A practical synthesis of N α -Fmoc protected l-threo-β-hydroxyaspartic acid derivatives for coupling via α- or β-carboxylic group

Abstract  

A simple and practical general synthetic protocol towards orthogonally protected tHyAsp derivatives fully compatible with Fmoc solid-phase peptide synthetic methodology is reported. Our approach includes enantioresolution of commercially available d,l-tHyAsp racemic mixture by co-crystallization with l-Lys, followed by ion exchange chromatography yielding enantiomerically pure l-tHyAsp and d-tHyAsp, and their selective orthogonal protection. In this way N ^α -Fmoc protected tHyAsp derivatives were prepared ready for couplings via either α- or β-carboxylic group onto the resins or the growing peptide chain. In addition, coupling of tHyAsp via β-carboxylic group onto amino resins allows preparation of peptides containing tHyAsn sequences, further increasing the synthetic utility of prepared tHyAsp derivatives.     

Life cycle assessment of the manufacture of lactide and PLA biopolymers from sugarcane in Thailand

Background  

l-lactide is the monomer for the polymer poly-l-lactic acid (PLLA). PLLA can be made from renewable resources, and is used in an increasing amount of applications. The biopolymer PLLA is one type of polymer of the family of polylactic acids (PLAs). Purac produces l-lactide and d-lactide, and supports partners with know-how to produce their own PLA from lactide. This life cycle assessment (LCA) study supporting market development presents the eco-profile of lactides and PLA biopolymers.     

α-(1 → 3)-D-glucans from fruiting bodies of selected macromycetes fungi and the biological activity of their carboxymethylated products

Purpose of work  

To show biological activity of carboxymethylated α-(1 → 3)-d-glucans isolated from the selected macromycetes fungi on human tumor and normal cells.     

Amino acid racemization reveals differential protein turnover in osteoarthritic articular and meniscal cartilages

Introduction  

Certain amino acids within proteins have been reported to change from the L form to the D form over time. This process is known as racemization and is most likely to occur in long-lived low-turnover tissues such as normal cartilage. We hypothesized that diseased tissue, as found in an osteoarthritic (OA) joint, would have increased turnover reflected by a decrease in the racemized amino acid content.     

The effects of polymer phase ratio and feeding strategy on solid–liquid TPPBs for the production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylacetylcarbinol from benzaldehyde using <Emphasis Type="Italic">Candida utilis</Emphasis>

Purpose of Work  

To increase the bioproduction of l-phenylacetylcarbinol (PAC), a precursor molecule in the synthesis of the decongestants ephedrine and pseudoephedrine and which suffers from substrate, product, and by-product inhibition, by ensuring that the delivery of the substrate, benzaldehyde, is maintained within a strict concentration window.     

Synthesis of γ-aminobutyric acid by expressing <Emphasis Type="Italic">Lactobacillus brevis</Emphasis>-derived glutamate decarboxylase in the <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> strain ATCC 13032

Purpose of work  

Purpose of this work is to synthesize γ-aminobutyric acid by glutamate-producing species expressing Lactobacillus brevis-derived glutamate decarboxylase genes, i.e. recombinant Corynebacterium glutamicum strains, which directly convert endogenous l-glutamate precursor into γ-aminobutyric acid (GABA) through single-step fermentation.     

SPRIT: Identifying horizontal gene transfer in rooted phylogenetic trees

Background  

Phylogenetic trees based on sequences from a set of taxa can be incongruent due to horizontal gene transfer (HGT). By identifying the HGT events, we can reconcile the gene trees and derive a taxon tree that adequately represents the species' evolutionary history. One HGT can be represented by a rooted Subtree Prune and Regraft (RSPR) operation and the number of RSPRs separating two trees corresponds to the minimum number of HGT events. Identifying the minimum number of RSPRs separating two trees is NP-hard, but the problem can be reduced to fixed parameter tractable. A number of heuristic and two exact approaches to identifying the minimum number of RSPRs have been proposed. This is the first implementation delivering an exact solution as well as the intermediate trees connecting the input trees.     

