Synthesis and NMR elucidation of novel pentacycloundecane‐based peptides

The synthesis and NMR elucidation of two novel pentacycloundecane (PCU)‐based peptides are reported. The PCU cage amino acids were synthesised as racemates and the incorporation of the cage amino acid with (S)‐natural amino acids produced diastereomeric peptides. The diastereomeric ‘cage’ peptides were separated using preparative HPLC and the NMR elucidation of these PCU containing peptides are reported for the first time. The ¹H and ¹³C NMR spectra showed series of overlapping signals of the cage skeleton and that of the peptide, making it extremely difficult to resolve the structure using one‐dimensional NMR techniques only. The use of two‐dimensional NMR techniques proved to be a highly effective tool in overcoming this problem. Copyright © 2010 John Wiley & Sons, Ltd.     

LC and LC-MS Separation of Peptides on Macrocyclic Glycopeptide Stationary Phases: Diastereomeric Series and Large Peptides

Previous work on the LC separation of peptides had shown that macrocyclic glycopeptide stationary phases to be selective for peptides of five to thirteen amino acids in length. In this work, the selectivity of the teicoplanin stationary phase is compared to that of a C18 stationary phase for seven diastereomeric enkephalin peptides. The teicoplanin stationary phase separated all seven diastereomeric enkephalin peptides in a single chromatographic run. The insertion of d-amino acids into the primary enkephalin sequence produced areas of hydrophobicity that influenced retention order on the C18 stationary phase. However, analogous trends are not observed on the teicoplanin stationary phase, which is more polar and structurally diverse. Optimization of the mobile phase and the use of a step-gradient for the enkephalin separation on the teicoplanin stationary phase is discussed. Also, the selectivity of macrocyclic glycopeptide stationary phases for peptides of 14, 28, 30, and 36 amino acids also is investigated and compared to separation on a C18 stationary phase. A method for eluting peptides with multiple basic amino acids, which tend to be strongly retained on the macrocyclic glycopeptide stationary phases, is presented.     

Chiral separation of amide conjugates of jasmonic acid by liquid chromatography

Summary Synthetic amide conjugates of (−)-jasmonic acid and its (+)-enantiomer were resolved by means of chiral liquid chromatography. The diastereomeric pairs prepared by chemical reaction of (±)-jasmonic acid with a series of (S)- or (R)-amino acids and with some (S)-amino acid alcohols were completely separated on Chiralpak AS using a mixture of n-hexane/2-propanal as mobile phase. The retention data indicate that the (−)-jasmonic acid conjugates eluted faster than those of the (+)-enantiomer, independent on the configuration of the bound amino acid. Likewise, enantiomeric derivatives of (±)-jasmonic acid and non-chiral amino acids were completely separated on the chiral stationary phase and showed the same elution sequence. The resolution factors,Rs, were found to range between 1.13 and 6.64. The separated compounds were chiropatically analyzed by measurement of the circular dichroism. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Separation of Free Amiimo Acids on Reverse Stationary Phases Using an Alkyl Sulfonate Salt as a Mobile Phase Additive

Abstract

Alkylsulfonate (RSO₃ ^?) salts were evaluated as mobile phase additives for the separation of free amino acids on reverse stationary phases using an acidic mobile phase where the amino acids are cations. The enhanced amino acid retention is the result of two major interactions, one being retention of the RSO₃ ^? salt on the stationary phase and the other an ion exchange selectivity between the amino acid analyte cation and the RSO₃ ^? countercation, or other countercations in the mobile phase. Major mobile phase variables are: type and concentration of RSO₃ ^? salt (the studies focused on C₈SO₃ ^? salts), presence of organic modifier, type of countercation present, and mobile phase pH and ionic strength. Alkyl modified silica and polystyrenedivinyl-benzene copolymeric reverse stationary phases were compared. A mobile phase gradient, increasing per cent organic modifier was shown to be best, is necessary for separating complex mixtures of polar and nonpolar or basic amino acids. The procedure is applicable to the identification and/or determination of amino acids in mixtures or in peptides after hydrolysis.     

