Resolution Of Amino Acids Research Articles

Synthesis and characterization of macroporous silica modified with optically active poly[N‐(oxazolinylphenyl)acrylamide] derivatives for potential application as chiral stationary phases

AbstractEnantiopure acrylamide derivatives, (S)‐N‐[o‐(4‐methyl‐4,5‐dihydro‐1,3‐oxazol‐2‐yl)phenyl]acrylamide and (R)‐N‐[o‐(4‐phenyl‐4,5‐dihydro‐1,3‐oxazol‐2‐yl)phenyl]acrylamide, were synthesized through the acylation of chiral 2‐oxazolinylanilines. The radical polymerization of the chiral monomers was carried out with (3‐mercaptopropyl)trimethoxysilane as a chain‐transfer agent to obtain the corresponding optically active prepolymers with a trimethoxysilyl group. By immobilizing the prepolymers on porous silica gel via the grafting‐to method, we prepared a new chiral stationary phase (CSP) and characterized it by elemental analysis, thermogravimetry, and Fourier transform infrared spectroscopy. The enantioseparation capacities of the CSPs were evaluated with high‐performance liquid chromatography toward several racemic compounds, including 1,1′‐bi‐2‐naphthol, benzoin, 2‐amino‐1‐butanol, and loxoprofen sodium under the normal‐phase mode. The results indicate that the CSPs exhibited improved chromatographic performances compared to their brush‐type analogs obtained by the alternative grafting‐from approach. Also, the column packed with poly{(R)‐N‐[o‐(4‐phenyl‐4,5‐dihydro‐1,3‐oxazol‐2‐yl)phenyl]acrylamide}‐bonded silica was found to have an extent of enantioselectivity in the chiral resolution of some unmodified amino acids with reversed‐phase eluents. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Enantioseparation of amino acids, α‐hydroxy acids, and dipeptides by ligand‐exchange CEC using silica‐based chiral stationary phases

This work deals with the application of silica-based ligand-exchange chiral stationary phases (CSPs) for the enantioseparation of underivatized amino acids, alpha-hydroxy acids, and dipeptides with packed CEC. Two different possibilities of preparing silica-based CSPs are presented. One phase contains L-4-hydroxyproline chemically bonded via a spacer to 3 mum silica material. The other approach makes use of N-decyl-L-4-hydroxyproline dynamically coated on a reversed-phase packed capillary. Dynamical coating of reversed-phase material represents a simple alternative to prepare CSP. A comparison of the chemically bonded phase with the dynamically coated CSP by means of resolution of complex-forming analytes is presented. The chemically bonded phase was found to be superior to the dynamically coated phase in terms of resolution of amino acids and dipeptides. However, the dynamically coated CSP was found to be especially suitable for the separation of alpha-hydroxy acids. Both techniques are applicable for enantiomer purity tests.

Quantum dots laser desorption/ionization MS: multifunctional CdSe quantum dots as the matrix, concentrating probes and acceleration for microwave enzymatic digestion for peptide analysis and high resolution detection of proteins in a linear MALDI‐TOF MS

We report the first use of functionalized cadmium selinide quantum dots (CdSe QDs) with 11-mercaptoundecanoic acid (MUA) as the matrix for the selective ionization of proteins with high resolution and rapid analysis of amino acids and peptides by using quantum dots laser desorption/ionization mass spectrometry (QDLDI-MS). The mercaptocarboxylic groups of CdSe QDs have been known to be an effective affinity probe to interact with the biomolecules at low abundance level. Using these QDs as the matrix, sensitivity of the method was greatly enhanced and the LOQ of peptides was found to be 100 pM with RSD <10%. The QDLDI-MS is capable for the selective ionization of insulin, lysozyme and myoglobin with high resolution, which is not observed with sinapic acid (SA) as the matrix. The QDLDI-MS technique offers many advantages for the analysis of amino acids, peptides and proteins with regard to simplicity, rapidity, sensitivity and the mass spectra were generated in the presence of signal suppressors such as urea and Trition X-100. In addition, the CdSe QDs have been successfully applied as preconcentrating probes for the analysis of digested peptides in lysozyme from chicken egg white by microwave-assisted enzymatic digestion. This indicates that the QDs are able to absorb radiation from microwave and their ability to trap peptides from microwave-digested lysozyme. These results demonstrate that the CdSe QDs are promising candidates for the selective ionization of the analytes with an accurate platform to the rapid screening of biomolecules.

