Synthesis of peptides employing 9-fluorenylmethyl chloroformate as a coupling agent

Summary The synthesis of peptides employing 9-fluorenylmethyl chloroformate (Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtained in good yield (70–95%). Furthermore, both the¹H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.     

1-(t-Butyldimethylsilyloxy) benzotriazole (TBDMS-OBt): A new and novel reagent for the synthesis of peptides

Synthesis and use of 1-(t-butyldimethylsilyloxy)benzotriazole (TBDMS-OBt) in the coupling of Fmoc-amino acid chlorides to amino free amino acid esters in homogeneous solution phase is described. The coupling required no addition of base and was fast and racemization free. Work up and isolation of products were easy. Yield, purity and ¹H NMR analysis of peptides, synthesised by this method, were satisfactory.     

1-(t-Butyldimethylsilyloxy) benzotriazole (TBDMS-OBt): A new and novel reagent for the synthesis of peptides

Summary Synthesis and use of 1-(t-butyldimethylsilyloxy)benzotriazole (TBDMS-OBt) in the coupling of Fmoc-amino acid chlorides to amino free amino acid esters in homogeneous solution phase is described. The coupling required no addition of base and was fast and racemization free. Work up and isolation of products were easy. Yield, purity and¹H NMR analysis of peptides, synthesised by this method, were satisfactory.     

Application of tritium high resolution NMR spectroscopy to analysis of tritium-labelled amino acids and peptides

Summary A method has been developed for the qualitative and quantitative analysis of complex isotopic mixtures of tritium-labelled amino acids and peptides by using high resolution³H NMR spectroscopy at 266.8 MHz. Determined were tritium distribution in alanine, glycine, tryptophan and 4-hydroxyproline amino acids, as well as in glycine and valine residues of peptides. Approaches have been worked out for the determination of spin coupling constants and isotope chemical shifts for the strongly coupled nonequivalent atoms of the methylene groups.     

Unexpected aspartimide formation during coupling reactions using Asp(OAl) in solid-phase peptide synthesis

Summary Succinimide ring closure is a well-documented side reaction in the synthesis of certain Asp-containing peptides. This side reaction is typically acid-or base-catalyzed, and its occurrence during coupling reactions has not been previously noted. This unforeseen manifestation of aspartimide formation was detected while exploring a new strategy for side-chain to side-chain lactam formation on a solid support to synthesizecyclo[D-Asp², Dap⁵]dynorphin A-(1-11) amide. The availability of allyl protecting groups, which provide an additional level of orthogonality in solid-phase peptide synthesis, was very appealing for use in preparing this conformationally constrained analogue. We found that the allyl ester (OAl) was not sufficient protection from this side reaction in this susceptible D-Asp²-Gly³ sequence. Remarkably, the aspartimide formation appeared to occur during the coupling reaction in the absence of base if excess coupling reagent was present.     

Standard dimensions forcis N-methyl peptide unit and flexibility ofcis peptide units

The mean dimensions of thecis N-methyl peptide unit have been arrived at by analysing the crystal structure data on compounds containing such units. These dimensions can be used as standard in conformational studies on cyclic peptides. While the bonds meeting at C are almost coplanar, those meeting at N show a slight pyramidal disposition. A comparison of the dimensions of the normal and N-methylatedcis peptide units show that there are perceptible differences in the parameters connected with N. In addition, the flexibility of thecis peptide unit has been analysed by studying the distribution of the parameters in different classes of compounds such as cyclic di, tri and higher peptides. The salient features are: (i) The angle C^αCN in cyclic dipeptide and the angle C^δNC^α in higher peptides tend to be lower, when the peptide unit is associated with a prolyl residue; (ii) in cyclic tripeptides the internal anglesviz., C^αCN and CNC^α are significantly larger thereby increasing the intra-annular space; (iii) the bond C^α-C is distinctly shorter when it occurs in cyclic dipeptides. The results lead to the conclusion that thecis peptide unit takes up aneed-based flexibility in its dimension.     

