Probing Microbiome Genotoxicity: A Stable Colibactin Provides Insight into Structure-Activity Relationships and Facilitates Mechanism of Action Studies.

Colibactin is a genotoxic metabolite produced by commensal-pathogenic members of the human microbiome that possess the clb (aka pks) biosynthetic gene cluster. clb+ bacteria induce tumorigenesis in models of intestinal inflammation and have been causally linked to oncogenesis in humans. While colibactin is believed underlie these effects, it has not been possible to study the molecule directly due to its instability. Herein, we report the synthesis and biological studies of colibactin 742 (4), a stable colibactin derivative. We show that colibactin 742 (4) induces DNA interstrand-cross-links, activation of the Fanconi Anemia DNA repair pathway, and G2/M arrest in a manner similar to clb+E. coli. The linear precursor 9, which mimics the biosynthetic precursor to colibactin, also recapitulates the bacterial phenotype. In the course of this work, we discovered a novel cyclization pathway that was previously undetected in MS-based studies of colibactin, suggesting a refinement to the natural product structure and its mode of DNA binding. Colibactin 742 (4) and its precursor 9 will allow researchers to study colibactin's genotoxic effects independent of the producing organism for the first time.

Chemical Reduction of NiII Cyclam and Characterization of Isolated NiI Cyclam with Cryogenic Vibrational Spectroscopy and Inert-Gas-Mediated High-Resolution Mass Spectrometry.

NiII cyclam (cyclam = 1,4,8,11-tetraazacyclotetradecane) is an efficient catalyst for the selective reduction of CO2 to CO. A crucial elementary step in the proposed catalytic cycle is the coordination of CO2 to a NiI cyclam intermediate. Isolation and spectroscopic characterization of this labile NiI species without solvent has proven to be challenging, however, and only partial IR spectra have previously been reported using multiple photon fragmentation of ions generated by gas-phase electron transfer to the NiII cyclam dication at 300 K. Here, we report a chemical reduction method that efficiently prepares NiI cyclam in solution. This enables the NiI complex to be transferred into a cryogenic photofragmentation mass spectrometer using inert-gas-mediated electrospray ionization. The vibrational spectra of the 30 K ion using both H2 and N2 messenger tagging over the range 800-4000 cm-1 were then measured. The resulting spectra were analyzed with the aid of electronic structure calculations, which show strong method dependence in predicted band positions and small molecule activation. The conformational changes of the cyclam ligand induced by binding of the open shell NiI cation were compared with those caused by the spherical, closed-shell LiI cation, which has a similar ionic radius. We also report the vibrational spectrum of a NiI cyclam complex with a strongly bound O2 ligand. The cyclam ligand supporting this species exhibits a large conformational change compared to the complexes with weakly bound N2 and H2, which is likely due to significant charge transfer from Ni to the coordinated O2.

Base-Directed Photoredox Activation of C-H Bonds by PCET.

Photoredox catalysis using proton-coupled electron transfer (PCET) has emerged as a powerful method for bond transformations. We previously employed traditional chemical oxidants to achieve multiple-site concerted proton-electron transfer (MS-CPET) activation of a C-H bond in a proof-of-concept fluorenyl-benzoate substrate. As described here, photoredox oxidation of the fluorenyl-benzoate follows the same rate constant vs driving force trend determined for thermal MS-CPET. Analogous photoredox catalysis enables C-H activation and H/D exchange in a number of additional substrates with favorably positioned bases. Mechanistic studies support our hypothesis that MS-CPET is a viable pathway for bond activation for substrates in which the C-H bond is weak, while stepwise carboxylate oxidation and hydrogen atom transfer likely predominate for stronger C-H bonds.

Characterization of the non-covalent docking motif in the isolated reactant complex of a double proton-coupled electron transfer reaction with cryogenic ion spectroscopy.

The solution kinetics of a proton-coupled electron transfer reaction involving two-electron oxidation of a Ru compound with concomitant transfer of two protons to a quinone derivative have been interpreted to indicate the formation of a long-lived intermediate between the reactants. We characterize the ionic reactants, products, and an entrance channel reaction complex in the gas phase using high-resolution mass spectrometry augmented by cryogenic ion IR photodissociation spectroscopy. Collisional activation of this trapped entrance channel complex does not drive the reaction to products but rather yields dissociation back to reactants. Electronic structure calculations indicate that there are four low-lying isomeric forms of the non-covalently bound complex. Comparison of their predicted vibrational spectra with the observed band pattern indicates that the C=O groups of the ortho-quinone attach to protons on two different -NH2 groups of the reactant scaffold, exhibiting strong O-H-N contact motifs. Since collisional activation does not lead to the products observed in the liquid phase, these results indicate that the reaction most likely proceeds through reorientation of the H-atom donor ligand about the metal center.

