- Binding Methylarginines and Methyllysines as Free Amino Acids: A Comparative Study of Multiple Host Classes**
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Methylated free amino acids are an important class of targets for host-guest chemistry that have recognition properties distinct from those of methylated peptides and proteins. We present comparative binding studies for three different host classes that are each studied with multiple methylated arginines and lysines to determine fundamental structure-function relationships. The hosts studied are all anionic and include three calixarenes, two acyclic cucurbiturils, and two other cleft-like hosts, a clip and a tweezer. We determined the binding association constants for a panel of methylated amino acids using indicator displacement assays. The acyclic cucurbiturils display stronger binding to the methylated amino acids, and some unique patterns of selectivity. The two other cleft-like hosts follow two different trends, shallow host (clip) following similar trends to the calixarenes, and the other more closed host (tweezer) binding certain less-methylated amino acids stronger than their methylated counterparts. Molecular modelling sheds some light on the different preferences of the various hosts. The results identify hosts with new selectivities and with affinities in a range that could be useful for biomedical applications. The overall selectivity patterns are explained by a common framework that considers the geometry, depth of binding pockets, and functional group participation across all host classes.
- Warmerdam, Zoey,Kamba, Bianca E.,Le, My-Hue,Schrader, Thomas,Isaacs, Lyle,Bayer, Peter,Hof, Fraser
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- Direct monitoring of biocatalytic deacetylation of amino acid substrates by1H NMR reveals fine details of substrate specificity
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Amino acids are key synthetic building blocks that can be prepared in an enantiopure form by biocatalytic methods. We show that thel-selective ornithine deacetylase ArgE catalyses hydrolysis of a wide-range ofN-acyl-amino acid substrates. This activity was revealed by1H NMR spectroscopy that monitored the appearance of the well resolved signal of the acetate product. Furthermore, the assay was used to probe the subtle structural selectivity of the biocatalyst using a substrate that could adopt different rotameric conformations.
- De Cesare, Silvia,McKenna, Catherine A.,Mulholland, Nicholas,Murray, Lorna,Bella, Juraj,Campopiano, Dominic J.
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supporting information
p. 4904 - 4909
(2021/06/16)
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- DISCOVERY, TOTAL SYNTHESIS, AND BIOACTIVITY OF DOSCADENAMIDES
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The invention is directed towards compounds (e.g., Formulae (I)-(IX)), their mechanism of action, processes to prepare the compounds, methods of activating quorum sensing signaling activity, and methods of treating diseases and disorders using the compounds described herein (e.g., Formulae (I)-(IX)).
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Page/Page column 81; 84-85
(2021/02/05)
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- Development of a Raltegravir-based Photoaffinity-Labeled Probe for Human Immunodeficiency Virus-1 Integrase Capture
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Photoaffinity labeling (PAL) is one of the upcoming and powerful tools in the field of molecular recognition. It includes the determination of dynamic parameters, such as the identification and localization of the target protein and the site of drug binding. In this study, a photoaffinity-labeled probe for full-length human immunodeficiency virus-1 integrase (HIV-1 IN) capture was designed and synthesized, following the structure of the FDA-approved drug Raltegravir. This photoprobe was found to retain the HIV IN inhibitory potential in comparison with its parent molecule and demonstrates the ability to label the HIV-1 IN protein. Putative photoprobe/inhibitor binding sites near the catalytic site were then identified after protein digestion coupled to mass and molecular modeling analyses.
- Pala, Nicolino,Esposito, Francesca,Tramontano, Enzo,Singh, Pankaj Kumar,Sanna, Vanna,Carcelli, Mauro,Haigh, Lisa D.,Satta, Sandro,Sechi, Mario
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supporting information
p. 1986 - 1992
(2020/11/09)
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- Preparation and characterization of a new open-tubular capillary column for enantioseparation by capillary electrochromatography
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In order to use the enantioseparation capability of cationic cyclodextrin and to combine the advantages of capillary electrochromatography (CEC) with open-tubular (OT) column, in this study, a new OT-CEC, coated with cationic cyclodextrin (1-allylimidazolium-β-cyclodextrin [AI-β-CD]) as chiral stationary phase (CSP), was prepared and applied for enantioseparation. Synthesized AI-β-CD was characterized by infrared (IR) spectrometry and mass spectrometry (MS). The preparation conditions for the AI-β-CD-coated column were optimized with the orthogonal experiment design L9(34). The column prepared was characterized by scanning electron microscopy (SEM) and elemental analysis (EA). The results showed that the thickness of stationary phase in the inner surface of the AI-β-CD-coated columns was about 0.2 to 0.5?μm. The AI-β-CD content in stationary phase based on the EA was approximately 2.77?mmol·m?2. The AI-β-CD-coated columns could separate all 14 chiral compounds (histidine, lysine, arginine, glutamate, aspartic acid, cysteine, serine, valine, isoleucine, phenylalanine, salbutamol, atenolol, ibuprofen, and napropamide) successfully in the study and exhibit excellent reproducibility and stability. We propose that the column, coated with AI-β-CD, has a great potential for enantioseparation in OT-CEC.
- Li, Yingjie,Tang, Yimin,Qin, Shili,Li, Xue,Dai, Qiang,Gao, Lidi
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p. 283 - 292
(2019/02/05)
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- Biocatalytic Reversal of Advanced Glycation End Product Modification
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Advanced glycation end products (AGEs) are a heterogeneous group of molecules that emerge from the condensation of sugars and proteins through the Maillard reaction. Despite a significant number of studies showing strong associations between AGEs and the pathologies of aging-related illnesses, it has been a challenge to establish AGEs as causal agents primarily due to the lack of tools in reversing AGE modifications at the molecular level. Herein, we show that MnmC, an enzyme involved in a bacterial tRNA-modification pathway, is capable of reversing the AGEs carboxyethyl-lysine (CEL) and carboxymethyl-lysine (CML) back to their native lysine structure. Combining structural homology analysis, site-directed mutagenesis, and protein domain dissection studies, we generated a variant of MnmC with improved catalytic properties against CEL in its free amino acid form. We show that this enzyme variant is also active on a CEL-modified peptidomimetic and an AGE-containing peptide that has been established as an authentic ligand of the receptor for AGEs (RAGE). Our data demonstrate that MnmC variants are promising lead catalysts toward the development of AGE-reversal tools and a better understanding of AGE biology.
- Kim, Nam Y.,Goddard, Tyler N.,Sohn, Seungjung,Spiegel, David A.,Crawford, Jason M.
