Size makes a difference: Chiral recognition in complexes of fenchone with cyclodextrins studied by means of NMR titration

Gibbs energies of complex formation between enantiomers of bicyclic terpenoid, fenchone, and naturally occurring cyclodextrins, βCD and γCD, were determined by means of ¹³C and ¹H nuclear magnetic resonance (NMR) titration data. These results were compared with the corresponding data obtained previously for the diastereomeric fenchone/αCD complexes. The size of the inner cavity of host molecules significantly influences stoichiometry, association constants, and enantiomeric differentiation of the studied complexes. These complementary data allow us to discuss qualitatively the influence of the host size on the guest–host interactions. A method of the simultaneous use of titration data collected for several resonances of different isotopes in the determination of association constants was worked out and thoroughly analyzed. Comparison of the results of global data analyses with weighted means of individual ones revealed that both these approaches are equally trustworthy.     

Affinities of Shiga toxins 1 and 2 for univalent and oligovalent Pk-trisaccharide analogs measured by electrospray ionization mass spectrometry

The binding stoichiometry and affinities of the Shiga toxins, Stx1 and Stx2, for a series of uni- and oligovalent analogs of the Pk-trisaccharide were measured using the direct electrospray ionization mass spectrometry (ES-MS) assay. Importantly, it is shown that, for a given ligand, Stx1 and Stx2 exhibit similar affinities. The binding data suggest a high degree of similarity in the spatial arrangement and structural characteristics of the Pk binding sites in Stx1 and Stx2. The results confirm that both toxins recognize the alpha-D-Galp(1-->4)-beta-D-Galp(1-->4)-beta-D-Glcp carbohydrate motif of the cell surface glycolipid Gb3. This, taken together with the results of the chemical mapping study, suggests that the nature of the Pk binding interactions with Stx1 and Stx2 are similar. The affinities of Stx1-B(5) and Stx2 for the multivalent ligands reveals that site 2 of Stx2, which shares the same spatial arrangement as site 2 in Stx1, is the primary Pk binding site and that site 1 of Stx1 and of Stx2 can also participate in Pk binding.     

High-performance liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry for analysis of Angelica sinensis

An HPLC-PAD-API/MS method for analysing the chemical constituents of Angelica sinensis (A. sinensis) has been developed. ESI and APCI spectra, in both positive ion (PI) and negative ion (NI) modes, provided very useful information concerning the molecular weights of detected compounds. By comparing the retention times, UV spectra, mass spectra and molecular weights of detected compounds with those published in literature, 15 constituents of A. sinensis could be tentatively identified. This technique involving combined MS information may provide an objective, reliable and rapid analytical method for the quality control and database research of traditional Chinese medicines.     

Chiral separation and discrimination of catechin by sinorhizobial octasaccharides in capillary electrophoresis and C NMR spectroscopy

Succinoglycan, a sinorhizobial exopolysaccharide produced by Sinorhizobium meliloti, is composed of an octasaccharide subunit. S. meliloti produces both high-molecular-weight and low-molecular-weight (M_r < 10,000) succinoglycans which consist of monomers, dimers, or trimers. Succinoglycan monomers were isolated and further purified in the monomer series (M1, M2, and M3) by the degree of succinylation. We used sinorhizobial octasaccharides (M1, M2, and M3) as chiral additives in capillary electrophoresis (CE) for chiral separation of catechin and also as chiral shift reagents with ¹³C NMR spectroscopy for chiral discrimination of catechin. Chiral separation of catechin took place when sinorhizobial octasaccharides (M2 and M3) were added to the background electrolyte (BGE) in CE. NMR signal splittings were also observed in the interactions of sinorhizobial octasaccharides with the enantiomers of catechin. Both chiral separation and discrimination of catechin depend on the presence of succinate substituents of the linear monomeric octasaccharide in CE and NMR spectroscopy, suggesting that succinylation of sinorhizobial octasaccharide is decisive for the effective chiral separation and discrimination of catechin.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry

A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.     

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 μg protein) or preparative (>250 μg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.     

Chiral resolutions of (9-anthryl)methoxyacetic acid and (9-anthryl)hydroxyacetic acid by capillary electrophoresis

The resolutions of (9-anthryl)methoxyacetic acid (9AMAA) and (9-anthryl)hydroxyacetic acid (9AHAA) were performed by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as a chiral selector. Various factors affecting migration time and resolutions of these compounds were investigated with a run voltage of 20 kV, column temperature 20 degrees C and 20 mM Tris-H(3)PO(4) buffer (pH 6.5) containing 5 mM HP-beta-CD for 9AMAA, or 10 mM HP-beta-CD for 9AHAA, (+/-)-9AMAA and (+/-)-9AHAA were successfully separated at Rs 3.27 and 1.92, respectively.     

