Aspartic Acid Enantiomers Research Articles

Aspartic acid enantiomer quantification using ultraperformance liquid chromatography–tandem mass spectroscopy combined with deuterium‐chloride hydrolysis to improve age estimation in Antarctic minke whale Balaenoptera bonaerensis

AbstractAspartic acid racemization is a useful tool for estimating age in marine mammals; however, acid hydrolysis during sample preparation may increase the D‐ to L‐enantiomer ratio. To improve age estimation in Antarctic minke whale (Balaenoptera bonaerensis), we optimized the quantification of aspartic acid enantiomers in ocular lens by using ultraperformance liquid chromatography–tandem mass spectrometry combined with sample preparation by hydrolysis with deuterium chloride. Using this approach, we determined D/L in fetal and adult whale lens, examined the degree of racemization induced by acid hydrolysis, and constructed equations for age estimation. In the fetal lens, D/L obtained after hydrolysis with HCl or DCl were positively correlated with gestation age. Estimates of D/L at birth using a DCl‐hydrolysis model were much lower than those obtained using a HCl‐hydrolysis model, indicating that racemization during hydrolysis with HCl reduces the precision of subsequently derived age estimates. Furthermore, the D/L0 unaffected by Asp‐D generated during hydrolysis was estimated. The loge[(1 + D/L)/(1 − D/L)] value was exponentially increased with increasing earplug‐derived age, and the standard error of the age estimates after hydrolysis with DCl was lower than that after HCl. We conclude that our method can provide precise age estimates without methodological bias.

Bovine serum albumin-coated titanium dioxide modified electrochemical interface for enantioselective discrimination of D/L-aspartic acid.

Due to the similar physicochemical properties, the discrimination of chiral isomers faces huge challenges in drug production and biochemical analysis. Herein, the bovine serum albumin-coated titanium dioxide (bovine serum albumin [BSA]/TiO2 ) was modified as a novel electrochemical interface for efficient, simple, and enantioselective discrimination of aspartic acid enantiomers (D/L-Asp) based on the electrochemical impedance spectroscopy (EIS). Utilizing the structural characteristics of large cavity and high specific surface area, TiO2 material provided sufficient space for adequate loading of BSA. The BSA/TiO2 electrochemical interface was successfully fabricated to support abundant chiral recognition sites. The enantioselective discrimination of D/L-Asp was achieved on the interface with a good linear relationship against the impedance difference in the concentration range from 1 to 1000 nM with the detection limit of 0.37 nM for L-Asp and 0.94 nM for D-Asp, reaching the identification coefficient (Ic = KL /KD ) of 1.85. The proposed interface is easy to form with a stable formation of BSA in TiO2 microporous architecture, which maintained the desired stability and reproducibility. For the unknown racemic solution, Ic levels of different enantio-ratios of D/L-Asp were effectively obtained to evaluate the chiral percentage of racemic sample. The possible mechanism of chiral recognition by density function theory (DFT) was confirmed with a stronger adsorption to L-Asp in accordance with our experiment results, reinforcing the validity of our presented interface. The BSA/TiO2 electrochemical interface with robust enantioselective discrimination of D/L-Asp has great potential for the practical application in pharmaceutical surveillance and food security.

3. Easier access to “fossil gelatin” and quality check by amino acid racemization

Gelatin in fossil bones is the most important source for radiocarbon, stable isotopes, ZooMS (Zooarchaeology by Mass Spectrometry) and Amino Acid Racemization (AAR) studies. The isolation and cleaning of fossil gelatin is a time consuming procedure and can introduce contaminations, which, as a consequence, can produce erroneous results.By synthesizing and optimizing a “Molecularly Imprinted Polymer” (MIP) for gelatin this isolation procedure can be faster and more specific.This first application of MIP on fossil gelatin is described. By optimizing the sample preparation over all steps from getting fossil bone powder (ca. 20 mg), isolating the fossil gelatin by MIP, and analyzing the amino acids, especially the enantiomers of aspartic acid from the fossil gelatin by gas chromatography (GC), results are available within one day.These results include a measure of the quality of the fossil gelatin by the pattern of the single amino acids and the ratios of defined amino acids from the GC chromatogram. At the same time, the AAR can give an estimate of the relative age of the fossil bones.