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p. 2402 - 2410
(2019/08/12)
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- Discovery and Total Synthesis of Doscadenamide A: A Quorum Sensing Signaling Molecule from a Marine Cyanobacterium
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Quorum sensing (QS) plays a critical role in the regulation of bacterial pathogenesis. Doscadenamide A (1a) was isolated from a marine cyanobacterium, its structure elucidated by NMR, and its activity linked to QS induction. The total synthesis of 1a was developed, and the absolute configuration confirmed through comparison of the isolated natural product with synthetic diastereomers. Our preliminary investigation indicated that 1a could activate QS signaling in a LasR-dependent manner.
- Liang, Xiao,Matthew, Susan,Chen, Qi-Yin,Kwan, Jason C.,Paul, Valerie J.,Luesch, Hendrik
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supporting information
p. 7274 - 7278
(2019/09/07)
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- Chiral Metal–Organic Framework Hollow Nanospheres for High-Efficiency Enantiomer Separation
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Chiral ZIF-8 hollow nanospheres with d-histidine as part of chiral ligands (denoted as H-d-his-ZIF-8) were prepared for separation of (±)-amine acids. Compared to bulk d-his-ZIF-8 without a hollow cavity, the prepared H-d-his-ZIF-8 showed 15 times higher separation capacity and higher ee values of 90.5 % for alanine, 95.2 % for glutamic acid and 92.6 % for lysine, respectively.
- Wang, Xiaoshi,Zhu, Yanan,Liu, Jian,Liu, Chang,Cao, Changyan,Song, Weiguo
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p. 1535 - 1538
(2018/06/26)
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- Covalent Organic Frameworks with Chirality Enriched by Biomolecules for Efficient Chiral Separation
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The separation of racemic compounds is important in many fields, such as pharmacology and biology. Taking advantage of the intrinsically strong chiral environment and specific interactions featured by biomolecules, here we contribute a general strategy is developed to enrich chirality into covalent organic frameworks (COFs) by covalently immobilizing a series of biomolecules (amino acids, peptides, enzymes) into achiral COFs. Inheriting the strong chirality and specific interactions from the immobilized biomolecules, the afforded biomolecules?COFs serve as versatile and highly efficient chiral stationary phases towards various racemates in both normal and reverse phase of high-performance liquid chromatography (HPLC). The different interactions between enzyme secondary structure and racemates were revealed by surface-enhanced Raman scattering studies, accounting for the observed chiral separation capacity of enzymes?COFs.
- Zhang, Sainan,Zheng, Yunlong,An, Hongde,Aguila, Briana,Yang, Cheng-Xiong,Dong, Yueyue,Xie, Wei,Cheng, Peng,Zhang, Zhenjie,Chen, Yao,Ma, Shengqian
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supporting information
p. 16754 - 16759
(2018/11/27)
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- Chromatographic Resolution of α-Amino Acids by (R)-(3,3'-Halogen Substituted-1,1'-binaphthyl)-20-crown-6 Stationary Phase in HPLC
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Three new chiral stationary phases (CSPs) for high-performance liquid chromatography were prepared from R-(3,3'-halogen substituted-1,1'-binaphthyl)-20-crown-6 (halogen = Cl, Br and I). The experimental results showed that R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 (CSP-1) possesses more prominent enantioselectivity than the two other halogen-substituted crown ether derivatives. All twenty-one α-amino acids have different degrees of separation on R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6-based CSP-1 at room temperature. The enantioselectivity of CSP-1 is also better than those of some commercial R-(1,1'-binaphthyl)-20-crown-6 derivatives. Both the separation factors (α) and the resolution (Rs) are better than those of commercial crown ether-based CSPs [CROWNPAK CR(+) from Daicel] under the same conditions for asparagine, threonine, proline, arginine, serine, histidine and valine, which cannot be separated by commercial CR(+). This study proves the commercial usefulness of the R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 chiral stationary phase.
- Wu, Peng,Wu, Yuping,Zhang, Junhui,Lu, Zhenyu,Zhang, Mei,Chen, Xuexian,Yuan, Liming
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supporting information
p. 1037 - 1042
(2017/07/25)
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- Purification, structural characterization and bioactivity evaluation of a novel proteoglycan produced by Corbicula fluminea
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A novel proteoglycan, named CFPS-11, was isolated from Corbicula fluminea, which is a food source of freshwater bivalve mollusk. CFPS-11 had an average molecular weight of 807.7 kDa and consisted of D-glucose and D-glucosamine in a molar ratio of 12.2:1.0. The protein moiety (~5%) of CFPS-11 was covalently bonded to the polysaccharide chain in O-linkage type through both serine and thereonine residues. The polysaccharide chain of CFPS-11 was composed of (1 → 4)-α-D-glucopyranosyl and (1 → 3,6)-α-D-glucopyranosyl residues, which branched at O-6. The branch chain consisted of (1 →)-α-D-glucopyranosyl and (1 →)-α-D-N-acetylglucosamine residues. CFPS-11 exhibited significant antioxidant activity in a dose-dependent manner and remarkable inhibition activities against α-amylase and α-glucosidase by in vitro assays. These findings indicated that the CFPS-11 from C. fluminea has the potential for development as a health food ingredient.
- Yan, Jing-Kun,Wang, Yao-Yao,Qiu, Wen-Yi,Wu, Li-Xia,Ding, Zhi-Chao,Cai, Wu-Dan
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- A method for preparing DL-lysine
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The invention provides a method for preparing DL-lysine with chiral lysine salt as a raw material. The method specifically comprises the following steps: dissolving chiral lysine salt in an acetic acid water solution, adding salicylaldehyde or benzaldehyde as a catalyst, carrying out heating and racemization, removing solvents through vacuum distillation after racemization is completed, washing a residual solid with ethanol, obtaining DL-lysine salt, removing salt with an ion exchange column and carrying out concentration and decoloration, thus obtaining a DL-lysine solid. The preparation method has the advantages that the preparation method is lower in production cost and simple in process; pollution is not easily caused in the production process; the racemization rate of L-lysine hydrochloride can be 100%; the purity of the obtained DL-lysine finished product is more than 98%.
- -
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Paragraph 0032; 0033
(2017/04/03)
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- Colorimetric and fluorescence probe for the detection of nano-molar lysine in aqueous medium
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A single crystal X-ray structurally characterized BODIPY based probe, THBPY, derived from 4-hydroxy-5-isopropyl-2 methyl-isophthalaldehyde, detects nano-molar lysine in aqueous medium. In the presence of lysine, THBPY visibly changes its color and fluorescence profile due to the formation of a stable imine bond. A distinctive color change allows for facile discrimination over other amino acids in a wide range of concentrations of lysine. The detection limit for lysine is 0.001 μM by a fluorescence method and 0.01 μM by a colorimetric method. The probe shows good reversibility for multiple uses and cleanly discriminates between lysine and other amino acids. Density functional theoretical studies closely resemble experimental results.
