Peptide synthesis using o-nitrophenylsulfenyl N-carboxy alpha-amino acid anhydrides. Sequential oligopeptides having alternate gamma-methyl L-glutamyl and L-phenylalanyl residues.

A series of sequential oligopeptides having the sequence alternating γ-methyl L -glutamyl and L -phenylalanyl residues have been successfully prepared by a rapid method involving the reaction of o-nitrophenylsulfenyl N-carboxy α-amino acid anhydrides with amino acid and peptide esters. The sequential oligopeptides, which are interesting from a conformational aspect, were obtained in optical pure forms above 74% yields. This result demonstrates that the o-nitrophenylsulfenyl N-carboxy α-amino acid anhydride method is especially useful for easy synthesis of protected oligopeptides with o-nitrophenylsulfenyl group.     

DNA adducts of ortho-toluidine in human bladder

Background: 4-Aminobiphenyl (4-ABP) and o-toluidine are known human bladder carcinogens, but only 4-ABP-releasing DNA adducts are known.

Methods: Determination of 4-ABP and o-toluidine-releasing DNA adducts in epithelial and submucosal bladder tissues of sudden death victims (SDV: n?=?46), and bladder tumours (n?=?12) by gas chromatography/mass spectrometry.

Results: Above background, 4 and 11 of 12 tumour samples contained adducts of 4-ABP (0.057?±?0.125?fmol/µg DNA) and o-toluidine (8.72?±?4.49?fmol/µg DNA), respectively. Lower adduct levels were present in both epithelial and submucosal bladder tissues of SDV (4-ABP: 0.011?±?0.022 and 0.019?±?0.047?fmol/µg DNA; o-toluidine: 0.24?±?0.63 and 0.27?±?0.70?fmol/µg DNA).

Conclusion: Detection of o-toluidine-releasing DNA adducts support the carcinogenicity of o-toluidine in the human bladder.     

A method for hydrolyzing and determining the amino acid compositions of picomole quantities of proteins in less than 3 hours

A method is described for quantitatively hydrolyzing proteins in 45 min and for analyzing the hydrolysates by high-performance liquid chromatography in an additional 52 min. The α-amino acids were detected by the fluorescence of their o-phthaldialdehyde derivatives. Ten picomoles of each of the commonly occuring α-amino acids could be reliably determined. The method described yielded OPA-ethanethiolamino acid derivatives that were stable for $\text{1}\text{h}$ h and the HPLC method produced a better separation than previously published methods.     

Yellowing and photosynthetic decline of barley primary leaves in response to atmospheric CO2 enrichment

The photosynthetic response of barley (Hordeum vulgare L. cv. Brant) primary leaves was studied as a function of chlorosis induced by CO₂ enrichment. Leaf yellowing, measured as changes of chlorophyll a and b, was more extensive in controlled environments at elevated (680 ± 17 µl l^?1) than at ambient (380 ± 21 µl l^?1) CO₂. Stomatal conductance of primary leaves was decreased by growth in elevated CO₂ between 11 and 18 days after sowing (DAS) when measured at both 380 and 680 µl l^?1 CO₂. Internal leaf CO₂ concentration (C_i) was also lower for elevated- compared to ambient-CO₂-grown primary leaves between 11 and 14 DAS. Results suggest that non-stomatal factors were responsible for the decreased photosynthetic rates of elevated- compared to ambient-CO₂-grown primary leaves 18 DAS. Various photochemical measurements, including quantum absorptance (α), minimal (F₀), maximal (F_m), and variable (F_v) chlorophyll fluorescence, as well as the F_v/F_m ratio, were significantly decreased 18 DAS in the elevated- compared to ambient-CO₂ treatment. Photochemical (qP) and nonphotochemical (qN) chlorophyll fluorescence quenching coefficients of 18-day-old primary leaves did not differ between CO₂ treatments. Photosynthetic electron transport rates of photosystem II were slightly lower for elevated- compared to ambient-CO₂-grown primary leaves 18 DAS. Concentrations of α-amino N (i.e. free amino acids) in barley primary leaves were increased by CO₂ enrichment 10 DAS, but subsequently, α-amino N decreased in association with photosynthetic decline. Total acid protease activity was greater in elevated- than in ambient-CO₂-grown leaves 18 DAS. The above findings suggest that photoinhibition and premature senescence were factors in the CO₂-dependent yellowing of barley primary leaves.     

