Construction of heterocycle-fused tetrahydrocarbazoles through a formal [3 + 3]-annulation of 2-indolylmethanols with para-quinone methides.

A metal-free approach for the synthesis of tetrahydroindolo[2,3-b]carbazoles has been developed through an acid-mediated one-pot [3 + 3]-annulation of 2-indolylmethanols and 3-indolyl-substituted para-quinone methides. This operationally simple protocol allowed us to prepare many unsymmetrical tetrahydroindolo[2,3-b]carbazoles in good to excellent yields with a broad substrate scope. This concept was also elaborated to the synthesis of tetrahydrothieno[2,3-b]carbazoles and tetrahydrothieno[3,2-b]carbazoles.

TfOH-Catalyzed Intramolecular Annulation of 2-(Aryl)-Phenyl-Substituted p-Quinone Methides under Continuous Flow: Total Syntheses of Selaginpulvilin I and Isoselagintamarlin A.

In this article, we describe a convenient method to access 9-aryl fluorene derivatives through a TfOH-catalyzed intramolecular 1,6-conjugate arylation of 2-(aryl)-phenyl-substituted p-quinone methides (QMs) under continuous flow using the microreaction technique. This method was found to be very effective for most of the p-QMs, and the corresponding 9-aryl fluorene derivatives were obtained in moderate to excellent yields. Moreover, this protocol was further elaborated to the first total syntheses of selaginpulvilin I and isoselagintamarlin A.

Construction of Oxygen- and Nitrogen-based Heterocycles from p-Quinone Methides.

In the last few years, there has been an explosive growth in the area of para-quinone methide (p-QM) chemistry. This boom is actually due to the unique reactivity pattern of p-QMs, and also their remarkable synthetic applications. In fact, p-QMs serve as synthons for unsymmetrical diaryl- and triarylmethanes, and also for the construction of diverse range of carbocycles and heterocycles. In the last few years, a wide range of structurally complex heterocyclic frameworks could be accessed through the synthetic transformations of structurally modified stable p-QMs. Therefore, the main focus of this review article is to cover the recent advancements in the transition-metal, Lewis acid and base-catalyzed/mediated synthetic transformations of the stable p-quinone methides (p-QMs) to oxygen- and nitrogen-containing heterocycles.

Optimizing secretory expression of recombinant human interferon gamma from Kluyveromyces lactis.

Biosimilar/biotherapeutic production is becoming a major area of focus for a big chunk of biotechnology industry. Easy licensing and already approved status for clinical use have given it a boost. In the present study, recombinant human interferon gamma (IFNG) was expressed for the first time in Kluyveromyces lactis expression system and its expression was optimized by varying growth parameters and carbon source concentration with the aim of increasing recombinant protein production level. Human IFNG gene was cloned in the genomic DNA of K. lactis by homologous recombination and under unoptimized conditions in shake flask, IFNγ protein was secreted in the fermentation medium at a level of 175 µg/L quantified by ELISA assay. After the optimization of expression conditions using one-variable-at-a-time technique, expression level was enhanced by 2.2-folds. Substrate inhibition studies revealed that up to 80 g/L of lactose is well tolerated by K. lactis cells for its growth but more than 80 g/L of lactose causes remarkable reduction in biomass production.

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis.

The present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box-Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80 g/L lactose, 10.353 g/L yeast extract, and 15 mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881 µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1 mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.

Genetic and substrate-level modulation of Bacillus subtilis physiology for enhanced extracellular human interferon gamma production.

Human interferon-gamma (hIFNG) production is limited by various gene-level bottlenecks including translation, protein folding, and secretion which depends upon the physiological state of the organism. In this study gene-level and substrate-level modulations have been used to control Bacillus subtilis physiology for >15 fold extracellular soluble hIFNG production. Two variants of the native human interferon-gamma gene (hifng) were designed and synthesized, namely, cohifnghis and cohifng having codon adaptation index 25.33 and 26.89% higher than the native gene, respectively. BScoIFNG and BScoIFNGhis with ΔG of -100.0 and -113.7 kcal mol-1 resulted in 30 and 6.5% higher hIFNG compared to the native gene in complex medium. BScoIFNG produced 1.53 fold higher hIFNG using glucose-based defined medium as compared to the complex medium by modulating the physiological parameter growth rate from 0.35 to 0.26 hr-1. Further modulatory effect of various phosphotransferase transport system (PTS) and no-PTS sugars, sugar alcohols, and organic acids was quantified on the physiology of B. subtilis WB800N for extracellular hIFNG production. Sorbitol and glycerol emerged as the best hIFNG producers with lowest growth and substrate consumption rates. BScoIFNG produced maximum 3.15 mg L-1 hIFNG at 50 g L-1 glycerol with highest hIFNG yield (Yp/x = 0.136) and lowest substrate uptake rate (qs = 0.26).

