Hydroxy Amino Acids Research Articles

Propargyloxycarbonyl as a protecting group for the side chains of serine, threonine and tyrosine

Propargyloxycarbonyl group is used as a protecting group for the hydroxyl groups of serine, threonine and tyrosine. The propargyloxycarbonyl derivatives of these hydroxy amino acids are stable to acidic and basic reagents commonly employed in peptide synthesis. The deprotection of the O-Poc derivatives using tetrathiomolybdate does not affect commonly used protecting groups such as N-Boc, N-Cbz, N-Fmoc, methyl and benzyl esters. The di-and tripeptides synthesized using O-Poc derivatives of serine, threonine and tyrosine are stable, isolable compounds and give the hydroxy peptides in good yields when treated with tetrathiomolybdate.

O-nitrates of hydroxyamino acids serine and threonine

The synthesis of O-nitrates of natural hydroxyamino acids serine and threonine was described for the first time.

Isolation of 8′-Phosphate Ester Derivatives of Amicoumacins: Structure-activity Relationship of Hydroxy Amino Acid Moiety

Two new compounds, 8'-phospho derivatives of amicoumacins A and B, were isolated from the culture broth of a strain of Bacillus pumilus together with amicoumacins A and B. Their structures were elucidated on the basis of spectroscopic methods and alkaline phosphatase treatments. Comparison of the antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) of these compounds suggested that C-8' hydroxyl and C-12' amide group of amicoumacin A played a critical role for anti-MRSA activity.

Systematic Analysis of Proteoglycan Modification Sites in Caenorhabditis elegans by Scanning Mutagenesis

Proteoglycan modification is essential for development and early cell division in Caenorhabditis elegans. The specification of proteoglycan attachment sites is defined by the Golgi enzyme polypeptide xylosyltransferase. Here we evaluate the substrate specificity of this xylosyltransferase for its downstream targets by using reporter proteins containing proteoglycan modification sites from C. elegans syndecan/SDN-1. The N terminus of the SDN-1 contains a Ser-Gly proteoglycan site at Ser(71), flanked by potential mucin and N-glycosylation sites. However, Ser(71) was exclusively used as a proteoglycan site in vivo, based on mapping studies with a Ser(71) reporter protein, glycosyltransferase RNA interference, and co-expression of worm polypeptide xylosyltransferase. To elucidate the substrate requirements of this enzyme, a library of 42 point mutants of the Ser(71) reporter was expressed in tissue culture. The nematode proteoglycan modification site in SDN-1 required serine (not threonine), two flanking glycine residues (positions -1 and +1), and either one proximal acidic N-terminal amino acid (positions -4, -3, and -2) or a pair of distal N-terminal acidic amino acids (positions -6 and -5). C-terminal acidic amino acids, although present in many proteoglycan modification sites, had minimal impact on xylosylation at Ser(71). Proline inhibited glycosylation when present at -1, +1, or +2. The position of glycine, proline, and acidic amino acids allows the glycosylation machinery to discriminate between mucin and proteoglycan modification sites. The key residues that define proteoglycan modification sites also function with the Drosophila polypeptide xylosyltransferase, indicating that the specificity in the glycosylation process is evolutionarily conserved. Using a neural network method, a preliminary proteoglycan predictor has been developed.

GC‐MS detection of chiral markers in cocoa beans of different quality and geographic origin

