Electrospray Ionization Mass Spectrometry Experiments Research Articles

Observation of Solvent-Dependence in the Mechanism of Neutral-Catalyzed Isomerization of para-Aminobenzoic Acid Protomers.

Proton-transfer reactions are commonplace during electrospray ionization (ESI) mass spectrometry experiments and are often responsible for imparting charge to analyte molecules. Multiple protonation-site isomers (protomers) can arise for polyfunctional molecules and these isomers can interconvert via solvent-mediated proton transfer reactions during various stages of the ESI process. Studying the populations and interconversion of protonation isomers provides key insight into the ESI process, ion-molecule interactions, and ion dissociation mechanisms. An archetype molecule to study protomer interconversion fundamentals in this context is para-aminobenzoic acid (pABA), where both the amino and carboxylic acid protomers are typically formed under ESI and the mechanisms for interconversion are still under refinement. Using ion-trap mass spectrometry reaction kinetics (2.5 mTorr, 300 K), this study examines gas-phase interconversion catalysis of pABA protomers by seven neutral species, which are commen solvents and additives used for ESI: water, formic acid, methanol, ethanol, propanol, ammonia, and acetonitrile. Three distinct reaction cases are reported: (i) formic acid, methanol, ethanol, propanol, and ammonia each catalyze the interconversion between the amino and carboxylic acid protomers via a n = 1 solvent-molecule vehicle mechanism; (ii) for water, however, a n = 6 adduct complex is detected and this suggests that the observed protomer interconversion occurs through a Grotthuss mechanism, in accord with literature reports; (iii) acetonitrile inhibits proton transfer by the formation of particularly stable n = 1 and 2 adduct complexes. The second-order rate constants for the protomer interconversion are observed to increase in the following order: H2O < HCO2H < MeOH < EtOH < PrOH < NH3. Potential energy schemes are reported for all neutral-catalyzed proton transfer reactions using the DSD-PBEP86-D3(BJ)/aug-cc-pVDZ level of theory. A central transition state, which connects the protonation site adducts, is shown to be the key rate-limiting step. The energy of this transition state is sensitive to the proton affinity of the neutral solvent, and this is supported by the correlation between the reaction rate and the solvent proton affinity.

Gas-phase F-atom migration reaction of deprotonated N-fluoroarenesulfonamides.

Recently N-Fluoroarenesulfonamides (ArSO2 NHF) were found to be promising precursors for the preparation of N-fluorobenzenesulfonimide derivatives without applying F2 . However, very few studies have discussed the mass spectrometric behaviors of ArSO2 NHF with N-F structure. In this article, we applied high-resolution electrospray ionization tandem mass spectrometry (HR-ESI-MS/MS) to study the effect on the mass spectrometric behaviors of ArSO2 NHF after the introduction of the F-atom to the N-atom of ArSO2 NH2 . High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) experiments showed that ArSO2 NHF produced only good signals in negative ion mode, and the dominating product ion SO2 F- at m/z 83 was observed in all HR-ESI-MS/MS of ArSO2 NF- with different substituents in the Ar group. The formation of the product ion SO2 F- was proof of the gas-phase F-atom migration rearrangement from the N-atom to the S-atom in ESI-MS/MS of ArSO2 NF- . To fully explain the gas-phase reaction mechanism from ArSO2 NF- to SO2 F- , we studied the HR-ESI-MS/MS of deprotonated ArSO2 NHF and also performed theoretical calculations. Both results confirmed that ArSO2 NF- first underwent Smiles rearrangement to yield intermediate I (INT1) ArNFSO2 - , and then the F-atom of ArNFSO2 - migrated from the N-atom to the S-atom to form intermediate II (INT2) ArN- SO2 F, which finally dissociated to SO2 F- at m/z 83 with loss of a neutral nitrene (ArN). All these results showed that the formation of the product ion SO2 F- from ArSO2 NF- was a common and intrinsic gas-phase reactivity of ArSO2 NF- .

