Charge Detection Mass Spectrometry for Megadalton Polymer Characterization and Measurement of Electrospray-Generated Charged Droplet Dynamics with a New "Direct Visualization of the Rayleigh Limit" Approach to Aid in m/z Calibration.

A charge detector has been constructed and mounted inside the vacuum housing of a commercial mass spectrometer (Micromass-Waters Quattro I, Waters Corp., Manchester, UK). The in-house built single-pass charge detector is composed of a designed, complete electronics system that includes a low-noise charge amplifier. Communication to the data acquisition system was enabled, and analog and digital filters were devised, followed by their tuning and programming. Data treatment scripts for data analysis and plotting were automated, and the assembled system was calibrated and tested. The instrument has an acquisition speed of ∼200 detection events/s, and it permits detection down to ∼510 charges (= three times RMS noise) for a single measured particle. The charge detector was employed to determine the oligomer distribution of a megadalton polymer, polyethylene glycol (PEG). The PEG size distribution exhibits a maximum at ∼ m/z 5910 with the oligomeric population mass distribution peaking near 4.45 MDa. In studies of methanol droplet dynamics, "charge vs time-of-flight" plots enabled clear visualization of the zone near the Rayleigh limit to droplet charging. The highest population of methanol droplets near the Rayleigh limit carried 5000-7000 charges. This corresponds to droplet weights of 10-20 GDa, with the high-end tail extending above 70 GDa. This visualization of the most highly charged droplets (that bear numbers of charges near those defined by the Rayleigh equation) was exploited as a calibration aid for our charge detector, which lacks a means of precisely defining ion energy. A maximum m/z error of -12.3% was calculated for the method, i.e., less than the potential error in assigning the true level of charging of the most highly charged droplets relative to the Rayleigh limit. With these limitations in mind, the introduced method will provide a new means for aiding the calibration of m/z values in charge detectors.

Bond-Dissociation Energies to Probe Pyridine Electronic Effects on Organogold(III) Complexes: From Methodological Developments to Application in π-Backdonation Investigation and Catalysis.

In this work, we report on the synthesis of several organogold(III) complexes based on 4,4'-diterbutylbiphenyl (C^C) and 2,6-bis(4-terbutylphenyl)pyridine (C^N^C) ligands and bond with variously substituted pyridine ligands (pyrR). Altogether, 33 complexes have been prepared and studied with mass spectrometry using higher-energy collision dissociation (HCD) in an Orbitrap mass spectrometer. A complete methodology including the kinetic modeling of the dissociation process based on the Rice-Ramsperger-Kassel-Marcus (RRKM) statistical method is proposed to obtain critical energies E0 of the pyrR loss for all complexes. The capacity of these E0 values to describe the pyridine ligand effect is further explored, at the same time as more classical descriptors such as 1H pyridinic NMR shift variation upon coordination and Au-NpyrR bond length measured by X-ray diffraction. An extensive theoretical work, including density functional theory (DFT) and domain-based local pair natural orbital coupled-cluster theory (DLPNO-CCSD(T)) methods, is also carried out to provide bond-dissociation energies, which are compared to experimental results. Results show that dissociation energy outperforms other descriptors, in particular to describe ligand effects over a large electronic effect range as seen by confronting the results to the pyrR pKa values. Further insights into the Au-NpyrR bond are obtained through an energy decomposition analysis (EDA) study, which confirms the isolobal character of Au+ with H+. Finally, the correlation between the lability of the pyridine ligands toward the catalytic efficiency of the complexes could be demonstrated in an intramolecular hydroarylation reaction of alkyne. The results were rationalized considering both pre-catalyst activation and catalyst reactivity. This study establishes the possibility of correlating dissociation energy, which is a gas-phase descriptor, with condensed-phase parameters such as catalysis efficiency. It therefore holds great potential for inorganic and organometallic chemistry by opening a convenient and easy way to evaluate the electronic influence of a ligand toward a metallic center.

Mechanism of Alkyne Hydroarylation Catalyzed by (P,C)-Cyclometalated Au(III) Complexes.

