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LTX-401 is an oncolytic amino acid derivative with potential immunogenic properties. Here, we demonstrate that LTX-401 selectively destroys the structure of the Golgi apparatus, as determined by means of ultrastructural analyses and fluorescence microscopic observation of cells expressing Golgi-targeted GFP reporters. Subcellular fractionation followed by mass spectrometric detection revealed that LTX-401 selectively enriched in the Golgi rather than in mitochondria or in the cytosol. The Golgi-dissociating agent Brefeldin A (BFA) reduced cell killing by LTX-401 as it partially inhibited LTX-401-induced mitochondrial release of cytochrome c and the activation of BAX. The cytotoxic effect of LTX-401 was attenuated by the double knockout of BAX and BAK, as well as the mitophagy-enforced depletion of mitochondria, yet was refractory to caspase inhibition. LTX-401 induced all major hallmarks of immunogenic cell death detectable with biosensor cell lines including calreticulin exposure, ATP release, HMGB1 exodus and a type-1 interferon response. Moreover, LTX-401-treated tumors manifested a strong lymphoid infiltration. Altogether these results support the contention that LTX-401 can stimulate immunogenic cell death through a pathway in which Golgi-localized LTX-401 operates upstream of mitochondrial membrane permeabilization.

Studies describe formation of the lithium enolate of N-(4-methoxybenzyloxy)azetidin-2-one (1) and characterization of representative aldol reactions with aldehydes and ketones. Diastereoselectivity features the production of anti-aldol adducts from α,β-unsaturated ketones and α-branched aliphatic aldehydes. The stereoselectivity is rationalized via closed, six-membered transition-state arrangements leading to the formation of Felkin-Anh and anti-Felkin products. Examples illustrate the direct incorporation of monocyclic β-lactams into a variety of molecular architectures. The utility of 1 as an enolate synthon of homoglycine (β-alanine) is illustrated by the efficient synthesis of novel β-amino acid derivatives, including complex 4-hydroxy-2-pyridinones.

The oxidation of the aniline dimer, p-aminodiphenylamine (PADPA), with Trametes versicolor laccase and O2 in an aqueous solution of pH 3.5 is controlled by negatively charged AOT (sodium bis(2-ethylhexyl) sulfosuccinate) vesicles. With vesicles, a product resembling polyaniline in its emeraldine salt form (PANI-ES) is obtained, in contrast to the reaction without vesicles where no such product is formed. To understand this observation, the product distribution and structures from the reaction with and without vesicles were determined by using partially selectively deuterated PADPA as a starting material and analyzing the products with HPLC-MS. We found that in the presence of vesicles the main product is obtained in about 50% yield, which is the N-C-para-coupled PADPA dimer that has spectroscopic properties of PANI-ES, as determined by time-dependent density functional theory (TD-DFT) calculations. A secondary reaction route leads to longer PADPA oligomers that must contain a phenazine core. Without vesicles, PADPA and its products undergo partial hydrolysis, but in the presence of vesicles, hydrolysis does not occur. Because molecular dynamics (MD) simulations show that the main intermediate oxidation product is embedded within the vesicle membrane, where the water content is very low, we propose that the microenvironment of the vesicle membrane protects the oxidation products from unwanted hydrolysis.

The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis, where it has so far been underexploited. d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis During our studies on alternative inhibitors of the d-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro-d-alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d-alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development., (Copyright © 2016 Prosser et al.)

By exploiting orthogonal hydrogen bonding involving supramolecular synthons and hydrophobic/hydrophilic interactions, a new series of simple organic salt based hydrogelators derived from pyrene butyric acid and its β-alanine amide derivative, and various primary amines has been achieved. The hydrogels were characterised by microscopy, table-top rheology and dynamic rheology. FTIR, variable-temperature (1) H NMR and emission spectroscopy established the role of various supramolecular interactions such as hydrogen bonding and π-π stacking in hydrogelation. Single-crystal X-ray diffraction (SXRD) studies supported the conclusion that orthogonal hydrogen bonding involving amide-amide and primary ammonium monocarboxylate (PAM) synthons indeed played a crucial role in hydrogelation. The hydrogels were found to be stimuli-responsive and were capable of sensing ammonia and adsorbing water-soluble dye (methylene blue). All the hydrogelators were biocompatible (MTT assay in RAW 264.7 cells), indicating their suitability for use in drug delivery., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel pseudo cyclic penta-β-peptide composed of a β-naphthylalanine, two β-alanines, and a sequence of ethylenediamine-succinic acid (CP5ES) is synthesized and investigated on peptide nanotube (PNT) formation. When the PNT is formed with the maximum number of intermolecular hydrogen bonds between the cyclic peptides, the sequence enables the alignment of the side chains, naphthyl groups, on one side of the PNT. Microscopic and spectroscopic observations of CP5ES crystals reveal that CP5ES forms rod- or needle-shaped molecular assemblies showing exciton coupling of the Cotton effect and predominant monomer emission, which are different from a reference cyclic tri-β-peptide composed of a β-naphthylalanine and two β-alanines. Insertion of a sequence of ethylenediamine-succinic acid into β-amino acids in the cyclic skeleton is therefore suggested to be effective to make the side chains aligning on one side of the PNT. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

