Rapid complexing of oxoacylglycerols with amino acids peptides and aminophospholipids

We prepared model Schiff bases from 2-[9-oxo]nonanoyl glycerol (2-MAG-ALD) and various amino compounds. 2-MAG-ALD was obtained by pancreatic lipase hydrolysis of trioleoyl glycerol and reductive ozonolysis of the resulting 2-monooleoyl glycerol. The reaction products were purified by thin-layer chromatography. Schiff bases were synthesized in greater than 50% yield by reacting 2-MAG-ALD with twofold molar excess of valine, Nα-acetyl-l-lysine methyl ester and the tripeptides glycyl-glycyl-glycine, glycyl-glycyl-histidine, and glycyl-histidyl-lysine in aqueous methanol and with 1-palmitoyl-2-stearoyl glycerophosphoethanolamine (PE) in chloroform.methanol for 16 h at room temperature. Prior to analysis the bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4°C. Reaction products were analyzed by high-performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS). Reduced Schiff bases of 2-MAG-ALD with PF and amino acids were analyzed by normal-phase HPLC/ESI/MS and those with peptides by reversed-phase HPLC/ESI/MS. Single adducts were obtained in all cases and both the α-amino group of valine and the ε-amino group of Nα-acetyl-l-lysine methyl ester were reactive. Molecular ions of reaction products were the only detected ions in the negative ionization mode, whereas in the positive ion mode sodiated molecular ions were also detected. The present study suggests that 2-MAG-ALD may form Schiff base adducts with amino compounds in other aqueous media, such as the intestinal lumen and in the hydrophobic environment of cell membranes.     

MALDI Q-TOF CID MS for Diagnostic Ion Screening of Human Milk Oligosaccharide Samples

Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.     

Development of an LC–MS/MS Method for the Analysis of Free Sphingoid Bases Using 4-Fluoro-7-nitrobenzofurazan (NBD-F)

The molecular species of sphingoid bases were tagged with the fluorescent amino group reagent, 4-fluoro-7-nitrobenzofurazan (NBD-F). The NBD-sphingoid bases were analyzed by a highly selective and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) technique capable of reliable detection of several fmol of the derivatives. Lipid extracts from plant samples were derivatized with NBD-F, and all nine species of free sphingoid bases present in plant sphingolipids were separated and quantified for the first time; a complete baseline resolution was achieved for cis-8 and trans-8 isomers of sphingoid bases by reversed phase HPLC on a C₁₈ column. The extraction and derivatization procedures and LC–MS/MS method can facilitate the progress of the studies for seeking the active components of sphingoid bases species in response to biological challenges.     

Compositional Analysis of Wax Esters in Human Meibomian Gland Secretions by Direct Infusion Electrospray Ionization Mass Spectrometry

Wax esters (WE) are one of the predominant lipid types in human meibomian gland secretions (meibum) and account for 40–50 % of total meibum lipids. Recently, we managed to quantify 51 isomeric groups of intact WE in normal human meibum samples by direct infusion electrospray ionization mass spectrometry (ESI–MS), with each WE peak in the MS spectrum corresponding to one isomeric group (Chen et al, Invest Ophthalmol Vis Sci 54(8):5730–53, 2013). However, the information of the isomeric composition in each group was not obtained. In this study, tandem mass spectrometry (MS/MS) was applied to quantify relative amounts of these isomers using the intensities of the corresponding diagnostic ions after appropriate correction and normalization. This data was combined with the previous obtained mole fraction of each isomeric group to total WE in human meibum to determine the corresponding percentage of each isomer. A total of 23 of the most abundant WE peaks of different molecular weights (corresponding to 85.3 % of the total amount of WE) in human meibum were studied and resulted in quantification of 92 WE species. The quantitative information of composition of WE in human meibum will help better understand their role in the tear film.     