Hidden role of intermolecular proton transfer in the anomalously diffuse vibrational spectrum of a trapped hydronium ion.

We report the vibrational spectra of the hydronium and methyl-ammonium ions captured in the C3v binding pocket of the 18-crown-6 ether ionophore. Although the NH stretching bands of the CH3NH3+ ion are consistent with harmonic expectations, the OH stretching bands of H3O+ are surprisingly broad, appearing as a diffuse background absorption with little intensity modulation over 800 cm-1 with an onset ∼400 cm-1 below the harmonic prediction. This structure persists even when only a single OH group is present in the HD2O+ isotopologue, while the OD stretching region displays a regular progression involving a soft mode at about 85 cm-1 These results are rationalized in a vibrationally adiabatic (VA) model in which the motion of the H3O+ ion in the crown pocket is strongly coupled with its OH stretches. In this picture, H3O+ resides in the center of the crown in the vibrational zero-point level, while the minima in the VA potentials associated with the excited OH vibrational states are shifted away from the symmetrical configuration displayed by the ground state. Infrared excitation between these strongly H/D isotope-dependent VA potentials then accounts for most of the broadening in the OH stretching manifold. Specifically, low-frequency motions involving concerted motions of the crown scaffold and the H3O+ ion are driven by a Franck-Condon-like mechanism. In essence, vibrational spectroscopy of these systems can be viewed from the perspective of photochemical interconversion between transient, isomeric forms of the complexes corresponding to the initial stage of intermolecular proton transfer.

Cooperatively enhanced hydrogen bonds in ionic liquids: closing the loop with molecular mimics of hydroxy-functionalized cations.

We address the cooperative hydrogen bonding interactions in play between the ionic constituents of ionic liquids (ILs) with particular attention to those involving the attractive interactions between two cations in the system 1-(2-hydroxyethyl)pyridinium tetrafluoroborate [HEPy][BF4]. This is accomplished by comparing the temperature-dependent linear infrared spectra of [HEPy][BF4] with that of the molecular mimic of its cation, 2-phenylethanol (PhenEthOH). We then explored the structural motifs of these H-bonded configurations at the molecular level by analyzing the cryogenic ion vibrational predissociation spectroscopy of cold (∼35 K) gas phase cluster ions with quantum chemical methods. The analysis of the OH stretching bands reveals the formation of the various binding motifs ranging from the common +OHBF4- interaction in ion-pairs (c-a) to the unusual +OH+OH interaction (c-c) in linear and cyclic, homodromic H-bonding domains. Replacing ion-pairs by the molecular (neutral) analogue of the IL cation also results in the formation of positively charged cyclic motifs, with the bands of the gas phase cationic cyclic tetramer (HEPy+)(PhenEthOH)3 appearing quite close to those assigned previously to cyclic tetramers in the liquid. These conclusions are supported by density functional theory (DFT) calculations of the cationic and neutral clusters as well as the local structures in the liquid. Our combined experimental and theoretical approach for the gas and the liquid phases provides important insight into the competition between differently H-bonded and charged constituents in liquids.

Spectroscopic Evidence for an Attractive Cation-Cation Interaction in Hydroxy-Functionalized Ionic Liquids: A Hydrogen-Bonded Chain-like Trimer.

We address the formation of hydrogen bonded domains among the cationic constituents of the ionic liquid (IL) 1-(3-hydroxypropyl)pyridinium tetrafluoroborate [HPPy][BF4 ] by means of cryogenic ion vibrational predissociation spectroscopy of cold (ca. 35 K) gas-phase cluster ions and quantum chemistry. Specifically, analysis of the OH stretching bands reveals a chain-like OH⋅⋅⋅OH⋅⋅⋅OH⋅⋅⋅BF4 - binding motif involving the three cations in the cationic quinary cluster ion (HPPy+ )3 (BF4 - )2 . Calculations show that this cooperative H-bond attraction compensates for the repulsive Coulomb forces and results in stable complexes that successfully compete with those in which the OH groups are predominantly attached to the counter anions. Our combined experimental and theoretical approach provides insight into the cooperative effects that lead to the formation of hydrogen bonded domains involving the cationic constituents of ILs.

