HPLC with chiral stationary phase for separation and kinetics study of aspartic acid epimerization in Peroxiredoxin 2 active site peptide.

Amino acid epimerization, a process of converting L-amino acids to D-amino acids, will lead to modification in the protein structure and, subsequently, its biological function. This modification causes no change in protein m/z and may be overlooked during protein analysis. Aspartic Acid Epimerization (AAE) is faster than other amino acids and could be accelerated by free radicals and peroxides. In this work, a novel and site-specific HPLC method using a chiral stationary phase for determining the AAE in the active site model peptide (AP) of Peroxiredoxin 2 has been developed and validated. The developed method showed good linearity (1 - 200 µg/mL) and recoveries of the limit of quantification (LOQ), low, medium, and high concentrations were between 85% and 115%. The Kinetics of AAE in AP were studied using the developed method, and the results showed that when ascorbic acid and Cu2+ coexisted, the AP epimerized rapidly. The AAE extent increased with time and was positively correlated with hydrogen peroxide generation.

Isolation and characterization of seven neovibsane-type diterpenoids from Viburnum odoratissimum and their neuroblastoma cell protective effects.

Seven undescribed neovibsane-type diterpenoids (1-7) were isolated from the leaves of Viburnum odoratissimum. Their planar structures and relative configurations were elucidated based on a combination of 1D and 2D NMR analysis. The absolute configurations were confirmed by Rh2(OCOCF3)4-induced ECD analysis and comparison of experimental and TDDFT-calculated ECD spectrum. Based on the empirical results of the ECD of in situ formed Rh-complexes, rapid determination of the absolute configuration of C-14 within vibsane-type diterpenoids was proposed. In addition, 3 exhibited a high neuroblastoma cell protective effect of 81.8 % at 50 µM (the control group showed a neuroblastoma cell protective effect of 56.2 % at 50 µM).

Separation and simultaneous estimation of enantiomers and Diastereomers of muscarinic receptor antagonist Solifenacin using stability-indicating Normal-phase HPLC technique with chiral stationary phase amylose tris-(3,5-dimethylphenylcarbamate).

The R,S-enantiomer impurity and diastereomer impurities (S,S-isomer and R,R-isomer) of the solifenacin (S,R-enantiomer) were effectively separated and quantified simultaneously utilizing normal-phase high-performance liquid chromatography with a chiral stationary phase consisting of amylose tris (3,5-dimethylphenylcarbamate) coated on silica-gel (Chiralpak, AD-H). The enantiomeric and stereo-selective separation was achieved within a run time of 35 minutes using a mobile phase of 'n-hexane, ethanol, and diethylamine' in an isocratic elution mode with a detection wavelength of 220 nm. The validation attributes assessed were accuracy (which showed excellent recoveries between 97.5% and 100.4%) and linearity (which was proven in the range of 0.081-1.275 µg.mL-1 , with a linear regression of 0.999). The stress testing experiments proved that the developed methodology by the HPLC technique has stability-indicating characteristics, as all closely eluting peak pairs were separated well with a resolution of 2.3 and without any interference. The proposed methodology was highly efficient in separating and simultaneously determining the chiral impurities (enantiomers and diastereomers) of the solifenacin in the release and stability sample analyses of drug substances and tablets in pharmaceutical formulations.

Synthesis of Novel Phosphorus-Containing Derivatives of 1,3,4-Trimethylglycoluril via the Birum-Oleksyszyn Reaction.

This work presents the synthesis of a new compound, 1-[aryl-(diphenylphosphono)methyl]-3,4,6-trimethylglycolurils, via the interaction of benzaldehyde and its mononitro- and monohydroxyderivatives with 1,3,4-trimethylglycoluril and triphenylphosphite. By varying the reaction conditions and the catalysts, the obtained product yields ranged from satisfactory to good. The diastereomers formed during the reaction were separated by semipreparative HPLC on the C18 stationary phase. The isolated diastereomers were characterized by 1H, 13C, and 31P NMR, and the structures of the diastereomers were confirmed using a single-crystal X-ray crystal structure analysis and quantum chemical calculations.

Unraveling the Effect of Stereoisomerism on Mobility-Stretchability Properties of n-Type Semiconducting Polymers with Biobased Epimers as Conjugation Break Spacers.

