New synthetic catecholate-type siderophores with triamine backbone

New analogues of triscatecholate siderophores based on linear or tripodal triamines with or without spacer groups or lipophilic and hydrophilic substituents were synthesized. The catecholate moieties were prepared in OH-forms, as acetylated compounds or masked as 8-methoxycarbonyloxy-2,4-dioxo-1,3-benzoxazine derivatives. Some of the new compounds were active as siderophores tested by growth promotion assays using various Gram-negative bacteria and mycobacteria under iron limitation and by CAS-assay. Structure-activity-correlations have been studied.     

Novel catecholate-type siderophore analogs based on a myo-inositol scaffold

A novel 1,3,5-triamino-myo-inositol derivative ispresented as a readily available scaffold for the design oftripodal siderophore mimetics.Based on this scaffold, various hexadentate catecholate-type siderophoreanalogs weresynthesized by attaching the catechols to the inositol scaffold via spacer units of differentstruc-tureand length. The potential to tune the polarity of the inositol containing siderophoreanalogs has also beendemonstrated by varying the protection group strategy. The siderophoreactivity of the prepared siderophoreanalogs was examined by cross-feeding tests withvarious Gram-negative bacteria and mycobacteria.     

New artificial siderophores based on a monosaccharide scaffold

New artificial catecholate siderophores with methyl -d-glucopyranoside as scaffold were synthesized. The dihydroxy- or di(acetoxy)benzoyl moieties were attached either directly or via aminopropyl spacer groups, to the carbohydrate scaffold. The siderophore activity of the prepared siderophore analogs was examined by a growth promotion assay using various Gram-negative bacteria and mycobacteria and by the CAS-assay.     

Trishydroxamates and triscatecholates based on monosaccharides and myo-inositol as artificial siderophores

New trishydroxamates and triscatecholates based on methyl -D-glucopyranoside, methyl -D-galactopyranoside, methyl -D-ribopyranoside and methyl -D-xylopyranoside as well as on 1,3,5-tri-O-benzyl-myo-inositol were synthesized. N-Methylsuccinohydroxamate, N-methylglutarohydroxamate and their O-benzoyl derivatives were used as hydroxamate moieties. 2,3-Dihydroxybenzoyl derivatives and acylated compounds as well as 2,3- and 3,4-dihydroxybenzylidenehydrazino derivatives, partly with spacer groups, were utilized as catecholate components. The siderophore activity of the prepared siderophore analogues was examined by a growth promotion assay with various Gram-negative bacteria and mycobacteria and by the CAS-assay. Some trishydroxamates and triscatecholates showed siderophore activity on Gram-negative bacteria and triscatecholates on mycobacteria. Iron complexes of the trishydroxamates act as siderophores for all types of iron transport mutants. The recognition and uptake specificity of these compounds was studied by E. coli siderophore receptor and iron transport mutants. Structure activity correlations are discussed.     

Catecholates and mixed catecholate hydroxamates as artificial siderophores for mycobacteria

Different mono-, bis- or triscatecholates and mixed mono- or biscatecholate hydroxamates were synthesized as potential siderophores for mycobacteria. Siderophore activity was tested by growth promotion assays using wild type strains and iron transport mutants of mycobacteria as well as Gram-negative bacteria. Some triscatecholates and biscatecholate hydroxamates were active in mutants of Mycobacterium smegmatis deficient in mycobactin and exochelin biosynthesis or exochelin permease, respectively, indicating an uptake route independent of the exochelin/mycobactin pathway. Structure activity relationships were studied. Ampicillin conjugates of some of these compounds were inactive against mycobacteria but active against Gram-negative bacteria.     

New synthetic siderophores and their beta-lactam conjugates based on diamino acids and dipeptides

Linking of siderophores to antibiotics improves the penetration and therefore increases the antibacterial activity of the antibiotics. We synthesized the acylated catecholates and hydroxamates as siderophore components for antibiotic conjugates to reduce side effects of unprotected catecholate and hydroxamate moieties. In this paper, we report on bis- and tris-catecholates and mixed catecholate hydroxamates based on diamino acids or dipeptides. These compounds were active as siderophores in a growth promotion assay under iron limitation. Most of the conjugates with beta-lactams showed high in vitro activity against Gram-negative bacteria especially Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Stenotrophomonas maltophilia. The compounds with enhanced antibacterial activity use active iron uptake routes to penetrate the bacterial outer membrane barrier, demonstrated by assays with mutants deficient in components of the iron transport system. Correlation between chemical structure and biological activity was studied.     

