High resolution quadrupole time of flight mass spectrometry in pharmaceutical bioanalysis

This cumulative PhD thesis put forward several aspects of the analysis of biomolecules employing high resolution quadrupole time of flight mass spectrometry (HR-QTOF-MS). Particular analytical challenges in the context of the analysis of various classes of analytes, i.e. amino acids and peptides, oligonucleotide complexes, triterpenoid esters, intact proteins have been addressed and new analytical solutions by either liquid chromatographic separation or HR-QTOF-MS, respectively, their hyphenation have been suggested. This PhD-Thesis is comprised of four thematically distinct parts: The first part dealt with the stereoselective analysis of amino acids and peptides. In one study, the complete stereoconfiguration of an antimicrobial active lipopeptide, poaeamide, was determined. Lipopeptides are typically synthesized by a non-ribosomal enzymatic peptide synthesis machinery. As result, they frequently contain several D-amino acids providing hydrolysis resistance towards target organism peptidases. As lipopeptides are of general interest for research on and development of antimicrobial compounds, complete structural elucidation is essential, which encompasses also determination of the absolute configurations of the amino acids constituting the respective peptide, which is presented in Publication II. The analysis strategy enveloped the incomplete hydrolysis of the peptide yielding overlapping sequence fragments, micro-scale preparative liquid chromatography and stereoconfiguration analysis of hydrolysis fragments by chiral GC-MS, ultimately providing determination of the stereoconfigurations of its comprising amino acids enantiomers. As the latter cannot be distinguished by mass spectrometry alone, HR-QTOFMS has been hyphenated with appropriate enantioselective chromatography using cinchonan carbamate based chiral stationary phases. This work involved the optimization of the chromatographic and MS conditions and the demonstration of the feasibility of aforementioned phases in providing complementary chromatographic selectivity when compared to RP and HILIC type phases, in detail emphasized in Publication VIII. A major challenge was the determination of configurations of amino acids with more than one stereogenic centers (Thr/allo-Thr, Ile/allo-Ile) and those also present as constitutional isomers (Leu/Ile). A particular intricacy solved by the analysis strategy of a combined LC and GC approach employing reversed phase (RP), hydrophilic interaction (HILIC) and enantioselective stationary phases was the localization of D-Leu discovered in the peptide, as its position was disguised by presence of several Leu residues in the lipopeptide. To advance insight into the enantioselective interaction between the employed LC stationary phases and amino acids and derivatives thereof, Publication I reported the application of quantitative structure-retention and structure-enantioselectivity relationships to quantitatively study enantioselective molecular recognition mechanisms. By employing a Free-Wilson type generalized linear modelling approach, Publication I validated hypotheses that describe binding energy contribution of individual molecular moieties as being linear independent of each other. Major contribution to retention of the analytes could be attributed to pi-interacting derivatization groups, a finding that stands in congruence to experimental findings reported amongst others by Publication II. With mass spectrometry today representing the chromatographic detection method of choice and consequently entailing the desideratum of stationary phases compatible with this technology, the first part of this thesis was concluded by Publication VII enhancing mass spectrometric compatibility of the employed chiral stationary phases. With a hydrolysis stable crosslinked methylpolysiloxane type surface chemistry, also providing a scaffold for various surface ligand modifications by the employed thiol-ene click chemistry, significant enhancement of mass spectrometric compatibility could be demonstrated. Using the enantioselective cinchonan carbamate based chiral stationary phases as an example ligand, ameliorated phase stability and resultant enhancement in mass spectrometric sensitivity was assessed and confirmed by high resolution quadrupole time of flight mass spectrometry. In the second part, challenging analysis, both from chromatographic and mass spectrometric perspective, of regioisomers of pentacyclic triterpenoid fatty acid esters instable even under soft ionizing conditions was addressed by Publication V. Novel esters of triterpenoids with anti-inflammatory potential, amongst them mixed esters of faradiol, myristic and palmitic acid could be confirmed to be present in extracts of by employment of orthogonal analysis methods, namely NMR, GC-MS and LC-HR-QTOF-MS. In order to address the challenging liquid chromatographic separation of mixed regioisomeric diesters, molecular shape selective chromatography was employed using C30-type RP-stationary phases tailored for the task. Chromatographic and mass spectrometric requirements, the latter stemming from the astonishing instability of the analytes during ionization in the presence of water, even under the soft ionization conditions encountered in electrospray (ESI) or atmospheric pressure chemical ionization (APCI), could be harmonized by application of a non-aqueous binary eluent system, cold LC column temperatures facilitating entropic optimization of regioisomer separation and adequate application and tuning of parameters of APCI-QTOFMS for sensitivity, mass accuracy and resolution. The third part was dedicated to intact protein mass spectrometric analysis (Publications IV IX X and XI). Employing the Sciex 5600+ TripleTOFs capability in mass accuracy, mass resolution and sensitivity even for large molecular species by adequate mass spectrometric and chromatographic method development, analytical questions revolving around analysis of intact proteins could be addressed, including antibody characterization and, in a straightforward approach demonstrating mass accuracy and resolution of QTOF, direct confirmation of attachment and correct target location of covalent kinase inhibitors with sub kDa molecular weight to >40kDa protein targets. Finally, the fourth part of this thesis includes two studies (Publications III and XII) that examine interaction between G4-DNA-selective ligands and G4-DNA-quadruplexes, the latter representing an in vivo form of a DNA that is of oncological research interest as it is frequently encountered in promoter regions of oncogenes. Ligands specifically binding to this DNA form is subject of research aimed at cancer imaging or potential anticancer drugs. Study of such non-covalent complexes in solution is preferably performed by NMR. However, NMR spectra interpretation is both regularly and in case of Publication III and XII severely hampered by extensive peak broadening and overlapping as consequence of fast to intermediate exchange rates relative to the NMR chemical shift timescale of ligands occupying different binding sites Fluorescence titration, employed as orthogonal method in both studies, also could not unequivocally unveil the stoichiometry of the complexes studied in the two publications. The author’s contribution was the development of a native electrospray ionization high resolution quadrupole time of flight mass spectrometry (ESI-HRQTOF) method to elucidate stoichiometry and binding mode of aforementioned complexes. The intricacy to address for both studies was the provision of mass spectrometric method capable of mapping non-covalent complex stoichiometries and properties from solution to the vacuum of the mass spectrometric ion path without distortion for example of secondary structure or ligand binding by the ionization process, by the atmosphere to vacuum transition or flight through the ion path. The native-ESI-QTOF-MS method developed was capable of providing these requirements for noncovalent DNA-ligand complexes of several kDa molecular mass, yet still allowing to quantitively monitor specific binding of very low molecular (e.g. ammonium NH4+) species to the complex. Overall, the studies summarized in this Thesis, demonstrated the great utility and wide area of application of high-resolution quadrupole time of flight mass spectrometry, either in its hyphenated form with liquid chromatography or as direct infusion-MS, to solve challenging analytical questions in the context of (bio)pharmaceutical analysis