Synthesis of <Emphasis Type="SmallCaps">L</Emphasis>-ascorbic acid in the phloem

Background  

Although plants are the main source of vitamin C in the human diet, we still have a limited understanding of how plants synthesise L-ascorbic acid (AsA) and what regulates its concentration in different plant tissues. In particular, the enormous variability in the vitamin C content of storage organs from different plants remains unexplained. Possible sources of AsA in plant storage organs include in situ synthesis and long-distance transport of AsA synthesised in other tissues via the phloem. In this paper we examine a third possibility, that of synthesis within the phloem.     

Synthesis of mono- and disaccharide amino-acid derivatives for use in solid phase peptide synthesis

N-Fluorenylmethyloxycarbonyl-protected serine and threonine derivatives, carryingO-glycosidically - or -linked peracetylated -d-Galp-(1–3)-d-GalNAcp carbohydrate chains, were prepared. These derivatives are intended for use in solid phase glycopeptide synthesis. Suitably protected mono- and disaccharide thioglycosides were used as carbohydrate intermediates. These were activated by treatment with bromine to give the glycosyl bromides, which were then used in silver triflate-promoted glycosidations ofN-fluorenylmethyloxycarbonyl amino-acid phenacyl esters. Removal of the phenacyl esters with zinc gave the target free acids.     

Long-distance transport of <Emphasis Type="SmallCaps">L</Emphasis>-ascorbic acid in potato

Background  

Following on from recent advances in plant AsA biosynthesis there is increasing interest in elucidating the factors contributing to the L-ascorbic acid (AsA) content of edible crops. One main objective is to establish whether in sink organs such as fruits and tubers, AsA is synthesised in situ from imported photoassimilates or synthesised in source tissues and translocated via the phloem. In the current work we test the hypothesis that long-distance transport is involved in AsA accumulation within the potato tuber, the most significant source of AsA in the European diet.     

Agaroids from New Zealand members of the Gracilariaceae (Gracilariales,Rhodophyta) — a novel dimethylated agar

Polysaccharide extracts from four New Zealand members of the Gracilariaceae have been characterized by ¹³C-NMR spectroscopy and GLC analysis of alditol acetate derivatives prepared using a new double hydrolysis-reduction procedure. All were based on variously substituted repeating disaccharide units of agarobiose and 20% of its precursor containing l-galactose-6-sulfate. Gracilaria truncata yielded a firm gelling agar with 67% methylation on the 6-position of the d-galactose residues. The other extracts belong to a new class of agar molecules having methylation on both the 6-position of the d-galactose units and the 2-position of the l-sugar units. The Curdiea coriacea polysaccharide displayed this double methylation almost completely ( 96 %); the alkali-modified polymer thus had only two free hydroxy-groups per disaccharide repeat unit, yet still gave a firm gel. The Curdiea flabellata and Melanthalia abscissa extracts had this double methylation pattern but to a lesser extent, and additional xylosyl branch units on up to 18% of the repeating disaccharide units.     

Physiological comparison of d-cysteine desulfhydrase of Escherichia coli with 3-chloro-d-alanine dehydrochlorinase of Pseudomonas putida CR 1-1

d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.     

The xylogalactan sulfate from Chondria macrocarpa (Ceramiales,Rhodophyta)

A structure is proposed for the complex xylogalactan sulfate from Chondria macrocarpa. The hot-water extract of C. macrocarpa was desulfated or alkali-treated and Smith degraded. Constituent sugars and their substitution patterns were identified using a modified Hakamori methylation procedure suited to sulfated polysaccharides and a double hydrolysis-reduction protocol that yielded derivatives from all of the sugar residues, including the labile 3,6-anhydrogalactosyl residues. The polymer has an agar-type backbone of alternating 3-linked \-d- and 4-linked -L-galactopyranosyl units. The d-residues are partially sulfated on O-2 (50%) and O-6 (20–30%). About 40% of the l-residues are present as the 3,6-anhydride and 25% as its precursor l-galactose 6-sulfate. A significant proportion of the remaining l-galactosyl residues have both a d-xylopyranosyl substituent on O-3 and a sulfate ester on O-6 and are stable to alkali.     

