Enantiomeric Mixtures Research Articles

5'-Noraristeromycin Repurposing: Well-known S-Adenosyl-L-homocysteine Hydrolase Inhibitor As a Potential Drug Against Leukemia

5ˊ-Noraristeromycin as a racemic mixture of enantiomers was found to exhibit a pronounced cytotoxic effect on leukemia cells; IC50 for the Jurkat, K562, and THP-1 cell lines was 7.3, 1.3, and 3.7 μM, respectively. The general toxicity of 5'-noraristeromycin was studied in experiments on white mice upon single-dose intragastric administration; toxicometric parameters were determined, and the clinical and pathomorphological presentation of acute intoxication was studied. LD50 of the substance was shown to be 63.2 (52.7÷75.8) mg/kg; LD16, 44.7 mg/kg, and LD84, 89.4 mg/kg. Administration of the substance at a dose within the studied dose range is accompanied by systemic damage to the internal organs and tissues of the experimental animals.

(+) Anatoxin-a elicits differential survival, photolocomotor behavior, and gene expression in two alternative vertebrate models

Anatoxin-a is a globally occurring, yet understudied, chiral cyanobacterial toxin that threatens public health and the environment. It has led to numerous dog. livestock and bird poisonings and although it has been studied in rodent models, comparatively little research has occurred in aquatic species. To advance a comparative toxicology understanding of this toxin in alternative vertebrate models, developing zebrafish and fathead minnow were exposed to environmentally relevant and elevated levels (13–4400 μg/L) of (+) anatoxin-a to examine potential mortality and sublethal effects, including photolocomotor behavior and gene expression responses. We observed significantly higher mortality (p < 0.05) in fathead minnows exposed to ≥ 1400 μg/L (65 – 83 % survival versus 97 % in controls). Locomotor response profiles for zebrafish typically displayed hypoactivity after exposure to (+) anatoxin-a in both light and dark periods, while hyperactivity of fathead minnows was observed at the lowest treatment level, but only in light conditions. Gene expression in zebrafish was significantly (p < 0.05) downregulated for mbp, which is associated with myelin sheath formation, and elavl3, which is involved in neurogenesis, along with cyp3a65 and gst, two genes related to phase I and II metabolism. However, no significant (p > 0.05) transcriptional changes were observed in the fathead minnow model. These differential responses between commonly employed species employed as alternative vertebrate models in toxicology research and chemicals risk assessments highlight the need for more comparative studies to understand sensitivities and variations in organismal response. Furthermore, we identified higher mortality, refractory behavioral effects, and gene expression in (+) anatoxin-a exposed fish when compared to previously reported (±) anatoxin-a (racemic 50:50 enantiomer mixture) studies, which is frequently used as a surrogate chemical for experimental work. Our findings identify the importance of understanding species and enantiomer specific effects of natural toxins.

New Active Ingredients for Sustainable Modern Chemical Crop Protection in Agriculture.

Today, the agrochemical industry faces enormous challenges to ensure the sustainable supply of high-quality food, efficient water use, low environmental impact, and the growing world population. The shortage of agrochemicals due to consumer perception, changing needs of farmers and ever-changing regulatory requirements is higher than the number of active ingredients that are placed on the market. The introduction of halogen atoms into an active ingredient molecule offers the opportunity to optimize its physico-chemical properties such as molecular lipophilicity. As early as 2010, around four-fifths of modern agrochemicals on the market contained halogen atoms. In addition, it becomes clear that modern agrochemicals have increasingly complex molecular structures with one or more stereogenic centers in the molecule. Today, almost half of modern agrochemicals are chiral molecules (herbicides, insecticides/acaricides/nematicides ≪ fungicides) and most of them consist of mixtures such as racemic mixtures of enantiomers, followed by mixtures of diastereomers and mixtures of pure enantiomers. Therefore, it is important that halogen-containing substituents or stereogenic centers are considered in the structural optimization of the active ingredients to ultimately develop sustainable agrochemicals in terms of efficacy, ecotoxicology, ease of use and cost-effectiveness.