Targets for covalent protein modification by 4‐hydroxynonenal/4‐hydroxyhexenal‐mediated carbonyl stress in the mitochondria

Protein carbonylation is associated with various neurodegenerative diseases. Characterization of selectivity of 4‐hydroxy‐2‐nonenal (HNE) and 4‐hydroxy‐2‐hexenal (HHE) modifications, major components of carbonyl stress, helps to understand its impact on protein function and/or activity, as well as its effects on downstream targets or other interacting proteins. Rat brain mitochondrial proteins modified by reactive carbonyl species representing end‐products of lipid peroxidation such as HNE and HHE were investigated using high performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS). Shotgun‐based approach involving conventional data‐dependent acquisition on a hybrid linear ion trap‐Fourier transform ion cyclotron resonance mass spectrometer was employed after prior enrichment of HNE‐/HHE‐modified peptides by solid‐phase hydrazide strategy. Selective enrichment of substoichiometric levels of modified peptides from complex mixtures followed by LC‐MS/MS identified several peptides that were modified with HNE or HHE with amino acid resolution. Additionally, bioinformatics (Ingenuity Pathway Analysis) was used to identify biological pathways, networks, and functions significantly altered by such modifications. This research was supported by the grant AG025384 from the National Institutes of Health.

The effect of the copper(II) salt anion in the Chiral Ligand-Exchange Chromatography of amino acids

Due to the strong involvement in numerous biochemical processes, the separation and resolution of amino acids is a continuously challenging task. Among the experimental parameters affecting the performances in a Chiral Ligand-Exchange Chromatography (CLEC) environment, the effect of the copper(II) salt counter-ion has received very limited attention. Aimed at evaluating the Cu(II) counter-ion effect upon the overall chromatographic performances when the S-trityl-( R)-cysteine is adopted as the chiral selector for the coated stationary phase, a number of both organic (acetate, formate, trifluoromethane-sulfonate) and inorganic (bromide, chloride, nitrate, perchlorate, sulfate) salts has been engaged for the enantiomer separation of a selected set of aliphatic (Allo-Ile, Ile, Leu, Nor-Leu, Nor-Val, Val) and aromatic (AIDA, ATIDA, His, Phe, Phg, Tyr) amino acids. By varying the physico-chemical nature of the Cu(II) anion, a pronounced impact upon the resolution factor ( R S ) has been observed. Even if to a lesser extent, the enantioseparation factor values ( α) underwent variation, as well. A molecular modelling investigation has also been carried out as a rationalization attempt of the observed chromatographic outcomes.

Morphology of Neurofilament Protrusions: Sequence-Based Modeling of Neurofilament Brush