Osteoarthritis (OA), one of the most widespread musculoskeletal joint diseases among the aged, is characterized by the progressive loss of articular cartilage and continuous changes in subchondral bone. The exact pathogenesis of osteoarthritis is not completely clear. In this work, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) in combination with multivariate statistical analysis was applied to analyze the metabolic profiling of subchondral bone from 42 primary osteoarthritis patients. This paper described a modified two-step method for extracting the metabolites of subchondral bone from primary osteoarthritis patients. Finally, 68 metabolites were identified to be significantly changed in the sclerotic subchondral bone compared with the non-sclerotic subchondral bone. Taurine and hypotaurine metabolism and beta-alanine metabolism were probably relevant to the sclerosis of subchondral bone. Taurine, L-carnitine, and glycerophospholipids played a vital regulation role in the pathological process of sclerotic subchondral bone. In the sclerotic process, beta-alanine and L-carnitine might be related to the increase of energy consumption. In addition, our findings suggested that the intra-cellular environment of sclerotic subchondral bone might be more acidotic and hypoxic compared with the non-sclerotic subchondral bone. In conclusion, this study provided a new insight into the pathogenesis of subchondral bone sclerosis. Our results indicated that metabolomics could serve as a promising approach for elucidating the pathogenesis of subchondral bone sclerosis in primary osteoarthritis. Graphical Abstract Metabolic analysis of osteoarthritis subchondral bone.

Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.

2,3-Diaminopropionic acid (DapA), a medicinal amino acid, is used for the first time to prepare a DapA cross-linked graphene sponge (DCGS) for hemostasis treatment. In a comparison with the reported ethanediamine (EDA) cross-linked graphene sponge (CGS), this carboxyl-functionalized DCGS can not only quickly absorb plasma, but also stimulate erythrocytes and platelets to change their normal form and structure at the interface, which largely affects a cell's metabolism and biofunction, thus further promoting blood coagulation. Whole blood clotting and rat-tail amputation tests indicated that on the basis of the additional interfacial stimulation, the hemostatic efficiency of the DCGS has been significantly improved in comparison with that of the CGS control (P < 0.05). In-depth insight revealed that the increased oxidation degree and the negative charge density play the crucial rule in the enhanced hemostatic performance. The chiral effect contributes mainly to the selective adhesion of erythrocytes and platelets rather than practical hemostasis. Nevertheless, this presentation demonstrated that, on the premise of keeping the fast absorbability, this is an effective method to improve the hemostatic efficiency by enhancing the cell/graphene interface interaction.

Apoptosis is a genetically encoded cell death program that involves different processes occurring on molecular and sub-cellular levels. Here we report on its new features--the increased accumulation of fluorescent carbon nanoparticles (CDots) in cells and their changed distribution within cell interior, which can witness on altered mechanisms of their translocation through the membrane. The comparative studies of living (intact) and apoptotic cells were provided with two cell lines (HeLa, Vero) using two types of fluorescent nanoparticles ("violet" and "blue" CDots). In all studied cases the images of living and apoptotic cells were different; the apoptotic cells incorporated larger number of CDots resulting in their much brighter images. These nanoparticles are distributed in cell cytoplasm, however, when the cells are fixed and treated with detergent, their nucleus is also labeled. Flow cytometry allows distinguishing the sub-populations of living and apoptotic cells in their cultures and suggests a very cheap and easy way to characterize them.

Acrylamide found in thermal-treated foods has led to an intensive and persistent research effort, since it is a neurotoxic, genotoxic and probable carcinogenic compound to humans. Plant polyphenols are the most abundant antioxidants in human diet. Several researches indicated that the polyphenols affected the acrylamide formation during heating. However, the controversial effects of the polyphenols on acrylamide formation were related to their structure, concentrations, and antioxidant capacity, as well as reaction condition. Polyphenols can inhibit acrylamide formation through trapping of carbonyl compounds and preventing against lipid oxidation, while some special polyphenols can enhance the acrylamide content by providing carbonyl groups, accelerating the conversion from 3-aminopropionamide (3-APA) to acrylamide and inhibiting acrylamide elimination. This review concludes the effects of polyphenols in the Maillard reaction and food systems conducted so far, aimed to give an overview on the role of plant polyphenols in acrylamide formation and elimination., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Kojic acid is a natural antifungal and antibacterial agent that has been extensively studied for its tyrosinase inhibitory and metal coordination properties. Tyrosinase is a metalloenzyme with two copper ions in the active site. It is widely accepted that the tyrosinase inhibitory activity of kojic acid is related to its ability to coordinate metals. Over the past five years, we have used kojic acid to synthesize new and efficient bis-kojic acid chelators of iron and aluminium. In parallel, we investigated whether the de novo designed ligands could interfere with proper tyrosinase functioning. The present study combines our experience with inhibition and coordination studies of the new ligand: Kojic-βAla-Kojic. Research aimed at the assembly of a new potent tyrosinase inhibitor was based on the well-known crystal structure of the enzyme. Two questions were whether two kojic acids could act better than one and to what extent the length and kind of linker could ameliorate metal coordination, and inhibitory activity. Our results show that Kojic-βAla-Kojic has high affinity for Fe(III), Al(III), Zn(II), and Cu(II) and strong tyrosinase inhibitory effect and it can be proposed for use in industrial and pharmaceutical applications., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Thermodynamic and transport properties of aqueous solutions are very useful in the elucidation of solute-solvent and solute-solute interactions, which help to understand the hydration and taste behavior of solutes. The densities and viscosities of L-glycine, β-alanine and L-leucine have been determined in water and in aqueous solutions of sodium propionate (NaP) and calcium propionate (CaP) at temperatures 298.15 and 308.15K. From these data, apparent molar volumes (V2,ϕ), viscosity B-coefficients and corresponding transfer parameters (ΔtrV2,ϕo and ΔtrB) have been calculated. The dB/dT values suggest that L-glycine and β-alanine act as structure-breaker, while L-leucine acts as structure-maker both in water and in aqueous solutions of NaP and CaP. The decrease in hydration number and change in taste behavior have also been observed with increasing concentration of the cosolute., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Release of lipopolysaccharide (LPS) (endotoxin) from bacteria into the bloodstream may cause serious unwanted stimulation of the host immune system. Some but not all antimicrobial peptides can neutralize LPS-stimulated proinflammatory responses. Salt resistance and serum stability of short antimicrobial peptides can be boosted by adding β-naphthylalanine to their termini. Herein, significant antiendotoxin effects were observed in vitro and in vivo with the β-naphthylalanine end-tagged variants of the short antimicrobial peptides S1 and KWWK., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Many synthetic strategies are available for preparing well-defined conjugates of peptides/proteins and polymers. Most reports on this topic involve coupling methoxy poly(ethylene glycol) to therapeutic proteins, a process referred to as PEGylation, to increase their circulation lifetime and reduce their immunogenicity. Unfortunately, the major dissuading dogma of PEGylation is that, in many cases, polymer modification leads to significant (or total) loss of activity/function. One approach that is gaining momentum to address this challenge is to release the native protein from the polymer with time in the body (releasable PEGylation). This contribution will present the state-of-the-art of this rapidly evolving field, with emphasis on the chemistry behind the release of the peptide/protein and the means for altering the rate of release in biological fluids. Linkers discussed include those based on the following: substituted maleic anhydride and succinates, disulfides, 1,6-benzyl-elimination, host-guest interactions, bicin, β-elimination, biodegradable polymers, E1cb elimination, β-alanine, photoimmolation, coordination chemistry, zymogen activation, proteolysis, and thioesters.