The development of intrinsically stretchable n-type semiconducting polymers has garnered much interest in recent years. In this study, three biobased dianhydrohexitol epimers of isosorbide (ISB), isomannide (IMN), and isoidide (IID), derived from cellulose, were incorporated into the backbone of a naphthalenediimide (NDI)-based n-type semiconducting polymer as conjugation break spacers (CBSs). Accordingly, three polymers were synthesized through the Migita-Kosugi-Stille coupling polymerization with NDI, bithiophene, and CBSs, and the mobility-stretchability properties of these polymers were investigated and compared with those of their analogues with conventional alkyl-based CBSs. Experimental results showed that the different configurations of these epimers in CBSs sufficiently modulate the melt entropies, surface aggregation, crystallographic parameters, chain entanglements, and mobility-stretchability properties. Comparable ductility and edge-on preferred stacking were observed in polymers with endo- or exo-configurations in IMN- and IID-based polymers. By contrast, ISB with endo-/exo-configurations exhibits an excellent chain-realigning capability, a reduced crack density, and a proceeding bimodal orientation under tensile strain. Therefore, the ISB-based polymer exhibits high orthogonal electron mobility retention of (53 and 56)% at 100% strain. This study is one of the few examples where biobased moieties are incorporated into semiconducting polymers as stress-relaxation units. Additionally, this is the first study to report on the effect of stereoisomerism of epimers on the morphology and mobility-stretchability properties of semiconducting polymers.

The differentiation of N-butyl pentylone isomers using GC-EI-MS and NMR.

Forensic laboratories are faced with an ever-expanding seized drug landscape including the increasing prevalence of novel psychoactive substances (NPS), such as synthetic cathinones, that have varying potencies and scheduling. This study demonstrates a combined gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) and nuclear magnetic resonance (NMR) spectroscopy approach for the differentiation of N-butyl pentylone isomers based on distinct retention times, characteristic EI mass spectra, and NMR characterization. Retention time reproducibility was assessed from 60 replicate measurements for each isomer over the course of a month. In addition, the effect of the mass spectrometer tune and the stability of an identified characteristic ion ratio using spectral data from ± 1 scan on either side of the peak apex were also statistically assessed using Welch's ANOVA testing. The presence of diastereomers for N-sec-butyl pentylone was identified using the developed GC-EI-MS method, which was confirmed using one-dimensional and two-dimensional NMR spectroscopy. The retention time reproducibility of the chromatographic method was ± 0.076% or less over the course of a month. An identified characteristic ion ratio between the abundance of the fragment ion at m/z 128 and the fragment ion at m/z 72 enabled the differentiation of the four N-butyl pentylone isomers, even when accounting for the effect of the mass spectrometer tune and mass spectral scans used to calculate the characteristic ion ratio. The 95% confidence interval mean abundance ratio of the fragment ions at m/z 128 and m/z 72 was 17.14 ± 0.14 for N-butyl pentylone, 6.44 ± 0.05 for N-isobutyl pentylone, 3.38 ± 0.02 for N-sec-butyl pentylone, and 0.75 ± 0.01 for N-tert-butyl pentylone. These results highlight the capabilities of a combined GC-EI-MS and NMR approach for the differentiation and characterization of synthetic cathinone isomers.

Characterization of antisense oligonucleotide and guide ribonucleic acid diastereomers by hydrophilic interaction liquid chromatography coupled to mass spectrometry.

Oligonucleotides have become an essential modality for a variety of therapeutic approaches, including cell and gene therapies. Rapid progress in the field has attracted significant research in designing novel oligonucleotide chemistries and structures. Beyond their polar nature, the length of large RNAs and presence of numerous diastereomers for phosphorothioate (PS)-modified RNAs pose heightened challenges for their characterization. In this study, the stereochemistry of a fully-modified antisense oligonucleotide (ASO) and partially-modified guide RNAs (gRNAs) was investigated using HILIC and orthogonal techniques. The profiles of three lots of a fully-modified ASO with PS linkages were compared using ion-pairing RPLC (IPRP) and HILIC. Interestingly, three isomer peaks were partially resolved by HILIC for two lots while only one peak was observed on the IPRP profile. Model oligonucleotides having the same sequence of the five nucleotides incorporated to the 3'-end of the gRNA but differing in their number and position of PS linkages were investigated by HILIC, IPRP, ion mobility spectrometry-mass spectrometry (IM-MS) and nuclear magnetic resonance (NMR). An strategy was ultimately designed to aid in the characterization of gRNA stereochemistry. Ribonuclease (RNase) T1 digestion enabled the characterization of gRNA diastereomers by reducing their number from 32 at the gRNA intact level to 4 or 8 at the fragment level. To our knowledge, this is the first time that HILIC has successfully been utilized for the profiling of diastereomers for various oligonucleotide formats and chemical modifications.