The structure of XIAP BIR2: understanding the selectivity of the BIR domains.

XIAP, a member of the inhibitor of apoptosis family of proteins, is a critical regulator of apoptosis. Inhibition of the BIR domain-caspase interaction is a promising approach towards treating cancer. Previous work has been directed towards inhibiting the BIR3-caspase-9 interaction, which blocks the intrinsic apoptotic pathway; selectively inhibiting the BIR2-caspase-3 interaction would also block the extrinsic pathway. The BIR2 domain of XIAP has successfully been crystallized; peptides and small-molecule inhibitors can be soaked into these crystals, which diffract to high resolution. Here, the BIR2 apo crystal structure and the structures of five BIR2-tetrapeptide complexes are described. The structural flexibility observed on comparing these structures, along with a comparison with XIAP BIR3, affords an understanding of the structural elements that drive selectivity between BIR2 and BIR3 and which can be used to design BIR2-selective inhibitors.

A Base-Mediated Approach Towards Dihydrofuro[2,3-b]Benzofurans from 2-Nitrobenzofurans and 1,3-Dicarbonyls.

A straight-forward approach for the synthesis of a dihydrofuro[2,3-b]benzofuran derivatives has been achieved through a base-mediated Michael addition of 1,3-dicarbonyls to 2-nitrobenzofurans followed by intramolecular cyclization. A variety of 1,3-dicarbonyls, including cyclic as well as trifluoromethylated ones, have been subjected to react with 2-nitrobenzofurans under optimal conditions, and the respective dihydrofuro[2,3-b]benzofurans could be accessed in moderate to excellent yields.

Base-Catalyzed 1,6-Conjugate Addition of Nitroalkanes to p-Quinone Methides under Continuous Flow.

A mild base-catalyzed protocol for the synthesis of substituted nitroalkane derivatives has been developed under continuous flow using a microreaction technique. This transformation basically involves the 1,6-conjugate addition of nitroalkanes to p-quinone methides, leading to the substituted nitroalkanes in good to excellent yields.

Re-engineering of an Escherichia coli K-12 strain for the efficient production of recombinant human Interferon Gamma.

The Escherichia coli phosphoglucose isomerase (pgi) mutant strain GALG20 was developed previously from wild-type K12 strain MG1655 for increased plasmid yield. To investigate the potential effects of the pgi deletion/higher plasmid levels on recombinant human Interferon Gamma (IFN-γ) production, a detailed network of the central metabolic pathway (100 metabolites, 114 reactions) of GALG20 and MG1655 was constructed. Elementary mode analysis (EMA) was then performed to compare the phenotypic spaces of both the strains and to check the effect of the pgi deletion on flux efficiency of each metabolic reaction. The results suggested that pgi deletion increases amino acid biosynthesis and flux efficiency towards IFN-γ synthesis by 11%. To further confirm the qualitative prediction that the pgi mutation favours recombinant human IFN-γ expression, GALG20 and MG1655 were lysogenised, transformed with a plasmid coding for IFN-γ and tested alongside with BL21(DE3) for their expression capabilities in shake flask experiments using complex media. IFN-γ gene expression was analysed by quantifying plasmid and mRNA copy number per cell and IFN-γ protein production level. Specific IFN-γ yields confirmed the in silico metabolic network predictions, with GALG20(DE3) producing 3.0-fold and 1.5-fold more IFN-γ as compared to MG1655(DE3) and BL21(DE3), respectively. Most of the total IFN-γ was expressed as inclusion bodies across the three strains: 95% in GALG20(DE3), 97% in BL21(DE3) and 72% in MG1655(DE3). The copy number of mRNA coding for IFN-γ was found to be higher in GALG20(DE3) as compared to the other two strains. Overall, these findings show that GALG20(DE3) has the potential to become an excellent protein expression strain.