Exploring the Gas-Phase Activation and Reactivity of a Ruthenium Transfer Hydrogenation Catalyst by Experiment and Theory in Concert.

This study elucidates structures, activation barriers, and the gas-phase reactivity of cationic ruthenium transfer hydrogenation catalysts of the structural type [(η6-cym)RuX(pympyr)]+. In these complexes, the central ruthenium(+II) ion is coordinated to an η6-bound p-cymene (η6-cym), a bidentate 2-R-4-(2-pyridinyl)pyrimidine ligand (pympyr) with R = NH2 or N(CH3)2, and an anion X = I-, Br-, Cl-, or CF3SO3-. We present infrared multiple-photon dissociation (IR-MPD) spectra of precursors (before HCl loss) and of activated complexes (after HCl loss), which elucidates C-H activation as the key step in the activation mechanism. A resonant two-color IR-MPD scheme serves to record several otherwise "dark" bands and enhances the validity of spectral assignments. We show that collision-induced dissociation (CID)-derived activation energies of the [(η6-cym)RuX(pympyr)]+ (R = N(CH3)2) complexes depend crucially on the anion X. The obtained activation energies for the HX loss correlate well with quantum chemical activation barriers and are in line with the HSAB concept. We further elucidate the reaction of the activated complexes with D2 under single-collision conditions. Quantum mechanical simulations substantiate that the resulting species represent analogues for hydrido intermediates formed after abstraction of H+ and H- from isopropanol, as postulated for the catalytic cycle of transfer hydrogenation by us before.

Alkali-Controlled C-H Cleavage or N-C Bond Formation by N2-Derived Iron Nitrides and Imides.

Formation of N-H and N-C bonds from functionalization of N2 is a potential route to utilization of this abundant resource. One of the key challenges is to make the products of N2 activation reactive enough to undergo further reactions under mild conditions. This paper explores the strategy of "alkali control," where the presence of an alkali metal cation enables the reduction of N2 under mild conditions, and then chelation of the alkali metal cation uncovers a highly reactive species that can break benzylic C-H bonds to give new N-H and Fe-C bonds. The ability to "turn on" this C-H activation pathway with 18-crown-6 is demonstrated with three different N2 reduction products of N2 cleavage in an iron-potassium system. The alkali control strategy can also turn on an intermolecular reaction of an N2-derived nitride with methyl tosylate that gives a new N-C bond. Since the transient K(+)-free intermediate reacts with this electrophile but not with the weak C-H bonds in 1,4-cyclohexadiene, it is proposed that the C-H cleavage occurs by a deprotonation mechanism. The combined results demonstrate that a K(+) ion can mask the latent nucleophilicity of N2-derived nitride and imide ligands within a trimetallic iron system and points a way toward control over N2 functionalization.

Capture of CO2 by a Cationic Nickel(I) Complex in the Gas Phase and Characterization of the Bound, Activated CO2 Molecule by Cryogenic Ion Vibrational Predissociation Spectroscopy.

We describe a systematic method for the preparation and spectroscopic characterization of a CO2 molecule coordinated to an activated bisphenoidal nickel(I) compound containing a tetraazamacrocyclic ligand in the gas phase. The resulting complex was then structurally characterized by using mass-selected vibrational predissociation spectroscopy. The results indicate that a highly distorted CO2 molecule is bound to the metal center in an η(2)-C,O coordination mode, thus establishing an efficient and rational method for the preparation of metal-activated CO2 for further studies using ion chemistry techniques.

Synthesis, Characterization, and Nitrogenase-Relevant Reactions of an Iron Sulfide Complex with a Bridging Hydride.

The FeMoco of nitrogenase is an iron-sulfur cluster with exceptional bond-reducing abilities. ENDOR studies have suggested that E4, the state that binds and reduces N2, contains bridging hydrides as part of the active-site iron-sulfide cluster. However, there are no examples of any isolable iron-sulfide cluster with a hydride, which would test the feasibility of such a species. Here, we describe a diiron sulfide hydride complex that is prepared using a mild method involving C-S cleavage of added thiolate. Its reactions with nitrogenase substrates show that the hydride can act as a base or nucleophile and that reduction can cause the iron atoms to bind N2. These results add experimental support to hydride-based pathways for nitrogenase.

Thermodynamics of water dimer dissociation in the primary hydration shell of the iodide ion with temperature-dependent vibrational predissociation spectroscopy.

