Well-Defined Pre-Catalysts in Amide and Ester Bond Activation.

Over the past few decades, transition metal catalysis has witnessed a rapid and extensive development. The discovery and development of cross-coupling reactions is considered to be one of the most important advancements in the field of organic synthesis. The design and synthesis of well-defined and bench-stable transition metal pre-catalysts provide a significant improvement over the current catalytic systems in cross-coupling reactions, avoiding excess use of expensive ligands and harsh conditions for the synthesis of pharmaceuticals, agrochemicals and materials. Among various well-defined pre-catalysts, the use of Pd(II)-NHC, particularly, provided new avenues to expand the scope of cross-coupling reactions incorporating unreactive electrophiles, such as amides and esters. The strong σ-donation and tunable steric bulk of NHC ligands in Pd-NHC complexes facilitate oxidative addition and reductive elimination steps enabling the cross-coupling of broad range of amides and esters using facile conditions contrary to the arduous conditions employed under traditional catalytic conditions. Owing to the favorable catalytic activity of Pd-NHC catalysts, a tremendous progress was made in their utilization for cross-coupling reactions via selective acyl C⁻X (X=N, O) bond cleavage. This review highlights the recent advances made in the utilization of well-defined pre-catalysts for C⁻C and C⁻N bond forming reactions via selective amide and ester bond cleavage.

Optimized upstream and downstream process conditions for the improved production of recombinant human asparaginase (rhASP) from Escherichia coli and its characterization.

The present work elucidates the production of recombinant human asparaginase (rhASP) under optimized fermentation and downstream processes in Escherichia coli. The maximum biomass yield of 6.7 g/L was achieved with fed-batch fermentation. The highest rhASP inclusion bodies recovery yield (91%) was achieved with the optimized lysis conditions. The 8.0 M urea at pH 8.5 has shown efficient solubilization (94%) of rhASP inclusion bodies. The refolding efficiency of rhASP increased at pH 8.5 (84%) and temperature 25°C (86%). The diluted rhASP solution was concentrated and partially purified (92%) using cross flow filtration. A single step ion exchange chromatography is successfully achieved the maximum purity of ≥ 97%. The molecular mass of purified rhASP is confirmed as 34.1 kDa by mass spectrometry. The secondary structure of rhASP is characterized by FT-IR spectroscopy based on the structural elements. Finally, cell proliferative assay of purified rhASP is signifies the similar biological activity over the standard.

Efficient and easily scalable protein folding strong anion exchange chromatography for renaturation and simultaneous purification of recombinant human asparaginase from E. coli.

Recombinant proteins are revolutionizing present day therapeutics. They are generally expressed as insoluble inclusion bodies in the E. coli and mis-folding, loss of protein, and high cost of down streaming are the hurdles in their recovery. For the first time, we are reporting the refolding with simultaneous purification of rhASP in E. coli using a single step utilizing protein folding-strong anion exchange chromatography (PF-SAX). The purification method is also standardized for optimal concentration of solution additives, pH, and mobile phase composition. The results showed purification of rhASP with anion exchange chromatography was effective. Phosphate buffer and slightly alkaline pH produced significant recovery yields and purity profiles. The effect of solution additives such as arginine, glycerol, TMAO, sorbitol, dextran, glutamate, and fructose on rhASP renaturation is also investigated. Significant results were achieved using arginine-TMAO combination in terms of purity, recovery yield and specific activity of 99%, 78%, and 210 IU/mg, respectively. The work concludes that PF-SAX refolding method is superior to other conventional methods and it can be applied to large scale purification of rhASP produced in E. coli. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1036-1044, 2018.

Bismuth-Catalyzed Synthesis of 2-Substituted Quinazolinones.

The bismuth-catalyzed oxidative condensation of aldehydes with 2-aminobenzamide under aerobic conditions is reported using ethanol as the solvent. Good to excellent isolated yields (68-95%) of the corresponding 2-substituted quinazolinones were obtained under mild reaction conditions with excellent functional group tolerance. The quinazolinones were further functionalized to afford N-allylated quinazolinones, 2-aminopyridine derivatives, and annulated polyheterocyclic compounds via transition-metal catalyzed reactions.

Indium-Mediated Stereoselective Allylation.

