- Detection of Localized Hepatocellular Amino Acid Kinetics by using Mass Spectrometry Imaging of Stable Isotopes
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Mass spectrometry imaging (MSI) simultaneously detects and identifies the spatial distribution of numerous molecules throughout tissues. Currently, MSI is limited to providing a static and ex vivo snapshot of highly dynamic systems in which molecules are constantly synthesized and consumed. Herein, we demonstrate an innovative MSI methodology to study dynamic molecular changes of amino acids within biological tissues by measuring the dilution and conversion of stable isotopes in a mouse model. We evaluate the method specifically on hepatocellular metabolism of the essential amino acid l-phenylalanine, associated with liver diseases. Crucially, the method reveals the localized dynamics of l-phenylalanine metabolism, including its in vivo hydroxylation to l-tyrosine and co-localization with other liver metabolites in a time course of samples from different animals. This method thus enables the dynamics of localized biochemical synthesis to be studied directly from biological tissues.
- Arts, Martijn,Soons, Zita,Ellis, Shane R.,Pierzchalski, Keely A.,Balluff, Benjamin,Eijkel, Gert B.,Dubois, Ludwig J.,Lieuwes, Natasja G.,Agten, Stijn M.,Hackeng, Tilman M.,van Loon, Luc J. C.,Heeren, Ron M. A.,Olde Damink, Steven W. M.
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- Transmucosal delivery of leucine enkephalin: Stabilization in rabbit enzyme extracts and enhancement of permeation through mucosae
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Leucine enkephalin (Tyr-Gly-Gly-Phe-Leu; Leu-Enk) is a naturally occurring peptide that has been shown to have pain modulating properties. To evaluate the feasibility of using various absorptive mucosae as a route of systemic delivery, the stability of Leu-Enk and the effect of enzyme inhibitors (e.g., amastatin, EDTA, and thimerosal) on stabilization and permeation of Leu-Enk through rabbit mucosae in the presence of dihydrofusidates were investigated. Enzymes in the nasal, rectal, and vaginal mucosae were extracted and Leu-Enk (50 μg/mL) was added to each of the enzyme extracts and incubated to determine the kinetics and mechanism of degradation. The rate of degradation in the extracts in the absence of inhibitors followed the order: rectal > vaginal > nasal. Whereas EDTA had the best stabilizing effect on Leu-Enk, thimerosal was the best stabilizer for the degradation intermediates. A combination of amastatin (50 μM), EDTA (5 mM), and thimerosal (50 μM) had the greatest stabilizing effect on Leu-Enk and its degradation intermediates. For permeation studies, each mucosa was mounted onto a Valia-Chien permeation cell with Leu-Enk (200 μg/mL) in isotonic phosphate buffer (as donor solution). The enhancers used for the study were sodium tauro-dihydrofusidate (STDHF), sodium glycodihydrofusidate (SGDHF), and phosphato-dihydrofusidate (PHDHF). The greatest effect was achieved by PHDHF for all the mucosae. STDHF had a significant effect only on the rectal permeation, whereas SGDHF had significant effects on rectal and vaginal mucosae. Mechanisms by which the dihydrofusidates enhance permeation may involve micelle formation. Thus, the use of enzyme inhibitors and dihydrofusidates in combination has made transmucosal delivery of Leu-Enk a viable option.
- Sayani,Chun,Chien
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- Resolution of amino acids in a mixture of 2-methyl-2-propanol/water (19:1) catalyzed by alcalase via in situ racemization of one antipode mediated by pyridoxal 5-phosphate
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Procedures for the conversion of a racemic amino acid into the L-enantiomer by the alcalase catalyzed resolution of the amino acid ester in 2-methyl-2-propanol/water (19:1) simultaneously with the pyridoxal 5-phosphate-catalyzed racemization of the unhydrolyzed antipode have been developed.
- Chen,Huang,Wang
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- Kinetic of adsorption and of photocatalytic degradation of phenylalanine effect of pH and light intensity
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Phenylalanine (Phe) was chosen to study the TiO2 photocatalytic degradation of amino acids, which are at the origin of the formation of odorous compounds after chlorination. The photocatalytic degradation has been investigated in aqueous solutions containing TiO2 suspensions as photocatalyst, in order to assess the influence of various parameters, such as adsorption, initial concentration, pH and radiant flux on the photocatalytic process. Results showed no correlation between dark adsorption and photocatalytic degradation. A multilayer kinetic was observed in the dark with a monolayer corresponding to less that 1% of OH covered, whereas Langmuir-Hinshelwood model seems to modelize the photocatalytic disappearance of Phe. However, even if the form of the curve is similar to L-H model, the degradation of phenylalanine is not a kinetic of L-H as we could plan it by considering the adsorption of the phenylalanine in the dark. The study of the mineralization of carbon and nitrogen showed that nitrogen atoms were predominantly photoconverted into NH4+ and a total mineralization of nitrogen and carbon seems occur. The identification of the by-products by LC-MS reveal mono- and di-hydroxylation and nitrogen-carbon (N-C) cleavage. The effect of pH showed an increase of adsorption under acid pH but a decrease of disappearance rate. The more efficient degradation was found at basic pH. The evolution of hydroxylated compounds of phenylalanine as a function of conversion revealed the presence of more hydroxylated compounds at natural pH and at basic pH compared to acid pH suggesting a modification of mechanism with solution pH. The effect of the radiant flux evaluated under different initial concentration of phenylalanine allowed us to determine that Κ increases by increasing the radiant flux, whereas Κ decreases or remains constant from about a value of 3.5 mW/cm2. The disappearance rate as a function of radiant flux has been showed to reach a maximal value corresponding to a maximal quantum yield of 1.6%.
- Elsellami,Vocanson,Dappozze,Puzenat,Pa?sse,Houas,Guillard
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- A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom
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Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.
- Siigur, Ene,T?nism?gi, Külli,Trummal, Katrin,Samel, Mari,Vija, Heiki,Aasp?llu, Anu,R?nnholm, Gunilla,Subbi, Juhan,Kalkkinen, Nisse,Siigur, Jüri
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- Photolysis of phenylalanine in the presence of oxidized carbon nanotubes
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Photolyses at 254 nm of phenylalanine (Phe) in aqueous solutions, were carried out in the presence of oxidized carbon nanotubes modified by the reaction with SO2 (mNTO). Kinetics of the photolyses were followed by UV spectrophotometry at 220 nm, and the products were characterized by HPLC, XPS, and 13C-SSNMR. The ratio of the initial rates of photolysis in the presence and absence of mNTO, k/ko, showed a systematic decrease. The photolytic decay of Phe occurs with minor formation of tyrosine. The mass of nanotubes produced an exponential attenuation of the photolytic decomposition of Phe. Total carbon analyses (TCA) showed no inorganic carbon formation after the photolyses. The first-order rate constant of photofunctionalization of mNTO by the insertion of phenylalanine onto the nanotube matrix was calculated from TCA to be kin = 30.1 min-1. Comparison of the XPS spectra of the mNTO before and after the photolysis, using the atom inventory technique, suggests the insertion of Phe along with the extrusion of a sulfide radical anion (?S-) which undergo subsequent oxidation to SO42-. The obtained results show the effects of mNTO on the photolysis of Phe and provide a new method of photofunctionalization of carbon materials, modified by the intermediates of the reduction of SO2, with an organic moiety.
- Humeres, Eduardo,De Souza, Eduardo Pinheiro,Debacher, Nito Angelo,Moreira, Regina De F.P.M.,Lopes, Cristiane Nunes,Fernndez, Ma Isabel,Santaballa, J. Arturo,Canle, Moiss L.,Schreiner, Wido H.,Aliev, Abil E.
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- Optimisation of the retroracemisation procedure for α-amino acids using (S)-2-[(N-alkylprolyl)amino]benzophenones, recyclable chiral axiliaries
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The retroracemisation procedure developed by Belokon and coworkers has been re-examined using a variety of new (S)-2-[N-alkylprolyl)amino]benzophenone chiral auxiliaries. It has been found that (S)-2-[(N-benzylprolyl)amino] and (S)-2-[(N-1-(naphthalenyl-1-methyl)prolyl)amino] benzophenones ((S)-BPB and (S)-NPB) when used in conjunction with Ni(NO3)2·6H2O and a racemic α-amino acid preferentially form a single diastereoisomer in the presence of a mild base such as sodium methoxide. Decomposition of this complex under acidic conditions leads to the isolation of the (S)-amino acid in good yield, and in 55 to 99% e.e. The retroracemisation abilities of a polymer supported form of the (S)-BPB ligand have also been investigated and preliminary results for this are presented here.
- De, Binod B.,Thomas, Neil R.
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- Hydroxylation of Phenylalanine by Aqueous H2O2 in the Presence of an Artificial Water-soluble Iron Porphyrin Complex
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Phenylalanine is hydroxylated by aqueous H2O2 in the presence of a catalytic amount of an artificial water-soluble iron porphyrin complex (1) to give monohydroxylated and dihydroxylated products, i.e., tyrosine and dihydroxyphenylalanine, which are obtained in good yields.
- Shimidzu, Takeo,Iyoda, Tomokazu,Kanda, Naoya
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- IMPROVED DEPROTECTION IN SOLID PHASE PEPTIDE SYNTHESIS: REMOVAL OF PROTECTING GROUPS FROM SYNTHETIC PEPTIDES BY AN SN2 MECHANISM WITH LOW CONCENTRATIONS OF HF IN DIMETHYLSULFIDE
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The use of a low-concentration HF in dimethylsulfide (1:3, v/v) for the removal of protecting groups from synthetic peptides has been found to be efficient and to eliminate several side reactions associated with the high-concentration HF in anisole (9:1, v/v) normally employed in peptide synthesis.The mechanism of the low-concentration HF cleavage is SN2, in contrast to the SN1 mechanism of the high-concentration HF cleavage, and consequently the reaction proceeds without generation of carbocation intermediates.
- Tam, James P.,Heath, William F.,Merrifield, R. B.
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- Enzymatic Reactions in Aqueous-Organic media. XVII. Optical Resolution of Amino Acid Esters by Enzymatic Hydrolysis in Organic Solvents
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Enantiospecific hydrolysis of DL-tyrosine ethyl ester was carried out using α-chymotrypsin (CT) as a catalyst in organic solvents, and L-tyrosine was obtained with high optical purity.Activity and enantiospecificity of CT were found to be greatly altered by water content in the reaction media, and generally high enantiospecificity was observed at low water contents.Many of natural and unnatural amino acid esters were resolved by hydrolysis in organic solvents using CT, subtilisin Carlsberg, or subtilisin BPN' as catalysts.