A method for examining the biochemical composition of dental tissues in human teeth

In medical literature there is no single detailed standard for studying the biochemical composition of a tooth. This circumstance led to the development of a proprietary method for determining the enantiomers of aspartic acid in hard dental tissues for use in the further study of the relationship between the amino acid composition of dental tissues and the biological age of humans. Dental tissue samples obtained from living individuals aged from 20 to 50 years were studied by chromatography-mass spectrometry. The results of the study allowed us to put forward a number of practical recommendations on the standardization of the procedure for the preparation of dental tissue samples and the parameters of chromatography-mass spectrometry research.

Highly sensitive and selective hyphenated technique (molecularly imprinted polymer solid-phase microextraction–molecularly imprinted polymer sensor) for ultra trace analysis of aspartic acid enantiomers

The present work is related to combination of molecularly imprinted solid-phase microextraction and complementary molecularly imprinted polymer-sensor. The molecularly imprinted polymer grafted on titanium dioxide modified silica fiber was used for microextraction, while the same polymer immobilized on multiwalled carbon nanotubes/titanium dioxide modified pencil graphite electrode served as a detection tool. In both cases, the surface initiated polymerization was found to be advantageous to obtain a nanometer thin imprinted film. The modified silica fiber exhibited high adsorption capacity and enantioselective diffusion of aspartic acid isomers into respective molecular cavities. This combination enabled double preconcentrations of d- and l-aspartic acid that helped sensing both isomers in real samples, without any cross-selectivity and matrix complications. Taking into account 6×104-fold dilution of serum and 2×103-fold dilution of cerebrospinal fluid required by the proposed method, the limit of detection for l-aspartic acid is 0.031ngmL−1. Also, taking into account 50-fold dilution required by the proposed method, the limit of detection for d-aspartic acid is 0.031ngmL−1 in cerebrospinal fluid.

Growth inhibition of calcium oxalate monohydrate crystal by linear aspartic acid enantiomers investigated by in situatomic force microscopy

The inhibitory effect of linear enantiomers of L- and D-Asp6 on the growth of calcium oxalate monohydrate crystal has been investigated using in situ atomic force microscopy. The inhibitory magnitude of D-Asp6 on the growth of the [00] step on the (010) face is about 10% larger than that of L-Asp6. While no chiral effect is observed or expected on the growth of the [0] step on the (01) face by both enantiomers, their inhibitory effect on this step is much stronger than that on the [00] step on the (010) face. In both cases, the step morphology indicates that these enantiomers create the impurity pinning along the steps, while the dependence of step speed on supersaturation shows that they also produce a reduction of the step kinetic coefficients. Analysis of the step speed data within the context of an existing model for step pinning and kink blocking shows that the major impact of Asp6 is to block active kink sites. The larger inhibition of the [00] step growth by D-Asp6 over L-Asp6 and the substantially larger inhibition of the [0] step over the [00] step by both enantiomers both result from larger affinity for adsorption to the (010) face and the (01) face, respectively. This is because the larger adsorption leads to a higher density of blocking kink sites along the steps. The estimated difference in binding energy of L- and D-Asp6 to the respective faces from the kinetics model is consistent with the trend predicted by our molecular modeling of the enantiomer binding to the faces.

Electrochemically imprinted molecular recognition sites on multiwalled carbon-nanotubes/pencil graphite electrode surface for enantioselective detection of d- and l-aspartic acid

Molecularly imprinted polymeric nano-materials (thickness 2.03nm) for aspartic acid enantiomers were electrochemically synthesized onto multi-walled carbon nanotubes immobilized pencil graphite electrode surface. The molecular recognition in this work was based on doping/de-doping characteristics of the conducting polymer, poly (indole-3-acetic acid), wherein the template (aspartic acid) acted as a dopant which could easily be ejected from the polymer backbone after over-oxidation. This resulted in molecular cavities complementary to the template in the imprinted polymer texture. The transduction was made via differential pulse anodic stripping voltammetric detection of d- and l-aspartic acid using respective sensors. The detection limits (S/N=3) for d- and l-aspartic acid were found to be 0.025 and 0.016μM, respectively in aqueous solutions. Enantioselective analysis of l-aspartic acid was validated in real samples that suggested practicability of the proposed sensor for the evaluation of this bioactive molecule as a disease biomarker in clinical settings, without any cross-reactivity and false-positives.