- Adhikari, Susanta,Ghosh, Avijit,Mandal, Sandip,Guria, Subhajit,Banerjee, Prajna Paramita,Chatterjee, Ansuman,Das, Debasis
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supporting information
p. 10688 - 10694
(2016/11/30)
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- COMPOSITIONS AND METHODS FOR THE PREPARATION OF KIDNEY PROTECTIVE AGENTS COMPRISING AMIFOSTINE AND AMINO ACIDS
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This invention relates to composition and method of preparation of AminoMedix? comprising of Amifostine, at least one amino acid (Arginine, Lysine, Histidine) with or without other pharmaceutically active compounds. The AminoMedix? composition can be applied for kidney protection during therapy using radiolabeled and non-radiolabeled compounds, contrast agents, chemotherapeutics, antibiotics and drugs showing nephrotoxic effect.
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- A novel thyroglobulin-binding lectin from the brown alga Hizikia fusiformis and its antioxidant activities
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A lectin (HFL) was isolated from the brown alga, Hizikia fusiformis, through ion exchange on cellulose DE52 and HPLC with a TSK-gel G4000PWXL column. SDS-PAGE showed that HFL had a molecular mass of 16.1 kDa. The HPLC (with a TSK-gel G4000PWXL column) indicated that HFL is a tetramer in its native state. The total carbohydrate content was 41%. Glucose, galactose and fucose were the monosaccharide units of HFL, and the normalized mol% values were 6, 14 and 80, respectively. HFL contains a large amount of the acidic amino acid, Asx. The β-elimination reaction suggested that the oligosaccharide and peptide moieties of HFL may belong to the N-glucosidic linkage. The amino acid sequences, of about five segments of HFL, were acquired by MALDI-TOF/TOF, and the sequences have no homology with other lectins. HFL was found to agglutinate sheep erythrocytes. The hemagglutination activity was inhibited by thyroglobulin, from bovine thyroid, but not by any of the monosaccharides tested. The lectin reaction was independent of the presence of the divalent cation Ca2+. HFL showed free radical scavenging activity against hydroxyl, DPPH and ABTS+ radicals.
- Wu, Mingjiang,Tong, Changqing,Wu, Yue,Liu, Shuai,Li, Wei
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- The Role of a Nonribosomal Peptide Synthetase in l -Lysine Lactamization during Capuramycin Biosynthesis
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Capuramycins are one of several known classes of natural products that contain an l-Lys-derived l-α-amino-caprolactam (l-ACL) unit. The α-amino group of l-ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP-independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP-dependent amide-bond-forming catalyst responsible for the biosynthesis of the remaining amide bond present in l-ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l-Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS-mediated mechanism is employed in the biosynthesis of other l-ACL-containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS-derived peptides that contain an unmodified l-Lys residue.
- Liu, Xiaodong,Jin, Yuanyuan,Cui, Zheng,Nonaka, Koichi,Baba, Satoshi,Funabashi, Masanori,Yang, Zhaoyong,Van Lanen, Steven G.
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p. 804 - 810
(2016/05/19)
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- Rapid, effective deprotection of tert-butoxycarbonyl (Boc) amino acids and peptides at high temperatures using a thermally stable ionic liquid
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A method for high temperature Boc deprotection of amino acids and peptides in a phosphonium ionic liquid is described. The ionic liquid had low viscosity, high thermal stability and demonstrated a beneficial effect. The study extended the possibility for extraction of water soluble polar organic molecules using ionic liquids. Trace water significantly improved product purity and yield, while only 2 equiv. TFA led to deprotection within 10 min. The trityl group was also deprotected.
- Bhawal, Sumit S.,Patil, Rahul A.,Armstrong, Daniel W.
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p. 95854 - 95856
(2015/11/24)
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- Enantiospecific C-H Activation Using Ruthenium Nanocatalysts
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The activation of C-H bonds has revolutionized modern synthetic chemistry. However, no general strategy for enantiospecific C-H activation has been developed to date. We herein report an enantiospecific C-H activation reaction followed by deuterium incorporation at stereogenic centers. Mechanistic studies suggest that the selectivity for the α-position of the directing heteroatom results from a four-membered dimetallacycle as the key intermediate. This work paves the way to novel molecular chemistry on nanoparticles.
- Taglang, Céline,Martínez-Prieto, Luis Miguel,Del Rosal, Iker,Maron, Laurent,Poteau, Romuald,Philippot, Karine,Chaudret, Bruno,Perato, Serge,Sam Lone, Ana?s,Puente, Céline,Dugave, Christophe,Rousseau, Bernard,Pieters, Grégory
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supporting information
p. 10474 - 10477
(2015/09/02)
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- Site-specific labeling of synthetic peptide using the chemoselective reaction between N-methoxyamino acid and isothiocyanate
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Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N-terminus, leaving side chain amino group intact. The synthetic human β-defensin-2 carrying MeOGly at its N-terminus or the side chain amino group of Lys10 reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides.
- Hara, Toshiaki,Purwati, Euis Maras,Tainosyo, Akira,Kawakami, Toru,Hojo, Hironobu,Aimoto, Saburo
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p. 765 - 769
(2015/09/21)
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- Isolation, Characterization, and Synthesis of the Barrettides: Disulfide-Containing Peptides from the Marine Sponge Geodia barretti
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Two disulfide-containing peptides, barrettides A (1) and B (2), from the cold-water marine sponge Geodia barretti are described. Those 31 amino acid residue long peptides were sequenced using mass spectrometry methods and structurally characterized using NMR spectroscopy. The structure of 1 was confirmed by total synthesis using the solid-phase peptide synthesis approach that was developed. The two peptides were found to differ only at a single position in their sequence. The three-dimensional structure of 1 revealed that these peptides possess a unique fold consisting of a long β-hairpin structure that is cross-braced by two disulfide bonds in a ladder-like arrangement. The peptides are amphipathic in nature with the hydrophobic and charged residues clustered on separate faces of the molecule. The barrettides were found not to inhibit the growth of either Escherichia coli or Staphylococcus aureus but displayed antifouling activity against barnacle larvae (Balanus improvisus) without lethal effects in the concentrations tested. (Figure Presented).
- Carstens, Bodil B.,Rosengren, K. Johan,Gunasekera, Sunithi,Schempp, Stefanie,Bohlin, Lars,Dahlstr?m, Mia,Clark, Richard J.,G?ransson, Ulf
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p. 1886 - 1893
(2015/09/08)
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- METHODS FOR IMPROVING HEALTH IN CANINES
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A method of improving health in a canine includes administering to the canine a nutritional supplement comprising an amino acid secretagogue composition, which stimulates the pituitary gland of the canines to produce growth hormone. The nutritional supplement may be administered orally. The nutritional supplement may comprise L-arginine hydrochloride, Oxo-proline, L-lysine hydrochloride, and cysteine. When desired, the nutritional supplement may consist essentially of L-arginine hydrochloride, Oxo-proline, L-lysine hydrochloride, N-acetyl-L-cysteine, L-glutamine, and schizonepeta powder.