Stereoselective indium-mediated organic reactions have enjoyed tremendous growth in the last 25 years. This is in part due to the insensitivity of allylindium to moisture, affording facile and practical reaction conditions coupled with outstanding functional group tolerance and minimal side reactions. Despite the plethora of articles about allylindium, there is much yet to be discovered and exploited for efficient and sustainable synthesis. In this Account, we describe indium-mediated synthetic methods for the preparation of chiral amines with the aim to present a balance of practical method development, novel asymmetric chemistry, and mechanistic understanding that impact multiple chemical and materials science disciplines. In 2005, we demonstrated the indium-mediated allylation of chiral hydrazones with complete diastereoselectivity (>99:1) and quantitative yields. Further, we revealed the first example of enantioselective indium-mediated allylation of hydrazones using catalytic (R)-3,3'-bis(trifluoromethyl)-BINOL ligands to afford homoallylic amines with high enantioselectivity. The use of enantiopure perfluoroalkylsulfonate BINOLs greatly improved the indium-mediated allylation of N-acylhydrazones with exquisite enantiocontrol (99% yield, 99% ee). This laboratory has also investigated indium-mediated asymmetric intramolecular cyclization in the presence of amino acid additives to deliver biologically relevant chromanes with excellent diastereoselectivity (dr >99:1). The effect of amino acid additives (N-Boc-glycine) was further investigated during the indium-mediated allylation of isatins with allyl bromide to yield homoallylic alcohols in excellent yields in a short time with a wide range of functional group tolerance. Critical mechanistic insight was gained, and evidence suggests that the additive plays two roles: (1) to increase the rate of formation of allylindium from allyl bromide and In(0) and (2) to increase the nucleophilicity of the allylindium reagent, probably through disruption of aggregates and coordination to the metal. We recently reported the palladium-catalyzed umpolung allylation of hydrazones with allyl acetates in the presence of indium(I) iodide (InI) with excellent diastereoselectivity (up to 99:1). The conversion was found to be inversely proportional to the phosphine concentration, providing insight into the mechanism of the critical redox transmetalation process that has implications for other Pd-catalyzed umpolung-type allylation processes. A detailed overview of the work in our lab is presented with the intention of stimulating further research interest in organoindium chemistry and its application in organic synthesis.

Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae

Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of recombinant human antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT by S. cerevisiae, the biomass and rhAT concentration were analyzed through fed-batch fermentation process. Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration (312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency (99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95 and 94%, respectively. The concentrated solution was further purified by the single step ion exchange chromatography with the recovery yield and purity of 55 and >98%, respectively. The purified rhAT was characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and Liquid chromatography mass spectrometry (LC-MS) analysis. The biological activity of rhAT was analyzed as heparin cofactor to meet the therapeutic grade applications. Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease.

Efficient single step chromatographic purification of recombinant human antithrombin (rhAT) from Saccharomyces cerevisiae.

Antithrombin (AT) is a glycoprotein that inactivates the several physiological target enzymes of coagulation system. The effect of purification strategies plays a crucial role in getting maximum recovery of yield, purity and biological activity of recombinant human antithrombin (rhAT). In the present work, the task of purifying rhAT from Saccharomyces cerevisiae BY4741 has been carried out using two different approaches such as cross flow filtration (CFF) system and chromatography methods. In the first approach, the protein was concentrated and partially purified through CFF to achieve maximum recovery yield and purity of 87 and 94 %, respectively. In the second approach, purification involved a single step chromatography with various types of ion exchange and size exclusion resins to analyze the maximum rhAT recovery yield and purity. From the experimental results, it has been observed that the size exclusion chromatography (SEC) technique with Superose 12 matrix was suitable for the purification of rhAT and achieved the maximum recovery yield and purity of 51 and 97 %, respectively. Further, to acquire a high recovery yield and purity of rhAT, the effect of various chromatographic conditions such as mobile phase, mobile phase pH, flow rate, sample volume and sample concentration were also investigated. Under the optimal chromatographic conditions, rhAT was significantly recovered and purified in a single step with maximum recovery yield, purity and biological activity of 67, 99 % and 410 IU/L, respectively. Based on these investigations, it was concluded that SEC with Superose 12 matrix was a more suitable and a potential method for the purification of rhAT.

Characterization of recombinant human granulocyte colony-stimulating factor expression by FT-IR spectroscopy: Studies on thermal induction and media formulation on the stability of the protein secondary structure.