The role of the delocalized α,α′-carbanion in vitamin B6 and Al(III) catalyzed transamination of α-amino and α-keto acids

The rates of the transamination reactions of α-amino acids and α-keto acids were followed by measurement of the 200 MHz proton NMR spectra of solution species as a function of time. Reaction systems measured in D₂O at 10 °C consisted of 1:1:1 molar ratios of pyridoxal:α-amino acid:Al(III) or pyridoxamine:α-keto acid:Al(III). Amino and keto acids employed are alanine, α-aminoisobutyric acid, valine, phenylglycine, pyruvic acid, and α-ketobutyric acid. A negatively charged deprotonated Schiff base coordinated to Al(III) was detected in all systems that undergo transamination (i.e., except α-aminoisobutyric acid). The intermediate resembles the aldimine Al(III) chelate with NMR resonances shifted upfield in accordance with its greater negative charge. Its equilibrium concentration is reached in the time required to reach transamination equilibrium and is maintained in solution at a ca. 10–20% of the aldimine Schiff base concentration.     

Δ1-Pyrroline-5-carboxylate: The product of proline dehydrogenase from Cucurbita moschata cotyledons

The product of oxidation of proline by pumpkin proline dehydrogenase reacted with o-aminobenzaldehyde to give a yellow compound that had an absorption spectrum similar to that obtained from chemically synthesized Δ¹-pyrroline-5-carboxylate. The product of the proline dehydrogenase reaction and synthetic Δ¹-pyrroline-5-carboxylate had identical R_f values. Both authentic Δ¹-pyrroline-5-carboxylate and the product of the enzyme gave a pink colour with acid ninhydrin on paper chromatograms and both had identical elution patterns on Dowex 50(H⁺) columns. Neither synthetic Δ¹-pyrroline-5-carboxylate nor the product of proline-dehydrogenase produced γ-amino butyrate with hydrogen peroxide.     

A proposed solution for the determination of the ionization constants of sets of ionizinggroups in proteins

A mixture of acids with known pK′ and in a known concentration was dissolved to simulate the ionic character of hemoglobin. The titration curves of the mixture were obtained in water and 3.6 M KCl at 20°C. These curves were subjected to analysis by a reiterative curve-fitting procedure to determine if one could evaluate both the group ionization constants and the numbers of groups. This approach was successful in that the calculated parameters were within the experimental error encountered in obtaining these constants on each of the model compounds. However, analysis for the electrostatic interaction parameter w indicated that there was a possible effect on the ionization of formic acid, pyridine, imidazole, and the α-amino group of glycylglycine in water and on imidazole and the α-amino group of glycylglycine in 3.6 M KCl.     

Antibiotic potential of plant growth promoting rhizobacteria (PGPR) against Sclerotium rolfsii