Solution conformation study of substance P methyl ester and [Nle10]-neurokinin A (4–10) by nmr spectroscopy

High-resolution proton spectra at 500 MHz of two tachykinin peptides, substance P methyl ester (SPOMe) and [Nle¹⁰]-neurokinin A (4–10), have been obtained in dimethylsulfoxide (DMSO), and for SPOMe, also in 2, 2, 2-trifluoroethanol (TFE)/water mixtures. Complete chemical shift assignments for these peptides were made based on two-dimensional (2D) nmr techniques, correlated spectroscopy and total COSY. J coupling measurement and nuclear Overhauser effect spectroscopy (NOESY) were then used to determine the conformation of these peptides in the various solvents. Based on the J coupling, NOE correlations, and temperature coefficients of the NH resonances, it is concluded that these two peptides exist in DMSO at room temperature as a mixture of conformers that are primarily extended. For SPOMe in TFE/water with high TFE content, however, helical structures are found to be present, and they become quite clear at temperatures between 270 and 280 K. The variation of the ¹³C chemical shifts of the C_α (the secondary shift) with TFE contents corroborates this conclusion. The NOE and C_α shifts show that the main helical region for SPOMe lies between ⁴P and ⁹G. The C-terminus segment L? M? NH₂ is found to be quite flexible, which appears to be quite common for neurokinin-1 selective peptides. © 1993 John Wiley & Sons, Inc.     

1H-1H and 13C-13C vicinal coupling constants and amino acid side chain conformation in peptides

The vicinal coupling constants ¹³C′-¹³C^γ were measured in aspartic acid and phenylalanine (85 % ¹³C enrichment) as free amino acids and in the peptides Asp-Pro and Gly-Pro-Phe. These coupling constants used in connection with those between the α -and the β-protons provide the unambiguous assignment of rotamers I and II in the Asp and Phe side chains. The method is generally applicable to other amino acids and residues even in large peptides. A possible set of J_g^c,c and J_t^c,c values is proposed for the use of carbon 13-carbon 13 vicinal coupling constants in the side chain conformational studies of amino acid residues with a free carboxyl group.     

Maillard Glycation of Peptides Containing the (N αHis)Ac Chelator for 99mTc(CO)3 Labeling

The retro-N ^α-carboxymethyl histidine moiety, short (N ^αHis)Ac, functions as an efficient chelator for the ^99mTc(CO)₃ core which allows the labeling of the peptides with a very high specific activity. However as a general consequence of the neutral [^99mTc(CO)₃] (N ^αHis)Ac-complex, undesired accumulation in kidney and liver may be high. In order to improve the pharmacokinetic properties of the radiolabeled peptides containing this chelate, attempts have been made to conjugate a carbohydrate using the Maillard reaction. Since the (N ^αHis)Ac moiety has an unusually reactive N ^α, various chemical strategies have been explored to perform the Maillard reaction followed by the Amadori rearrangement on peptides containing this chelator. This indicated that a selective protection of the secondary nitrogen in the chelator is necessary.Australian Peptide Conference Issue.     

Radioenzymatic determination of CMP-N-acetylsialic acid and free N-acetylsialic acid in biological material

Free N-acetylsialic acid (NeuNAc) and CMP-N-acetylsialic acids (CMP-NeuNAc) are extracted from freeze-clamped or liquid nitrogen-frozen biological material by sequential extraction with cold acetone and acetone/water. [¹⁴C]NeuNAc and [¹⁴C]CMP-NeuNAc (20,000 dpm each) are added to the frozen material to correct for small losses occurring during the subsequent steps. NeuNAc and CMP-NeuNAc are separated by anion-exchange chromatography. CMP-NeuNAc is hydrolyzed with formic acid and again chromatographed on an ion-exchange column. The NeuNAc-containing fractions (representing free NeuNAc and CMP-NeuNAc) are converted to [¹⁴C]CMP-NeuNAc in the presence of [¹⁴C]CTP and CMP-NeuNAc synthetase. [¹⁴C]CMP-NeuNAc is separated by paper chromatography and the radioactivity measured by liquid scintillation counting. The amount of NeuNAc is calculated from a calibration curve obtained with NeuNAc standards. The small amounts of [¹⁴C]NeuNAc and [¹⁴C]CMP-NeuNAc added initially do not interfere with the final assay. The method gives reliable values down to 50 pmol/assay, but the sensitivity can be easily increased by a factor of 10. Recoveries, with NeuNAc and CMP-NeuNAc added to biological extracts, were 98.3 and 98.5% for NeuNAc and CMP-NeuNAc, respectively. With this method values of 61.2 ± 12.8 and 24.4 ± 5.2 nmol/g wet wt were found in rat liver for free NeuNAc and CMP-NeuNAc, respectively. Values for free NeuNAc found in human blood plasma were 600 ± 476 and 373 ± 180 pmol/g plasma for healthy persons and patients with breast cancer, respectively. Free CMP-NeuNAc could not be found in plasma.     