Four new diarylheptanoids and two new terpenoids from the fruits of Alpinia oxyphylla and their anti-inflammatory activities

Four previously unreported diarylheptanoids (1a/1b-2a/2b), one undescribed sesquiterpenoid (8), one new diterpenoid (12), and twelve known analogs were isolated from the fruits of Alpinia oxyphylla. The structural elucidation of these compounds was achieved through a comprehensive analysis of spectroscopic data, single-crystal X-ray diffraction, electronic circular dichroism (ECD), and modified Mosher's method. Enantiomeric mixtures (1a/1b, 2a/2b, 3a/3b, 4a/4b, and 5a/5b) were separated on a chiral column using acetonitrile-water mixtures as eluents. Among them, compounds 3a/3b and 4a/4b were isolated as optically pure enantiomers in the initial chiral separation. Furthermore, most of the isolates were evaluated for their inhibitory effects against the production of nitric oxide (NO) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Interestingly, 2 and 4 showed significant inhibitory activities against NO production with IC50 values of 33.65 and 9.88 μmol·L-1 (hydrocortisone: IC50 34.26 μmol·L-1), respectively. Additionally, they also partially reduced the secretion of IL-6.

Sustainable solutions for direct TLC enantioseparation with in-home thought-out, prepared/modified chiral stationary phases.

TLC is used globally, yet less attention has been paid to TLC (in enantioseparation) despite its advantages. The present paper describes/reviews successfully practiced direct approaches of 'chiral additive in achiral stationary phase' (as an application of in-home thought out, prepared, tested, and modified chiral stationary phase), 'pre-mixing of chiral reagent with the enantiomeric mixture' (an approach using both achiral phases during chromatographic separation) and 'chiral additive in mobile phase', and chiral ligand exchange for enantioseparation of DL-amino acids, their derivatives, and some active pharmaceutical ingredients. It provided efficient enantioseparation, quantitative determination, and isolation of native forms via in-situ formation of non-covalent diastereomeric pair. The mechanism of enantioseparation in these approaches has been discussed along with the isolation and establishment of the structure of diastereomers. This may help chemists gain useful insights into fields outside their specialization and the experts get brief accounts of recent key developments, providing solutions for sustainable development of less expensive methods for control of enantiomeric purity and isolation of native enantiomers.

Enhancing chiral molecule detection: A weak measurement approach utilizing two-dimensional information

This study addresses the critical need for high purity chiral molecules in biological systems by overcoming the challenges associated with the quantitative detection of chiral molecules and their enantiomeric mixtures. We developed an innovative detection approach that leverages the two-dimensional information gleaned from natural optical rotation (NOR) and Faraday optical rotation (FOR) under magnetic fields in chiral molecules, combined with an ultrahigh-resolution weak measurement sensor. This novel weak measurement system achieves unparalleled accuracy in detecting spin angles, with a precision of 1.86 × 10−5°. Notably, our method introduces no chemical reactions or interference with the substances under test. It offers enhanced discrimination capabilities through the dual-dimensional analysis of both natural and Faraday optical rotation, alongside a simple and compact sensor design. Conclusively, our study introduces a novel, high-precision, and multi-dimensional optical detection paradigm for chiral molecules. By incorporating Faraday rotation in the presence of a magnetic field, we expand the informational dimensionality accessible to the original weak measurement sensor, facilitating the quantitative analysis of chiral molecules and their enantiomers. This breakthrough not only furnishes a novel instrument for the exploration and development of chiral pharmaceuticals but also propels the advancement of weak measurement sensing technology forward.

β-CD@AgNPs with peroxisase-like activity for colorimetric determination of chiral tryptophan

Different enantiomer forms of amino acids play different roles in multifarious fields, and improper use will cause irreversible effects. Therefore, the identification of chiral amino acids is a vital issue in the field of pharmaceutical analysis. Herein, a chiral sensing system of β-cyclodextrin coated silver nanoparticle (β-CD@AgNPs) with peroxidase-like activity was designed for the fast and efficient colorimetric identification of tryptophan (Trp) enantiomers based on the difference in binding capacity between D/L-Trp and β-CD. The results showed the satisfactory linearity for detecting D/L-Trp over the concentration range from 0.2 to 4 mM with a LOD of 0.16 and 0.18 mM, respectively. Moreover, the absorbance increased linearly with the rise of D-Trp concentration percentage in the Trp enantiomer mixture. The proposed method avoided the use of natural enzymes and improved the stability due to the protective effect of cyclodextrin, which provided a new idea for selective colorimetric recognition and detection of D/L-Trp based on cyclodextrin.

