Stereoselective Synthesis of (3R)‐3‐Alkyl‐4,1‐Benzoxazepine‐2,5‐Diones

Novel 3‐alkyl‐4,1‐benzoxazepine‐2,5‐diones were synthesized in good ee exploiting the chiral pool methodology, an economical way of asymmetric synthesis. Various anthranilic acids are coupled with different α‐haloacids to afford N‐acylated anthranilic acid intermediates which undergo cyclization to (3R)‐3‐alkyl‐4,1‐benzoxazepines‐2,5‐diones. Chirality 25:865–870, 2013. © 2013 Wiley Periodicals, Inc.     

Photoluminescence imaging of europium (III)‐doped γ‐Al2O3 nanofiber structures

Aluminium oxide (Al₂O₃) has widely been used for catalysts, insulators, and composite materials for diverse applications. Herein, we demonstrated if γ‐Al₂O₃ was useful as a luminescence support material for europium (Eu) (III) activator ion. The hydrothermal method and post‐thermal treatment at 800°C were employed to synthesize Eu(III)‐doped γ‐Al₂O₃ nanofibre structures. Luminescence characteristics of Eu(III) ions in Al₂O₃ matrix were fully understood by taking 2D and 3D‐photoluminescence imaging profiles. Various sharp emissions between 580 to 720 nm were assigned to the ⁵D₀→⁷F_J (J = 0, 1, 2, 3, 4) transitions of Eu(III) activators. On the basis of X‐ray diffraction crystallography, Auger elemental mapping and the asymmetry ratio, Eu(III) ions were found to be well doped into the γ‐Al₂O₃ matrix at a low (1 mol%) doping level. A broad emission at 460 nm was substantially increased upon higher (2 mol%) Eu(III) doping due to defect creation. The first 3D photoluminescence imaging profiles highlight detailed understanding of emission characteristics of Eu(III) ions in Al oxide‐based phosphor materials and their potential applications.     

Posttranslational oxidative modification of (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA) leads to improved degradation of 2,4‐dichlorophenoxyacetate (2,4‐D)

Microbial activities and the versatility gained through adaptation to xenobiotic compounds are the main biological forces to counteract environmental pollution. The current results present a new adaptive mechanism that is mediated through posttranslational modifications. Strains of Delftia acidovorans incapable of growing autochthonously on 2,4‐dichlorophenoxyacetate (2,4‐D) were cultivated in a chemostat on 2,4‐D in the presence of (R)‐2‐(2,4‐dichlorophenoxy)propionate. Long‐term cultivation led to enhanced 2,4‐D degradation, as demonstrated by improved values of the Michaelis–Menten constant K_m for 2,4‐D and the catalytic efficiency k_cat/K_m of the initial degradative key enzyme (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA). Analyses of the rdpA gene did not reveal any mutations, indicating a nongenetic mechanism of adaptation. 2‐DE of enzyme preparations, however, showed a series of RdpA forms varying in their pI. During adaptation increased numbers of RdpA variants were observed. Subsequent immunoassays of the RdpA variants showed a specific reaction with 2,4‐dinitrophenylhydrazine (DNPH), characteristic of carbonylation modifications. Together these results indicate that posttranslational carbonylation modified the substrate specificity of RdpA. A model was implemented explaining the segregation of clones with improved degradative activity within the chemostat. The process described is capable of quickly responding to environmental conditions by reversibly adapting the degradative potential to various phenoxyalkanoate herbicides.     

Computationally designed β‐turn foldamers of γ‐peptides based on 2‐(aminomethyl)cyclohexanecarboxylic acid

The γ‐peptide β‐turn structures have been designed computationally by the combination of chirospecific γ 2 , 3 ‐residues of 2‐(aminomethyl)cyclohexanecarboxylic acid (γAmc₆) with a cyclohexyl constraint on the C^α?C^β bond using density functional methods in water. The chirospecific γAmc₆ dipeptide with the (2S,3S)‐(2R,3R) configurations forms a stable turn structure in water, resembling a type II′ turn of α‐peptides, which can be used as a β‐turn motif in β‐hairpins of Ala‐based α‐peptides. The γAmc₆ dipeptide with homochiral (2S,3S)‐(2S,3S) configurations but different cyclohexyl puckerings shows the capability to be incorporated into one of two β‐turn motifs of gramicidin S. The overall structure of this gramicidin S analogue is quite similar to the native gramicidin S with the same patterns and geometries of hydrogen bonds. Our calculated results and the recently observed results may imply the wider applicability of chirospecific γ‐peptides with a cyclohexyl constraint on the backbone to form various peptide foldamers. © 2012 Wiley Periodicals, Inc. Biopolymers 97:1018–1025, 2012.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Cytotoxic (9βH)‐Pimarane and (9βH)‐17‐Norpimarane Diterpenes from the Tuber of Icacina trichantha

