Two novel chiral ionic liquids are synthesized as the chiral selector. Racemates of amines, amino alcohols, and amino acids could generate enantioselective precipitate with multicomponent self-assemblies under mild conditions. The approach allows for enantioseparation with good yields (79–94%) and excellent ee’s (>95%).

There are two challenges in oligonucleotide detection by liquid chromatography coupled with mass spectrometry (LC-MS), the serious ion suppression effects caused by ion-pair reagents and the low detection sensitivity in positive mode MS. In this study, highly concentrated alcohol vapors were introduced into an enclosed electrospray ionization chamber, and oligonucleotides could be well detected in negative mode MS even with 100 mM triethylammonium acetate (TEAA) as an ion-pair reagent. The MS signal intensity was improved 600-fold (for standard oligonucleotide dT15) by the isopropanol vapor assisted electrospray, and effective ion-pair LC separation was feasibly coupled with high-sensitive MS detection. Then, oligonucleotides were successfully detected in positive mode MS with few adducts by propanoic acid vapor assisted electrospray. The signal intensity was enhanced more than 10-fold on average compared with adding acids into the electrospray solution. Finally, oligonucleotides and peptides or histones were simultaneously detected in MS with little interference with each other. Our strategy provides a useful alternative for investigating the biological functions of oligonucleotides.

This paper describes a simple method to determine the absolute configuration of amino acids residues in peptides by mass spectrometry using a newly developed pair of mass-tagged chiral probes without the requirement of reference standards. A pair of benzylicaldehyde probes, 1-(S)-1H in S configuration and 2-(R)-2D in deuterium-labeled R configuration with the ratio of 1:1, were synthesized for in situ condensation with amino acid residues and transformed into a pair of stereodynamic imine products. The characteristic intensity difference observed in mass spectrometry can be used to determine the absolute configuration and to quantify the enantiomeric composition of chiral amino acid residues. Significant chiral recognition ability was achieved for 18 natural chiral amino acids and for one β-amino acid by comparing the ion intensity ratio of imine products I[1-(S)-1H-AA]– to I[2-(R)-2D-AA]–. For 16 kinds of amino acids, the L form of the amino acids was more reactive with 1-(S)-1H, while D configuration amino acids preferred to react with 2-(R)-2D. However, for three kinds of amino acid, the opposite result was obtained. The configurations of the residues in the peptides, Phe-Tyr-Ala, D-Phe-Tyr-Ala, Val-Pro-Phe-D-Leu-Met, Val-Pro-Phe-Leu-D-Met, as well as in a natural peptide with unknown chirality were determined by acid hydrolysis followed by the present method. In addition, molecular modeling results illustrate that the recognition process is mainly controlled by kinetic factors. Using the new probes coupled with a mass spectrometry approach avoids time-consuming workup and separation steps. We expect that the probes could be applied as tools to determine the absolute configuration of amino acid residues in proteins in future research.

A copper catalyzed direct α-oxyacylation of ketones with carboxylic acids has been developed. Various acids including aromatic and aliphatic acids are well tolerated, and the scope of ketones is also investigated. This protocol provides a convenient and benign method for the synthesis of α-acyloxycarbonyl compounds by using oxygen as the oxidant. In addition, we have investigated the role of copper and trapped the key intermediate to confirm the mechanism.

Among gas-phase dissociation reactions, double bond cleavage reaction appears to happen extremely rare, especially in the case of CC double bond. In the dissociation reaction of protonated 2-benzylidenecyclopentanones in tandem mass spectrometry, the formation of benzyl cations was observed, resulting from the cleavage of Cα=Cβ double bonds, which is different from the general cleavage route seen in most α, β-unsaturated ketone cases. A combined experimental and theoretical investigation on intramolecular hydrogen transfers was carried out to illustrate the mechanisms. The external proton is initially localized on the carbonyl oxygen (the thermodynamically-preferred protonation site). Upon collisional activation, the mobile proton stepwise migrates to the Cα position to achieve the reduction and subsequent cleavage of the Cα=Cβ double bond. The stepwise proton transfer is achieved via intramolecular proton-transport catalysis with the assistance of the phenyl ring. The ortho position of the phenyl accepts the proton from the carbonyl oxygen via a 1,6-H shift, and then donates it to the Cα stepwise. The conventional 1,3-H shift from the carbonyl oxygen to the Cα position can be excluded in this case due to its significant energy barrier. Further isotope-labeling experiments are applied to confirming the reaction mechanism. Last but not least, the scope-expansion experiments indicates that the aromatic and cycloalkanonyl moieties play a crucial roles in the cleavage reaction of Cα=Cβ double bond.Download high-res image (205KB)Download full-size image

•Structure and bioactivity of BTp1 from B. ternatum were probed for the first time.•Based on 3AQ-CHCA, MALDI could be used to detect BTp1 with high Mw directly.•The backbone of BTp1 was deduced quickly based on enzyme hydrolysis and MALDI-TOF.•BTp1 could greatly promote the release of NO from RAW 264.7 without cytotoxicity.As a folk medicine, Botrychium ternatum has been used for thousands of years in China. In the present work, a water soluble polysaccharide BTp1 was extracted and purified from B. ternatum. Based on the MALDI matrix 3-aminoquinoline-α-cyano-4-hydroxycinnamic acid, the molecular weight of BTp1 was determined to be 11638 Da directly. Monosaccharide analysis showed that BTp1 was composed of arabinose (Ara). Combining enzymatic hydrolysis and subsequent MALDI-TOF analysis, a linear backbone of BTp1, consisted of (1 → 5)-linked α-l-Araf, was inferred quickly. Then according to NMR experiments, the whole structure of BTp1 was established. The repeating unit of BTp1 was deduced as a linear backbone with branches at O-2, O-3 and its neighboring O-2 positions terminated with (1→)-linked α-l-Araf, respectively. The immunomodulatory assay exhibited that BTp1 could significantly enhance the viability and promote the release of NO in RAW 264.7 cells, suggesting that BTp1 could be a potential immunomodulatory agent in pharmacological fields.

•Novel ILMs, DHB/N-methylaniline (DHB/N-MA) and DHB/N-ethylaniline, have been synthesized for the first time.•Novel ILMs were used to detect both the oligosaccharide and polysaccharide efficiently.•Using DHB/N-MA, quantitative analysis of oligosaccharide mixtures was achieved sensitively.Analysis of carbohydrates based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is still challenging and researchers have been devoting themselves to efficient matrices discovery. In the present study, the design, synthesis, qualitative and quantitative performance of non-derivative ionic liquid matrices (ILMs) were reported. DHB/N-methylaniline (N-MA) and DHB/N-ethylaniline (N-EA), performing best for carbohydrate detection, have been screened out. The limit of detection for oligosaccharide provided by DHB/N-MA and DHB/N-EA were as low as 10 fmol. DHB/N-MA and DHB/N-EA showed significantly higher ion generation efficiency than DHB. The comparison of capacity to probe polysaccharide between these two ILMs and DHB also revealed their powerful potential. Their outstanding performance were probably due to lower proton affinities and stronger UV absorption at λ = 355 nm. What is more, taking DHB/N-MA as an example, quantitative analysis of fructo-oligosaccharide mixtures extracted and identified from rice noodles has been accomplished sensitively using an internal standard method. Overall, DHB/N-MA and DHB/N-EA exhibited excellent performance and might be significant sources as the carbohydrate matrices.Download high-res image (209KB)Download full-size image

A novel task-specific ionic liquid derived from L-phenylalaninol was prepared as an enantioselective fluorescent sensor for the first time. Fluorescent chiral ionic liquid 1 (FCIL1) is found to exhibit highly enantioselective fluorescence enhancements toward both aromatic and non-aromatic chiral amino alcohols. When (S)-FCIL1 was treated with the enantiomers of phenylalaninol, a great fluorescence enhancement at 349 nm could be observed and the value of the enantiomeric fluorescence difference (ef) is 5.92. This demonstrated that the chiral sensor (S)-FCIL1 exhibited an excellent enantioselective response behaviour to D-phenylalaninol. Besides that, both the fluorescence intensity at 349 nm (I349) and the ratio of I349 to I282 depend linearly on the concentration of amino alcohols. Both the concentration and the enantiomeric composition could be determined by using the chiral ionic liquid. Differently, the sensor treated with the enantiomers of 2-amino-1-butanol showed an opposite result: the fluorescence intensity of the S-enantiomer is higher than that of the R-enantiomer. Furthermore, the size of the substituents on the chiral carbon might be important for the enantioselective fluorescent response.

A mild hemin catalytic system for sulfonium ylide generation via a metal carbenoid and a subsequent [2,3]-sigmatropic rearrangement reaction in aqueous solvent is well-established, with the assistance of cyclodextrin (CD) and Triton X-100. The protocol displays high catalytic activity with a broad substrate scope of aryl/alkyl allyl sulfides and diazo reagents, affording homoallyl sulfide products in up to 99% yield. Notably, this catalytic system is successful for water-insoluble allyl sulfides but ineffective for allyl amines or allyl ethers. Moreover, an unprecedented cascade reaction of sulfonium ylide formation, [2,3]-sigmatropic rearrangement and C–H insertion was reported.