Impact of Non-Covalent Interactions of Chiral Linked Systems in Solution on Photoinduced Electron Transfer Efficiency.

It is well-known that non-covalent interactions play an essential role in the functioning of biomolecules in living organisms. The significant attention of researchers is focused on the mechanisms of associates formation and the role of the chiral configuration of proteins, peptides, and amino acids in the association. We have recently demonstrated the unique sensitivity of chemically induced dynamic nuclear polarization (CIDNP) formed in photoinduced electron transfer (PET) in chiral donor-acceptor dyads to non-covalent interactions of its diastereomers in solutions. The present study further develops the approach for quantitatively analyzing the factors that determine the association by examples of dimerization of the diastereomers with the RS, SR, and SS optical configurations. It has been shown that, under the UV irradiation of dyads, CIDNP is formed in associates, namely, homodimers (SS-SS), (SR-SR), and heterodimers (SS-SR) of diastereomers. In particular, the efficiency of PET in homo-, heterodimers, and monomers of dyads completely determines the forms of dependences of the CIDNP enhancement coefficient ratio of SS and RS, SR configurations on the ratio of diastereomer concentrations. We expect that the use of such a correlation can be useful in identifying small-sized associates in peptides, which is still a problem.

Discovery of a Potent Highly Biased MOR Partial Agonist among Diastereomeric C9-Hydroxyalkyl-5-phenylmorphans.

All possible diastereomeric C9-hydroxymethyl-, hydroxyethyl-, and hydroxypropyl-substituted 5-phenylmorphans were synthesized to explore the three-dimensional space around the C9 substituent in our search for potent MOR partial agonists. These compounds were designed to lessen the lipophilicity observed with their C9-alkenyl substituted relatives. Many of the 12 diastereomers that were obtained were found to have nanomolar or subnanomolar potency in the forskolin-induced cAMP accumulation assay. Almost all these potent compounds were fully efficacious, and three of those chosen for in vivo evaluation, 15, 21, and 36, were all extremely G-protein biased; none of the three compounds recruited beta-arrestin2. Only one of the 12 diastereomers, 21 (3-((1S,5R,9R)-9-(2-hydroxyethyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), was a MOR partial agonist with good, but not full, efficacy (Emax = 85%) and subnanomolar potency (EC50 = 0.91 nM) in the cAMP assay. It did not have any KOR agonist activity. This compound was unlike morphine in that it had a limited ventilatory effect in vivo. The activity of 21 could be related to one or more of three well-known theories that attempt to predict a dissociation of the desired analgesia from the undesirable opioid-like side-effects associated with clinically used opioids. In accordance with the theories, 21 was a potent MOR partial agonist, it was highly G-protein biased and did not attract beta-arrestin2, and it was found to have both MOR and DOR agonist activity. All the other diastereomers that were synthesized were either much less potent than 21 or had either too little or too much efficacy for our purposes. It was also noted that a C9-methoxymethyl compound with 1R,5S,9R stereochemistry (41) was more potent than the comparable C9-hydroxymethyl compound 11 (EC50 = 0.65 nM for 41 vs. 2.05 nM for 11). Both 41 and 11 were fully efficacious.

Separation of flavonoid isomers by cyclic ion mobility mass spectrometry.