Synthesis and biological activity of new glycyrrhizic acid conjugates with amino acids and dipeptides

New glycyrrhizic acid (GA) conjugates were synthesized with the use of tert-butyl esters of amino acids or benzyl esters of dipeptides; they contained two residues of L-amino acids (Met, Phe, Pro, and Ile or dipeptides Gly-Leu and Gly-Phe). Activation of GA carboxy groups was carried out with the help of N-hydroxysuccinimide, N,N′-dicyclohexylcarbodiimide, or N-hydroxybenzotriazole with dicyclohexylcarbodiimide. A proline-containing GA derivative is a low-toxic substance; it raises the level of agglutinins by 3.7 times in the blood of mice and 3 times that of hemolysins compared with the control. Dipeptide GA derivatives possess an expressed anti-HIV-1 activity in cultures of MT-4 cells and are 90-70 times less cytotoxic than azidothymidine. The selectivity index of the compounds exceeds those of GA by 110 and 34 times, respectively.     

Direct specific rotation measurements of amino acids,dipeptides, and tripeptides by laser‐based polarimetry

This paper addresses direct HPLC detection of amino acids, di and tripeptides via laser‐based polarimetry. Mass detection limits typically range from 0.5 to 50 μg injected on‐column, corresponding to concentration levels from 0.1 to 1 mg/ml. These levels are 1 to 2 orders of magnitude lower than measurements with conventional polarimetry, implying that concentration‐dependent phenomena (e.g., solute dimerization) are minimized. This is the first published report of the specific rotation of the di and tripeptides of Ala and Phe. The relationship between pH and specific rotation of selected amino acids, peptides, and lysozyme is described. Chirality 11:187–194, 1999. © 1999 Wiley‐Liss, Inc.     

基于氨基酸和二肽组成的蛋白质四级结构分类研究

基于最近邻居算法,从蛋白质一级序列出发,利用蛋白质序列氨基酸组成、二肤组成以及混合组成方法对蛋白质单聚体、二聚体、三聚体、四聚体、五聚体、六聚体和八聚体进行分类研究。结果表明:采用二肽组成编码方法的预洲效果最好,Jackknife检验和独立测试集检验的总体预测精度分别达到90.83%和95.48%,比相同数据集上基于伪氨基酸组成和组分耦合预测的方法提高了12和15个百分点;特别是对于五聚体蛋白,预测精度分别提高了90和50个百分点;说明二肽组成对于蛋白质四级结构分类研究是一种非常有效的特征提取方法。     

Screening and development of enzymes for determination and transformation of amino acids

ABSTRACT

The high stereo- and substrate specificities of enzymes have been utilized for micro-determination of amino acids. Here, I review the discovery of l-Phe dehydrogenase and its practical use in the diagnosis of phenylketonuria in more than 5,400,000 neonates over two decades in Japan. Screening and uses of other selective enzymes for micro-determination of amino acids have also been discussed. In addition, novel enzymatic assays with the systematic use of known enzymes, including assays based on a pyrophosphate detection system using pyrophosphate dikinase for a variety of l-amino acids with amino-acyl-tRNA synthetase have been reviewed. Finally, I review the substrate specificities of a few amino acid-metabolizing enzymes that have been altered, using protein engineering techniques, mainly for production of useful chemicals, thus enabling the wider use of natural enzymes.     

l-threo- and l-erythro-2-amino-3-hydroxyhex-4-ynoic acids: New amino acids from Tricholomopsis rutilans

2-Amino-3-hydroxyhex-4-ynoic acid, reported previously from Tricholomopsis rutilans, was shown to be a mixture of its threo- and erythro-forms. They were separated from each other and characterized by elementary analysis, optical rotation, TLC, IR, NMR spectra, catalytic hydrogenation, and by chemical synthesis. Their configurations were determined by the comparison of their hydrogenation products with known threo- and erythro-2-amino-3-hydroxyhexanoic acids.     

Synthesis and biological activity of tuftsin, its analogue and conjugates containing muramyl dipeptides or nor-muramyl dipeptides.

Several conjugates of muramyl dipeptide (MDP) or nor-muramyl dipeptide (nor-MDP) with tuftsin were synthesized. Conjugates 8a-f were prepared by acylation of protected tuftsin with the isoglutamine carboxyl group of MDP or nor-MDP 2a-f. Also tuftsin analogue 6 (H-Thr-Lys-Pro-Arg(NO2)-OH) was obtained. All synthesized compounds were investigated at the Medical University of Gdansk. The biological activity of the examined compounds was estimated using in vitro cultures of human monocytes and lymphocytes. The substances displayed cytotoxic effects, as was revealed in the viability tests performed. The effects were most probably mediated by the induction of an oxidative burst in monocytes and the stimulation of redox enzymes in lymphocytes. In addition, the analogues turned out to be efficient stimulators of TNFalpha and IL6 secretion by monocytes and lymphocytes. Nevertheless, the secretion of cytokines did not affect the viability of the leukocyte population used in the experiments.The beneficial properties of the compounds examined (mainly 6, 3, 8a and 8c), which implies their usefulness as potential therapeutic agents, are connected with their rapid start of action and more efficient effects compared with tuftsin alone. An in vivo assay on animal models will be performed.     

