Rate Of Racemization Research Articles

Investigation of stereochemical stability of 2-, 3- and 4-chloromethcathinones in various biological matrixes

The stereochemical stability of the popular drugs of abuse 2-, 3- and 4-chloromethcathinone was studied in the mobile phase used for the isolation of their enantiomers by high-performance liquid chromatography, as well as in various biological matrixes such as whole blood, saliva and urine. For 2-, 3-, and 4-chloromethcathinones the rate constants and half-lives of their first order racemization reaction was assessed. It was found that at 25 °C the racemization rate constant decreases in the order 2-CMC > 3-CMC > 4-CMC while their stereochemical stability in biological matrixes decreases in the order urine > saliva > whole blood. This information must be considered for the adequate storage of purified enantiomers in the collected fractions, as well as in the studies focused on their enantioselective transformation in the human body.

A dynamic kinetic resolution catalyzed by immobilized lipase coupled with a novel grinding immobilized oxovanadium：Kinetic model and performance

A dual immobilized lipase and oxovanadium co-catalyzed dynamic kinetic resolution (DKR) system was established and successfully applied to synthesize (R)-1-(3-Methoxyphenyl) acetate in batch, resulting in a key chiral intermediate of (S)-rivastigmine with highly yield (97.6 %), e.e (99.01 %) and selectivity Sel (99.4 %). Herein, oxovanadium was immobilized onto diatomite via grinding immobilization for the first time to access a novel immobilized oxovanadium (V-D) used to catalyze an in situ racemization of undesired enantiomer substrate. The racemization rate of hydrated vanadyl sulfate was found for the first time to be much higher than that of vanadyl sulfate without crystalline water because water molecules in the former may form ion clusters of solvation membranes to disperses the charge density of positive carbon ions and promote their stability. This grinding immobilization approach may become a promising alternative for the immobilization of metals, especially precious metals due to its low cost, high efficiency, easily separated and large reaction interface. The kinetic model of the kinetic resolution catalyzed by LA-MR was developed, which may obey the sequential mechanism with both substrate and product inhibition, while the in-situ racemization reaction catalyzed by V-D may comply with power law.

Characterization of different-sized human αA-crystallin homomers and implications to Asp151 isomerization.

Site-specific modifications of aspartate residues spontaneously occur in crystallin, the major protein in the lens. One of the primary modification sites is Asp151 in αA-crystallin. Isomerization and racemization alter the crystallin backbone structure, reducing its stability by inducing abnormal crystallin-crystallin interactions and ultimately leading to the insolubilization of crystallin complexes. These changes are considered significant factors in the formation of senile cataracts. However, the mechanisms driving spontaneous isomerization and racemization have not been experimentally demonstrated. In this study, we generated αA-crystallins with different homo-oligomeric sizes and/or containing an asparagine residue at position 151, which is more prone to isomerization and racemization. We characterized their structure, hydrophobicity, chaperone-like function, and heat stability, and examined their propensity for isomerization and racemization. The results show that the two differently sized αA-crystallin variants possessed similar secondary structures but exhibited different chaperone-like functions depending on their oligomeric sizes. The rate of isomerization and racemization of Asp151, as assessed by the deamidation of Asn151, was also found to depend on the oligomeric sizes of αA-crystallin. The predominant isomerization product via deamidation of Asn151 in the different-sized αA-crystallin variants was L-β-Asp in vitro, while various modifications occurred around Asp151 in vivo. The disparity between the findings of this in vitro study and in vivo studies suggests that the isomerization of Asp151 in vivo may be more complex than what occurs in vitro.

Synthesis, structure and stereodynamics of atropisomeric N-chloroamides.

Atropisomeric N-chloroamides were efficiently accessed by electrophilic halogenation of ortho-substituted secondary anilides. The stereodynamics of atropisomerism in these novel scaffolds was interrogated by detailed experimental and computational studies, revealing that racemization is correlated with amide isomerization. The stereoelectronic nature of the amide was shown to significantly influence racemization rates, with potentially important implications for other C-N atropisomeric scaffolds.

Unlocking same-sign CPL: solvent effects on spectral form and racemisation kinetics in nine-coordinate chiral europium(III) complexes.