The strong temperature dependence of the I(-)·(H2O)2 vibrational predissociation spectrum is traced to the intracluster dissociation of the ion-bound water dimer into independent water monomers that remain tethered to the ion. The thermodynamics of this process is determined using van't Hoff analysis of key features that quantify the relative populations of H-bonded and independent water molecules. The dissociation enthalpy of the isolated water dimer is thus observed to be reduced by roughly a factor of three upon attachment to the ion. The cause of this reduction is explored with electronic structure calculations of the potential energy profile for dissociation of the dimer, which suggest that both reduction of the intrinsic binding energy and vibrational zero-point effects act to weaken the intermolecular interaction between the water molecules in the first hydration shell. Additional insights are obtained by analyzing how classical trajectories of the I(-)·(H2O)2 system sample the extended potential energy surface with increasing temperature.

Characterization of the Oxazolone and Macrocyclic Motifs in the bn (n = 2-5) Product Ions from Collision-Induced Dissociation of Protonated Oligoglycine Peptides with Isomer-Selective, Cryogenic Vibrational Spectroscopy.

Collision-induced dissociation (CID) of small, protonated peptides leads to the formation of b-type fragment ions that can occur with several structural motifs driven by different covalent intramolecular bonding arrangements. Here, we characterize the so-called "oxazolone" and "macrocycle" bn ion structures that occur upon CID of oligoglycine peptides (Gn) ions (n = 2-6). This is determined by acquiring the vibrational band patterns of the cryogenically cooled, D2-tagged bn ions obtained using isomer-selective, two-color IR-IR photobleaching and analyzing them with predicted (DFT) harmonic spectra for the candidate structures. Both oxazolone and macrocyclic isomers are formed by b4, whereas only oxazolone species are created for b2 and b3 and the macrocycle is created for b5. As such, n = 4 corresponds to the minimum size where both Oxa and MC forms are present.

Chain Length Dependence of Hydrogen Bond Linkages between Cationic Constituents in Hydroxy-Functionalized Ionic Liquids: Tracking Bulk Behavior to the Molecular Level with Cold Cluster Ion Spectroscopy.

Hydroxy functionalization of cations in ionic liquids (ILs) can lead to formation of contacts between their OH groups [so-called (c-c) interactions]. One class of these linkages involves cooperatively enhanced hydrogen bonds to anionic partners that are sufficiently strong to overcome the repulsion between two positively charged centers. Herein, we clarify how the propensity for the formation of (c-c) contacts depends on the alkyl chain length between two cationic rings and their OH groups by analyzing the temperature-dependent IR spectra of bulk ILs as well as the vibrational predissociation spectra of ∼35 K complexes comprised of two cations and one anion. This study compares the behavior of two cationic derivatives with ethyl and propyl chains complexed with two different anions: bis(trifluoromethylsulfonyl)imide and tetrafluoroborate. Only the bulk ILs with the longer chain propyl derivative [HPMPip+ = 1-(3-hydroxypropyl)-1-methylpiperidinium] display (c-c) interactions. Molecular-level aspects of this docking arrangement are revealed by analyzing the OH stretching fundamentals displayed by the ternary complexes.

Integration of High-Resolution Mass Spectrometry with Cryogenic Ion Vibrational Spectroscopy.

We describe an instrumental configuration for the structural characterization of fragment ions generated by collisional dissociation of peptide ions in the typical MS2 scheme widely used for peptide sequencing. Structures are determined by comparing the vibrational band patterns displayed by cryogenically cooled ions with calculated spectra for candidate structural isomers. These spectra were obtained in a linear action mode by photodissociation of weakly bound D2 molecules. This is accomplished by interfacing a Thermo Fisher Scientific Orbitrap Velos Pro to a cryogenic, triple focusing time-of-flight photofragmentation mass spectrometer (the Yale TOF spectrometer). The interface involves replacement of the Orbitrap's higher-energy collisional dissociation cell with a voltage-gated aperture that maintains the commercial instrument's standard capabilities while enabling bidirectional transfer of ions between the high-resolution FT analyzer and external ion sources. The performance of this hybrid instrument is demonstrated by its application to the a1, y1 and z1 fragment ions generated by CID of a prototypical dipeptide precursor, protonated L-phenylalanyl-L-tyrosine (H+-Phe-Tyr-OH or FY-H+). The structure of the unusual z1 ion, nominally formed after NH3 is ejected from the protonated tyrosine (y1) product, is identified as the cyclopropane-based product is tentatively identified as a cyclopropane-based product.