The dimerization of semisynthetic eremomycin derivatives studied by the electrospray ionization mass spectrometry and its effect on their antibacterial activity

The dimerization constants for glycopeptide antibiotics vancomycin, ristocetin, and eremomycin and nine semisynthetic eremomycin derivatives were determined by the electrospray ionization mass spectrometry; the constants for natural antibiotics turned out to be close to those previously determined by NMR. No correlation between these dimerization constants and antibacterial activities of all the compounds toward the clinical strains of Gram-positive bacteria was found.     

枯草芽孢杆菌JA产生的脂肽类抗生素－iturin A的纯化 及电喷雾质谱鉴定

枯草芽孢杆菌JA产生的抗生素对植物病原真菌具有广谱抗性,明确抗生素的种类是进一步研究的基础.用6mol/L盐酸沉淀JA菌株的去菌体培养基,再用甲醇抽提获得抗生素的粗提物.利用反相HPLC系统,将粗提物过Diamonsil C18柱,收集有抗小麦赤霉病等病原真菌活性的化合物1、2.运用电喷雾质谱法(ESI/MS)测得其分子量分别为1042.4D和1056.5D.再利用碰撞诱导解离(CID)技术获得化合物的典型结构特征离子碎片,结果表明分子量为1042.4D的化合物一级结构为Pro-Asn-Tyr-βAA-Asn-Tyr-Asn-Gln(βAA为14个碳原子的氨基脂肪酸),属于脂iturin A.化合物1、2为相差一个亚甲基(-CH2)的iturin A同系物.研究结果提供了一种从枯草芽孢杆菌发酵液中快速分离纯化和鉴定脂肽类抗生素iturin A的新方法.     

Analytical characterisation of crude extracts from an African Ancistrocladus species using high-performance liquid chromatography and capillary electrophoresis coupled to ion trap mass spectrometry

The analysis by HPLC, CE and CE-MS/MS of root bark extracts of a, so far undescribed, Central-African Ancistrocladus species (family Ancistrocladaceae) is described. Owing to the complexity of the extract, the application of reversed-phase HPLC resulted in a partially incomplete separation of the naphthylisoquinoline alkaloids, whilst CE using a non-aqueous buffer proved to be a very valuable complementary method for a first characterisation of the crude extract. By performing additional CE-MS/MS experiments, in combination with parallel isolation studies and structural elucidation using conventional methods, six alkaloidal substances present in the plant could be identified.     

Optical resolution and enantiomeric purity determination of the analgesic cizolirtine

The optical resolution of (±)‐cizolirtine was accomplished with excellent results (>99% ee) by means of crystallization with (+)‐ or (−)‐di‐p‐toluoyltartaric acid. The optical purity of the samples was controlled by three independent methods: ¹H NMR, capillary electrophoresis (CE) (using β‐cyclodextrins as chiral resolving agents), and HPLC (using a glycoproteic column). The use of a rapid analytical technique like ¹H NMR for estimating the relative amounts of each enantiomer, together with the high sensitivity of CE, afforded a convenient strategy for monitoring the entire process leading to enantiopure compounds. Chirality 11:63–69, 1999. © 1999 Wiley‐Liss, Inc.     

In vitro probe acylcarnitine profiling assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry predicts severity of patients with glutaric aciduria type 2

Glutaric aciduria type 2 (multiple acyl-CoA dehydrogenase deficiency, MAD) is a multiple defect of mitochondrial acyl-CoA dehydrogenases due to a deficiency of electron transfer flavoprotein (ETF) or ETF dehydrogenase. The clinical spectrum are relatively wide from the neonatal onset, severe form (MAD-S) to the late-onset, milder form (MAD-M). In the present study, we determined whether the in vitro probe acylcarnitine assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry (MS/MS) can evaluate their clinical severity or not. Incubation of cells from MAD-S patients with palmitic acid showed large increase in palmitoylcarnitine (C16), whereas the downstream acylcarnitines; C14, C12, C10 or C8 as well as C2, were extremely low. In contrast, accumulation of C16 was smaller while the amount of downstream metabolites was higher in fibroblasts from MAD-M compared to MAD-S. The ratio of C16/C14, C16/C12, or C16/C10, in the culture medium was significantly higher in MAD-S compared with that in MAD-M. Loading octanoic acid or myristic acid led to a significant elevation in C8 or C12, respectively in MAD-S, while their effects were less pronounced in MAD-M. In conclusion, it is possible to distinguish MAD-S and MAD-M by in vitro probe acylcarnitine profiling assay with various fatty acids as substrates. This strategy may be applicable for other metabolic disorders.     