De Novo Designed Cell-Penetrating Peptide Self-Assembly Featuring Distinctive Tertiary Structure.

Recent attention has focused on the de novo design of proteins, paralleling advancements in biopharmaceuticals. Achieving protein designs with both structure and function poses a significant challenge, particularly considering the importance of quaternary structures, such as oligomers, in protein function. The cell penetration properties of peptides are of particular interest as they involve the penetration of large molecules into cells. We previously suggested a link between the oligomerization propensity of amphipathic peptides and their cell penetration abilities, yet concrete evidence at cellular-relevant concentrations was lacking due to oligomers' instability. In this study, we sought to characterize oligomerization states using various techniques, including X-ray crystallography, acceptor photobleaching Förster resonance energy transfer (FRET), native mass spectrometry (MS), and differential scanning calorimetry (DSC), while exploring the function related to oligomer status. X-ray crystallography revealed the atomic structures of oligomers formed by LK-3, a bis-disulfide bridged dimer with amino acid sequence LKKLCLKLKKLCKLAG, and its derivatives, highlighting the formation of hexamers, specifically the trimer of dimers, which exhibited a stable hydrophobic core. FRET experiments showed that LK-3 oligomer formation was associated with cell penetration. Native MS confirmed higher-order oligomers of LK-3, while an intriguing finding was the enhanced cell-penetrating capability of a 1:1 mixture of l/d-peptide dimers compared to pure enantiomers. DSC analysis supported the notion that this enantiomeric mixture promotes the formation of functional oligomers, crucial for cell penetration. In conclusion, our study provides direct evidence that amphipathic peptide LK-3 forms oligomers at low nanomolar concentrations, underscoring their significance in cell penetration behavior.

Bionic nanopore recognition receptors for single-molecule enantioselectivity studies of chiral drugs

BackgroundEnantiodiscrimination of chiral drugs is critical for understanding physiological phenomena and ensuring medical safety. Although enantiomers of these drugs share identical physicochemical properties, they exhibit significant differences in pharmacodynamic, pharmacokinetic, and toxicological properties due to the differences in their three-dimensional shapes. Therefore, the development of effective methods for chiral recognition is of great significance and has been a hot topic in chemo/biological studies. ResultsIn this study, we designed a recognition receptor comprising a α-hemolysin (α-HL) nanopore and sulfobutyl ether-β-cyclodextrin (SBEβCD) for identifying the enantiomers of the antidepressant duloxetine at the single-molecule level. Chiral molecules were discriminated based on the different current blockages within the recognition receptor. The results indicated a strong interaction between R-duloxetine and the recognition receptor. By combining the experimental data and molecular docking results, we explored the recognition mechanism of the designed nanopore recognition receptor for chiral drug molecules. It was found that hydrophobic and electrostatic interactions play key roles in chiral recognition. Additionally, by comparing the binding kinetics of enantiomers to cyclodextrins in confined nanospace and bulk solution, we found that enantiomeric identification was better facilitated in the confined nanospace. Finally, the enantiomeric excess (ee) of the enantiomeric duloxetine mixture was measured using this recognized receptor. SignificanceThis strategy has the advantages of low cost, high sensitivity, and no need for additional derivative modifications, providing a new perspective on the development of chiral recognition sensors with excellent enantioselectivity in drug design, pharmaceuticals, and biological applications.