Differentiation of aminohydroxypropanesulfonic acid structural isomers using tandem mass spectrometry-based methods

d-Cysteinolic acid (D-CA) is an important metabolite within the biosulfur cycle. A structural isomer, (R)-3-amino-2-hydroxypropanesulfonate ((R)-AHPS), is less common in nature but potentially can be misidentified as D-CA due to their many shared physical properties. To support confident assignment of these two isomers by use of mass spectrometry alone, this study explores the fragmentation reactions of their [M+H]+ and [M−H]− ions using collision-induced dissociation (CID). Electrospray ionization mass spectrometry (ESI-MS) experiments were conducted on authentic standards using an ion trap mass spectrometer, while a triple-quadrupole (QqQ) mass spectrometer was used in the selective reaction monitoring (SRM) mode to record energy-resolved CID. Density-functional theory (DFT) calculations were carried out at the M06/6-31+G* level of theory to study gas-phase fragmentation mechanisms. The data generated revealed kinetically-controlled fragmentations involving participation of neighboring amino groups in the positive ion mode. Negative ion mode MS analysis could distinguish the structural isomers through different collision energy-resolved results for m/z 95 product ions, CH3SO3−. DFT calculations revealed an enthalpy (ΔH) (Gibbs energy (ΔG)) gap of 31.8 (31.4) kJ/mol between transition state barriers of a concerted mechanism for D-CA, and a more preferred stepwise mechanism for (R)-AHPS.

Furanonaphthoquinones, Diterpenes, and Flavonoids from Sweet Marjoram and Investigation of Antimicrobial, Bacterial Efflux, and Biofilm Formation Inhibitory Activities.

The chloroform extract of Origanum majorana exhibited high antibacterial and antifungal activities against 12 bacterial and 4 fungal strains; therefore, it was subjected to bioassay-guided isolation to afford six compounds (1-6). The structures were determined via one- and two-dimensional nuclear magnetic spectroscopy and high-resolution electrospray ionization mass spectrometry experiments. The compounds were identified as furanonaphthoquinones [majoranaquinone (1), 2,3-dimethylnaphtho[2,3-b]furan-4,9-dione (2)], diterpenes [19-hydroxyabieta-8,11,13-trien-7-one (3), 13,14-seco-13-oxo-19-hydroxyabieta-8-en-14-al (4)], and flavonoids [sterubin (5) and majoranin (6)]. Compounds 1 and 2 were first obtained from a natural source and compounds 3 and 4 were previously undescribed. Majoranaquinone (1) exhibited a high antibacterial effect against 4 Staphylococcus, 1 Moraxella, and 1 Enterococcus strains (MIC values between 7.8 μM and 1 mM). In the efflux pump inhibition assay, majoranaquinone (1) showed substantial activity in Escherichia coli ATCC 25922 strain. Furthermore, 1 was found to be an effective biofilm formation inhibitor on E. coli ATCC 25922 and E. coli K-12 AG100 bacteria. Our findings proved that bioactivities of majoranaquinone (1) significantly exceed those of the essential oil constituents; therefore, it should also be considered when assessing the antimicrobial effects of O. majorana.

Polyporenic Acids from the Mushroom Buglossoporus quercinus Possess Chemosensitizing and Efflux Pump Inhibitory Activities on Colo 320 Adenocarcinoma Cells.

Polyporenic acids N-R (1-5), five novel 24-methylene lanostane triterpenes along with seven known polyporenic acids (6-12), were identified from the fruiting bodies of Buglossoporus quercinus. The isolation of compounds 1-12 was performed by a combination of multistep flash chromatography and reversed-phase high-performance liquid chromatography (HPLC). The structure determination was carried out by extensive spectroscopic analysis, including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) experiments. The isolated fungal metabolites were investigated for their antiproliferative activity in vitro by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on the resistant Colo 320 human colon adenocarcinoma cell line expressing P-glycoprotein (ABCB1). The lanostane triterpenes exerted moderate antiproliferative activity with IC50 values in the range of 20.7-106.2 μM. A P-glycoprotein efflux pump modulatory test on resistant Colo 320 cells highlighted that fungal metabolites 3, 5, 8, and 10-12 have the ability to inhibit the efflux pump activity of cancer cells. Moreover, the drug interactions of triterpenes with doxorubicin were studied by the checkerboard method. Compounds 3-4, and 7-12 interacted in a synergistic manner, while an outstanding potency was detected for compound 9, which was defined as strong synergism (CI = 0.276). The current study reveals that B. quercinus is a remarkable source of fungal steroids with considerable chemosensitizing activity on cancer cells.