The Fourier-transform infrared (FT-IR) spectroscopic approach has been employed to understand the recombinant human G-CSF (rhG-CSF) protein accumulation, secondary structure, and thermal stability in Escherichia coli grown under a temperature shift strategy (37 and 28°C) in various media formulations. The choline + sodium pyruvate (37°C) and sodium pyruvate (28°C) formulations have shown the highest inclusion body (IB) accumulation of 0.41 and 0.46 mg/mL, respectively. Furthermore, insights on the structure of the rhG-CSF within IBs and intact cells have been investigated through secondary structure analysis. Thermal stability experiments were also carried out to explain the pattern of the second derivative structure of rhG-CSF. The studies showed that choline + sodium pyruvate formulation has preserved the protein secondary structure even at 82°C. Overall, the FT-IR spectroscopic technique can also be adopted to accelerate the characterization of other recombinant therapeutic proteins of E. coli origin.

In vitro refolding with simultaneous purification of recombinant human parathyroid hormone (rhPTH 1-34) from Escherichia coli directed by protein folding size exclusion chromatography (PF-SEC): implication of solution additives and their role on aggregates and renaturation.

Recombinant proteins are frequently hampered by aggregation during the refolding and purification process. A simple and rapid method for in vitro refolding and purification of recombinant human parathyroid hormone (rhPTH 1-34) expressed in Escherichia coli with protein folding size exclusion chromatography (PF-SEC) was developed in the present work. Discrete effects of potential solution additives such as urea, polypolyethylene glycol, proline, and maltose on the refolding with simultaneous purification of rhPTH were investigated. The results of individual additives indicated that both maltose and proline had remarkable influences on the efficiency of refolding with a recovery yield of 65 and 66% respectively. Further, the synergistic effect of these additives on refolding was also explored. These results demonstrate that the additive combinations are more effective for inhibiting protein aggregation during purification of rhPTH in terms of recovery yield, purity, and specific activity. The maltose and proline combination system achieved the highest renatured rhPTH having a recovery yield of 78%, a purity of ≥99%, and a specific activity of 3.31 × 10(3) cAMP pM/cell respectively, when compared to the classical dilution method yield (41%) and purity (97%). In addition, the role of maltose and proline in a combined system on protein aggregation and refolding has been explained. The molecular docking (in silico) scores of maltose (-10.91) and proline (-9.0) support the in vitro results.

Improved Production and Characterization of Recombinant Human Granulocyte Colony Stimulating Factor from E. coli under Optimized Downstream Processes.

This work reports the upstream and downstream process of recombinant human granulocyte colony stimulating factor (rhG-CSF) expressed in Escherichia coli BL21 (DE3)pLysS. The fed batch mode was selected for the maximum output of biomass (6.4g/L) and purified rhG-CSF (136mg/L) under suitable physicochemical environment. The downstream processing steps viz., recovery, solubilization, refolding and concentration were optimized in this study. The maximum rhG-CSF inclusion bodies recovery yield (97%) was accomplished with frequent homogenization and sonication procedure. An efficient solubilization (96%) of rhG-CSF inclusion bodies were observed with 8M urea at pH 9.5. Refolding efficiency studies showed maximum refolding ⩾86% and ⩾84% at 20°C and pH 9 respectively. The renatured protein solution was concentrated, clarified and partially purified (⩾95%) by the cross flow filtration technique. The concentrated protein was further purified by a single step size exclusion chromatography with ⩾98% purity. The characterization of purified rhG-CSF molecular mass as evidenced by SDS-PAGE, western blot and LC/MS analysis was shown to be 18.8kDa. The secondary structure of rhG-CSF was evaluated by the CD spectroscopic technique based on the helical structural components. The biological activity of the purified rhG-CSF showed a similar activity of cell proliferation with the standard rhG-CSF. Overall, the results demonstrate an optimized downstream process for obtaining high yields of biologically active rhG-CSF.

Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF.

A growth inhibitory model with SO(x) influenced effective growth rate for estimation of algal biomass concentration under flue gas atmosphere.

A theoretical model for the prediction of biomass concentration under rice husk flue gas emission has been developed. The growth inhibitory model (GIM) considers the CO2 mass transfer rate, the critical SOx concentration and its role in pH-based inter-conversion of bicarbonate. The calibration and subsequent validation of the growth profile of Nannochloropsis limnetica at 2% and 10% (v/v) CO2 showed that the predicted values were consistent with the measured values, with r(2) being 0.96 and 0.98, respectively, and p<0.001 in both cases. The constants used in the GIM for the prediction of biomass have been justified using sensitivity analysis. GIM applicability was defined as ±30% of the calibrated flow rate (3.0 L min(-1)). This growth model can be applied to predict algal growth in photo-bioreactors treated with flue gas in the generation of biomass feed stock for biofuel production.