A metal-free cascade arylation/aryl migration/desulfonylation of N-phenyl-N-(phenylsulfonyl)methacrylamide is described. The in situ generated diazonium salts from anilines and t-BuONO are used as aryl precursors. This process provides an efficient strategy for the synthesis of α-all-carbon quaternary stereocenters amides. A radical mechanism was proposed for this transformation.

•A novel task-specific ionic liquid functionalized gold nanoparticle was successfully prepared.•This material was successfully applied to recognizing five amino acids with Cu(II) through distinctive color changes.•The proposed strategy was successfully used to analyze the histidine in real samples.In this study, a novel task-specific ionic liquid functionalized gold nanoparticle (TSIL-GNP) was successfully prepared and applied in the recognition of amino acids. Particularly, the surface of GNP was modified with the ionic liquid containing carbamido and ester group via thiol, which was characterized by Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The stability of this material in aqueous solution improves apparently and can remain unchanged for more than three months. The effect of pH was also discussed in this study. Attractive ionic interaction would effectively weaken intensity of the covalent coupling between the metal ion and the functional groups of amino acids. Thus, TSIL-GNP was successfully applied to recognizing serine, aspartic acid, lysine, arginine, and histidine in the presence of Cu2+ through distinctive color changes. Suspension would be generated once a spot of cysteine was added into the GNPs solution. Results indicated that it had a good linear relationship between extinction coefficients and concentration of amino acids in a wide range of 10−3–10−6 M. Moreover, the proposed strategy was successfully used to analyze the histidine in urinary samples. In brief, TSIL-GNP is a suitable substrate for discrimination of five amino acids in a rapid and simple way without sophisticated instruments.

Counter-current chromatography (CCC) is a quick preparative separation technique with immiscible liquid-liquid solvent systems. This paper reviews recent research progress in the instruments improvement of CCC and screening of solvent systems. Moreover, some applications and prospective about chiral resolution by CCC are discussed. Finally, the content and goal of further research in this field are also presented.This paper reviews recent research progress in the instrument improvement of counter-current chromatography and screening of solvent systems. Furthermore, some applications and prospective about chiral resolution by CCC are discussed.

The interaction of antitumor drug, cisplatin (cis-[PtCl2(NH3)2], CDDP) with insulin from porcine pancreas has been investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high resolution hybrid ion trap/time-of-flight mass spectrometry (MALIDI-TOF/TOF–MS and ESI-IT/TOF MS). The MALDI-TOF/TOF–MS results demonstrated that the presence of cisplatin complex resulted in the reduction of the disulfide bond in porcine pancreas after the incubations of the two substances were performed in vitro. It indicated that the presence of cisplatin would destroy the native configuration of insulin, which may lead to the inactivation of insulin. High resolution mass values and the characteristic isotopic pattern of the platinated insulin ions allowed the analysis of platinated mono-, di- and triadducts of cisplatin and insulin in the incubations under different conditions. The laser-induced dissociation of the monoadduct obtained in MALDI source was carried out and one platinum was found to bind to insulin B chain was determined. The platinum binding sites were further identified to be the N terminus (B chain), cysteine 7 (B chain) and cysteine 19 (B chain) residues by electrospray ionization tandem mass spectrometry. The identification of the interaction between insulin and cisplatin broadens the horizon of the knowledge in the interaction of the proteins and metallodrugs.

The fragmentation of halogen-substituted protonated amines and quaternary ammonium ions (R1R2R3N+CH2(CH2)nX, where X = F, Cl, Br, I, n = 1, 2, 3, 4) was studied by electrospray ionization tandem mass spectrometry. A characteristic fragment ion (R1R2R3N+X) resulting from halogen transfer was observed in collision-induced dissociation. A new mechanism for the intramolecular halogen transfer was proposed that involves a reactive intermediate, [amine/halonium ion]. A potential energy surface scan using DFT calculation for CH2–N bond cleavage process of protonated 2-bromo-N,N-dimethylethanamine supports the formation of this intermediate. The bromonium ion intermediate-involved halogen transfer mechanism is supported by an examination of the ion/molecule reaction between isolated ethylenebromonium ion and triethylamine, which generates the N-bromo-N,N,N-triethylammonium cation. For other halogens, Cl and I also can be involved in similar intramolecular halogen transfer, but F cannot be involved. With the elongation of the carbon chain between the halogen (bromine as a representative example) and amine, the migration ability of halogen decreases. When the carbon chain contains two or three CH2 units (n = 1, 2), formal bromine cation transfer can take place, and the transfer is easier when n = 1. When the carbon chain contains four or five CH2 units (n = 3, 4), formal bromine cation transfer does not occur, probably because the five- and six-membered cyclic bromonium ions are very stable and do not donate the bromine to the amine.

A method for the copper-catalyzed regioselective β-functionalization of tertiary amines with thiophenols has been developed. The control experiments and primary studies show that a thiyl radical is involved in the reaction, and the method provides a novel and direct approach to synthesize C(sp3)–S bonds without a directing group under ligand-free conditions.

•We have designed a new MALDI-ISD strategy to identify the protein mutation site.•4-Methyl-3-(pent-4-yn-1-yl)-imidazol-3-ium was synthesized to derive the protein.•The protein after derivatization directly occurred ISD with the normal matrix CHCA.•The protein after derivatization produced much larger range of fragments ions.Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) can be considered as state of the art in the field of proteins and peptides analysis. In this work, we have designed an ionic liquid derivative strategy to obtain abundant fragment ions in MALDI in-source decay (ISD) and used the analysis of angiogenin with mutation in the fortieth (K40I) as an instance. Firstly, we have synthesized two types of ionic liquids, 3-allyl-4-methyl-1H-imidazol-3-ium and 4-methyl-3-(pent-4-yn-1-yl)-1H-imidazol-3-ium. Then in the light-catalyzed reaction, the alkenyl ionic liquid can open the disulfide bond of K40I protein and add to the thiol. And the derived protein can process in-source decay under the effect of ionic liquid group to produce c–z type ions. Additionally this fragmentation is potentiated to support widely range of fragment ions which can cover the location of mutation. Our results have supplied a new top-down method about how to analyze the mutation or even post-translational modification of proteins in MALDI mass spectrometry.

A concise strategy to improve the p-NPP (p-nitrophenyl palmitate) catalytic activity and enantioselectivity towards secondary alcohols of Pseudomonas cepacia lipase (PcL) has been described. The PcL was modified by I3−, N-acetyl imidazole (NAI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and ethylenediamine (EDA) in the absence or presence of n-hexane, respectively. After being modified by the four modification reagents, the enantioselectivity (E value) of the PcL towards secondary alcohols was enhanced by 2- to 4-fold. The catalytic activity of EDA-PcL was increased by about 6-fold. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of modified PcL showed that Tyr4, Tyr29, Tyr45, Tyr95, Asp36 and Asp55 were the modified sites. When Tyr29 was modified, the E value of PcL towards secondary alcohols was largely improved. MALDI-TOF-MS characterization and molecular dynamics simulation of the lipase indicated that Tyr29 located inside the catalytic cavity had a significant impact on the E value. The strong steric hindrance of acetyl and iodine ion to the groups on the chiral center of the substrates is responsible for the improvement. In addition, the enhancement of hydrophobicity on the surface of the lipase due to the sidechain replacement of Asp with uncharged hydrophobic groups also improved the E value.

The benzylsilver cation which emerges from the collisional dissociation of silver(I)–N-benzylbenzamide complexes was characterized by deuterium-labeling experiments, theoretical calculations, breakdown curves and substituent effects. The nucleophilic attack of the carbonyl oxygen on an α-hydrogen results in the generation of the benzylsilver cation, which is competitive to the AgH loss with the α-hydrogen.

•The enantiomeric separation of the racemic amino acids with aqueous two-phase systems was performed for the first time.•New ionic liquids containing functional groups acted as both solvent and enantioseparation substrate.•The mechanism was studied by 1H NMR and DFT calculations.•Racemic amino acids were separated with some e.e. values by the same procedures.Aqueous two-phase systems (ATPS) based on hydrophilic ionic liquid (IL) and inorganic salt solution were designed and prepared for the enantiomeric separation of racemic amino acids. Two different kinds of hydrophilic ionic liquids (IL-1 and IL-2) containing functional groups were synthesized to separate racemic amino acids. Preliminary experiments showed that d-enantiomer of amino acids cooperatively interacted with ILs, which pushed d-enantiomer to remain in the bottom IL-rich phase. By contrast, l-enantiomer was transferred into the top Na2SO4-rich phase. The enantioselectivity of IL-1 was better than that of IL-2 because of their different intermolecular interactions. Various factors influencing separation efficiency were also systematically investigated including extraction time, IL volume and temperature. Furthermore, the mechanism was studied by 1H NMR and DFT calculations, which showed that the hydrogen bond between the carboxylate and amide groups and the resonance-assisted hydrogen bond between amino and hydroxyl groups conditioned the movement between the residues and ILs. Finally, IL-1 was validated with other general amino acids by the same procedures based on ATPS.