Diserinol Isophthalamide: A Novel Reagent for Complexation with Biomolecular Anions in Electrospray Ionization Mass Spectrometry

Transferring biomolecules from solution to vacuum facilitates a detailed analysis of molecular structure and dynamics by isolating molecules of interest from a complex environment. However, inherent in the ion desolvation process is the loss of solvent hydrogen bonding partners, which are critical for the stability of a condensed-phase structure. Thus, transfer of ions to vacuum can favor structural rearrangement, especially near solvent-accessible charge sites, which tend to adopt intramolecular hydrogen bonding motifs in the absence of solvent. Complexation of monoalkylammonium moieties (e.g., lysine side chains) with crown ethers such as 18-crown-6 can disfavor structural rearrangement of protonated sites, but no equivalent ligand has been investigated for deprotonated groups. Herein we describe diserinol isophthalamide (DIP), a novel reagent for the gas-phase complexation of anionic moieties within biomolecules. Complexation is observed to the C-terminus or side chains of the small model peptides GD, GE, GG, DF-OMe, VYV, YGGFL, and EYMPME in electrospray ionization mass spectrometry (ESI-MS) studies. In addition, complexation is observed with the phosphate and carboxylate moieities of phosphoserine and phosphotyrosine. DIP performs favorably in comparison to an existing anion recognition reagent, 1,1'-(1,2-phenylene)bis(3-phenylurea), that exhibits moderate carboxylate binding in organic solvent. This improved performance in ESI-MS experiments is attributed to reduced steric constraints to complexation with carboxylate groups of larger molecules. Overall, diserinol isophthalamide is an effective complexation reagent that can be applied in future work to study retention of solution-phase structure, investigate intrinsic molecular properties, and examine solvation effects.

Online protein unfolding characterized by ion mobility electron capture dissociation mass spectrometry: cytochrome C from neutral and acidic solutions.

Electrospray ionization mass spectrometry (ESI-MS) experiments, including ion mobility spectrometry mass spectrometry (ESI-IMS-MS) and electron capture dissociation (ECD) of proteins ionized from aqueous solutions, have been used for the study of solution-like structures of intact proteins. By mixing aqueous proteins with denaturants online before ESI, the amount of protein unfolding can be precisely controlled and rapidly analyzed, permitting the characterization of protein folding intermediates in protein folding pathways. Herein, we mixed various pH solutions online with aqueous cytochrome C for unfolding and characterizing its unfolding intermediates with ESI-MS charge state distribution measurements, IMS, and ECD. The presence of folding intermediates and unfolded cytochrome c structures were detected from changes in charge states, arrival time distributions (ATDs), and ECD. We also compared structures from nondenaturing and denaturing solution mixtures measured under "gentle" (i.e., low energy) ion transmission conditions with structures measured under "harsh" (i.e., higher energy) transmission. This work confirms that when using "gentle" instrument conditions, the gas-phase cytochrome c ions reflect attributes of the various solution-phase structures. However, "harsh" conditions that maximize ion transmission produce extended structures that no longer correlate with changes in solution structure.

Atomistic Modeling of Jet Formation in Charged Droplets.

The first atomistic simulations that reveal the mechanism of Rayleigh fission are presented. It is demonstrated that simple ion or macroion ejection takes place through droplet deformation from a spherical into a distinct "tear" shape that contains a conical protrusion. We assert that the latter state is a free-energy minimum along an order parameter that measures the degree of droplet asphericity. The charged droplet's long-time evolution proceeds by alternating between the two minima above and below the critical value that are reached through solvent evaporation and ion ejection, respectively. For the first time, this mechanism allows one to explain the nature of the progeny droplets and the percentage of charge lost during fission. The cone half angle is estimated and found to be in good agreement with the value predicted from the solution of the electrostatic equation for the dielectric liquid. It is found that the conical deformation is independent of the effect of electrohydrodynamic forces reported in experiments. Contrary to the experimental observations of two diametrically opposite jets for droplets suspended in the electric field, we find that a single jet is formed at the Rayleigh limit. The study provides insight into the mechanism of capture of a macroion in jets appearing in electrospray ionization mass spectrometry (ESI-MS) experiments and may explain the tolerance of the ESI-MS spectrum to salt contamination of the sample.

