The Ni(I) hydrogen oxidation catalyst [Ni(PCy2NtBu2)2]+ (1+; PCy2NtBu2= 1,5bis(tert-butyl)-3,7-dicyclo-hexyl-1,5-diaza-3,7-diphosphacychlooctane) has been studied using a combination of EPR techniques (X-, Q-, and D-band; electron-nuclear double resonance, hyperfine sublevel correlation spectroscopy), X-ray crystallography, and density functional theory (DFT) calculations. Crystallographic and DFT studies indicate that the molecular structure of 1+ is highly symmetrical. EPR spectroscopy has allowed determination of the electronic g-tensor and the spin density distribution on the ligands, and revealed that the Ni(I) center does not interact strongly with the potentially coordinating solvents acetonitrile and butyronitrile. The EPR spectra and magnetic parameters of 1+ are found to be distinctly different from those for the related compound [Ni(PPh2NPh2)2]+ (4+). One significant contributor to these differences is that the molecular structure of 4+ is unsymmetrical, unlike that of 1+. DFT calculations on derivatives in which the R and R′ groups are systematically varied have allowed elucidation of structure/substituent relationships and their corresponding influence on the magnetic resonance parameters.

The reactivity of 1-hydroxybenzoyl triazole (HOBt) esters with the carboxylate functionality present in peptides is demonstrated in the gas phase with a doubly deprotonated dianion. The reaction forms an anhydride linkage at the carboxylate site. Upon ion trap collisional induced dissociation (CID) of the modified peptide, the resulting spectrum shows a nominal loss of the mass of the reagent and a water molecule. Analogous phenomenology was also noted for model peptide cations that likely contain zwitterionic/salt-bridged motifs in reactions with a negatively charged HOBt ester. Control experiments indicate that a carboxylate group is the likely reactive site, rather than other possible nucleophilic sites present in the peptide. These observations suggest that HOBt ester chemistry may be used as a chemical probe for the presence and location of carboxylate groups in net positively-charged polypeptide ions. As an illustration, deprotonated sulfobenzoyl-HOBt was reacted with the [M+7H] 7+ ion of ubiquitin. The ion was shown to react with the reagent and CID of the covalent reaction product yielded an abundant [M+6H-H 2 O] 6+ ion. Comparison of the CID product ion spectrum of this ion with that of the water loss product generated from CID of the unmodified [M+6H] 6+ ion revealed the glutamic acid at residue 64 as a reactive site, suggesting that it is present in the deprotonated form.

Nucleophilic substitution covalent modification ion/ion reactions were carried out in a linear quadrupole ion trap between the doubly protonated peptides KGAIL­KGAILR, RAR­ARAA, and RKR­ARAA and isomers of either singly deprotonated 3- or 4-sulfobenzoic acid (n-SBA) esterified with either N-hydroxysuccinimide (NHS) or 1-hydroxy-7-aza-benzotriazole (HOBt). The cation/anion attachment product, through which the covalent reaction occurs, was isolated and subjected to dipolar DC (DDC) activation to generate covalently modified product over the ranges of DDC activation energies and times. The resulting survival yields were used to determine reaction rates, and Tolmachev’s effective ion temperature was used to extract Arrhenius and Eyring activation parameters. It was found that the kinetics determined under these conditions are highly sensitive to the identities and locations of the nucleophilic sites on the peptides, the leaving groups on the reagent, and the location of the attachment sites on the reagent and analyte. Depending upon the identity of the analyte/reagent combination, significant variations in activation energy or entropy (or both) were both found to underlie the measured rate differences. The determination of dissociation kinetics under DDC conditions and application of Tolmachev’s effective ion temperature treatment enables unique insights into the dynamics of gas-phase covalent bond formation via ion/ion reactions.

Native mass spectrometry (MS) is usually conducted in the positive-ion mode; however, in some cases, it is advantageous to use the negative-ion polarity. Challenges associated with native MS using ensemble measurements (i.e., the measurement of many ions at a time as opposed to the measurement of the charge and the mass-to-charge ratio of individual ions) include narrow charge state distributions with the potential for an overlap in neighboring charge states. These issues can either compromise or preclude confident charge state (and hence mass) determination. Charge state determination in challenging instances can be enabled via the attachment of multiply charged ions of opposite polarity. Multiply charged ion attachment facilitates the resolution of charge states and generates mass-to-charge (m/z) information across a broad m/z range. In this work, we demonstrated the attachment of multiply charged cations to anionic complexes generated under native MS conditions. To illustrate the flexibility available in selecting the mass and charge of the reagents, the 15+ and 20+ charge states of horse skeletal muscle apomyoglobin and the 20+ and 30+ charge states of bovine carbonic anhydrase were demonstrated to attach to model complex anions derived from either β-galactosidase or GroEL. The exclusive attachment of reagent ions is observed with no evidence for proton transfer, which is the key for the unambiguous interpretation of the post-ion/ion reaction product ion spectrum. To illustrate the application to mixtures of complex ions, the 10+ charge state of bovine ubiquitin was attached to mixtures of anions generated from the 30S and 50S particles of the Escherichia coli ribosome. Six and five major components were revealed, respectively. In the case of the 50S anion population, it was shown that the attachment of two 30+ cations of carbonic anhydrase revealed the same information as the attachment of six 10+ cations of ubiquitin. In neither case was the intact 50S particle observed. Rather, particles with different combinations of missing components were observed. This work demonstrated the utility of multiply charged cation attachment to facilitate charge state assignments in native MS ensemble measurements of heterogeneous mixtures.

Strong electrostatic interactions such as zwitterionic pairing between oppositely charged amino acids are common in the condensed phase at neutral pH and can play a large role in determining the conformational landscape of peptides and proteins. Whether such interactions are possible in the absence of solvent however, has been previously debated. Growing experimental evidence suggests that these interactions are indeed possible in isolated gas phase ions and may give rise to unique fragmentation upon UV irradiation. In this study we use a series IR-UV-IR triple resonance techniques performed at 10 K to investigate the influences of these electrostatic interactions on the electronic and structural properties of model YGRXR ( X = gly, asp) pentapeptide systems and their methyl ester counterparts. The initial electronic spectra, under single UV photon conditions, of model systems which may possess zwitterionic pairing hardly show discrete electronic transitions, rather a broad absorbtion which mainly gives rise to tyrosine side chain cleavage is observed. Upon methylation of the carboxylate functional groups, which prevents zwitterionic interactions, the cold action spectra become well resolved such that sharp electronic transitions due to the ππ* transition of the tyrosine aromatic ring are observed. Using an UV-IR double resonance scheme it is possible to enhance the tyrosine side chain cleavage after an initial UV excitation, provided that the IR laser is fixed on a vibrational. Under these conditions the Franck Condon progressions for the non-methyl esterified systems become clearly observable. These initial results suggest that local excitation of the chromophore may couple to the autoionizing state responsible for electron detachment, similar to the mechanism postulated for photoinduced electron detachment from gas phase anions. Using IR-UV-IR triple resonance, conformer specific IR spectra can be taken for zwitterionic systems despite the large "off-resonance" absorbtion. A comparison of the IR spectra reveal that the +1 charge states are more prone to form zwitterionic interactions than the +2. Harmonic-level vibrational frequency calculations will be performed on candidate structures and compared to experimental spectra such that the influences of zwitterionic ionic pairing on the 3-dimensional structure can be directly compared to conformations without such parings.