Mass spectrometry-based gas phase intramolecular benzyl migration in sparsentan, a novel endothelin and angiotensin II receptor antagonist.

We report a collision-induced dissociation (CID) based gas phase rearrangement study using quadrupole time-of-flight mass spectrometry coupled with liquid chromatography on a novel endothelin and angiotensin II receptor antagonist, sparsentan. We performed tandem mass spectrometry to identify precursor and fragment ion relationships and assigned structures for major fragment ions. We propose a benzyl migration mechanism based on bond length measurements in density functional theory (B3LYP/6-31+G*) optimized geometries of protonated sparsentan and its m/z 547 fragment. Protonated sparsentan undergoes loss of ethanol, which yields a resonance-stabilized benzylic cation with m/z 547, which further fragments into m/z 353 via benzyl migration, where the benzylic cation migrates to one of the nucleophilic nitrogen atoms followed by proton transfer from the sulfonamide nitrogen to a carbonyl oxygen, resulting in a neutral loss of mass 194. Further fragmentation of m/z 353 results in m/z 258, which undergoes radical and neutral loss to yield m/z 193 and 194, respectively. The proposed mechanism of generation of m/z 353 was confirmed by CID of deuterated sparsentan. Considering the importance of gas phase rearrangements of organic molecules in structural identifications as well as the novelty of the molecule, these findings will be helpful for future studies to predict gas phase benzyl migration in sparsentan analogs and for degradation product and metabolite identification of sparsentan and its analogs using LC-MS.

Ion Mobility and Fourier Transform Ion Cyclotron Resonance Collision Cross Section Techniques Yield Long-Range and Hard-Sphere Results, Respectively.

We determined collision cross section (CCS) values for singly and doubly charged cucurbit[n]uril (n = 5-7), decamethylcucurbit[5]uril, and cyclohexanocucurbit[5]uril complexes of alkali metal cations (Li+-Cs+). These hosts are relatively rigid. CCS values calculated using the projection approximation (PA) for computationally modeled structures of a given host are nearly identical for +1 and +2 complexes, with weak metal ion dependence, whereas trajectory method (TM) calculations of CCS for the same structures consistently yield values 7-10% larger for the +2 complexes than for the corresponding +1 complexes and little metal ion dependence. Experimentally, we measured relative CCS values in SF6 for pairs of +1 and +2 complexes of the cucurbituril hosts using the cross-sectional areas by Fourier transform ion cyclotron resonance ("CRAFTI") method. At center-of-mass collision energies <∼30 eV, CRAFTI CCS values are sensitive to the relative binding energies in the +1 and +2 complexes, but at collision energies >∼40 eV (sufficient that ion decoherence occurs on essentially every collision) that dependence is not evident. Consistent with the PA calculations, these experiments found that the +2 complex ions have CCS values ranging between 94 and 105% of those of their +1 counterparts (increasing with metal ion size). In contrast, but consistent with the TM CCS calculations, ion mobility measurements of the same complexes at close to thermal energies in much less polarizable N2 find the CCS of +2 complexes to be in all cases 9-12% larger than those of the corresponding +1 complexes, with little metal ion dependence.

Collision Cross-Section Measurements of Collision-Induced Dissociation Precursor and Product Ions in an FTICR-MS and an IM-MS: A Comparative Study.