Analytical techniques, such as liquid chromatography coupled to mass spectrometry (LC-MS) or nuclear magnetic resonance (NMR), are widely used for characterization of complex mixtures of (isomeric) proteins, carbohydrates, lipids, and phytochemicals in food. Food can contain isomers that are challenging to separate, but can possess different reactivity and bioactivity. Catechins are the main phenolic compounds in tea; they can be present as various stereoisomers, which differ in their chemical properties. Currently, there is a lack of fast and direct methods to monitor interconversion and individual reactivity of these epimers (e.g. epicatechin (EC) and catechin (C)). In this study, cyclic ion mobility mass spectrometry (cIMS-MS) was explored as a potential tool for the separation of catechin epimers. Formation of sodium and lithium adducts enhanced IMS separation of catechin epimers, compared to deprotonation and protonation. Baseline separation of the sodium adducts of catechin epimers was achieved. Moreover, we developed a fast method for the identification and semi-quantification of cIMS-MS separated catechin epimers. With this method, it is possible to semi-quantify the ratio between EC and C (1:5 to 5:1, within 50-1200 ng mL-1) in food samples, such as tea. Finally, the newly developed approach for cIMS-MS separation of flavonoids was demonstrated to be successful in separation of two sets of positional isomers (i.e. morin, tricetin, and quercetin; and kaempferol, fisetin, luteolin, and scutellarein). To conclude, we showed that both epimers and positional isomers of flavonoids can be separated using cIMS-MS, and established the potential of this method for challenging flavonoid separations.

Diastereomeric multi-chiral pendant groups: Their key role in stimuli-responsive polymeric responses.

Chiral information transmission in helical polymers bearing multi-chiral pendant groups is usually determined by the absolute configuration of the first chiral center. The second chiral residue usually has low-to-null influence in the macromolecular handedness of the polymer, due to its remote position respect to the polyene main chain. Here, we demonstrate how the stimuli responsive properties of diastereomeric polymers, obtained by changing the absolute configuration of the second chiral center, are different due to the unlike properties of diastereoisomers.

Effect of relative configuration of TEMPO-type nitroxides on ascorbate reduction.

2,2,6,6-Tetramethylpiperidin-N-oxyl (TEMPO)-type nitroxides are susceptible to bioreduction, leading to a loss of radical properties. Although it has been reported that the steric and electronic environments around the N-O moiety of nitroxides affect the reduction, how the relative configuration of nitroxide derivatives alters it is unclear. In this study, we investigated the effect of diastereomers on the radical properties of C2- and C4-disubstituted TEMPO-type nitroxides. We succeeded in isolating the diastereomers of the studied nitroxides for the first time. In addition, we compared the reactivities of nitroxide derivatives with different substituents at the C2 and C4 positions toward ascorbate reduction. We found that the bulky substituents at both C2 and C4 and the electronic effect of C4 affected the reduction of the isomers. C2- and C4-disubstituted nitroxides were administered to mice for electron spin resonance imaging to assess bioreduction in the brain. Similar to the reactivity to reduction in vitro, a difference in the bioreduction of diastereomers was observed in brain tissues. Our research strongly indicates that bioreduction can be controlled by changing the relative configuration, which can be used in the design of nitroxide derivatives for biological applications.

Drug testing for mitragynine and kratom: Analytical challenges and medico-legal considerations.

Mitragyna speciosa, known as kratom, is a tropical tree native to Southeast Asia that has long been used to increase energy and in traditional medicine. Kratom leaves contain several indole alkaloids including mitragynine, mitraciliatine, speciogynine, and speciociliatine, which have the same molecular formula and connectivity, but different spatial arrangements (i.e., diastereomers). A routine liquid-chromatographic-high-resolution mass-spectrometric (LC-HRMS) multi-analyte method for addictive and herbal drugs in urine did not separate mitragynine from speciogynine and speciociliatine. Separation and individual measurement of the four diastereomers was possible with an improved LC method. All diastereomers were detected in 29 patient urine samples who tested positive for mitragynine with the routine method, albeit at variable absolute amounts and relative proportions. The presence of all diastereomers rather than individual substances indicated that they originated from the intake of kratom (i.e., plant material). Speciociliatine dominated in most samples (66%), whereas mitragynine and mitraciliatine were the highest in 17% each. A kratom product (powdered plant material) marketed in Sweden contained all diastereomers with mitragynine showing the highest level. In Sweden, there are signs of an increasing use of kratom in society, based on the results from drug testing, the number of poisons center consultations on intoxications, and customs seizure statistics. Because there may be health risks associated with kratom use, including dependence, serious adverse reactions, and death, analytical methods should be able to identify and quantify all diastereomers. In Sweden, this is important from a legal perspective, as only mitragynine is classified, whereas the other three diastereomers, and kratom (plant material), are not.