High performance liquid chromatographic (HPLC) analysis of culture filtrates of plant growth promoting rhizobacteria (PGPR) and medium of inhibitory zone of interaction of Sclerotium rolfsii with PGPR, viz. Pseudomonas aeruginosa, Pseudomonas fluorescens 4, Pseudomonas fluorescens 4 (new) and Pseudomonas sp. varied from sample to sample. In all the culture filtrates of PGPRs, P. aeruginosa had nine phenolic acids in which ferulic acid (14.52 μg/ml) was maximum followed by other phenolic acids. However, the culture filtrates of P. fluorescens 4 had six phenolic acids with maximum ferulic acid (20.54 μg/ml) followed by indole acetic acid (IAA), caffeic, salicylic, o-coumeric acid and cinnamic acids. However, P. fluorescens 4 culture filtrate had seven phenolic acids in which salicylic acid was maximum (18.03 μg) followed by IAA, caffeic, vanillic, ferulic, o-coumeric and cinnamic acids. Pseudomonas sp. also showed eight phenolic acids where caffeic acid (2.75 μg) was maximum followed by trace amounts of ferulic, salicylic, IAA, vanillic, cinnamic, o-coumeric and tannic acids. The analysis of antibiosis zone of PGPRs showed fairly rich phenolic acids. A total of nine phenolic acids were detected in which caffeic acid was maximum (29.14 μg/g) followed by gallic (17.64 μg/g) and vanillic (3.52 μg/g) acids but others were in traces. In P. aeruginosa, antibiosis zone had seven phenolic acids where IAA was maximum (3.48 μg/g) followed by o-coumeric acid (2.08 μg/g), others were in traces. The medium of antibiosis zone of P. fluorescens 4 and P. fluorescens 4 new had eight phenolic acids in which IAA was maximum with other phenolic acids in traces.     

Investigation of Pyrazine Formation Pathways in Glucose-Ammonia Model Systems

Model systems of d-glucose and ammonia with metal ions, oxygen, antioxidants, and sodium hydroxide were reacted at 100°C (solution temp.) for 2~18 hr to investigate pyrazine formation pathways. Pyrazines identified in these model systems were unsubstituted-, 2-methyl-, 2,5-dimethyl-, 2,6-dimethyl-, 2-ethyl-, 2,3-dimethyl-, 2-ethyl-5-methyl-, 2-ethyl-6-methyl-, 2,3,5-trimethyl-, 2-ethyl-3-methyl-, 2-vinyl-, 2-ethyl-3,5-dimethyl-, 2-ethyl-3,6-dimethyl- and ethyl vinyl-. Results show that α-amino carbonyl compounds acted as intermediates to form various pyrazines. For example, 2-methylpyrazine may be formed from the condensation of α-amino-α-hydroxy acetaldehyde and α-amino acetone following the elimination of a water molecule.

We propose that the formation of a pyrazine ring from dihydropyrazine is due to the dehydration of hydroxy dihydropyrazine rather than the dehydrogenation of dihydropyrazine. This explains the formation of an alkyl group (e.g., an ethyl group) from the sugar moiety.

We propose ten α-amino carbonyl intermediates from the alkylpyrazines which we obtained, and their formation schemes are discussed.     

Synthesis of N-protected peptides by the o-nitrophenylsulfenyl group using o-nitrophenylsulfenyl N-carboxy α-amino acid anhydrides

N-protected peptides, which are important intermediates as a carboxyl component in the fragment condensation method, have been prepared in high yields by the reaction of o-nitrophenylsulfenyl (Nps) N-carboxy α-amino acid anhydrides with unprotected peptides and amino acids in aqueous organic systems. An Nps hexapeptide ester was prepared by the fragment condensation of an Nps tripeptide with a tripeptide ester. It was demonstrated that the synthesis of unprotected peptides by the NCA method, followed by N-protection by the Nps-NCA, is a rapid and very useful method for preparing Nps peptides.     

Efficient production of phenylpropionic acids by an amino-group-transformation biocatalytic cascade

Phenylpropionic acids are commonly used in the synthesis of pharmaceuticals, cosmetics, and fine chemicals. However, the synthesis of phenylpropionic acids faces the challenges of high cost of substrates and a limited range of products. Here, we present an artificially designed amino-group-transformation biocatalytic process, which uses simple phenols, pyruvate, and ammonia to synthesize diverse phenylpropionic acids. This biocatalytic cascade comprises an amino-group-introduction module and three amino-group-transformation modules, and operates in a modular assembly manner. Escherichia coli catalysts coexpressing enzymes from different modules achieve whole-cell simultaneous one-pot transformations of phenols into the corresponding phenylpropionic acids including (S)-α-amino acids, α-keto acids, (R)-α-amino acids, and (R)-β-amino acids. With cofactor recycling, protein engineering, and transformation optimization, four (S)-α-amino acids, four α-keto acids, four (R)-α-amino acids, and four (R)-β-amino acids are synthesized with good conversion (68–99%) and high enantioselectivities (>98%). Therefore, the amino-group-transformation concept provides a universal and efficient tool for synthesizing diverse products.     