Over the last 5-10 years, gold(III) catalysis has developed rapidly. It often shows complementary if not unique features compared to gold(I) catalysis. While recent work has enabled major synthetic progress in terms of scope and efficiency, very little is yet known about the mechanism of Au(III)-catalyzed transformations and the relevant key intermediates have rarely been authenticated. Here, we report a detailed experimental/computational mechanistic study of the recently reported intermolecular hydroarylation of alkynes catalyzed by (P,C)-cyclometalated Au(III) complexes. The cationic (P,C)Au(OAcF)+ complex (OAcF = OCOCF3) was authenticated by mass spectrometry (MS) in the gas phase and multi-nuclear NMR spectroscopy in solution at low temperatures. According to density functional theory (DFT) calculations, the OAcF moiety is κ2-coordinated to gold in the ground state, but the corresponding κ1-forms featuring a vacant coordination site sit only slightly higher in energy. Side-on coordination of the alkyne to Au(III) then promotes nucleophilic addition of the arene. The energy profiles for the reaction between trimethoxybenzene (TMB) and diphenylacetylene (DPA) were computed by DFT. The activation barrier is significantly lower for the outer-sphere pathway than for the alternative inner-sphere mechanism involving C-H activation of the arene followed by migratory insertion. The π-complex of DPA was characterized by MS. An unprecedented σ-arene Au(III) complex with TMB was also authenticated both in the gas phase and in solution. The cationic complexes [(P,C)Au(OAcF)]+ and [(P,C)Au(OAcF)(σ-TMB)]+ stand as active species and off-cycle resting state during catalysis, respectively. This study provides a rational basis for the further development of Au(III) catalysis based on π-activation.

TUTORIAL: ION ACTIVATION IN TANDEM MASS SPECTROMETRY USING ULTRA-HIGH RESOLUTION INSTRUMENTATION.

Tandem mass spectrometry involves isolation of specific precursor ions and their subsequent excitation through collision-, photon-, or electron-mediated activation techniques in order to induce unimolecular dissociation leading to formation of fragment ions. These powerful ion activation techniques, typically used in between mass selection and mass analysis steps for structural elucidation, have not only found a wide variety of analytical applications in chemistry and biology, but they have also been used to study the fundamental properties of ions in the gas phase. In this tutorial paper, a brief overview is presented of the theories that have been used to describe the activation of ions and their subsequent unimolecular dissociation. Acronyms of the presented techniques include CID, PQD, HCD, SORI, SID, BIRD, IRMPD, UVPD, EPD, ECD, EDD, ETD, and EID. The fundamental principles of these techniques are discussed in the context of their implementation on ultra-high resolution tandem mass spectrometers. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

[(C C)Au(N N)]+ Complexes as a New Family of Anticancer Candidates: Synthesis, Characterization and Exploration of the Antiproliferative Properties.

A library of eleven cationic gold(III) complexes of the general formula [(C C)Au(N N)]+ when C C is either biphenyl or 4,4'-ditertbutyldiphenyl and N N is a bipyridine, phenanthroline or dipyridylamine derivative have been synthesized and characterized. Contrasting effects on the viability of the triple negative breast cancer cells MDA-MB-231 was observed from a preliminary screening. The antiproliferative activity of the seven most active complexes were further assayed on a larger panel of human cancer cells as well as on non-cancerous cells for comparison. Two complexes stood out for being either highly active or highly selective. Eventually, reactivity studies with biologically meaningful amino acids, glutathione, higher order DNA structures and thioredoxin reductase (TrxR) revealed a markedly different behavior from that of the well-known coordinatively isomeric [(C N C)Au(NHC)]+ structure. This makes the [(C C)Au(N N)]+ complexes a new class of organogold compounds with an original mode of action.

Exploring Phosphine Electronic Effects on Molybdenum Complexes: A Combined Photoelectron Spectroscopy and Energy Decomposition Analysis Study.

In organometallic chemistry, especially in the catalysis area, accessing the finest tuning of a catalytic reaction pathway requires a detailed knowledge of the steric and electronic influences of the ligands bound to the metal center. Usually, the M-L bond between a ligand and metal is depicted by the Dewar-Chatt-Duncanson model involving two opposite interactions, σ-donor and π-acceptor effects of the ligand. The experimental evaluation of these effects is essential and complementary to in-depth theoretical approaches that are able to provide a detailed description of the M-L bond. In this work, we present a study of LMo(CO)5 complexes with L being various tertiary phosphine ligands by means of mass-selected high-resolution photoelectron spectroscopy (PES) performed with synchrotron radiation, DFT, and energy decomposition analyses (EDA) combined with the natural orbitals for chemical valence (NOCV) analysis. These methods enable a separated access of the σ-donor and π-acceptor effects of ligands by probing either the electronic configuration of the complex (PES) or the interaction of the ligand with the metal (EDA). Three series of PR3 ligands with various electronic influences are investigated: the strong donating alkyl substituents (PMe3, PEt3, and PiPr3), the intermediate PPhxMe(3-x) (x = 0-3) set, and the PPhxPyrl(3-x) set (x = 0-3 with Pyrl being the strong electron withdrawing pyrrolyl group C4H4N). For each complex, their adiabatic and vertical ionization energies (IEs) could be determined with a 0.03 eV precision. Experiment and theory show an excellent agreement, either for the IE determination or electronic effect analysis. The ability to interpret the spectra is shown to depend on the character of the ligand. "Innocent" ligands provide the spectra that are the most straightforward to analyze, whereas the "non-innocent" ligands (which are ionized prior to the metal center) render the analysis more difficult due to an increased number of molecular orbitals in the energy range considered. A very good linear correlation is finally found between the measured adiabatic ionization energies and the interaction energy term obtained by EDA for each of these two types of ligands, which opens interesting perspective for the prediction of ligand characters.