Structural Motifs in Cold Ternary Ion Complexes of Hydroxyl-Functionalized Ionic Liquids: Isolating the Role of Cation-Cation Interactions.

We address the competition between intermolecular forces underlying the recent observation that ionic liquids (ILs) with a hydroxyl-functionalized cation can form domains with attractive interactions between the nominally repulsive positively charged constituents. Here we show that this behavior is present even in the isolated ternary (HEMIm+)2NTf2- complex (HEMIm+ = 1-(2-hydroxyethyl)-3-methylimidazolium) cooled to about 35 K in a photodissociation mass spectrometer. Of the three isomers isolated by double resonance techniques, one is identified to exhibit direct contact between the cations. This linkage involves a cooperative H-bond wherein the OH group on one cation binds to the OH group on the other, which then attaches to the basic N atom of the anion. Formation of this motif comes at the expense of the usually dominant interaction of the acidic C(2)H group on the Im ring with molecular anions, as evidenced by isomer-dependent shifts in the C(2)H vibrational fundamentals.

Mechanistic Studies on Ruthenium(II)-Catalyzed Base-Free Transfer Hydrogenation Triggered by Roll-Over Cyclometalation.

The synthesis of 2-substituted pyridine-pyrimidine ligands and their complexation with arene ruthenium(II) chloride moieties is reported. Depending on the electronic and steric influences of the ligand, the catalysts undergo CH activation by roll-over cyclometalation. This process opens up the route to the catalytic transfer hydrogenation of ketones with isopropanol as the hydrogen source under base-free and mild conditions. Barriers related to the roll-over cyclometalation process can be determined experimentally by collision-induced dissociation ESI mass spectrometry. They are supported by DFT calculations and allow the classification of the ligands according to their electronic and steric properties, which is also in accordance with critical bond parameters derived from X-ray structure data. DFT calculations furthermore reveal that the formation of a ruthenium(II) hydrido species is plausible through ß-hydride elimination from isopropanol.

Identification and Partial Structural Characterization of Mass Isolated Valsartan and Its Metabolite with Messenger Tagging Vibrational Spectroscopy.

Recent advances in the coupling of vibrational spectroscopy with mass spectrometry create new opportunities for the structural characterization of metabolites with great sensitivity. Previous studies have demonstrated this scheme on 300 K ions using very high power free electron lasers in the fingerprint region of the infrared. Here we extend the scope of this approach to a single investigator scale as well as extend the spectral range to include the OH stretching fundamentals. This is accomplished by detecting the IR absorptions in a linear action regime by photodissociation of weakly bound N2 molecules, which are attached to the target ions in a cryogenically cooled, rf ion trap. We consider the specific case of the widely used drug Valsartan and two isomeric forms of its metabolite. Advantages and challenges of the cold ion approach are discussed, including disentangling the role of conformers and the strategic choices involved in the selection of the charging mechanism that optimize spectral differentiation among candidate structural isomers. In this case, the Na+ complexes are observed to yield sharp resonances in the high frequency NH and OH stretching regions, which can be used to easily differentiate between two isomers of the metabolite. Graphical Abstract ᅟ.

Trapping and Structural Characterization of the XNO2·NO3- (X = Cl, Br, I) Exit Channel Complexes in the Water-Mediated X- + N2O5 Reactions with Cryogenic Vibrational Spectroscopy.

The heterogeneous reaction of N2O5 with sea spray aerosols yields the ClNO2 molecule, which is postulated to occur through water-mediated charge separation into NO3- and NO2+ ions followed by association with Cl-. Here we address an alternative mechanism where the attack by a halide ion can yield XNO2 by direct insertion in the presence of water. This was accomplished by reacting X-(D2O)n (X = Cl, Br, I) cluster ions with N2O5 to produce ions with stoichiometry [XN2O5]-. These species were cooled in a 20 K ion trap and structurally characterized by vibrational spectroscopy using the D2 messenger tagging technique. Analysis of the resulting band patterns with DFT calculations indicates that they all correspond to exit channel ion-molecule complexes based on the association of NO3- with XNO2, with the NO3- constituent increasingly perturbed in the order I > Br > Cl. These results establish that XNO2 can be generated even when more exoergic reaction pathways involving hydrolysis are available and demonstrate the role of the intermediate [XN2O5]- in the formation of XNO2.