High synergistic antibacterial,antibiofilm, antidiabetic and antimetabolic activity of Withania somnifera leaf extract-assisted zinc oxide nanoparticle

Nanotechnology is currently gaining immense attention to combat food borne bacteria, and biofilm. Diabetes is a common metabolic disease affecting majority of people. A better therapy relies on phytomediated nanoparticle synthesis. In this study, W. somnifera leaf extract-assisted ZnO NPs (Ws-ZnO NPs) was synthesized and characterized. From HR-TEM analysis, it has been found that the hexagonal wurtzite particle is 15.6 nm in size and − 12.14 mV of zeta potential. A greater antibacterial effect of Ws-ZnO NPs was noticed against E. faecalis and S. aureus at 100 µg mL⁻¹. Also, the biofilm of E. faecalis and S. aureus was greatly inhibited at 100 µg mL⁻¹ compared to E. coli and P. aeruginosa. The activity of α-amylase and α-glucosidase enzyme was inhibited at 100 µg mL⁻¹ demonstrating its antidiabetic potential. The larval and pupal development was delayed at 25 µg mL⁻¹ of Ws-ZnO NPs. A complete mortality (100%) was recorded at 25 µg mL⁻¹. Ws-ZnO NPs showed least LC₅₀ value (9.65 µg mL⁻¹) compared to the uncoated ZnO NPs (38.8 µg mL⁻¹) and leaf extract (13.06 µg mL⁻¹). Therefore, it is concluded that Ws-ZnO NPs are promising to be used as effective antimicrobials, antidiabetic and insecticides to combat storage pests.

     

Cholera Toxin Induces Cyclic AMP-Independent Down-Regulation of Gsα and Sensitization of α2-Autoreceptors in Chick Sympathetic Neurons

Abstract: The role of the stimulatory GTP-binding protein (G_S) in the α₂-autoinhibitory modulation of noradrenaline release was investigated in cultured chick sympathetic neurons. The α₂-adrenoceptor agonist UK 14,304 caused a concentration-dependent reduction of electrically evoked [³H]noradrenaline release with half-maximal effects at 14.0 ± 5.5 nM. In neurons treated with 100 ng/ml cholera toxin for 24 h, the half-maximal concentration was lowered to 3.2 ± 1.4 nM without changes in the maximal effect of UK 14,304. The pretreatment with cholera toxin also increased the inhibitory action of 10 nM UK 14,304 when compared with the inhibition of noradrenaline release in untreated cultures derived from the same cell population. In cultures treated with either 10 µM forskolin or 100 µM 8-bromo-cyclic AMP, neither the half-maximal concentration nor the maximal effect of UK 14,304 was altered. Cholera toxin, forskolin, and 8-bromo-cyclic AMP all induced an increase in spontaneous outflow and a reduction in electrically evoked overflow, effects not observed after a pretreatment with dideoxyforskolin. Exposure of neurons to cholera toxin, but not to forskolin or 8-bromo-cyclic AMP, induced a translocation of α-subunits of G_s (G_sα) from particulate to soluble fractions and led ultimately to a complete loss of G_sα from the neurons. In contrast, no effect was seen on the distribution of either α-subunits of G_i- or G_o-type G proteins or of β-subunits. These results indicate that cholera toxin causes a selective, cyclic AMP-independent down-regulation of G_sα. This down-regulation of G_sα is associated with the sensitization of α₂-autoreceptors.     