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Page/Page column 3
(2014/04/03)
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- Peptide-nanofiber-supported palladium nanoparticles as an efficient catalyst for the removal of N-terminus protecting groups
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Sonication-induced tryptophan- and tyrosine-based peptide bolaamphiphile nanofibers have been used to synthesize and stabilize Pd nanoparticles under physiological conditions. The peptide bolaamphiphile self-assembly process has been thoroughly studied by using several spectroscopic and microscopic techniques. The stiffness of the soft hydrogel matrix was measured by an oscillatory rheological experiment. FTIR and circular dichroism (CD) experiments revealed a hydrogen-bonded β-sheet conformation of peptide bolaamphiphile molecules in a gel-phase medium. The π-π stacking interactions also played a crucial role in the self-assembly process, which was confirmed by fluorescence spectroscopy. Electron (SEM and TEM) and atomic force microscopy (AFM) studies showed that the peptide bolaamphiphile molecules self-assemble into nanofibrillar structures. Pd nanoparticles were synthesized in the hydrogel matrix in which redox-active tryptophan and tyrosine residues reduce the metal ions to metal nanoparticles. The size of the Pd nanoparticles are in the range of 3-9 nm, and are stabilized by peptide nanofibers. The peptide-nanofiber- supported Pd nanoparticles have shown effective catalytic activity for the removal of N-terminus protecting groups of amino acids and peptides.
- Maity, Indrajit,Manna, Manoj K.,Rasale, Dnyaneshwar B.,Das, Apurba K.
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p. 413 - 420
(2014/04/03)
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- A mild removal of Fmoc group using sodium azide
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A mild method for effectively removing the fluorenylmethoxycarbonyl (Fmoc) group using sodium azide was developed. Without base, sodium azide completely deprotected Nα-Fmoc-amino acids in hours. The solvent-dependent conditions were carefully studied and then optimized by screening different sodium azide amounts and reaction temperatures. A variety of Fmoc-protected amino acids containing residues masked with different protecting groups were efficiently and selectively deprotected by the optimized reaction. Finally, a biologically significant hexapeptide, angiotensin IV, was successfully synthesized by solid phase peptide synthesis using the developed sodium azide method for all Fmoc removals. The base-free condition provides a complement method for Fmoc deprotection in peptide chemistry and modern organic synthesis. Graphical Abstract: [Figure not available: see fulltext.]
- Chen, Chun-Chi,Rajagopal, Basker,Liu, Xuan Yu,Chen, Kuan Lin,Tyan, Yu-Chang,Lin, Fui,Lin, Po-Chiao
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p. 367 - 374
(2014/03/21)
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- Biochemical characterisation and assessment of fibril-forming ability of collagens extracted from Bester sturgeon Huso huso × Acipenser ruthenus
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Collagens purified from Bester sturgeon organs were characterised biochemically, and their fibril-forming abilities and fibril morphologies formed in vitro clarified. Yields of collagens were 2.1%, 11.9%, 0.4%, 18.1%, 0.4%, 0.8% and 0.03% (collagen dry weight/tissue wet weight) from scales, skin, muscle, swim bladder, digestive tract, notochord and snout cartilage, respectively. Using SDS-PAGE and amino acid composition analyses, collagens from scales, skin, muscle, the swim bladder and digestive tract were characterised as type I, and collagens from the notochord and snout cartilage as type II. Denaturation temperatures of the collagens, measured using circular dichroism, were 29.6, 26.8, 29.0, 32.9, 31.6 and 36.3 °C in scales, skin, muscle, swim bladder, digestive tract, and notochord, respectively. For fibril formation, swim bladder and skin collagen showed a more rapid rate of increase in turbidity, a shorter time to attain the maximum turbidity, and formed thicker fibrils compared with porcine tendon type I collagen.
- Zhang, Xi,Ookawa, Mika,Tan, Yongkai,Ura, Kazuhiro,Adachi, Shinji,Takagi, Yasuaki
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p. 305 - 312
(2014/05/06)
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- Efficient one-step preparation of γ-aminobutyric acid from glucose without an exogenous cofactor by the designed Corynebacterium glutamicum
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Lactobacillus plantarum CCTCC M209102 efficiently produces γ-aminobutyric acid (GABA) from l-glutamate, in which glutamate decarboxylase and pyridoxal kinase are involved in the transformation. Pyridoxal kinase catalyzes ATP-dependent phosphorylation of pyridoxal to produce pyridoxal-5′-phosphate, which is the cofactor required for glutamate decarboxylase to biotransform GABA from l-glutamate. Corynebacterium glutamicum G01 is a good producer of l-glutamate from glucose. However, it cannot yield GABA from l-glutamate due to the absence of glutamate decarboxylase and pyridoxal kinase. In this work, to realize the efficient one-step preparation of GABA from glucose without exogenous pyridoxal-5′-phosphate, the metabolic module from L-glutamate to GABA based on glutamate decarboxylase and pyridoxal kinase in L. plantarum was grafted into C. glutamicum. To further improve the GABA production, the pathways to by-product pools of L-arginine, L-proline and L-lysine were blocked using the insertional mutation technique. The engineered C. glutamicum APLGGP carrying argB::tacgad, proB::tacgad and dapA::tacplk could efficiently convert glucose into GABA in one-step without an exogenous co-factor. In fed-batch cultures, the recombinant C. glutamicum APLGGP produced 70.6 g L-1 GABA at 30 °C and 70 h through a two-stage pH control strategy. To our knowledge, this is the highest reported GABA production using glucose as a substrate, and this designed C. glutamicum should be an excellent candidate for producing GABA on an industrial scale. This work is expected to pave the way to redesign the bioreactor for efficient one-step biosynthesis of GABA from glucose without an exogenous co-factor. the Partner Organisations 2014.
- Zhang, Rongzhen,Yang, Taowei,Rao, Zhiming,Sun, Hongmei,Xu, Meijuan,Zhang, Xian,Xu, Zhenghong,Yang, Shangtian
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p. 4190 - 4197
(2014/11/08)
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- SEPARATING AGENT AND MANUFACTURING METHOD THEREOF
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An embodiment of the present invention is a separating agent wherein a group represented by a chemical formula of: or a group represented by a chemical formula of: is introduced on a surface thereof.
- -
-
Paragraph 0067; 0068; 0069; 0070; 0071; 0072; 0101; 0102
(2015/01/07)
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- Surugamides A-E, cyclic octapeptides with four D-amino acid residues, from a marine streptomyces sp.: LC-MS-aided inspection of partial hydrolysates for the distinction of D- and L-amino acid residues in the sequence
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Surugamides A-E (1-5), cyclic octapeptides with four d-amino acid residues, were isolated from the broth of marine-derived Streptomyces sp. Their planar structures were determined by analyses of spectroscopic data, and the absolute configuration of constituent amino acid residues was determined by the Marfey's method. Differentiation of d-Ile and l-Ile in the sequence was established by chiral analysis of fragment peptides obtained from the partial hydrolysate, whose identification was conducted by LC-MS/MS.
