobacterium smegmatis. We further showed that this self-regulation mechanism could be widespread in other mycobacteria, which might be beneficial for those slow-growing mycobacteria to adapt to the nitrogen-starvation environments such as within human macrophages for M. tuberculosis.

Construction of Acyclic All-Carbon Quaternary Stereocenters and 1,3-Nonadjacent Stereoelements via Organo/Metal Dual Catalyzed Asymmetric Allenylic Substitution of Aldehydes.

A method for the asymmetric construction of functionalized acyclic all-carbon quaternary stereocenters and 1,3-nonadjacent stereoelements has been developed via organo/metal dual catalyzed asymmetric allenylic substitution of branched and linear aldehydes, by developing an unknown acyclic secondary-secondary diamine as the enabling organocatalyst. Although it is believed that secondary-secondary diamines are difficult to be used as the organocatalysts in organo/metal dual catalysis, this study demonstrates that such diamines can be successfully combined with a metal catalyst in organo/metal dual catalysis. Our study enables the asymmetric construction of two important classes of motifs which were previously difficult to access, axially chiral allene-containing acyclic all-carbon quaternary stereocenters and 1,3-nonadjacent stereoelements bearing allenyl axial chirality and central chirality, in good yields with high enantio- and diastereoselectivity.

Enantioselective synthesis of N-alkylindoles enabled by nickel-catalyzed C-C coupling.

Enantioenriched N-alkylindole compounds, in which nitrogen is bound to a stereogenic sp3 carbon, are an important entity of target molecules in the fields of biological, medicinal, and organic chemistry. Despite considerable efforts aimed at inventing methods for stereoselective indole functionalization, straightforward access to a diverse range of chiral N-alkylindoles in an intermolecular catalytic fashion from readily available indole substrates remains an ongoing challenge. In sharp contrast to existing C-N bond-forming strategies, here, we describe a modular nickel-catalyzed C-C coupling protocol that couples a broad array of N-indolyl-substituted alkenes with aryl/alkenyl/alkynyl bromides to produce chiral N-alkylindole adducts in single regioisomeric form, in up to 91% yield and 97% ee. The process is amenable to proceed under mild conditions and exhibit broad scope and high functional group compatibility. Utility is highlighted through late-stage functionalization of natural products and drug molecules, preparation of chiral building blocks.

Synthesizing carbonyl furan derivatives by a dehydrogenative coupling reaction.

Herein, we report the development of an efficient green procedure for synthesizing carbonyl furan derivatives by dehydrogenative coupling of furfuryl alcohol with carbonyl compounds. The reaction is performed under mild reaction conditions in the presence of iPrPNP-Mn as the catalyst and a weak base (Cs2CO3). A range of ketones and aldehydes were efficiently diversified with furfuryl alcohol to afford furyl-substituted saturated ketones, and α,ß-unsaturated ketones and aldehydes in good isolated yields.

Palladium-Catalyzed Intermolecular Asymmetric Dearomative Annulation of Phenols with Vinyl Cyclopropanes.

Herein, we report the Pd(0)-catalyzed intermolecular asymmetric dearomative [3 + 2] annulation of phenols with vinyl cyclopropanes via in situ generated ortho-quinone methide intermediates. A series of highly functionalized spiro-[5,6] bicycles which bear three contiguous stereogenic centers including one all-carbon quaternary were obtained with excellent stereoselectivities. Density functional theory (DFT) calculations indicate that the reactions were controlled by thermodynamics.

Efficient Synthesis of C3-Alkylated and Alkenylated Indoles via Manganese-Catalyzed Dehydrogenation.

The catalytic dehydrogenation of alcohols is essential for the sustainable production of valuable products. This provides a new strategy for green organic synthesis in chemical industries. Herein, we describe a manganese-based catalytic system that enables the efficient synthesis of C3-alkylated indoles from benzyl alcohols and indoles via the borrowing hydrogen process. Furthermore, dehydrogenative coupling of 2-arylethanols and indoles yields C3-alkenylated indoles. Meanwhile, reacting 2-aminophenethanol instead of indoles can also obtain the corresponding indole products with high selectivity under the same conditions.

Enantioselective [3 + 2] Cycloaddition of Vinylcyclopropanes with Alkenyl N-Heteroarenes Enabled by Palladium Catalysis.

The first catalytic enantioselective [3 + 2] cycloaddition reaction between vinylcyclopropanes and alkenyl N-heteroarenes in the presence of LiBr and a Pd(0)/SEGPHOS complex was developed. LiBr plays a key role in improving the reactivity of alkenyl N-heteroarenes as a mild Lewis acid.

Dual C(sp3)-H Functionalization of Cyclic Ethers via Singlet Oxygen-Mediated Ring Opening and Ring Closing.