Enantiomeric purity determination of lactofen formulations through Chemometric Deconvolution of partially overlapped chromatographic profiles

Lactofen is a chiral restricted-use herbicide, classified as toxicity class I by the Environmental Protection Agency (EPA), commonly used to control broadleaved weeds. While the observed herbicidal activity mostly derives from the (S)-(+)-enantiomer, formulations have been manufactured as a mixture of both enantiomers. In this work, we present a quantitative method for the enantiomeric determination of lactofen in commercial formulations based on the development of a second-order calibration to assist the selectivity of chiral separation under reversed-phase liquid chromatographic mode coupled to diode array detection. Chromatographic conditions were aimed to minimize analysis time and solvent consumption. This approach is feasible because second-order calibration allows for the quantification of overlapping bands and analytes in the presence of interferences. Chromatographic-spectral matrices were processed by unfolded partial least-squares coupled to residual bilinearization. The proposed methodology was evaluated through validation samples, achieving relative errors of predictions of 2–4 %. Finally, commercial formulations were subjected to minimal sample treatment and analyzed, and figures of merit for real samples were determined. The described methodology results suitable for the determination of enantiomeric purity and quality control in commercial lactofen formulations.

Dimerization of 9-Phenyl-ferroceno[2,3]indenylmethyl Radicals: Electrochemical and Spectroelectrochemical Studies.

We report on three new 9-phenyl-substituted ferroceno[2,3]indenylmethylium dyes 1 + -3 + with electron-donating (OMe, Me) or electron-withdrawing (CF3) substituents. Complexes 1 + -3 + exist as racemic mixtures of Rp and Sp enantiomers. Pyramidalization at the methyl C atom in the precursor carbinol species 1-OH-3-OH or the corresponding one-electron reduced radicals induces a second stereocenter, as the 9-phenyl substituent may reside in an endo or an exo position. Indeed, alcohol 2-OH crystallizes as a racemate of Rp,S and Sp,R isomers. Cationic complexes 1 + -3 + are of deep green color and show intense electronic absorption in the visible. The oxidation and reduction processes are thoroughly investigated by means of cyclic voltammetry and UV/vis/NIR spectroelectrochemistry, the latter showing their electrochromic behavior. T-dependent EPR spectroscopy, EPR spin counting at 20 °C, as well as the UV/vis/NIR spectra of the reduced samples suggest that the one-electron reduced, neutral radicals dimerize nearly quantitatively (≥99.98%). Chemical reduction of 2 + furnished an isomeric mixture of dimeric 2-2. As was shown by cyclic voltammetry and UV/vis/NIR spectroelectrochemistry, the latter dimer redissociates to monomers 2 + upon oxidation, thereby closing a reversible cycle of redox-induced C-C bond making and breaking.

Differences in enantiomeric diffusion can lead to selective chiral amplification

A fundamental question associated with chirality is how mixtures containing equal amounts of interconverting enantiomers can spontaneously convert to systems enriched in only one of them. Enantiomers typically have similar chemical properties, but can exhibit distinct reactivity under specific conditions, and these differences can be used to bias the system's composition in favor of one enantiomer. Transport properties are also expected to differ for enantiomers in chiral solvents, but the role of such differences in chiral symmetry breaking has not been clarified yet. In this work, we develop a theoretical framework to show that asymmetry in diffusion properties can trigger a spontaneous and selective symmetry breaking in mixtures of enantiomers. We derive a generic evolution equation for the enantiomeric excess in a chiral solvent. This equation shows that the relative stability of homochiral domains is dictated by the difference of diffusion coefficients of the two enantiomers. Consequently, deracemization toward a specific enantiomeric excess can be achieved when this difference is large enough. These results hold significant implications for our understanding of chiral symmetry breaking.

Spiral packing and chiral selectivity in model membranes probed by phase-resolved sum-frequency generation microscopy

Since the lipid raft model was developed at the end of the last century, it became clear that the specific molecular arrangements of phospholipid assemblies within a membrane have profound implications in a vast range of physiological functions. Studies of such condensed lipid islands in model systems using fluorescence and Brewster angle microscopies have shown a wide range of sizes and morphologies, with suggestions of substantial in-plane molecular anisotropy and mesoscopic structural chirality. Whilst these variations can significantly alter many membrane properties including its fluidity, permeability and molecular recognition, the details of the in-plane molecular orientations underlying these traits remain largely unknown. Here, we use phase-resolved sum-frequency generation microscopy on model membranes of mixed chirality phospholipid monolayers to fully determine the three-dimensional molecular structure of the constituent micron-scale condensed domains. We find that the domains possess curved molecular directionality with spiralling mesoscopic packing, where both the molecular and spiral turning directions depend on the lipid chirality, but form structures clearly deviating from mirror symmetry for different enantiomeric mixtures. This demonstrates strong enantioselectivity in the domain growth process and indicates fundamental thermodynamic differences between homo- and heterochiral membranes, which may be relevant in the evolution of homochirality in all living organisms.