- Takada, Kentaro,Ninomiya, Akihiro,Naruse, Masato,Sun, Yi,Miyazaki, Masayuki,Nogi, Yuichi,Okada, Shigeru,Matsunaga, Shigeki
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p. 6746 - 6750
(2013/07/26)
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- Using the 9-BBN group as a transient protective group for the functionalization of reactive chains of α-amino acids
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Achieving chemoselectivity is a longstanding challenge in chemical synthesis. This problem has been addressed using different approaches, but a definitive solution is still pending. For instance, in peptide chemistry, particularly with amino acids containing side chains functionalities with reactivity patterns similar to the main functional groups, such as aspartic and glutamic acids, and lysine and ornithine, specific semi-permanent protecting groups have been employed. The use of 9-borabicyclo[3.3.1]nonane (9-BBN-H) as a transient protective group for the selective protection of α-amino acids, which allows the chemoselective manipulation of the functional groups embedded in the side chains of the molecule, is described.
- Sanchez, Adrian,Calderon, Ernesto,Vazquez, Alfredo
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p. 1364 - 1372
(2013/07/05)
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- Identification of new peptide amides as selective cathepsin L inhibitors: The first step towards selective irreversible inhibitors?
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A small library of peptide amides was designed to profile the cathepsin L active site. Within the cathepsin family of cysteine proteases, the first round of selection was on cathepsin L and cathepsin B, and then selected hits were further evaluated for binding to cathepsin K and cathepsin S. Five highly selective sequences with submicromolar affinities towards cathepsin L were identified. An acyloxymethyl ketone warhead was then attached to these sequences. Although these original irreversible inhibitors inactivate cathepsin L, it appears that the nature of the warhead drastically impact the selectivity profile of the resulting covalent inhibitors.
- Torkar, Ana,Lenar?i?, Brigita,Lah, Tamara,Dive, Vincent,Devel, Laurent
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supporting information
p. 2968 - 2973
(2013/06/27)
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- In situ deprotection and incorporation of unnatural amino acids during cell-free protein synthesis
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The S30 extract from E. coli BL21 Star (DE3) used for cell-free protein synthesis removes a wide range of α-amino acid protecting groups by cleaving α-carboxyl hydrazides; methyl, benzyl, tert-butyl, and adamantyl esters; tert-butyl and adamantyl carboxamides; α-amino form-, acet-, trifluoroacet-, and benzamides and sidechain hydrazides and esters. The free amino acids are produced and incorporated into a protein under standard conditions. This approach allows the deprotection of amino acids to be carried out in situ to avoid separate processing steps. The advantages of this approach are demonstrated by the efficient incorporation of the chemically intractable (S)-4-fluoroleucine, (S)-4,5- dehydroleucine, and (2S,3R)-4-chlorovaline into a protein through the direct use of their respective precursors, namely, (S)-4-fluoroleucine hydrazide, (S)-4,5-dehydroleucine hydrazide, and (2S,3R)-4-chlorovaline methyl ester. These results also show that the fluoroand dehydroleucine and the chlorovaline are incorporated into a protein by the normal biosynthetic machinery as substitutes for leucine and isoleucine, respectively. Copyright
- Arthur, Isaac N.,Hennessy, James E.,Padmakshan, Dharshana,Stigers, Dannon J.,Lesturgez, Stéphanie,Fraser, Samuel A.,Liutkus, Mantas,Otting, Gottfried,Oakeshott, John G.,Easton, Christopher J.
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p. 6824 - 6830
(2013/06/26)
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- SEPARATING AGENT FOR CHROMATOGRAPHY
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A separating agent for chromatography is provided that is useful for the separation of specific compounds, e.g., for the optical resolution of amino acids. This separating agent for chromatography provides a higher productivity and contains a crown ether-like cyclic structure and optically active binaphthyl. This separating agent for chromatography containing a crown ether-like cyclic structure and optically active binaphthyl is provided by introducing a substitution group for binding to carrier into a specific commercially available 1,1′-binaphthyl derivative that has substituents at the 2, 2′, 3, and 3′ positions, then introducing a crown ether-like cyclic structure, and subsequently chemically bonding the binaphthyl derivative to the carrier through the substitution group for binding to carrier.
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Paragraph 0074; 0075
(2013/08/15)
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- Identification and characterization of a periplasmic aminoacyl- phosphatidylglycerol hydrolase responsible for Pseudomonas aeruginosa lipid homeostasis
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Background: Continuous adaptation of the bacterial membrane is required in response to changing environmental conditions. Results: Pseudomonas aeruginosa ORF PA0919 codes for an alanyl-phosphatidylglycerol hydrolase that is anchored to the periplasmic surface of the inner membrane. Conclusion: The elucidated enzymatic activity implies a new regulatory circuit for the fine tuning of cellular alanyl-phosphatidylglycerol concentrations. Significance: Lipid homeostasis is crucial for understanding antimicrobial susceptibility. Specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine (or with lysine) was shown to render various organisms less susceptible to antimicrobial agents and environmental stresses. In this study, we make use of the opportunistic pathogen Pseudomonas aeruginosa to decode ORF PA0919-dependent lipid homeostasis. Analysis of the polar lipid content of the deletion mutant ΔPA0919 indicated significantly enlarged levels of alanyl-PG. The resulting phenotype manifested an increased susceptibility to several antimicrobial compounds when compared with the wild type. A pH-dependent PA0919 promoter located within the upstream gene PA0920 was identified. Localization experiments demonstrated that the PA0919 protein is anchored to the periplasmic surface of the inner bacterial membrane. The recombinant overproduction of wild type and several site-directed mutant proteins in the periplasm of Escherichia coli facilitated a detailed in vitro analysis of the enzymatic PA0919 function. A series of artificial substrates (p-nitrophenyl esters of various amino acids/aliphatic acids) indicated enzymatic hydrolysis of the alanine, glycine, or lysine moiety of the respective ester substrates. Our final in vitro activity assay in the presence of radioactively labeled alanyl-PG then revealed hydrolysis of the aminoacyl linkage, resulting in the formation of alanine and PG. Consequently, PA0919 was termed alanyl-PG hydrolase. The elucidated enzymatic activity implies a new regulatory circuit for the appropriate tuning of cellular alanyl-PG concentrations.