Enzymatic Conversion of Unnatural Amino Acids by Yeast D‐Amino Acid Oxidase

Unnatural amino acids, particularly synthetic α‐amino acids, are becoming crucial tools for modern drug discovery research. In particular, this application requires enantiomerically pure isomers. In this work we report on the resolution of racemic mixtures of the amino acids d,l ‐naphthylalanine and d,l ‐naphthylglycine by using a natural enzyme, D ‐amino acid oxidase from the yeast Rhodotorula gracilis. A significant improvement of the bioconversion is obtained using a single‐point mutant enzyme designed by a rational approach. With this D ‐amino acid oxidase variant the complete resolution of all the unnatural amino acids tested was obtained: in this case, the bioconversion requires a shorter time and a lower amount of biocatalyst compared to the wild‐type enzyme. The simultaneous production of the corresponding α‐keto acid, a possible precursor of the amino acid in the L ‐form, improves the significance of the procedure.     

NMR-spektroskopische Untersuchungen zum Komplexbildungsverhalten von Hydrazonen des Thiocarbazinsäure-O-methylesters mit Zink(II)-Ionen

Complexes of Zn(II) with Schiff Bases of Hydrazine-O-methylcarbothionates – NMR-Spectroscopic Investigations Schiff bases of hydrazine-O-methylcarbothionates react as bi- or tridentate ligands with Zn(II) ions forming neutral metal complexes ( 1a-1g ). ¹H-, ¹³C- and ¹⁵N-NMR-spectra of these complexes show the existence of configurational isomers of the CN-double bond. Furthermore, depending on the ligands 4 different coordination patterns with the donor atoms N₂S₂, N₄, N₂O₂ and NOS are demonstrated.     

Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of <Emphasis Type="Italic">N</Emphasis>-Acyl Moiety of Sphingomyelin

Sphingomyelin (ceramide‐phosphocholine, CerPCho) is a common sphingolipid in mammalian cells and is composed of phosphorylcholine and ceramide as polar and hydrophobic components, respectively. In this study, a qualitative liquid chromatography‐electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS/MS) analysis is proposed in which CerPCho structures were assigned based on product ion spectra corresponding to sphingosylphosphorylcholine and N‐acyl moieties. From MS/MS/MS analysis of CerPCho, we observed product ion spectra of the N‐acyl fatty acids as [RCO₂]^? ions as well as sphingosylphosphorylcholine. A calibration curve for CerPCho was constructed using two stable isotopically labeled CerPCho species and then used to quantify the CerPCho species in HeLa cells as a proof‐of‐principle study. The present study proposes an accurate method for quantifying and assigning structures to each CerPCho species in crude biologic samples by LC–ESI–MS/MS/MS analysis.     

Maillard反应及其产物在烟草加香中的作用

制备了甘氨酸等18种单一氨基酸与葡萄糖进行的美拉德反应产物以及复合氨基酸粉．动物水解蛋白与葡萄糖进行的美拉德反应产物,筛选出了两组适合于烤烟加香的混合氨基酸(混合氨基酸A,混合氨基酸B),并以混合氨基酸A为例．初步研究了反应条件(pH、反应温度等)对反应产物性质(如香味特征)的影响。     

Multidimensional mass spectrometry methods for the structural characterization of cyclic polymers

Several synthetic methods have been developed for the tailored preparation of cyclic macromolecules due to their unique physical and chemical properties. Unequivocal characterization of the macrocyclic architectures has remained challenging, however, because isomeric linear structures often exist, or the spectral features of linear vs. cyclic chains are similar. To address this problem, multidimensional mass spectrometry (MS) techniques have been evaluated for the separation and identification of polymeric macrocycles. Tandem mass spectrometry (MS²) is found to be ideally suitable for the differentiation of linear and cyclic architectures whose molecular ions exhibit distinct fragmentation characteristics. Conversely, differences in macromolecular sizes and shapes can be exploited to identify the correct architecture by ion mobility mass spectrometry (IM-MS). A third option, chromatographic separation (LC) before MS analysis, is available for the detection of cyclics in complex mixtures. The capabilities of these techniques and combinations thereof are demonstrated with specific covalent or supramolecular (co)polymers.     

