Determination of N-centered stereochemistry in N22-methylated chlorophyll-a derivatives and their epimer-dependent optical spectra.

An N-centered epimeric mixture of chlorophyll-a derivatives methylated at the inner nitrogen atom was separated by reverse-phase high-performance liquid chromatography. Circular dichroism (CD) spectroscopic analyses of the epimerically pure N22-methyl-chlorins revealed that the minor first-eluted and major second-eluted stereoisomers were (22S)- and (22R)-configurations, respectively. Their visible absorption and CD spectra in solution were dependent on the N22-stereochemistry. The epimer-dependent spectral changes were independent of the substituents at the peripheral 3-position of the core chlorin chromophore.

Two-dimensional sequential selective comprehensive chiral×reversed-phase liquid chromatography of synthetic phosphorothioate oligonucleotide diastereomers.

In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of Rp- and Sp-diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.

Influence of oligonucleotides structures for separation of diastereomers by capillary electrophoresis method using polyvinylpyrrolidone 1,300,000.

In the field of oligonucleotides drug discovery, phosphorothioate (PS) modification has been recognized as an effective tool to overcome the nuclease digestion, and generates 2n of possible diastereomers, where n equals the number of PS linkages. However, it is also well known that differences in drug efficacy and toxicity are caused by differences in stereochemistry of oligonucleotides. Therefore, the development of a high-resolution analytical method that enables stereo discrimination of oligonucleotides is desired. Under this circumstance, capillary electrophoresis (CE) using polyvinylpyrrolidone (PVP) is considered as one of the useful tools for the separation analysis of diastereomers. In this study, we evaluated the several oligonucleotides with the structural diversities in order to understand the separation mechanism of the diastereomers by CE. Especially, five kinds of 2'-moieties were deeply examined by CE with PVP 1,300,000 polymer solution. We found that different trend of the peak shapes and the peak resolution were observed among these oligonucleotides. For example, the better peak resolution was observed in 6 mer PS3-DNA compared to the rigid structure of 6 mer PS3-LNA. As for this reason, the computational simulation revealed that difference of accessible surface area caused by the steric structure of thiophosphate in each oligonucleotide is one of the key attributes to explain the separation of the diastereomers. In addition, we achieved the separation of sixteen peak tops of the diastereomers in 6 mer PS4-DNA, and the complete separation of fifteen diastereomers in 6 mer PS4-RNA. These knowledge for the separation of the diastereomers by CE will be expected to the quality control of the oligonucleotide drugs.

Separation of the diastereomers of phosphorothioated siRNAs by anion-exchange chromatography under non-denaturing conditions.

In recent years, several small interfering RNA (siRNA) therapeutics have been approved, and most of them are phosphorothioate (PS)-modified for improving nuclease resistance. This chemical modification induces chirality in the phosphorus atom, leading to the formation of diastereomers. Recent studies have revealed that Sp and Rp configurations of PS modifications of siRNAs have different biological properties, such as nuclease resistance and RNA-induced silencing complex (RISC) loading. These results highlight the importance of determining diastereomeric distribution in quality control. Although various analytical approaches have been used to separate diastereomers (mainly single-stranded oligonucleotides), it becomes more difficult to separate all of them as the number of PS modifications increases. Despite siRNA exhibits efficacy in the double-stranded form, few reports have examined the separation of diastereomers in the double-stranded form. In this study, we investigated the applicability of non-denaturing anion-exchange chromatography (AEX) for the separation of PS-modified siRNA diastereomers. Separation of the four isomers of the two PS bonds tended to improve in the double-stranded form compared to the single-stranded form. In addition, the effects of the analytical conditions and PS-modified position on the separation were evaluated. Moreover, the elution order of the Sp and Rp configurations was confirmed, and the steric difference between them, i.e., the direction of the anionic sulfur atom, appeared to be important for the separation mechanism in non-denaturing AEX. Consequently, all 16 peak tops of the four PS modifications were detected in one sequence, and approximately 30 peak tops were detected out of 64 isomers of six PS bonds, indicating that non-denaturing AEX is a useful technique for the quality control of PS-modified siRNA therapeutics.

Non-enzymatic reactions in biogenesis of fungal natural products.

Fungi have long been regarded as abundant sources of natural products (NPs) exhibiting significant biological activities. Decades of studies on the biosynthesis of fungal NPs revealed that most of the biosynthetic steps are catalyzed by sophisticated enzymes encoded in biosynthetic gene clusters, whereas some reactions proceed without enzymes. These non-enzymatic reactions complicate biosynthetic analysis of NPs and play important roles in diversifying the structure of the products. Therefore, knowledge on the non-enzymatic reactions is important for elucidating the biosynthetic mechanism. This review focuses on non-enzymatic reactions we recently encountered during biosynthetic studies of four types of NPs (viridicatins, Sch210972, lentopeptins, and lentofuranine).