•Antioxidant activities of three resveratrol dimers were testified against DPPH, hydroxyl radical, superoxide anion, and singlet oxygen.•They are all efficient DPPH scavengers and selective singlet oxygen quenchers.•Pallidol can activate Nrf2 to upregulate the expression of phase 2 enzymes while parthenocissin A and quadrangularin A cannot.•Pallidol is proved to be a bifunctional antioxidant and the other two are direct antioxidants.Resveratrol monomer (Res) and its oligomers are considered as nutritional components distributed in edible plants. Three naturally occurring resveratrol dimers, namely parthenocissin A (Par), quadrangularin A (Qua) and pallidol (Pal), were synthesized and evaluated for their ability to scavenge reactive oxygen species (ROS) and to activate the transcription factor Nrf2, which regulates cellular antioxidant systems. In vitro studies with different ROS and radical assay models showed that all the three dimers are strong DPPH quenchers and selective singlet oxygen (1O2) scavengers (IC50 = 4.90, 1.05 and 5.50 μM, respectively). However, they were ineffective against hydroxyl radical (OH) or superoxide anion (O2−). Exposing the dimers to an antioxidant response element (ARE) reporter cell line revealed that only pallidol was able to activate Nrf2 at 30 μM, while parthenocissin A and quadrangularin A had no significant effect on Nrf2. Our data demonstrates the distinct difference between reservatrol monomer and its dimers in activating the Nrf2/ARE signalling pathway.

In this study, we report a gas-phase benzyl anion transfer via intramolecular aromatic nucleophilic substitution (SNAr) during the course of tandem mass spectrometry of deprotonated N-(phenylsulfonyl)-benzeneacetamide. Upon collisional activation, the formation of the initial ion/neutral complex ([C6H5CH2−/C6H5SO2NCO]), which was generated by heterolytic cleavage of the CH2–CO bond, is proposed as the key step. Subsequently, the anionic counterpart, benzyl anion, is transferred to conduct the intra-complex SNAr reaction. After losing neutral HNCO, the intermediate gives rise to product ion B at m/z 231, whose structure is confirmed by comparing the multistage spectra with those of deprotonated 2-benzylbenzenesulfinic acid and (benzylsulfonyl)benzene. In addition, intra-complex proton transfer is also observed within the complex [C6H5CH2−/C6H5SO2NCO] to generate product ion C at m/z 182. The INC-mediated mechanism was corroborated by theoretical calculations, isotope experiments, breakdown curve, substituent experiments, etc. This work may provide further understanding of the physicochemical properties of the gaseous benzyl anion.

Rationale: overalkylation often appears during the proteolytic digestion process when using iodoacetamide (IAM) to protect the produced side chain thiol of Cys from disulfide bonds. However, side reactions between IAM and other functional groups of amino acids have not been investigated completely. Here, matrix-assisted laser desorption ionization equipped with time of fight analyzer tandem mass spectrometry (MALDI-TOF/TOF MS) has been used to identify overalkylation sites and compare the reactivity of different amino acid residues accurately and rapidly. Methods: in this work, H13A protein, an angiogenin mutant with a mutation from His to Ala at the thirteenth site was analyzed by the bottom-up method and the overalkylation sites were determined by MALDI tandem mass spectrometry. Results: MS results show two or more acetyl groups can be attached to the tryptic peptides. MS/MS results further show the attached acetyl is produced by a reaction between IAM and the side chain of Asp, Lys or His. This result indicates side reactions of IAM with other amino acids and the tendency seems to be Cys > His > Asp > Lys. Conclusions: besides the side chain thiol of Cys, the excess IAM could also react with the side chain of other amino acids such as Asp, Lys and His, etc. The clarified mechanism of this phenomenon can help to avoid disturbances of the interference peaks produced by side reactions and also contribute to the improvement of the method by accurately choosing tryptic peptides.

The fragmentations of argentinated N-allylbenzamides have been exhaustively studied through collision-induced dissociation and through deuterium labeling. The intriguing elimination of AgOH is certified as the consequence of intramolecular cyclization between terminal olefin and carbonyl carbon following proton transfer to carbonyl oxygen, rather than simple enolization of amide. Linear free energy correlations and density functional theory (DFT) calculations were performed to understand the competitive relationship between AgOH loss and AgH loss, which results from the 1,2-elimination of α-hydrogen (to the amido nitrogen) with the silver.

The protonation site of para-dimethylaminobenzoic acid (p-DMABA) was investigated using atmospheric pressure ionization methods (ESI and APCI) coupled with collision-induced dissociation (CID), nuclear magnetic resonance (NMR), and computational chemistry. Theoretical calculations and NMR experiments indicate that the dimethyl amino group is the preferred site of protonation both in the gas phase and aqueous solution. Protonation of p-DMABA occurs at the nitrogen atom by ESI independent of the solvents and other operation conditions under typical thermodynamic control. However, APCI produces a mixture of the nitrogen- and carbonyl oxygen-protonated p-DMABA when aprotic organic solvents (acetonitrile, acetone, and tetrahydrofuran) are used, exhibiting evident kinetic characteristics of protonation. But using protic organic solvents (methanol, ethanol, and isopropanol) in APCI still leads to the formation of thermodynamically stable N-protonated p-DMABA. These structural assignments were based on the different CID behavior of the N- and O-protonated p-DMABA. The losses of methyl radical and water are the diagnostic fragmentations of the N- and O-protonated p-DMABA, respectively. In addition, the N-protonated p-DMABA is more stable than the O-protonated p-DMABA in CID revealed by energy resolved experiments and theoretical calculations.

Unexpected dimethylsulfinyl anions (I), generated in situ from the superbase system CsOH–DMSO, was found to be a highly active catalyst for controllable nitrile hydration reactions in water, which selectively afforded the versatile amides via interesting Cs-activated I-catalyzed direct and indirect hydration mechanisms involving an O-transfer process from DMSO onto the nitriles.

Copper-mediated direct S–N formation using readily available starting materials via an oxygen-activated radical process has been developed. This method provides a novel and direct approach for synthesis of sulfonamides under air conditions.

Cu+–benzyne complexes bearing bidentate nitrogen ligands were synthesized in the gas phase for the first time using electrospray ionization mass spectrometry. The addition reactivity of copper-stabilized benzyne with amines was studied in the ion trap analyzer. The structures of products were identified by comparing their MSn data with authentic compounds obtained from another generation route.

•A convenient method based on a simple chiral probe for rapid enantiomer determination in mass spectrometry was developed.•This method is applied for enantiomer determination both qualitatively and quantitatively.•The method has great potential for chirality screening in both chemical solvent and biological solution.A tandem mass spectrometry method for high-sensitivity qualitative and quantitative discrimination of chiral amino compounds is conducted. The method is based on a chemical derivation process that uses a simple reagent, L-1-(phenylsulfonyl)pyrrolidine-carbonyl chloride, as the probe. The method is applicable in both organic solutions and biological conditions. Twenty-one pairs of enantiomer containing amino acids, amino alcohols, and amines are used to produce diastereomers using the probe via in situ reaction for 20 s at room temperature. The resulting diastereomers are successfully recognized based on the relative peak intensities of their fragments in positive mode, with the chiral recognition ability values ranging from 0.35 to 3.83. The L/D ratio of Pro spiked at different concentrations (enantiomeric excess) in both acetonitrile and dog plasma is determined by establishing calibration curves. This method achieves a lower limit of quantification of 50 pmol in analyzing amino acids using an extract ion chromatograph. The relative standard deviation for both qualitative and quantitative results is <5%. Thus, the present method is demonstrated as a new and practical technique of rapidly and sensitively determining enantiomers of amino compounds.

In collisional activation of argentinated N-arylmethyl-pyridin-2-ylmethanimine, a neutral molecule of AgNH2 is eliminated, carrying one hydrogen from the methylene and the other one from the ortho position (relative to the ipso carbon) of the aryl ring. Taking argentinated N-benzyl-pyridin-2-ylmethanimine for example, the proposition that the AgNH2 loss results from intramolecular arylmethyl transfer combined with cyclodeamination is rationalized by deuterium labeling experiments, blocking experiments, and theoretical calculations. The structure of the final product ion from loss of AgNH2 was confirmed further by multistage mass spectrometry.

In this study, the gas-phase fragmentations of protonated N-benzylbenzaldimines were investigated by electrospray ionization tandem mass spectrometry (ESI-MSn). Upon collisional activation, several characteristic fragment ions are produced and their fragmentation mechanisms are rationalized by electrophilic aromatic substitution accompanied by benzyl cation transfer. (1) For N-(p-methoxybenzylidene)-1-phenylmethanimine, concomitant with a loss of HCN, a product ion at m/z 121 was observed. It is proposed to be generated from electrophilic substitution at the ipso-position by transferring benzyl cation rather than cleavage of the C-N double bond. (2) For N-(m-methoxybenzylidene)-1-phenylmethanimine, a product ion at m/z 209 was obtained, corresponding to the elimination of NH3 carrying two hydrogens from the two aromatic rings respectively. This process can be rationalized by two sequential electrophilic substitutions and cyclodeamination reaction based on the benzyl cation transfer. Deuterium-labeled experiments, density functional theory (DFT) calculation and substituent effect results also corroborate the proposed mechanism.