Sustained off-resonance irradiation-cross-sectional areas by Fourier transform ion cyclotron resonance mass spectrometry (SORI-CRAFTI) is an FTICR-MS strategy to collisionally activate precursor ions and then measure their ion-neutral collision cross sections, as well as those of selected products, at the same time. We benchmarked SORI-CRAFTI using protonated leucine-enkephalin, to excellent agreement (typically within 1-2%) with previous studies performed via collision-induced dissociation-ion mobility (CID-IMS). SORI-CRAFTI was then applied to alkali metal-cationized leucine-enkephalin and compared with CID-IMS via precursor/product cross-section ratios. Qualitative agreement between SORI-CRAFTI and CID-IMS was excellent (again, usually within 1-2%); however, neither SORI-CRAFTI nor CID-IMS could determine if metalated leucine-enkephalin was present in its canonical or zwitterionic form. When SORI-CRAFTI was used on [2.2.2]-cryptand+Cs+, SORI activation resulted in a 5% decrease in collision cross section, consistent with migration of the externally bound Cs+ into the cryptand's cavity and similar to the cross section observed when electrospraying from an isopropanol-rich solvent. Thus, SORI-CRAFTI is useful for studying gas-phase ion chemistry of small- to medium-sized molecules and host-guest systems.

Prototypical Allosterism in a Simple Ditopic Ligand: Gas-Phase Topologies of Cucurbit[n]uril·n-Alkylammonium Complexes Controlled by Binding in the Second Site.

We have employed mass spectrometry, ion mobility, and computational techniques to characterize complexes of n-alkylammonium ions with cucurbit[5]uril (CB[5]) and cucurbit[6]uril (CB[6]) ligands in the gas phase. Nonrotaxane structures are energetically preferred and experimentally observed for all CB[5] complexes. Pseudorotaxane structures are computationally favored and experimentally observed for [CB[6]·n-alkylammonium]+ complexes, but the addition of a second cation (proton, alkali metal ion, another alkylammonium ion, or guanidinium) on the opposite rim of CB[6] causes sufficiently unfavorable steric interactions that n-pentylammonium and longer chains no longer remain threaded through the CB[6] cavity; nonrotaxane topologies are then favored. This provides a very simple example of negative allosteric interactions and molecular structure switching in these complexes.

Multi-CRAFTI: Relative Collision Cross Sections from Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Line Width Measurements.

Determination of collision cross sections (CCS) using the cross-sectional areas by the Fourier transform ion cyclotron resonance (CRAFTI) technique is limited by the requirement that accurate pressures in the trapping cell of the mass spectrometer must be known. Experiments must also be performed in the energetic hard-sphere regime such that ions decohere after single collisions with neutrals; this limits application to ions that are not much more massive than the neutrals. To mitigate these problems, we have resonantly excited two (or more) ions of different m/z to the same center-of-mass kinetic energy in a single experiment, subjecting them to identical neutral pressures. We term this approach "multi-CRAFTI". This facilitates measurement of relative CCS without requiring knowledge of the pressure and enables determination of absolute CCS using internal standards. Experiments with tetraalkylammonium ions yield CCS in reasonable agreement with the one-ion-at-a-time CRAFTI approach and with ion mobility spectrometry (IMS) when differences in collision energetics are taken into account (multi-CRAFTI generally yields smaller CCS than does IMS due to the higher collision energies employed in multi-CRAFTI). Comparison of multi-CRAFTI and IMS results with CCS calculated from structures computed at the M06-2X/6-31+G* level of theory using projection approximation or trajectory method values, respectively, indicates that the computed structures have CCS increasingly smaller than the experimental CCS as m/z increases, implying the computational model overestimates interactions between the alkyl arms. For ions that undergo similar collisional decoherence processes, relative CCS reach constant values at lower collision energies than do absolute CCS values, suggesting a means of increasing the accessible upper m/z limit by employing multi-CRAFTI.

Halide Size-Selective Binding by Cucurbit[5]uril-Alkali Cation Complexes in the Gas Phase.