The phosphylated butyrylcholinesterase-derived tetrapeptide GlyGluSerAla proves exposure to organophosphorus agents with enantioselectivity.

We herein present for the first time the phosphylated (*) tetrapeptide (TP)-adduct GlyGluSer198*Ala generated from butyrylcholinesterase (BChE) with proteinase K excellently suited for the verification of exposure to toxic organophosphorus nerve agents (OPNA). Verification requires bioanalytical methods mandatory for toxicological and legal reasons. OPNA react with BChE by phosphonylation of the active site serine residue (Ser198) forming one of the major target protein adducts for verification. After its enzymatic cleavage with pepsin, the nonapeptide (NP) PheGlyGluSer*AlaGlyAlaAlaSer is typically produced as biomarker. Usually OPNA occur as racemic mixtures of phosphonic acid derivatives with the stereocenter at the phosphorus atom, e.g. (±)-VX. Both enantiomers react with BChE, but the adducted NP does not allow their chromatographic distinction. In contrast, the herein introduced TP-adducts appeared as two peaks when using a stationary reversed phase (1.8 µm) in micro-liquid chromatography-electrospray ionisation tandem-mass spectrometry (µLC-ESI MS/MS) analysis. These two peaks represent diastereomers of the (+)- and (-)-OPNA adducted to the peptide that comprises chiral L-amino acids exclusively. Concentration- and time-dependent effects of adduct formation with (±)-VX and its pure enantiomers (+)- and (-)-VX as well as with (±)-cyclosarin (GF) were investigated in detail characterising enantioselective adduct formation, stability, ageing and spontaneous reactivation. The method was also successfully applied to samples from a real case of pesticide poisoning as well as to samples of biomedical proficiency tests provided by the Organisation for the Prohibition of Chemical Weapons.

Enantio- and Diastereoselective Mannich Reactions of ß-Dicarbonyls by Second Stage Diastereoconvergent Crystallization.

The synthesis of chiral α-monosubstituted-ß-dicarbonyls is a challenging task in asymmetric catalysis due to the rapid, typically uncontrolled, product racemization or epimerization under most reaction conditions. For this reason, diastereoselective additions of unsubstituted ß-dicarbonyls to π-electrophiles are unusual. Herein, we disclose a simple catalytic crystallization-driven enantio- and diastereoselective Mannich reaction for the synthesis of stereodefined α-monosubstituted-ß-keto esters, dissymmetric ß-diesters, dissymmetric ß-diketones, and ß-keto amides that productively leverages product epimerization in solution. Mechanistic studies suggest a scenario where the initial enantioselective, diastereodivergent skeletal assembly is catalyzed by a chiral tertiary amine organocatalyst, which then facilitates second stage crystallization-induced diastereoconvergence to provide the challenging α-stereocenter in excellent stereoselectivity.

Cistadesertosides B-E, Four New Diastereomeric Lignan Glycosides from the Stems of Cultural Cistanche deserticola in Tarim Desert.

Four previously undescribed diastereomeric lignan glycosides, namely cistadesertosides B-E (1-4) were isolated from the stems of cultural Cistanche deserticola in Tarim desert. The structures of these compounds were elucidated on the basis of extensive spectroscopic analyses, including IR, HR-ESI-MS, 1D and 2D NMR, circular dichroism (CD) data and chemical degradation. The in vitro anti-inflammatory activity of the isolates was also investigated. It showed that compounds 3 and 4 exhibited potential effects with IC50 values of 21.17 µM and 26.97 µM, respectively (positive control quercetin, IC50 , 10.01 µM).

Diastereomer Resolution of M4 L6 Coordination Cages by Ultra-High-Resolution Ion-Mobility Mass Spectrometry.

Coordination cages can be used for enantio- and regioselective catalysis and for the selective sensing and separation of isomeric guest molecules. Here, stereoisomers of a family of coordination cages are resolved using ultra-high-resolution cyclic ion-mobility mass spectrometry (cIM-MS). The observed ratio of diastereomers is dependent on both the metal ion and counter ion. Moreover, the point groups can be assigned through complementary NMR experiments. This method enables the identification and interrogation of the individual isomers in complex mixtures of cages which cannot be performed in solution. Furthermore, these techniques allow the stability of individual isomers within the mixture to be probed, with the T-symmetric isomers in this case shown to be more robust than the C3 and S4 analogues.