PCR Incorporation of Polyoxometalate Modified Deoxynucleotide Triphosphates and Their Application in Molecular Electrochemical Sensing of Yersinia pestis.

Redox-labeled nucleotides are of increasing interest for the fabrication of next generation molecular tools and should meet requirements of being thermally stable, sensitive, and compatible with polymerase-mediated incorporation while also being electrochemically discriminable. The synthesis and characterization of Keggin and Dawson polyoxometalate-deoxynucleotide (POM-dNTP) bioconjugates linked through 7-deaza-modified purines is described. The modified POM-dNTPs were used for polymerase-based amplification of a DNA sequence specific for Yersinia pestis and the amplified DNA detected using an electrochemical DNA sensor. This highlights the potential of polyoxometalates as thermally stable, sensitive and polymerase-compatible redox labels for exploitation in bioanalytical applications.

Lithium Amide Protected against Hydrolysis by Aggregated Lithium Halides: An MS + DFT Investigation.

Supported by mass spectrometry experiments, DFT computations indicate that the lithium amide of a 3-aminopyrrolidine (lithium benzhydryl(1-benzylpyrrolidin-3-yl)amide, 1-Li) is protected, up to a certain limit, against hydrolysis when it is aggregated with a strongly polar partner such as LiCl, LiBr, or MeLi.

Cellular response to alkylating agent MNNG is impaired in STAT1-deficients cells.

The SN 1 alkylating agents activate the mismatch repair system leading to delayed G2 /M cell cycle arrest and DNA repair with subsequent survival or cell death. STAT1, an anti-proliferative and pro-apoptotic transcription factor is known to potentiate p53 and to affect DNA-damage cellular response. We studied whether STAT1 may modulate cell fate following activation of the mismatch repair system upon exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Using STAT1-proficient or -deficient cell lines, we found that STAT1 is required for: (i) reduction in the extent of DNA lesions, (ii) rapid phosphorylation of T68-CHK2 and of S15-p53, (iii) progression through the G2 /M checkpoint and (iv) long-term survival following treatment with MNNG. Presence of STAT1 is critical for the formation of a p53-DNA complex comprising: STAT1, c-Abl and MLH1 following exposure to MNNG. Importantly, presence of STAT1 allows recruitment of c-Abl to p53-DNA complex and links c-Abl tyrosine kinase activity to MNNG-toxicity. Thus, our data highlight the important modulatory role of STAT1 in the signalling pathway activated by the mismatch repair system. This ability of STAT1 to favour resistance to MNNG indicates the targeting of STAT1 pathway as a therapeutic option for enhancing the efficacy of SN1 alkylating agent-based chemotherapy.

Chiral Phosphate in Rhodium-Catalyzed Asymmetric [2+2+2] Cycloaddition: Ligand, Counterion, or Both?

Investigations based on NMR spectroscopy, mass spectrometry, and DFT calculations shed light on the metallic species generated in the rhodium-catalyzed asymmetric [2+2+2] cycloaddition reaction between diynes and isocyanates with the chiral phosphate TRIP. The catalytic mixture comprising [{Rh(cod)Cl}2 ], 1,4-diphenylphosphinobutane (dppb), and Ag(S)-TRIP actually gives rise to two species, both having an effect on the stereoselectivity. One is a rhodium(I) complex in which TRIP is a weakly coordinating counterion, whereas the other is a bimetallic Rh/Ag complex in which TRIP is a strongly coordinating X-type ligand.

Biofunctionalization of Polyoxometalates with DNA Primers, Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products.