We report data that suggest complexes with alkali cations capping the portals of cucurbit[5]uril (CB[5]) bind halide anions size-selectively as observed in the gas phase: Cl- binds inside the CB[5] cavity, Br- is observed both inside and outside, and I- binds weakly outside. This is reflected in sustained off-resonance irradiation collision-induced dissociation (SORI-CID) experiments: all detected Cl- complexes dissociate at higher energies, and Br- complexes exhibit unusual bimodal dissociation behavior, with part of the ion population dissociating at very low energies and the remainder dissociating at significantly higher energies comparable to those observed for Cl-. Decoherence cross sections measured in SF6 using cross-sectional areas by Fourier transform ion cyclotron resonance techniques for [CB[5] + M2X]+ (M = Na, X = Cl or Br) are comparable to or less than that of [CB[5] + Na]+ over a wide energy range, suggesting that Cl- or Br- in these complexes are bound inside the CB[5] cavity. In contrast, [CB[5] + K2Br]+ has a cross section measured about 20% larger than that of [CB[5] + Na]+, suggesting external binding that may correspond with the weakly bound component seen in SORI. While I- complexes with alkali cation caps were not observed, alkaline earth iodides with CB[5] yielded complexes with cross sections 5-10% larger than that of [CB[5] + Na]+, suggesting externally bound iodide. Geometry optimization at the M06-2X/6-31+G* level of ab initio theory suggests that internal anion binding is energetically favored by approximately 50-200 kJ mol-1 over external binding; thus, the externally bound complexes observed experimentally must be due to large energetic barriers hindering the passing of large anions through the CB[5] portal, preventing access to the interior. Calculation of the barriers to anion egress using MMFF//M06-2X/6-31+G* theory supports this idea and suggests that the size-selective binding we observe is due to anion size-dependent differences in the barriers.

Barriers for Extrusion of a Guest from the Interior Binding Cavity of a Host: Gas Phase Experimental and Computational Results for Ion-Capped Decamethylcucurbit[5]uril Complexes.

Factors affecting the extrusion of guests from metal ion-capped decamethylcucurbit[5]uril (mc5) molecular container complexes are investigated using both collision-induced dissociation techniques and molecular mechanics simulations. For guests without polar bonds, the extrusion barrier increases with increasing guest volume. This is likely because escape of larger guests requires more displacement of the metal ion caps and, thus, more disruption of the ion-dipole interactions between the ion caps and the electronegative rim oxygens of mc5. However, guests larger than the optimum size for encapsulation displace the ion caps prior to collision-induced dissociation, resulting in less stable complexes and lower dissociation thresholds. The extrusion barriers obtained for guests with polar bonds are smaller than those obtained for similarly sized guests without polar bonds. This is likely because the partial charges on the guest allow electrostatic interactions with the ion cap and rim oxygens of mc5 during extrusion, thus stabilizing the extrusion transition state and reducing the extrusion barrier. Results from this study demonstrate simple principles to consider for designing host-guest complexes with specific guest-loss behaviors. Similar trends are observed between the experimental and computational results, demonstrating that molecular mechanics simulations can be used to approximate the relative stability of mc5 molecular container complexes and likely those of other similar complexes.

Quantitative Collision Cross-Sections from FTICR Linewidth Measurements: Improvements in Theory and Experiment.

Two corrections to the equation used in the cross-sectional areas by Fourier transform ion cyclotron resonance ("CRAFTI") technique are identified. In CRAFTI, ion collision cross-sections are obtained from the pressure-dependent ion linewidths in Fourier transform mass spectra. The effects of these corrections on the accuracy of the cross-sections obtained using the CRAFTI technique are evaluated experimentally using the 20 biogenic amino acids and several crown ether complexes with protonated alkyl monoamines. Good absolute agreement is obtained between the CRAFTI cross-sections and the corresponding cross-sections obtained using both static drift ion mobility spectrometry and computational simulations. These results indicate that the CRAFTI cross-sections obtained using the updated equation presented here are quantitatively descriptive of the size and shape of the gas-phase ions. Cross-sections that differ by less than 3% are measured for the isobaric isomers n-butylamine and tert-butylamine complexed with the crown ethers. This level of precision is similar to what has been achieved previously using traveling wave ion mobility devices. These results indicate that CRAFTI can be used to probe subtle structural differences between ions with approximately the same precision as that achieved in traveling wave ion mobility devices. Graphical Abstract ᅟ.