Three (9βH)‐pimaranes, 1, 2 , and 3 , and two (9βH)‐17‐norpimaranes, 4 and 5 , belonging to a rare compound class in nature, were obtained from the tubers of Icacina trichantha for the first time. Compound 1 is a new natural product, and 2 – 5 have been previously reported. The structures were elucidated based on NMR and MS data, and optical rotation values. The absolute configurations of (9βH)‐pimaranes were unambiguously established based on X‐ray crystallographic analysis. Full NMR signal assignments for the known compounds 2, 4 , and 5 , which were not available in previous publications, are also reported. All five isolates displayed cytotoxic activities on MDA‐MB‐435 cells (IC₅₀ 0.66–6.44 μM ), while 2, 3 , and 4 also exhibited cytotoxicities on HT‐29 cells (IC₅₀ 3.00–4.94 μM ).     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Syntheses and β‐adrenergic binding affinities of (R)‐ and (S)‐fluoronaphthyloxypropanolamines

The β‐adrenergic receptors mediate several physiological processes including heart rate (β₁), bronchodilation (β₂), and lipolysis (β₃). Therefore, selectivity is important for a possible therapeutic agent acting via these receptors. Aryloxypropanolamines are β‐receptor agonists or antagonists, depending on the aryl group and its substituents. We therefore hypothesized that fluorine substitution on the aromatic ring in this class could lead to significant biological effects because of the unique chemical characteristics of fluorine. Because the target compound has a chiral center, we set out to synthesize the two enantiomers so that effects of stereochemistry on biological activity could be evaluated. Syntheses of the enantiomers were performed starting with commercially available fluoronaphthalene and subsequent use of the chiral synthon (2R)‐ or (2S)‐glycidyl 3‐nitrobenzenesulfonate, depending on the desired enantiomer. High‐pressure liquid chromatography (HPLC) methods were used to characterize %ee. Each enantiomer was synthesized. They exhibited nanomolar binding activities on β‐adrenergic receptors. The (S)‐enantiomer was found to be up to 310 times more potent than the (R). It was also found to be about five‐fold more selective for β₂‐ than for β₁‐receptors. The current report demonstrates the importance of stereochemistry for the fluoroaromatic β‐receptor ligands. Chirality 11:144–148, 1999. © 1999 Wiley‐Liss, Inc.     

High yield and cost‐effective production of poly(γ‐glutamic acid) with Bacillus subtilis

Poly(γ‐glutamic acid) (γ‐PGA) is a promising biopolymer with many potential industrial and pharmaceutical applications. To reduce the production costs, the effects of yeast extract and L ‐glutamate in the substrate for γ‐PGA production were investigated systematically at shake flask scale. The results showed that lower concentrations of yeast extract (40 g/L) and L ‐glutamate (30 g/L) were beneficial for the cost‐effective production of γ‐PGA in the formulated medium. By maintaining the glucose concentration in the range of 3–10 g/L via a fed‐batch strategy in a 10‐L fermentor, the production of γ‐PGA was greatly improved with the highest γ‐PGA concentration of 101.1 g/L, a productivity of 2.19 g/L·h and a yield of 0.57 g/g total substrate, which is about 1.4‐ to 3.2‐fold higher than those in the batch fermentation. Finally, this high‐density fermentation process was successfully scaled up in a 100‐L fermentor. The present work provides a powerful approach to produce this biopolymer as a bulk chemical in large scale.     