We present a combined experimental and theoretical study of the fragmentation of doubly positively charged β-alanine molecules in the gas phase. The dissociation of the produced dicationic molecules, induced by low-energy ion collisions, is analysed by coincidence mass spectrometric techniques; the coupling with ab initio molecular dynamics simulations allows rationalisation of the experimental observations. The present strategy gives deeper insights into the chemical mechanisms of multiply charged amino acids in the gas phase. In the case of the β-alanine dication, in addition to the expected Coulomb explosion and hydrogen migration processes, we have found evidence of hydroxyl-group migration, which leads to unusual fragmentation products, such as hydroxymethyl cation, and is necessary to explain some of the observed dominant channels.

β-Alaninium tetrafluoroborate crystallizes in the monoclinic system (space group P21/n, Z=4). The asymmetric unit contains one β-alaninium cation and one tetrafluoroborate anion, in which the fluorine atoms are disordered. All β-alaninium cations are connected with the symmetry related cations via an inversion center, thus forming dimeric centrosymmetric β-Ala(+)···β-Ala(+) cations. The O···O distance (2.698(2)Å) in the dimeric cation is relatively long compared to known distances of previously reported salts with A(+)···A(+) type dimeric cations. The infrared and Raman spectra are studied., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Taurine is one of the osmolytes that maintain the structure of proteins in cells exposed to denaturing environmental stressors. Recently, cryoelectron tomographic analysis of eukaryotic cells has revealed that their cytoplasms are crowded with proteins. Such crowding conditions would be expected to hinder the efficient folding of nascent polypeptide chains. Therefore, we examined the role of taurine on the folding of denatured and reduced lysozyme, as a model protein, under a crowding condition. The results confirmed that taurine had a better effect on protein folding than did β-alanine, which has a similar chemical structure, when the protein to be folded was present at submillimolar concentration. NMR analyses further revealed that under the crowding condition, taurine had more interactions than did β-alanine with the lysozyme molecule in both the folded and denatured states. We concluded that taurine improves the folding of the reduced lysozyme at submillimolar concentration to allow it to interact more favorably with the lysozyme molecule. Thus, the role of taurine, as an osmolyte in vivo, may be to assist in the efficient folding of proteins.

Grape composition affects wine flavour and aroma not only through varietal compounds, but also by influencing the production of volatile compounds by yeast. C9 and C12 compounds that potentially influence ethyl ester synthesis during fermentation were studied using a model grape juice medium. It was shown that the addition of free fatty acids, their methyl esters or acyl-carnitine and acyl-amino acid conjugates can increase ethyl ester production in fermentations. The stimulation of ethyl ester production above that of the control was apparent when lower concentrations of the C9 compounds were added to the model musts compared to the C12 compounds. Four amino acids, which are involved in CoA biosynthesis, were also added to model grape juice medium in the absence of pantothenate to test their ability to influence ethyl and acetate ester production. β-Alanine was the only one shown to increase the production of ethyl esters, free fatty acids and acetate esters. The addition of 1 mg∙L(-1) β-alanine was enough to stimulate production of these compounds and addition of up to 100 mg∙L(-1) β-alanine had no greater effect. The endogenous concentrations of β-alanine in fifty Cabernet Sauvignon grape samples exceeded the 1 mg∙L(-1) required for the stimulatory effect on ethyl and acetate ester production observed in this study.

A new methodology for the synthesis of side chain mono- or bis-methylated Fmoc-Dap, -Dab and -Orn amino acids was developed by probing the reactivity of commercially available Fmoc amino acids.