Collision Cross Sections for 20 Protonated Amino Acids: Fourier Transform Ion Cyclotron Resonance and Ion Mobility Results.

We report relative dephasing cross sections for the 20 biogenic protonated amino acids measured using the cross sectional areas by Fourier transform ion cyclotron resonance (CRAFTI) technique at 1.9 keV in the laboratory reference frame, as well as momentum transfer cross sections for the same ions computed from Boltzmann-weighted structures determined using molecular mechanics. Cross sections generally increase with increasing molecular weight. Cross sections for aliphatic and aromatic protonated amino acids are larger than the average trend, suggesting these side chains do not fold efficiently. Sulfur-containing protonated amino acids have smaller than average cross sections, reflecting the mass of the S atom. Protonated amino acids that can internally hydrogen-bond have smaller than average cross sections, reflecting more extensive folding. The CRAFTI measurements correlate well with results from drift ion mobility (IMS) and traveling wave ion mobility (TWIMS) spectrometric measurements; CRAFTI results correlate with IMS values approximately as well as IMS and TWIMS values from independent measurements correlate with each other. Both CRAFTI and IMS results correlate well with the computed momentum transfer cross sections, suggesting both techniques provide accurate molecular structural information. Absolute values obtained using the various methods differ significantly; in the case of CRAFTI, this may be due to errors in measurements of collision gas pressure, measurement of excitation voltage, and/or dependence of cross sections on kinetic energy. Graphical Abstract ᅟ.

Linewidth pressure measurement: a new technique for high vacuum characterization.

Pressure measurement is often the limiting factor in the accuracy of quantitative ion-molecule experiments. We present a new method for pressure measurement based on analysis of pressure-limited Fourier transform ion cyclotron resonance (FTICR) linewidths for well-characterized collisions of Ar(+) with Ar. The kinetic energy dependence of Ar(+)/Ar collision cross sections is well-described using a single-parameter fitting procedure, which results in pressure measurements in good agreement with those from a cold cathode tube and from measurement of total ion signal following electron impact ionization. The new method avoids problems inherent in ionization-based methods, such as those arising from differences in ionization potential or perturbations to the pressure that occur during electron ionization of the gas to be measured, and should be applicable in the trapping cells of FTICR and Orbitrap mass spectrometers.

Resorcinarene-based cavitands with chiral amino acid substituents for chiral amine recognition.

Resorcinarene-based deep cavitands alanine methyl resorcinarene acid (AMA), alanine undecyl resorcinarene acid (AUA) and glycine undecyl resorcinarene acid (GUA), which contain chiral amino acids, have been synthesized. The upper rim of the resorcinarene host is elongated with four identical substituents topped with alanine and glycine groups. The structures of the new resorcinarenes were elucidated by nuclear magnetic resonance (NMR), mass spectrometry (MS) and the sustained off-resonance irradiation collision induced dissociation (SORI-CID) technique in FTICR-MS. These studies revealed that eight water molecules associate to the cavitand, two for each alanine group. The alanine substituent groups are proposed to form a kite-like structure around the resorcinarene scaffold. The binding of AMA, AUA, and GUA with chiral R- and S-methyl benzyl amines was studied by (1)H NMR titration, and compared to that of a binary L-tartaric acid and the monoacid phthalyl alanine (PA). The results show that these compounds interact with amine guests; however, with four carboxylic acid groups, they bind several amine molecules strongly while the binary L-tartaric acid only binds one amine guest strongly. The simple compound PA, which contains one carboxylic group, shows weak binding to the amines. The (1)H NMR titration of AUA with primary, secondary, and tertiary chiral amines showed that it can discriminate between these three types of amines and showed chiral discrimination for chiral secondary amines.

Collision cross sectional areas from analysis of Fourier transform ion cyclotron resonance line width: a new method for characterizing molecular structure.