Screening of synthesis conditions for the development of a radium ion-imprinted polymer using the dummy template imprinting approach

In this study, a selective and specific sorbent for radium extraction was developed for the first time using ion-imprinted polymer technology. A novel and original approach was used in order to screen the best synthesis conditions. After identifying the best monomer candidates, porogen, and complexation time from electrospray ionization mass spectrometry experiments, solubility tests, and conductimetry experiments, five ion-imprinted polymers (IIPs) were synthesized by bulk polymerization using Ba2+ as a dummy template ion. Non-imprinted polymers (NIPs) were similarly prepared but without template ions. Polymers were packed in solid phase extraction (SPE) cartridges for their characterization. Composition of the percolation and washing solutions (e.g. buffer concentration, nature and pH, and ethanol proportion) was varied to achieve the highest retention for the template ion while minimizing that of interfering ions. The retention, selectivity (retention on IIP versus on NIP), and specificity (targeted ions versus interfering ions) properties of the different polymers were compared by measuring the recovery yields in the SPE collected fractions by inductively coupled plasma mass spectrometry (ICP-MS). The IIP synthesized in acetonitrile/dimethylsulfoxyde (1/1, v/v) using Ba2+ as template ion, vinylphosphonic acid as complexing monomer, and styrene and divinylbenzene as co-monomer and cross-linker showed both selectivity and specificity. It retained strongly Ra2+ while having the potential to isolate it from alkali metals, metalloids and some transition metals.

Chemical Investigation of Tetradium ruticarpum Fruits and Their Antibacterial Activity against Helicobacter pylori.

The fruit of Tetradium ruticarpum, known as Evodiae Fructus, is a traditional herbal medicine used to treat gastric and duodenal ulcers, vomiting, and diarrhea. The traditional usage can be potentially associated with the antibacterial activity of T. ruticarpum fruits against Helicobacter pylori. However, so far, the antibacterial activity of T. ruticarpum fruits and antibacterial components against H. pylori has not been investigated despite the traditional folk use. The current study was conducted to investigate the bioactive chemical components of T. ruticarpum fruits and evaluate their antibacterial activity against H. pylori. Phytochemical investigation of the EtOH extract of T. ruticarpum fruits led to the isolation and identification of nine compounds (1–9), including phellolactone (1), the absolute configuration of which has not yet been determined. The chemical structures of the isolated compounds were elucidated by analyzing the spectroscopic data from one-dimensional (1D) and two-dimensional (2D) NMR and high-resolution electrospray ionization mass spectrometry (HR-ESIMS) experiments. Specifically, the absolute configuration of compound 1 was established by the application of computational methods, including electronic circular dichroism (ECD) calculation and the NOE/ROE-based interproton distance measurement technique via peak amplitude normalization for the improved cross-relaxation (PANIC) method. In the anti-H. pylori activity test, compound 3 showed the most potent antibacterial activity against H. pylori strain 51, with 94.4% inhibition (MIC50 and MIC90 values of 22 and 50 μM, respectively), comparable to that of metronidazole (97.0% inhibition, and MIC50 and MIC90 values of 17 and 46 μM, respectively). Moreover, compound 5 exhibited moderate antibacterial activity against H. pylori strain 51, with 58.6% inhibition (MIC50 value of 99 μM), which was higher than that of quercetin (34.4% inhibition) as a positive control. Based on the bioactivity results, we also analyzed the structure–activity relationship of the anti-H. pylori activity. Conclusion: These findings demonstrated that T. ruticarpum fruits had antibacterial activity against H. pylori and could be used in the treatment of gastric and duodenal ulcers. Meanwhile, the active compound, 1-methyl-2-(8E)-8-tridecenyl-4(1H)-quinolinone (3), identified herein also indicated the potential application in the development of novel antibiotics against H. pylori.