The fragmentation of b3 ions derived from protonated Arg-Xxx-Asp-Ala-Ala (Xxx = Ala, Asp, Glu, Cys) and Arg-Xxx-Glu-Ala-Ala was investigated by electrospray ionization tandem mass spectrometry (MSn) with collision-induced dissociation. A particular ion, which is 1 Da less than b2 ion, is shown to be the c2-H2O ion. The mechanism for its formation involved the aspartic acid in the third position easily losing anhydride to form a c2 ion, which then lost water to form an eight-membered ring of azacyclooctane derivative under the participation of the guanidine of the N-terminal arginine. However, this phenomenon was not observed when the aspartic acid was replaced by glutamic acid. The Amber program was used to determine the conformation of the original c2 residue from the dynamic energy perspective, and then density functional theory-based calculations and changing N-terminal amino acid from arginine to phenylalanine supported this mechanism.

Protonation and alkali-metal cation adduction are the most important ionization processes in soft-ionization mass spectrometry. Studies on the fragmentation mechanism of protonated and alkali-metal-cationized compounds in tandem mass spectrometry are essential and helpful for structural analysis. In some cases, it was often observed that a compound attached by different alkali-metal cations (or proton) exhibits similar fragmentation patterns but the relative abundances of product ions are different. This difference was considered to derive from the different electrostatic interactions of alkali-metal cations (or the bonded effect of proton) with the analyte. The alkali-metal cation with a smaller ionic radius shows stronger electrostatic interaction with the molecule because of its higher charge density. In addition, the bonded effect of the proton is stronger than the electrostatic interaction of the alkali-metal cation. In the present study, which used McLafferty-type rearrangements of even-electron ions ([M + Cat]+, Cat = H, Li, Na, K) as model reactions, the effect of cation size in mass spectrometric fragmentation reactions is highlighted. These considerations were also successfully applied to interpret the similar but distinct fragmentation behavior of proton and alkali-metal cation adducts of a synthetic compound (2-(acetamido(phenyl)methyl)-3-oxobutanoate) and a drug (entecavir).

•Eight triterpenoids were isolated from Lysimachia parvifolia for the first time.•Four compounds showed significant cytotoxicities on tested human cancer cell lines.•Cytotoxic activity is directly proportional to the number of sugars.•Hydroxylation of C-16 and hydroxymethylation of Me-23 could improve the cytotoxicity.Five new oleanane-type triterpenoids, including two aglycones, 13β-28-epoxy-3β,22α,23-trihydroxyolean-16-one (1) and 13β-28-epoxy-22α,23-dihydroxyolean-3,16-dione (2), and three glycosides, anagalligenone-3-O-α-l-arabinopyranoside (3), anagalligenone-3-O-[β-d-glucopyranosyl(1 → 4)-α-l-arabinopyranoside] (4) and anagalligenone-3-O-[β-d-xylopyranosyl(1 → 2)-β-d-glucopyranosyl(1 → 4)-α-l-arabinopyranoside] (5), were isolated from the aerial parts of Lysimachia parvifolia, together with three known oleanane-type triterpenoid glycosides (6–8). The structures of the new compounds were subsequently elucidated by spectroscopic analysis and their cytotoxicities evaluated against six human cancer cell lines. Compounds 5–8 exhibited significant cytotoxicities against all the cell lines tested, with IC50 values lower than 10 μM. The possible mechanism of action of compound 6 was also studied.Five new oleanane-type triterpenoids, together with three known oleanane-type triterpenoid glycosides were isolated from “Lysimachia parvifolia” for the first time and some of them showed significant cytotoxicities.

Horseradish Peroxidase (HRP) is a commercially available and prevalently used peroxidase with no specific substrate binding domain. However, after being incorporated with different metal cations, new catalytic functions were found in biomimetic oxidation of resveratrol. Based on the results of screening, Ca, Cu, Fe and Mn incorporated enzymes showed distinctive effects, either decomposition or dimerization products were observed.

The fragmentation reactions of Cinchona alkaloid-based quaternary ammonium cations were characterized by collision-induced dissociation mass spectrometry. Besides the common elimination of water and the formation of benzyl cations, the title compounds show two distinct fragmentation reactions resulting from intramolecular benzyl cation transfer. In one channel, the benzyl cation is transferred from the initial site (nitrogen) to the hydroxyl group leading to elimination of the corresponding benzyl alcohol. In another channel, precursor ion first dissociates into an intermediate ion–neutral complex consisting of the benzyl cation and the neutral partner. The intra-complex electrophilic aromatic substitution through benzyl cation attack leads to the formation of protonated benzylquinoline. Such dissociation occurs after the benzyl group migrates from a site with the highest benzylation nucleophilicity (nitrogen) to a different one (hydroxylic oxygen or aromatic ring carbon), which is a typical example of dissociative benzyl cation transfer.Graphical abstractHighlights► Benzyl cation transfers from the initial site to the hydroxyl group leading to loss of the corresponding benzyl alcohol. ► The intra-complex electrophilic aromatic substitution through benzyl cation attack leads to the formation of protonated benzylquinoline. ► We found two typical examples of dissociative benzyl cation transfer.

For p-(dimethylamino)chalcone (p-DMAC), the N atom is the most basic site in the liquid phase, whereas the O atom possesses the highest proton affinity in the gas phase. A novel and interesting observation is reported that the N- and O-protonated p-DMAC can be competitively produced in atmospheric pressure chemical ionization (APCI) with the change of solvents and ionization conditions. In neat methanol or acetonitrile, the protonation is always under thermodynamic control to form the O-protonated ion. When methanol/water or acetonitrile/water was used as the solvent, the protonation is kinetically controlled to form the N-protonated ion under conditions of relatively high infusion rate and high concentration of water in the mixed solvent. The regioselectivity of protonation of p-DMAC in APCI is probably attributed to the bulky solvent cluster reagent ions (SnH+) and the analyte having different preferred protonation sites in the liquid phase and gas phase.

This paper reports here Nazarov cyclization and oxo-Diels–Alder reaction induced by the naked silver cation in the collisional activation of silver(I)/chalcone complexes, which lead to the final elimination of AgOH and AgH, respectively. Deuterium labeling experiments, DFT calculations, and the substituent effect were utilized to confirm the reaction mechanisms.

Collision-induced dissociation (CID) of protonated N-benzylindoline and its derivatives was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Elimination of benzene was observed besides hydride transfer and electron transfer reactions. D-labeling experiments and accurate mass determinations of the product ions confirm that the external proton is retained in the fragment ion, and the elimination reaction was proposed to be initiated by benzyl cation transfer rather than proton transfer. Benzyl cation transfer from the nitrogen atom to one of the sp2-hybridized carbon atoms in the indoline core is the key step, and subsequent proton transfer reaction leads to the elimination of benzene. Density functional theory (DFT)-based calculations were performed and the computational results also support the benzyl cation/proton transfer mechanism.

A selective, concise and green biomimetic synthesis strategy of seven resveratrol dimers from natural resveratrol monomer as starting material is described. By succinct adjustment of environmental pH, the enzyme used, horseradish peroxidase (HRP), can perform in three distinctly different patterns and five dimers form with considerable selectivity. Two other derivative dimers are subsequently synthesized.

The polar compounds such as alkaloid compounds are important bioactive components in traditional Chinese medicines. In present study, a comprehensive method for separation and analysis of polar compounds from the polar fraction of traditional Chinese medicine Stephania yunnanensis was established. Both the major components and minor components were analyzed by counter-current chromatography combined with liquid chromatography tandem mass spectrometry (LC–MSn). From 50 mg polar fraction of crude extract, 15.2 mg corydine and 4.8 mg stepharine with purities over 90% were successfully separated via a polar solvent system n-butanol: methanol: water (4:1:5, v/v) with 10 mM NaOH as an additive in the lower phase, in one step operation. Their structures were further identified by 1H NMR and FTICR–MS. Besides, three minor components were identified by HPLC–MSn based on the fragmentation behavior of the purified compounds.

In mass spectrometry of protonated N-phenylcinnamides, the carbonyl oxygen is the thermodynamically most favorable protonation site and the added proton is initially localized on it. Upon collisional activation, the proton transfers from the carbonyl oxygen to the dissociative protonation site at the amide nitrogen atom or the α-carbon atom, leading to the formation of important reactive intermediates. When the amide nitrogen atom is protonated, the amide bond is facile to rupture to form ion/neutral complex 1, [RC6H4CHCHCO+/aniline]. Besides the dissociation of the complex, proton transfer reaction from the α-carbon atom to the nitrogen atom within the complex takes place, leading to the formation of protonated aniline. The presence of electron-withdrawing groups favored the proton transfer reaction, whereas electron-donating groups strongly favored the dissociation (aniline loss). When the proton transfers from the carbonyl oxygen to the α-carbon atom, the cleavage of the Cα–CONHPh bond results in another ion/neutral complex 2, [PhNHCO+/RC6H4CHCH2]. However, in this case, electron-donating groups expedited the proton transfer reaction from the charged to the neutral partner to eliminate phenyl isocyanate. Besides the cleavage of the Cα–CONHPh bond, intramolecular nucleophilic substitution (a nucleophilic attack of the nitrogen atom at the β-carbon) and stepwise proton transfer reactions (two 1,2-H shifts) also take place when the α-carbon atom is protonated, resulting in the loss of ketene and RC6H5, respectively. In addition, the H/D exchanges between the external deuterium and the amide hydrogen, vinyl hydrogens and the hydrogens of the phenyl rings were discovered by D-labeling experiments. Density functional theory-based (DFT) calculations were performed to shed light on the mechanisms for these reactions.