生理活性氨基酸Schiff碱的合成与表征

用天冬酰胺分别与水杨醛、邻香草醛、2 ,4 二羟基苯甲醛在甲醇中反应 ,然后加入异丙醇析出产物的方法合成了三种氨基酸Schiff碱 ,产物收率分别为 84 3 %、81 2 %、76 7%。用元素分析、红外光谱、紫外光谱等手段对所合成的Schiff碱进行了表征。采用EPR技术对所合成的Schiff碱清除超氧阴离子自由基 (O2 ·)的性能进行了研究 ,结果表明 ,所合成的氨基酸Schiff碱质量浓度为 5kg/m3 时 ,对O2 ·的清除率分别达到 6 3 4%、6 2 .0 %、84 2 % ,具有明显的生理活性。     

Protection of mild steel corrosion with Schiff bases in 0.5 M H2SO4 solution

Three new Schiff bases, viz., N,N′-ethylen-bis (salicylidenimine) [S₁], N,N′-isopropylien-bis (salicylidenimine) [S₂], and N-acetylacetone imine, N′-(2-hydroxybenzophenone imine) ortho-phenylen [S₃] have been investigated as corrosion inhibitors for mild steel in 0.5 M H₂SO₄ using Tafel polarization and electrochemical impedance spectroscopy (EIS). The three Schiff bases function as good inhibitors reaching inhibition efficiencies of ∼97-98% at 300 ppm concentration. The fraction <theta> of the metal surface covered by the inhibitor is found to increase with inhibitor concentration. Of the three Schiff bases, the S2 shows better efficiency than the other two Schiff bases. The adsorption of the inhibitor follows Langmuir isortherm. Thermodynamic calculations indicate the adsorption to be physical in nature.     

Novel Poly(Styrene) Attached Schiff Bases for Uptake Mn(II) and Ni(II) Ions and as Antimicrobial Agent against Micrococcus luteus

In this study, three novel poly(styrene) attached Schiff bases (AMP–SB) were synthesized from condensation reaction of (aminomethyl)polystyrene with 2-hydroxy-5-bromobenzaldehyde, 2-hydroxy-5-methylbenzaldehyde and 2-hydroxy-5-chlorobenzaldehyde for uptake from solutions containing Mn(II) and Ni(II) ions and as antimicrobial agent against Micrococcus luteus. Furthermore coordination polymers were synthesized including Mn(II) and Ni(II) ions for understand to geometric structure of uptake ions. Polymers attached Schiff bases and coordination polymers were characterized by means of elemental analyses, magnetic measurements, FTIR, UV-GB, TG/DTA and ¹H-NMR. The Ni(II) and Mn(II) contents in the metal-containing polymer complexes were determined by the ICP-MS technique. Antibacterial activities of the polymers attached Schiff bases and their complexes were studied by the well-diffusion method against M. luteus (ATCC-9341).     

Identification of Conjugated Pentadecadienals as Sex Pheromone Components of the Sphingid Moth, Dolbina tancrei

Homologs of bombykal, (10E,12Z)-10,12-hexadecadienal, have been reported to be sex pheromones or sexual attractants of several species of sphingid moths. In this study, we identified novel bombykal analogs as sex pheromone components from a Japanese sphingid moth, Dolbina tancrei. Staudinger (Sphingidae: Lepidoptera). Sex pheromone gland extracts from calling female moths were subjected to gas chromatography/electroantennograhic detection (GC/EAD), gas chromatography/mass spectrometry (GC/MS), and gas chromatography (GC) analyses. GC/EAD analyses showed two active components in the crude pheromone extracts. GC/MS analysis determined these two components to be pentadecadienals. GC/MS of their MTAD derivatives showed conjugated double bonds at the 9- and 11-positions, indicating 9,11-pentadecadienals. The isomeric configurations of these candidates were determined by comparison of their Kováts retention indices with those of synthetic compounds. Field bioassays with the four isomers of 9,11-pentadecadienal and their mixtures confirmed that the two sex pheromone components of D. tancrei are (9E,11Z)-9,11-pentadecadienal and (9Z,11Z)-9,11-pentadecadienal, with the highest male catches observed for a 90:10 blend. This is the first report of 9,11-pentadecadienals as sex pheromone components in lepidopteran species.     