- Tomiuchi, Yoshimasa,Ohshima, Kouji,Kise, Hideo
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- Evaluation of degradation pathways for plasmid DNA in pharmaceutical formulations via accelerated stability studies
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The stability of highly purified supercoiled plasmid DNA formulated in simple phosphate or Tris-buffered saline solutions has been characterized to establish the overall degradation processes that occur during storage in aqueous solution. Plasmid DNA stability was monitored during accelerated stability studies (at 50°C) by measurements of supercoiled, open-circle, and linear DNA content, as well as the accumulation of apurinic sites and 8- hydroxydeoxyguanosine residues over time. The effects of formulation pH, demetalation, metal ion chelators, and ethanol (hydroxyl radical scavenger) on the supercoiled content of plasmid DNA during storage at 50°C were also determined. The results indicate that free radical oxidation may be a major degradative process for plasmid DNA in pharmaceutical formulations unless specific measures are taken to control it by the addition of free radical scavengers, specific metal ion chelators, or both. The generation of hydroxyl radicals in phosphate-buffered saline was confirmed by examining the hydroxylation of phenylalanine over time by reverse phase high-performance liquid chromatography. Ethanol was found to enhance plasmid DNA stability and to inhibit the hydroxylation of phenylalanine; both observations are consistent with the known ability of ethanol to serve as a hydroxyl radical scavenger. Moreover, the combination of ethylenediamine tetraacetic acid (EDTA) and ethanol had a synergistic enhancing effect on DNA stability. However, the metal ion chelator diethylenetriaminepentaacetic acid (DTPA) was as potent as the combination of EDTA and ethanol for enhancing the stability of plasmid DNA. By controlling free radical oxidation with EDTA and ethanol, the rate constants of plasmid DNA degradation by means of depurination and β-elimination were then determined, allowing accurate predictions of DNA storage stability as a function of formulation pH and temperature. The ability to predict plasmid DNA storage stability in the absence of free radical oxidation should prove to be a valuable tool for the design of stable pharmaceutical formulations of plasmid DNA. (C) 2000 Wiley-Liss, Inc.
- Evans, Robert K.,Xu, Zheng,Bohannon, Kathryn E.,Wang, Bei,Bruner, Mark W.,Volkin, David B.
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- L-tyrosine β-naphthylamide is a potent competitive inhibitor of tyramine n-(hydroxycinnamoyl)transferase in vitro
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L-Tyrosine β-naphthylamide, a synthetic substrate designed to measure tyrosine aminopeptidase activity, is a potent inhibitor of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) purified from elicited tobacco cell-suspension cultures. T
- Negrel, Jonathan,Javelle, Francine
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- Phosphorylation of enkephalins enhances their proteolytic stability
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Pharmacological action of enkephalins as opioid peptides is limited because of their rapid degradation by endoproteases. A novel approach is used in this study to prolong the life of those peptides. Phosphorylation of N-terminal tyrosine residue is found to have a profound influence in improving the stability of [Met]enkephalin and [Leu]enkephalin against the action of aminopeptidase M. Whereas, breakdown of [Met]enkephalin and [Leu]enkephalin is essentially complete in less than one min when incubated at 37°C with purified aminopeptidase M (EC 3.4.11.2; substrate:enzyme = 1:0.1) in Tris buffer (pH 7.02), the corresponding phospho analogs are still detected 60 min after start of incubation. The rate of disappearance of phospho-[Met]enkephalin and phospho-[Leu]enkephalin follows first-order kinetics with half-lives of 7.3 and 8.8 min, respectively.
- Dass, Chhabil,Mahalakshmi
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- Enantioselective reductive amination of α-keto acids to α-amino acids by a pyridoxamine cofactor in a protein cavity
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Adipocyte lipid binding protein (ALBP) is a small 131 residue protein with a simple architecture that consists of two orthogonal planes of β-sheet secondary structure. This protein binds a variety of fatty acids in a large cavity formed between the two sheets such that the bound ligands are completely enclosed within the protein. In this paper, the synthesis of an ALBP conjugate (ALBP-PX) containing a pyridoxamine cofactor attached to a thiol within the protein interior is described. The conjugate was characterized by mass spectrometry, UV/vis spectroscopy, and gel filtration chromatography. ALBP-PX reductively aminates a number of alkyl, aryl, and side chain functionalized α-keto acids to α-amino acids with enantioselectivities as high as 94% ee.
- Kuang, Hao,Brown, Matthew L.,Davies, Ronald R.,Young, Eva C.,Distefano, Mark D.
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- Anti-inflammatory amino acid derivatives from the ascidian Herdmania momus
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Four new amino acid derivatives, herdmanines A-D (1-4), were isolated from the marine ascidian Herdmania momus. Herdmanines A-C contain the unusual d-form of arginine. Compounds 3 and 4 had a moderate suppressive effect on the production of NO, with IC50 values of 96 and 9 μM, respectively. These compounds were found to inhibit the mRNA expression of iNOS. The inhibitory activities on the production and mRNA expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were evaluated.
- Li, Jian Lin,Han, Sang Chul,Yoo, Eun Sook,Shin, Sook,Hong, Jongki,Cui, Zheng,Li, Huayue,Jung, Jee H.
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- Iotrochamides A and B, antitrypanosomal compounds from the Australian marine sponge Iotrochota sp.
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Bioassay-guided isolation of the CH2Cl2/MeOH extract from the Australian sponge Iotrochota sp. resulted in the purification of two new N-cinnamoyl-amino acids, iotrochamides A (1) and B (2). The chemical structures of 1 and 2 were determined by 1D/2D NMR and MS data analyses. Compounds 1 and 2 were shown to inhibit Trypanosoma brucei brucei with IC 50 values of 3.4 and 4.7 μM, respectively.
- Feng, Yunjiang,Davis, Rohan A.,Sykes, Melissa L.,Avery, Vicky M.,Quinn, Ronald J.
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- Free-radical Formation in the Pulse-radiolysis Oxidation of Inactive Escherichia coli Met-R2 Ribonucleotide Reductase
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The regeneration of a tyrosyl radical from the inactive Escherichia coli met-R2 enzyme using pulse radiolytically generated azide radicals N3(.) has been studied.Tryptophan and tyrosine amino acid residues on the protein are reactive towards N3(.) with the formation of either a tryptophan radical (peak at 510 nm) or a tyrosyl radical (peak at 410 nm).The combined rate constant (19 deg C) obtained for the formation of both these deprotonated species in N2O-saturated solution, = 0.012 M, pH 7 (40 mM phosphate), and I = 0.100 M, is 1.75 x 1E9 M-1 s-1.Biphasic decay of the tryptophan radical is then observed, rate constants 2.9 x 1E3 and 7.3 x 1E2 s-1, which are assigned as intramolecular electron-transfer steps.In the faster of these the tryptophan radical receives an electron from a tyrosine to give a tyrosyl radical product.The tyrosyl radicals formed in both the primary and secondary processes decay within 1 s in contrast to the much longer-lived tyrosyl-122 radical present in active R2 enzyme.Similar results were obtained on pulse radiolysis of the mutant Tyr122Phe R2 protein.Thus the stable Tyr-122 radical form of R2 does not appear to be formed in any of these reactions, and the specificity of the regeneration of the radical in the native enzyme involving reaction of the diiron(II) site with O2 is highlighted.
- Lam, Kin-Yu,Govindaraju, K.,Han, Joo-Yeon,Salmon, G. Arthur,Sykes, A. Geoffrey
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- Substrate profiling of protein tyrosine phosphatase PTP1B by screening a combinatorial peptide library
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Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. To date, the in vivo substrates and physiological functions of PTPs remain poorly defined. In this work, we have developed a novel combinatorial library method to systematically determine the substrate specificity of PTPs. A one-bead-one-compound peptide library containing five randomized residues, Fmoc-XXXXXpYAA (where X is norleucine or 17 proteinogenic amino acids excluding Tyr, Cys, and Met), was chemically synthesized on 90-μm TentaGel resin by the split-and-pool method. Limited treatment of the library with a PTP removed the phosphoryl group from beads that carry the most preferred substrates. The exposed tyrosine side chain was selectively oxidized into an orthoquinone by the treatment with tyrosinase in the presence of atmospheric oxygen. The orthoquinone was then selectively derivatized with biotin-hydrazide, followed by on-bead colorimetric assay. The positive beads were isolated and individually sequenced by partial Edman degradation/mass spectrometry to give the most preferred substrates of the PTP. Screening of the pY library against PTP1B confirmed the previously reported PTP1B specificity, but also revealed a second class of previously unrecognized peptide substrates. Several selected peptides were individually synthesized and assayed against PTP1B and the kinetic data confirmed the screening results. This method is generally applicable to studying the substrate specificity of other PTPs. Copyright
- Garaud, Mathieu,Pei, Dehua
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- Ianthesines A-D, four novel dibromotyrosine-derived metabolites from a marine sponge, Ianthella sp.
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Novel dibromotyrosine-derived metabolites, ianthesines A, B, C, and D, were isolated from an Australian marine sponge of the genus Ianthella sp. Their structures were elucidated using chemical and spectroscopic techniques. Ianthesines A, B, and D were derived from two dibromotyrosines. Ianthesine C is a tetramer possessing eight bromine atoms and its molecular weight is 1606. Ianthesines B-D showed Na,K-ATPase inhibitory activity in the range of 50-440 μM, whereas ianthesine A is inactive. (C) 2000 Elsevier Science Ltd.
- Okamoto, Yoshihiro,Ojika, Makoto,Kato, Shigemasa,Sakagami, Youji
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- Kinetic characterization of O-phospho-L-tyrosine phosphohydrolase activity of two fungal phytases
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Fungal phytases belonging to "histidine acid phosphatase" or HAP class of phosphohydrolases that catalyze the hydrolysis of phytic acid could also hydrolyze O-phospho-L-tyrosine, which is also called phosphotyrosine. Two phytases from Aspergillus niger and Aspergillus awamori with pH optima 2.5 were tested for phosphotyrosine hydrolase activity; both enzymes cleaved the phosphomonoester bond of phosphotyrosine efficiently at acidic pH. The K m for phosphotyrosine ranged from 465 to 590 μM as opposed to 135 to 160 μM for phytate. The Vmax, however, is 2-4 times higher for phosphotyrosine than it is for phytate. The catalytic efficiency of phytase for phosphotyrosine is on the same order as it is for phytate (3.5 × 10 6 to 1.6 × 107 M-1 s-1); the pH versus activity profile for phosphotyrosine is, however, different from what it is for phytate. The temperature optima shifted 5°C higher to 70°C when phosphotyrosine was used as the substrate. Taken together, the kinetic data show that fungal HAPs that are known as PhyB are capable of cleaving the phosphomonoester bond in phosphotyrosine. This is the first time that phosphotyrosine phosphatase (PTPase) activity has been reported for the subgroup of HAP known as phytase.