Coprisamides A and B (1 and 2) were isolated from a bacterium in the gut of the dung beetle Copris tripartitus. Spectroscopic analysis revealed that the planar structures of 1 and 2 are novel cyclic heptapeptides bearing unusual units, such as β-methylaspartic acid and 2,3-diaminopropanoic acid branched to valine and 2-heptatrienyl cinnamic acid. Absolute configurations were established by chemical derivatization and chiroptical spectroscopy. The coprisamides displayed significant activity for induction of quinone reductase.

In combined quantum mechanical/molecular mechanical (QM/MM) free energy calculations, it is often advantageous to have a frozen geometry for the quantum mechanical (QM) region. For such multiple-environment single-system (MESS) cases, two schemes are proposed here for estimating the polarization energy: the first scheme, termed MESS-E, involves a Roothaan step extrapolation of the self-consistent field (SCF) energy; whereas the other scheme, termed MESS-H, employs a Newton-Raphson correction using an approximate inverse electronic Hessian of the QM region (which is constructed only once). Both schemes are extremely efficient, because the expensive Fock updates and SCF iterations in standard QM/MM calculations are completely avoided at each configuration. They produce reasonably accurate QM/MM polarization energies: MESS-E can predict the polarization energy within 0.25 kcal/mol in terms of the mean signed error for two of our test cases, solvated methanol and solvated β-alanine, using the M06-2X or ωB97X-D functionals; MESS-H can reproduce the polarization energy within 0.2 kcal/mol for these two cases and for the oxyluciferin-luciferase complex, if the approximate inverse electronic Hessians are constructed with sufficient accuracy.

The efficient synthesis of calix[6]cryptothiourea 6 was achieved through a two-step sequence that involves a key [1+1] macrocyclization step. It was shown by NMR spectroscopy that this heteroditopic receptor can bind zwitterions in protic media with an outstanding selectivity for β-alanine betaine G5, which is likely due to a high complementarity between the two partners. This result constitutes a rare example of cavity complexation of a zwitterion by a calix[6]arene. In comparison with the parent urea-based receptors, 6 behaves as a much more efficient host for betaines. This strengthening of the binding properties is due to the better preorganization of the tripodal hydrogen-bonding cap as well as to the higher acidity of the thiourea groups and their poor ability to self-associate. Remarkably, host 6 is able to perform solid-liquid as well as liquid-liquid extraction of G5. Finally, 6 provides an excellent structural model for the binding site of glycine betaine G4 encountered in natural systems., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Novel FMDP amides 4-6 have been synthesized and tested against Candida strains. The anticandidal activity has been confined only to Candida albicans. Anticandidal activity of the tested amides has correlated with their inhibitory activity of glucosamine-6-phosphate synthase in cell free extract from C. albicans., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Total synthesis of biologically active cyclodepsipeptide destruxin E using solid- and solution-phase synthesis is described. The solid-phase synthesis of destruxin E was initially investigated for the efficient synthesis of destruxin analogues. Peptide elongation from polymer-supported β-alanine was efficiently performed using DIC/HOBt or PyBroP/DIEA, and subsequent cleavage from the polymer-support under weakly acidic conditions furnished a cyclization precursor in moderate yield. Macrolactonization of the cyclization precursor was smoothly performed using 2-methyl-6-nitrobenzoic anhydride (MNBA)/4-(dimethylamino)pyridine N-oxide (DMAPO) to afford macrolactone in moderate yield. Finally, formation of the epoxide in the side chain via three steps provided destruxin E, and the stereochemistry of the epoxide was determined to be S. Its diastereomer, epi-destruxin E, was also synthesized in the same manner used to synthesize the natural product. The stereochemistry of the epoxide was critical for the V-ATPase inhibition; natural product destruxin E exhibited 10-fold more potent V-ATPase inhibition than epi-destruxin E. Next, the scalable synthesis of destruxin E for in vivo study was also performed via solution-phase synthesis. The scalable synthesis of a key component, (S)-HA-Pro-OH, was achieved using osmium-catalyzed diastereoselective dihydroxylation with (DHQD)2PHAL as a chiral ligand; peptide synthesis using Cbz-protected amino acid derivatives furnished the cyclization precursor on a gram-scale. Macrolactonization smoothly provided the macrolactone without forming a dimerized product, even at 6 mM, and the synthesis of destruxin E was achieved via three steps on a gram scale in high purity (>98%).

Dendritic Cells (DCs), the most potent antigen-presenting cells, play a critical role in the detection of invading pathogens, which are recognized also by multiple carbohydrate-specific receptors. Among them, DC-SIGN is one of the best characterized, with high-mannose and Lewis-type glycan specificity. In this study, we present a potent DC-SIGN targeting device developed using gold nanoparticles functionalized with α-fucosyl-β-alanyl amide. The nanoparticles bound to cellular DC-SIGN and induced internalization as effectively as similar particles coated with comparable amounts of Lewis(X) oligosaccharide. They were found to be neutral toward dendritic cell maturation and IL-10 production, thus envisaging a possible use as targeted imaging tools and antigen delivery devices.