The bioconjugation of polyoxometalates (POMs), which are inorganic metal oxido clusters, to DNA strands to obtain functional labeled DNA primers and their potential use in electrochemical detection have been investigated. Activated monooxoacylated polyoxotungstates [SiW11 O39 {Sn(CH2 )2 CO}](8-) and [P2 W17 O61 {Sn(CH2 )2 CO}](6-) have been used to link to a 5'-NH2 terminated 21-mer DNA forward primer through amide coupling. The functionalized primer was characterized by using a battery of techniques, including electrophoresis, mass spectrometry, as well as IR and Raman spectroscopy. The functionality of the POM-labeled primers was demonstrated through hybridization with a surface-immobilized probe. Finally, the labeled primers were successfully used in the polymerase chain reaction (PCR) and the PCR products were characterized by using electrophoresis.

A Lithium Amide Protected Against Protonation in the Gas Phase: Unexpected Effect of LiCl.

In cold THF and in the presence of LiCl, a lithium pyrrolidinylamide forms a 1:1 mixed aggregate, which is observed directly by ESI-MS. Gas-phase protonation of this species leads to selective transfer of H(+) to the chlorine, suggesting that LiCl shields the amide nitrogen and prevents its direct protonation.

Vacuum ultraviolet photoionization study of gas phase vitamins A and B1 using aerosol thermodesorption and synchrotron radiation.

Gas-phase studies of biomolecules are often difficult to initiate because of the thermolability of these systems. Such studies are nevertheless important to determine fundamental intrinsic properties of the molecules. Here we present the valence shell photoionization of gas-phase vitamins A and B1 close to their ionization threshold. The study was performed by means of an aerosol thermodesorption source coupled to an electron/ion coincidence spectrometer and synchrotron radiation (SOLEIL facility, France). Ion yield curves were recorded for both molecules over a few electronvolt energy range and the threshold photoelectron spectrum was also obtained for vitamin A. Some fundamental properties were extracted for both ions such as adiabatic and the three first vertical ionization energies of retinol (IEad = 6.8 ± 0.2 eV and IEvert = 7.4, 8.3, and 9.2 eV) and dissociation appearance energies for the main fragment ions of vitamin B1. Analysis of the data was supported by ab initio calculations which show a very good agreement with the experimental observations.

The Pauson-Khand mechanism revisited: origin of CO in the final product.

The mechanism of the Pauson-Khand reaction has been studied by mass spectrometry and it has been found, through ion-molecule reaction with (13) CO, that the carbon monoxide incorporated into the product cyclopentenone is one that has been retained within the complex. Theoretical and kinetic calculations support this finding, which provides a complementary explanation for the effect of Pauson-Khand promoters.

Enantioselective Reduction of Noncovalent Complexes of Amino Acids with CuII via Resonant Collision-Induced Dissociation: Collision Energy, Activation Duration Effects, and RRKM Modeling.

Formation of noncovalent complexes is one of the approaches to perform chiral analysis with mass spectrometry. Enantiomeric distinction of amino acids (AAs) based on the relative rate constants of competitive fragmentations of quaternary copper complexes is an efficient method for chiral differentiation. Here, we studied the complex [CuII,(Phe,PhG,Pro-H)]+ (m/z 493) under resonant collision-induced dissociation conditions while varying the activation time. The precursor ion can yield two main fragments through the loss of the non-natural AA phenylglycine (PhG): the expected product ion [CuII,(Phe,Pro-H)]+ (m/z 342) and the reduced product ion [CuI,(Phe,Pro)]+ (m/z 343). Enantioselective reduction describes the difference in relative abundance of these ions, which depends on the chirality of the precursor ion: the formation of the reduced ion m/z 343 is favored in homochiral complexes (DDD) compared to heterochiral complexes (such as LDD). Energy-resolved mass spectrometry data show that reduction, which arises from rearrangement, is favored at a low collision energy (CE) and long activation time (ActT), whereas direct cleavage preferentially occurs at a high CE and short ActT. These results were confirmed with kinetic modeling based on RRKM theory. For this modeling, it was necessary to set a pre-exponential factor as a reference, so that the E0 values obtained are relative values. Interestingly, these simulations showed that the critical energy E0 required to form the reduced ion is comparable in both homochiral and heterochiral complexes. However, the formation of product ion m/z 342 through direct cleavage is associated with a lower E0 in heterochiral complexes. Consequently, enantioselectivity would not be caused by enhanced reduction in homochiral complexes but rather by direct cleavage being favored in heterochiral complexes.

Energetics of key Au(III)-substrate adducts relevant to catalytic hydroarylation of alkynes.