Ligand exchange reactions of [Ni2+ M (SCH3)−]+ (m/z 315, CI-a), (M = ArCH = NNHC(=S)SCH3), with the residual ESI solvent molecules in the ion trap have been investigated by electrospray ionization (ESI) mass spectrometry and quantum calculation. The intermediates, including [Ni2+ M (SCH3)− (CH3CN)]+ (m/z 356) and [Ni2+ (M−H)−]+ (m/z 267), in both the associative and the dissociative pathways have been obtained in the tandem MS, indicating that the two mechanistic pathways are efficient in the ligand exchange reactions. The electronic effect of the phenyl substituent on the reactivity of [Ni2+ (M*−H)−]+ has also been investigated by mass selection in the MS4 experiments. Intra-complex proton transfer to the anion ligand (SCH3)− is essential for the ligand exchange reactions. D-labeling experiments confirmed that the transferring proton comes from either the thioamide proton, or the phenyl proton via Csp2-H activation leading to cyclometallated complexes. DFT calculations at uB3LYP/6-31+G(2d, p) level gave the key species and the potential energy surfaces for the model reaction system of CI-a and acetonitrile.

In this study, the fragmentation reactions of various N-benzylammonium and N-benzyliminium ions were investigated by electrospray ionization mass spectrometry. In general, the dissociation of N-benzylated cations generates benzyl cations easily. Formation of ion/neutral complex intermediates consisting of the benzyl cations and the neutral fragments was observed. The intra-complex reactions included electrophilic aromatic substitution, hydride transfer, electron transfer, proton transfer, and nucleophilic aromatic substitution. These five types of reactions almost covered all the potential reactivities of benzyl cations in chemical reactions. Benzyl cations are well-known as Lewis acid and electrophile in reactions, but the present study showed that the gas-phase reactivities of some suitably ring-substituted benzyl cations were far richer. The 4-methylbenzyl cation was found to react as a Brønsted acid, benzyl cations bearing a strong electron-withdrawing group were found to react as electron acceptors, and para-halogen-substituted benzyl cations could react as substrates for nucleophilic attack at the phenyl ring. The reactions of benzyl cations were also related to the neutral counterparts. For example, in electron transfer reaction, the neutral counterpart should have low ionization energy and in nucleophilic aromatic substitution reaction, the neutral counterpart should be piperazine or analogues. This study provided a panoramic view of the reactions of benzyl cations with neutral N-containing species in the gas phase.

Collision-induced dissociation (CID) of Li+ adducts of three sets of compounds that contains an amide bond, including 2-(4, 6-dimethoxypyrimidin-2-ylsulfanyl)-N-phenylbenzamide, its derivatives and simpler structures was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Observed fragment ions include those that reflect loss of LiOH. Other product ions result from the Smiles rearrangement and direct C–S bond cleavage. MS/MS of H/D exchange products demonstrated occurrence of a 1,3-H shift from the amide nitrogen atom to the phenyl ring of these compounds. The LiOH loss from Li+ adducts of amides was further examined by CID of [M + Li]+ ions of N-phenylbenzamide and N-phenylcinnamide. Loss of LiOH was essentially the sole fragmentation reaction observed for the former. For the latter, both losses of LiOH and H2O were discovered. The presence of electron-donating substituents of the phenyl ring of these compounds was found to facilitate elimination of LiOH, while that loss was retarded by electron-withdrawing substituents. Proposed fragment ion structures were supported by elemental compositions deduced from ultrahigh resolution Fourier transform ion cyclotron resonance tandem mass spectrometry (FTICR-MS/MS) m/z value determinations. Density functional theory-based (DFT) calculations were performed to evaluate potential mechanisms for these reactions.

In collisional activation of protonated N-benzylaniline, the benzene loss from the benzyl moiety is actually not the result of dissociative proton transfer (PT). In fact, benzyl cation transfer (BCT) from the nitrogen to the anilinic ring (ortho or para position) is the key step for benzene loss. Such dissociation occurs only after the benzyl group migrating from the site with the highest benzylation nucleophilicity (nitrogen) to a different one (aromatic ring carbon), which is described as dissociative benzyl cation transfer.

Two previously unknown resveratrol trimers named wilsonols A–B, as well as a resveratrol tetramer named wilsonol C, were isolated from Vitis wilsonae Veitch, together with 12 known oligostilbenes. Their chemical structures have been elucidated by detailed analyses of 1D and 2D NMR spectroscopic data, as well as chemical evidence obtained via either catalysis with HRP (horseradish peroxidase) and H2O2 (hydrogen peroxide), acid, or UV irradiation. During the chemical processes, a biomimetic resveratrol tetramer named diviniferin B that has not been found in nature was obtained. These oligostilbenes showed potent scavenging abilities towards DPPH radicals and selective quenching effects on 1O2 radicals. Furthermore, the biogenetic transformations between the 16 oligostilbenes have been elaborated chemically to provide a comprehensive mechanism of the antioxidative defense system in this plant species.Graphical abstractPhytochemical investigation of Vitis wilsonae Veitch resulted in isolation of three oligostilbenes (1–3) and a biomimetic oligostilbene 4, whose structures were identified by 1D, 2D NMR spectroscopic and chemical means. All compounds showed selective quenching capacities towards 1O2.Highlights► HPLC/ESI-MSn-guided isolation has been utilized to obtain compounds of different molecular masses. ► Four compounds including a biomimetic one were obtained from Vitis wilsonae. ► A biogenetic transformation has been achieved by HRP–H2O2, acid, and UV irradiation treatment. ► The compounds showed selective quenching effects towards 1O2 compared with less effect on O2·− and OH. ► The compounds showed potent inhibitory effects in response to oxidative stress caused by 1O2.

The dissociation chemistry of primary fragment ions from the protonated proline-containing tripeptides glycylprolylglycine, prolylglycylglycine, and prolylprolylglycine was investigated by electrospray ionization multi-stage mass spectrometry. Calculations showed the a2 ions generated from b2 ions were cyclic, which is energetically more favorable than the linear form. The prolyl residue in the structure affected the energy hypersurface of the dissociation reaction from the b2 ion to the a2 ion. In the fragmentation of a2 ions, the iminium-imine complex corresponding to loss of CO from the a2 ion was suggested to be an ion-neutral complex (INC). The a1 ion was generated from direct separation of this INC, and the internal iminium ion, which was absent in PGG, was generated from another INC that was formed from the first INC via proton-bridged complex-mediated intramolecular proton transfer. Although these intermediates are unstable, their existence is supported by experiments and density functional theory calculations.

During the positive-ion mode electrospray ionization mass spectrometry analysis of 1,4-diphenyl-3-benzoyl-1,4-dihydropyridines with methanol as the solvent, the major ion generated is one mass unit lower than the analyte, which can be rationalized as the oxidative aromatization of 1,4-dihydropyridines to pyridine cations.

An on-line column-switching counter-current chromatography (CCC) with solid-phase trapping interphase is presented in this paper. The large volume injection is avoided using solid-phase trapping interphase. Thereby, totally different chemical composition biphasic solvent system can be utilized to enhance system orthogonality. In the present work, a 140 mL-capacity CCC instrument was used in the first dimension, and a parallel three-coil CCC centrifuge (40 mL each coil) was used in the second dimensional separation allowing three injections at the same time. With biphasic solvent system composed of n-hexane: ethyl acetate: methanol: water (1:1:1:1, v/v), five well-separated fractions were obtained in the first dimension. Two fractions were online concentrated and further separated in the second dimension with solvent system composed of methyl tert-butyl ether: acetonitrile: water (2:2:3, v/v), where trifluoroacetic acid (10 mM) was added to the upper organic phase as a retainer and triethylamine (10 mM) to the aqueous mobile phase as an eluter. Four hydroxyanthraquinones were successfully separated and purified from Chinese medicinal plant Rheum officinale in only one step.

The dissociation chemistry of the ortho-, meta- or para-isomers of protonated S-methyl methoxyl- (or chloro-) benzenylmethylenehydrazine dithiocarboxylate, RPhCHN–NHC(S)–SCH3 (R = MeO– or Cl–), has been investigated by collision induced dissociation experiments and DFT theoretical calculations. The three methoxyl-substituted isomers were easily differentiated according to the different abundance of the characteristic ion at m/z 136, resulting from the varying reactivity of the (NSC)SCH3 elimination. This fragmentation is triggered by the positive charge upon protonation on the imine N2. Relative to the meta isomer, the positive charge on N2 in the para isomer is dispersed due to the electron donating resonance of the methoxyl group, which leads to higher energy barrier in the dissociation reaction and the less abundant product ion (m/z 136) in the MS/MS. (NSC)SCH3 elimination of the ortho- isomer is further suppressed due to both the resonance effect and the “ortho effect” (an intramolecular hydrogen bond), with much higher energy barrier and extremely lower abundance of the fragment ion (m/z 136, 0.4%). The chloro substituted isomers, however, are short of the above positional effects due to the weak electronic effect of the substituent, and share the similar tandem mass spectrum.The ortho, meta or para methoxyl-substituted isomers were differentiated by ESI tandem MS according to the different abundance of the fragment ion (m/z 136), due to the different positional effect on the (NSC)SCH3 elimination.

