Effects of fluidity on the ensemble structure of a membrane embedded α‐helical peptide

Melittin, the main hemolytic component of honeybee venom, is unfolded in an aqueous environment and folds into an α‐helical conformation in a lipid environment. Membrane fluidity is known to affect the activity and structure of melittin. By combining two structurally sensitive optical methods, circular dichroism (CD) and deep‐ultraviolet resonance Raman spectroscopy (dUVRR), we have identified distinct structural fluctuations in melittin correlated with increased and decreased 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine bilayer fluidities. CD spectra have reduced intensity at temperatures above 22°C and high concentrations of the cholesterol analog 5α‐cholestan‐3β‐ol indicating distortions in the α‐helical structure under these conditions. No increase in the amide S is observed in the temperature‐dependent dUVRR spectra, suggesting an increase in 3₁₀‐helical structure with increasing temperatures above 22°C. However, incorporation of 25 mol% 5α‐cholestan‐3β‐ol resulted in a small increase in the amide S intensity indicating partial unfolding of melittin. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 895–902, 2014.     

Lipid‐dependent pore formation by antimicrobial peptides arenicin‐2 and melittin demonstrated by their proton transfer activity

This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, β‐structural arenicin‐2 and α‐helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light‐induced electrochemical proton gradient ?ΔpH. Addition of several nanomoles of the peptides lowers ?ΔpH that is proximately indicative of the pore formation. The quantitative analysis of sigmoidal dependences of ?pH on the peptides concentration was carried out using liposomes prepared from PC, PC/PE, PC/PE/PI and PC/PG. Substitution of PC‐containing liposomes with PE‐containing ones, having negative spontaneous curvature, reduced the PTA of α‐helical melittin and increased that of β‐structural arenicin‐2. This result indicates an essential difference in the pore formation by these peptides. Further increase of PTA in response to arenicin‐2 (in contrast to melittin) was observed in the liposomes prepared from PC/PE/PI. The data analysis leads to the conclusion that PTA is influenced by (i) efficiency of the pore assemblage, which depends on the structure of pore‐forming peptides, and the spontaneous curvature of lipids and (ii) the presence of mobile protons in the polar head groups of phospholipids. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

NMR studies on thermal stability of α‐helix conformation of melittin in pure ethanol and ethanol–water mixture solvents

Thermal stability of the α‐helix conformation of melittin in pure ethanol and ethanol–water mixture solvents has been investigated by using NMR spectroscopy. With increase in water concentration of the mixture solvents (from 0 wt% to ~71.5 wt%) as well as temperature (from room temperature to 60 °C), the intramolecular hydrogen bonds formed in melittin are destabilized and the α‐helix is partially uncoiled. Further, the hydrogen bonds are found to be more thermally stable in pure ethanol than in pure methanol, suggesting that their stability is enhanced with increase in the size of the alkyl groups of alcohol molecules. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and Olfactory Characterization of Silicon‐Containing Derivatives of the Acyclic Lily‐of‐the‐Valley Odorant 5,7,7‐Trimethyl‐4‐methylideneoctanal

5‐Methyl‐4‐methylidene‐6‐(trimethylsilyl)hexanal ( 1b ), a sila analog of the acyclic lily‐of‐the‐valley odorant 5,7,7‐trimethyl‐4‐methylideneoctanal ( 1a ), and the Si‐containing derivatives 2 – 6 were prepared in multistep syntheses, starting from Cl₃SiH and Cl₂SiMe₂, respectively. Compounds 1b, 2 – 6 , and their new precursors were characterized by elemental analyses (C, H, N) and NMR spectroscopic studies (¹H, ¹³C, ¹⁵N, and ²⁹Si). To gain more information about the structure? odor correlation in the family of lily‐of‐the‐valley or ‘muguet’ odorants, C/Si analogs 1a / 1b and derivatives 2 – 6 were evaluated for their olfactory properties.     

HS‐SPME‐GC‐FID method for detection and quantification of Bacillus cereus ATCC 10702 mediated 2‐acetyl‐1‐pyrroline

A rapid micro‐scale solid‐phase micro‐extraction (SPME) procedure coupled with gas‐chromatography with flame ionized detector (GC‐FID) was used to extract parts per billion levels of a principle basmati aroma compound “2‐acetyl‐1‐pyrroline” (2‐AP) from bacterial samples. In present investigation, optimization parameters of bacterial incubation period, sample weight, pre‐incubation time, adsorption time, and temperature, precursors and their concentrations has been studied. In the optimized conditions, detection of 2‐AP produced by Bacillus cereus ATCC10702 using only 0.5 g of sample volume was 85 μg/kg. Along with 2‐AP, 15 other compounds produced by B. cereus were also reported out of which 14 were reported for the first time consisting mainly of (E)?2‐hexenal, pentadecanal, 4‐hydroxy‐2‐butanone, n‐hexanal, 2–6‐nonadienal, 3‐methoxy‐2(5H) furanone and 2‐acetyl‐1‐pyridine and octanal. High recovery of 2‐AP (87 %) from very less amount of B. cereus samples was observed. The method is reproducible fast and can be used for detection of 2‐AP production by B. cereus. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1356–1363, 2014     