- Ullah, Abul H. J.,Sethumadhavan, Kandan,Mullaney, Edward J.
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- A Novel Cyclodepsipeptide, HA23, from a Fusarium sp
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(Matrix Presented) HA 23, a novel cyclodepsipeptide (1) of mixed peptide-polyketide origins, was isolated from a fungal isolate of a Fusarium sp. The structure was determined from 1D and 2D NMR and mass spectral data.
- Feng, Yunjiang,Blunt, John W.,Cole, Anthony L. J.,Munro, Murray H. G.
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- Aromatic hydroxylations by flavins: Evidence on direct attack of phenylalamine by flavin radical species
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In 0.05 - 12.0 N acidic solutions, the 5-ethyl-3-methyllumiflavosemiquinone 5 (and/or 5H+) spontaneously arose from the corresponding flavinium cation 4. Raising the temperature from 20 to 50°C, considerably increased the reaction rates with no significant changes in the yields of 5 (5H+). The spontaneous one-electron reduction of 4 requires a coupling with a one-electron oxidation of another flavin such as 5-ethyl-4(a)-hydroxy-3-methyl-4(a),5-dihydroflavin pseudobase 1. The latter, being in equilibrium with 4, can be oxidized to give the transient 5-ethyl-4(a)-hydroxy-3-methyllumiflavin radical 2. This is the protonated form of a flavinoxy radical 3(a,b), a product of a homolysis of the O-O bond in a dihydroflavin hydroperoxide. As an alternative to the homolysis mentioned, the one-electron oxidation of 1 provides the principle to develop a new hydroxylating model system that does not require a dihydroflavin hydroperoxide as a starting compound. Using phenylalanine as a test substrate, the anaerobic formation of tyrosine and its o- and m-hydroxyphenylalanine isomers was established. This achievement is a strong experimental support for the hypothesis that flavin radical species like 2 may directly attack an aromatic. Evidence was obtained on some accumulation of an intermediate that is not a hydroxycyclohexadienyl radical. It was shown to react in a secondary, oxidative chain reaction, remarkably increasing the yields of aromatic hydroxylation without any further supply of flavin.
- Mager,Tu
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- Cavity effects on the enantioselectivity of chiral amido[4]resorcinarene stereoisomers
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The dramatic effects of the size and hydrophilic/hydrophobic properties of cavities on the intrinsic reaction kinetics and dynamics is shown by the gas-phase reaction of (R)-(-)-2-butylamine and complexes of stereoisomeric amido[4]resorcinarene hosts with aromatic amino acids. The graph shows the kinetic plots of the base-induced loss of L-Phe (green), L-Tyr (red), and L-dopa (blue).
- Botta, Bruno,Subissati, Deborah,Tafi, Andrea,Delle Monache, Giuliano,Filippi, Antonello,Speranza, Maurizio
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- Reversibility of Charge Transfer between Tryptophan and Tyrosine
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Whereas tryptophan radicals oxidise tyrosine over a wide range of pH values, the one-electron reduction potential of the electron-deficient tryptophan radical at pH 7 is only 0.093 V more positive than that of the corresponding tyrosine radical, so that the reaction proceeds in reverse in strongly acid and alkaline solution.
- Butler, John,Land, Edward J.,Pruetz, Walter A.,Swallow, A. John
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- Rapid and controllable hydrogen/deuterium exchange on aromatic rings of α-amino acids and peptides
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Novel hydrogen/deuterium exchange for aromatic α-amino acids and their corresponding peptides were performed through the use of deuterated trifluoromethanesulfonic acid (TfOD). Detailed analysis of the exchange revealed that equal hydrogen/deuterium excha
- Murai, Yuta,Wang, Lei,Masuda, Katsuyoshi,Sakihama, Yasuko,Hashidoko, Yasuyuki,Hatanaka, Yasumaru,Hashimoto, Makoto
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- Identification of phenylalanine 3-hydroxylase for meta -tyrosine biosynthesis
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Phenylalanine hydroxylase (PheH) is an iron(II)-dependent enzyme that catalyzes the hydroxylation of aromatic amino acid l-phenylalanine (l-Phe) to l-tyrosine (l-Tyr). The enzymatic modification has been demonstrated to be highly regiospecific, forming proteinogenic para-Tyr (p-Tyr) exclusively. Here we biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr) from Phe. Subsequent mutagenesis studies revealed that two residues in the active site of Phe3H (Cys187 and Thr202) contribute to C-3 rather than C-4 hydroxylation of the phenyl ring. This work sets the stage for the mechanistic and structural study of regiospecific control of the substrate hydroxylation by PheH.
- Zhang, Wenjun,Ames, Brian D.,Walsh, Christopher T.
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- Characterization of diketopiperazine heterodimers as potential chemical markers for discrimination of two dominant black aspergilli, Aspergillus niger and Aspergillus tubingensis
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Black aspergilli are distributed worldwide and represent one of the most prolific sources of metabolites with biomedical and agrochemical interests. However, due to their similar morphological characteristics and insufficient molecular identification, the taxonomic classification of black aspergilli remains ill-defined. The production of specialised metabolites is often unique for species among black aspergilli and could be used as diagnostic chemical markers for species identification. In this study, chemical investigation of Aspergillus tubingensis OUCMBIII 143291 led to the discovery of the diagnostic chemical marker asperazine, a complex diketopiperazine heterodimer, as well as two previously undescribed analogues, asperazine B and C. In addition, an undescribed 2-benzylpyridin-4(1H)-one-containing amide, pestalamide D, along with four known related metabolites were isolated. Their chemical structures, including their absolute configurations, were established on the basis of comprehensive spectral analysis and chiral HPLC analysis of the acidic hydrolysates. Asperazines B and C can serve as potential chemical markers for distinguishing A. tubingensis from A. niger, two representative species of black aspergilli that are usually incorrectly identified. Moreover, the isolated compounds were evaluated for their antifungal activity against eight phytopathogenic fungi including Alternaria alternata, A. brassicae, Botrytis cinerea, Colletotrichum lagenarium, Fusarium oxysporum, Gaeumannomyces graminis, Penicillium digitatum, and Valsa mali. Pestalamide D exhibited significant activities against B. cinerea, C. lagenarium, and V. mali, with MIC values of 4, 8, and 8 μg/mL, respectively, compared with the positive controls carbendazim (MICs = 8, 4, and 4 μg/mL) and prochloraz (MICs = 8, 8, and 4 μg/mL). The results of this study reveal two additional chemical markers and provide a powerful tool for the rapid identification of black aspergilli.
- Deng, Ning,Li, Wei,Ren, Guang-Wei,Wang, Xiao-Qiang,Wang, Xiu-Fang,Xu, Ce,Xu, Kuo,Yuan, Xiao-Long,Zhang, Peng
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- Cytotoxicity of okadaic acid and kinetic characterization of protein tyrosine phosphatase activity in V79 fibroblasts
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Protein phosphorylation and dephosphorylation mediate signal transduction events that control several important cellular processes. The aim of this work was to determine some kinetic properties of a protein phosphatase obtained from non-metabolizing V79 Chinese hamster cells. The effect of okadaic acid, a tumour promoter, on these cell cultures was also determined. Phosphatase activity was assayed in V79 cells which were lysed with 0.1 M imidazole buffer (pH 7.4). Enzyme activity was determined using p-nitrophenylphosphate and tyrosine phosphate (TyrP) as substrates. Maximum phosphatase activity was obtained at pH7·4. The apparent K(m) (Michael's constant) and specificity constant for TyrP were 0·06 nm and 57, respectively. Phosphatase activity was inhibited by 100μM pCMB (p-chloro-mercuribenzoate; 70%), 10 mM fluoride (30%), 10 mM phosphate (40%), 100 μM m-vanadate (80%) and 100 μM o-vanadate (90%). Tartrate (5 mM) had no effect. The low K(m) value for TyrP and the high level of inhibition by vanadate suggest that the V79 phosphatase is a protein tyrosine phosphatase. The cytotoxic effect of okadaic acid was evaluated and the IC50 was 15, 20, 35 and 45 nM for nucleic acid content, neutral red uptake, MTT and protein phosphatase assays, respectively. These results agree with the kinetic data, indicating that the V79 phosphatase is a protein tyrosine phosphatase, as enzyme activity was stimulated when the cells were treated with okadaic acid. Serine/threonine protein phosphatases are completely inhibited by okadaic acid. The phosphatase enzyme system may be useful for studying cellular adaptation to apoptosis, oxidative stress and other conditions.
- Aoyama,Melo,Granjeiro,Haun,Ferreira
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- Effects of three peptidase inhibitors, amastatin, captopril and phosphoramidon, on the hydrolysis of [Met5]-enkephalin-Arg6-Phe7 and other opioid peptides
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The contents of [Met5]-enkephalin-Arg6-Phe7 (met-enk-RF) and its six hydrolysis products: Y, YG, YGG, YGGF, YGGFM, and YGGFMR were estimated after incubating met-enk-RF with either a guinea-pig ileal or striatal membrane fraction for various times at 37°C. After 45 min incubation with either ileal or striatal membranes, met-enk-RF was completely hydrolyzed, yielding Y as the major product. Incubation with either membrane preparation for 60 min in the presence of the aminopeptidase inhibitor amastatin hydrolyzed 90 or 92% of met-enk-RF, respectively, with YGG being the major product. If the dipeptidyl carboxypeptidase I inhibitor captopril is also included in the incubation, met-enk-RF hydrolysis decreases by about half for both membranes, with YGG remaining the major product. Inclusion of three peptidase inhibitors, amastatin, captopril, and phosphoramidon (inhibition of endopeptidase-24.11) further reduced met-enkhydrolysis, with 87% or more remaining intact. This shows that met-enk-RF was mainly hydrolyzed by three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I and phosphoramidon-sensitive endopeptidase-24.11, in both ileal and striatal membranes. Additionally, estimations of [Leu5]-enkephalin (leu-enk), α- and β-neoendorphins (α- and β-neoends), and dynorphin B (dyn B) contents after incubating the individual peptides with striatal membrane for 60 min in the presence of the three peptidase inhibitors showed that 98, 32, 5, and 23%, respectively, remained intact. Our previous studies together with the data obtained here show that one group of endogenous opioid peptides: met-enk, leu-enk, met-enk-RF, met-enk-RGL, and dyn A-(1-8) are largely or almost exclusively hydrolyzed by the three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I, and phosphoramidon-sensitive endopeptidase-24.11, and indicate that an unidentified fourth enzyme(s) is involved in the hydrolysis of another group of peptides: α-neoend, β-neoend, and dyn B.