Physicochemical Studies of 4-Substituted N-(2-Mercaptophenyl)-Salicylideneimines: Corrosion Inhibition of Mild Steel in an Acid Medium

A series of 4-substituted N-(2-mercaptophenyl)salicylideneimine Schiff bases were synthesized and investigated for corrosion inhibition of mild steel in hydrochloric acid medium. Inhibition through adsorption mechanism is proposed for these inhibitors, which is well supported by electrochemical impedance spectroscopy, the Langmuir adsorption isotherm and Scanning Electron Microscope morphologies of inhibited and uninhibited mild steel specimens. The negative ?G _ads indicates the spontaneous adsorption of the inhibitor on a mild steel surface. Among all the examined inhibitors, 5-bromo-N-(2-mercaptophenyl)salicylideneimine showed a higher inhibition efficiency. In order to reveal the usefulness of these Schiff bases as corrosion inhibitors under various circumstances, weight loss measurements were performed at various temperatures, acid concentrations and immersion times.     

Preparation and characterization of glucosylated aminoglycerophospholipids

Natural aminophospholipids were isolated from egg yolk and from human red blood cells. Glucosylated ethanolamine and serine phosphatides were prepared by exposing synthetic and natural aminophospholipids to glucose for 3–18 h at pH 7.4. The glucosylation products were resolved from parent phospholipids by normal-phase high-performance liquid chromatography and were identified by on-line mass spectrometry with an electrospray interface. The soft ionization method allowed us to detect the glucosylation products as molecular ions of the Schiff bases. The Schiff bases could be stabilized by sodium cyanoborohydride reduction. The molecular species of the ethanolamine and serine phosphatides reacted in proportion to their molar concentration in the mixtures. The yields of the glucosylation products varied with time of reaction and the concentration of glucose in the medium. At 50 mM glucose and 0.6 mg/mL phosphatidylethanolamine, 20% of the aminophospholipid was glycated in 18 h at 37°C.     

Free L-Lysine and Its Methyl Ester React with Glyoxal and Methylglyoxal in Phosphate Buffer (100 mM,pH 7.4) to Form Nε-Carboxymethyl-Lysine,Nε-Carboxyethyl-Lysine and Nε-Hydroxymethyl-Lysine

Glyoxal (GO) and methylglyoxal (MGO) are highly reactive species formed in carbohydrate metabolism. N^ε-Carboxymethyllysine (CML) and N^ε-carboxyethyllysine (CEL) are considered to be the advanced glycation end-products (AGEs) of L-lysine (Lys) with GO and MGO, respectively. Here, we investigated the reaction of free L-lysine (Lys) with GO and MGO in phosphate buffer (pH 7.4) at 37 °C and 80 °C in detail in the absence of any other chemicals which are widely used to reduce Schiff bases. The concentrations of Lys, GO and MGO used in the experiments were 0.5, 2.5, 5.0, 7.5 and 10 mM. The reaction time ranged between 0 and 240 min. Experiments were performed in triplicate. The concentrations of remaining Lys and of CML and CEL formed in the reaction mixtures were measured by stable-isotope dilution gas chromatography-mass spectrometry (GC-MS). Our experiments showed that CML and CEL were formed at higher concentrations at 80 °C compared to 37 °C. CML was found to be the major reaction product. In mixtures of GO and MGO, MGO inhibited the formation of CML from Lys (5 mM) in a concentration-dependent manner. The highest CML concentration was about 300 µM corresponding to a reaction yield of 6% with respect to Lys. An addition of Lys to GO, MGO and their mixtures resulted in strong reversible decreases in the Lys concentration up to 50%. It is assumed that free Lys reacts rapidly with GO and MGO to form many not yet identified reaction products. Reaction mixtures of Lys and MGO were stronger colored than those of Lys and GO, notably at 80 °C, indicating higher reactivity of MGO towards Lys that leads to polymeric colored MGO species. We have a strong indication of the formation of N^ε-(hydroxymethyl)-lysine (HML) as a novel reaction product of Lys methyl ester with MGO. A mechanism is proposed for the formation of HML from Lys and MGO. This mechanism may explain why Lys and GO do not react to form a related product. Preliminary analyses show that HML is formed at higher concentrations than CEL from Lys methyl ester and MGO. No Schiff bases or their hydroxylic precursors were identified as reaction products. In their reactions with Lys, GO and MGO are likely to act both as chemical oxidants on the terminal aldehyde group to a carboxylic group (i.e., R-CHO to R-COOH) and as chemical reductors on labile Schiff bases (R-CH=N-R to R-CH₂-NH-R) presumably via disproportionation and hydride transfer. Our study shows that free non-proteinic Lys reacts with GO and MGO to form CML, CEL and HML in very low yield. Whether proteinic Lys also reacts with MGO to form HML residues in proteins remains to be investigated. The physiological occurrence and concentration of HML in biological fluids and tissues and its relation to CML and CEL are elusive and warrant further investigations in health and disease. Chemical synthesis and structural characterization of HML are expected to advance and accelerate the scientific research in this topic.     