Development of a novel method for analysing N-acetyl-DL-leucine enantiomers in human fingernail by UPLC-ESI-MS/MS and the evaluation in diabetes mellitus.

BACKGROUND: Recent research has been reported that N-acetyl-leucine content is significantly reduced in the saliva of diabetic patients, but no reports of detection in human nails have been found. This study aims to develop a novel method for the chiral separation of N-acetyl-DL-leucine (Ac-DL-Leu) in human fingernails to investigate the differences between healthy volunteers (HVs), prediabetes (PDs) and diabetic patients (DPs), and to verify its effectiveness in early warning of diabetes. METHOD: Chiral resolution was performed using DBD-Apy pre-column derivatization on a C18 column (2.1 × 150 mm, 1.9 µm) at 40 °C, and detected by UPLC-ESI-MS/MS. RESULTS: The resolution and the limit of detection (LOD) of Ac-DL-Leu were 1.75 and 1.50 fmol, respectively. The linear range of Ac-DL-Leu was 10-2000 fmol and the determination coefficient (R2) was above 0.9997. The recovery of Ac-DL-Leu in human nails was 96.92-105.69 %. The contents of Ac-D-Leu and Ac-L-Leu were analyzed in 18 HVs, 13 PDs and 16 DPs fingernails. The results showed that their contents were significantly lower in DPs than in PDs and HVs (p < 0.0001). CONCLUSIONS: A method for evaluating the effectiveness of Ac-DL-Leu enantiomers in human fingernails as a biomarker for diabetes was firstly developed.

Total Synthesis of Hemerocallisamine I Paved by Gram-Scale Synthesis of (2S,4S)-4-Hydroxyglutamic Acid Lactone.

Total synthesis of the 2-formylpyrrole alkaloid hemerocallisamine I is presented, both in racemic and enantiopure form. Our synthetic strategy involves (2S,4S)-4-hydroxyglutamic acid lactone as the key intermediate. Starting from an achiral substrate, the target stereogenic centers were introduced by means of crystallization-induced diastereomer transformation (CIDT) in a highly stereoselective fashion. A Maillard-type condensation was crucial to constructing the desired pyrrolic scaffold.

Rapid discrimination of enantiomers by ion mobility mass spectrometry and chemical theoretical calculation: Chiral mandelic acid and its derivatives.

Because R/S-mandelic acids (MA) and their derivatives are critical starting materials or intermediates in the synthesis of chiral drugs, their chirality discrimination is important. In this study, R/S-MA and its derivatives, including R/S-2-phenylpropionic acid (2-PPA), R/S-methoxyphenylaceticacid (MPA), and R/S-2-hydroxy-4-phenylbutyric acid (HPBA), were accurate simultaneous mobility-discriminated by forming diastereomer complexes for the first time, which were obtained by simply mixing with cyclodextrins (α, ß, γ-CD) and transition-metal ions (Mn2+, Fe2+, Co2+, Ni2+, Cu2+, and Zn2+). The mass spectra revealed non-covalent diastereomer complexes formed by CD, enantiomers, and metal ions, and ion-mobility spectrometry (IMS) was performed for 109 pairs of complexes. Significant chiral discrimination was observed in the formed diastereomeric complexes, and their separation peak-to-peak resolution (Rp-p) for the enantiomers depended on the transition metal ion type. In most cases, the Rp-p value gradually increases with CD size, with quaternary complexes having the largest Rp-p value. The greatest chiral distinctions of 2-PPA, MA, MPA, and HPBA were obtained by the diastereomeric complex ions of [(2-PPA)(α)2+Zn2+-H]+, [(MA)(α)2+Zn2+-H]+, [(MPA)2(ß)+Co2+-H]+, and [(HPBA)(α)2+Fe2+-H]+, with Rp-p values of 1.35, 1.57, 1.70, and 0.71, respectively. Furthermore, the favorable conformation and collisional cross section (CCS) value of the different [CD + R/S-MA + Cu-H]+ complexes were measured using chemical theoretical calculations to detail their intermolecular interaction, revealing that [α-CD + R/S-MA + Cu-H]+ has two favored gas complexes, and the CCS calculated were consistent with the TIMS observed. In addition, R2 > 0.99 was obtained for the relative quantification of the chiral enantiomers. Overall, the proposed method provides a promising strategy for distinguishing the enantiomers of MA and their derivatives, with the advantages of simplicity, speed, and accuracy, without the need for complex chemical derivatization or chromatographic separation.