(P,C)-cyclometalated Au(III) complexes have shown remarkable ability to catalyze the intermolecular hydroarylation of alkynes. Evidence of an outer-sphere mechanism has been provided in a previous study and is confirmed here by analysing the experimental data and DFT calculations. In this work, we propose evaluation of critical energies of dissociation of Au(III) complexes with different substrates via energy-resolved mass spectrometry (ERMS) experiments and kinetic modelling. The kinetic model is based on a multi-collisional approach. On the one hand, the classification confirms the mechanism previously proposed; on the other hand, it supports the collisional model and its application to particularly fragile adducts.

Formation and characterization of gaseous adducts of carbon dioxide to magnesium, (CO2)MgX- (X=OH, Cl, Br).

A good fix: the structure and chemical reactivity of a reduced form of CO(2) bonded to magnesium, XMg(η(2)-O(2)C)(-), is reported. Upon reaction with water it loses CO, while it adds CH(3) upon reaction with alkyl halides, thereby signifying nucleophilicity of the carbon atom in XMg(η(2)-O(2)C)(-) in S(N)2 reactions.

Benchmarking higher energy collision dissociation (HCD) by investigation of binding energies of gas-phase host-guest complexes of hemicryptophane cages.

Synthesis of host molecules that feature well-defined characteristics for molecular recognition of guest molecules is often a major aim of synthetic host-guest (H-G) chemistry. A key consideration in evaluating the selectivity of hosts and the affinities of guests is the measurement of binding energies of obtained H-G complexes. In contrast to nuclear magnetic resonance (NMR) or fluorescence measurements that are capable of measuring binding strengths in solution, mass spectrometry offers the opportunity to measure gas-phase binding energies. Presented in this article is a higher energy collision dissociation (HCD) approach for determining critical energies of dissociation of H-G complexes. Experiments were performed on electrospray ionization (ESI)-generated H-G pairs in an LTQ-XL/Orbitrap hybrid instrument. The presented HCD approach requires preliminary calibration of the internal energy distribution of generated ions that was achieved by the use of activation parameters that were known from previous low-energy collision-induced dissociation (low-energy CID) experiments. Internal energy deposition was modeled based on a truncated Maxwell-Boltzmann distribution and characteristic temperature (Tchar ). Using this method, critical energies of dissociation were determined for 10 H-G biologically relevant complexes of the heteroditopic hemicryptophane cage host (Host). Obtained results are compared with those found previously by low-energy CID. The use of this HCD technique is relatively straightforward, although its implementation does require knowledge (or a presumption) about the Arrhenius pre-exponential factor of the complexes to obtain their critical energies of dissociation.

A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB.

BACKGROUND: The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear. RESULTS: The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death. CONCLUSIONS: The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.

Gold- and platinum-catalyzed cycloisomerization of enynyl esters versus allenenyl esters: an experimental and theoretical study.

Ester-way to heaven: Unexpected formation of bicyclo[3.1.0]hexene 4 was the main focus of combined experimental and theoretical studies on the Au-catalyzed cycloisomerization of branched dienyne 1 (see scheme), which provided better understanding of the mechanistic details governing the cyclization of enynes bearing a propargylic ester group.Experimental and theoretical studies on Au- and Pt-catalyzed cycloisomerization of a branched dienyne with an acetate group at the propargylic position are presented. The peculiar architecture of the dienyne precursor, which has both a 1,6- and a 1,5-enyne skeleton, leads, in the presence of alkynophilic gold catalysts, to mixtures of bicyclic compounds 3, 4, and 5. Formation of unprecedented bicyclo[3.1.0]hexene 5 is the main focus of this study. The effect of the ancillary ligand on the gold center was examined and found to be crucial for formation of 5. Further mechanistic studies, involving cyclization of an enantioenriched dienyne precursor, (18)O-labeling experiments, and DFT calculations, allowed an unprecedented reaction pathway to be proposed. We show that bicyclo[3.1.0]hexene 5 is likely formed by a 1,3-OAc shift/allene-ene cyclization/1,2-OAc shift sequence, as calculated by DFT and supported by Au-catalyzed cyclization of isolated allenenyl acetate 7, which leads to improved selectivity in the formation of 5. Additionally, the possibility of OAc migration from allenyl acetates was supported by a trapping experiment with styrene that afforded the corresponding cyclopropane derivative. This unprecedented generation of a vinyl metal carbene from an allenyl ester supports a facile enynyl ester/allenenyl ester equilibrium. Further examination of the difference in reactivity between enynyl acetates and their corresponding [3,3]-rearranged allenenyl acetates toward Au- and Pt-catalyzed cycloisomerization is also presented.