The identification of binding sites for small molecules in genomic DNA is important in various applications. Previously, we demonstrated rapid transcriptional activation by our small molecule SAHA-PIP. However, it was not clear whether the strong biological effects exerted by SAHA-PIP were attributable to its binding specificity. Here, we used high-throughput sequencing (Bind-n-seq) to determine the binding specificity of SAHA-PIPs. Sequence specificity bias was determined for SAHA-PIPs (3 and 4), and this showed enhanced 6 bp sequence-specific binding compared with hairpin PIPs (1 and 2). This finding allowed us to investigate the role of the β-alanine that links SAHA to PIP, and led in turn to the design of ββ-PIPs (5 and 6), which showed enhanced binding specificity. Overall, we demonstrated the importance of β-moieties for the binding specificity of PIPs and the use of cost-effective high-throughput screening of these small molecules for binding to the DNA minor groove., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Activated protein C resistance assays can detect factor V Leiden with high accuracy, depending on the method used. Factor Xa inhibitors such as rivaroxaban and direct thrombin inhibitors including dabigatran, argatroban, and bivalirudin can cause falsely normal results. Lupus anticoagulants can cause incorrect results in most current assays. Assays that include dilution into factor V-deficient plasma are needed to avoid interference from factor deficiencies or elevations, which can arise from a wide variety of conditions such as warfarin, liver dysfunction, or pregnancy. The pros and cons of the currently available assays are discussed., (© 2014 Wiley Periodicals, Inc.)

Background: Specific mass spectrometry and direct activated factor X (Xa)- and thrombin inhibition assays do not allow determination of the reversal of anticoagulant effects of non-vitamin K direct oral anticoagulants (NOACs) by prothrombin complex concentrate (PCC). The objective of this study was the evaluation of the applicability of a variety of commercially available global coagulation assays in analyzing the reversal of NOAC anticoagulation by PCC., Methods: Plasma and whole blood were spiked with apixaban or dabigatran and PCC was added to these samples. Prothrombin time (PT), modified PT (mPT), activated partial prothrombin time (APTT), thrombography (CAT method) and thromboelastography (ROTEM, TEG) were performed., Results: Assays triggered by contact activation (APTT, INTEM) did not show inhibitor reversal by PCC. Assays triggered by tissue factor (TF) showed NOAC type and NOAC concentration dependent anticoagulation reversal effects of PCC ranging from partial normalization to overcorrection of the following parameters: clotting or reaction time (PT, mPT TEG-TF, EXTEM, FIBTEM); angle in thromboelastography (TEG-TF); thrombin generation (CAT) lag time, endogenous thrombin potential (ETP) and peak thrombin. Extent of reversal was assay reagent dependent. ETP (5 pM TF) was the only parameter showing complete reversal of anticoagulation by PCC for all NOACs ranging from 200 to 800 μg/L., Conclusions: ETP fits with the concept that reversal assessment of NOAC anticoagulation by PCC should be based on measurements on the clotting potential or thrombin generating potential of the plasma or whole blood patient sample. Low sensitivity of ETP for NOACs and its correlation with bleeding are issues that remain to be resolved.

Drosophila Ebony is a β-alanyl biogenic amine synthetase with proven function in cuticle and in glia of the nervous system. It is closely related to nonribosomal peptide synthetases (NRPSs), which typically consist of at least an adenylation, a peptidyl carrier protein and a peptide bond forming condensation domain. Besides its role in cuticle formation, Ebony is in most glia of the brain thought to convert biogenic amines to β-alanyl conjugates. If the metabolization of the neurotransmitter histamine to β-alanyl histamine requires a fast reaction in visual signal transduction, Ebony must be able to fulfill this requirement. Since NRPSs are in general slowly acting multi-modular protein machineries, the enigma of how Ebony quickly facilitates this inactivation remains a key question for understanding its role in vision. To quantitatively analyze the reaction kinetics, we used phosphopantetheinylated holo-Ebony prepared from Baculovirus infected Sf9 cells. Kinetic parameters for the loading reaction, e.g. the formation of β-alanyl-Ebony thioester, complied with those of slow NRPSs. In contrast, single-turnover analysis of the last reaction step, peptide bond formation between pre-activated β-alanyl Ebony thioester and histamine, revealed a very rapid conjugation reaction. This biphasic nature of activity identifies Ebony as a novel type of NRPS related molecule that combines a slow amino acid activation phase with a very fast product formation step., (© 2014 FEBS.)

Helices are important structural/recognition elements in proteins and peptides. Stability and conformational differences between helices composed of α- and β-amino acids as scaffolds for mimicry of helix recognition has become a theme in medicinal chemistry. Furthermore, helices formed by β-amino acids are experimentally more stable than those formed by α-amino acids. This is paradoxical because the larger sizes of the hydrogen-bonding rings required by the extra methylene groups should lead to entropic destabilization. In this study, molecular dynamics simulations using the second-generation force field, AMOEBA (Ponder, J.W., et al., Current status of the AMOEBA polarizable force field. J Phys Chem B, 2010. 114(8): p. 2549-64.) explored the stability and hydrogen-bonding patterns of capped oligo-β-alanine, oligoalanine, and oligoglycine dodecamers in water. The MD simulations showed that oligo-β-alanine has strong acceptor+2 hydrogen bonds, but surprisingly did not contain a large content of 3(12) -helical structures, possibly due to the sparse distribution of the 3(12) -helical structure and other structures with acceptor+2 hydrogen bonds. On the other hand, despite its backbone flexibility, the β-alanine dodecamer had more stable and persistent <3.0 Å hydrogen bonds. Its structure was dominated more by multicentered hydrogen bonds than either oligoglycine or oligoalanine helices. The 3(1) (PII) helical structure, prevalent in oligoglycine and oligoalanine, does not appear to be stable in oligo-β-alanine indicating its competition with other structures (stacking structure as indicated by MD analyses). These differences are among the factors that shape helical structural preferences and the relative stabilities of these three oligopeptides., (© 2014 Wiley Periodicals, Inc.)

