Recombinant granulocyte colony-stimulating factor (filgrastim): Optimization of conjugation conditions with polyethylene glycol

With the purpose of creating an active prolonged-release pharmaceutical substance, modification of the recombinant human granulocyte colony-stimulating factor G-CSF (filgrastim) with polyethylene glycol (PEG, molecular mass 21.5 kDa) has been performed. The method for the preparation of the filgrastim PEG derivative intended to develop and scale-up the technological manufacturing process is described. Protein modification with PEG was performed by selective covalent attachment of the ??-methyl-PEG-propionaldehyde molecule to the ??-amino group of the N-terminal of the methionine amino acid residue of the recombinant G-CSF. The selected reaction conditions provide no less than 85% product yield of the total protein, a high protein concentration in the reaction mixture (more than 9 mg/mL) and allow us to reduce PEG consumption on the protein terminal ??-amino group basis. RP HPLC and MALDI mass spectrometry data demonstrate that the preparation is modified by PEG at the N-terminal residue and contains no more than 10% of products with the higher degree of modification.     

Microwave-assisted synthesis of new benzimidazole derivatives with lipase inhibition activity

Abstract

A practical protocol has been used for the synthesis of benzimidazoles. The reaction of iminoester hydrochlorides of phenylacetic with 4,5-dichloro-1,2-phenylenediamine under microwave irradiation leads to the benzimidazole derivatives with good yields and in short reaction times. After the synthesis of benzimidazoles, we synthesized ester and hydrazide derivatives under microwave irradiation with good yields. All compounds were evaluated with regard to pancreatic lipase activity and 3b, 3c, 5a and 6a showed lipase inhibition at various concentrations.     

Chemo-enzymatic asymmetric synthesis of S-citalopram by lipase-catalyzed cyclic resolution and stereoinversion of quaternary stereogenic center

A chemo-enzymatic synthesis method of S-citalopram was developed to overcome the disadvantage of relatively low selectivity of enzyme towards tertiary alcohols. The combination of kinetic resolution, cyclic resolution and stereoinversion synthesis was successfully applied in the asymmetric synthesis of the S-citalopram. Using the kinetic model to predict the cyclic resolution, R-diol with high ee value was obtained by controlling the conversion rate. Subsequently, the unwanted R-diol was inverted to S-citalopram by stereoinversion of chiral quaternary center with 98.0 % yield and ee value of 91.0 %. Based on dynamic simulation and experiments, the kinetic resolution was scaled up from 10 mL to 1 L and 14 L, gradually. There was no significant scale-up effect and the dynamic simulation result fitted the experimental data well, with an error of 12.5 and 14.0 %, respectively. This chemo-enzymatic synthesis route is a promising model system for the production of pharmaceuticals with the chiral tertiary alcohols intermediate.     

Inhibition of aminoacyl-tRNA synthetases by p-chloroamphetamine and its role in protein synthesis inhibition

p-Chloroamphetamine inhibited to some degree all amino acid-dependent pyrophosphate-exchange activities which could be detected in a rabbit reticulocyte extract. A detailed kinetic analysis of the reaction catalyzed by reticulocyte leucyl-tRNA synthetase demonstrated that the inhibitor affected only amino acid binding. Less rigorous studies of other synthetases from both rabbit reticulocyte and Escherichia coli could be similarly interpreted, suggesting that this compound interacts in a common manner with these several enzymes. The contribution of such effects to the inhibition of protein synthesis by the drug was investigated using cell-free translation systems in which rates of amino acid incorporation were limited to varying degrees by the synthesis and availability of aminoacyl-tRNA. In a wheat germ system programmed with globin mRNA, in which levels of amino acids and aminoacyl-tRNAs were shown to limit the rate of protein synthesis, the inhibition produced by p-chloroamphetamine could be partially reversed by increasing the concentration of the limiting amino acid. In a reticulocyte lysate, in which amino acid concentrations were not limiting, inhibition failed to show an amino acid-reversible component. Thus, while the inhibition of aminoacyl-tRNA synthetases by amphetamines can be shown in some cases to play a role in the effects of these compounds on in vitro protein synthesis, other sites of interference with initiation and/ or elongation reactions may predominate, depending on the construction of the system under study.     