- Hiranuma, Toyokazu,Kitamura, Ken,Taniguchi, Takao,Kobayashi, Tomomi,Tamaki, Raita,Kanai, Masayuki,Akahori, Kazuhito,Iwao, Kayoko,Oka, Tetsuo
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- Ingredients contribute to variation in production of reactive oxygen species by areca quid
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Areca quid (AQ) chewing has been implicated an independent risk factor for the development of oral cancer. Taiwanese areca quid (AQ) refers to a combination of areca nut (AN), lime, and inflorescence of Piper betle Linn. (IPB) or Piper betle leaf (PBL). Studies of AQ in other countries reported that AN extract combined with lime generates reactive oxygen species (ROS), such as hydroxyl radical (HO? ), known to be a contributing factor in oral mucosa damage. To determine whether HO? is formed in the oral cavity during AQ chewing, the formation of meta -tyrosine ( m -Tyr) and ortho -tyrosine ( o -Tyr) from l -phenylalanine (Phe) was confirmed. It was demonstrated that combined aqueous extracts of AN, lime, metal ions (such as Cu 2+ and Fe 2+ ), and IPB or PBL produced HO?. Thus, the yield of HO? significantly increases when higher amounts of IPB or lime are added and also when Cu 2+ and Fe 2+ are increased. Further, the omission of any one of these ingredients significantly reduces the formation of HO ?. Our results found that chewing AQ with IPB generated significantly higher HO? than chewing AQ with PBL, and may result in greater oxidative damage to the surrounding oral mucosa. Copyright Taylor & Francis Group, LLC.
- Chen, Ping-Ho,Tsai, Chi-Cheng,Lin, Ying-Chu,Ko, Ying-Chin,Yang, Yi-Hsin,Shieh, Tien-Yu,Ho, Pei-Shan,Li, Chien-Ming,Min-Shan Ko, Albert,Chen, Chung-Ho
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- OXIDATION OF L-PHENYLALANINE BY THE MODIFIED UDENFRIEND SYSTEM
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L-phenylalanine is oxidized by oxygen into 3,4-dihydroxyphenylalanine (DOPA) at 40 deg C with Fe(2+) EDTA as a catalyst.For the reduction of Fe(3+) species, electrons are released from the cathode of an electrochemical cell.
- Blanchard, M.,Bouchoule, C.,Djaneye-Boundjou, G.,Canesson, P.
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- Interaction of mercury and copper on papain and their combined inhibitive determination
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Influence and interaction of mercury ion (Hg2+) and copper ion (Cu2+) on papain activity in casein hydrolysis were investigated. Single Hg2+ or Cu2+ at low concentrations induced an increase in papain activity, but decreased it at high concentrations, confirming a typical hormesis phenomenon. The interaction of Hg2+ and Cu2+ at various concentration combinations showed that the binary interaction of 10-8mol/L Cu2+ and 10-6mol/L Hg2+ (Binary union S) buffer was of synergistic nature, while 10-4mol/L Cu2+ and 10-4mol/L Hg2+ (Binary union I) buffer was of competitive inhibition. The conformational changes in papain structure due to the interaction of binary metal ions were studied by ATR-FTIR, UV-vis and intrinsic fluorescence spectroscopies, also the changes of papain catalytic behavior were studied through kinetic analysis. Decreasing of α-helix content with increasing in intermolecular β-sheet aggregates content in Binary union I buffer resulted in an inactivation of papain activity by 57.2% and lower affinity for casein. On the contrary, papain activity increased with α-helix content increasing and intermolecular β-sheet aggregates content decreasing in Binary union S buffer. The competitive interaction between Cu2+ and Hg2+ on papain activity was found at higher concentrations (≥10-4mol/L), and the inhibition of the binary metal ions on papain was of a noncompetitive type.
- Liu, Xue-Ying,Zeng, Hong-Yan,Liao, Meng-Chen,Feng, Bo,Gohi, Bi Foua Claude Alain
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- Minor constituents from the tubers of Gymnadenia conopsea
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Four new minor constituents including two cyclodipeptides (1 and 2) and two cyclopentene derivatives (3 and 4), together with four known cyclodipeptides, have been isolated from an ethanolic extract of the tubers of Gymnadenia conopsea. Their structures i
- Zi, Jia-Chen,Lin, Sheng,Zhu, Cheng-Gen,Yang, Yong-Chun,Shi, Jian-Gong
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- A novel hydroxylation of aromatics in a flavin-initiated chain reaction
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In aqueous acidic solutions, the 5-ethyl-3-methyllumiflavinium cation 5 was spontaneously transformed into the dihydroflavin pseudobase radical 6 and the flavosemiquinone 7. In attacking an aromatic like phenylalanine, 6 was mainly reconverted to 5 which, ultimately, could lead to an anaerobic accumulation of 7 in yields of 95 ± 5%. In the immediate presence of O2 and/or H2O2, 7 was rapidly and continuously reoxidized to 5 which, consequently, gave a continued production of 6 increasing the hydroxylating ability of the system. The oxidants also had a second distinct effect in converting an intermediate X in a chain reaction to further increase the yields of hydroxyphenylalanines. As illustrated by the results obtained with O2 and H2O2 under comparable conditions, the efficiency of this chain reaction proved to be significantly influenced by the nature of the oxidant. These new findings imply that hydroxyl radicals arising in a homolysis of the O-O bond in a dihydroflavin hydroperoxide should not be taken for granted as the primary attacking species in the hydroxylation of an aromatic. From a practical point of view it is noticed that the hydroxylating ability of the new flavin / H2O2 system surpasses that of any other known, flavin-free chemical system.
- Mager, Humphrey I. X.,Tu, Shiao-Chun
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- An antifungal tetrapeptide from the culture of Penicillium canescens
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A new tetrapeptide D-Phe-L-Val-D-Val-L-Tyr (1), along with three known diketopiperazines and pseurotin A, were isolated from the culture of Penicillium canescens, collected from pollen from beehives, in a screening for new antimicrobial products from unex
- Bertinetti, Brenda V.,Pena, Nora I.,Cabrera, Gabriela M.
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- Formation of a hydroxyl radical from riboflavin sodium phosphate by photo-illumination
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Photo-illumination of riboflavin sodium phosphate (Rp) with phenylalanine produced significant levels of o-tyrosine, m-tyrosine and p- tyrosine as hydroxylated products. The hydroxylation of Rp was pH-dependent, and the maximum rate was around pH 4.5. Replacement of air with nitrogen prevented the formation of tyrosine isomers while the addition of superoxide dismutase or catalase to this system prevented hydroxylation. The tyrosine formation by the system was significantly prevented by hydroxyl radical (HO·) scavengers such as potassium iodide, potassium bromide, thiourea and sodium formate. No free iron and cupric ions were detected in the reaction mixture by inductively-coupled plasma atomic emission spectrometry. The above results suggest that the formation of HO· may occur in the photochemical reaction system in the presence of Rp under aerobic conditions, and that a superoxide radical and hydrogen peroxide may be involved in HO· formation.
- Ishimitsu, Susumu,Mishima, Ikuko,Tsuji, Sumiko,Shibata, Tadashi
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- High-performance liquid chromatographic analysis of pulmonary metabolites of Leu- and Met-enkephalins in isolated perfused rat lung
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A high-performance liquid radiochromatographic analytical system has been developed which allows the determination of [3H]Leu-enkephalin, [3H]Met-enkephalin and their potential metabolites [3H]TyrGlyGlyPhe, [3H]TyrGlyGly, [3H]TyrGly and [3H]tyrosine. Using this procedure, the biotransformation of each of the above enkephalins after 20 min of recirculating transit through isolated perfused rat lungs resulted in the formation of two major metabolites: tyrosine and TyrGlyGly in each case. The results indicate that significant metabolism of enkephalins may occur in the pulmonary circulation.
- Crooks,Krechniak,Olson,Gillespie
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- Simple conversion of fully protected amino acids to zwitterions
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An operationally simple and efficient method under mild acidic conditions was developed to convert fully protected amino acids to the corresponding zwitterions without either isoelectric precipitation or ion exchange chromatography.
- Hao, Junliang,Reinhard, Matt,Henry, Steven S.,Seest, Eric P.,Belvo, Matthew D.,Monn, James A.
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- Enzyme immobilization on smart polymers: Catalysis on demand
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A new approach for the synthesis of hydrogel films with thermo-sensitive enzymatic activity is reported. Pepsin (PEP) was covalently immobilized on thermo-responsive hydrogels by radical polymerization in the presence of N-isopropylacrylamide and poly-(ethylene glycol) dimethacrylate 750, acting as functional monomer and crosslinking agent, respectively. Hydrogels showing lower critical solution temperatures between 32.9 and 36.1 °C were synthesized by UV-irradiation of reaction batches differing in the PEP/monomers ratio. The derivatization degree of the hydrogels was expressed as mg of PEP per gram of matrix and found to be in the range of 6 to 11% as assessed by Lowry method. Scanning electron microscopy analysis and water affinity evaluation allowed to highlight the porous morphology and thermo-responsivity of hydrogels as a function of temperature. Using bovine serum albumin as a substrate, kinetics parameters were determined by Lineweaver-Burk plots and the catalyst efficiency evaluated. The influence of temperature on enzyme activity, as well as the thermal stability and reusability of devices, were also investigated.
- Cirillo, Giuseppe,Nicoletta, Fiore Pasquale,Curcio, Manuela,Spizzirri, Umile Gianfranco,Picci, Nevio,Iemma, Francesca
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- A blocked diketo form of avobenzone: Photostability, photosensitizing properties and triplet quenching by a triazine-derived UVB-filter
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Novel sunscreens are required providing active protection in the UVA and UVB regions. On the other hand, there is an increasing concern about the photosafety of UV filters, as some of them are not sufficiently photostable. Avobenzone is one of the most fr
- Paris, Cecilia,Lhiaubet-Vallet, Virginie,Jimenez, Oscar,Trullas, Carles,Miranda, Miguel Angel
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- Squamins C–F, four cyclopeptides from the seeds of Annona globiflora
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Four cyclic octapeptides, squamins C–F, were isolated from the seeds of Annona globiflora Schltdl. These compounds share part of their amino acid sequence, -Pro-Met(O)-Tyr-Gly-Thr-, with previously reported squamins A and B. Their structures were determined using NMR spectroscopic techniques together with quantum mechanical calculations (QM-NMR), ESI-HRMS data and a modified version of Marfey's chromatographic method. All compounds showed cytotoxic activity against DU-145 (human prostate cancer) and HeLa (human cervical carcinoma) cell lines. Clearly, A. globiflora is an important source of bioactive molecules, which could promote the sustainable exploitation of this undervalued specie.