Background: Direct oral anticoagulants (DOACs) are prescribed for systemic anticoagulation. Fixed doses are recommended, but dose individualization may be warranted. Functional coagulation assays may be available, but their use requires knowledge of the drug taken. To provide alternative methodology for guiding dosage, we have developed and validated a liquid chromatography-mass spectrometric assay for apixaban, dabigatran, edoxaban, and rivaroxaban at the concentrations attained during therapy., Methods: Samples, calibrators, and internal quality controls (100 μL) were mixed with internal standard solution (50 μg/L both dabigatran-13C6 and rivaroxaban-13C6 in acetonitrile) and, after centrifugation (16,400g, 4 minutes), supernatant (100 μL) was injected onto a Cyclone-C18-P-XL TurboFlow column. Analytes were focused onto an Accucore PhenylHexyl (2.1 × 100 mm, 2.6 μm) analytical column and eluted using a methanol + acetonitrile (1 + 1):aqueous ammonium acetate (10 mmol/L) gradient. Data were acquired using high-resolution mass spectrometry in full-scan mode (100-2000 m/z) with data-dependent fragmentation to confirm peak identity. Calibration was linear (1-500 μg/L all analytes)., Results: Total analysis time was 6 minutes. Intra-assay imprecision (% RSD) at 1 μg/L was 2.6%, 4.2%, 17.3%, and 9.5% for apixaban, dabigatran, edoxaban, and rivaroxaban, respectively. Mean recovery was 96%-101%. No signal suppression or enhancement was observed. Apixaban, dabigatran, and rivaroxaban were stable over 3 freeze-thaw cycles, after storage at room temperature, and at 2-8°C for up to 2 weeks. Edoxaban was stable over 3 freeze-thaw cycles but showed a mean deterioration of 16% if stored at 2-8°C (2 weeks) and of 18% and 70% (1 day and 2 weeks, respectively) at room temperature., Conclusions: The method is suitable for high-throughput therapeutic drug monitoring of DOACs. The acquisition of full scan data allows for the retrospective identification of metabolites. The method can be used to identify a particular DOAC if information on the drug taken is lacking.

A new fluorescent label N-[4′-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-β-alanine (7AF) was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The 7AF label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms.

Three new branched peptides, namely, H-Gly-Dap(H-Gly)-Gly-NH2 (3G), H-His-Dap(H-His)-Gly-NH2 (2HG), and H-Gly-Dap(H-Gly)-His-NH2 (2GH), where Dap stands for the 2,3-diaminopropionic acid residue, were synthesized by solid phase procedures. Because of the junction at Dap these peptides have three available pending arms for metal chelation. The complex formation between these peptides and 1 equiv of Cu(2+) was investigated as a function of pH by potentiometry ultraviolet-visible absorption, circular dichroism, and X-band electron paramagnetic resonance spectroscopy in aqueous medium. Our results clearly demonstrate that cooperation between all three peptide arms essentially contributes to the stability of copper(II) complexes.

Background: Dabigatran etexilate (DE) is an oral direct thrombin inhibitor used to prevent strokes in patients with atrial fibrillation. No licensed DE antidote is currently available. We hypothesized that active site-mutated S195A thrombin (S195A-IIa) and/or its trypsinized derivative (γT -S195A-IIa) would sequester dabigatran, the active form of DE, and reduce its anticoagulant effects., Objective: To assess active site-mutated S195A or γT -S195A-IIa as dabigatran reversal agents in vitro and in vivo., Methods: Diluted thrombin time (dTT) assays were performed using human or murine plasma containing dabigatran, combined with S195A-IIa, γT -S195A-IIa or FPR-chloromethyl ketone-treated thrombin (FPR-IIa). Bleeding times were determined in anesthetized DE-treated mice also receiving γT -S195A-IIa or vehicle 15 min prior to tail transection. The time to occlusion of carotid arteries of DE-treated mice also receiving S195A-IIa, γT -S195A-IIa, prothrombin complex concentrate (PCC) or vehicle, 15 min prior to topical FeCl3 , was determined using Doppler ultrasound., Results: γT-S195A-IIa reduced dTT values of dabigatran-containing human and murine plasma more effectively than S195-IIa; FPR-IIa had no effect. A dose of 13 mg kg(-1) DE abrogated occlusive thrombus formation in the carotid arteries of FeCl3 -treated mice; γT -S195A-IIa (6 mg kg(-1) ) or PCC (14.3 IU kg(-1) ), but not saline vehicle or S195A-IIa (6 mg kg(-1) ), was equally effective in restoring thrombus formation. Bleeding times of mice treated with 60 mg kg(-1) DE and γT -S195A-IIa (6 mg kg(-1) ) or saline vehicle did not differ., Conclusions: Our data suggest that γT -S195A-IIa decreases the anticoagulant effects of dabigatran in vitro and is partially effective at restoring hemostasis-related thrombus formation in DE-treated mice in vivo., (© 2014 International Society on Thrombosis and Haemostasis.)