Neurofilaments are essential cytoskeletal filaments that impart mechanical integrity to nerve cells and regulate the cross-sectional area of axons. They are assembled from three distinct molecular weight proteins (NF-L, NF-M, NF-H) bound to each other laterally forming 10 nm diameter filamentous rods along with side-arm extensions. These protrusions contain an abundance of charged amino acid residues. The charged side-arms are considered to mediate the interactions between neighboring filaments, regulating interfilament spacing, and hence axonal caliber. The precise mechanism by which neurofilament protrusions regulate axonal diameter remains unsettled. In particular, the role of individual proteins has remained to be a matter of debate. Computer modeling is instrumental in demonstrating the details about the structural arrangement of individual protrusions. The present study employs Monte Carlo Simulations of neurofilament to reveal the role played by individual side-arms. The simulations are conducted under different phosphorylation state by making use of physically motivated 3D model of neurofilament brush. The model consists of a neurofilament backbone along with side-arm extensions that are distributed according to the stoichiometry of the three subunits. The side-arms are modeled at amino acid resolution with each amino acid represented by a hard sphere along with the corresponding charge valence. The phosphorylation states are specified through charges assigned to the serine amino acids of the Lys-Ser-Pro (KSP) repeat motifs of the side-arms. The equilibrium structure of the neurofilament brush has been studied via the model that maintained the proper charge distributions and grafting density of neurofilament side arms. It has been found that in spite of extensive phosphorylation sites present on NF-H, the tails of the medium sized neurofilament subunit (NF-M) is more elongated than NF-H tails. This suggests that NF-M protrusions are more critical in regulating neurofilament spacings and axonal caliber.

Chitosan chiral ligand exchange membranes for sorption resolution of amino acids

The concept of chiral ligand exchange is employed in the present study to achieve the chiral resolution of tryptophan (Trp) enantiomers by using chitosan (CS) membrane in a sorption resolution mode and copper(II) ion as the complexing ion. CS porous membranes are prepared by freeze-drying method (CS-LT) and sol–gel process at high temperature (CS-HT), respectively, to investigate their sorption resolution characteristics. The proposed CS chiral ligand exchange membranes exhibit good chiral resolution capability. Meanwhile the sorption selectivity of the CS membranes is found to be reversed from L-selectivity at low copper(II) ion concentration to D-selectivity at high copper(II) ion concentration, which is attributable to the stability difference between the copper(II)– L-Trp and copper(II)– D-Trp complexes. Moreover, the CS-HT membrane shows better performance with respect to both sorption selectivity and sorption capability than the CS-LT membrane, which mainly results from its more amorphous structures compared with the more crystalline structures of the CS-LT membrane. The superiority of sorption capability of the CS-HT membrane is also attributable to its larger specific surface area than that of the CS-LT membrane. The results obtained in this study are conducive to the design and fabrication of chiral ligand exchange membranes for enantiomer separation in sorption mode.

Chirality conversion and enantioselective extraction of amino acids by imidazolium-based binol-aldehyde

A novel imidazolium-based binol receptor 4 has been synthesized and used as a chirality conversion reagent for general amino acids with higher d-form selectivity compared to other guanidinium-based receptors. Favorable solubility in chloroform enabled 4 as an effective chiral extractant for the resolution of racemic amino acids.

Application of statistical design for the optimization of amino acid separation by reverse-phase HPLC

Modified resolution and overall separation factors used to quantify the separation of complex chromatography systems are described. These factors were proven to be applicable to the optimization of amino acid resolution in reverse-phase (RP) HPLC chromatograms. To optimize precolumn derivatization with phenylisothiocyanate, a 2 5–1 fractional factorial design in triplicate was employed. The five independent variables for optimizing the overall separation factor were triethylamine content of the aqueous buffer, pH of the aqueous buffer, separation temperature, methanol/acetonitrile concentration ratio in the organic eluant, and mobile phase flow rate. Of these, triethylamine concentration and methanol/acetonitrile concentration ratio were the most important. The methodology captured the interaction between variables. Temperature appeared in the interaction terms; consequently, it was included in the hierarchic model. The preliminary model based on the factorial experiments was not able to explain the response curvature in the design space; therefore, a central composite design was used to provide a quadratic model. Constrained nonlinear programming was used for optimization purposes. The quadratic model predicted the optimal levels of the variables. In this study, the best levels of the five independent variables that provide the maximum modified resolution for each pair of consecutive amino acids appearing in the chromatograph were determined. These results are of utmost importance for accurate analysis of a subset of amino acids.