- Sosa-Rueda, Javier,Domínguez-Meléndez, Vanihamin,Ortiz-Celiseo, Araceli,López-Fentanes, Fernando C.,Cuadrado, Cristina,Fernández, José J.,Daranas, Antonio Hernández,Cen-Pacheco, Francisco
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- Highly Stable Zr(IV)-Based Metal-Organic Frameworks for Chiral Separation in Reversed-Phase Liquid Chromatography
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Separation of racemic mixtures is of great importance and interest in chemistry and pharmacology. Porous materials including metal-organic frameworks (MOFs) have been widely explored as chiral stationary phases (CSPs) in chiral resolution. However, it remains a challenge to develop new CSPs for reversed-phase high-performance liquid chromatography (RP-HPLC), which is the most popular chromatographic mode and accounts for over 90% of all separations. Here we demonstrated for the first time that highly stable Zr-based MOFs can be efficient CSPs for RP-HPLC. By elaborately designing and synthesizing three tetracarboxylate ligands of enantiopure 1,1′-biphenyl-20-crown-6, we prepared three chiral porous Zr(IV)-MOFs with the framework formula [Zr6O4(OH)8(H2O)4(L)2]. They share the same flu topological structure but channels of different sizes and display excellent tolerance to water, acid, and base. Chiral crown ether moieties are periodically aligned within the framework channels, allowing for stereoselective recognition of guest molecules via supramolecular interactions. Under acidic aqueous eluent conditions, the Zr-MOF-packed HPLC columns provide high resolution, selectivity, and durability for the separation of a variety of model racemates, including unprotected and protected amino acids and N-containing drugs, which are comparable to or even superior to several commercial chiral columns for HPLC separation. DFT calculations suggest that the Zr-MOF provides a confined microenvironment for chiral crown ethers that dictates the separation selectivity.
- Jiang, Hong,Yang, Kuiwei,Zhao, Xiangxiang,Zhang, Wenqiang,Liu, Yan,Jiang, Jianwen,Cui, Yong
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supporting information
p. 390 - 398
(2021/01/13)
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- Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii
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Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/ reducing conditions) of nickel-affinity purified protein revealed the presence of a nearhomogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 × 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., 'nonspecific' substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.
- Smiley-Moreno, Elizabeth,Smith, Douglas,Yu, Jieh-Juen,Cao, Phuong,Arulanandam, Bernard P.,Chambers, James P.
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- Method for photolysis of amido bonds
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The invention discloses a method for photo-splitting amido bonds, wherein the method is mild in reaction condition and can realize splitting of amido bonds by using illumination. The method for photo-splitting the amido bonds comprises the following steps: reacting 2,4-dinitrofluorobenzene with an amino group of a substance which contains alpha amino acid at the tail end and is shown as a structural formula I to generate a compound 1 represented by a structural formula II; and under light irradiation, carrying out amido bond cleavage reaction on the compound 1, wherein R1 is a side chain group of alpha-amino acid, and R2 is aryl, aliphatic hydrocarbon, -CH(R)-COOH or polypeptide.
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Paragraph 0046; 0048-0049; 0098-0101
(2021/06/26)
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- Powerful Steroid-Based Chiral Selector for High-Throughput Enantiomeric Separation of α-Amino Acids Utilizing Ion Mobility-Mass Spectrometry
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Stereospecific recognition of amino acids (AAs) plays a crucial role in chiral biomarker-based diagnosis and prognosis. Separation of AA enantiomers is a long and tedious task due to the requirement of AA derivatization prior to the chromatographic or electrophoretic steps which are also time-consuming. Here, a mass-tagged chiral selector named [d0]/[d5]-estradiol-3-benzoate-17β-chloroformate ([d0]/[d5]-17β-EBC) with high reactivity and good enantiomeric resolution in regard to AAs was developed. After a quick and easy chemical derivatization step of AAs using 17β-EBC as the single chiral selector before ion mobility-mass spectrometry analysis, good enantiomer separation was achieved for 19 chiral proteinogenic AAs in a single analytical run (~2 s). A linear calibration curve of enantiomeric excess was also established using [d0]/[d5]-17β-EBC. It was demonstrated to be capable of determining enantiomeric ratios down to 0.5% in the nanomolar range. 17β-EBC was successfully applied to investigate the absolute configuration of AAs among peptide drugs and detect trace levels of-AAs in complex biological samples. These results indicated that [d0]/[d5]-17β-EBC may contribute to entail a valuable step forward in peptide drug quality control and discovering chiral disease biomarkers.
- Li, Yuling,Zhou, Bowen,Wang, Keke,Zhang, Jing,Sun, Wenjian,Zhang, Li,Guo, Yinlong
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p. 13589 - 13596
(2021/10/21)
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- Rational engineering ofAcinetobacter tandoiiglutamate dehydrogenase for asymmetric synthesis ofl-homoalanine through biocatalytic cascades
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l-Homoalanine, a useful building block for the synthesis of several chiral drugs, is generally synthesized through biocascades using natural amino acids as cheap starting reactants. However, the addition of expensive external cofactors and the low efficiency of leucine dehydrogenases towards the intermediate 2-ketobutyric acid are two major challenges in industrial applications. Herein, a dual cofactor-dependent glutamate dehydrogenase fromAcinetobacter tandoii(AtGluDH) was identified to help make full use of the intracellular pool of cofactors when using whole-cell catalysis. Through reconstruction of the hydrophobic network between the enzyme and the terminal methyl group of the substrate 2-ketobutyric acid, the strict substrate specificity ofAtGluDH towards α-ketoglutarate was successfully changed, and the activity obtained by the most effective mutant (K76L/T180C) was 17.2 times higher than that of the wild-type protein. A three-enzyme co-expression system was successfully constructed in order to help release the mass transfer restriction. Using 1 Ml-threonine, which is close to the solubility limit, we obtained a 99.9% yield ofl-homoalanine in only 3.5 h without adding external coenzymes to the cascade, giving 99.9% ee and a 29.2 g L?1h?1space-time yield. Additionally, the activities of the engineeredAtGluDH towards some other hydrophobic amino acids were also improved to 1.1-11.2 fold. Therefore, the engineering design of some dual cofactor-dependent GluDHs could not only eliminate the low catalytic activity of unnatural substrates but also enhance the cofactor utilization efficiency of these enzymes in industrial applications.
- Diao, Shiqing,Jiang, Shuiqin,Liu, Yan,Sun, Yangyang,Wang, Hualei,Wang, Liuzhu,Wei, Dongzhi
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p. 4208 - 4215
(2021/06/30)
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- Simultaneous Preparation of (S)-2-Aminobutane and d -Alanine or d -Homoalanine via Biocatalytic Transamination at High Substrate Concentration
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(S)-2-Aminobutane, d-alanine, and d-homoalanine are important intermediates for the production of various active pharmaceutical ingredients and food additives. The preparation of these small chiral amine or amino acids with high water solubility still demands searching for efficient methods. In this work, we identified an ω-transaminase (ω-TA) from Sinirhodobacter hungdaonensis (ShdTA) that catalyzed the kinetic resolution of racemic 2-aminobutane at a concentration of 800 mM using pyruvate as the amino acceptor, leading to the simultaneous isolation of enantiopure (S)-2-aminobutane and d-alanine in 46% and 90% yield, respectively. In addition, (S)-2-aminobutane (98% ee) and d-homoalanine (99% ee) were isolated in 45% and 93% yield, respectively, in the kinetic resolution of racemic 2-aminobutane at a concentration of 400 mM coupled with deamination of l-threonine by threonine deaminase. We thus developed a biocatalytic process for the practical synthesis of these valuable small chiral amine and d-amino acids.
- Li, Jianjiong,Wang, Yingang,Wu, Qiaqing,Yao, Peiyuan,Yu, Shanshan,Zhu, Dunming
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supporting information
(2022/03/01)
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- Biocascade Synthesis of L-Tyrosine Derivatives by Coupling a Thermophilic Tyrosine Phenol-Lyase and L-Lactate Oxidase
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A one-pot biocascade of two enzymatic steps catalyzed by an l-lactate oxidase and a tyrosine phenol-lyase has been successfully developed in the present study. The reaction provides an efficient method for the synthesis of l-tyrosine derivatives, which exhibits readily available starting materials and excellent yields. In the first step, an in situ generation of pyruvate from readily available bio-based l-lactate catalyzed by a highly active l-lactate oxidase from Aerococcus viridans (AvLOX) was developed (using oxygen as oxidant and catalase as hydrogen peroxide removing reagent). Pyruvate thus produced underwent C–C coupling with phenol derivatives as acceptor substrate using specially designed thermophilic tyrosine phenol-lyase mutants from Symbiobacterium toebii (TTPL). Overall, this cascade avoids the high cost and easy decomposition of pyruvate and offered an efficient and environmentally friendly procedure for l-tyrosine derivatives synthesis.
- Jiang, Yiqi,Ju, Shuyun,Li, Guosi,Lian, Jiazhang,Lin, Jianping,Wu, Mianbin,Xue, Hailong,Yang, Lirong
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supporting information
(2020/02/25)
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- Investigations of conformational structures and activities of trypsin and pepsin affected by food colourant allura red
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Herein, binding interactions of food colourant allura red (AR) with trypsin and pepsin were comparably investigated for deep revelations of conformational structures and activities of proteinases affected by food colourant. Various results indicated that
- Huang, Shan,Li, Haimei,Liang, Jiandan,Luo, Huajian,Xiao, Qi,Yang, Jing
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- Scope and limitations of reductive amination catalyzed by half-sandwich iridium complexes under mild reaction conditions
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The conversion of aldehydes and ketones to 1° amines could be promoted by half-sandwich iridium complexes using ammonium formate as both the nitrogen and hydride source. To optimize this method for green chemical synthesis, we tested various carbonyl substrates in common polar solvents at physiological temperature (37 °C) and ambient pressure. We found that in methanol, excellent selectivity for the amine over alcohol/amide products could be achieved for a broad assortment of carbonyl-containing compounds. In aqueous media, selective reduction of carbonyls to 1° amines was achieved in the absence of acids. Unfortunately, at Ir catalyst concentrations of 1 mM in water, reductive amination efficiency dropped significantly, which suggest that this catalytic methodology might be not suitable for aqueous applications where very low catalyst concentration is required (e.g., inside living cells).
- Nguyen, Dat P.,Sladek, Rudolph N.,Do, Loi H.
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supporting information
(2020/07/15)
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- Hydrogen Bond Assisted l to d Conversion of α-Amino Acids
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l to d conversion of unactivated α-amino acids was achieved by solubility-induced diastereomer transformation (SIDT). Ternary complexes of an α-amino acid with 3,5-dichlorosalicylaldehyde and a chiral guanidine (derived from corresponding chiral vicinal diamine) were obtained in good yield as diastereomerically pure imino acid salt complexes and were hydrolysed to obtain enantiopure α-amino acids. A combination of DFT computation, NMR spectroscopy, and crystal structure provide detailed insight into how two types of strong hydrogen bonds assist in rapid epimerization of the complexes that is essential for SIDT.