Understanding the factors that shape the circularly polarised luminescence (CPL) emission profiles of europium(III)-based CPL emitters to have specific sign properties, e. g. monosignate individual CPL transitions, is key to design novel complexes for applications ranging from advanced security inks to bio-probes for live cell imaging. In order to correlate structure and spectral characteristics, a photophysical and kinetic investigation has been conducted on a series of coordinatively saturated nine-coordinate europium(III) systems based on 1,4,7-triazacyclononane. We highlight that lanthanide emission is sensitive to changes in the ligand field by showing the linear dependence of total emission intensity ratios as a function of solvent polarity, for europium(III) complexes displaying an internal charge transfer (ICT) excited state. This sensitivity increases by a factor of 20 when studying changes in CPL spectra, rendering these complexes accurate probes of local polarity. Solvent polarity, solvent-specific effects, and the nature of the chromophores' coordinating donor atoms strongly influence the kinetic stability of europium(III) complexes with respect to enantiomer interconversion. Notably, we show that the choice of donor groups to coordinating to europium(III) and the nature and polarity of the solvent affects the rate of racemisation, leading to systems with very long half-lives at room temperature in non-polar media.

Interrogating the configurational stability of atropisomers.

Atropisomers are molecules whose stereogenicity arises from restricted rotation about a single bond. They are of current importance because of their applications in catalysis, medicine and materials science. The defining feature of atropisomeric molecules is that their stereoisomers are related to one another by bond rotation: as a result, evaluating their configurational stability (i.e., the rate at which their stereoisomers interconvert) is central to any work in this area. Important atropisomeric scaffolds include C-C linked biaryls, such as the ligand BINAP and the drug vancomycin, and C-N linked amine derivatives such as the drug telenzepine. This article focuses on the three most widely used experimental methods that are available to measure the rate of racemization in atropisomers, namely: (i) kinetic analysis of the racemization of an enantioenriched sample, (ii) dynamic HPLC and (iii) variable-temperature NMR. For each technique, an explanation of the theory is set out, followed by a detailed experimental procedure. A discussion is also included of which technique to try when confronted with a new molecular structure whose properties are not yet known. None of the three procedures require complex experimental techniques, and all can be performed by using standard analytical equipment (NMR and HPLC). The time taken to determine a racemization rate depends on which experimental method is required, but for a new compound it is generally possible to measure a racemization rate in <1 d.

A comparison between incremental lines of cementum and aspartic acid racemization in age estimation: histological and biochemical study

BackgroundAge estimation is a vital aspect of the identification of an individual in forensic science. Teeth are one of the toughest structures in the human body and generally survive postmortem destruction. So, they can therefore be used more reliably than any other morphological or histological trait of the adult skeleton for estimating age.Aim of the studyThe present work aimed to compare cementum incremental lines and aspartic acid racemization in age estimation among Egyptian subjects.MethodsSixty-four subjects were assigned to two groups: males and females (each with 32 cases) to determine the effect of sex as a possible factor that might affect age estimation. For each group, in all subjects, age was estimated using histologic examination of ground, unstained section teeth by light and polarized microscopes, in addition to estimating the rate of aspartic acid racemization in the collagen of dentin by using the HPLC method.ResultsThere was a statistically significant correlation between each method of age estimation and chronological age. However, the strongest degree of correlation was observed for the racemization method, with a correlation coefficient of r = 0.99 (95% CI 0.98 to 0.99), followed by the light method, r = 0.94 (95% CI 0.90 to 0.96), and the polarized method, r = 0.93 (95% CI 0.88 to 0.95).ConclusionsBoth methods, cementum incremental lines and aspartic acid racemization, could help in age estimation, but aspartic acid racemization is more reliable and accurate.

Amino acid racemization in Neogloboquadrina pachyderma and Cibicidoides wuellerstorfi from the Arctic Ocean and its implications for age models