Antigen 85C-mediated acyl-transfer between synthetic acyl donors and fragments of the arabinan

Antigen 85 (ag85) is a complex of acyltransferases (ag85A–C) known to play a role in the mycolation of the d-arabino-d-galactan (AG) component of the mycobacterial cell wall. In order to better understand the chemistry and substrate specificity of ag85, a trehalose monomycolate mimic p-nitrophenyl 6-O-octanoyl-β-d-glucopyranoside (1) containing an octanoyl moiety in lieu of a mycolyl moiety was synthesized as an acyl donor. Arabinofuranoside acceptors, methyl α-d-arabinofuranoside (2), methyl β-d-arabinofuranoside (3), and methyl 2-O-β-d-arabinofuranosyl-α-d-arabinofuranoside (9) were synthesized to mimic the terminal saccharides found on the AG. The acyl transfer reaction between acyl donor 1 and acceptors 2, 3, and 9 in the presence of ag85C from Mycobacterium tuberculosis (M. tuberculosis) resulted in the formation of esters, methyl 2, 5-di-O-octanoyl-α-d-arabinofuranoside (10), methyl 5-O-octanoyl-β-d-arabinofuranoside (11), and methyl 2-O-(5-O-octanoyl-β-d-arabinofuranosyl)-5-O-octanoyl-α-d-arabinofuranoside (12) in 2 h, 2 h and 8 h, respectively. The initial velocities of the reactions were determined with a newly developed assay for acyltransferases. As expected, the regioselectivity corresponds to mycolylation patterns found at the terminus of the AG in M. tuberculosis. The study shows that d-arabinose-based derivatives are capable of acting as substrates for ag85C-mediated acyl-transfer and the acyl glycoside 1 can be used in lieu of TMM extracted from bacteria to study ag85-mediated acyl-transfer and inhibition leading to the better understanding of the ag85 protein class. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of a mannose-6-phosphate isomerase from Geobacillus thermodenitrificans that converts monosaccharides

A recombinant mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (GTMpi) isomerizes aldose substrates possessing hydroxyl groups oriented in the same direction at the C2 and C3 positions such as the d- and l-forms of ribose, lyxose, talose, mannose, and allose. The activity of GTMpi for d-lyxose isomerization was optimal at pH 7.0, 70°C and 1 mM Co²⁺. Under these conditions, the k _cat and K _m values were 74,300 s⁻¹ and 390 mM for d-lyxose and 28,800 s⁻¹ and 470 mM for l-ribose, respectively. The half-lives of the enzyme at 60, 65, and 70°C were 388, 73, and 27 h, respectively. GTMpi catalyzed the conversion of d-lyxose to d-xylulose with a 38% conversion yield after 3 h, and converted l-ribose to l-ribulose with a 29% conversion yield.     

Stepwise engineering of a <Emphasis Type="Italic">Pichia pastoris</Emphasis> D-amino acid oxidase whole cell catalyst

Background  

Trigonopsis variabilis D -amino acid oxidase (Tv DAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of Tv DAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris Tv DAO whole-cell biocatalyst.     