Self-Assembly of Macrocyclic Triangles into Helicity-Opposite Nanotwists by Competitive Planar over Point Chirality.

While helix has elegant biomimetic structures and functionalities, it still remains a big question how the nanoscale helicity evolved from the molecular chiral building blocks across length scales. Herein, macrocyclic triangles composed of achiral edges and chiral vertices were rationally designed, in which the planar chirality emerged due to the restriction of edge rotation by intermolecular stackings and led to a unique chiral self-assembly. In contrast to the solution systems where the chiroptical property is exclusively dominated by the point chiral vertices, the emerged planar chirality was found to control the chiral self-assembly, resulting the nanotwist with the handedness determined by the planar chirality. Our work unveiled the self-assembly behaviors of macrocyclic conformers for the first time and provided a deep understanding on the macrocyclic chirality evolution including the excited-state chirality.

Analytical techniques for characterizing diastereomers of phosphorothioated oligonucleotides.

Oligonucleotides have emerged as powerful therapeutics for treating diverse diseases. To fully unlock the therapeutic potential of oligonucleotides, there is still a great need to further improve their drug-like properties. Numerous chemical modifications have been explored to achieve this goal, with phosphorothioation being one of the most widely used strategies. However, phosphorothioate modification produces diastereomers that are reported to have different properties and performances, demanding detailed characterization of these diastereomers. Here we provide an overview of phosphorothioated oligonucleotide diastereomers, covering their origin and configurations, physicochemical and pharmacological properties, and stereo-selective chemical synthesis, followed by a summary of currently available analytical techniques for characterizing these diastereomers, with a focus on liquid chromatography-based approaches, including ion-pair reversed-phase liquid chromatography, anion exchange chromatography, mixed-mode chromatography, and hybrid approaches. Non-chromatographic techniques, such as capillary electrophoresis, spectroscopy and other methods, are also being reviewed.

Separation and detection of D-/L-serine by conventional HPLC.

D-serine has a role as an endogenous allosteric agonist of N-methyl-D-aspartate (NMDA) receptor in the mammalian brain. In this study, we present a detailed description of our method that measures D-/L-serine by using conventional high performance liquid chromatography (HPLC). • We reacted D-serine and L-serine with ortho-phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) to form diastereomeric isoindole derivatives, then we separated and detected them by conventional reversed phase HPLC with electrochemical detector (ECD). • We present typical measurement data of rat brain homogenate as an example of a convenient, appropriate method for measuring brain concentrations of D-serine. • Since many peaks appear in biological samples, we confirmed that the peaks were derived from serine by treating the sample with D-amino oxidase and catalase to decompose D-serine. As a results, one peak disappeared, suggesting that it is derived from D-serine.

Stereochemical switch driven by crystallization: Interplay between stoichiometry and configuration of the products.

An intriguing example of a crystallization-induced stereochemical switch in the configuration of aza-Michael reaction products is described. Depending on both the stereochemical purity and stoichiometric ratio of the chiral amine used, the reaction delivers crystalline diastereomers of a different stereochemistry. The optically pure diastereomer smoothly converts to its racemic epimer salt upon the addition of a complementary chiral amine.

Diastereomer- and enantiomer-selective accumulation and depuration of 1,2-dibromo-4-(1,2-dibromoethyl) cyclohexanes (DBE-DBCHs) and 1,2,5,6-tetrabromocyclooctanes (TBCOs) in earthworms (Eisenia fetida).

Due to the regulation of hexabromocyclododecane (HBCD), much attention has been paid to its potential substitutes, 1,2-dibromo-4-(1,2-dibromoethyl) cyclohexane (DBE-DBCH) and 1,2,5,6-tetrabromocyclooctane (TBCO). DBE-DBCH and TBCO contain several diastereomers and enantiomers, which may exhibit different environmental behaviors and biological effects. In this study, the accumulation and depuration of individual DBE-DBCH and TBCO diastereomers by earthworms (Eisenia fetida) from diastereomer-contaminated soils were evaluated. The accumulation and depuration kinetics of DBE-DBCH and TBCO diastereomers followed one-compartment first-order kinetics. The biota soil accumulation factor (BSAF) of ß-DBE-DBCH (2.74 goc glip-1) was 1.26 times that of α-DBE-DBCH (2.18 goc glip-1), while the BSAF of ß-TBCO (2.15 goc glip-1) was 1.62 times that of α-TBCO (1.3 goc glip-1), showing the diastereomer-specific accumulation of DBE-DBCH and TBCO. DBE-DBCH and TBCO diastereomers appeared to be transformed in earthworm-soil systems; however, no evidence of bioisomerization of the four diastereomers in earthworms was found, and no potential metabolites of debromination and hydroxylation were detected. Furthermore, the selective enrichment of E1-α-DBE-DBCH and E1-ß-DBE-DBCH (E1 represents the first enantiomer eluted) occurred in earthworms as the enantiomer fractions (EFs) for α-DBE-DBCH (0.562-0.763) and ß-DBE-DBCH (0.516-0.647) were significantly greater than those in the technical products (0.501 for α-DBE-DBCH and 0.497 for ß-DBE-DBCH, p < 0.05), especially in the depuration stage. The results demonstrated the diastereomer- and enantiomer-selective accumulation of DBE-DBCH and the diastereomer-selective accumulation of TBCO in the earthworm.