Abstract. We report the results of amino acid racemization (AAR) analyses of aspartic acid (Asp) and glutamic acid (Glu) in the planktic Neogloboquadrina pachyderma, and the benthic Cibicidoides wuellerstorfi, foraminifera species collected from sediment cores from the Arctic Ocean. The cores were retrieved at various deep-sea sites of the Arctic, which cover a large geographical area from the Greenland and Iceland seas (GIS) to the Alpha and Lomonosov ridges in the central Arctic Ocean. Age models for the investigated sediments were developed by multiple dating and correlation techniques, including oxygen isotope stratigraphy, magnetostratigraphy, biostratigraphy, lithostratigraphy, and cyclostratigraphy. The extent of racemization (D/L values) was determined on 95 samples (1028 subsamples) and shows a progressive increase downcore for both foraminifera species. Differences in the rates of racemization between the species were established by analysing specimens of both species from the same stratigraphic levels (n=21). Aspartic acid (Asp) and glutamic acid (Glu) racemize on average 16 ± 2 % and 23 ± 3 % faster, respectively, in C. wuellerstorfi than in N. pachyderma. The D/L values increase with sample age in nearly all cases, with a trend that follows a simple power function. Scatter around least-squares regression fits are larger for samples from the central Arctic Ocean than for those from the Nordic Seas. Calibrating the rate of racemization in C. wuellerstorfi using independently dated samples from the Greenland and Iceland seas for the past 400 ka enables estimation of sample ages from the central Arctic Ocean, where bottom water temperatures are presently relatively similar. The resulting ages are older than expected when considering the existing age models for the central Arctic Ocean cores. These results confirm that the differences are not due to taxonomic effects on AAR and further warrant a critical evaluation of existing Arctic Ocean age models. A better understanding of temperature histories at the investigated sites, and other environmental factors that may influence racemization rates in central Arctic Ocean sediments, is also needed.

Synthesis and Atropisomeric Properties of Benzoazepine-Fused Isoindoles.

Atropisomeric, bench-stable benzoazepine-fused isoindoles were synthesized via oxidation from isoindoline precursors. Using the isoindoles 5d-f as models, the stereochemistry and conformational folding of the systems were examined. Chiral UHPLC was used to analyze the rate of racemization and calculate the Gibbs free energy of enantiomerization (ΔG‡Enant). X-ray crystallography, 1H NMR spectroscopy, and DFT calculations were used to elucidate the three axes of chirality and clarify the structural factors contributing to ΔG‡Enant. Tandem rotation around the axes of chirality precludes the formation of diastereomers, with rotational restriction of the Caryl-Nsulfonamide bond determined as the moderator of atropisomeric stability in the system, affected primarily by steric hindrance as well as by π-stacking interactions facilitated by the folded conformation of the sulfonamide over the isoindole moiety.

Enantioseparation and racemization of 3-fluorooxindoles.

Fluorinated oxindoles are frequently used building blocks in asymmetric synthesis and represent an important scaffold found in a variety of biologically relevant compounds. While it is understood that incorporation of fluorine atoms into organic molecules can improve their pharmacological properties, the impact on the configurational stability of chiral organofluorines is still underexplored. In this study, semipreparative HPLC enantioseparations of five oxindoles were carried out, and the resulting enantiomerically enriched solutions were used to investigate base promoted racemization kinetics at room temperature. It was found that incorporation of fluorine at the chiral center increases the configurational stability, while substitutions on the aromatic ring and at the lactam moiety also have significant effects on the rate of racemization, which generally follows reversible first-order reaction kinetics.

Chromatographic analysis of hard tooth tissue to determine the age of personality

The biochemical approach for age assessment is most appropriate in forensic medicine, as racemization of aspartic acid in bones and teeth is closely related to human biological age. The aim of the study is to assess the biochemical parameters of aspartic acid in human teeth, which can be implemented into forensic practice in Russia. Samples of dentin in amount of 20, taken from the teeth of subjects aged between 16 and 76, were examined. Chromatographic analysis of the samples was performed on a gas chromatograph using chiral column. Statistical data processing showed that the relative squared peak of D-aspartic acid has a strong correlation with human biological age. Data, obtained from the Russian population study, demonstrate the applicability of chromatography for forensic purposes. It should be noted that the approach to the racemization rate estimation in the hard tooth tissue was performed using standard laboratory equipment, which allows to easily implement this method in forensic medical practice.

Comparison of racemization rates between vital and endodontically treated teeth for age estimation