Inhibitory Effects of Cysteine and Cysteine Derivatives on Germination of Sporangiospores and Hyphal Growth of Different Zygomycetes

The in vitro antifungal activity of cysteine (d- and l-cysteine) and its four derivatives (l-cysteine-methyl-ester, N-acetyl-cysteine, N-isobutyryl-d-cysteine, and N-isobutyryl-l-cysteine) were investigated on 20 fungal isolates representing 16 genera (Absidia, Actinomucor, Backusella, Gilbertella, Micromucor, Mortierella, Mucor, Mycotypha, Phycomyces, Rhizomucor, Rhizopus, Saksenaea, Syncephalastrum, Thamnostylum, Umbellopsis, and Zygorynchus). The inhibitory potential of different concentrations of these compounds, ranging from 0.625 to 10 mM, were investigated on the germination of sporangiospores as well as on hyphal extension, using broth microdilution method and agar plate test. Treatment with cysteine and its derivatives resulted in a strong inhibition in most studied strains. At 10 mM of compounds, complete blockage of growth was observed for some isolates. Sensitive species exhibited severe changes in colony morphology in the presence of 10 mM l-cysteine, N-acetyl-cysteine, and N-isobutyryl-l-cysteine. Microscopic observations revealed that 10 mM N-acetyl-cysteine induced dramatic modifications in the structural organization of the hyphae. Results suggest that cysteine and its derivatives have a therapeutic potential against fungal infections caused by Zygomycetes species.     

A comparative study of recombinant and native frutalin binding to human prostate tissues

Background  

Numerous studies indicate that cancer cells present an aberrant glycosylation pattern that can be detected by lectin histochemistry. Lectins have shown the ability to recognise these modifications in several carcinomas, namely in the prostate carcinoma, one of the most lethal diseases in man. Thus, the aim of this work was to investigate if the α-D-galactose-binding plant lectin frutalin is able to detect such changes in the referred carcinoma. Frutalin was obtained from different sources namely, its natural source (plant origin) and a recombinant source (Pichia expression system). Finally, the results obtained with the two lectins were compared and their potential use as prostate tumour biomarkers was discussed.     

On the Manner of Cyclization of <Emphasis Type="Italic">N</Emphasis>-Acylated Aspartic and Glutamic Acid Derivatives

Abstract  

When synthesizing arylpiperazine library modified with N-acylated amino acid derivatives (e.g., cyclized aspartic acid, cyclized glutamic acid, proline) we wished to rapidly determine the way of cyclization of N-acylated glutamic acid derivatives. During concomitant cleavage and cyclization two alternative routes were possible—either formation of six-member imide (glutarimide) or five-member lactam. Application of MS/MS and ¹H NMR method allowed us to establish that cyclization of N-acylated glutamic acid derivatives preceded to lactams—N-acylated pyroglutamic acid derivatives.     

In vitro study of the antioxidative properties of the glucose derivatives against oxidation of plasma components

Oxidative stress has been implicated in the pathogenesis of variety of diseases. Since the endogenous antioxidant defense may be not adequate to counteract the enhanced generation of oxidants, a growing interest in research for exogenous nutrients has been observed. The present study was designed to assess in vitro the antioxidative properties of the glucose derivatives: calcium d-glucarate, d-gluconic acid lactone, and sodium d-gluconate (0.5–3 mM) in the protection of plasma proteins and lipids, against the damage caused by 0.1 mM peroxynitrite (ONOO⁻). Exposure of plasma to ONOO⁻ resulted in carbonyl groups increase, 3-nitrotyrosine (3-NT) formation, reduction in thiol groups, and enhanced lipid peroxidation. d-Gluconic acid lactone and sodium d-gluconate effectively decreased 3-NT formation; the antinitrative action of calcium d-glucarate was less effective. In plasma samples incubated with ONOO⁻ and tested compounds, the level of carbonyl groups was decreased in comparison to plasma samples treated only with ONOO⁻. The level of protein −SH groups and glutathione was significantly higher in the presence of glucose derivatives than in plasma samples treated with ONOO⁻ only. All the tested compounds had the inhibitory effect on the peroxynitrite-induced plasma lipids peroxidation. The results obtained from our work indicate that calcium d-glucarate, d-gluconic acid lactone, and sodium d-gluconate may partly protect plasma proteins and lipids against peroxynitrite-induced damages.