- Chin, Jik,Fu, Rui,Lough, Alan J.,So, Soon Mog
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supporting information
p. 4335 - 4339
(2020/02/11)
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- D-Phenylglycine aminotransferase (d-PhgAT)-substrate scope and structural insights of a stereo-inverting biocatalyst used in the preparation of aromatic amino acids
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Enantiopure amines are key building blocks in the synthesis of many pharmaceuticals, so a route to their production is a current goal for biocatalysis. The stereo-inverting d-phenylglycine aminotransferase (d-PhgAT), isolated from Pseudomonas stutzeri ST-201, catalyses the reversible transamination from l-glutamic acid to benzoylformate, yielding α-ketoglutarate and d-phenylglycine (d-Phg). Detailed kinetic analysis revealed a range of amine donor and acceptor substrates that allowed the synthesis of enantiopure aromatic d-amino acids at a preparative scale. We also determined the first X-ray crystal structure of d-PhgAT with its bound pyridoxal 5′-phosphate (PLP) cofactor at 2.25 ? resolution. A combination of structural analysis and site-directed mutagenesis of this class III aminotransferase revealed key residues that are potentially involved in the dual substrate recognition, as well as controlling the stereo-inverting behaviour of d-PhgAT. Two arginine residues (Arg34 and Arg407) are involved in substrate recognition within P and O binding pockets respectively. These studies lay the foundation for further enzyme engineering and promote d-PhgAT as a useful biocatalyst for the sustainable production of high value, aromatic d-amino acids. This journal is
- Akhtar, M. Kalim,Campopiano, Dominic J.,De Cesare, Silvia,Loake, Gary J.,Marles-Wright, Jon,Serpico, Annabel
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p. 6533 - 6543
(2020/11/13)
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- Colistin sulfate chiral stationary phase for the enantioselective separation of pharmaceuticals using organic polymer monolithic capillary chromatography ?
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A new functionalized polymer monolithic capillary with a macrocyclic antibiotic, namely colistin sulfate, as chiral selector was prepared via the copolymerization of binary monomer mixtures consisting of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) in porogenic solvents namely 1-propanol and 1,4-butanediol, in the presence of azobisiso-butyronitrile (AIBN) as initiator and colistin sulfate. The prepared capillaries were investigated for the enantioselective nano-LC separation of a group of racemic pharmaceuticals, namely, α- and β-blockers, anti-inflammatory drugs, antifungal drugs, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, antihistaminics, anticancer drugs, and antiarrhythmic drugs. Acceptable separation was achieved for many drugs using reversed phase chromatographic conditions with no separation achieved under normal phase conditions. Colistin sulfate appears to be useful addition to the available macrocyclic antibiotic chiral phases used in liquid chromatography.
- Fouad, Ali,Shaykoon, Montaser Sh.A.,Ibrahim, Samy M.,El-Adl, Sobhy M.,Ghanem, Ashraf
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- Heterologous production of asperipin-2a: Proposal for sequential oxidative macrocyclization by a fungi-specific DUF3328 oxidase
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Asperipin-2a is a ribosomally synthesized and post-translationally modified peptide isolated from Asperigillus flavus. Herein, we report the heterologous production of asperipin-2a and determination of its absolute structure. Notably, the characteristic bicyclic structure was likely constructed by a single oxidase containing the DUF3328 domain.
- Ye, Ying,Ozaki, Taro,Umemura, Myco,Liu, Chengwei,Minami, Atsushi,Oikawa, Hideaki
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supporting information
p. 39 - 43
(2019/01/04)
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- Photocatalysis Enables Visible-Light Uncaging of Bioactive Molecules in Live Cells
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The photo-manipulation of bioactive molecules provides unique advantages due to the high temporal and spatial precision of light. The first visible-light uncaging reaction by photocatalytic deboronative hydroxylation in live cells is now demonstrated. Using Fluorescein and Rhodamine derivatives as photocatalysts and ascorbates as reductants, transient hydrogen peroxides were generated from molecular oxygen to uncage phenol, alcohol, and amine functional groups on bioactive molecules in bacteria and mammalian cells, including neurons. This effective visible-light uncaging reaction enabled the light-inducible protein expression, the photo-manipulation of membrane potentials, and the subcellular-specific photo-release of small molecules.
- Wang, Haoyan,Li, Wei-Guang,Zeng, Kaixing,Wu, Yan-Jiao,Zhang, Yixin,Xu, Tian-Le,Chen, Yiyun
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supporting information
p. 561 - 565
(2019/01/04)
-
- Direct Synthesis of Free α-Amino Acids by Telescoping Three-Step Process from 1,2-Diols
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A practical telescoping three-step process for the syntheses of α-amino acids from the corresponding 1,2-diols has been developed. This process enables the direct synthesis of free α-amino acids without any protection/deprotection step. This method was also effective for the preparation of a 15N-labeled α-amino acid. 1,2-Diols bearing α,β-unsaturated ester moieties afforded bicyclic α-amino acids through intramolecular [3 + 2] cycloadditions. A preliminary study suggests that the resultant α-amino acids are resolvable by aminoacylases with almost complete selectivity.
- Inada, Haruki,Shibuya, Masatoshi,Yamamoto, Yoshihiko
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supporting information
p. 709 - 713
(2019/01/25)
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- Electrosynthesis of amino acids from biomass-derivable acids on titanium dioxide
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Seven amino acids were electrochemically synthesized from biomass-derivable α-keto acids and NH2OH with faradaic efficiencies (FEs) of 77-99% using an earth-Abundant TiO2 catalyst. Furthermore, we newly constructed a flow-Type electrochemical reactor, named a "polymer electrolyte amino acid electrosynthesis cell", and achieved continuous production of alanine with an FE of 77%.
- Fukushima, Takashi,Yamauchi, Miho
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supporting information
p. 14721 - 14724
(2019/12/24)
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- Highly selective synthesis of d-amino acids from readily available l-amino acids by a one-pot biocatalytic stereoinversion cascade
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d-Amino acids are key intermediates required for the synthesis of important pharmaceuticals. However, establishing a universal enzymatic method for the general synthesis of d-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we constructed and optimized a cascade enzymatic route involving l-amino acid deaminase and d-amino acid dehydrogenase for the biocatalytic stereoinversions of l-amino acids into d-amino acids. Using l-phenylalanine (l-Phe) as a model substrate, this artificial biocatalytic cascade stereoinversion route first deaminates l-Phe to phenylpyruvic acid (PPA) through catalysis involving recombinant Escherichia coli cells that express l-amino acid deaminase from Proteus mirabilis (PmLAAD), followed by stereoselective reductive amination with recombinant meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH) to produce d-phenylalanine (d-Phe). By incorporating a formate dehydrogenase-based NADPH-recycling system, d-Phe was obtained in quantitative yield with an enantiomeric excess greater than 99%. In addition, the cascade reaction system was also used to stereoinvert a variety of aromatic and aliphatic l-amino acids to the corresponding d-amino acids by combining the PmLAAD whole-cell biocatalyst with the StDAPDH variant. Hence, this method represents a concise and efficient route for the asymmetric synthesis of d-amino acids from the corresponding l-amino acids.
- Zhang, Danping,Jing, Xiaoran,Zhang, Wenli,Nie, Yao,Xu, Yan
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p. 29927 - 29935
(2019/10/01)
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- Artificial Biocatalytic Cascade with Three Enzymes in One Pot for Asymmetric Synthesis of Chiral Unnatural Amino Acids
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Two biocatalytic reactions, transamination catalyzed by transaminases and reductive amination catalyzed by amino acid dehydrogenases, can be used for asymmetric synthesis of optically pure unnatural amino acids. However, although transaminases show a great diversity and broad substrate spectrum, most transaminase reactions are reversible, while amino acid dehydrogenases catalyze reductive amination irreversibly but with strict substrate specificity. Accordingly, herein we developed a tri-enzyme one-pot reaction system to exploit the respective advantages of transaminases and amino acid dehydrogenases, while overcoming the disadvantages of each. In this work, representatives of all four subgroups of transaminases coupled with different amino acid dehydrogenases to produce five l- and four d- unnatural amino acid products, using ammonia and the co-enzyme NAD(P)H, which is regenerated by a robust alcohol dehydrogenase with 2-propanol as cheap cosubstrate. The complete conversion and high enantiopurity (ee > 99 %) of the products, demonstrated it as an ideal alternative for asymmetric synthesis of chiral amino acid compounds.
- Zhou, Haisheng,Meng, Lijun,Yin, Xinjian,Liu, Yayun,Xu, Gang,Wu, Jianping,Wu, Mianbin,Yang, Lirong
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supporting information
p. 6470 - 6477
(2019/11/02)
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- Click-to-Release from trans-Cyclooctenes: Mechanistic Insights and Expansion of Scope from Established Carbamate to Remarkable Ether Cleavage
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The bioorthogonal cleavage of allylic carbamates from trans-cyclooctene (TCO) upon reaction with tetrazine is widely used to release amines. We disclose herein that this reaction can also cleave TCO esters, carbonates, and surprisingly, ethers. Mechanistic studies demonstrated that the elimination is mainly governed by the formation of the rapidly eliminating 1,4-dihydropyridazine tautomer, and less by the nature of the leaving group. In contrast to the widely used p-aminobenzyloxy linker, which affords cleavage of aromatic but not of aliphatic ethers, the aromatic, benzylic, and aliphatic TCO ethers were cleaved as efficiently as the carbamate, carbonate, and esters. Bioorthogonal ether release was demonstrated by the rapid uncaging of TCO-masked tyrosine in serum, followed by oxidation by tyrosinase. Finally, tyrosine uncaging was used to chemically control cell growth in tyrosine-free medium.
- Versteegen, Ron M.,ten Hoeve, Wolter,Rossin, Raffaella,de Geus, Mark A. R.,Janssen, Henk M.,Robillard, Marc S.
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p. 10494 - 10499
(2018/08/17)
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- Using the 3-Diethylaminobenzyl Group as a Photocage in Aqueous Solution
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We have demonstrated that the 3-diethylaminobenzyl (DEABn) photolabile protecting group (PPG) is an effective and structurally simple PPG for releasing molecules in aqueous environment. In general, the photoreaction is clean, and the released substrate and the PPG product, i.e., 3-diethylaminobenzyl alcohol, are obtained in high yield. The clean photoreaction can also be achieved under mild ambient conditions with sunlight, while the reactant is stable under indoor lighting. Release of two substrates from one PPG chromophore in aqueous solution has been demonstrated to be feasible. We have also compared the uncaging properties of the DEABn and the widely used o-nitrobenzyl (o-NB) group, given their comparable structural simplicity. With its clean and efficient photochemical reaction, DEABn should find wide applications, including in the basic and applied research areas where o-NB and its various derivatives are widely used.