M(rac-N-benzyl Asp)(H2O)] (M = Co, Ni): Noncentrosymmetric Coordination Polymeric Chains with Racemic Chiral Ligands

The hydrothermal reaction of fumaric acid, benzylamine, and metal salts yielded M[(rac-N-benzyl-Asp)(H(2)O)] (M = Co, Ni), 1 and 2, and Ni[(rac-N-benzyl-Asp)(H(2)O)(3)]·H(2)O 3. Under mild hydrothermal conditions, Michael addition of benzylamine to fumaric acid led to the formation of a racemic mixture of N-benzyl aspartic acid enantiomers. The noncentrosymmetric structures of 1 and 2 consist of one-dimensional polymeric chains in which metal cations are bridged by d- and l-N-benzyl aspartate anions alternating along the chain. The centrosymmetric structure of 3 is composed of discrete Ni[(rac-N-benzyl-Asp)(H(2)O)(3)] units that are connected by hydrogen bonds into layers. The single layers are homochiral but are hydrogen bonded to similar homochiral layers that contain the N-benzyl aspartate with the opposite handedness. Compounds 1 and 2 showed second harmonic generation (SHG), and their magnetic and thermodynamic properties are described.

Identification of Aspartic Acid Enantiomers Based on Molecularly Imprinted Polyaniline

AbstractAffinity sites for L‐aspartic acid (L‐Asp) in polyaniline (PAn) were created by two successive processes: first, L‐Asp was simply added as template molecules during the polymerization of aniline; second, L‐Asp incorporated in PAn backbone was extracted by solvent. PAn with cavities complementary to L‐Asp template molecules has been utilized for enantioselective recognition of L‐ and D‐Asp. Enantioselectivity of the imprinted PAn could be attributed to the cavities in the imprinted PAn which was complementary to L‐Asp templates both in shape and in positioning of groups. Also in this paper, the structural changes before and after extraction of L‐Asp templates were revealed by Fourier‐transform infrared (FTIR) spectrum.

Stacking and separation of aspartic acid enantiomers under discontinuous system by capillary electrophoresis with light-emitting diode-induced fluorescence detection

We describe the stacking and separation of d- and l-aspartic acid (Asp) by capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF). In the presence of cyanide, d- and l-Asp were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form fluorescent derivatives prior to CE-LEDIF. The separation of NDA-derivatized d- and l-Asp was accomplished using a discontinuous system – buffer vials contained a solution of 0.6% poly(ethylene oxide) (PEO), 150 mM sodium dodecyl sulfate (SDS), and 60 mM hydroxypropyl-β-cyclodextrin (Hp-β-CD), while a capillary was filled with a solution of 150 mM SDS and 60 mM Hp-β-CD. The role of PEO, Hp-β-CD, and SDS is to act as a concentrating media, as a chiral selector, and as a pseudostationary phase, respectively. This discontinuous system could be employed for the stacking of 600 nL of NDA-derivatized d- and l-Asp without the loss of chiral resolution. The stacking mechanism is mainly based on the difference in viscosity between sample zone and PEO as well as SDS sweeping. The limits of detection at signal-to-noise of 3 for d- and l-Asp were down to 2.4 and 2.5 × 10 −10 M, respectively. Compared to normal sample injection volume (25 nL), this stacking approach provided a 100- and 110-fold improvement in the sensitivity of d- and l-Asp, respectively. This method was further applied for determining d- and l-Asp in cerebrospinal fluid, soymilk, and beer.