Simultaneous chiral analysis of amphetamine‐type stimulants and ephedrine by capillary electrophoresis coupled to time‐of‐flight mass spectrometry

This study focused on the chiral characteristics of methamphetamine seizures in Shanghai for inferring the synthetic pathways of drugs. Capillary electrophoresis coupled to time‐of‐flight mass spectrometry was used for simultaneous chiral separation of amphetamine‐type stimulants and ephedrine, including S(+)‐amphetamine/R(?)‐amphetamine, S(+)‐methamphetamine/R(?)‐methamphetamine, (±)‐MDA (3,4‐methylenedioxyamphetamine), (±)‐MDMA (3,4‐methylenedioxymethamphetamine), (±)‐MDEA (3,4‐methylenedioxy‐N‐ethylamphetamine), d,l‐N‐ethylamphetamine, methylephedrine/methylpseudoephedrine, and 1S,2R(+)‐ephedrine/(?)‐ephedrine. The running buffer was 50‐mM ammonium formate (pH 2.2 was adjusted by 1‐M formic acid) containing 0.26% highly sulfated γ‐cyclodextrin as the chiral selector. All enantiomers were well resolved within 40 minutes by capillary electrophoresis at 20 kV in an uncoated fused‐silica capillary (50‐μm I.D. × 375‐μm O.D. × 90‐cm length) and detected by micro time‐of‐flight mass spectrometry. Twenty seized methamphetamine samples were determined by the established method. They were classified into two groups through their chiral characteristics.     

Studies on the stability of diester-diterpenoid alkaloids from the genus Aconitum L. by high performance liquid chromatography combined with electrospray ionisation tandem mass spectrometry (HPLC/ESI/MSn)

The stability of diester-diterpenoid alkaloids (DDA) from plants of the genus Aconitum L. has been studied in different solvents and pH buffers. The HPLC/ESIMS method for analysing the concentration of DDA was established and DDA's decomposition products were elucidated by HPLC/ESI-MS/MS(n). In different solvents, e.g. dichloromethane, ether, methanol and distilled water, the decomposition pathways of DDA are quite different and their difference in stabilities depends on the difference of their structures, in which substituents at the N atom and substituents at C-3 are different. The pyrolytic products of DDA, such as deacetoxy aconitine-type alkaloids, have been observed in the above solvents, whereas 8-methoxy-14-benzoyl aconitine-type alkaloids have been obtained only in methanol. Furthermore, the experimental results demonstrate that the stability of DDA depends on pH values of the buffer. Aconine as hydrolysate has been only found in pH 10.0 buffer, and the other hydrolysates and the pyrolyzates of DDA, such as benzoylaconine and deacetoxy aconitine, have been observed in all pH aqueous solutions. The decomposition pathways of DDA in buffers are related to the substituent on the C-3 position. The decomposition pathway of aconitine is similar to that of mesaconitine, but different from that of hypaconitine.     

Spectroscopic, mass spectrometry, and semiempirical investigation of a new ester of Monensin A with ethylene glycol and its complexes with monovalent metal cations

A new ester of Monensin A with ethylene glycol (MON2) has been synthesized by a new method and its ability to form complexes with Li+, Na+, and K+ cations has been studied by ESI MS, 1H and 13C NMR, FT-IR, and PM5 semiempirical methods. It is demonstrated that MON2 forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by intramolecular hydrogen bonds in which the OH groups are always involved. In the structure of MON2 the oxygen atom of the C=O ester group is involved in very weak bifurcated intramolecular hydrogen bonds with two hydroxyl groups, whereas in the complexes of MON2 with monovalent metal cations the C=O ester group is not engaged in any intramolecular hydrogen bonds. The structures of the MON2 and its complexes with Li+, Na+, and K+ cations are visualized and discussed in detail.     

Secondary phosphine oxides: tautomerism and chiral recognition monitored by multinuclear NMR spectroscopy of their Rh2[(R)-MTPA]4 adducts

Six secondary phosphine oxides and their tautomeric equilibria as free ligands and in the presence of an equimolar amount of the chiral dirhodium complex Rh* are described and discussed. Discrimination of enantiomers is easily possible by inspecting the (31)P NMR resonances; some (1)H and (13)C NMR resonances are useful as well. H/D exchange of the acidic protons in the phosphine oxides takes place with acetone-d(6), the solvent additive, after some hours but does not obscure the chiral recognition experiment. (103)Rh,(31)P coupling constants are discussed briefly. Decomposition of ligand molecules in 1:1-Rh*-adducts occurs slowly but completely.     

Chiral phospholene and phospholane chalcogenides: stereochemistry and chiral recognition by multinuclear NMR spectroscopy of their Rh2[(R)-MTPA]4 adducts

Full NMR spectral assignments of the phospholene chalcogenides 1-12 are presented and their stereochemistry proven. The enantiomeric ratio of any of these compounds can be monitored easily by adding one mole equivalent of the chiral auxiliary Rh(2)[(R)-MTPA](4) (MTPA-H identical with Mosher's acid) and subsequent NMR inspection. Some surprisingly large diastereomeric signal dispersion is observed in the (1)H NMR spectra of the adducts, leading to the conclusion that intramolecular anisotropy interaction between groups inside the ligand molecules exists. The dependence of dispersion effects on the nature of the chalcogenide atom is investigated.