- Ding, Xiong,Wang, Pengfei
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p. 7459 - 7466
(2018/05/29)
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- New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites
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Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.
- Ballet, Caroline,Correia, Mário S. P.,Conway, Louis P.,Locher, Theresa L.,Lehmann, Laura C.,Garg, Neeraj,Vujasinovic, Miroslav,Deindl, Sebastian,L?hr, J.-Matthias,Globisch, Daniel
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p. 6233 - 6239
(2018/08/07)
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- A Bioinspired Synthesis of Polyfunctional Indoles
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Polyfunctional indoles bearing substituents at C5 and C6 are prevalent in natural products, pharmaceuticals, agrochemicals, and materials. Owing to the remoteness of the C5 and C6 positions, indoles of this family can be difficult to prepare, and often require multistep syntheses. Herein, we describe a concise process that converts simple derivatives of tyrosine into 5,6-difunctionalized indoles by direct oxidation of C?H, N?H, and O?H bonds. Our work draws inspiration from the biosynthetic polymerization of tyrosine to make melanin pigments, but makes an important departure to provide well-defined indole heterocycles.
- Huang, Zheng,Kwon, Ohhyeon,Huang, Haiyan,Fadli, Aziz,Marat, Xavier,Moreau, Magali,Lumb, Jean-Philip
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supporting information
p. 11963 - 11967
(2018/09/11)
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- Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family
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Flavin-dependent enzymes catalyze many oxidations, including formation of ring structures in natural products. The gene cluster for biosynthesis of fumisoquins, secondary metabolites structurally related to isoquinolines, in the filamentous fungus Aspergillus fumigatus harbors a gene that encodes a flavoprotein of the amine oxidase family, termed fsqB (fumisoquin biosynthesis gene B). This enzyme catalyzes an oxidative ring closure reaction that leads to the formation of isoquinoline products. This reaction is reminiscent of the oxidative cyclization reported for berberine bridge enzyme and tetrahydrocannabinol synthase. Despite these similarities, amine oxidases and berberine bridge enzyme–like enzymes possess distinct structural properties, prompting us to investigate the structure–function relationships of FsqB. Here, we report the recombinant production and purification of FsqB, elucidation of its crystal structure, and kinetic analysis employing five putative substrates. The crystal structure at 2.6 ? resolution revealed that FsqB is a member of the amine oxidase family with a covalently bound FAD cofactor. N-methyl-dopa was the best substrate for FsqB and was completely converted to the cyclic isoquinoline product. The absence of the meta-hydroxyl group, as e.g. in L-Nmethyl-tyrosine, resulted in a 25-fold lower rate of reduction and the formation of the demethylated product L-tyrosine, instead of a cyclic product. Surprisingly, FsqB did not accept the D-stereoisomer of N-methyltyrosine, in contrast to N-methyl-dopa, for which both stereoisomers were oxidized with similar rates. On the basis of the crystal structure and docking calculations, we postulate a substrate-dependent population of distinct binding modes that rationalizes stereospecific oxidation in the FsqB active site.
- Lahham, Majd,Pavkov-Keller, Tea,Fuchs, Michael,Niederhauser, Johannes,Chalhoub, Gabriel,Daniel, Bastian,Kroutil, Wolfgang,Gruber, Karl,Macheroux, Peter
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p. 17021 - 17032
(2018/11/21)
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- One-Pot Enzymatic Synthesis of d-Arylalanines Using Phenylalanine Ammonia Lyase and l-Amino Acid Deaminase
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The phenylalanine ammonia-lyase (AvPAL) from Anabaena variabilis catalyzes the amination of substituent trans-cinnamic acid (t-CA) to produce racemic d,l-enantiomer arylalanine mixture owing to its low stereoselectivity. To produce high optically pure d-arylalanine, a modified AvPAL with high d-selectivity is expected. Based on the analyses of catalytic mechanism and structure, the Asn347 residue in the active site was proposed to control stereoselectivity. Therefore, Asn347 was mutated to construct mutant AvPAL-N347A, the stereoselectivity of AvPAL-N347A for d-enantiomer arylalanine was 2.3-fold higher than that of wild-type AvPAL (WtPAL). Furthermore, the residual l-enantiomer product in reaction solution could be converted into the d-enantiomer product through stereoselective oxidation by PmLAAD and nonselective reduction by reducing agent NH3BH3. At optimal conditions, the conversion rate of t-CA and optical purity (enantiomeric excess (eeD)) of d-phenylalanine reached 82% and exceeded 99%, respectively. The two enzymes displayed activity toward a broad range of substrate and could be used to efficiently synthesize d-arylalanine with different groups on the phenyl ring. Among these d-arylalanines, the yield of m-nitro-d-phenylalanine was highest and reached 96%, and the eeD exceeded 99%. This one-pot synthesis using AvPAL and PmLAAD has prospects for industrial application.
- Zhu, Longbao,Feng, Guoqiang,Ge, Fei,Song, Ping,Wang, Taotao,Liu, Yi,Tao, Yugui,Zhou, Zhemin
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- Bio-inspired enantioselective full transamination using readily available cyclodextrin
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The mimics of vitamin B6-dependent enzymes that catalyzed an enantioselective full transamination in the pure aqueous phase have been realized for the first time through the establishment of a new “pyridoxal 5′-phosphate (PLP) catalyzed non-covalent cyclodextrin (CD)-keto acid inclusion complexes” system, and various optically active amino acids have been obtained.
- Zhang, Shiqi,Li, Guangxun,Liu, Hongxin,Wang, Yingwei,Cao, Yuan,Zhao, Gang,Tang, Zhuo
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p. 4203 - 4208
(2017/02/05)
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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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p. 1037 - 1042
(2017/07/25)
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- Biocatalytic stereoinversion of d-: Para -bromophenylalanine in a one-pot three-enzyme reaction
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Halogenated derivatives of phenylalanine can be used as cross-coupling reagents for making drug-like molecules, and pure enantiomers of these precursors are therefore highly desirable. In our exploration of enzymatic routes to simplify the deracemisation process, the application of two enzymes, d-amino acid transaminase and phenylalanine dehydrogenase, both from Lysinibacillus sphaericus, has given promising results for the stereo-inversion of d-enantiomers of para-bromophenylalanine as the model substrate and also p-chloro/fluorophenylalanine and tyrosine. The addition of a coenzyme recycling system using ethanol and alcohol dehydrogenase reduced the amount of coenzyme needed for the reaction catalysed by phenylalanine dehydrogenase, reducing cost and permitting efficient and complete conversion of the racemic amino acids to the l-enantiomer. Relative proportions of the enzymes were optimized. The high purity of the l-enantiomer, with an ee over 99%, and the ease of the process make it an ideal alternative for deracemisation of the studied compounds.
- Khorsand, Fahimeh,Murphy, Cormac D.,Whitehead, Andrew J.,Engel, Paul C.
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p. 503 - 510
(2017/08/14)
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- Stylissamide I, a new cyclic heptapeptide from an okinawan marine sponge Stylissa sp.
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A new cyclic heptapeptide, stylissamide I (1), was isolated from an Okinawan marine sponge Stylissa sp. The structure of stylissamide I (1) was elucidated to be cyclo-(L-Tyr1-L-Tyr2-L-Tyr3-L-Pro1-L-Pro2-L-Val-L-Pro3) by extensive spectral analyses and Marfey's method. Stylissamide I (1) showed antifungal activity against Aspergillus Niger.
- Kubota, Takaaki,Nakamura, Kenta,Kurimoto, Shin-Ichiro,Sakai, Kanae,Fromont, Jane,Gonoi, Tohru,Kobayashi, Jun'ichi
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p. 799 - 806
(2017/07/28)
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- Identification of Cyclic Depsipeptides and Their Dedicated Synthetase from Hapsidospora irregularis
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Seven cyclic depsipeptides were isolated from Hapsidospora irregularis and structurally characterized as the calcium channel blocker leualacin and six new analogues based on the NMR and HRESIMS data. These new compounds were named leualacins B-G. The absolute configurations of the amino acids and 2-hydroxyisocaproic acids were determined by recording the optical rotation values. Biological studies showed that calcium influx elicited by leualacin F in primary human lobar bronchial epithelial cells involves the TRPA1 channel. Through genome sequencing and targeted gene disruption, a noniterative nonribosomal peptide synthetase was found to be involved in the biosynthesis of leualacin. A comparison of the structures of leualacin and its analogues indicated that the A2 and A4 domains of the leualacin synthetase are substrate specific, while A1, A3, and A5 can accept alternative precursors to yield new molecules.
- Zhang, Shuwei,Qiu, Yixing,Kakule, Thomas B.,Lu, Zhenyu,Xu, Fuchao,Lamb, John G.,Reilly, Christopher A.,Zheng, Yong,Sham, Shing Wo Simon,Wang, Wei,Xuan, Lijiang,Schmidt, Eric W.,Zhan, Jixun
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p. 363 - 370
(2017/03/08)
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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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- Palladium-Triggered Chemical Rescue of Intracellular Proteins via Genetically Encoded Allene-Caged Tyrosine
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Chemical de-caging has emerged as an attractive strategy for gain-of-function study of proteins via small-molecule reagents. The previously reported chemical de-caging reactions have been largely centered on liberating the side chain of lysine on a given protein. Herein, we developed an allene-based caging moiety and the corresponding palladium de-caging reagents for chemical rescue of tyrosine (Tyr) activity on intracellular proteins. This bioorthogonal de-caging pair has been successfully applied to unmask enzymatic Tyr sites (e.g., Y671 on Taq polymerase and Y728 on Anthrax lethal factor) as well as the post-translational Tyr modification site (Y416 on Src kinase) in vitro and in living cells. Our strategy provides a general platform for chemical rescue of Tyr-dependent protein activity inside cells.
- Wang, Jie,Zheng, Siqi,Liu, Yanjun,Zhang, Zhaoyue,Lin, Zhi,Li, Jiaofeng,Zhang, Gong,Wang, Xin,Li, Jie,Chen, Peng R.
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p. 15118 - 15121
(2016/12/06)
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- Characterization of aromatic aminotransferases from Ephedra sinica Stapf
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Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using l-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.
- Kilpatrick, Korey,Pajak, Agnieszka,Hagel, Jillian M.,Sumarah, Mark W.,Lewinsohn, Efraim,Facchini, Peter J.,Marsolais, Frédéric
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p. 1209 - 1220
(2016/04/26)
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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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- Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase
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D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD’s invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD’s chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2′-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD’s activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.