Amino acid racemization of dentin (AAR) is among the most precise methods for age estimation in unidentified adult cadavers. Although vital teeth are generally used for this technique, cases often have endodontically treated (Endo) teeth only. Therefore, the aim of this preliminary pilot study was to determine the applicability of Endo teeth by comparing AAR rates between vital and Endo teeth. Thirty-six dentin samples from vital teeth and 18 dentin samples from Endo teeth were analyzed, including the maxillary first premolar, maxillary second molar, and mandibular second premolar. Standard calibration curves for the mandibular second premolar and maxillary second molar were compared among vital teeth, Endo teeth, and mixed vital/Endo teeth. Discrepancies between estimated and actual ages were assessed by analyzing the AAR rates for the maxillary first premolar between vital and Endo teeth. The AAR rates for Endo teeth were higher than those for vital teeth in both the mandibular second premolar and maxillary second molar, and the correlation of the standard calibration curve for vital teeth only was highest (r = 0.982836; r = 0.92011467), followed by mixed (r = 0.949579; r = 0.76158) and Endo teeth only (r = 0.896082; r = 0.744991). In the maxillary first premolar, discrepancies were more than 10 years for all 5 Endo teeth, based on the standard calibration curve of vital teeth. The present study suggests that the AAR rates for Endo teeth might be affected by variation in dentin structure between vital and Endo teeth.

Chiral Amplification through the Interplay of Racemizing Conditions and Asymmetric Crystal Growth.

Amplification of enantiomeric excesses (ee) is routinely observed during chiral crystallization of conglomerate crystals for which the enantiomers undergo racemization in solution. Although routes comprising a combination of crystal growth and dissolution are frequently used to obtain enantiopure molecules, crystal growth by itself has rather been considered as a source of enantiomeric erosion and discounted as a potential source of enantiomeric amplification. Counterintuitively, we here demonstrate striking enantiomeric amplification during crystal growth for clopidogrel and tert-leucine precursors. Based on a mechanistic framework, we identify that the interplay between racemization and crystal growth rates elicits this surprising effect. The asymmetric amplification of the solid-phase ee can be enhanced by increasing the mass of grown material relative to the product such that small amounts of seeds of only 60% ee already result in virtually exclusive growth of the majority phase. These results impact our understanding of asymmetric amplification mechanisms during crystallization and offer a tangible basis for practical production of enantiopure molecules.

A Novel Stereospecific Bioluminescent Assay for Detection of Endogenous d-Cysteine.

The presence of endogenous d-stereoisomers of amino acids in mammals dispels a long-standing dogma about their existence. d-Serine and d-aspartate function as novel neurotransmitters in mammals. However, the stereoisomer with the fastest, spontaneous in vitro racemization rate, d-cysteine, has not been reported. We utilized a novel, stereospecific, bioluminescent assay to identify endogenous d-cysteine in substantial amounts in the eye, brain, and pancreas of mice. d-Cysteine is enriched in mice embryonic brains at day E9.5 (4.5 mM) and decreases progressively with development (μM levels). d-Cysteine is also present in significantly higher amounts in the human brain white matter compared with gray matter. In the luciferase assay, d-cysteine conjugates with cyano hydroxy benzothiazole in the presence of a base and reducing agent to form d-luciferin. d-Luciferin, subsequently, in the presence of firefly luciferase and ATP, emits bioluminescence proportional to the concentration of d-cysteine. The assay is stereospecific and allows the quantitative estimation of endogenous d-cysteine in tissues in addition to its specificity for d-cysteine. Future efforts aimed at bioluminescent in vivo imaging of d-cysteine may allow a more noninvasive means of its detection, thereby elucidating its function.

Can amino acid racemization be utilized for fish age validation?

We investigated the relationship between aspartic acid d:l ratios and otolith-derived age estimates in Gulf of Mexico red snapper, Lutjanus campechanus (ages 1–26 years; R2 = 0.89) and Caribbean yellowtail snapper, Ocyurus chrysurus (ages 2–17 years; R2 = 0.84). The estimated racemization rate was 0.61 × 10−3 year−1 for red snapper and 1.28 × 10−3 year−1 for yellowtail snapper, reflecting temperature differences between study regions. Mean jackknifed error in ages predicted from aspartic acid d:l was 1.70 ± 0.39 years for red snapper and 1.57 ± 0.41 years for yellowtail snapper. Results suggest amino acid racemization may be an effective tool for direct age estimation and potentially age validation in fishes.

Shortcut Model for Batch Preferential Crystallization Coupled with Racemization for Conglomerate-Forming Chiral Systems.