Use of the pH Memory Effect in Lyophilized Proteins to Achieve Preferential Methylation of α-Amino Groups

It is demonstrated that the pH memory effect can be used to control the ionization state of amino groups in lyophilized proteins and hence their chemical reactivity toward modifying reagents. When proteins were lyophilized from aqueous solutions at pH values between 6 and 7 and reacted in vacuo with iodomethane, the α-amino groups were found to be either preferentially or selectively trimethylated. Reaction with ¹³C-labeled iodomethane permitted detection and identification of individual trimethylated α-amino groups by ¹³C-NMR spectroscopy as distinct peaks in the spectral region between 52 and 57 ppm. There was adequate sensitivity to detect minor resonances of free α-amino groups arising from proteolysis of the major protein or from protein impurities. The resonances of the trimethylated α-amino groups in standard amino acids and peptides are sufficiently close to those in the derivatized protein to make a tentative identification of the N-terminal amino acid. It is also demonstrated that advantage can be taken of the pH memory effect to use the preferential ¹³C-methylation of amino groups to verify whether a protein has a free or blocked amino terminus.     

Honeydew amino acids in relation to sugars and their role in the establishment of ant-attendance hierarchy in eight species of aphids feeding on tansy (Tanacetum vulgare)

Abstract. The ratio of the concentration of honeydew total amino acids to total sugars in the honeydew of eight species of aphids, all feeding on tansy, Tanacetum vulgare (L.), was determined and correlated with honeydew production and ant‐attendance. The honeydew of the five ant‐attended aphid species [Metopeurum fuscoviride (Stroyan), Trama troglodytes (v. Hayd), Aphis vandergooti (Börner), Brachycardus cardui (L.), Aphis fabae (Scopoli)] was rich in total amino acids, ranging from 12.9 to 20.8 nmol µL^?1 compared with the unattended aphid Macrosiphoniella tanacetaria (Kalt.) with only 3 nmol µL^?1. Asparagine, glutamine, glutamic acid and serine (all nonessential amino acids) were the predominant amino acids in the honeydew of all species. The total concentration of amino acids in the phloem sap of tansy was much higher (78.7 nmol µL^?1) then in the honeydew samples, and the predominant amino acids were glutamate (34.3%) and threonine (17.7%). A somewhat unexpected result was the finding that those aphid species with the highest total amino acid concentration in the honeydew always had the highest concentration of sugars. The lowest amino acid–sugar combined value was 104–28.8 nmol µL^?1 in the non ant‐attended species M. tanacetaria, and the highest value was an average of 270–89.9 nmol µL^?1 for the three most intensely attended aphid species M. fuscoviride, A. vandergooti and T. troglodytes. There is no evidence that any single amino acid or group of amino acids in the honeydew acted as an attractant for ant‐attendance in these eight aphid species. The richness of the honeydew (rate of secretion × total concentration of sugars), along with the presence of the attractant sugar melezitose, comprised the critical factors determining the extent of ant‐attendance of the aphids feeding on T. vulgare. The high total amino acid concentration in sugar‐rich honeydews can be explained by the high flow‐through of nutrients in aphids that are particularly well attended by ants.     

Design and synthesis of cell selective α/β-diastereomeric peptidomimetic with potent in vivo antibacterial activity against methicillin resistant S. Aureus