RK-270D and E, Oxindole Derivatives from Streptomyces sp. with Anti-Angiogenic Activity.

A chemical investigation of a culture extract from Streptomyces sp. RK85-270 led to the isolation and characterization of two new oxindoles, RK-270D (1) and E (2). The structures of 1 and 2 were determined by analyzing spectroscopic and spectrometric data from 1D and 2D NMR and High-resolution electrospray ionization mass spectrometry (HRESIMS) experiments. Compound 1 exhibited anti-angiogenic activities against human umbilical vein endothelial cells (HUVECs) without cytotoxicity. Results of Western blot analysis revealed that 1 inhibits VEGF-induced angiogenesis in the HUVECs via VEGFR2/ p38 MAPK-mediated pathway.

An electrospray ionization study on complexes of amylin with Cu(II) and Cu(I).

Human amylin (hIAPP) is one of a number of different peptides known to be responsible for the formation of amyloid fibrils in the pancreas of subjects with Type 2 diabetes mellitus. It was recognized that metal ions such as Cu(II) are implicated in the aggregation process of amyloidogenic peptides. However, the role of Cu(II) ions in the aggregation and dyshomeostasis of amylin has been controversial. Considering that most of the research reported in the literature pertain to the interactions between Cu(II) and amylin, we thought of interest to compare the interactions of Cu(II) and Cu(I) ions with amylin by electrospray ionization (ESI) mass spectrometry and collisional experiments, to elucidate possible differences in structural aspects of the complexes so formed. The ESI mass spectra of solutions containing hIAPP and Cu(I) or Cu(II) ions show the formation of hIAPP-Cu complexes. In both cases, M + Cu ions with three and four positive charges are detected. However, a series of fragment ions, absent in the ESI spectrum of untreated hIAPP, become detectable. Some of them are common for both Cu(I) and Cu(II) complexes, whereas others are specific for the complexes containing Cu in different oxidation states. Some fragments imply the involvement of residues His18, Ser19, Ser20, Asn21, and Asn22 in the complex formation, but the detection of the fragment b22 3+ indicates the presence of copper ions in a different position. This suggests different interaction sites between Cu(II) and Cu(I) and hIAPP. In contrast to Cu(II) complex, in the Cu(I) complex, some peculiar structures are present, corresponding to the cleavage of Asn-Asn peptidic bond and to [b30 + Cu(I)]4+ and [b28 + Cu(I)]4+ species. These results are in agreement with the coordination vacancy in [Cu(I)-(peptide)] species, which promotes Cu(I) interaction with additional neighboring donors (mainly N-histidine, and also S-methionine or other groups depending on the peptide conformation) through formation of trigonal T-shaped intermediates.

Spectroscopic Studies of Synthetic Methylaluminoxane: Structure of Methylaluminoxane Activators.

Hydrolysis of trimethylaluminum (Me3 Al) in polar solvents can be monitored by electrospray ionization mass spectrometry (ESI-MS) using the donor additive octamethyltrisiloxane [(Me3 SiO)2 SiMe2 , OMTS]. Using hydrated salts, hydrolytic methylaluminoxane (h-MAO) features different anion distributions, depending on the conditions of synthesis, and different activator contents as measured by NMR spectroscopy. Non-hydrolytic MAO was prepared using trimethylboroxine. The properties of this material, which contains incorporated boron, differ significantly from h-MAO. In the case of MAO prepared by direct hydrolysis, oligomeric anions are observed to rapidly form, and then more slowly evolve into a mixture dominated by an anion with m/z 1375 with formula [(MeAlO)16 (Me3 Al)6 Me]- . Theoretical calculations predict that sheet structures with composition (MeAlO)n (Me3 Al)m are favoured over other motifs for MAO in the size range suggested by the ESI-MS experiments. A possible precursor to the m/z 1375 anion is a local minimum based on the free energy released upon hydrolysis of Me3 Al.