We demonstrate a technique for determining molecular collision cross sections via measuring the variation of Fourier transform ion cyclotron resonance (FTICR) line width with background damping gas pressure, under conditions where the length of the FTICR transient is pressure limited. Key features of our method include monoisotopic isolation of ions, the pulsed introduction of damping gas to a constant pressure using a pulsed leak valve, short excitation events to minimize collisions during the excitation, and proper choice of damping gas (Xe is superior to He). The measurements are reproducible within a few percent, which is sufficient for distinguishing between many structural possibilities and is comparable to the uncertainty in cross sections calculated from computed molecular structures. These techniques complement drift ion mobility measurements obtained on dedicated instruments. They do not require a specialized instrument, but should be easily performed on any FTICR mass spectrometer equipped with a pulsed leak valve.

Influence of charge repulsion on binding strengths: experimental and computational characterization of mixed alkali metal complexes of decamethylcucurbit[5]uril in the gas phase.

Theory and experiment demonstrate that Coulombic repulsion plays a dominant role in the strength of binding a second cation to a rigid, ditopic host.

Supramolecular modification of ion chemistry: modulation of peptide charge state and dissociation behavior through complexation with cucurbit[n]uril (n = 5, 6) or alpha-cyclodextrin.

Electrospray Fourier transform ion cyclotron resonance mass spectrometry, ion mobility spectrometry, and computational methods were utilized to characterize the complexes between lysine or pentalysine with three prototypical host molecules: alpha-cyclodextrin (alpha-CD), cucurbit[5]uril (CB[5]), and cucurbit[6]uril (CB[6]). Ion mobility measurements show lysine forms externally bound, singly charged complexes with either alpha-CD or CB[5], but a doubly charged complex with the lysine side chain threaded through the host cavity of CB[6]. These structural differences result in distinct dissociation behaviors in collision-induced dissociation (CID) experiments: the alpha-CD complex dissociates via the simple loss of intact lysine, whereas the CB[5] complex dissociates to yield [CB[5] + H(3)O](+), and the CB[6] complex loses neutral NH(3) and CO, the product ion remaining a doubly charged complex. These results are consistent with B3LYP/6-31G* binding energies (kJ mol(-1)) of D(Lys + H(+)-alpha-CD) = 281, D(Lys + H(+)-CB[5]) = 327, and D(Lys + 2H(2+)-CB[6]) = 600. B3LYP/6-31G* geometry optimizations show complexation with alpha-CD stabilizes the salt bridge form of protonated lysine, whereas complexation with CB[6] stabilizes doubly protonated lysine. Complexation of the larger polypeptide pentalysine with alpha-CD forms a nonspecific adduct: no modification of the pentalysine charge state distribution is observed, and dissociation occurs via the simple loss of alpha-CD. Complexation of pentalysine with the cucurbiturils is more specific: the observed charge state distribution shifts higher on complexation, and fragmentation patterns are significantly altered relative to uncomplexed pentalysine: C-terminal fragment ions appear that are consistent with charge stabilization by the cucurbiturils, and the cucurbiturils are retained on the fragment ions. Molecular mechanics calculations suggest CB[5] binds to two protonated sites on pentalysine without threading onto the peptide and that CB[6] binds two adjacent protonated sites via threading onto the peptide.

One ring to bind them all: shape-selective complexation of phenylenediamine isomers with cucurbit[6]uril in the gas phase.