Two new ent-kaurane-type diterpene diastereomers isolated from Coffea canephora.

Phytochemical investigation of the trunks of Coffea canephora yielded two new ent-kaurane diterpene diastereomers, which have been named coffecanepholide A, ent-3ß,16ß,17-trihydroxykauran-18-al (1) and coffecanepholide B, ent-3ß,16ß,17-trihydroxykauran-19-al (2). Structural elucidation and configurational assignment were deduced from extensive spectroscopic NMR/HRESIMS analysis and by comparison with the spectral data of the literature relevant structures. The isolated compounds were assayed for in vitro inhibitory activities against α-glucosidase. Structure 2 showed the α-glucosidase inhibitory activity with an IC50 value of 294.7 ± 0.9 µM, while compound 1 exhibited inactivity. In addition, the docking results revealed that structure 2 can form more interactions with amino acid residues at the active site of α-glucosidase, which gave a more negative binding energy (-9.56 kcal/mol) compared with 1 (-8.60 kcal/mol). This observation might be responsible for a better activity of 2 against α-glucosidase.

Computational insights into the stereo-selectivity of catechins for the inhibition of the cancer therapeutic target EGFR kinase.

The epidermal growth factor receptor (EGFR) plays a crucial role in regulating cellular growth and survival, and its dysregulation is implicated in various cancers, making it a prime target for cancer therapy. Natural compounds known as catechins have garnered attention as promising anticancer agents. These compounds exert their anticancer effects through diverse mechanisms, primarily by inhibiting receptor tyrosine kinases (RTKs), a protein family that includes the notable member EGFR. Catechins, characterized by two chiral centers and stereoisomerism, demonstrate variations in chemical and physical properties due to differences in the spatial orientation of atoms. Although previous studies have explored the membrane fluidity effects and transport across cellular membranes, the stereo-selectivity of catechins concerning EGFR kinase inhibition remains unexplored. In this study, we investigated the stereo-selectivity of catechins in inhibiting EGFR kinase, both in its wild-type and in the prevalent L858R mutant. Computational analyses indicated that all stereoisomers, including the extensively studied catechin (-)-EGCG, effectively bound within the ATP-binding site, potentially inhibiting EGFR kinase activity. Notably, gallated catechins emerged as superior EGFR inhibitors to their non-gallated counterparts, revealing intriguing binding trends. The top four stereoisomers exhibiting high dock scores and binding energies with wild-type EGFR comprise (-)-CG (-)-GCG (+)-CG, and (-)-EGCG. To assess dynamic behavior and stability, molecular dynamics simulations over 100 ns were conducted for the top-ranked catechin (-)-CG and the widely investigated catechin (-)-EGCG with EGFR kinase. This study enhances our understanding of how the stereoisomeric nature of a drug influences inhibitory potential, providing insights that could guide the selection of specific stereoisomers for improved efficacy inexisting drugs.

Screening of Novel Antimicrobial Diastereomers of Azithromycin-Thiosemicarbazone Conjugates: A Combined LC-SPE/Cryo NMR, MS/MS and Molecular Modeling Approach.

A well-known class of antibacterials, 14- and 15-membered macrolides are widely prescribed to treat upper and lower respiratory tract infections. Azithromycin is a 15-membered macrolide antibiotic possessing a broad spectrum of antibacterial potency and favorable pharmacokinetics. Bacterial resistance to marketed antibiotics is growing rapidly and represents one of the major global hazards to human health. Today, there is a high need for discovery of new anti-infective agents to combat resistance. Recently discovered conjugates of azithromycin and thiosemicarbazones, the macrozones, represent one such class that exhibits promising activities against resistant pathogens. In this paper, we employed an approach which combined LC-SPE/cryo NMR, MS/MS and molecular modeling for rapid separation, identification and characterization of bioactive macrozones and their diastereomers. Multitrapping of the chromatographic peaks on SPE cartridges enabled sufficient sample quantities for structure elucidation and biological testing. Furthermore, two-dimensional NOESY NMR data and molecular dynamics simulations revealed stereogenic centers with inversion of chirality. Differences in biological activities among diastereomers were detected. These results should be considered in the process of designing new macrolide compounds with bioactivity. We have shown that this methodology can be used for a fast screening and identification of the macrolide reaction components, including stereoisomers, which can serve as a source of new antibacterials.