Fermented cocoa beans (Theobroma cacao L., Sterculiaceae) from different countries of origin (Ecuador, Ghana, Trinidad) and cocoa beans roasted under defined conditions (industrial roasting; 150-220 degrees C for 20 min, dry roasting in conventional oven) were analyzed for their contents of certain chiral hydroxy acids, catechins, and amino acids. Cocoa beans are fermented, dried, and industrially transformed by roasting for the production of chocolate, cocoa powders, and other cocoa-related products. Fermentation and roasting conditions influence the contents of chiral compounds such as hydroxy acids, amino acids, and polyphenols, depending on technological procedures as well as some technical parameters. The aim of this work was to check if the content and nature of the named chiral compounds present both in fermented and roasted cocoa beans could be related to the traditional parameters used to classify the variety of seeds and the degree of fermentation. The extent of racemization of amino acids in fermented cocoa beans was low while it slowly increased during roasting, depending on the temperature applied. L-lactic acid was always higher than the D-form while citric acid was generally the most abundant hydroxy acid detected in beans. A correlation was found between polyphenol content and degree of fermentation, while epimerization of (-)-epicatechin to (+)-catechin was observed during roasting. On the whole, results showed that several chiral compounds could be considered as good quality markers for cocoa seeds and cocoa-related products of different quality and geographic origin.

“Click Peptides” — Chemical Biology‐Oriented Synthesis of Alzheimer′s Disease‐Related Amyloid β Peptide (Aβ) Analogues Based on the “O‐Acyl Isopeptide Method”

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Analysis of hydroxyproline isomers and hydroxylysine by reversed-phase HPLC and mass spectrometry

Collagens, the most abundant mammalian proteins, contain a high content of hydroxylated amino acids, such as, 3- and 4- cis-/ trans-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl). Whereas the global content of 4-Hyp was studied by amino acid analysis, no technique to determine all five hydroxyamino acids simultaneously in collagens has been reported. Here, we report the separation of all five hydroxyamino acids as well as two Hyp epimers from all other proteinogenic amino acids after derivatization with N 2-(5-fluoro-2,4-dinitrophenyl)- l-valine amide ( l-FDVA) by RPC-UV-ESI-MS. The general applicability of this method is shown for three Hyp-containing peptides as well as collagen type I.

“Click Peptides”—Chemical Biology‐Oriented Synthesis of Alzheimer's Disease‐Related Amyloid β Peptide (Aβ) Analogues Based on the “O‐Acyl Isopeptide Method”

A clear understanding of the pathological mechanism of amyloid beta peptide (Abeta) 1-42, a currently unexplained process, would be of great significance for the discovery of novel drug targets for Alzheimer's disease (AD) therapy. To date, though, the elucidation of these Abeta1-42 dynamic events has been a difficult issue because of uncontrolled polymerization, which also poses a significant obstacle in establishing experimental systems with which to clarify the pathological function of Abeta1-42. We have recently developed chemical biology-oriented pH- or phototriggered "click peptide" isoform precursors of Abeta1-42, based on the "O-acyl isopeptide method", in which a native amide bond at a hydroxyamino acid residue, such as Ser, is isomerized to an ester bond, the target peptide subsequently being generated by an O-N intramolecular acyl migration reaction. These click peptide precursors did not exhibit any self-assembling character under physiological conditions, thanks to the presence of the one single ester bond, and were able to undergo migration to give the target Abeta1-42 in a quick and easy, one-way (so-called "click")conversion reaction. The use of click peptides could be a useful strategy to investigate the biological functions of Abeta1-42 in AD through inducible activation of Abeta1-42 self-assembly.

O—N Intramolecular Alkoxycarbonyl Migration of Typical Protective Groups in Hydroxyamino Acids.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Identification of Tyrosine Residues Critical for the Function of an Ion-coupled Multidrug Transporter

Aromatic residues may play several roles in integral membrane proteins, including direct interaction with substrates. In this work, we studied the contribution of tyrosine residues to the activity of EmrE, a small multidrug transporter from Escherichia coli that extrudes various drugs across the plasma membrane in exchange with protons. Each of five tyrosine residues was replaced by site-directed mutagenesis. Two of these residues, Tyr-40 and Tyr-60, can be partially replaced with hydroxyamino acids, but in the case of Tyr-40, replacement with either Ser or Thr generates a protein with modified substrate specificity. Replacement of Tyr-4 with either Trp or Phe generates a functional transporter. A Cys replacement at this position generates an uncoupled protein; it binds substrate and protons and transports the substrate downhill but is impaired in uphill substrate transport in the presence of a proton gradient. The role of these residues is discussed in the context of the published structures of EmrE.