Single electron transfer (SET) via ion/neutral complex (INC) was proposed and confirmed to be the key step in the formation of N-centered odd-electron ions from fragmentation of protonated even-electron ions in the present study. Upon collisional activation, the model compounds, protonated N,N′-dibenzylpiperazine and protonated N-benzylpiperazines initially dissociated to form intermediate INCs consisting of N-benzylpiperazine (or piperazine) and benzyl cation. In these ion/neutral complexes, SET reaction and direct separation as well as other reactions were observed and characterized experimentally and theoretically. Density functional theory calculations demonstrated that the energy requirement for homolysis of the precursor ion was so large that it could not be achieved, whereas the heterolytic dissociation followed by electron transfer via INC was energetically preferred. The SET process occurred only when the radical products were more stable than the separation products. The energy barrier for SET in the compounds studied was roughly estimated by comparison with other competing reactions. When the INC contained electron donor with lower ionization energy and electron acceptor with higher electron affinity, the SET reaction was more efficient.

2-(Phenylthio)phenols were successfully synthesized from simple phenols and aromatic halides by using dimethyl sulfoxide as the oxidant. The transformation was accomplished via tandem copper(I)-catalyzed C−S coupling/C−H functionalization employing the CuI/L [L = (E)-3-(dimethylamino)-1-(2-hydroxyphenyl)prop-2-en-1-one] catalyst system. The mechanism of the reaction was elucidated based on an isotope labeling strategy.

An integrated online column-switching countercurrent chromatography (CCC) with a solid-phase trapping/preconcentration interface is presented. The interface is systematically evaluated in terms of sorbent type, column size, and kinetic factor from the view of the unique CCC process. Results indicate that satisfactory trapping efficiency can be achieved using a 25 mm × 10 mm i.d. column packed with Oasis HLB materials. In addition to the analyte focusing effect, large volume injection is avoided, thereby allowing the use of totally different biphasic liquid systems to enhance the system orthogonality. The present integrated system simply combines the liquid and solid supports and is successfully applied in a one-step preparative separation of four antioxidative compounds from the ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia L. (Rubiaceae), exhibiting great advantages in peak resolution, peak capacity, and instrument integration compared with conventional CCC separations.

Resveratrol and its oligomers, abundantly present in wine grapes, are believed to be effective phytoalexins for the phenomenon “French paradox” partially by virtue of their powerful antiradical properties. EPR spin-trapping technique was utilized, demonstrating all polyphenols were selective 1O2 quenchers but not effective •OH and O2•¯ scavengers. On the basis of the HPLC-ESI-MS2 analysis for the simulated reactions of polyphenols with 1O2, the molecular weights of the resulting photochemical products were 14 or 28 Da higher than those of their substrates. No fragment C2H2O (42 Da), which was rather distinctive of the resorcinol rings in these cases, had been observed, whereas their MS/MS spectra displayed characteristic neutral fragments including carbon monoxide (CO, 28 Da) and 2-hydroxy[1,4]benzoquinone (C6H4O3, 124 Da). Finally, PM3 semiempirical calculations and HR-FTICR-MS experiments were performed, supporting the assertion that their quenching mechanism involved physical and chemical pathways. Chemical quenching underwent an endoperoxide intermediate form to generate quinones.

The dissociation chemistry of the protonated S-methyl benzenylmethylenehydrazine dithiocarboxylate, PhCHNNHC(S)SCH3, has been investigated by collision-induced dissociation (CID) mass spectrometry experiments in combination with density functional theory (DFT) calculations. Eliminations of H2S, CH3SH and (NSC)SCH3 were the three fragmentation reactions observed in the tandem mass spectra, witnessed by the MS/MS analysis of native 34S-isotopic ion and the D-labeling CID-MS experiment. Of the three fragmentations, both the added proton and the internal thiocarbamide hydrogen shift to the fragment ion (m/z 106) in the dissociation of losing (NSC)SCH3, while both of them shift to the neutral fragment H2S to generate the minor product ion at m/z 177. In the case of the feasible fragmentation process of CH3SH elimination, one of the proton/the thiocarbamide hydrogen migrates to the fragment ion at m/z 163, and the other migrates to the neutral specie. Calculated results show that thiocarbamide sulfur (S5) is the most thermodynamically favored position for protonation. The mechanisms of these reactions were postulated according to the theoretical results, and the reaction energy profiles were also constructed. These results indicated that fragmentation of the protonated molecule was viewed as a result of the coordinated migration of both the external proton and the thiocarbamide hydrogen.The added proton and the thiocarbamide hydrogen have different transferring directions in the fragmentations: fragment ion, neutral product, or both.

In this paper, five isoquinoline alkaloids were successfully separated from a crude extract of Stephania yunnanensis using pH-zone-refining counter-current chromatography in single-step. With a two-phase solvent system composed of methyl-tert-butyl ether (MtBE)–acetonitrile–water (2:2:3, v/v) where triethylamine (10 mM) was added to the upper organic phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.4 g crude extract, 68.7 mg isocorydine, 78.2 mg corydine, 583.4 mg tetrahydropalmatine, 36.3 mg N-methylasimilobine, and 47.3 mg anonaine were separated with purities over 90%. Their structures were identified by 1H NMR, 13C NMR, ESI-MS data.

The application of ionic liquids based microwave-assisted extraction (ILMAE) was successfully developed for extracting three alkaloids N-nornuciferine, O-nornuciferine, and nuciferine from lotus leaf. Seven kinds of 1-alkyl-3-methylimidazolium with different cations and anions were investigated in this work and 1.0 M 1-hexyl-3-methylimidazolium bromide ([C6MIM]Br) solution was selected as solvent. In addition, the microwave parameters including irradiation power, extraction time and solid–liquid ratio were optimized. Compared with the regular MAE and conventional heat-reflux extraction (HRE), the proposed approach exhibited higher efficiency (0.9–43.7% enhanced) and shorter extraction time (from 2 h to 2 min), which indicated ILMAE was an efficient, rapid and simple sample preparation technique. Moreover, the proposed method was validated by the linearity, reproducibility, and recovery experiments. Good linearity was observed with the regression coefficients (r2) between 0.9998 and 0.9999. The recoveries of all methods were in the range of 94.6% and 105.5% with RSD lower than 6.6%, which indicated that the proposed method was credible.

By applying water as the reaction medium, the one-pot synthesis of 1,2-disubstituted benzimidazoles has been achieved in excellent efficiency and selectivity at room temperature viatrimethylsilyl chloride promoted reaction of o-phenylenediamine with aldehyde. This green catalyst system has also been successfully extended to the synthesis of quinoxalines via the reaction of o-phenylenediamine with α-bromoketone. Water displayed a specific functionality in mediating the selectivity, and remarkable advantages over organic solvents in terms of yields as well as in the work up procedure of the reactions.

A novel and facile cascade Biginelli-like assembly employing enaminone, aldehyde and urea/thiourea has been developed, which provides a highly chemo- and regioselective synthesis of new dihydropyrimidinones, 1,3-thiazines and chromones by altering particular functional groups in the reactants.

In countercurrent chromatography (CCC) the choice of the liquid system is the heart of any separation. It corresponds to the selection of the mobile phase and the stationary phase at the same time. Any change in one phase composition induces a change in the other phase composition which renders the choice of the appropriate liquid system difficult and lengthy. A scale of compositions of the heptane−ethyl acetate−methanol−water quaternary liquid system was referred to by letters from A to Z and called the Arizona (AZ) liquid system. Each composition of the AZ system has the same heptane/ethyl acetate and methanol/water volume ratios. It is shown that there is a continuous polarity change from the hydrophilic A composition (ethyl acetate−water) to the hydrophobic Z (heptane−methanol) mixture by measuring the distribution constant KD of a known test mixture. For all compounds, the log KD is linearly increasing with the water content of the lower aqueous phase of the composition used. The slopes of the log KD versus percent H2O have very different values which means that the chromatographic selectivity changes with liquid system compositions. The AZ system was associated to the elution−extrusion method to design a procedure to identify rapidly the appropriate solvent composition able to fractionate correctly a complex natural extract. With the use of an integrated three-coil CCC column (40 mL each coil) able to test three AZ compositions in parallel, it is shown that the optimum AZ composition is found in half a day using less than a liter total volume of solvents. Two natural extracts are rapidly screened using the proposed protocol. An extract of Piper longum L. of intermediate polarity was fractionated in five usable portions using the 3/2/3/2 (Q) composition of the AZ system. A polar extract of Polygonum cuspidatum was also separated in five fractions using the 1/6/1/6 (D) composition. In both cases, a 140 mL CCC column was used for a direct scale-up transfer with the same liquid system. Purified fractions were subjected to an antioxidant activity assay and liquid chromatography with UV and mass spectrometry detection (LC-UV/MS) analysis to determine the molecular weight, number, and quantity of compounds in the active fractions. Four fractions of P. cuspidatum showed excellent antioxidant activity. They were rapidly produced at the milligram level by the 140 mL CCC column and fractionated by semipreparative high-performance liquid chromatography (HPLC) in individual compounds that were each identified by NMR and MS and reevaluated for confirmation of bioactivity. The rapid screening CCC protocol associated to the preparative capability of CCC allows for a fast identification and characterization of active compounds in natural products.