Quantification of glycated N‐terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry

A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1‐bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC‐MS/MS. In this study, 1‐deoxyfructosyl‐VHLTPE and VHLTPE, which are N‐terminal peptides of the β‐chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1‐bromobutane and analyzed with LC‐MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non‐glycated peptide. Copyright © 2015 John Wiley & Sons, Ltd.     

Direct Thin – Layer Chromatographic Separation of Enantiomers of Six selected Amino Acids Using 2-O-[(R)-2-Hydroxypropyl]-β-CD as a Mobile Phase Additive

Abstract

Compared with β-Cyclodextrin (β-CD), 2-O-[_(R)-2-hydroxypropyl]-β-CD (A) is more sensitive for chiral recognition. When (A) was used as the chiral mobile phase additive, some racemic amino acids were directly separated on silica gel TLC for the first time.     

A New Bonded-Phase for High Performance Liquid Chromatographic Analysis

Abstract

A silica bonded-phase with carboxylic acid functionality and a side chain of three carbon atoms has been synthesized for HPLC analysis. Mixtures of nucleic acid bases, PTH-amino acids, phospholipids and ethanolic extract of dry ginger powder were analyzed to demonstrate its performance. Nucleic acid bases and PTH-amino acids were separated by cation-exchange mechanism whereas phospholipids experienced combination of cation-exchange and normal-phase resolution. The resolution of gingerols in the dry ginger powder was achieved by reversed-phase mechanism.     

Synthesis and NMR elucidation of pentacycloundecane-derived hydroxy acid peptides as potential anti-HIV-1 agents

The synthesis and NMR elucidation of eight novel peptides incorporating the pentacycloundecane (PCU)-derived hydroxy acid are reported. The PCU cage amino acids were synthesized as racemates and the incorporation of the PCU-derived hydroxy acid with natural (S)-amino acids produced inseparable diastereomeric peptides. A series of overlapping signals from the cage and that of the peptide side chain was observed in the ¹H- and ¹³C-NMR spectra, complicating the elucidation thereof. Two-dimensional NMR techniques proved to be a very useful tool in overcoming these difficulties. These compounds are potential HIV protease inhibitors.     

Reversed Phase Thin Layer Chromatography of Amino Acids

Abstract

The use of reversed phase layers for the thin layer chromatography of amino acids is described. Only when a modifier was added to the mobile phase was clear separation of the amino acids achieved. Ion paring with trifluoroacetic acid overcame problems with streaking and poor separation on C₂ or C₁₈ reversed phase layers. All amino acids could not be separated with a single mobile phase. Thus, three different combination of acetonitrile-0.4% trifluoroacetic acid were used to separate eighteen amino acids with derivatization. No derivative was required.     

Laterally attached liquid-crystalline polymers as stationary phases. Effect of temperature in RP HPLC and comparison with a commercial C18 stationary phase

Summary Specific side-on-fixed liquid-crystalline polymers (SOLCP) have been synthesized for use in silica-modified stationary phases in high-performance liquid chromatography (HPLC). The mesogenic side group of the SOLCP is composed of three benzoate-type phenyl rings with terminal alkoxy chains and is laterally linked to a polysiloxane backbone via an alkyl ester spacer arm. The dependence of the logarithm of the retention factor on the reciprocal temperature showed that the liquid-crystalline anisotropic order was conserved in the small pores (200 ? diameter) of the silica gel. The first-order nematic-isotropic transition is lost and probably becomes second-order. Adsorption enthalpies for the liquid-crystalline stationary phases have been measurement for three polycyclic aromatic hydrocarbon isomers (ortho-terphenyl, triphenylene, and chrysene) and compared with those for a commercial C₁₈ phase. The adsorption enthalpies never exceeded 30 kJ mol⁻¹, i.e. ten times the thermal agitation energy,RT. They were always less on the SOLCP stationary phase than on the C₁₈ column, emphasizing the more rigid structure of the liquid crystalline phase and its mechanism based upon adsorption. Better separation of steroids, pesticides and amino acids were obtained with the LCP-coated silica than the commercial bonded C₁₈ column. Four small peptides were successfully separated by using pure water as mobile phase.     