Mathematical modelling of amino acid resolution catalyzed by l-amino acid oxidases from Crotalus adamanteus and Crotalus atrox

Amino acid resolution was studied in this paper. l-Methionine and dl-methionine were used as substrates for the two snake venom l-amino acid oxidases (from Crotalus adamanteus and Crotalus atrox). Even though these enzymes have similar background, they exhibit differences as well as similarities. It was found that both enzymes are active towards l-methionine as substrate ( V m = 0.78 mmol min −1 g −1) and have similar affinities ( K m L - met ( C. adamanteus) = 0.19 mM, K m L - met ( C. atrox) = 0.25 mM). Both enzymes were inhibited in the presence of 2- oxo-4-methylthiobutyric acid ( K i 2 - oxo ( C. adamanteus) = 0.83 mM, K i 2 - oxo ( C. atrox) = 2.04 mM), a reaction product of l-methionine oxidative deamination. l-Amino acid oxidase from C. atrox was found to be inhibited by the substrate as well. l-Amino acid oxidase from C. adamanteus was inhibited by d-enantiomer of l-methionine ( K i D - met = 1.41 mM ). Both enzymes were successful in l-methionine oxidation in the batch reactor (100% conversion). This was not the case in the continuously operated enzyme membrane reactor where enzyme deactivation occurred.

Optical resolution of phenylalanine using D‐Phe‐imprinted poly(acrylic acid‐co‐acrylonitrile) membrane—Racemate solution concentration effect

AbstractA novel nanoporous molecularly imprinting copolymer [Poly(acrylic acid‐co‐acrylonitrile)] membrane (DICMI) has been synthesized by the wet phase inversion method, using D‐Phenylalanine as template, for the optical resolution of amino acid. The membrane prepared is intended for the selective separation of phenylalanine. The racemate solutions of different concentrations (10‐ppm, 50‐ppm, 100‐ppm, and 200‐ppm) were filtered through the membrane fixed in an ultrafiltration kit at 98 kPa pressure to investigate the concentration effect on optical resolution of amino acid using DICMI. Adsorption selectivities of 0.37, 0.45, 0.57, and 0.71 were achieved after 25 ml of filtration of 10‐ppm, 50‐ppm, 100‐ppm, and 200‐ppm racemate solutions respectively. Scanning electron microscopy was used to study the morphological structure of membrane. Chemical structure was studied by Fourier transmission infrared spectroscopy. POLYM. COMPOS., 2008. © 2008 Society of Plastics Engineers

Mapping the Binding Site of Thiopeptide Antibiotics by Proximity-Induced Covalent Capture

Proximity-induced covalent capture (PICC) has been established for the investigation of ligand binding to composite protein/oligonucleotide target complexes. The RNA-induced attachment of the thiopeptides Thiostrepton and Nosiheptide to engineered Cys mutants of the ribosomal protein L11 was highly position selective and allowed mapping of their binding site at amino acid resolution.

Resolution of non-proteinogenic amino acids via microbial lipase-catalyzed enantioselective transesterification

A number of non-proteinogenic amino acids bearing aliphatic side chains were resolved with moderate to good enantioselectivities (E = 15–42) through the Burkholderia cepacia lipase-catalyzed enantioselective transesterification of the 2,2,2-trifluoroethyl esters of their N-benzyloxycarbonyl derivatives with methanol as a nucleophile in diisopropyl ether.Key words: non-proteinogenic amino acids, microbial lipases, Burkholderia cepacia lipase, 2,2,2-trifluoroethyl ester, enantioselective transesterification, enantioselectivity.