Isoform‐specific cleavage of 14‐3‐3 proteins in apoptotic JURL‐MK1 cells

The proteins of 14‐3‐3 family are substantially involved in the regulation of many biological processes including the apoptosis. We studied the changes in the expression of five 14‐3‐3 isoforms (β, γ, ε, τ, and ζ) during the apoptosis of JURL‐MK1 and K562 cells. The expression level of all these proteins markedly decreased in relation with the apoptosis progression and all isoforms underwent truncation, which probably corresponds to the removal of several C‐terminal amino acids. The observed 14‐3‐3 modifications were partially blocked by caspase‐3 inhibition. In addition to caspases, a non‐caspase protease is likely to contribute to 14‐3‐3's cleavage in an isoform‐specific manner. While 14‐3‐3 γ seems to be cleaved mainly by caspase‐3, the alternative mechanism is essentially involved in the case of 14‐3‐3 τ, and a combined effect was observed for the isoforms ε, β, and ζ. We suggest that the processing of 14‐3‐3 proteins could form an integral part of the programmed cell death or at least of some apoptotic pathways. J. Cell. Biochem. 106: 673–681, 2009. © 2009 Wiley‐Liss, Inc.     

Physiological Importance of Poly‐(R)‐3‐hydroxybutyrates

Poly‐(R)‐3‐hydroxybutyrates (PHB), linear polymers of (R)‐3‐hydroxybutyrate, are components of all biological cells in which short polymers (<200 monomer residues) are covalently attached to certain proteins and/or noncovalently associated with polyphosphates – inorganic polyphosphate (polyP), RNA, and DNA. The low concentrations, lack of unusual atoms or functional groups, and flexible backbones of this complexed PHB, referred to as cPHB, make them invisible to many analytical procedures; whereas other physical properties – water‐insolubility, high intrinsic viscosity, temperature sensitivity, multiple bonding interactions with other molecules – make them requisite participants in vital physiological processes as well as contributors to the development of certain diseases.     

Biotransformation of (−)‐(R)‐α‐Phellandrene: Antimicrobial Activity of Its Major Metabolite

Terpene derivatives converted by microbial biotransformation constitute an important resource for natural pharmaceutical, fragrance, and aroma substances. In the present study, the monoterpene α‐phellandrene was biotransformed by 16 different strains of microorganisms (bacteria, fungi, and yeasts). The transformation metabolites were initially screened by TLC and GC/MS, and then further characterized by NMR spectroscopic techniques. Among the six metabolites characterized, 6‐hydroxypiperitone, α‐phellandrene epoxide, cis‐p‐menth‐2‐en‐1‐ol, and carvotanacetone, which originated from (?)‐(R)‐α‐phellandrene, are reported for the first time in this study. Additionally, the substrate and the metabolite 5‐p‐menthene‐1,2‐diol were subjected to in vitro antibacterial and anticandidal tests. The metabolite showed moderate‐to‐good inhibitory activities (MICs=0.125 to >4 mg/ml) against various bacteria and especially against Candida species in comparison with its substrate (?)‐(R)‐α‐phellandrene and standard antimicrobial agents.     

Binding Pockets and Permeation Channels for Dioxygen through Cofactorless 3‐Hydroxy‐2‐methylquinolin‐4‐one 2,4‐Dioxygenase in Association with Its Natural Substrate, 3‐Hydroxy‐2‐methylquinolin‐4(1H)‐one. A Perspective from Molecular Dynamics Simulations

This work describes an investigation of pathways and binging pockets (BPs) for dioxygen (O₂) through the cofactorless oxygenase 3‐hydroxy‐2‐methylquinolin‐4‐one 2,4‐dioxygenase in complex with its natural substrate, 3‐hydroxy‐2‐methylquinolin‐4(1H)‐one, in aqueous solution. The investigation tool was random‐acceleration molecular dynamics (RAMD), whereby a tiny, randomly oriented external force is applied to O₂ in order to accelerate its movements. In doing that, care was taken that the external force only continues, if O₂ moves along a direction for a given period of time, otherwise the force changed direction randomly. Gates for expulsion of O₂ from the protein, which can also be taken as gates for O₂ uptake, were found throughout almost the whole external surface of the protein, alongside a variety of BPs for O₂. The most exploited gates and BPs were not found to correspond to the single gate and BP proposed previously from the examination of the static model from X‐ray diffraction analysis of this system. Therefore, experimental investigations of this system that go beyond the static model are urgently needed.