Asymmetrically simultaneous synthesis of L‐homophenylalanine and N6‐protected‐2‐oxo‐6‐amino‐hexanoic acid by engineered Escherichia coli aspartate aminotransferase

L ‐Homophenylalanine (L ‐HPA) and N⁶‐protected‐2‐oxo‐6‐amino‐hexanoic acid (N⁶‐protected‐OAHA) can be used as building blocks for the manufacture of angiotensin‐converting enzyme inhibitors. To synthesize L ‐HPA and N⁶‐protected‐OAHA simultaneously from 2‐oxo‐4‐phenylbutanoic acid (OPBA) and N⁶‐protected‐L ‐lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site‐directed mutagenesis and their catalytic activities were investigated. Three kinds of N⁶‐protected‐L ‐lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70±0.01 U/mg protein and 0.67±0.02 U/mg protein to 2‐amino‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐lysine) and 2‐amino‐6‐(2,2,2‐trifluoro‐acetylamino)‐hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC‐lysine to L ‐HPA and 2‐oxo‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L ‐HPA and BOC‐OAHA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Application of NMR spectroscopy for assignment of the absolute configuration of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one

In order to assign the absolute configurations of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one ( 2a , 2b ), their esters ( 5a , 5b , 5c , 5d ) with (R)‐ or (S)‐2‐methoxyphenylacetic acid ( 4a , 4b ) have been synthesized. The absolute configurations of these compounds have been determined on the basis of NOESY correlations between the protons of the tert‐butyl group and the cyclopentane fragment of the molecules. The crucial part of this analysis was assignment of the absolute configuration at C‐5. Additionally, by calculation of the chemical shift anisotropy, δ^RS, for the relevant protons, it was also possible to confirm the absolute configurations at the C‐2 centres of compounds 2a , 2b and 5a , 5b , 5c , 5d . Chirality, 25:422–426, 2013.© 2013 Wiley Periodicals, Inc.     

Solvent selection and optimization of α‐chymotrypsin‐catalyzed synthesis of N‐Ac‐Phe‐Tyr‐NH2 using mixture design and response surface methodology

A peptide, N‐Ac‐Phe‐Tyr‐NH₂, with angiotensin I‐converting enzyme (ACE) inhibitor activity was synthesized by an α‐chymotrypsin‐catalyzed condensation reaction of N‐acetyl phenylalanine ethyl ester (N‐Ac‐Phe‐OEt) and tyrosinamide (Tyr‐NH₂). Three kinds of solvents: a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic‐aqueous solvent (Tris‐HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N‐Ac‐Phe‐Tyr‐NH₂ could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N‐Ac‐Phe‐Tyr‐NH₂, so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

1‐Aminobenzocyclobutene‐1‐phosphonic Acid and Related Compounds as Inhibitors of Phenylalanine Ammonia‐Lyase

Five new geminal aminocycloalkanephosphonic acids ( 4 – 8 ) containing both an aromatic ring and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat phenylalanine ammonia‐lyase (PAL). Within the set of compounds which are related to 2‐aminoindane‐2‐phosphonic acid (AIP, 3 ), a known powerful inhibitor of PAL, racemic 1‐aminobenzocyclobutene‐1‐phosphonic acid ( 4 ), was six times weaker than AIP as an in vitro inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of phenylalanine‐derived anthocyanin synthesis in buckwheat.     

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N‐terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP‐like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β‐ d ‐galactosidase‐labeled Gly‐γAbu‐SDKP as a marker antigen, an anti‐rabbit IgG‐coated immunoplate as a bound/free separator and 4‐methylumbelliferyl‐β‐ d ‐galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP‐IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter‐assay and 13.3 and 7.8% for intra‐assay comparisons. Plasma AcSDKP‐IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The special optical properties and application of the heterocyclic compound diethyl 6‐anilino‐5H‐2,3‐dithia‐5,7‐diazacyclopenta(cd)indene‐1,4‐dicarboxylate