A rapid approach has been developed for screening trace level compounds with antitumor activities based on their interactions with microtubules. This interaction can be quantified with liquid chromatography (LC) by measuring the difference of bioactive compound's concentration before and after the formation of compound–microtubule complexes in the fast dialyzers. To test the effectiveness of this approach, several antitumor drugs such as colchicine, taxol, daunorubicin, and a non-antitumor reagent ketoprofen were used. Results indicate that the antitumor constituents can be identified without any disturbance, and the inactive components can be excluded. This screening method was then successfully applied to some potential antitumor compound mixtures, and the active compounds could be separated and screened rapidly. The binding activities measured were consistent with their cytotoxicity assays. This integrative approach is rapid and convenient for screening, isolating, and analyzing potential antitumor active compounds from a mixture.

Traditional Chinese medicines (TCMs) have attracted much attention in recent years. Elution-extrusion and/or back-extrusion counter-current chromatography (EECCC/BECCC) both take full advantage of the liquid nature of the stationary phase. They effectively extend the solute hydrophobicity window that can be studied and rendered the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, a popular traditional Chinese medicine, Evodia rutaecarpa, was used as the target complex mixture for extrusion CCC separations. With a carefully selected biphasic liquid system (n-hexane/ethyl acetate/methanol/water, 3/2/3/2, v/v) and optimized conditions (VCM = VC, mobile phase flow rate: 3 mL/min in descending mode, sample loading: 100 mg), five fractions could be obtained in only 100 min on a 140-mL capacity CCC instrument using both elution- and back-extrusion methods. Each fraction was analyzed and identified compared with the data of major standards using LC/MS. Moreover, the performance of both extrusion protocols was systematically compared and summarized. EECCC could be operated continuously and was found extremely suitable for high-throughput separation; however, post-column addition of a clarifying reagent is recommended to smooth the UV-signal during the extrusion process. Considering BECCC, the practical operation is very simple by just switching a 4-port valve to change the flow direction. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode so that solutes with small and medium distribution constants have been eluted. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column, mix together and produce a broad peak showing in the mobile phase eluting after the stationary phase extrusion. Compared with classical CCC or other preparative separation tools, extrusion CCC approaches exhibit distinguished superiority in the modernization process of traditional Chinese medicines.

The three-component sequential reaction of α,β-unsaturated aldehydes, amines, and enaminones proceeded smoothly to give 1,3,4-trisubstituted 1,4-dihydropyridines in aqueous DMF. Moreover, the unexpected regioselective formation of 1,2-dihydropyridines has been observed for the first time in such an approach. On the basis of a systematic study, the novel regioselectivity could be assigned both to steric and electronic effects originating from the amine partner.

The liquid chromatography/electrospray tandem mass spectrometry method was developed for analyses and characterization of oleanane-type saponins in the roots of Stephanotis mucronata. Collision-induced dissociation have been performed in order to elucidate the degradation pathways for the [M+Na]+ ions and their predominant product ions. The following characteristic fragmentation was observed from the title saponins: when the substituent (except hydroxyl group) located at C21 position, direct loss of terminal sugar residue from the [M+Na]+ ion was observed; when the substituent (except hydroxyl group) linked to C22 position, the loss of the terminal sugar cannot occur. To further confirm the structures, offline Fourier transform ion cyclotron resonance tandem mass spectrometry (FTICR-MSn) was performed. The structures of four proposed saponins have been identified by 1D and 2D NMR experiments. In this study, 12 saponins were identified. Among them, to our knowledge, four saponins were reported in this plant for the first time and four saponins were presumed to be new compounds. Hence, the described method has wide applicability to rapidly screen and provide structural information of oleanane-type saponins in crude materials.

The main feature of counter-current chromatography (CCC) is that the stationary phase is a liquid as well as the mobile phase. The retention volumes of solutes are directly proportional to their distribution coefficients KD in the biphasic liquid system used in the CCC column. Solutes with high KD coefficients are highly retained in the column. The back-extrusion method (BECCC) uses the fact that the liquid stationary phase, that contains the retained solutes, can be easily moved. Switching the column inlet and outlet ports without changing the liquid phase used as the mobile phase causes the rapid collapse of the two immiscible liquid phases inside the column, the previously stationary phase being gathered at the new column outlet. Then this previously stationary liquid phase is extruded outside the CCC column carrying the retained solutes. The back-extrusion method is tested with a standard mixture of five compounds and compared with the recently described elution–extrusion method. It is shown that the chromatographic resolution obtained during the back-extrusion step is good because the solute band broadening is minimized as long as the solute is located inside the “stationary” phase. However, a major drawback of the BECCC method is that all solutes are split between the liquid phases according to their distribution ratios when the CCC column equilibrium is broken. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode, eluting solutes with small and medium distribution ratios. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column and produce a broad peak showing after the stationary phase extrusion.

Elution-extrusion counter-current chromatography (EECCC) takes full advantages of the liquid nature of the stationary phase. It effectively extends the solute hydrophobicity window that can be studied and renders the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, EECCC was used to screen the crude ethanol extract of Zingiber cassumunar and to isolate milligram-amounts of bioactive components. The two column volume (2VC) EECCC method was applied to rapidly optimize the composition of the biphasic liquid system in both reversed- and normal-phase separation mode. With the n-hexane/ethyl acetate/methanol/water 1/1/1/1 (v/v) system, 100 mg of crude Z. cassumunar extract were fractionated on a 140 mL-capacity semi-preparative hydrodynamic CCC column and 0.5 g on a 1600 mL column for large-scale preparation. Satisfactory separation efficiency was achieved in both cases, producing milligram-amounts of four phenylbutenoids over 90% pure and of a mixture of diastereoisomers (phenylbutenoid dimers). However, the global throughputs of the two columns were 8 and 11 mg/h, not very different. This is due to the fact that the 1600 mL column could not retain the liquid stationary phase as well as the smaller 140 mL column. It was necessary to work at much lower flow rate than calculated. Methanol was added as a post-column clarifying reagent for stable continuous UV detection. A lipophilic biphasic liquid system composed of n-hexane/acetonitrile/water (5/3/2, v/v) allowed to resolve the pair of diastereoisomers with the larger preparative instrument producing 35 mg of the (±)-trans form 99.1% pure and 28 mg of the (±)-cis isomer 98.1% pure. Compared with classical elution, the EECCC approach exhibits strong separation efficiency and great potential to be a high-throughput separation technique in the case of complex samples.

Thermophilic proteins show substantially higher intrinsic thermal stability than their mesophilic counterparts. Amino acid composition is believed to alter the intrinsic stability of proteins. Several investigations and mutagenesis experiment have been carried out to understand the amino acid composition for the thermostability of proteins. This review presents some generalized features of amino acid composition found in thermophilic proteins, including an increase in residue hydrophobicity, a decrease in uncharged polar residues, an increase in charged residues, an increase in aromatic residues, certain amino acid coupling patterns and amino acid preferences for thermophilic proteins. The differences of amino acids composition between thermophilic and mesophilic proteins are related to some properties of amino acids. These features provide guidelines for engineering mesophilic protein to thermophilic protein.

A facile method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC/(+)ESI-MSn) has been established for the analysis of polyoxypregnane glycosides in the stems of Marsdenia tenacissima. The data reveals the ability of MSn in the structural elucidation of polyoxypregnane glycosides including the nature of the polyoxypregnane core, the kinds of the substituents and the types of sugar residues. Offline Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is also performed to assign accurate elemental compositions. In this study, eighteen polyoxypregnane glycosides have been investigated. Among these components, five compounds are unambiguously identified as Marsdenoside K, Tencissoside A, B, C and D; two compounds are established as novel compounds based on mass spectral data; and the other eleven compound's structures are tentatively proposed. Furthermore, breakdown curves are constructed to distinguish five pairs of isomers among these eighteen compounds. As far as our knowledge, this is the first report on identification of polyoxypregnane glycosides in the stems of M. tenacissima by HPLC/ESI-MSn directly, which could save time and material consuming efforts in traditional phytochemistry analyses.

A two-dimensional counter-current chromatographic system (2D-CCC) for preparative isolation and purification of three prenylflavonoids from Artocarpus altilis is presented. An upright CCC instrument (CCC1, total capacity: 1600 ml) was used as the first dimension. Effluent of interest from CCC1 was collected on-line into a 30 ml sample loop by a laboratory-prepared column-switching interface and introduced into a high-speed CCC instrument (CCC2, total capacity: 210 ml) for the second dimension separation. With this 2D-CCC system and a pair of two-phase solvent systems composed of n-hexane–ethyl acetate–methanol–water (5:5:7:3 and 5:5:6.5:3.5, v/v/v/v), which had been selected by high-speed CCC, about a 500 mg amount of the crude extract was separated, yielding 9 mg of compound 1, 28 mg of compound 2 and 78 mg of compound 3. The purities of the three prenylflavonoids were 98.7 (1), 98.3 (2) and 97.2% (3), respectively, as determined by HPLC analysis. Their chemical structures were identified by electrospray ionization MS, 1H NMR and 13C NMR.