Copper(II) complexes of lipophilic aminoglycoside derivatives for the amino acid enantiomeric separation by ligand-exchange liquid chromatography

In this paper, a new class of ligand-exchange chiral stationary phase (LE-CSP) based on the copper complexes of lipophilic aminoglycoside derivatives was reported. Different stationary phases were developed by coating reversed-phase liquid chromatography supports with three neamine derivatives carrying a lipophilic octadecyl chain at the 4′, 5 and 6 positions, respectively. The enantioselective ability of these LE neamine-based CSPs was evaluated and the 4′-derivative coated column was found to be the most interesting one for the amino acid resolution. The effects of the variation of several chromatographic parameters on the enantioseparation were evaluated in order to identify the analysis optimal conditions.

Engineered dehydrogenase biocatalysts for non-natural amino acids: efficient isolation of the d-enantiomer from racemic mixtures

With a view to their use in the kinetic resolution of racemic non-natural amino acids, five variants of the enzyme L-phenylalanine dehydrogenase, the wild-type enzyme from Bacillus sphaericus and four active-site mutants, have been tested with a range of amino acids. In each case, the rates of reaction with 0.2 mM L-amino acid and with the racemic mixture at 0.4 mM were compared, so that the starting concentration of the active substrate was kept constant. Although the D-amino acids are not substrates, they were inhibitory in all cases. The extent of inhibition, however, varied greatly from compound to compound and among the mutants. With the N145L mutant and DL 4-O-Me-Phe, the equimolar D-enantiomer gave 83.2% inhibition, and with the wild-type enzyme there was 86.7% inhibition with racemic norleucine. By contrast, with these same substrates the N145V mutant showed less than 9% and 24% inhibition respectively. The N145A mutant was selected for use with DL-4-Cl-Phe. The pH was decreased from the enzyme's optimum of 10.4 to 9.5 to minimise breakdown of the coenzyme NAD(+), and the coenzyme was recycled by molecular oxygen with the assistance of a commercial diaphorase. Reaction on a 200 micromole scale in 20 ml ethanolamine HCl buffer, pH 9.5, with 25 microg N145A enzyme and 100 microg diaphorase, was monitored by chiral HPLC. The L-isomer was removed to an extent of >99% after 40 h, with the D-isomer peak undiminished. The pure D-isomer was isolated from the reaction mixture in 85% overall yield after ion-exchange chromatography.

A Self-Consistent Field Analysis of the Neurofilament Brush with Amino-Acid Resolution

Using the numerical model of Scheutjens and Fleer we investigated, on a self-consistent field level, the equilibrium structure of the neurofilament brush formed by the projection domains of NF-H, NF-M, and NF-L proteins. Although the actual amino-acid sequences in the projection domains are coarse-grained, the different (realistic) solubilities of amino-acid residues and the specific distribution of its intrinsic charges inside the arms of the NF proteins are taken explicitly into account. We collect strong evidence that the electrostatic interactions are a dominant force that controls the NF brush structure. There exists a remarkable spatial separation of the H, M, and L tails. In a dephosphorylated NF we found confined and flowerlike conformations for the H and M projection domains, respectively. We demonstrate that the ionization of KSP repeats in NF proteins triggers a conformational transition in the H tail that leads to the expulsion of its terminal (KEP) domain to the periphery of the NF brush. We argue that the phosphorylation of the NF proteins in axons can both increase the interfilament distance and stabilize cross bridges between neurofilaments.

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

Many substrates of ERK2 contain a D-site, a sequence recognized by ERK2 that is used to promote catalysis. Despite lacking a canonical D-site, the substrate Ets-1 is displaced from ERK2 by peptides containing one. This suggests that Ets-1 may contain a novel or cryptic D-site. To investigate this possibility a protein footprinting strategy was developed to elucidate ERK2-ligand interactions. Using this approach, single cysteine reporters were placed in the D-recruitment site (DRS) of ERK2 and the resulting ERK2 proteins subjected to alkylation by iodoacetamide. The ability of residues 1-138 of Ets-1 to protect the cysteines from alkylation was determined. The pattern of protection observed is consistent with Ets-1 occupying a hydrophobic binding site within the DRS of ERK2. Significantly, a peptide derived from the D-site of Elk-1, which is known to bind the DRS, exhibits a similar pattern of cysteine protection. This analysis expands the repertoire of the DRS on ERK2 and suggests that other targeting sequences remain to be identified. Furthermore, cysteine-footprinting is presented as a useful way to interrogate protein-ligand interactions at the resolution of a single amino acid.