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25°C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l⁻¹, respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties. Journal of Industrial Microbiology & Biotechnology (2001) 26, 235–240. Received 02 August 2000/ Accepted in revised form 15 December 2000     

Ocellatins: New Antimicrobial Peptides from the Skin Secretion of the South American Frog <Emphasis Type="Italic">Leptodactylus ocellatus</Emphasis> (Anura: Leptodactylidae)

The emergence, in recent years, of microbial resistance to commonly used antibiotics has aroused a search for new naturally occurring bactericidal and fungicidal agents that may have clinical utility. In the present study, three new antimicrobial peptides were purified from the electrical-stimulated skin secretion of the South American frog Leptodactylus ocellatus by reversed-phase chromatographic procedures. Ocellatin 1 (¹GVVDILKGAGKDLLAHLVG ISEKV²⁵-CONH²), ocellatin 2 (¹GVVDILKGAGKDLLAHLVGKISEKV²⁵-CONH₂) and ocellatin 3 (¹GVLDILKNAAKNILAHAAEQI²¹-CONH₂) are structurally related peptides. These peptides present hemolytic activity against human erythrocytes and are also active against Escherichia coli. Ocellatins exhibit significant sequence similarity to other amphibian antimicrobial peptides, mainly to brevinin 2ED from Rana esculenta.     

Control of diferulate formation in dicotyledonous and gramineous cell-suspension cultures

Primary cell wall polysaccharides of some plants carry ester-linked feruloyl groups that can be oxidatively dimerised both within the protoplast and after secretion into the apoplast. Apoplastic dimerisation has been postulated to form inter-polysaccharide cross-links, contributing to wall assembly, but this role remains conjectural. By feeding cultured cells with [¹⁴C]cinnamate, we monitored the kinetics of polysaccharide-binding and subsequent dimerisation of ¹⁴C-labelled feruloyl groups. Cultured maize and spinach cells took up [¹⁴C]cinnamate more rapidly than barley, Arabidopsis, Acer, tomato and rose cultures. Maize and spinach cells rapidly formed [¹⁴C]feruloyl-polysaccharides and, simultaneously, low-M_r [¹⁴C]feruloyl esters. When all free [¹⁴C]cinnamate had been consumed, there followed a gradual recruitment of radiolabel from the low-M_r pool into the polysaccharide fraction. A proportion of the [¹⁴C]feruloyl-polysaccharides was sloughed into the culture medium, the rest remaining wall-bound. Some of the polysaccharide-bound [¹⁴C]feruloyl groups were coupled to form dehydrodiferulates. At least six putative isomers of [¹⁴C]dehydrodiferulate were formed both rapidly (thus intra-protoplasmically) and gradually (thus mainly apoplastically). These data do not support the hypothesis that intra-protoplasmic dimerisation yields predominantly one isomer (8–5′-dehydrodiferulate). In maize, apoplastic coupling was much more extensive in 7-day old than in 2-day-old cultures; indeed, in 2-day-old cultures apoplastic coupling could not be evoked even by exogenous H₂O₂, suggesting strong control of peroxidase action by apoplastic factors. When apoplastic coupling was minimised by exogenous application of peroxidase-blockers (iodide, dithiothreitol and cysteine), a higher proportion of the secreted [¹⁴C]feruloyl-polysaccharides was sloughed into the medium. This observation lends support to the hypothesis that feruloyl coupling contributes to wall assembly.     

Analysis of the peptide carrier in the scutellum of barley embryos by photoaffinity labelling

The preparation of a phenylalanine analogue containing an azido group and its incorporation into dipeptides is described. Peptides modified in this way are taken up into barley (Hordeum vulgare L.) scutella via the previously characterized peptide-transport system. Photoactivation of modified peptides in the presence of isolated scutella resulted in irreversible inhibition of peptide uptake in a concentration-dependent manner. Transport of other solutes which share a common mechanism of energy coupling, but which are transported via distinct carriers, was not inhibited after photo-derivatization of scutella with the modified peptides. Derivatization of isolated scutellar tissue with a ¹⁴C-labelled peptide analogue, resulted in incorporation of label into two proteins of M_r = 54000 and 41000. Scutellar tissue from early-germinating seeds, which do not show active peptide uptake, did not incorporate label into these polypeptides. It is concluded that these proteins are components of the barley peptide-transport system.Abbreviations Ala alanine - Gly glycine - PAGE polyacrylamide gel electrophoresis - Phe phenylalanine - Pro proline - SDS sodium dodecyl sulphate This work was supported by a grant from the Agricultural and Food Research Council.     