Biosynthesis of food constituents: Amino acids: 4. Non-protein amino acids - a review

This review article gives a brief survey of the principal pathways that lead to the biosynthesis of the most important non-protein amino acids occurring in foods and feeds. These amino acids have been divided into the following groups: 3-amino acids and 4-amino acids, N-substituted amino acids, alicyclic amino acids, hydroxyamino acids, sulfur-containing amino acids, basic amino acids, and taurine.  

Phosphorylation and glycosylation interplay: Protein modifications at hydroxy amino acids and prediction of signaling functions of the human β3 integrin family

Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.

Pir Proteins of Saccharomyces cerevisiae Are Attached to β-1,3-Glucan by a New Protein-Carbohydrate Linkage

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

O−N Intramolecular Alkoxycarbonyl Migration of Typical Protective Groups in Hydroxyamino Acids

O-N Intramolecular alkoxycarbonyl (carbonate-carbamate) migration was found to occur as a common reaction of hydroxyamino acids under mild basic aqueous conditions with no formation of side products. Carbonate protective groups migrate to produce amino-protected carbamate derivatives of hydroxyamino acids with high efficiency and purity.

Ab initio studies of the conformers and conformational distribution of the gaseous hydroxyamino acid threonine

Systematic and extensive conformational search has been performed to characterize the gas-phase threonine structures. A total of 1296 unique trial structures were generated by allowing for all combinations of internal single-bond rotamers. All the trial structures were optimized at the B3LYP/6-311G* level of the theory and then subjected to further optimization at the B3LYP/6-311++G** level. A total of 71 conformers were found and their rotational constants, dipole moments, zero-point vibrational energies, harmonic frequencies and vertical ionization energies of all the conformers were determined. Single-point energies were also calculated at the MP2/6-311G(2df,p) and B3LYP/6-311G(2df,p) levels. Characteristic H-bonding types were classified and listed for all the conformers. The conformational distributions of gaseous threonine at various temperatures were calculated.

Phosphorylation of WASP by the Cdc42-associated Kinase ACK1: DUAL HYDROXYAMINO ACID SPECIFICITY IN A TYROSINE KINASE

ACK1 is a nonreceptor tyrosine kinase that associates specifically with Cdc42. Relatively few ACK1 substrates and interacting proteins have been identified. In this study, we demonstrated that ACK1 phosphorylates the Wiskott-Aldrich syndrome protein (WASP), a Cdc42 effector that plays an important role in the formation of new actin filaments. ACK1 and WASP interact in intact cells, and overexpression of ACK1 promotes WASP phosphorylation. Phosphorylation of WASP in vitro was enhanced by the addition of Cdc42 or phosphatidylinositol 4,5-biphosphate, presumably due to release of the autoinhibitory interactions in WASP. Surprisingly, when we mapped the sites of WASP phosphorylation, we found that ACK1 possesses significant serine kinase activity toward WASP (directed at Ser-242), as well as tyrosine kinase activity directed at Tyr-256. A serine peptide derived from the Ser-242 WASP phosphorylation site is also a substrate for ACK1. ACK1 expressed in bacteria retained its serine kinase activity, eliminating the possibility of contamination with a copurifying kinase. Serine phosphorylation of WASP enhanced the ability of WASP to stimulate actin polymerization in mammalian cell lysates. Thus, the tyrosine kinase ACK1 acts as a dual specificity kinase toward this substrate. In contrast to other dual specificity kinases that more closely resemble Ser/Thr kinases, ACK1 is a tyrosine kinase with an active site that can accommodate both types of hydroxyamino acids in substrates.