- Arendt, Wiebke,Groenewold, Maike K.,Hebecker, Stefanie,Dickschat, Jeroen S.,Moser, Juergen
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p. 24717 - 24730
(2013/09/23)
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- Champacyclin, a new cyclic octapeptide from Streptomyces strain C42 isolated from the Baltic Sea
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New isolates of Streptomyces champavatii were isolated from marine sediments of the Gotland Deep (Baltic Sea), from the Urania Basin (Eastern Mediterranean), and from the Kiel Bight (Baltic Sea). The isolates produced several oligopeptidic secondary metabolites, including the new octapeptide champacyclin (1a) present in all three strains. Herein, we report on the isolation, structure elucidation and determination of the absolute stereochemistry of this isoleucine/leucine (Ile/Leu = Xle) rich cyclic octapeptide champacyclin (1a). As 2D nuclear magnetic resonance (NMR) spectroscopy could not fully resolve the structure of (1a), additional information on sequence and configuration of stereocenters were obtained by a combination of multi stage mass spectrometry (MSn) studies, amino acid analysis, partial hydrolysis and subsequent enantiomer analytics with gas chromatography positive chmical ionization/electron impact mass spectrometry (GC-PCI/EI-MS) supported by comparison to reference dipeptides. Proof of the head-to-tail cyclization of (1a) was accomplished by solid phase peptide synthesis (SPPS) compared to an alternatively side chain cyclized derivative (2). Champacyclin (1a) is likely synthesized by a non-ribosomal peptide synthetase (NRPS), because of high content of (D)-amino acids. The compound (1a) showed antimicrobial activity against the phytopathogen Erwinia amylovora causing the fire blight disease of certain plants.
- Pesic, Alexander,Baumann, Heike I.,Kleinschmidt, Katrin,Ensle, Paul,Wiese, Jutta,Suessmuth, Roderich D.,Imhoff, Johannes F.
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p. 4834 - 4857
(2014/02/14)
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- Peptide bond hydrolysis catalyzed by the Wells-Dawson Zr(α 2-P2W17O61)2 polyoxometalate
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In this paper we report the first example of peptide hydrolysis catalyzed by a polyoxometalate complex. A series of metal-substituted Wells-Dawson polyoxometalates were synthesized, and their hydrolytic activity toward the peptide bond in glycylglycine (GG) was examined. Among these, the Zr(IV)- and Hf(IV)-substituted ones were the most reactive. Detailed kinetic studies were performed with the Zr(IV)-substituted Wells-Dawson type polyoxometalate K 15H[Zr(α2-P2W17O 61)2]·25H2O which was shown to act as a catalyst for the hydrolysis of the peptide bond in GG. The speciation of K 15H[Zr(α2-P2W17O 61)2]·25H2O which is highly dependent on the pD, concentration, and temperature of the solution, was fully determined with the help of 31P NMR spectroscopy and its influence on the GG hydrolysis rate was examined. The highest reaction rate (kobs = 9.2 (±0.2) × 10-5 min-1) was observed at pD 5.0 and 60 °C. A 10-fold excess of GG was hydrolyzed in the presence of K 15H[Zr(α2-P2W17O 61)2]·25H2O proving the principles of catalysis. 13C NMR data suggested the coordination of GG to the Zr(IV) center in K15H[Zr(α2-P2W 17O61)2]·25H2O via its N-terminal amine group and amide carbonyl oxygen. These findings were confirmed by the inactivity of K15H[Zr(α2-P2W 17O61)2]·25H2O toward the N-blocked analogue acetamidoglycylglycinate and the inhibitory effect of oxalic, malic, and citric acid. Triglycine, tetraglycine, and pentaglycine were also fully hydrolyzed in the presence of K15H[Zr(α2- P2W17O61)2]·25H2O yielding glycine as the final product of hydrolysis. K15H[Zr(α 2-P2W17O61)2] ·25H2O also exhibited hydrolytic activity toward a series of other dipeptides.
- Absillis, Gregory,Parac-Vogt, Tatjana N.
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p. 9902 - 9910,9
(2012/12/11)
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- Synthesis of monolysyl advanced glycation endproducts and their incorporation into collagen model peptides
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The synthesis of advanced glycation endproducts (AGEs), CML, CEL, and pyrraline and their incorporation into collagen model peptides is reported. AGEs are modified amino acids that form on proteins such as collagen and are thought to play a significant role in the pathogenesis of many diseases, particularly diabetes. The synthesis and incorporation of these compounds into synthetic peptides is a key step in developing model systems with which to investigate AGE-modified proteins.
- Woods, Tom M.,Kamalov, Meder,Harris, Paul W. R.,Cooper, Garth J. S.,Brimble, Margaret
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p. 5740 - 5743
(2013/01/15)
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- THERMO-RESPONSIVE HYDROGEL COMPOSITIONS
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A thermo-responsive hydrogel, including a biocompatible monomer and/or polymer having an amino acid side chain. The hydrogel is thermo-responsive at a physiological temperature, and can include, incorporate, or encapsulate a treatment agent, such as a drug composition, a biomolecule, and/or a nanoparticle. The hydrogel is useful in delivering the treatment agent. The hydrogel is in a first physicochemical state for administration to a mammal. The hydrogel is thermo-responsive at a physiological temperature of the mammal, and changes to a second physicochemical state that is more solid than the first physicochemical state. In the second physicochemical state the thermo-responsive hydrogel releases the treatment agent.
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- Lysine racemase from a lactic acid bacterium, Oenococcus oeni: Structural basis of substrate specificity
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Oenococcus oeni, a lactic acid bacterium, possesses a lysine racemase, which has a specific activity towards basic amino acids. A comparison of amino acid residues around the active site suggested that Ile222 and Tyr354 of the Geobacillus stearothermophilus alanine racemase, which shares 60% sequence similarity with lysine racemase, were replaced by Thr224 and Trp355 in the O. oeni lysine racemase. T224I/W355Y double mutations significantly decreased the activity of lysine racemase, whereas I222T/Y354W double mutations endowed alanine racemase with lysine racemization activity. These results suggest that the two residues play an important role in lysine racemization.
- Kato, Shiro,Hemmi, Hisashi,Yoshimura, Tohru
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p. 505 - 508
(2013/02/25)
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- A fluorescent probe for detection of histone deacetylase activity based on aggregation-induced emission
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A tetraphenylethylene-derivative fluorescent probe for the one-step detection of histone deacetylases (HDAC) was developed. The deacetylation of the probe triggers electrostatic interaction between the molecules and automatically leads to fluorescence enhancement based on aggregation-induced emission (AIE). The Royal Society of Chemistry 2012.