Monomeric complexes of Re(CO)3+ ion with tridentate N∩N∩O−ligands — Schiff base derivatives of salicylic aldehyde

Simple synthetic procedures, reactions of Re(CO)₅Cl with potentially tridentate N∩N∩OH ligands (Schiff bases prepared from aliphatic or aromatic amines and salicylic aldehyde) lead to formation of monomeric complexes of fac-Re(CO)₃⁺ ion. Three obtained complexes have been characterized by means of elemental analyses and IR, UV–vis, and EI-MS techniques. Molecular structures of the synthesized species were investigated using X-ray diffraction measurements. Depending on the nature of N∩N∩OH ligand the investigated Schiff bases form with fac-Re(CO)₃⁺ ion bidentate or tridentate chelates with N∩N, N∩O⁻ or N∩N∩O⁻ coordination types.     

Inhibition of copper corrosion by several Schiff bases in aerated halide solutions

The inhibitive action of three Schiff bases, N,N -o-phenylen-bis(3-methoxysalicylidenimine) (V–o-Ph–V), N,N -p-phenylen-bis (3-methoxysalicylidenimine) (V–p-Ph–V) and N-[(2-hydroxy-3-methoxyphenyl)methylene]-histidine (V-His), on copper corrosion in aerated 0.5 mol dm^–3 NaCl and NaBr solutions was investigated using EIS and steady-state polarization techniques. The inhibitor effectiveness depended strongly on the geometric structure of the Schiff bases. Among the three kinds of Schiff base used, the inhibition efficiency of V–o-Ph–V on copper corrosion was the highest, V–p-Ph–V the next and V-His the lowest. The Schiff bases inhibited the cathodic current more significantly than the anodic current. The different influences of V–o-Ph–V or V–p-Ph–V on the anodic and cathodic reactions led to the appearance of a low frequency capacitive loop in the impedance spectra. The inhibition action of the Schiff bases was due to their adsorption on the copper surface followed by complexation with Cu(I) or Cu(II) ions, forming a blocking barrier to copper corrosion.     

Mass Spectrometry Analysis Coupled with de novo Sequencing Reveals Amino Acid Substitutions in Nucleocapsid Protein from Influenza A Virus

Amino acid substitutions in influenza A virus are the main reasons for both antigenic shift and virulence change, which result from non-synonymous mutations in the viral genome. Nucleocapsid protein (NP), one of the major structural proteins of influenza virus, is responsible for regulation of viral RNA synthesis and replication. In this report we used LC-MS/MS to analyze tryptic digestion of nucleocapsid protein of influenza virus (A/Puerto Rico/8/1934 H1N1), which was isolated and purified by SDS poly-acrylamide gel electrophoresis. Thus, LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three substituted amino acid residues R452K, T423A and N430T in two tryptic peptides. The obtained results provided experimental evidence that amino acid substitutions resulted from non-synonymous gene mutations could be directly characterized by mass spectrometry in proteins of RNA viruses such as influenza A virus.     

质谱在多肽氨基酸序列分析中的应用

多肽氨基酸序列分析是蛋白质研究的重要内容;日臻完善的质谱技术在该项研究中起到了巨大的作用。