Synthesis and Antioxidative Properties of 1,2,3,4-Tetrahydropyridine Derivatives with Different Substituents in 4-Position.

Natural products are an excellent source of inspiration for the development of new drugs. Among them, betalains have been extensively studied for their antioxidant properties and potential application as natural food dyes. Herein, we describe the seven-step synthesis of new betalamic acid analogs without carboxy groups in the 2- and 6-position with an overall yield of ~70%. The Folin-Ciocalteu assay was used to determine the antioxidant properties of protected intermediate 21. Additionally, the five-step synthesis of betalamic acid analog 35 with three ester moieties was performed. Using NMR techniques, the stability of the obtained compounds towards oxygen was analyzed.

Total Syntheses of the Proposed Structure of Iriomoteolide-1a, -1b and Synthesis of Three Derivatives for Structural Studies.

Iriomoteolide-1a and iriomoteolide-1b are very potent cytotoxic agents, isolated from marine dinoflagellates. We carried out the enantioselective syntheses of the proposed structures of these natural products. However, our analysis of the NMR spectra of the synthetic iriomoteolide-1a and the natural products revealed that the structures of iriomoteolide-1a and iriomoteolide-1b were assigned incorrectly. Based upon our detailed analysis of the spectral data of the synthetic iriomoteolide-1a and the natural products, we rationally designed three diastereomers of the proposed structure of 1 in an effort to assign the correct structures. The key steps of our syntheses of the proposed structures of iriomoteolides involved a highly diastereoselective ene reaction, a carbocupration that utilized a Gilman reagent, a Julia-Kocienski olefination to couple fragments, and Yamaguchi macrolactonization to form the target macrolactone. This synthetic route was then utilized to carry out syntheses of three diastereomers to the proposed structure of 1. These diastereomeric structures show close similarities to natural iriomoteolide-1a; however, there were differences in their spectral data. While natural iriomoteolides exhibited potent cytotoxicies, our preliminary biological evaluation of synthetic iriomoteolide-1a, iriomoteolide-1b, and all three synthetic derivatives did not show any appreciable cytotoxic properties.

A High-Affinity 64Cu-Labeled Ligand for PET Imaging of Hepsin: Design, Synthesis, and Characterization.

Hepsin, a cell surface serine protease, is a potential biomarker for the detection of prostate cancer due to its high expression in prostate cancer but not in normal prostate. This study aimed to develop a radioligand for positron emission tomography (PET) imaging of hepsin. Six leucine-arginine (Leu-Arg) dipeptide derivatives (two diastereomers for each of three ligands) were synthesized and evaluated for their binding affinities and selectivity for hepsin. Based on the binding assay, a natCu-1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA)-conjugated ligand (3B) was selected for the development of a PET radioligand. [64Cu]3B was synthesized by labeling the DOTA-conjugated compound 11B with [64Cu]CuCl2 at 80 °C for 20 min. The radioligand was evaluated for prostate cancer cell binding and PET imaging in a prostate tumor mouse model. The results demonstrated that [64Cu]3B exhibited high binding to LNCaP cells, intermediate binding to 22Rv1 cells, and low binding to PC3 cells. PET studies of [64Cu]3B in mice, implanted with 22Rv1 and PC3 cells on each flank, revealed that the radioligand uptake was high and persistent in the 22Rv1 tumors over time, whereas it was low in PC3 tumors. The results of this study suggest that [64Cu]3B is a promising PET radioligand for hepsin imaging.

Synthesis and separation of diastereomeric N-(1-phenylethyl)amides of inherently chiral hydroxydibenzoyloxy-calix[4]arene acetic acids.

A preparative method for the synthesis of optically pure N-(1-phenylethyl)amides of inherently chiral (cR)- and (cS)-dibenzoyloxy-calix[4]arene acetic acids has been developed. Their absolute stereochemical configuration was determined by X-ray diffraction analysis.