A metal-free dual C(sp3)-H bond functionalization of saturated cyclic ethers via photooxidative singlet oxygen-mediated ring opening and ring closing has been developed, providing a method for generating hydrobenzofurans/pyrans/dioxins. Mechanistic studies have confirmed that ring-opening intermediates were effectively generated by singlet oxygen-mediated C(sp3)-H activation and efficiently reacted with aldehydes and activated methylene compounds to form a wide array of products with high diastereoselectivities (up to >95:5 dr). This study is a rare example of α,ß-dual C(sp3)-H bond functionalization of ethers.

Direct access to spirocycles by Pd/WingPhos-catalyzed enantioselective cycloaddition of 1,3-enynes.

Spirocycles play an important role in drug discovery and development. The direct, catalytic, and enantioselective synthesis of spirocycles from readily available starting materials and in an atom economic manner remains a highly sought-after task in organic synthesis. Herein, an enantioselective Pd-hydride-catalyzed cycloaddition method for the synthesis of spirocyclic compounds directly from two classes of commonly available starting materials, 1,3-enynes and cyclic carbon-hydrogen (C-H) bonds, is reported. The reactions employ a chiral Pd/WingPhos catalyst to both suppress the formation of bis-allenyl by-products and control the stereoselectivity. 1,3-Enynes are used as dielectrophilic four-carbon units in the cycloaddition reactions, which also enables an enyne substrate-directed enantioselectivity switch with good levels of stereocontrol. The present spirocycle synthesis tolerates a broad range of functional groups of 1,3-enyne substrates, including alcohols, esters, nitriles, halides, and olefins. A variety of diverse cyclic nucleophiles, including pharmaceutically important heterocycles and carbocycles, can be flexibly incorporated with spiro scaffolds.

In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract.

Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Here, we present a detailed protocol for measuring in vitro nitrate reductase (NaR) activity of NAS enzymes from Mycolicibacterium smegmatis crude extract using both physiological and non-physiological electron donors.

Developing a medium combination to attain similar glycosylation profile to originator by DoE and cluster analysis method.

Glycosylation is critical for monoclonal antibody production because of its impact on pharmacokinetics and pharmacodynamics. Modulation of glycan profile is frequently needed in biosimilar development. However, glycosylation profile is not a single value like that of cell culture titer, hence making it challenging for the Design of Experiment (DoE) methodology to be directly applied. In this study, a Her2-binding antibody was developed as a biosimilar to Herceptin. Cluster analysis was introduced to demonstrate the similarity of glycan profiles between the samples and the reference with specific value-distance. The glycosylation was subsequently optimized with the DoE method. Basal medium and feed medium were found to be the significant factors to the glycosylation pattern. Moreover, a combination of medium and feed strategy was developed to attain the most similar glycoprotein molecule to that of the originator biologic drug. This study may provide an additional option to evaluate multivariable factors and assess biosimilarity and/or comparability in monoclonal antibody production.

Desymmetrization of 1,3-Diones by Catalytic Enantioselective Condensation with Hydrazine.

We present herein an unprecedented desymmetrization of meso 1,3-diones by enantioselective intermolecular condensation. Under the catalysis by a chiral phosphoric acid, a range of readily available 1,3-diones undergo reaction with hydrazines to produce cyclic and acyclic keto-hydrazones bearing an all-carbon quaternary center in high efficiency and enantioselectivity. These compounds are also highly versatile for the preparation of multifunctional building blocks and heterocycles in excellent stereoselectivity.

Palladium-Catalyzed Asymmetric Direct Intermolecular Allylation of α-Aryl Cyclic Vinylogous Esters: Divergent Synthesis of (+)-Oxomaritidine and (-)-Mesembrine.

We demonstrate that α-aryl cyclic vinylogous esters are competent substrates in the direct intermolecular Pd-catalyzed asymmetric allylic alkylation, enabling a straightforward enantioselective synthesis of 6-allyl-6-aryl-3-ethoxycyclohex-2-en-1-ones, common motifs embedded in numerous structurally diverse natural products. As an initial demonstration of the utility of this protocol, the first catalytic enantioselective total synthesis of (+)-oxomaritidine and an improved five-step catalytic enantioselective synthesis of (-)-mesembrine have been completed divergently.

Similarity assessment by multivariate statistics method based on the distance between biosimilar and originator.

The development of biosimilar products or follow-on biologics has been flourishing in recent years because of their lower price than the originators. In this study, a multivariate data analysis method based on JMP software was proposed to assess the glycosylation pattern similarity of antibody candidates from different conditions in optimization experiments with a reference. A specific distance was generated by this method and indicated the glycoform similarity between the biosimilar and the reference. This method can be applied to analyze the similarity of other physicochemical and functional characteristics between follow-on biologics and originators. Then, the design of experimental methods can be realized to optimize the conditions of cell culture to attain similar antibody candidates. A higher concentration of GlcNAc added to the basal media made the glycan of the antibody more similar to the glycan of the reference in this study.