L-2,3-diaminopropionic acid (L-Dap) is an amino acid that is a precursor of antibiotics and staphyloferrin B a siderophore produced by Staphylococcus aureus. SbnA and SbnB are encoded by the staphyloferrin B biosynthetic gene cluster and are implicated in L-Dap biosynthesis. We demonstrate here that SbnA uses PLP and substrates O-phospho-L-serine and L-glutamate to produce a metabolite N-(1-amino-1-carboxyl-2-ethyl)-glutamic acid (ACEGA). SbnB is shown to use NAD(+) to oxidatively hydrolyze ACEGA to yield α-ketoglutarate and L-Dap. Also, we describe crystal structures of SbnB in complex with NADH and ACEGA as well as with NAD(+) and α-ketoglutarate to reveal the residues required for substrate binding, oxidation, and hydrolysis. SbnA and SbnB contribute to the iron sparing response of S. aureus that enables staphyloferrin B biosynthesis in the absence of an active tricarboxylic acid cycle., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ten accessions of grass pea (Lathyrus sativus L.) from different Sicilian sites, cultivated in the same environmental conditions, were analyzed for their morphological and productive parameters and for the content of two non-protein amino acids: α- and β-ODAP (α- and β-N-oxalyl-l-α,β-diaminopropionic acid). The β-isomer is the neurotoxin responsible for the neuron disease known as lathyrism. This analysis was carried out using two common analytical methodologies never applied in their determination, an HPLC separation with evaporative light scattering (ELS) as detector, and nuclear magnetic resonance (NMR). The content of the two isomers falls in the range reported for these compounds: 0.42-0.74 and 2.69-4.59 g/kg for α- and β-ODAP, respectively; and the two methods yield comparable results. High productivity and a high protein content were detected in three Sicilian accessions. Low β-ODAP content was found to be linked to accessions with heavier seeds and those originating at lower altitudes.

This research was aimed to investigate why chlorogenic acid, presents at high concentrations in some food raw material, influences acrylamide formation. In the asparagine/glucose Maillard reaction system (pH=6.8), addition of chlorogenic acid significantly increased acrylamide formation and inhibited its elimination. In contrast, the quinone derivative of chlorogenic acid decreased acrylamide formation. Three mechanisms may be involved for increasing acrylamide formation by chlorogenic acid. Firstly, it increased the formation of HMF, which acts as a more efficient precursor than glucose to form acrylamide. Secondly, it decreased activation energy for conversion of 3-aminopropionamide (3-APA) to acrylamide (from 173.2 to 136.6kJ/mol), and enhances deamination from 3-APA. And thirdly, it prevented attack of the produced acrylamide from free radicals by keeping high redox potential during the Maillard reaction., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells., (© 2013. Published by Elsevier B.V. All rights reserved.)

Background: Clinicians managing patients receiving the direct thrombin inhibitor dabigatran may benefit in being able to determine the amount of drug present in selected situations. This may include assessment of accumulation, concurrent drug interactions, or adequate removal from circulation. The ability to estimate the amount of dabigatran present using the chromogenic ecarin assay (ECA) requires further clarification., Objective: To describe the reliability of dabigatran measurements using a chromogenic ECA., Methods: This was an evaluation of the ECA method that incorporated assessment of imprecision, linearity, accuracy, carryover, and lower limits of detection or blank. Pooled normal plasma enriched with dabigatran at concentrations of 0, 25, 50, 75, 100, 125, 150, 200, 300, 400, and 500 ng/mL were sent blinded to 3 laboratories in the United States to compare our ECA results with those of laboratories reporting dilute thrombin time methods (HEMOCLOT thrombin inhibitor assay) for measuring dabigatran. Trough and peak levels from 35 patients were also compared with mass spectrophotometry for assessing ECA accuracy., Results: The within-run or day-to-day imprecision was less than 10%, with high linearity (R (2) = 0.989) and high degree of accuracy (R (2) = 0.985; slope = 0.908) for levels ranging between 18 and 470 ng/mL and no carryover at 0 ng/mL noted. The ECA approach appeared to be more reliable at lower dabigatran concentrations., Conclusions: The chromogenic ECA appears to be an effective approach to determine the amount of dabigatran present. Further insights are necessary to determine how it can be used to reduce thromboembolic or bleeding complications in patients receiving dabigatran.

In this study, hydroxyapatite (HAP) was surface-modified by the addition of β-alanine (β-Ala), and the ring-opening polymerization of γ-benzyl-L-glutamate-N-carboxy-anhydride (BLG-NCA) was subsequently initiated. HAP containing surface poly-γ-benzyl-L-glutamates (PBLG) was successfully prepared in this way. With the increase of PBLG content in HAP-PBLG, the solubility of HAP-PBLG increased gradually and it was ultimately soluble in chloroform. HAP-PLGA with surface carboxyl groups was obtained by the catalytic hydrogenation of HAP-PBLG. In the process of HAP modification, the morphology changes from rod to sheet and from flake to needle. The effect of BLG-NCA concentration on the character of hydroxyapatite-β-alanine-poly(γ-benzyl-L-glutamate) (HAP-PBLG) was investigated. The existence of amino acids on the HAP surfaces was confirmed in the resulting Fourier transform infrared (FTIR) spectra. The resulting powder X-ray diffraction patterns indicated that the crystallinity of HAP decreased when the ratio of BLG-NCA/HAP-NH2 increased to 20/1. Transmission electron microscopy (TEM) indicated that the particle size of HAP-PBLG decreased significantly and that the resulting particles appeared less agglomerated relative to that of the HAP-NH₂ crystals. Furthermore, ¹H-NMR spectra and FTIR spectra revealed that hydroxyapatite-β-alanine-poly (L-glutamic acid) (HAP-PLGA) was able to successfully bear carboxylic acid groups on its side chains.

Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes β-alanine as a substrate. Proteoliposomes containing purified VGAT transport β-alanine using Δψ but not ΔpH as a driving force. The Δψ-driven β-alanine uptake requires Cl(-). VGAT also facilitates Cl(-) uptake in the presence of β-alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates β-alanine uptake. These findings indicated that VGAT transports β-alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular β-alanine transporter. Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In the present study, we showed that proteoliposomes containing purified VGAT transport β-alanine using Δψ as a driving force. VGAT also facilitates Cl(-) uptake. Our findings indicated that VGAT functions as a vesicular β-alanine transporter., (© 2013 International Society for Neurochemistry.)

Molecular dynamics simulations of the polycationic antimicrobial peptide dendrimer (Leu)8(DapLeu)4(DapPhe)2DapLys-NH2 binding to membranes suggest that electrostatic interactions with the polyanionic lipopolysaccharide (LPS) and conformational flexibility of the 2,3-diaminopropanoic acid (Dap) branching units drive its selective insertion into microbial membranes.

Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was nonimmunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17, and 20 line the pore of assembled p22-T19R, S22W, and here we used 2,3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap substitutions. To gain further insights into the effects of Dap residues on the properties of the putative pore, we performed two-electrode voltage clamp electrophysiology on Xenopus oocytes exposed to p22-T19R, S22W or its Dap-modified analogues. We observed that Dap-substituted peptides also induced significantly larger voltage-dependent currents than the parent compound, but there was no apparent change in reversal potential upon replacement of external Na+, Cl- or K+, indicating that these currents remained nonselective. These results suggest that the introduction of positively charged side chains in predicted pore-lining residues does not improve anion-to-cation selectivity, but results in higher conductance, perhaps due to higher oligomerization numbers.

N-Methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that can recognize predetermined DNA sequences with high sequence specificity. As many eukaryotic promoter regions contain highly GC-rich sequences, it is valuable to synthesize and characterize Py-Im polyamides that recognize GC-rich motifs. In this study, we synthesized four hairpin Py-Im polyamides 1-4, which recognize 5'-GCGC-3' and investigated their binding behavior with surface plasmon resonance assay. Py-Im polyamides 2-4 contain two, one, and one β-alanine units, replacing the Py units of 1, respectively. The binding affinities of 2-4 to the target DNA increased 430, 390, and 610-fold, respectively, over that of 1. The association and dissociation rates of 2 to the target DNA were improved by 11 and 37-fold, respectively, compared with those of 1. Interestingly, the association and dissociation rates of 3 and 4 were higher than those of 2, even though the binding affinities of 2, 3, and 4 to the target DNA were comparable to each other. The binding affinity of 2 to DNA with a 2bp mismatch was reduced by 29-fold, compared with that to the matched DNA. Moreover, the binding affinities of 3 and 4 to the same mismatched DNA were reduced by 270 and 110-fold, respectively, indicating that 3 and 4 have greater specificities than 2 and are suitable as DNA-binding modules for engineered epigenetic regulation., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The prion protein coordinates copper with high affinity in the regions encompassing residues 92-99 (GGGTHSQW) and 106-115 (KTNMKHMAGA). Cu(II) binding to these sites involves the coordination of the His96/His111 imidazole ring and backbone deprotonated amides that precede the His residue. Such a coordination arrangement involves the formation of hexa- and penta-membered cycles that provide further stabilization of the metal-peptide complex. The purpose of the present study is to introduce a methylene group in the peptide backbone, to evaluate the impact of increasing the size of these cycles in Cu(II) binding. Thus, a β-alanine residue was inserted at different positions preceding the His residue in these prion fragments, and their Cu(II) coordination properties were assessed by UV-Visible absorption, circular dichroism, and electron paramagnetic resonance. Spectroscopic data show that the insertion of a methylene group leads to a completely different Cu(II) coordination that involves the His96/His111 imidazole ring and nitrogen or oxygen atoms provided by the peptide backbone towards the C-terminal. This study clearly shows that two main factors determine the nature of Cu(II)-peptide complexes involving an anchoring His residue and deprotonated amides from the backbone chain: i) the stabilization of Cu(II)-peptide complexes due to the formation of cyclic structures (i.e. chelate effect) and ii) the nature of the residues associated to the deprotonated amide groups that participate in metal ion coordination., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Fluorescent reporters are useful in vitro and in vivo probes of protein structure, function, and localization. Here we report that the fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), can be site-specifically incorporated into proteins in mammalian cells in response to the TAG codon with high efficiency using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. We further demonstrate that Anap can be used to image the subcellular localization of proteins in live mammalian cells. The small size of Anap, its environment-sensitive fluorescence, and the ability to introduce Anap at specific sites in the proteome by simple mutagenesis make it a unique and valuable tool in eukaryotic cell biology.

The efficacies of many antimicrobial peptides are greatly reduced under high salt concentrations, therefore limiting their use as pharmaceutical agents. Here, we describe a strategy to boost salt resistance and serum stability of short antimicrobial peptides by adding the nonnatural bulky amino acid β-naphthylalanine to their termini. The activities of the short salt-sensitive tryptophan-rich peptide S1 were diminished at high salt concentrations, whereas the activities of its β-naphthylalanine end-tagged variants were less affected.