- Routh, Satya Brata,Pawar, Komal Ishwar,Ahmad, Sadeem,Singh, Swati,Suma, Katta,Kumar, Mantu,Kuncha, Santosh Kumar,Yadav, Kranthikumar,Kruparani, Shobha P,Sankaranarayanan, Rajan
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- D-tyrosine a method for asymmetric synthesis of
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A provided asymmetric syntheses method for D-tyrosine comprises the following steps: performing a condensation reaction on p-hydroxybenzaldehyde and acetylglycine, then performing hydrolysis or alcoholysis to obtain a dehyddroamino acid or ester; and then utilizing rhodium to perform catalytic asymmetric hydrogenation, and performing hydrolysis to obtain a key intermediate D-tyrosine. According to the method, the whole process has no complex separation steps, the preparation technology is simple, a chromatography column is not needed, the reaction steps can be further reduced by utilizing rhodium catalysis asymmetric hydrogenation technology, the production cost is reduced, and the method is extremely suitable for industrialized batch production.
- -
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Paragraph 0057; 0058
(2019/02/02)
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- An easy 'Filter-and-Separate' method for enantioselective separation and chiral sensing of substrates using a biomimetic homochiral polymer
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We present a polyfluorene appended with protected l-glutamic acid that exhibited a reversible α-helix/β-sheet-like conformation and helical porous fibrous morphology mimicking the super-structure of proteins. The new homochiral polymer probe enabled efficient heterogeneous enantioselective separation and chiral sensing of a wide variety of substrates from their aqueous racemic mixture using an easy 'Filter-and-Separate' method.
- Senthilkumar,Asha
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supporting information
p. 8931 - 8934
(2015/05/27)
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- Biocatalytic One-Pot Synthesis of l-Tyrosine Derivatives from Monosubstituted Benzenes, Pyruvate, and Ammonia
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l-Tyrosine derivatives were obtained in >97% ee via a biocatalytic one-pot two-step cascade using substituted benzenes, pyruvate, and NH3 as starting materials. In the first step, monosubstituted arenes were regioselectively hydroxylated in the o-position by monooxygenase P450 BM3 (using O2 as oxidant with NADPH-recycling) to yield the corresponding phenols, which subsequently underwent C-C coupling and simultaneous asymmetric amination with pyruvate and NH3 using tyrosine phenol lyase to furnish l-DOPA surrogates in up to 5.2 g L-1. Instead of analytically pure arenes, crude aromatic gasoline blends containing toluene were used to yield 3-methyl-l-tyrosine in excellent yield (2 g L-1) and >97% ee.
- Dennig, Alexander,Busto, Eduardo,Kroutil, Wolfgang,Faber, Kurt
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p. 7503 - 7506
(2015/12/11)
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- Photoactivatable, biologically-relevant phenols with sensitivity toward 2-photon excitation
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Spatio-temporal release of biologically relevant small molecules provides exquisite control over the activation of receptors and signaling pathways. This can be accomplished via a photochemical reaction that releases the desired small molecule in response to irradiation with light. A series of biologically-relevant signaling molecules (serotonin, octopamine, capsaicin, N-vanillyl-nonanoylamide, estradiol, and tyrosine) that contain a phenol moiety were conjugated to the 8-bromo-7-hydroxyquinolinyl (BHQ) or 8-cyano-7-hydroxyquinolinyl (CyHQ) photoremovable protecting groups (PPGs). The CyHQ caged compounds proved sensitive toward 1PE and 2PE processes with quantum efficiencies of 0.2-0.4 upon irradiation at 365 nm and two-photon action cross sections of 0.15-0.31 GM when irradiated at 740 nm. All but one BHQ caged compound, BHQ-estradiol, were found to be sensitive to photolysis through 1PE and 2PE with quantum efficiencies of 0.30-0.40 and two photon cross sections of 0.40-0.60 GM. Instead of releasing estradiol, BHQ-estradiol underwent debromination.
- McLain, Duncan E.,Rea, Adam. C.,Widegren, Magnus B.,Dore, Timothy M.
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p. 2151 - 2158
(2015/12/04)
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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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- A thermodynamic insight into the recognition of hydrophilic and hydrophobic amino acids in pure water by aza-scorpiand type receptors
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Interactions of different hydrophilic (His, Asp, Glu,) and hydrophobic (Ala, Phe, Tyr, Trp) amino acids in water with a scorpiand aza-macrocycle (L1) containing a pyridine group in the ring and its derivative (L2) bearing a naphthalene group in the tail have been analysed by potentiometric and calorimetric measurements. Theoretical calculations corroborate that major attractive forces that hold the adduct together are hydrogen bonds and salt-bridges, even though other interactions such as π-stacking or NH+...π may contribute in the case of hydrophobic amino acids and L2. Calorimetric measurements indicate that the interactions between L1 and the different amino acids are principally driven by entropy, often associated with solvation/desolvation processes.
- Blasco, Salvador,Verdejo, Begoa,Bazzicalupi, Carla,Bianchi, Antonio,Giorgi, Claudia,Soriano, Concepcin,Garca-Espaa, Enrique
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supporting information
p. 843 - 850
(2015/02/19)
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- Anti-inflammatory activity of a new cyclic peptide, citrusin XI, isolated from the fruits of Citrus unshiu
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Ethnopharmacological relevance Citrus unshiu (Rutaceae) is an easy-peeling citrus fruit, which has been used as a traditional Korean medicine for improving skin elasticity, relieving fatigue and cough, and preventing bronchitis, flu, and various cancers. However, its active components associated with anti-inflammation and underlying mechanisms remain unknown. In this study, we investigated the active constituents from the fruits of Citrus unshiu and evaluated the anti-inflammatory activity in order to support the traditional usage of Citrus unshiu. Material and methods Repeated column chromatography, together with a semi-preparative HPLC purification was used to separate the bioactive constituent from the EtOAc soluble fraction of the EtOH extract of Citrus unshiu fruits. Anti-inflammatory effects of the isolated compounds on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW264.7 macrophage cells. Results A new cyclic peptide, citrusin XI (1), was isolated and identified from the fruits of Citrus unshiu. The structure of compound 1 was elucidated by spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR) (1H, 13C, COSY, HMQC and HMBC experiments), and high resolution (HR)-mass spectrometry, and its absolute configurations were further confirmed by the Marfeys method. Compound 1 decreased NO production in LPS-stimulated RAW264.7 cells in a dose-dependent manner with an IC50 value of 70 μM. Compound 1 suppressed NO production by decreasing iNOS expression but COX-2 expression was slightly associated with the reduction by compound 1 in LPS-induced RAW264.7 cells. Furthermore, compound 1 inhibited NF-κB activation by blocking IκBα degradation and NF-κB phosphorylation in LPS-stimulated RAW264.7 cells. Conclusions These results indicate that a new cyclic peptide, citrusin XI, from Citrus unshiu fruits has anti-inflammatory properties that inhibit the release of pro-inflammatory mediators. Compound 1 decreases NO production by decreasing iNOS expression and NF-κB activation associated with IκBα degradation and NF-κB phosphorylation in LPS-induced RAW264.7 cells. This is the first study to clarify the underlying mechanism of the anti-inflammatory effect exerted by a pure isolated compound from Citrus unshiu in LPS-stimulated RAW264.7 macrophage cells. The phytochemical, citrusin XI of Citrus unshiu may serve as lead compound in the design of new agents for preventing and treating inflammatory diseases.
- Noh, Hyung Jun,Hwang, Dukhyun,Lee, Eun Suk,Hyun, Jae Wook,Yi, Pyoung Ho,Kim, Geum Soog,Lee, Seung Eun,Pang, Changhyun,Park, Yong Joo,Chung, Kyu Hyuck,Kim, Gun Do,Kim, Ki Hyun
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p. 106 - 112
(2015/03/05)
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- Study of the stability of the 5-aminolevulinic acid tyrosine ester in aqueous solution
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Photodynamic therapy based on photoactivable porphyrins (PAPs) can treat various dermatological conditions. The side-effects as well as the non-selective or insufficient accumulation of PAPs in the targeted tissues limit performances. We studied the stability in solution at different temperatures (21 °C; 4 °C), different pH values (7.5; 2.0), and as a function of time of 5-aminolevulinic acid's Tyrosine-ester, a molecule presenting interesting properties to selectively produce PAPs in blood vessels after topical application. Solutions of this precursor can be kept up to 24 h at refrigerated temperatures and under acidic pH. At room temperature or physiological pH, they must be prepared minutes before their use. ARKAT-USA, Inc.
- Gay, Sandrine,Martoccia, Carla,Zellweger, Matthieu,Wang, Qian,Wagnieres, Georges
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p. 228 - 238
(2014/06/09)
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- Coordination properties of 3-functionalised β-cyclodextrins: Thermodynamic stereoselectivity of copper(II) complexes of the 3-histamine derivative and its exploitation in ligand-exchange capillary electrophoresis
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A histamine derivative of β-cyclodextrin functionalised at the secondary rim was synthesised and characterised by optical and NMR spectroscopy. Its binary systems both with proton and copper(II) were thermodynamically characterised through pH-metric potentiometry. In addition, the ternary systems with the enantiomers of tryptophan, phenylalanine and alanine were investigated. Thermodynamic stereoselectivity was observed for both the tryptophan and phenylalanine enantiomers. The properties of the synthesised cyclodextrin derivative as a chiral selector were verified in chiral ligand-exchange capillary electrophoresis (CLECE) towards the enantiomeric pairs of some amino acids. A β-cyclodextrin histamine-functionalised at the secondary rim was synthesised and characterised as a chiral selector of aromatic amino acids.
- Giuffrida, Alessandro,Cucinotta, Vincenzo,MacCarrone, Giuseppe,Messina, Marianna,Rizzarelli, Enrico,Vecchio, Graziella
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p. 377 - 383
(2014/01/23)
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- Chiral ligand-exchange resolution of underivatized amino acids on a dynamically modified stationary phase for RP-HPTLC
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The synthesis of Spi(τ-dec), derived from the selective alkylation of L-spinacine (4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid) at the τ-nitrogen of its heteroaromatic ring, with a linear hydrocarbon chain of 10 carbon atoms, is described here for the first time. Spi(τ-dec) was successfully employed in the past to prepare home-made chiral columns for chiral ligand-exchange high-performance liquid chromatography. In the present article a new method is described, using Spi(τ-dec) as a chiral selector in high-performance thin-layer chromatography (HPTLC): commercial hydrophobic plates were first coated with Spi(τ-dec) and then treated with copper sulfate. The performance of this new chiral stationary phase was tested against racemic mixtures of aromatic amino acids, after appropriate optimization of both the conditions of preparation of the plates and the mobile phase composition. The enantioselectivity values obtained for the studied compounds were higher than those reported in the literature for similar systems. The method employed here for the preparation of chiral HPTLC plates proved practical, efficient, and inexpensive. Chirality 26:313-318, 2014. 2014 Wiley Periodicals, Inc.
- Remelli, Maurizio,Faccini, Stefania,Conato, Chiara
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p. 313 - 318
(2014/06/09)
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