A31P NMR study of the interaction of amphibian antimicrobial peptides with the membranes of live bacteria

Amphibian skin is a rich source of peptides that are specificto pathogens and act by disrupting bacterial membranes. Threeantimicrobial peptides were isolated from the skin glands ofAustralian tree frogs, Litoria caerulea and Litoriagenimaculata. NMR spectroscopy was used to observe changesinduced by these peptides in the ³¹P resonances of bacterialmembranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics, disrupted the membranes ofBacillus cereus and Staphylococcus epidermidis (Gram-positive), leadingto an increase in the isotropic ³¹P NMR signal. Caerin 4.1, anarrow-spectrum antibiotic, however, did not affect the ³¹Pspectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents onthe membranes of live bacteria.     

Enzymatic peptide synthesis by new supported biocatalysts

Summary It is well known that proteases can be successfully used for peptide synthesis in aqueous medium or in water-containing organic solvents. Copolymerized of acrylated derivatives of-chymotrypsin and polyethylene glycol (PEG) have been prepared and used as biocatalysts for the synthesis of model peptides in organic solvents containing a very low quantity of water. Trypsin has been similarly treated and used as a new biocatalyst particularly for the coupling of non-specific substrates.     

99mTc标记丝状噬菌体肽库及其在正常小鼠体内的分布

利用噬菌体展示技术已选出了多条与靶结合的肽. 然而,即使是体内直接筛选得到的,肽与肿瘤或靶器官的体内结合并不理想. 为了更好地理解噬菌体在体内的循环, 通过MAG3 ^99mTc标记噬菌体肽库,研究了肽库在体内分布. 体内分布实验结果显示,^99mTc-噬菌体主要分布在肝和脾中,而心脏、肌肉、脑和胰腺这些器官或组织中的分布非常低. ^99mTc-噬菌体在胃、肠和骨中的累积,随着时间延长在不断升高,其他器官中的吸收则在不断降低. 从5 min到30 min,^99mTc-噬菌体在血中清除迅速. 当噬菌体在体内循环足够长的时间后,一些噬菌体颗粒可以穿透血管进入并内化在器官或组织中. 总之,为了筛选具有高特异性和亲和性的肽,应该根据靶器官和筛选部位的不同,在筛选前确定合适的噬菌体在体内的循环时间.     

Characterization of the glyceraldehyde 3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from the extremely halophilic archaebacterium Haloarcula vallismortis has been purified in a four step procedure to electrophoretic homogeneity. The enzyme is a tetramer with a relative molecular mass of 160000. It is strictly NAD⁺-dependent and exhibits its highest activity in 2 mol/l KCl at 45°C. Amino acid analysis and isoelectric focusing indicate an excess of acidic amino acids. Two parts of the primary sequence are reported. These peptides have been compared with glyceraldehyde 3-phosphate dehydrogenases from other archaebacteria, eubacteria and eucaryotes. The peptides show a high grade of similarity to glyceraldehyde 3-phosphate dehydrogenase from eucaryotes.Abbreviations BCA bicinchoninic acid - CTAB cetyltrimethyl ammonium bromide - DTE dithioerythritol - DTT dithiothreitol - GAP glyccraldehyde 3-phosphate - GAPDH glyceraldehyde 3-phosphate dehydrogenase     

A 31P NMR study of the interaction of amphibian antimicrobialpeptides with the membranes of live bacteria

Summary Amphibian skin is a rich source of peptides that are specific to pathogens and act by disrupting bacterial membranes. Three antimicrobial peptides were isolated from the skin glands of Australian tree frogs,Litoria caerulea andLitoria genimaculata. NMR spectroscopy was used to observe changes induced by these peptides in the³¹P resonances of bacterial membranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics disrupted the membranes ofBacillus cereus andStaphylococcus epidermidis (Gram-positive), leading to an increase in the isotropic³¹P NMR signal. Caerin 4.1, a narrow-spectrum antibiotic, however, did not affect the³¹P spectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents on the membranes of live bacteria.     

Spectral analysis of a series of partially protected and deprotected tetrapeptides, analogues of AS-I phytotoxin

Summary A series of six tetrapeptides, analogues of AS-I phytotoxin, pathogenic to sunflower, have been synthesized either in solution and/or by solid phase methods and have been tested for phytotoxic activity in various plants and cytotoxic activity in three cancer cell lines. These peptides were identified as model compounds by fast atom bombardment (FAB), plasma desorption (PD), electrospray ionization (ESI) mass spectrometry and by¹H,¹H-¹H,¹³C and¹H-¹³C NMR. The data presented show that in protected tetrapeptides the molecular ion was easily identified whereas some difficulties appeared with the fully deprotected peptides. NMR spectra are given.