The Origin of Life and the Crystallization of Aspartic Acid in Water

The unusual molecular complexation of the enantiomers of aspartic acid in water was discovered and proven by a solubility test, solution freezing point, crystallization kinetics, and the incubation time change. The transformation of a “conglomerate solution” (CS) to a “racemic compound solution” (RCS) was dependent on both temperature and time. The CS was the solution phase which produced conglomerate crystals, and the RCS was the solution phase which gave a racemic compound. Fourier transformed infrared spectroscopy and powder X-ray diffraction were used to characterize aspartic acid solids crystallized from those complex solution phases and to distinguish conglomerate crystals from a racemic compound. We found that it took more than 36 h at 25 °C and 5 h at 45 °C just to complete the solution phase transformation of the CS of aspartic acid to the RCS of aspartic acid. However, the presence of an equimolar of succinic acid could hinder the solution phase transformation of the CS of aspartic acid to the R...

2D separations on a 1D chip: gradient elution moving boundary electrophoresis—chiral capillary zone electrophoresis

A new method is described for two-dimensional (2D) separations using a microfluidic chip normally employed for single dimension electrophoresis. The method employs a combination of gradient elution moving boundary electrophoresis (GEMBE) and chiral capillary zone electrophoresis (CZE). The simplicity of the first dimension GEMBE method enables its implementation in the injection channel of a conventional electrophoresis chip, simplifying the design and operation of the device. The method was used for high resolution 2D chiral separations of a mixture of amino acids considered as possible signatures of extant or extinct life for solar system exploration. The enantiomers of aspartic acid, glutamic acid, serine, alanine, and valine were all resolved as well as glycine (achiral) and several unidentified impurities, giving an estimated peak capacity of 35 for the region between valine and glycine. The results highlight the need for high peak capacity separations for chiral amino acid analysis if accurate enantiomeric ratios are to be determined.

Racemization of aspartic acid from human dentin in the estimation of chronological age

The estimation of chronological age in cadavers, human remains and in living human beings by various methods is discussed. These methods, which are based on the age dependent non-enzymatic changes of l-form amino acids to d-form amino acids, mainly aspartic acid, are among the most reliable and accurate methods to date. Most of these methods use gas chromatography (GC). In this review, results of aspartic acid racemization in dentin at different targets are discussed. In addition, pre-considerations and guidelines are given for the selection of dentin from teeth. A pilot project was run to evaluate the efficiency of high performance liquid chromatography (HPLC) coupled with fluorescence detection. New buffer conditions were found to obtain stable derivatives of aspartic acid enantiomers for the estimation of racemization.

Method for on-line derivatization and separation of aspartic acid enantiomer in pharmaceuticals application by the coupling of flow injection with micellar electrokinetic chromatography

A novel, easy and accurate capillary electrophoresis (CE) coupled with flow injection (FI) method for the separation and determination of aspartic acid (Asp) enantiomers by on-line derivatization had been developed, and it had been applied to the real sample for the first time. The derivatization reagents were o-phthalaldehyde (OPA) and mercaptoethanol (ME), which were obtained easily, the chiral selector was β-cyclodextrin (β-CD), the micellar chemical was sodium dodecyl sulfate (SDS), and the modifier was methanol. By on-line derivatization, aspartic acid enantiomers were automatically and reproducibly converted to the ultraviolet (UV)-absorbing diastereoisomer derivates, which were separated by micellar electrokinetic chromatography (MEKC). According to the factors affecting the separation and sensitivity of aspartic acid enantiomer and other amino acids in the real sample, the pH value and concentration of the buffer, the concentration of β-CD and SDS, the volume percentage of the methanol (v/v) in the buffer, the applied voltage and the conversion time were selected as the investigating variates. Under the investigated separation conditions, d-aspartic acid ( d-Asp), l-aspartic acid ( l-Asp) and other four amino acids achieved the baseline separation in not only the standard mixture of amino acids but also the real sample (Compound Amino Acid Injection (6AA)). The repeatability (defined as relative standard deviation (RSD), n = 5) was 4.0% and 4.0% with peak area evaluation, and 4.2% and 3.7% with peak height evaluation for d-Asp and l-Asp in the real sample. Recovery at added standard levels of 1.0, 3.0 and 6.0 mM was 92%, 104% and 109%, respectively.

