Identification and Partial Structural Characterization of Mass Isolated Valsartan and Its Metabolite with Messenger Tagging Vibrational Spectroscopy
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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.
KeywordsVibrational spectroscopy Mass spectrometry IR-spectroscopy Metabolomics Metabolite Drug discovery Conformer differentiation
The practical consequence of this procedure is that spectra obtained in the laboratory can be directly compared with theoretical predictions for candidate structures, which are routinely available with electronic structure calculations of the linear absorption spectra of their equilibrium geometries .
We first evaluate the performance of messenger tagging IR spectroscopy by characterizing three commonly available modes for electrical charging of nominally neutral molecules in electrospray ionization (ESI) mass spectrometry: protonation, deprotonation, and complexation with Na+. We then demonstrate how structural details can be inferred from cold ion vibrational spectra when combined with conformer-selective, IR-IR double resonance spectroscopy as well as with site-specific isotopic substitution. We conclude by highlighting the ability of messenger tagging vibrational spectroscopy to reveal variations in the NH and OH stretching regions of the key hydroxyl groups in the natural metabolite II, as well as the synthetic, isomeric compound III, to test the efficacy of the method in the differentiation of structurally similar isomers.
For comparison, mass spectra (MS1) and collision induced dissociation (CID) mass spectra (MS2) were obtained using the linear ion trap of a Thermo Scientific LTQ Orbitrap XL Hybrid FT Mass Spectrometer (Waltham, MA, USA). Sample solutions of II and III in 50:50 methanol:water (same as those used for the spectroscopic measurements) were electrosprayed with the needle voltage held at ~4 kV. Ions were then guided to the ion trap, mass selected, and fragmented using He as a collision gas. The mass spectrometer was calibrated with Pierce LTQ ESI Positive Ion Calibration Solution, Thermo Fisher Scientific Inc.
Results and Discussion
Dependence of Vibrational Patterns on the Charging Method
One issue that immediately arises in the application of vibrational spectroscopy for structure elucidation is the choice of a charging scheme. In particular, messenger tagging vibrational spectroscopy is most effective if the key distinguishing features appear as sharp, well separated vibrational bands. Since the primary ring scaffolds are identical for oxidation products II and III, it would be valuable if the OH stretches of the hydroxyl groups in the two positions (C4 versus C5 of the valeroyl alkyl tail) could be used directly as reporters for compound identification. Such bands are often very diffuse in the condensed phase because of local perturbations arising from thermal fluctuations in the surrounding environment. In the analysis of the vibrational predissociation spectra, on the other hand, these features are often sharp and appear at frequencies that reflect the minimum energy conformations of the cold, isolated ions [12, 17]. In particular, the isomer-dependence of folded configurations with intra-molecular H-bonds promises to provide a natural mechanism to enhance differences in otherwise similar OH groups . These conformations will, in turn, be driven by the nature of the charging mechanism essential for the application of messenger tagging vibrational spectroscopy. As such, we first survey the spectra of parent molecule I when charged by the typical methods: protonation, deprotonation, and sodiation.
Remarks on Strategies for Specific Conformer and Band Assignments
We emphasize that our purpose here is not to determine the detailed ion-induced conformations from their spectra. Indeed, the conformationally flexible scaffold of this system, combined with the spectroscopic complexities that arise from ion-driven, intramolecular H-bonds (vide infra) often present profound challenges for assignments based on harmonic predictions. Even within this more limited scope, however, it is useful to determine the spectral pattern of all conformers that are generated in the ion source. We then demonstrate how we can experimentally identify features arising from the various functional groups, and then use this information to characterize the intramolecular interactions driving the observed structures.
To determine whether multiple conformers contribute to the I + Na+ spectrum in Figure 3b, we exploit the photochemical hole burning method [18, 23, 24, 25, 26]. This involves an IR-IR double resonance scheme carried out with two tunable IR lasers and three stages of mass selection (IR2MS3). The first MS stage isolates a parent mass for interaction with a scanning IR laser (the pump), and the second stage of MS separates the undissociated, tagged parent mass from the fragments generated by resonant excitation at the first laser crossing. A second IR laser (the probe) then interrogates the undissociated, tagged parent ion, and photofragments from this excitation are isolated with the third MS stage. To acquire a conformer-specific spectrum, the probe laser is fixed at a particular resonance, and the pump laser is scanned through the entire spectrum. In this way, whenever the pump laser excites a resonance that is also associated with the transition probed by the fixed probe laser, the probe fragment ion signal is decreased, and the complete spectrum associated with that species appears as dips in the probe laser signal because of the depletion in the population of this conformer by the scanning pump laser.