Bioluminescence of (R)-Cypridina Luciferin with Cypridina Luciferase.

Cypridina luciferin (CypL) is a marine natural product that functions as the luminous substrate for the enzyme Cypridina luciferase (CypLase). CypL has two enantiomers, (R)- and (S)-CypL, due to its one chiral center at the sec-butyl moiety. Previous studies reported that (S)-CypL or racemic CypL with CypLase produced light, but the luminescence of (R)-CypL with CypLase has not been investigated. Here, we examined the luminescence of (R)-CypL, which had undergone chiral separation from the enantiomeric mixture, with a recombinant CypLase. Our luminescence measurements demonstrated that (R)-CypL with CypLase produced light, indicating that (R)-CypL must be considered as the luminous substrate for CypLase, as in the case of (S)-CypL, rather than a competitive inhibitor for CypLase. Additionally, we found that the maximum luminescence intensity from the reaction of (R)-CypL with CypLase was approximately 10 fold lower than that of (S)-CypL with CypLase, but our kinetic analysis of CypLase showed that the Km value of CypLase for (R)-CypL was approximately 3 fold lower than that for (S)-CypL. Furthermore, the chiral high-performance liquid chromatography (HPLC) analysis of the reaction mixture of racemic CypL with CypLase showed that (R)-CypL was consumed more slowly than (S)-CypL. These results indicate that the turnover rate of CypLase for (R)-CypL was lower than that for (S)-CypL, which caused the less efficient luminescence of (R)-CypL with CypLase.

Tunable plasmonic superchiral light for ultrasensitive detection of chiral molecules.

The accurate detection, classification, and separation of chiral molecules are pivotal for advancing pharmaceutical and biomolecular innovations. Engineered chiral light presents a promising avenue to enhance the interaction between light and matter, offering a noninvasive, high-resolution, and cost-effective method for distinguishing enantiomers. Here, we present a nanostructured platform for surface-enhanced infrared absorption-induced vibrational circular dichroism (VCD) based on an achiral plasmonic system. This platform enables precise measurement, differentiation, and quantification of enantiomeric mixtures, including concentration and enantiomeric excess determination. Our experimental results exhibit a 13 orders of magnitude higher detection sensitivity for chiral enantiomers compared to conventional VCD spectroscopic techniques, accounting for respective path lengths and concentrations. The tunable spectral characteristics of this achiral plasmonic system facilitate the detection of a diverse range of chiral compounds. The platform's simplicity, tunability, and exceptional sensitivity holds remarkable potential for enantiomer classification in drug design, pharmaceuticals, and biological applications.

Identification of D- and L-phenylalanine enantiomeric mixtures by employing deep neural network models

Phenylalanine is an aromatic essential amino acid that exhibits the tendency to self-aggregate into fibrillar structures in its enantiomerically pure form. This observation was indicated as the underlying mechanism of phenylketonuria, which is a genetic condition associated with various neurological, physical, and developmental issues, characterized with phenylalanine buildup in the brain. The presence of D-phenylalanine was demonstrated previously to inhibit the formation of fibrils by L-phenlyalanine, indicating its potential use in phenylketonuria treatment. In this study, several combinations of D and L-phenylalanine were examined with the help of state-of-the-art deep learning methods for their fibril forming capacity, demonstrating the usefulness and accuracy of deep learning methods in distinguishing between different self-assembled structures.