Enlightening binding behaviour of sulfonatocalix[4]arene receptor with 2-acetoxybenzoic acid through the lens of experiments and theory

The inclusion behaviour of 2-acetoxy benzoic acid (ASA, aspirin) within the para-sulfonatocalix[4]arene (SCX4) host has been characterized employing UV–visible, 1H NMR, vibrational spectroscopy, electrospray ionization mass spectrometry (ESI–MS) and chromatography experiments in conjunction with the density functional theory. It has been shown that the ASA ⊂ SCX4 complex possesses 1:1 stoichiometry and is more stable toward oxidative conditions than in acidic and basic media compared to the free ASA guest. The complexation of SCX4 with the ASA emerges with signature in the ‘frequency up-shift’ of its Ar-OH (~14 cm−1) and SO3− (~36 cm−1) stretching vibrations whereas the carbonyl stretching vibration from the ester group engenders shift in the opposite direction (red shift) in the measured infrared spectra. These inferences are corroborated through the density functional theory.

Cyclodextrin Encapsulated pH Sensitive Dyes as Fluorescent Cellular Probes: Self-Aggregation and In Vitro Assessments.

We have designed and synthesized a series of novel, supramolecular, long-lived fluorescent probes based on the host-guest inclusion complexes formation between fluorescent indolizinyl-pyridinium salts and β-cyclodextrin. Fluorescence and electrospray ionisation mass spectrometry experiments, supported by theoretical molecular docking studies, were utilized in the monitoring of the inclusion complexes formation, evidencing the appearance of corresponding 1:1 and 1:2 species. Additionally, the influence of the guest molecule over the aggregation processes of the cyclodextrin inclusion complexes was investigated by transmission electron microscopy. The absence of cytotoxicity, cellular permeability, long-lived intracellular fluorescence, and in time specific accumulation within acidic organelles identified the investigated supramolecular entities as remarkable candidates for intracellular fluorescence probes. Co-staining experiments using specific organelle markers revealed the fact that, after a 24-h incubation period, the inclusion complexes accumulate predominantly in lysosomes rather than in mitochondria. This study opens new possibilities for a broad range of fluorescent dyes with solubility and high toxicity issues, able to form inclusion complexes with β-cyclodextrin, to be tested as intracellular fluorescence probes.

Structure and Stability of the Ionic Liquid Clusters [EMIM]n[BF4]n+1- (n = 1-9): Implications for Electrochemical Separations.

Precise functionalization of electrodes with size-selected ionic liquid (IL) clusters may improve the application of ILs in electrochemical separations. Herein we report our combined experimental and theoretical investigation of the IL clusters 1-ethyl-3-methylimidazolium tetrafluoroborate [EMIM]n[BF4]n+1- (n = 1-9) and demonstrate their selectivity and efficiency toward targeted adsorption of ions from solution. The structures and energies of the IL clusters, predicted with global optimization, agree with and help interpret the ion abundances and stabilities measured by high-mass-resolution electrospray ionization mass spectrometry and collision-induced dissociation experiments. The [EMIM][BF4]2- cluster, which was identified as the most stable IL cluster, was selectively soft-landed onto a working electrode. Electrochemical impedance spectroscopy revealed a lower charge transfer resistance on the soft-landed electrode containing [EMIM][BF4]2- compared with an electrode prepared by drop-casting of an IL solution containing the full range of IL clusters. Our findings indicate that specific IL clusters may be used to increase the efficiency of electrochemical separations by lowering the overpotentials involved.

A reactive probe for Co2+ ion detection based on a catalytic decomposition process and its fluorescence imaging in living cells.