Design of therapeutically viable antimicrobial peptides with cell selectivity against microorganisms is an important step towards the development of new antimicrobial agents. Here, we report four de novo designed, short amphipathic sequences based on a α-helical template comprising of Lys, Trp and Leu or their corresponding D-and/or β-amino acids. Sequence A-12 was protease susceptible whereas its α/β-diastereomeric analogue UNA-12 was resistant to trypsin and proteinase K up to 24 h. A-12 and UNA-12 exhibited broad-spectrum antibacterial activity (MIC: 2–32 µg/mL) against pathogens including methicillin resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE). Interestingly, A-12 was found to be most toxic (>50% haemolytic at 250 µg/mL) whereas UNA-12 was found to be non cytotoxic among the all analogues against hRBCs and human keratinocytes. Interaction studies with artificial membranes by tryptophan fluorescence and acrylamide quenching assay demonstrated A-12 interacted equally in bacterial as well as mammalian mimic membrane whereas UNA-12 was found to be more selective towards bacterial mimic membrane. Further microscopic tool has revealed membrane damaging ability of A-12 and UNA-12 with bactericidal mode of action against MRSA. Encouragingly, peptidomimetics analogue UNA-12 showed remarkable safety and efficacy against MRSA in in-vivo neutropenic mice thigh infection model. In summary, simultaneous replacement of the natural amino acids with D-/β-congeners is a promising strategy for designing of potent, cell selective and protease stable peptide based antibiotics.     

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids.     

Design and synthesis of new fused carbazole-imidazole derivatives as anti-diabetic agents: In vitro α-glucosidase inhibition,kinetic, and in silico studies

Twenty three fused carbazole–imidazoles 6a–w were designed, synthesized, and screened as new α-glucosidase inhibitors. All the synthesized fused carbazole-imidazoles 6a-w were found to be more active than acarbose (IC₅₀?=?750.0?±?1.5?µM) against yeast α-glucosidase with IC₅₀ values in the range of 74.0?±?0.7–298.3?±?0.9?µM. Kinetic study of the most potent compound 6v demonstrated that this compound is a competitive inhibitor for α-glucosidase (K_i value?=?75?µM). Furthermore, the in silico studies of the most potent compounds 6v and 6o confirmed that these compounds interacted with the key residues in the active site of α-glucosidase.     

Determination of phenformin hydrochloride using molecular imprinting technology coupled with flow-injection chemiluminescence

A novel method was developed using molecular imprinting technology (MIT) coupled with flow‐injection chemiluminescence (FI‐CL) for highly sensitive detection of phenformin hydrochloride (PH). The phenformin imprinted polymer was synthesized with methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate (EGDMA) as a cross‐linker. Newly synthesized molecularly imprinted polymer (MIP) particles were packed into a column as a selective recognition element for determination of PH. A CL method for the determination of PH was developed based on the CL reaction of PH with N‐bromosuccinimide sensitized by eosin Y in basic media. The optimization of detection conditions was investigated. The CL intensity responded linearly to the concentration of PH in the range 0.09–2.0 µg/mL, with a correlation coefficient of 0.9920. The detection limit was 0.031 µg/mL. The relative standard deviation for the determination of 1.0 µg/mL PH solution was 1.0% (n = 11). The method was applied to the determination of PH in urine samples, with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Laboratory and field evaluations of chemical and plant‐derived potential repellents against Culicoides biting midges in northern Spain

The efficacy of 23 compounds in repelling Culicoides biting midges (Diptera: Ceratopogonidae), particularly Culicoides obsoletus (Meigen) females, was determined by means of a Y‐tube olfactometer. The 10 most effective compounds were further evaluated in landing bioassays. The six most promising compounds (including chemical and plant‐derived repellents) were evaluated at 10% and 25% concentrations in field assays using Centers for Disease Control (CDC) light traps. At least three compounds showed promising results against Culicoides biting midges with the methodologies used. Whereas olfactometer assays indicated DEET at 1 µg/µL to be the most effective repellent, filter paper landing bioassays showed plant‐derived oils to be better. Light traps fitted with polyester mesh impregnated with a mixture of octanoic, decanoic and nonanoic fatty acids at 10% and 25% concentrations collected 2.2 and 3.6 times fewer midges than control traps and were as effective as DEET, which is presently considered the reference standard insect repellent. The best plant‐derived product was lemon eucalyptus oil. Although these have been reported as safe potential repellents, the present results indicate DEET and the mixture of organic fatty acids to be superior and longer lasting.