GAG Mimetic Libraries: Sulphated Peptide as Heparin-like Glycosaminoglycan Mimics in Their Interaction with FGF-1

Heparin and heparan sulphate (HS) are heterogenous, linear, polysulphated polysaccharides that are important in the regulation of a wide variety of biological processes including blood coagulation, in cell differentiation, adhesion, invasion, migration and development, and in tumor-related cellular events such as growth regulation and metastasis. In general, heparin/HS interacts with proteins mainly through ionic interactions between its negatively charged groups and positively charged groups on the proteins. From a mechanistic or therapeutic standpoint, it is attractive to design less complex charged molecules, other than oligosaccharides, as mimics of heparin. In an attempt to improve the accessibility of heparin mimics, it was assumed, provided that the correct charge topography could be achieved, that sulphated peptides might also act as mimics. Therefore, sulphated peptide combinatorial libraries were generated on solid support to identify novel polyanionic structures that mimic the role of heparin/HS in its binding to fibroblast growth factors (FGFs). Libraries were synthesised by direct sulphation of the peptide on solid phase or by using O-sulphonated building blocks during peptide synthesis. Quantitative solid-phase O-sulphonation of hydroxy amino acid residues in a peptide chain was effected by sulphur trioxide pyridine (SO3-Pyr) complex in anhydrous pyridine at 65 °C for 4 h. O-Sulphonated building blocks were successfully synthesised in solution and, after stabilisation of the sulphate group by complexion with tetrabutyl ammonium ions, were employed in the synthesis of sulphated peptide libraries, similar to those generated by direct O-sulphonation on solid supports. The libraries were incubated with fluorescent-labelled FGF-1, and analysis and sequence determination of active compounds was carried out using Edman degradation. Selected sulphated peptides from the screening were resynthesised and their affinity for FGF-1 (acidic FGF) was studied in solution competition assays using surface plasmon resonance. These studies showed that sulphated decapeptides do bind to FGF-1 and inhibit its binding to immobilised heparin in the low micromolar concentration range.

Biochemical and catalytic properties of two β-glycosidases purified from workers of the termite Macrotermes subhyalinus (Isoptera: Termitidae)

Two s-glycosidases were purified from the termite Macrotermes subhyalinus (Rambur) workers by chromatography on gel filtration, ion exchange and hydrophobic interaction columns. The preparations were shown to be homogeneous on Polyacrylamide gel. Both enzymes have a similar molecular mass (68 KDa) and optimum pH (5.4) but differ in optimal temperature and thermal stability. The s-glycosidases preferred s-fucosides to s-glucosides, s-galactosides and s-xylosides, and hydrolysed glucose-glucose-s-(l-4) linkages better than s-(l-3), s-(l-2) and s-(l-6) linkages. They did not hydrolyse saccharides such as melibiose, sucrose, lactose, xylobiose, melizitose, stachyose, lactose, raffinose, laminarin, arabinogalactan, carboxymethylcellulose, inulin, lichenan and starch. s-Glycosidase A and s-glycosidase B of M. subhyalinus workers are capable of catalysing transglucosylation reactions. The yields of glucosylation of hydroxyamino acid derivatives and phenylethanol, catalysed by the two enzymes in the presence of cellobiose as glucosyl donor, were lower than those reported previously with conventional sources of s-glycosidases. In addition, the optimum pH is different for the hydrolysis and trans glucosylation reactions.On the basis of this work, it is proposed that the physiological role of s-glycosidase A and s-glycosidase B of M. subhyalinus workers is the digestion of di- and oligosaccharides derived from hemicelluloses and celluloses.

The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

The orexin-1 receptor interacts with beta-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with beta-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of beta-arrestin-2-GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with beta-arrestin-2, studies in wild-type and beta-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a beta-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with beta-arrestin-2, and indicate an important role of beta-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation.

A Direct Intramolecular Asymmetric Catalytic Aldol Cyclodehydration of meso‐3,4‐Disubstituted‐1,6‐dialdehydes.

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