An enantioselective study of the behavior of the herbicide ethofumesate in agricultural soils: Impact of the addition of organoclays and biochar

Chiral pesticides that are still commercialized and incorporated into the environment as racemic mixtures of enantiomers require evaluation of the enantioselectivity of their biological activity and environmental fate processes for a better prediction of their field efficacy and environmental risks. In this work, we successfully separated the enantiomers of the chiral herbicide ethofumesate (ETFM), determined their absolute configuration, and characterized their herbicidal activity as well as their adsorption, degradation, enantiomerization, and leaching in Mediterranean agricultural soils. While the herbicidal activity of R-ethofumesate to the sensitive species Portulaca grandiflora was greater than that of S-ethofumesate, the adsorption, degradation, and leaching of the herbicide showed negligible enantioselectivity and enantiomer interconversion did not occur in soils. The adsorption of both enantiomers showed a positive correlation with the soil organic carbon content (r = 0.856, P = 0.015), and their degradation in soils occurred slowly (DT50 > 60 days) and at similar rates independent of their application as individual enantiomers or as a racemic mixture of enantiomers. The addition of three highly adsorptive materials to a scarcely adsorptive soil increased the adsorption of the enantiomers of ETFM and delayed their degradation without affecting the non-enantioselective character of the processes. As a result of their high adsorption capacity, the materials were highly effective in reducing the leaching of both enantiomers of ETFM through soil columns. The results of this work indicate that the application of single-enantiomer ETFM formulations, based on a higher herbicidal activity or a lower toxicity to non-target organisms of the formulated enantiomer, would reduce considerable exposure risks associated with incorporating into the environment the less favorable enantiomer, as this would show long persistence and high leaching potential in soils similar to its optical isomer.

Altering the mobile phase composition to enhance self-disproportionation of enantiomers in achiral chromatography

The influence of mobile phase composition on the efficiency of enantiomer separation by achiral chromatography (ACh) was investigated. The separation was induced by the phenomenon of self-disproportionation of enantiomers (SDE) triggered by their homo and hetero-chiral interactions in an achiral environment. Typically, SDE occurs in apolar mobile phases of weak elution strength, which causes the separation time to extend and the process productivity to deteriorate. To mitigate that effect, we altered the content of a strong solvent (modifier) in the mobile phase by use of a solvent gradient in which the target enantiomer was separated in the presence of the weak solvent, whereas the unresolved mixture of enantiomers was eluted by increasing the modifier content in the mobile phase. This enabled accelerating the solute elution while preserving the separation selectivity. The approach was examined for the separation of nonracemic mixtures of two structurally different compounds that exhibited the SDE effect in ACh, i.e., metalaxyl (MX) and methyl p-tolyl sulfoxide (MTSO). The target compound of the separation was the more abundant enantiomer in the enantiomeric mixture. The process realization was preceded by the determination of the effect of the modifier content on the separation yield for enantiomeric mixtures of MX and MTSO of different enantiomeric excess (ee). In the case of MX, yield of the pure target enantiomer varied from 2 %, for the maximum concentration of the modifier, to 45 % for the minimum modifier concentration and the largest ee used in the experiments. In the case of MTSO, the yield varied from minimum 40 % to maximum 66 %. To predict the process, we employed a dynamic model, in which underlying thermodynamic dependencies were implemented.

Determining enantiomeric excess utilizing ratiometric measurements of a deuterium labeled malonate by 1H NMR spectroscopy

Enantiomeric excess quantification is important in the synthesis of many small molecules. Esterase enzymes such as porcine liver esterase are known for their ability to generate high enantioselectivity. Many methods have been developed in order to measure the enantiomeric excess of a chiral mixture. Herein we report a new method of direct quantification by 1H NMR spectroscopy of an enantiomeric mixture using ratiometric measurements of an isotope labelled probe. Our method has been demonstrated to be comparable to that of HPLC and yield quantitative results with ease.

Two pairs of 2-pyrone enantiomers and a benzophenone analogue from the endophytic fungus Penicillium egyptiacum

Four new 2-pyrone derivatives, two pairs of enantiomers, (±)-egypyrone A [(±)-1] and (±)-egypyrone B [(±)-2], together with a new benzophenone analogue, orbiophenone B (3), were isolated from the endophytic fungus Penicillium egyptiacum. The enantiomeric mixtures (±)-1 and (±)-2 were separated through chiral HPLC, respectively. Their structures were elucidated by extensive analysis of spectroscopic data and the absolute configuration was determined by comparing the optical rotation of structurally similar molecule. Subsequently, the cytotoxic activities of (±)-1, (±)-2, and 3 against the U87 cell line were tested and no activity was observed at a concentration of 10 µM.