Biotechnological production of acetylated thymosin beta4

Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.     

Optimisation and Characterisation of Lipase-Catalysed Synthesis of a Kojic Monooleate Ester in a Solvent-Free System by Response Surface Methodology

Kojic acid is widely used to inhibit the browning effect of tyrosinase in cosmetic and food industries. In this work, synthesis of kojic monooleate ester (KMO) was carried out using lipase-catalysed esterification of kojic acid and oleic acid in a solvent-free system. Response Surface Methodology (RSM) based on central composite rotatable design (CCRD) was used to optimise the main important reaction variables, such as enzyme amount, reaction temperature, substrate molar ratio, and reaction time along with immobilised lipase from Candida Antarctica (Novozym 435) as a biocatalyst. The RSM data indicated that the reaction temperature was less significant in comparison to other factors for the production of a KMO ester. By using this statistical analysis, a quadratic model was developed in order to correlate the preparation variable to the response (reaction yield). The optimum conditions for the enzymatic synthesis of KMO were as follows: an enzyme amount of 2.0 wt%, reaction temperature of 83.69°C, substrate molar ratio of 1:2.37 (mmole kojic acid:oleic acid) and a reaction time of 300.0 min. Under these conditions, the actual yield percentage obtained was 42.09%, which is comparably well with the maximum predicted value of 44.46%. Under the optimal conditions, Novozym 435 could be reused for 5 cycles for KMO production percentage yield of at least 40%. The results demonstrated that statistical analysis using RSM can be used efficiently to optimise the production of a KMO ester. Moreover, the optimum conditions obtained can be applied to scale-up the process and minimise the cost.     

Synthesis of a conformationally constrained δ-amino acid building block

Conformationally restricted amino acids are important components in peptidomimetics and drug design. Herein, we describe the synthesis of a novel, non-proteinogenic constrained delta amino acid containing a cyclobutane ring, cis-3(aminomethyl)cyclobutane carboxylic acid (ACCA). The synthesis of the target amino acid was achieved in seven steps, with the key reaction being a base induced intramolecular nucleophilic substitution. A small library of dipeptides was prepared through the coupling of ACCA with proteinogenic amino acids.     

Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos.

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of amino acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for amino acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartmentation of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus laevis are 18 and 50–54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.     

Scale-up of pseudo solid-phase enzymatic synthesis of alpha-methyl glucoside acrylate

The successful scale-up of the enzymatic synthesis of alpha-methyl glucoside acrylate from laboratory-scale (milliliter) to pilot-scale (liter) was examined. Specifically, Candida antarctica lipase B (Novozym 435) was used as a biocatalyst to produce alpha-methyl glucoside acrylate via the transesterification of alpha-methyl glucoside (MG) with vinyl acrylate (VA) using acetone as a solvent. This is a pseudo-solid-phase synthesis; only a fraction of the alpha-methyl glucoside and the product are soluble in acetone. Molecular sieves were used to remove traces of water in the reaction medium and to increase enzyme stability by removing the acetaldehyde by-product. A general method was also developed to purify and recover the monoacrylate product from unreacted sugar and undesired diester by a simple crystallization and precipitation process.     

Dual-functional significance of ATM-mediated phosphorylation of spindle assembly checkpoint component Bub3 in mitosis and the DNA damage response