Chiral separation of dansyl amino acids in capillary electrophoresis using mono‐(3‐methyl‐imidazolium)‐β‐cyclodextrin chloride as selector

Enantioseparations of fourteen dansyl amino acids were achieved by using a positively-charged single-isomer beta-cyclodextrin, mono-(3-methyl-imidazolium)-beta-cyclodextrin chloride, as a chiral selector. Separation parameters such as buffer pH, selector concentration, separation temperature, and organic modifier were investigated for the enantioseparation in order to achieve the maximum possible resolution. Chiral separation of dansyl amino acids was found to be highly dependent on pH since the degree of protonation of these amino acids can alter the strength of electrostatic interaction and/or inclusion complexation between each enantiomer and chiral selector. In general, the chiral resolution of dansyl amino acids was enhanced at higher pH, which indicates that the carboxylate group on the analytes may interact with the imidazolium group of cationic cyclodextrin. For most analytes, a distinct maximum in enantioresolution was obtained at pH 8.0. Moreover, the chiral separation can be further improved by careful tuning of the separation parameters such as higher selector concentration (e.g. 10 mM), lower temperature, and addition of methanol. Enantioseparation of a standard mixture of these dansyl amino acids was further achieved in a single run within 30 min.

Uniformly aligned full-length membrane proteins in liquid crystalline bilayers for structural characterization.

High-resolution solid-state NMR spectra of three full-length membrane proteins uniformly aligned in lipid bilayers between glass slides are observed at high magnetic field. The resolution of the specific amino acid labeled samples shows promise for large membrane protein structure determination utilizing aligned samples and shows resonance patterns known as PISA wheels. The tilt angles of the transmembrane helices are extracted from the resonance patterns in PISEMA spectra.

Quantitative Monitoring of Extracellular Matrix Production in Bone Implants by 13C and 31P Solid-State Nuclear Magnetic Resonance Spectroscopy

We used (31)P and (13)C solid-state nuclear magnetic resonance (NMR) spectroscopy to detect and analyze the major organic and inorganic components (collagen type I and bioapatite) in natural rabbit bone and beta-tricalcium phosphate implants loaded with osteogenically differentiated mesenchymal stem cells. High-resolution solid-state NMR spectra were obtained using the magic-angle spinning (MAS) technique. The (31)P NMR spectra of bone specimens showed a single line characteristic of bone calcium phosphate. (13)C cross-polarization (CP) MAS NMR spectra of bone exhibited the characteristic signatures of collagen type I with good resolution for all major amino acids in collagen. Quantitative measurements of (13)C-(1)H dipolar couplings indicated that the collagen segments are very rigid, undergoing only small amplitude fluctuations with correlation times in the nanosecond range. In contrast, directly polarized (13)C MAS NMR spectra of rabbit bone were dominated by signals of highly mobile triglycerides. These quantitative investigations of natural bone may provide the basis for a quality control of various osteoinductive bone substitutes. We studied the formation of extracellular bone matrix in artificial mesenchymal stem cell-loaded beta-tricalcium phosphate matrices that were implanted into the femoral condyle of rabbits. The NMR spectra of these bone grafts were acquired 3 months after implantation. In the (31)P NMR spectra, beta-tricalcium phosphate and bone calcium phosphate could be distinguished quantitatively, allowing recording of the formation of the natural bone matrix. Further, (13)C CPMAS allowed detection of collagen type I that had been produced in the implants. Comparison with the spectroscopic data from natural bone allowed assessment of the quality of the bone substitute material.