The negative ions of deprotonated 2-(4, 6-dimethoxypyrimidin-2-ylsulfanyl)-N-phenylbenzamide and its derivatives are studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Upon collisional activation, the [M−H]− ions dissociate in two competitive pathways that can be considered as the gas-phase Smiles rearrangement reactions, giving rise to the characteristic fragment ions [M−H−C7H4OS]− and [M−H−C13H8NSR]− (R=substituent). Theoretical computations were invoked to shed light on the reaction mechanisms of the representative Compound 1 by the semiempirical PM3 method. These theoretical calculations show that the formation of [M−H−C13H8NSR]− (R=H for Compound 1) is more favorable. Furthermore, it is found that the intensities of the two product ions are strongly influenced by the position and the nature of the substituents. For the para-substituted compounds, the ln[(M−H−C7H4OS−)/(M−H−C13H8NSR−)] values are well correlated with the σp− substituent constants. In addition, the dependence of the intensity ratios of these two ions, ln[(M−H−C7H4OS−)/(M−H−C13H8NSR−)](R=CH3), on the collision energy can be used to distinguish the positional isomers.

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC/ESI–MS/MS) was used to identify C-21 steroidal glycosides with immunological activities in roots of Cynanchum chekiangense. In the MS/MS spectra, fragmentation reactions of the [M + Na]+ were recorded to provide structural information about the glycosyl and aglycone moieties. To further confirm the fragments structures, off-line Fourier transform ion cyclotron resonance tandem mass spectrometry (FT-ICR–MS/MS) was also performed. In the study, four known steroidal glycosides cynascyroside C, chekiangensosides A and B, glaucoside H, and four novel steroidal glycosides chekiangensosides C, D, E and chekiangensoside A isomer were identified based on mass spectral data, NMR spectral data and standards. This is the first report on identifying steroidal glycosides in roots of C. chekiangense by HPLC/ESI–MS/MS directly, which could save time and material consuming efforts in traditional phytochemistry analysis.

•Novel chiral ionic liquids were used as the chiral sensor for the first time.•Color changes and precipitate formation were visually determined.•Responsive mechanism and the structures of the precipitate were both discussed.•Other racemates were also successfully examined by this method.Novel task-specific ionic liquids derived from (S)-phenyl amino acid were prepared as a chiral sensor for the first time. It was found that the ionic liquids exhibited different chiral visual responses to the enantiomers. When (S)-tryptophan was treated with the sensor and Cu(II) in water, the clear solution would change to a sapphire suspension. Differently, another enantiomer could not produce the precipitate. Furthermore, the enantiomers could generate different colors in DMSO under the same conditions. The substantial difference contributes to the easy discrimination with visual determination. Four racemates were successfully tested with this strategy. The precipitation was isolated in the range of 90–94% yields after complete formation of the coordinative bonds, which could be as a simple way for enantioseparation. The responsive mechanism of the sensor was studied by NMR spectroscopy, FTIR and SEM. UV-vis spectrophotometer was used to monitor the colorimetric signal of the racemate with different e.e. values.

•A novel impurity was discovered and identified in fungicide pyraclostrobin.•The possible fragmentation mechanisms of pyraclostrobin and the impurity in electrospray ionization mass spectrometry was proposed.•A rapid and effective method for separation and identification of low-level impurities in bulk drug was established.Pyraclostrobin is one kind of new type methoxy acrylate fungicides that has been widely used in agriculture at present, with a lot of advantages including broad spectrum, high efficiency and high selectivity. In this work, a novel low-level impurity in the pyraclostrobin at about 0.2% was separated and characterized by high-performance liquid chromatography/tandem mass spectrometry (HPLC–MS). Firstly, the impurity was speculated to possess the same skeleton structure as the main product pyraclostrobin while the methyl group on the methyl ester was substituted to be CH2CH2Cl on the basis of the on-line multi-stage mass spectrometric behaviors compared with that of pyraclostrobin. Then the accurate molecular weight and element composition of target impurity was verified to be C20H19Cl2N3O4 by high resolution mass spectrometry. Finally, the proposed structure was further confirmed by the 1H NMR data.

In mass spectrometry of protonated N-phenylcinnamides, the carbonyl oxygen is the thermodynamically most favorable protonation site and the added proton is initially localized on it. Upon collisional activation, the proton transfers from the carbonyl oxygen to the dissociative protonation site at the amide nitrogen atom or the α-carbon atom, leading to the formation of important reactive intermediates. When the amide nitrogen atom is protonated, the amide bond is facile to rupture to form ion/neutral complex 1, [RC6H4CHCHCO+/aniline]. Besides the dissociation of the complex, proton transfer reaction from the α-carbon atom to the nitrogen atom within the complex takes place, leading to the formation of protonated aniline. The presence of electron-withdrawing groups favored the proton transfer reaction, whereas electron-donating groups strongly favored the dissociation (aniline loss). When the proton transfers from the carbonyl oxygen to the α-carbon atom, the cleavage of the Cα–CONHPh bond results in another ion/neutral complex 2, [PhNHCO+/RC6H4CHCH2]. However, in this case, electron-donating groups expedited the proton transfer reaction from the charged to the neutral partner to eliminate phenyl isocyanate. Besides the cleavage of the Cα–CONHPh bond, intramolecular nucleophilic substitution (a nucleophilic attack of the nitrogen atom at the β-carbon) and stepwise proton transfer reactions (two 1,2-H shifts) also take place when the α-carbon atom is protonated, resulting in the loss of ketene and RC6H5, respectively. In addition, the H/D exchanges between the external deuterium and the amide hydrogen, vinyl hydrogens and the hydrogens of the phenyl rings were discovered by D-labeling experiments. Density functional theory-based (DFT) calculations were performed to shed light on the mechanisms for these reactions.

Cu+–benzyne complexes bearing bidentate nitrogen ligands were synthesized in the gas phase for the first time using electrospray ionization mass spectrometry. The addition reactivity of copper-stabilized benzyne with amines was studied in the ion trap analyzer. The structures of products were identified by comparing their MSn data with authentic compounds obtained from another generation route.

A concise strategy to improve the p-NPP (p-nitrophenyl palmitate) catalytic activity and enantioselectivity towards secondary alcohols of Pseudomonas cepacia lipase (PcL) has been described. The PcL was modified by I3−, N-acetyl imidazole (NAI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and ethylenediamine (EDA) in the absence or presence of n-hexane, respectively. After being modified by the four modification reagents, the enantioselectivity (E value) of the PcL towards secondary alcohols was enhanced by 2- to 4-fold. The catalytic activity of EDA-PcL was increased by about 6-fold. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of modified PcL showed that Tyr4, Tyr29, Tyr45, Tyr95, Asp36 and Asp55 were the modified sites. When Tyr29 was modified, the E value of PcL towards secondary alcohols was largely improved. MALDI-TOF-MS characterization and molecular dynamics simulation of the lipase indicated that Tyr29 located inside the catalytic cavity had a significant impact on the E value. The strong steric hindrance of acetyl and iodine ion to the groups on the chiral center of the substrates is responsible for the improvement. In addition, the enhancement of hydrophobicity on the surface of the lipase due to the sidechain replacement of Asp with uncharged hydrophobic groups also improved the E value.

A copper catalyzed direct α-oxyacylation of ketones with carboxylic acids has been developed. Various acids including aromatic and aliphatic acids are well tolerated, and the scope of ketones is also investigated. This protocol provides a convenient and benign method for the synthesis of α-acyloxycarbonyl compounds by using oxygen as the oxidant. In addition, we have investigated the role of copper and trapped the key intermediate to confirm the mechanism.

Copper-mediated direct S–N formation using readily available starting materials via an oxygen-activated radical process has been developed. This method provides a novel and direct approach for synthesis of sulfonamides under air conditions.

A novel and facile cascade Biginelli-like assembly employing enaminone, aldehyde and urea/thiourea has been developed, which provides a highly chemo- and regioselective synthesis of new dihydropyrimidinones, 1,3-thiazines and chromones by altering particular functional groups in the reactants.

In this study, we report a gas-phase benzyl anion transfer via intramolecular aromatic nucleophilic substitution (SNAr) during the course of tandem mass spectrometry of deprotonated N-(phenylsulfonyl)-benzeneacetamide. Upon collisional activation, the formation of the initial ion/neutral complex ([C6H5CH2−/C6H5SO2NCO]), which was generated by heterolytic cleavage of the CH2–CO bond, is proposed as the key step. Subsequently, the anionic counterpart, benzyl anion, is transferred to conduct the intra-complex SNAr reaction. After losing neutral HNCO, the intermediate gives rise to product ion B at m/z 231, whose structure is confirmed by comparing the multistage spectra with those of deprotonated 2-benzylbenzenesulfinic acid and (benzylsulfonyl)benzene. In addition, intra-complex proton transfer is also observed within the complex [C6H5CH2−/C6H5SO2NCO] to generate product ion C at m/z 182. The INC-mediated mechanism was corroborated by theoretical calculations, isotope experiments, breakdown curve, substituent experiments, etc. This work may provide further understanding of the physicochemical properties of the gaseous benzyl anion.

The benzylsilver cation which emerges from the collisional dissociation of silver(I)–N-benzylbenzamide complexes was characterized by deuterium-labeling experiments, theoretical calculations, breakdown curves and substituent effects. The nucleophilic attack of the carbonyl oxygen on an α-hydrogen results in the generation of the benzylsilver cation, which is competitive to the AgH loss with the α-hydrogen.