Kinetically controlled preferential crystallization (PC) is a well-established elegant concept to separate mixtures of enantiomers of conglomerate-forming systems. Based on a smaller number of laboratory investigations, the key parameters of an available shortcut model (SCM) can be estimated, allowing for a rapid and reliable process design. This paper addresses a severe limitation of the method, namely, the limitation of the yield to 50%. In order to exploit the valuable counter enantiomer, the crystallization process is studied, coupled with a racemization reaction and a recycling step. It will be shown that the process integration can be performed in various ways. To quantify the different options in a unified manner and to provide a more general design concept, the SCM of PC is extended to include a kinetic model for the enzymatically catalyzed reaction. For illustration, model parameters are used, which characterize the resolution of the enantiomers of asparagine monohydrate and the racemization rate using an amino acid racemase. The theoretical study highlights the importance of exploiting the best stop time for batch operations in order to achieve the highest process productivity.

Reliability of aspartic acid racemization rate for chronological age estimation-a systematic review and meta-analysis.

The biochemical approach of dental age using aspartic acid racemization has been widely reported. The aim of this systematic review was to determine the reliability and accuracy of dental age estimation using aspartic acid racemization rate analysis. Eight research databases (PubMed, Google Scholar, Embase, Cochrane, MEDLINE, TRIP, Web of Science, and Scopus) were utilised to gather and assess published literatures in compliance with the PRISMA 2020 guidelines and reported in PROSPERO (CRD42020208877). This systematic review and meta-analysis focused solely on cross-sectional studies. The quality evaluation was performed using the GRADE system. The standardized mean difference between estimated and chronological age was meta-analyzed using the random effects model. The literature review yielded 213 studies, of which 26 were considered acceptable for inclusion in this report. Out of 26 studies, 19 presented sufficient evidence for meta-analysis and the remaining 7 were used to construct a qualitative review. According to the meta-analysis, premolar-related studies had the least variability (Tau2 = 0.23; I2 = 72%) among other tooth groups. A substantial degree of heterogeneity was found in every type of tooth. Only the premolar teeth had lower degree variability; thus, it is safe to believe that the premolar tooth is the best for this type of age estimation. It is recommended to develop population-specific mathematical equations to improve the accuracy of this age estimation approach.

Terahertz spectroscopy of temperature-induced transformation between glutamic acid, pyroglutamic acid and racemic pyroglutamic acid

Under heating conditions, L-Glutamic acid (L-Glu) can be dehydrated to form L-pyroglutamic acid (L-PGA), and L-PGA can racemize to form DL-PGA. Here, we characterized this transformation at different temperatures and times by terahertz time domain spectroscopy (THz-TDS). By Powder X-ray diffraction (PXRD), the validity of THz spectroscopy is verified. The results prove that the reaction rate of dehydration and racemization is significantly affected by temperature. The THz spectra divided the reactions into three stages. At 150–155 °C, the reaction changes drastically. Furthermore, we found that the absorption intensity at 0.97 and 1.55 THz has a good dependence on the reaction temperature and time, showing a non-linear relationship (R2 > 0.98). Our findings suggest that the chemical transformation and reaction rate can be sensitively probed by terahertz spectroscopy, which provides a potential method for the quantitative analysis of reaction products.

D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain

d-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examined in mammals. Using chiral high-performance liquid chromatography and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine-binding proteins in NPCs by immunoprecipitation with a d-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine-binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiologic regulator of NPC homeostasis in the developing brain.

Characterization of an Immobilized Amino Acid Racemase for Potential Application in Enantioselective Chromatographic Resolution Processes

Enantioselective resolution processes can be improved by integration of racemization. Applying environmentally friendly enzymatic racemization under mild conditions is in particular attractive. Owing to the variety of enzymes and the progress in enzyme engineering, suitable racemases can be found for many chiral systems. An amino acid racemase (AAR) from P. putida KT2440 is capable of processing a broad spectrum of amino acids at fast conversion rates. The focus of this study is the evaluation of the potential of integrating AAR immobilized on Purolite ECR 8309 to racemize L- or D-methionine (Met) within an enantioselective chromatographic resolution process. Racemization rates were studied for different temperatures, pH values, and fractions of organic co-solvents. The long-term stability of the immobilized enzyme at operating and storage conditions was found to be excellent and recyclability using water with up to 5 vol% ethanol at 20 °C could be demonstrated. Packed as an enzymatic fixed bed reactor, the immobilized AAR can be coupled with different resolution processes; for instance, with chromatography or with preferential crystallization. The performance of coupling it with enantioselective chromatography is estimated quantitatively, exploiting parametrized sub-models. To indicate the large potential of the AAR, racemization rates are finally given for lysine, arginine, serine, glutamine, and asparagine.