The heterocyclic compound diethyl 6‐anilino‐5H‐2,3‐dithia‐5,7‐diazacyclopenta(cd)indene‐1,4‐dicarboxylate (D1) was found to form highly emissive aggregates in polar solvents, and the aggregate emission can be tuned by the simple addition of water to a dimethylsulfoxide solution. A theoretical study based on Density functional theory (DFT) calculations, shows that intermolecular interactions of D1 with solvent may be potential factors in the fluorescence change. In addition, the phenyl ring in D1 plays an important role because of its response to solvent. In the non‐aggregated state, deprotonation of the N–H of D1 can proceed easily on the addition of base, and the deprotonated compound might interact with Ag⁺, resulting in a significant change in color and fluorescence quenching, which make it a potential chemosensor for the selective detection of trace amounts of Ag⁺. Copyright © 2013 John Wiley & Sons, Ltd.     

2H‐Pyran‐2‐one and 2H‐Furan‐2‐one Derivatives from the Plant Endophytic Fungus Pestalotiopsis fici

Two new α‐pyrones (=2H‐pyran‐2‐ones), ficipyrones A and B ( 1 and 2 , resp.), and two new α‐furanones (=2H‐furan‐2‐ones), ficifuranones A and B ( 3 and 4 , resp.), together with three known metabolites, antibiotic F 0368 ( 5 ), hydroxyseiridin ( 6 ), and hydroxyisoseiridin ( 7 ), were isolated from solid cultures of the plant endophytic fungus Pestalotiopsis fici. Their structures were elucidated primarily by NMR spectroscopy, and the absolute configuration of 1 was deduced from the circular‐dichroism (CD) data. Compound 1 showed antifungal activity against the plant pathogen Gibberella zeae (CGMCC 3.2873) with an IC₅₀ value of 15.9 μM .     

Cyclic‐di‐GMP and cyclic‐di‐AMP activate the NLRP3 inflammasome

The cyclic dinucleotides 3'‐5'diadenylate (c‐diAMP) and 3'‐5' diguanylate (c‐diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN‐Is) through the c‐diGMP‐binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL‐1β through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c‐diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen‐associated molecular patterns associated with intracellular infections.     

Resolution of 1‐n‐Butyl‐3‐Methyl‐3‐Phospholene 1‐Oxide With TADDOL Derivatives and Calcium Salts of O,O'‐Dibenzoyl‐(2R,3R)‐ or O,O'‐di‐p‐Toluoyl‐(2R,3R)‐tartaric Acid

The resolution methods applying (?)‐(4R,5R)‐4,5‐bis(diphenylhydroxymethyl)‐2,2‐dimethyldioxolane (“TADDOL”), (?)‐(2R,3R)‐α,α,α',α'‐tetraphenyl‐1,4‐dioxaspiro[4.5]decan‐2,3‐dimethanol (“spiro‐TADDOL”), as well as the acidic and neutral Ca²⁺ salts of (?)‐O,O'‐dibenzoyl‐ and (?)‐O,O'‐di‐p‐toluoyl‐(2R,3R)‐tartaric acid were extended for the preparation of 1‐n‐butyl‐3‐methyl‐3‐phospholene 1‐oxide in optically active form. In one case, the intermediate diastereomeric complex could be identified by single‐crystal X‐ray analysis. The absolute P‐configuration of the enantiomers of the phospholene oxide was also determined by comparing the experimentally obtained and calculated CD spectra. Chirality 26:174–182, 2014. © 2014 Wiley Periodicals, Inc.     

Enantiopure 4‐oxazolin‐2‐ones and 4‐methylene‐2‐oxazolidinones as chiral building blocks in a divergent asymmetric synthesis of heterocycles

Enantiopure 3‐((R)‐ and 3‐((S)‐1‐phenylethyl)‐4‐oxazoline‐2‐ones were evaluated as chiral building blocks for the divergent construction of heterocycles with stereogenic quaternary centers. The N‐(R)‐ or N‐(S)‐1‐phenylethyl group of these compounds proved to be an efficient chiral auxiliary for the asymmetric induction of the 4‐ and 5‐positions of the 4‐oxazolin‐2‐one ring through thermal and MW‐promoted nucleophilic conjugated addition to Michael acceptors and alkyl halides. The resulting adducts were transformed via a cascade process into fused six‐membered carbo‐ and heterocycles. The structure of the reaction products depended on the electrophiles and reaction conditions used. Alternative isomeric 4‐methylene‐2‐oxazolidinones served as chiral precursors for a versatile and divergent approach to highly substituted cyclic carbamates. DFT quantum calculations showed that the formation of bicyclic pyranyl compounds was generated by a diastereoselective concerted hetero‐Diels‐Alder cycloaddition.