An efficient method for the determination of enantiomeric purity of racemic amino acids using micellar chromatography,a green approach

A simple, sensitive and green micellar liquid chromatographic method (RP-HPLC) was developed for enantioseparation of four racemic amino acids, namely, (RS)-selenomethionine, (RS)-methionine, (RS)-cysteine and (RS)-penicillamine. An aqueous solution of sodium dodecyl sulfate and Brij-35 was prepared and used as mobile phase for HPLC analysis. Activated esters of (S)-ibuprofen, (S)-ketoprofen and (S)-levofloxacin were synthesized by reacting them with N-hydroxybenzotriazole. These esters were characterized by UV, IR, ¹HNMR, HRMS and elemental analysis. These chiral reagents (activated esters) were used for the synthesis of diastereomeric derivatives of the chosen amino acids. The diastereomeric derivatives were separated on a C₁₈ column by micellar liquid chromatography. Chromatographic conditions were optimized by varying concentration of surfactant in aqueous solution, and by varying the concentration and pH of the buffer. The green assessment score was calculated for the developed method (78, an excellent green method score). In addition, density functional theory calculations were performed, using Gaussian 09 rev. A.02 and hybrid density functional B3LYP with a 6-31G* basis set program, in order to develop lowest energy optimized structures of diastereomeric derivatives. The method was validated according to International Conference on Harmonization guidelines and the retention factor (k), selectivity factor (α), resolution factor (R_S) and limit of detection (0.295 ng ml⁻¹) and limit of quantification (0.896 ng ml⁻¹) were calculated.     

Separation of α‐ from β‐arylalanines by nickel nitrilotriacetate chromatography

A method is described to separate α‐ from β‐arylalanines by ligand exchange chromatography on a nickel nitrilotriacetate agarose column with UV monitoring of the effluent. Separate mixtures containing an α‐ and β‐arylalanine pair (1 mg of each) were individually loaded onto the nickel resin pre‐equilibrated with the mobile phase at room temperature, and the amino acids were eluted from the column with a gradient from pH 12.0–8.0. The β‐arylalanines eluted first, followed by the α‐isomers. The four α/β‐amino acid pairs tested were well separated with baseline resolution. An aliquot of each fraction was chemically treated to derivatize the amino acids to their N‐acyl methyl ester analogs, and their identities were confirmed by GC/MS analysis. The sample recovery was quantitative (>98%), and the column matrix was very resilient, as demonstrated by consistent separation of the solutes after ～100 preparative cycles.     

Design of silica microparticles with oligopeptide brushes and their interaction with proteins

Synthesis of small oligopeptide brushes (oligo(S-benzyl-l-cysteine)) onto polyelectrolyte functionalized silica microparticles was developed. Poly(vinyl amine) (PVAm) adsorbed from salt-free and KCl 10⁻¹ mol L⁻¹ aqueous solution onto silica microparticles was chemically and naturally cross-linked by epichlorohydrin and CO₂, respectively. After the adsorption of PVAm onto microporous silica particles and stabilization by cross-linking, five repeated coupling reactions of Boc-S-benzyl-l-cysteine were performed. To test the protein interactions with the newly designed surface, human serum albumin (HSA) has been selected as a model protein. X-ray photoelectron spectroscopy, total organic carbon, potentiometric and polyelectrolyte titrations, and electrokinetic analysis were employed to obtain information about the polyelectrolyte adsorption and the amount of the amino acid S-benzyl-l-cysteine that was covalently bound to the solid surface and for determination of the protein amount adsorbed onto functionalized surface. The amount of HSA adsorbed onto modified silica microparticles decreased in order: silica/PVAm-cross-linked (silica/PVAm-C) (8.00 mg g⁻¹) > silica/PVAm-C/S-benzyl-l-cysteine (6.34 mg g⁻¹) > silica (4.86 mg g⁻¹) > silica/PVAm-C/(S-benzyl-l-cysteine)₅ (1.86 mg g⁻¹).     