Drugs for chiral discrimination: Non-coded amino acids and dipeptides by asymmetric hydrogenation

The enantiomers of Propranolol, Pindolol, and Carazolol, well-known β-blockers, have been used to prepare cationic aminophosphine phosphinite rhodium complexes. Propraphos-Rh and Pindophos-Rh are very efficient catalysts in the asymmetric hydrogenation of N-Boc-protected unusual dehydroamino acid derivatives. Carazolol-Rh is less suitable in both activity and enantioselectivity. Under the same conditions, N-Boc-protected dehydrodipeptides are hydrogenated with diastereoselectivities between 70 and 90% de. Chirality 10:535–539, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis and reactivity of the monosaccharide esters of amino acids as models of teichoic acid fragment

The increasing prevalence of sepsis from Gram-positive bacterial pathogens necessitates further evaluation of the basic assumptions about the molecular pathogenesis of septic shock. Since diverse physiological functions of Gram-positive bacteria are controlled by the degree of esterification of teichoic acids with D-alanine, we examined the reactivity of monosaccharide esters in which anomerically free or protected D-glucose is linked through its C-6 hydroxy group to either phenylalanyl or tyrosyl residues as models for teichoic acid fragment. We show that the attached sugar moiety induces activation of the amino acid residue. Due to the enhanced reactivity of the NH₂ group in the monosaccharide esters studied, the formation of products generated by intramolecular and intermolecular glycation reactions is accelerated resulting in heterogeneous mixture of compounds. These findings suggest that, if similar adducts are formed by glycation of D-alanine in teichoic acid of Gram-positive bacteria, they should be examined as potential bioactive ligands or chemical message for infection.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

New branched amino acids for high affinity dendrimeric DC-SIGN ligands

A branched amino acid was synthesized from methyl glucopyranoside; this amino acid presents three amino groups protected by Fmoc and one acid group and can be used in classic peptide synthesis. In parallel, similar azido terminated blocks were synthesized.Successive coupling reaction and deprotection afforded dendrimers with up to 27 azido functional groups. As an example of application, d-mannose and l-fucose residues were linked through CuAAC coupling and resulting glycodendrimers were evaluated in their interaction with DC-SIGN using SPR competition assay.     

Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin—an enzyme that degrades the cell wall of Staphylococcus aureus—while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this “reactive handle” enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.     

Utilization of amino acids and dipeptides by Lactobacillus plantarum from orange in nutritionally stressed conditions

Aims:  To investigate amino acid and dipeptide utilization by Lactobacillus plantarum N4 isolated from orange peel, in a nutritionally depleted medium based on MRS (Mann, Rogosa, Sharpe).
Methods and Results:  In MRS with 0·1 g l⁻¹ of meat extract and without peptone and yeast extract, growth increased when essential and stimulatory amino acids and nonessential amino acid were added to the medium. Replacement of the essential amino acid, leucine, and the nonessential amino acid, glycine, by leucyl-leucine (Leu-Leu) and/or glycyl-glycine (Gly-Gly) significantly enhanced growth. Essential amino acids were mainly consumed and the dipeptides were almost completely used at the end of growth. Leucine and glycine accumulated internally from the peptides were higher than from the free amino acids. Glucose utilization increased in the media containing dipeptides compared with the medium containing free amino acids.
Conclusions:  In a N-depleted medium, Leu-Leu and/or Gly-Gly were more effective than the respective amino acids in supporting growth of the micro-organism. The more efficient internal accumulation of glycine and especially leucine from dipeptides confirmed the ability of the strain to assimilate mainly complex nitrogen molecules rather than simple ones.
Significance and Impact of the Study:  The ability of Lact. plantarum N4 to efficiently use dipeptides could contribute to spoilage development in the natural medium of the organism, orange juice.     

The accumulation and metabolism of amino acids by Biomphalaria glabrata, a freshwater pulmonate snail

Freshwater pulmonate snails (Biomphalaria glabrata), pre-treated under bacteriostatic conditions, were incubated in 10 ml of standard medium containing various U-¹⁴C-labelled amino acids at concentrations of 10 μM. Measurements of mass-specific accumulation rates (MSARs) based on HPLC and the accumulation of U-¹⁴C-labelled amino acids into snail tissues have shown unequivocally for the first time that freshwater snails achieved a net accumulation of all the amino acids tested, including aminoisobutyrate (AIB), aspartate, alanine and a mixture of 13 amino acids. There were no significant differences between the MSAR values determined by HPLC from those based on the use of radiolabelled amino acids, whereas MSAR values for control snails were negligible and significantly less. Incubation of snails in media containing radiolabelled aspartate and a mixture of amino acids showed that the accumulated amino acids were readily distributed through the snail’s tissues and then metabolized. The ecological and biochemical questions arising from the fact that freshwater snails are capable of net accumulation of exogenous amino acids at naturally occurring concentrations and subsequent metabolic conversion, contrary to widely held views, are addressed.