- Dhara, Koushik,Hori, Yuichiro,Baba, Reisuke,Kikuchi, Kazuya
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supporting information
p. 11534 - 11536
(2013/01/15)
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- Luminmycins A-C, cryptic natural products from Photorhabdus luminescens identified by heterologous expression in Escherichia coli
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The 18 kb silent luminmycin biosynthetic pathway from Photorhabdus luminescens was cloned into a vector by using the newly established linear-linear homologous recombination and successfully expressed in Escherichia coli. Luminmycins A-C (1-3) were isolated from the heterologous host, and their structures were elucidated using 2D NMR spectroscopy and HRESIMS. Luminmycin A is a deoxy derivative of the previously reported glidobactin A, while luminmycins B and C most likely represent its acyclic biosynthetic intermediates. Compound 1 showed cytotoxicity against the human colon carcinoma HCT-116 cell line with an IC50 value of 91.8 nM, while acyclic 2 was inactive at concentrations as high as 100 μg/mL.
- Bian, Xiaoying,Plaza, Alberto,Mueller, Rolf,Zhang, Youming
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p. 1652 - 1655,4
(2020/09/09)
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- New conjugates of muramyl dipeptide and nor-muramyl dipeptide linked to tuftsin and retro-tuftsin derivatives significantly influence their biological activity
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The synthesis and biological activity of new conjugates of muramyl dipeptide (MDP) and nor-muramyl dipeptide (nor-MDP) with tuftsin and retro-tuftsin derivatives containing isopeptide bond between e-amino group of lysine and carboxyl group of simple amino
- Dzierzbicka, Krystyna,Wardowska, Anna,Rogalska, Ma?gorzata,Trzonkowski, Piotr
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body text
p. 217 - 223
(2012/08/29)
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- Microbial enantioselective removal of the N-benzyloxycarbonyl amino protecting group
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In order to deprotect N-carbobenzoxy-l-aminoacids (Cbz-AA) and related compounds, a series of microorganisms was selected from soil by enrichment cultures with Cbz-l-Glu as sole nitrogen source. A lyophilized whole-cell preparation of two Arthrobacter sp. strains grown on Cbz-Glu or Cbz-Gly exhibited a high cleavage activity. The conditions of hydrolysis have been optimized and a quantitative enantioselective deprotection of several Cbz-dl-amino acids was obtained, as well as the deprotection of N-carbamoylester derivatives of several synthetic amino compounds. The preparation of Cbz-d-allylglycine and l-allylglycine in high yield and high optical purity is described as an application of this method.
- Maurs, Michele,Acher, Francine,Azerad, Robert
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- Powder storage container
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PROBLEM TO BE SOLVED: To prevent a sealing member from breaking away from a device body even when the sealing member is bent. SOLUTION: A toner replenishing type developing device 4Y, 4M, 4C or 4B comprises a developing device body 4a, and a sealing member 4e. The sealing member 4e comprises an insertion and removal part 41a which a toner replenishing pipe 13Y, 13M, 13C, or 13B is inserted into and removed from, and a bending part 41b which bends by the insertion of the replenishing pipe 13Y, 13M, 13C or 13B into the insertion and removal part 41a, and is restored to its original state by the removal of the toner replenishing pipe 13Y, 13M, 13C or 13B from the insertion and removal part 41a. The bending part 41b of the sealing member 4e is formed in such a manner that the thickness t2 thereof in the insertion direction of the toner replenishing pipe 13Y, 13M, 13C or 13B at the bending part 41b is smaller than the thickness t1 of the insertion and removal part 41a in the insertion direction of the toner replenishing pipe 13Y, 13M, 13C or 13B at the insertion and removal part 41a. COPYRIGHT: (C)2007,JPOandINPIT
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- Analysis of catalytic determinants of diaminopimelate and ornithine decarboxylases using alternate substrates
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Diaminopimelate decarboxylase (DAPDC) and ornithine decarboxylase (ODC) are pyridoxal 5′-phosphate dependent enzymes that are critical to microbial growth and pathogenicity. The latter is the target of drugs that cure African sleeping sickness, while the former is an attractive target for antibacterials. These two enzymes share the (β/α)8 (i.e., TIM barrel) fold with alanine racemase, another pyridoxal 5′-phosphate dependent enzyme critical to bacterial survival. The active site structural homology between DAPDC and ODC is striking even though DAPDC catalyzes the decarboxylation of a D stereocenter with inversion of configuration and ODC catalyzes the decarboxylation of an L stereocenter with retention of configuration. Here, the structural and mechanistic bases of these interesting properties are explored using reactions of alternate substrates with both enzymes. It is concluded that simple binding determinants do not control the observed stereochemical specificities for decarboxylation, and a concerted decarboxylation/proton transfer at Cα of the D stereocenter of diaminopimelate is a possible mechanism for the observed specificity with DAPDC.
- Fogle, Emily J.,Toney, Michael D.
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experimental part
p. 1113 - 1119
(2012/06/04)
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- Thermodynamical characteristics of the reaction of pyridoxal-5'-phosphate with L-amino acids in aqueous buffer solution
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The reaction of pyridoxal-5'-phosphate with L-isomers of alanine, lysine, arginine, aspartic acid, glutamic acid, and glycine in phosphate buffer solution was studied by absorption spectroscopy and the calorimetry of dissolution at physiological acidity of the medium (pH 7.35). The formation constants of Schiff bases during reactions and changes in Gibbs energy, enthalpy, and entropy were determined. It was shown that the formation constant of the Schiff base and its spectral properties depend on the nature of the bound amino acid. The progress of the reaction with a majority of amino acids is governed by the entropy factor due to the predominant role of the dehydration effect of the reaction center of amino acids during chemical reactions. The intramolecular electrostatic interaction of an ionized phosphate group with the positively charged amino group on the end of the chain of amino acid residue stabilizes the Schiff bases formed by lysine and arginine. The extinction coefficient of the base, equilibrium constant, and the exothermic effect of the reaction then increase. The excess negative charge on the end of the chain of amino acid residues of aspartic and glutamic acids destabilizes the molecule of the Schiff base. In this case, the equilibrium constant decreases and the endothermic effect of the reaction increases. Pleiades Publishing, Ltd., 2011.
- Barannikov,Badelin,Venediktov,Mezhevoi,Guseinov
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scheme or table
p. 16 - 20
(2011/06/18)
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- Modulation of the pharmacological activities of secretory phospholipase A2 from Crotalus durissus cascavella induced by naringin
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In this work we have characterized the action of the naringin, a flavonoid found in grapefruit and known for its various pharmacological effects, which include antioxidant, blood lipid lowering and anticancer activity, on the structure and biochemical activities of a secretory phospholipase A (sPLA2) from Crotalus durissus cascavella, an important protein involved in the releasinge of arachidonic acid in phospholipid membranes. sPLA2 was incubated with naringin (mol:mol) at 37 °C and a discrete reduction in the UV scanning signal and a modification of the circular dichroism spectra were observed after treatment with naringin, suggesting modifications of the secondary structure of the protein. This flavonoid was able to decrease enzymatic activity and some pharmacological effects, such as myonecrosis, platelet aggregation, and neurotoxic activity caused by sPLA2, however, the inflammatory effect was not affected by naringin. In addition, small angle X-ray scattering (SAXS) data were collected for sPLA2 and naringin-treated sPLA2 to evaluate possible modifications of the protein structure. These structural investigations have shown that sPLA2 is an elongated dimer in solution and after treatment with naringin a conformational change in the dimeric configuration was observed. Our results suggest that structural modification may be correlated with the loss of enzymatic activity and alterations in pharmacological properties.