Determination of inorganic acids by ion chromatography with n-tetradecylphosphocholine (zwitterionic surfactant) as the stationary phase and pure water as the mobile phase

A new ion chromatographic (IC) system, in which n-tetradecylphosphocholine (TDPC, a phosphobetaine type of zwitterionic surfactant) was used as the stationary phase, pure water as the mobile phase, and conductivity as the method of detection, has been developed for the determination of inorganic acids. Five model acids, HCl, HNO₃, HClO₄, H₂SO₄, and H₃PO₄, were separated to baseline and eluted in the order H₃PO₄ > HCl > HNO₃ > H₂SO₄ > HClO₄. When peak areas were plotted against the concentrations of the acids in samples, linear calibration curves were obtained. Ultimate determination limits were approximately 1 mmol L^–1, but the discrimination of the method between solutions of different concentration was better than 10 μmol L^–1 for those model analytes. Salts of divalent cations could also be separated, but they were eluted faster than the acids. No separation was observed for the salts of monovalent cations. This newly proposed approach is applicable to the simultaneous determination of the inorganic acids (produced by reactions of NO_x, SO_x, and HCl with water) in aerosols.     

Syntheses and CD‐Spectroscopic Investigations of Longer‐Chain β‐Peptides: Preparation by Solid‐Phase Couplings of Single Amino Acids,Dipeptides, and Tripeptides

The synthesis and CD‐spectroscopic analysis of eleven water‐soluble β‐peptides composed of all‐β³ or alternating β²‐ and β³‐amino acids is described. Different approaches for the efficient syntheses of longer‐chain β‐peptides (>9 residues) were investigated. They were synthesized on solid phase with Fmoc‐protected amino acids or Fmoc‐protected di‐ or tripeptide fragments (assembled using solution‐phase synthesis). The use of preformed fragments significantly increased the purity of the crude peptides and facilitated purification. Especially, the use of Fmoc‐protected β²/β³‐dipeptides for the synthesis of a ‘mixed' β²/β³‐nonapeptide proved to be remarkably effective, yielding the crude peptide in 95% purity and without detectable epimerization of the β²‐amino acid residues. This is a significant improvement over previously reported procedures for the solid‐phase synthesis of β‐peptides, and foreshadows that the field of β‐peptide research will now switch from synthesis to the design and study of complex functional ‘β‐proteins'.     

Explanation of Ionic Sequences in Various Phenomena. III. Salting-in and -out of Amino Acids

Abstract

Previous developed theories were applied in explaining the mechanism for the salting-in and -out of various amino acids. Glycine is salted-in according to the cationic sequences Li⁺ > Na⁺ > K⁺ > Rb⁺ and Ca²⁺ > Ba²⁺ > Sr²⁺. The ability of a cation to increase the solubility of an amino acid therefore corresponds to the destruction of the ion-ion bond between the - CO-₂and the -NH_{+ 2}group of the amino acid by forming an insoluble ion-ion bond between the added cation and the - CO?² group. This insolubilizing effect produces a positive charge on the amino acid. If, however, the anion of the added salt forms a relatively insoluble ion-ion bond with the -NH₊₂ group of the amino acid, then the effect is minimized because now both charges on the amino acid are reduced. Consequently, the more insoluble the cation amino acid salt and the more soluble the anion amino acid salt (or vice versa), the greater will be the salting-in effect. Titration of either charged group on the amino acid zwitterion has the same effect, since now the ion-ion bond of the amino acid is again destroyed. Aliphatic and carboxylic acid groups also effect the salting-in sequence, since these groups are salted-out by addition of salt when D^± < D_H2o. These mechanisms explain how leucine is first salted-out, then salted-in (at 4 M) and finally salted-out again (at 9 M) in LiCl solutions. Urea salts-in hydrophobic amino acids by increasing the dielectric constant and salts-out polar amino acids by increasing the interaction between the two charge groups on the amino acid. Glycine reverses the salting-in effect of NaCl on asparagine by competing for the Na⁺ ion.     