The acid group is more complicated, however, as is evident in trace 5c. 13C labeling at the central carbon atom of the carboxylate group displaces two bands: the sharp 1777 cm−1 feature at the expected location for a free acid functionality (v CO COOH , green), and the interloper (†, red) that was identified in the IR-IR hole burning study as arising from a second conformer. Thus, the acid group is unambiguously identified as the carrier of the † feature, which is red-shifted from that arising from a free acid by about 50 cm−1 and thus denoted v CO COOH bound . We note that a similar situation was observed earlier in the case of the SarSarH+ dipeptide spectrum , where an intramolecular H-bond to the acid carbonyl group in one of its conformers displayed a significant (24 cm−1) red-shift in the C=O stretching frequency.
Metabolite Identification through the OH Stretching Bands in the Vibrational Spectra of the Cold Na+ Complexes
To address the hypothetical issue where it is important to differentiate between structural isomers (in addition to the natural metabolite), we synthesized III with the hydroxyl group at the terminal C5 position (structure indicated on the lower right in Figure 1, with synthetic pathway detailed in SI). We note that these two compounds would present a challenging case for the widely used MS2 approach for metabolite identification , since they yield identical fragmentation patterns as illustrated in Figure S2.
The spectroscopic behavior of the hydroxylated derivatives emphasizes the fact that the differentiation arises from the local folded forms of conformers at low temperature. This also creates interesting possibilities for enhanced discrimination techniques where a strategic guest molecule is docked to the target species in a manner that optimizes host–guest interactions for a specific isomer. This yields an IR variant of molecular recognition methods that have been demonstrated in a mass spectrometric venue for chiral recognition [31, 32, 33].
We illustrated how messenger tagging vibrational spectroscopy can be used to differentiate metabolites in small molecule drug discovery within the context of structural mass spectrometry. The importance of the charging mechanism was explored by recording the vibrational spectra of protonated, deprotonated, and sodiated ions, with the result that only the sodiated ions yield clear bands associated with the NH, OH, and CO functionalities, whereas the other charging schemes yield intermolecular H-bonds, which complicate and obscure these features. The sodiated parent ion of Valsartan was observed in two conformations, which yield very different characteristic absorptions for the functional groups. These patterns were identified using conformer-selective IR-IR double resonance spectroscopy combined with site-specific isotopic substitution, and assigned to different classes of metal coordination and intramolecular H-bonding. We then obtained the vibrational spectra of the sodiated Valsartan metabolite and a synthetic isomer, which differ only in the addition of a hydroxyl group at either the C4 or C5 position of the valeroyl alkyl chain. The spectra of both sodiated derivatives were simpler than that of the parent as they exhibit only one of the conformers adopted by the sodiated parent. The sharp, free OH transitions of the hydroxyl groups are separated by over 160 cm−1, thus enabling a clear differentiation of the derivatives by coupling vibrational spectroscopy with the sensitivity of mass spectrometry. The large shift between the two fundamentals is discussed in terms of their likely intramolecular H-bonding interactions, thus highlighting the importance of quenching the internal energy of these conformationally flexible systems prior to analysis with vibrational spectroscopy. From these results, we suggest the potential role of neutral complex formation with strategically chosen partners based on molecular recognition as a way to further refine the selectivity afforded by cryogenic ion chemistry and spectroscopy.
M.A.J. thanks the National Science Foundation for support under grant number CHE-1465100. This work was supported in part by the Yale University Faculty of Arts and Sciences High Performance Computing Facility (and staff). A.B. acknowledges financial support from the National Science Foundation through the HBCU-UP award no. 1505095. We also thank Nan Yang and Chinh Duong for their work on the updated instrumental capabilities utilized in this experiment (Figure 2).
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