Age determination of minke whales (Balaenoptera acutorostrata) using the aspartic acid racemization technique

Eyeballs were collected from 31 female minke whales caught in the North Sea, Norwegian Sea and Barents Sea. The eye-lens nucleus was dissected out and the relative amount of D- and L- enantiomers of aspartic acid measured using high-performance liquid chromatography (HPLC). A racemization reaction changes the D/L aspartic acid relationship in the nucleus over time. The age of each whale was estimated using the racemization rate for fin whales along with the measured D/L ratio. The minimum age estimated was 0 years, while the maximum was 32 years. The SE of the age estimates ranged from 4.5 to 8.7 years. Age at sexual maturity was estimated to 5.8 ( - 3.0) years by regressing the number of ovulations versus the age estimate. Using the von Bertalanffy growth model, age at sexual maturity was estimated to 7.8 ( - 6.0) years, and age at physical maturity to 13 years. These estimates are consistent with other demographic data for the species. No systematic errors in the age estimates were detected, but the rac...

Determination of free aspartic acid enantiomers in rat brain by capillary electrophoresis with laser-induced fluorescence detection

Quantification of aspartic acid enantiomers in rat brain by using a chiral capillary electrophoresis procedure is described. Amino acids were pre-column derivatized with naphthalene-2,3-dialdehyde. Enantiomeric separation was achieved by micellar electrokinetic chromatography in the presence of methanol and β-cyclodextrin as chiral selector. The chiral separation was coupled with laser-induced fluorescence detection. Contents of d- and l-aspartic acids in rats at different stages of growth (from 1 day before birth to 90 days after birth) were determined. d-Aspartic acid was detected in all the brain tissue samples tested, but at different levels. In the cerebrum of rats 1 day before birth, d-aspartic acid was found to be at the highest concentration of 81 nmol/g wet tissue. The level of d-aspartic acid in rat brain falls rapidly after birth, while the l-aspartic acid level increases with age.

Determination of aspartic acid enantiomers in bio‐samples by capillary electrophoresis

Determination of aspartic acid enantiomers in bio‐samples by capillary electrophoresis

Determination of aspartic acid enantiomers in bio-samples by capillary electrophoresis.

Enantiomeric separation and detection of D,L-aspartic acid (Asp) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3, 6-tri-O-methyl)-beta-cyclodextrin(TM-beta-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-beta-CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D- and L-Asp determination since the other amino compounds migrated differently and D-Asp in bio-samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time.

Polystereoisomers with Two Stereogenic Centers of Malic Acid 2-Methylbutyl Ester Configurational Structure/Properties Relationship

Poly(β-malic acid alkyl esters) with two stereogenic centers have been synthesized by anionic ring-opening polymerization of racemic and optically active 4-[(2-methylbutyl)oxycarbonyl]-2-oxetanones and characterized. The two asymmetric centers have been introduced in the monomer by using malic acid or aspartic acid enantiomers as chiral synthons and 2-methyl-1-butyl alcohol enantiomers as lateral ester groups. By combining the different enantiomers during the β-substituted β-lactone synthesis route, it has been possible to prepare five stereoisomers which have conducted to five different stereocopolymers. The configurational structure of racemic and optically active poly(2-methylbutyl β-malates) has been determined by 13 C NMR analysis, and differentiation between polydiastereoisomers has been possible by using this spectroscopy method. Several main-chain and side-chain carbon atoms were stereosensitive to the presence of the two asymmetric centers per repeat unit, and the optical purities of main-chain and side-chain stereogenic centers have been deduced from 13 C NMR spectra. Thermal properties of the different polystereoisomers have been correlated with the configuration of the two chiral sites in the macromolecular chain. It has been shown that crystallinity was dependent on the configurational structure of the main chain and very poorly sensitive to the enantiomeric composition of the ester pendant group

Analysis of aspartic acid racemization : Evaluation of a chiral capillary gas chromatographic and a distereomeric high-performance liquid chromatographic method

A recently developed chiral gas chromatographic method and a diastereomeric high-performance liquid chromatographic method for the analysis of aspartic acid enantiomers in protein hydrolyzates have been evaluated. Although both techniques are fast and convenient, the latter is preferred because of its higher reproducibility and shorter analysis time. Furthermore, this method offers the possibility of on-line derivatization and analysis.