We examined complexes between cucurbit[6]uril and each of ortho-, meta-, and para-phenylenediamine using computational methods, Fourier transform ion cyclotron resonance mass spectrometry, and ion mobility spectrometry. These fundamental gas phase studies show that the lowest energy binding sites for ortho- and meta-phenylenediamine are on the exterior of cucurbit[6]uril, whereas para-phenylenediamine preferentially binds in the interior, in a pseudorotaxane fashion. This conclusion is based on reactivity of each of the complexes with tert-butylamine, where the ortho- and meta-phenylenediamine complexes exchange with tert-butylamine, whereas the para-phenylenediamine complex undergoes two slow additions without displacement. Further, under sustained off-resonance irradiation conditions, the ortho- and meta-phenylenediamine complexes fragment easily via losses of neutral phenylenediamine, whereas the para-phenylenediamine complex fragments at higher energies primarily via cleavage of covalent bonds in the cucurbituril. Finally, ion mobility studies show ion populations for the ortho- and meta-phenylenediamine complexes that primarily have collision cross sections consistent with external complexation, whereas the para-phenylenediamine complex has a collision cross section that is smaller, the same as that of protonated cucurbit[6]uril within experimental error. In agreement with experiment, computational studies indicate that at the HF/6-31G* and B3LYP/6-31G*//HF/6-31G* levels of theory external complexation is favored for ortho- and meta-phenylenediamine, whereas internal complexation is lower in energy for para-phenylenediamine. In contrast, MP2/6-31G*//HF-6-31G* calculations predict internal complexation for all three isomers.

Isotopic compositions and accurate masses of single isotopic peaks.

This paper presents and proves an algorithm to calculate the isotopic composition of individual (nominal) isotopic peaks. From this information one can calculate the accurate masses of isotopic peaks. This opens the way to use accurate mass measurements to determine chemical compositions of compounds using non-mono-isotopic peaks. The algorithm is computationally efficient and rigorously correct in the absence of roundoff error. Highly effective error correction strategies are described to detect and correct computational errors arising in practical calculations. Results from theoretical calculations of isotopic masses for a krypton inclusion complex agreed well with experimental measurements.

Incorporation of a venturi device in electrospray ionization.

Electrospray ionization has grown to be one of the most commonly used ionization techniques for mass spectrometry, and efforts continue to improve its performance. Typically, the sprayer tip must be very close to the entrance orifice of the mass spectrometer in order to maximize the conduction of ions from the sprayer into the mass spectrometer. However, because of space-charge repulsion, most ions never reach the sampling orifice. In this work, an industrial air amplifier, for which the working mechanism is based on venturi and coanda effects, was added between an electrospray ionization source and a time-of-flight mass spectrometer. When a series of reserpine solutions (0.5, 1.0, 5.0, and 10.0 microM) were monitored using mass spectrometry, an over 5-fold increase in m/z 609.3 ion intensity was measured for a separation distance of 14 mm between the electrospray tip and interface capillary inlet, as compared to when the electrospray tip was in its normal position 1 mm in front of the inlet without the amplifier. When a voltage was applied to the air amplifier to further assist in focusing the electrosprayed ions, an approximately 18-fold increase in m/z 609.3 ion intensity was obtained. In addition, a 34-fold reduction in method detection limit was observed.

Cucurbit[6]uril pseudorotaxanes: distinctive gas-phase dissociation and reactivity.

Cucurbit[6]uril forms a doubly charged complex with 1,4-butanediammonium cation that is observed using electrospray ionization Fourier transform mass spectrometry. Such 1:1 complexes are not observed for the smaller cucurbit[5]uril, which forms a 2:1 ammonium:cucurbituril complex instead. The 1:1 complex with cucurbit[6]uril is difficult to fragment via collisional activation; when it does fragment, both breakup of the cucurbituril cage and loss of the amine are observed. Further, the complex reacts with tert-butylamine via slow adduction. In contrast, nonrotaxane analogues (such as doubly charged 2:1 complexes of either protonated 1,4-butanediamine or protonated ethylenediamine with cucurbit[6]uril) fragment via easy loss of the intact amine upon collisional activation and react with tert-butylamine via rapid displacement of the original amine. On the basis of stoichiometry, fragmentation behavior, and reactivity, we conclude that the doubly charged complex of cucurbit[6]uril with 1,4-butanediammonium is a gas-phase pseudorotaxane.