A novel reactive fluorescent probe for cobalt ions was prepared based on integration of thiourea functional groups, coumarin, and naphthalimide fluorophores. There was no fluorescence observed for the probe itself, however, in the presence of cobalt ions, catalytic decomposition occurred for the probe and coumarin molecular fragments were produced that emitted blue fluorescence. This enabled the probe to be used as a 'turn on' reagent for detection of cobalt ions. Under physiological pH conditions and in appropriate solvent systems, an obvious fluorescence enhancement for cobalt ions was observed in selective experiments. Competition experiments indicated that cobalt ions could still induce fluorescence enhancement in the presence of other metal ions. Sensitivity experiments showed that the detection limit for cobalt ions was 6.0 nM. Dynamics research demonstrated that the catalytic process was a pseudo-first-order reaction and the reaction constant (kobs ) was calculated to be 1.49 × 10-2 min-1 . In addition, the mechanism of catalytic decomposition could be demonstrated using electrospray ionization mass spectrometry and thin layer chromatography experiments. Cell fluorescence imaging experiments demonstrated that the probe could be used to detect cobalt ions in living HeLa cells.

Iminodiacetic Acid as a Novel Metal-Binding Pharmacophore for New Delhi Metallo-β-lactamase Inhibitor Development.

The fungal natural product aspergillomarasmine A (AMA) has been identified as a noncompetitive inhibitor of New Delhi metallo-β-lactamase-1 (NDM-1) that inhibits by removing ZnII from the active-site. The nonselective metal-chelating properties and difficult synthesis and derivatization of AMA have hindered the development of this scaffold into a potent and selective inhibitor of NDM-1. Iminodiacetic acid (IDA) has been identified as the metal-binding pharmacophore (MBP) core of AMA that can be leveraged for inhibitor development. Herein, we report the use of IDA for fragment-based drug discovery (FBDD) of NDM-1 inhibitors. IDA (IC50 =120 μM) was developed into inhibitor 23 f (IC50 =8.6 μM, Ki =2.6 μM), which formed a ternary complex with NDM-1, as evidenced by protein thermal-shift and native-state electrospray ionization mass spectrometry (ESI-MS) experiments. Combining mechanistic analysis with inhibitor derivatization, the use of IDA as an alternative AMA scaffold for NDM-1 inhibitor development is detailed.

Evaluation of structurally different brominated flame retardants interacting with the transthyretin and their toxicity on HepG2 cells.

Brominated flame retardants (BFRs) are found at quantifiable levels in both humans and wildlife and may potentially cause a health risk. For BFRs and their derivatives, limited information regarding the relationship among the structure, binding affinity to the target protein and toxicity is currently available. In the present work, representative BFRs with different hydroxyl- or bromo-substituents, namely 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47), 3-hydroxy-2, 2', 4, 4'-tetrabromodiphenyl ether (3-OH-BDE-47) and tetrabromobisphenol A (TBBPA), were selected to investigate the interactions with transthyretin (TTR) by electrospray ionization mass spectrometry (ESI-MS) and cytotoxicity on HepG2 cells. It was noted that BDE-47 had a weak binding affinity to TTR, while 3-OH-BDE-47 and TBBPA had a stronger binding affinity than BDE-47 and thyroxine (T4). Hence, 3-OH-BDE-47 and TBBPA could affect the binding of TTR with its native ligand T4 by competitive binding to TTR, even at equal concentrations, which might be associated with BFR toxicity of endocrine disruption. Negative cooperativity was found for 3-OH-BDE-47 and TBBPA binding to TTR, similar to T4 with a well-established negatively cooperative binding mechanism. The tendency of toxic effects on HepG2 cells for these three BFRs was, 3-OH-BDE-47>TBBPA>BDE-47, and this order was in good agreement with the binding ability explored by ESI-MS experiments and molecular docking simulation. The observations obtained by this study demonstrate that the binding properties of these BFRs to TTR and their cytotoxicity are correlated with structure differentials and functional substituents.

Synthesis via A3 Coupling Reaction of Anthracene‐Propargylamine as a New Scaffold for the Interaction with DNA

AbstractA series of new anthracene‐propargylamine derivatives were obtained through an efficient synthesis based on the A3 coupling reaction. This set of planar molecules were designed to bind double stranded and G‐quadruplex DNA arrangements. Their interaction pattern of the small molecules with DNA was predicted by molecular docking and their theoretical pharmacokinetic properties were calculated, giving encouraging results in terms of drug‐likeness. The general preference for double stranded DNA suggested by docking experiments was supported by a set of electrospray ionization mass spectrometry (ESI‐MS) experiments.