Separation of peptide diastereomers using CEC and a hydrophobic monolithic column

A neutral hydrophobic monolith prepared by radical in situ copolymerization of lauryl methacrylate and ethylene dimethacylate has been evaluated for the CEC separation of diastereomers of small peptides using acidic mobile phases containing ACN as organic modifier. Using an acidic mobile phase, the peptides migrated due to their own electrophoretic mobility. Hydrophobic interactions with the stationary phase contributed to the separation. Peptide mobility and resolution increased with increasing the ACN content. Retention times increased with the pH of the mobile phase. Peak resolution increased with buffer pH and concentration. Di‐ and tripeptides composed only of L ‐configured amino acids migrated faster than peptides containing D ‐amino acids. A mixture of isomeric Asp tripeptides that could not be completely resolved by either CZE or HPLC as well as the 24mer peptides tetracosactide and ¹⁶[D ‐Lys]‐tetracosactide could also be separated by CEC on the hydrophobic monolith.     

Interaction of amino acids with the herbicides diquat and paraquat studied by charge-transfer chromatography

Summary The interaction of the amino acids and some peptides with the herbicides diquat and paraquat was determined by reversed-phase charge-transfer chromatography. Only charged amino acids and gluthathion (both reduced and oxidized forms) influenced the mobility of diquat and paraquat. The strength of interaction increased in the order: aspartic acid > gluthathione oxidized > gluthathione reduced > ornithine > cysteine glutamic acid > lysine. No significant difference was found between the effects of D and L forms of aspartic acid. Except cysteine the strength of interaction was similar on cellulose and on reversed-phase layers. The strength of interaction nonlinearly decreased with growing concentration of Ca²⁺ ions in the eluent proving the hydrophilic character of interaction. Glutamic acid-ornithine and glutamic acid-lysine mixtures did not show lower interactive strength than the single amino acids although interactions of commensurable strength were also observed between these amino acids. This finding supports the possible formation of dibasic amino acid-dicarboxylic amino acid-herbicid complexes of unknown stoichiometry.     

Separation of β-blocker and amino acid enantiomers on a mixed chiral sorbent modified with macrocyclic antibiotics eremomycin and vancomycin

A mixed chiral sorbent based on silica with immobilized macrocyclic antibiotics eremomycin and vancomycin was synthesized. A possibility of the separation of enantiomers of β-blockers (metoprolol, pindolol, alprenolol, oxprenolol, labetalol, and atenolol) and amino acids (tryptophan, phenylalanine, DOPA, methionine, and acetyl glutamic acid) on this chiral sorbent by HPLC was studied. The influence of the composition of the mobile phase (pH of buffer solution, its concentration, content of organic modifier, and its nature) on the retention times of β-blocker and amino acid enantiomers, selectivity, and resolution of peaks was studied. It was shown that the mixed chiral sorbent has enantioselectivity to both classes of compounds, while silica modified with vancomycin has no ability to the separation of enantiomers of non-derivatized amino acids, and silica modified with eremomycin has no ability to the separation of β-blocker enantiomers. High values of resolution for amino acids (max Rs > 4) and β-blockers (max Rs > 1) were obtained.     

Enantiomeric Separation of Non-Protein Amino Acids by Chiral Ligand-Exchange High-Performance Liquid Chromatography

Abstract

The enantiomeric separation by high-performance liquid chromatography of underivatized non-protein amino acids was investigated by using a column packed with octadecylsilanized silica coated with N,S-dioctyl-D-penicillamine as a chiral ligand-exchange phase (Sumichiral OA-5000). Excellent to good separations of enantiomers were achieved with a variety of nonprotein amino acids carrying aliphatic or aromatic side-chains by optimizing the amount (0-20%, v/v) of the organic component (2-propanol) and the concentration (1-5 mM) of the complexing metal ion (Cu²⁺) in the hydro-organic eluent.