Both the DNA damage response (DDR) and the mitotic checkpoint are critical for the maintenance of genomic stability. Among proteins involved in these processes, the ataxia–telangiectasia mutated (ATM) kinase is required for both activation of the DDR and the spindle assembly checkpoint (SAC). In mitosis without DNA damage, the enzymatic activity of ATM is enhanced; however, substrates of ATM in mitosis are unknown. Using stable isotope labeling of amino acids in cell culture mass spectrometry analysis, we identified a number of proteins that can potentially be phosphorylated by ATM during mitosis. This list is highly enriched in proteins involved in cell cycle regulation and the DDR. Among them, we further validated that ATM phosphorylated budding uninhibited by benzimidazoles 3 (Bub3), a major component of the SAC, on serine 135 (Ser135) both in vitro and in vivo. During mitosis, this phosphorylation promoted activation of another SAC component, benzimidazoles 1. Mutation of Bub3 Ser135 to alanine led to a defect in SAC activation. Furthermore, we found that ATM-mediated phosphorylation of Bub3 on Ser135 was also induced by ionizing radiation-induced DNA damage. However, this event resulted in independent signaling involving interaction with the Ku70–Ku80–DNA-PKcs sensor/kinase complex, leading to efficient nonhomologous end-joining repair. Taken together, we highlight the functional significance of the crosstalk between the kinetochore-oriented signal and double-strand break repair pathways via ATM phosphorylation of Bub3 on Ser135.     

A precise method for the quantitation of proteins taking into account their amino acid composition.

A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined.     

Repurposing benzimidazoles to fight Cryptococcus

The human diseases caused by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are associated with high rates of mortality and toxic or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Benzimidazoles are potentially attractive antifungal compounds that were introduced in clinical practice nearly 60 years ago to treat helminthic infections. In addition to being safe, their cost of treatment is extraordinarily low. Several studies suggested benzimidazoles as promising anticryptococcal agents combining low-cost and high antifungal efficacy. So far, anti-cryptococcal activities were demonstrated for 16 different benzimidazoles. In particular, albendazole, mebendazole, flubendazole, and fenbendazole have potent in vitro antifungal activity against C. neoformans and C. gattii. Mice lethally infected with C. neoformans and treated with fenbendazole had 100 % survival when the drug was administered intranasally. In this review, we discuss the potential of benzimidazoles as potential anti-cryptococcal agents, including a general literature overview, most recent findings, mechanism of antifungal action, costs, toxicity, and antifungal potential in vivo.     

Stepwise increase of thaxtomins production in Streptomyces albidoflavus J1074 through combinatorial metabolic engineering

Herbicide-resistance in weeds has become a serious threat to agriculture across the world. Thus, there is an urgent need for the discovery and development of herbicides with new modes of action. Thaxtomin phytotoxins are a group of nitrated diketopiperazines produced by potato common scab-causing phytopathogen Streptomyces scabies and other actinobacterial pathogens. They are generally considered to function as inhibitors of cellulose synthesis in plants, and thus have great potential to be used as natural herbicides. Generation of an overproducing strain is crucial for the scale-up production of thaxtomins and their wide use in agriculture. In the present study, we employed a stepwise strategy by combining heterologous expression, repressor deletion, activator overexpression, and optimization of fermentation media for high-level production of thaxtomins. The maximum yield of 728 mg/L thaxtomins was achieved with engineered Streptomyces albidoflavus J1074 strains in shake-flask cultures, and it was approximately 36-fold higher than S. albidoflavus J1074 carrying the unmodified cluster. Moreover, the yield of thaxtomins could reach 1973 mg/L when the engineered strain was cultivated in a small-scale stirred-tank bioreactor. This is the highest titer reported to date, representing a significant leap forward for the scale-up production of thaxtomins. Our study presents a robust, easy-to-use system that will be broadly useful for improving titers of bioactive compounds in many Streptomyces species.     

An Efficient and Epimerization Free Synthesis of C-Terminal Arylamides Derived from α-Amino Acids and Peptide Acids via T3P Activation

A high yield and rapid synthesis of enantiomerically pure N ^α -protected amino/peptide acid arylamides using n-propylphosphonic anhydride (T3P) in presence of N-methylmorpholine is described. The generality of the reaction has been studied for various N ^α -protected amino acids with diverse range of aromatic amines and coumarin derivatives.     