Photochemical Deracemization of a Medicinally-Relevant Benzopyran using an Oscillatory Flow Reactor.

Dynamic deracemization processes, such as crystallization-induced diastereomer transformations (CIDTs), offer the opportunity to combine racemization and resolution processes, to provide high yields of enantiomerically pure compounds. To date, few of these processes have incorporated photochemical racemization. By combining batch crystallization with a flow photoreactor for efficient irradiation, it is possible to perform such deracemization in an effective, scalable and high yielding manner. After applying design of experiment (DoE) principles and mathematical modelling, the most efficient parameter set could be identified, leading to excellent results in just 4 h reaction time: isolated yield of 82 % and assay ee of 96 %. Such photochemical racemization methods can serve to open new avenues for preparation of enantiomerically pure functional molecules on both small and industrially-relevant scales.

Protonation-Induced Chirality Drives Separation by Differential Ion Mobility Spectrometry.

Upon development of a workflow to analyze (±)-Verapamil and its metabolites using differential mobility spectrometry (DMS), we noticed that the ionogram of protonated Verapamil consisted of two peaks. This was inconsistent with its metabolites, as each exhibited only a single peak in the respective ionograms. The unique behaviour of Verapamil was attributed to protonation at its tertiary amino moiety, which generated a stereogenic quaternary amine. The introduction of additional chirality upon N-protonation of Verapamil renders four possible stereochemical configurations for the protonated ion: (R,R), (S,S), (R,S), or (S,R). The (R,R)/(S,S) and (R,S)/(S,R) enantiomeric pairs are diastereomeric and thus exhibit unique conformations that are resolvable by linear and differential ion mobility techniques. Protonation-induced chirality appears to be a general phenomenon, as N-protonation of 12 additional chiral amines generated diastereomers that were readily resolved by DMS.

Purification of N-acetylgalactosamine-modified-oligonucleotides using orthogonal anion-exchange and mixed-mode chromatography approaches.

N-acetylgalactosamine (GalNAc)-modified small interfering ribonucleic acids (siRNA) have shown promising outcomes for targeted siRNA delivery resulting in gene silencing in vivo; however, their structural complexity requires development of new purification methods to address high purity and recovery requirements. The current study evaluates complementary purification approaches using a mixed-mode Scherzo SS-C18 and anion-exchange (AEX) TSK-gel SuperQ-5PW for a range of single-stranded triantennary GalNAc-oligonucleotides. Initially, the semi-preparative mixed-mode support (10 × 250 mm, 3 µm) was compared against the preparative AEX analogue (21.5 × 300 mm, 13 µm), with the former affording double the recovery and higher purity of 95% over its AEX counterpart displaying 91% for a selected siRNA conjugate. An assortment of GalNAc-modified oligonucleotides was later purified using the mixed-mode resin revealing good recoveries (∼30-60%) and high purities of 90-94% ranging from straightforward to more challenging purifications. High sample loading in the 20 mg range was achieved, which was comparable with the larger preparative TSKgel SuperQ-5PW support. The Scherzo-SS-C18 resin also afforded some degree of resolution between diastereomers containing phosphorothioate functionalities. The TSKgel SuperQ-5PW support was later investigated to provide orthogonal separation selectivity to the Scherzo-SS-C18 column enabling purification of a selected, GalNAc-siRNA conjugate. The developed pH (8.5-11) and salt (0.3-0.7 M) gradients method provided enhanced separation selectivity between the free and conjugated siRNA, while minimizing formation of secondary structures and highlighting a complementary approach to deal with challenging purifications of oligonucleotide-GalNAc conjugates. Together, the use of AEX and mixed-mode columns provide much needed orthogonality to deal with complex GalNAc-modified oligonucleotides and potentially other upcoming modalities.