- Santos, Marcelo L.,Toyama, Daniela O.,Oliveira, Simone C. B.,Cotrim, Camila A.,Diz-Filho, Eduardo B. S.,Fagundes, Fabio H. R.,Soares, Veronica C. G.,Aparicio, Ricardo,Toyama, Marcos H.
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experimental part
p. 738 - 761
(2011/04/15)
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- Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties
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Aminopeptidase B (Ap-B) catalyzes the cleavage of arginine and lysine residues at the N-terminus of various peptide substrates. In vivo, it participates notably in the miniglucagon and cholecystokinin 8 processing, but the complete range of physiological functions of Ap-B remains to be discovered. Ap-B is a member of the M1 family of Zn2+-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX18E (Zn2+-binding site). In this study, mutagenesis and molecular modelling were used to investigate the enzymatic mechanism of Ap-B. Nineteen rat Ap-B mutants of the G 298XM300E301N302 motif and one mutant of the HEIS328HX18E motif were expressed in Escherichia coli. All mutations except G298P, G298S, and S328A abolished the aminopeptidase activity. The S328A mutant mimics the sequence of bovine Ap-B Zn2+-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G298S and G298P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl- anions. Moreover, the G298P mutant exhibits new substrate specificity. A structural analysis using circular dichroism, fluorescence spectroscopy, molecular modelling and dynamics was performed to investigate the role that residue G298 plays in the catalytic mechanism of Ap-B. Our results show that G298 is essential to Ap-B activity and participates to the substrate specificity of the enzyme.
- Pham, Viet-La?,Gouzy-Darmon, Cécile,Pernier, Julien,Hanquez, Chantal,Hook, Vivian,Beinfeld, Margery C.,Nicolas, Pierre,Etchebest, Catherine,Foulon, Thierry,Cadel, Sandrine
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experimental part
p. 730 - 741
(2012/05/04)
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- Stereochemical elucidation of new sagittamides CF from a didemnid ascidian
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Four new minor congeners, sagittamides CF, were isolated from an unidentified Didemnid tunicate that previously afforded sagittamides A and B. The structures were determined by interpretation of spectroscopic data, degradation to amino acids, and comparisons with sagittamide A. An unexpected change in relative configuration of the hexacetoxy C5C10 stereoelement is present in sagittamides D and F. A tentative assignment of configuration was possible through a systematic deduction based on analysis of 13C NMR data and symmetry considerations.
- Lievens, Sarah C.,Morinaka, Brandon I.,Molinski, Tadeusz F.
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experimental part
p. 935 - 941
(2011/08/09)
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- Glutamates 78 and 122 in the Active Site of Saccharopine Dehydrogenase Contribute to Reactant Binding and Modulate the Basicity of the Acid-Base Catalysts
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Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to give L-lysine and α-ketoglutarate. There are a number of conserved hydrophilic, ionizable residues in the active site, all of which must be important to the overall reaction. In an attempt to determine the contribution to binding and rate enhancement of each of the residues in the active site, mutations at each residue are being made, and double mutants are being made to estimate the interrelationship between residues. Here, we report the effects of mutations of active site glutamate residues, Glu78 and Glu122, on reactant binding and catalysis. Site-directed mutagenesis was used to generate E78Q, E122Q, E78Q/E122Q, E78A, E122A, and E78A/E122A mutant enzymes. Mutation of these residues increases the positive charge of the active site and is expected to affect the pKa values of the catalytic groups. Each mutant enzyme was completely characterized with respect to its kinetic and chemical mechanism. The kinetic mechanism remains the same as that of wild type enzymes for all of the mutant enzymes, with the exception of E78A, which exhibits binding of α-ketoglutarate to E and E·NADH. Large changes in V/K Lys, but not V, suggest that Glu78 and Glu122 contribute binding energy for lysine. Shifts of more than a pH unit to higher and lower pH of the pKa values observed in the V/KLys pH-rate profile of the mutant enzymes suggests that the presence of Glu78 and Glu122 modulates the basicity of the catalytic groups.
- Ekanayake, Devi K.,Andi, Babak,Bobyk, Kostyantyn D.,West, Ann H.,Cook, Paul F.
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experimental part
p. 20756 - 20768
(2011/04/18)
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- Structure, mechanism, and substrate profile for Sco3058: The closest bacterial homologue to human renal dipeptidase
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Human renal dipeptidase, an enzyme associated with glutathione metabolism and the hydrolysis of β-lactams, is similar in sequence to a cluster of ~400 microbial proteins currently annotated as nonspecific dipeptidases within the amidohydrolase superfamily. The closest homologue to the human renal dipeptidase from a fully sequenced microbe is Sco3058 from Streptomyces coelicolor. Dipeptide substrates of Sco3058 were identified by screening a comprehensive series of L-Xaa-L-Xaa, L-Xaa-D-Xaa, and D-Xaa-L-Xaa dipeptide libraries. The substrate specificity profile shows that Sco3058 hydrolyzes a broad range of dipeptides with a marked preference for an L-amino acid at the N-terminus and a D-amino acid at the C-terminus. The best substrate identified was L-Arg-D-Asp (kcat/Km = 7.6 x 105 M -1 s-1). The three-dimensional structure of Sco3058 was determined in the absence and presence of the inhibitors citrate and a phosphinate mimic of L-Ala-D-Asp. The enzyme folds as a (β/α)8 barrel, and two zinc ions are bound in the active site. Site-directed mutagenesis was used to probe the importance of specific residues that have direct interactions with the substrate analogues in the active site (Asp-22, His-150, Arg-223, and Asp-320). The solvent viscosity and kinetic effects of D2O indicate that substrate binding is relatively sticky and that proton transfers do not occurr during the rate-limiting step. A bell-shaped pH-rate profile for kcat and kcat/Km indicated that one group needs to be deprotonated and a second group must be protonated for optimal turnover. Computational docking of high-energy intermediate forms of L/D-Ala-L/D-Ala to the three-dimensional structure of Sco3058 identified the structural determinants for the stereochemical preferences for substrate binding and turnover.
- Cummings, Jennifer A.,Nguyen, Tinh T.,Fedorov, Alexander A.,Kolb, Peter,Xu, Chengfu,Fedorov, Elena V.,Shoichet, Brian K.,Barondeau, David P.,Almo, Steven C.,Raushel, Frank M.
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experimental part
p. 611 - 622
(2011/01/04)
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