Synthesis and bioorthogonal coupling chemistry of a novel cyclopentenone-containing unnatural tyrosine analogue

Herein we report the synthesis of a novel amino acid with orthogonal functionality to the natural amino acid side chains. Tyrosine was O-alkylated with a cyclic 5-membered α,β-unsaturated ketone ring (5). We have established that this amino acid analogue can undergo cycloaddition reactions in aqueous media with in situ generated nitrones. Nitrone formation occurred by micellar catalysis can undergo aqueous 1,3-dipolar cycloaddition reactions with the unnatural Tyr. We also performed a linear free energy analysis of the one pot bioconjugation reaction in water using cyclopentenone as a model for the Tyr analogue and seven different aryl nitrones. We found that the Hammett ρ value was −0.94, suggesting that the reaction occurs in a concerted fashion with a slight positive charge buildup in the transition state. The Hammett ρ value also suggests that the bioconjugation reaction is tolerant of different substituents and thus may be useful for introducing novel functionality into peptides and proteins containing the Tyr analogue 5. The aqueous 1,3-dipolar cycloaddition reactions, that use nitrones to trap the O-alkylated Tyr 5, establish a novel strategy for rapid, water compatible bioconjugation reactions.     

Inhibition of protein synthesis by products of lipid peroxidation

Effects of lipid peroxidation products on in vivo and in vitro protein synthesis have been studied. Malondialdehyde (MDA), a product, and a routinely used index of lipid peroxidation, inhibits in vivo protein synthesis in the two mosses, Tortula ruralis and Cratoneuron filicinum, and in pea (Pisum sativum) leaf discs. When wheat germ supernatant or poly(A)-rich mRNA of T. ruralis was incubated with MDA its subsequent activity in a cell-free protein-synthesizing system was reduced. When MDA was added directly to the in vitro protein-synthesizing mixture containing moss polyribosomes, the inhibition of amino acid incorporation was small. However, when simultaneous lipid peroxidation was allowed to occur along with in vitro protein synthesis there was a strong inhibition of amino acid incorporation and MDA accumulated in the reaction mixture indicating that products of lipid peroxidation other than, and apparently more toxic than, MDA were involved. It was concluded that lipid peroxidation inhibits protein synthesis probably by releasing toxic products which may react with and inactivate some components of the protein-synthesizing complex.     

Determination of the availability of carbodiimide-activated N-protected amino acids in solid phase peptide synthesis

A new method is described for the determination of the availability of carbodiimide-activated N-protected amino acids in solid phase peptide synthesis. The method involves the addition of a second nucleophile to a solid phase coupling reaction at different time intervals and measuring the amount of activated amino acid intercepted. Using the DCCI-mediated coupling reaction of Boc-Ala-OH and H-Gly-O-resin with H-Gly-O-tBu as the second nucleophile, it was determined that $\text{ca}$. 61% of the theoretical maximum amount of activated Boc-Ala-OH was available after 8 hr of reaction.     

Amino acid analysis with o-phthalaldehyde detection: effects of reaction temperature and thiol on fluorescence yields.

Chromatographic separation of amino acids with fluorometric detection of the o-phthalaldehyde-thiol reaction product offers an analytical system of high sensitivity for most amino acids. The fluorescence yield for amino acids having a fully substituted carbon atom in the α-position with respect to the amino group can be substantially improved by increasing the temperature of the reaction with o-phthalaldehyde-thiol and by substituting either ethanethiol or methanethiol for the more commonly used β-mercaptoethanol. The analytical sensitivity for these α-branched amino acids is thus brought into line with that for the other primary amino acids. A comparison of chromatograms obtained at reaction temperatures of 25 and 100°C allows recognition of amino acids of this type in complex mixtures by the substantial increase in fluorescence yield at 100°C.     

Preparation method forEscherichia coli S30 extracts completely dependent upon tRNA addition to catalyze cell-free protein synthesis

A simple method for depletingE. coli S30 extracts of endogenous tRNA has been developed. An ethanolamine-Sepharose^® column equilibrated with water selectively captured the tRNA molecules inE. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful forin vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.