Mastering analytical challenges for the characterization of pentacyclic triterpene mono- and diesters of Calendula officinalis flowers by non-aqueous C30 HPLC and hyphenation with APCI-QTOF-MS

Pentacyclic triterpene mono- and diesters have been isolated from Calendula officinalis flowers. GC–MS, APCI-Exactive Orbitrap HR-MS and NMR allowed to identify the triterpene skeleton in various samples (different triterpene mixtures from Calendula n-hexane extract). NMR provided evidence that triterpene diesters are present in the samples as well. However, the corresponding quasi-molecular ions could not be detected by APCI-Exactive Orbitrap HR-MS. Instability of triterpene diesters and loss of a fatty acid residue, respectively, in the ion-source made their MS detection challenging. Thus, a set of new APCI-QTOF-MS methods (using the TripleTOF 5600+ mass spectrometer) were developed which made it eventually possible to solve this problem and confirm the diester structures by MS via quasi-molecular ion [M + H]+ detection. Direct infusion APCI-QTOF MS experiments in MS/MS high sensitivity scan mode with low collision energy and multi-channel averaging acquisition (MCA) allowed the detection of quasi-molecular ions of triterpene diesters for the first time and unequivocally confirmed the presence of faradiol 3,16-dimyristate and -dipalmitate, as well as the corresponding mixed diesters faradiol 3-myristate,16-palmitate and faradiol 3-palmitate,16-myristate. Preferential loss of the fatty acid in 16-position made it possible to distinguish the mixed diesters by MS/MS spectra. Their chromatographic separations turned out to be challenging due to their bulkiness and extended molecular dimensions. However, separation could be achieved by an uncommon non-aqueous RPLC mode with an in-house synthesized C30 phase. Finally, two (U)HPLC-APCI-QTOF-MS methods with C18- and C30-based non-aqueous RPLC provided suitable, sensitive assays to monitor the presence of monoesters and diesters of various triterpenes (faradiol, maniladiol, arnidiol, arnitriol A and lupane-3β,16β,20-triol esters) in the n-hexane extract of C. officinalis with high mass resolution and good mass accuracy.Struktur- und Innovationsfonds Baden-Württemberg/[INST 37/821-1 FUGG]/SI-BW/AlemaniaGerman Research Foundation/[INST 37/821-1 FUGG]/DFG/AlemaniaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

Trisubstituted Imidazoles with a Rigidized Hinge Binding Motif Act As Single Digit nM Inhibitors of Clinically Relevant EGFR L858R/T790M and L858R/T790M/C797S Mutants: An Example of Target Hopping

The high genomic instability of non-small cell lung cancer tumors leads to the rapid development of resistance against promising EGFR tyrosine kinase inhibitors (TKIs). A recently detected triple mutation compromises the activity of the gold standard third-generation EGFR inhibitors. We have prepared a set of trisubstituted imidazoles with a rigidized 7-azaindole hinge binding motif as a new structural class of EGFR inhibitors by a target hopping approach from p38α MAPK inhibitor templates. On the basis of an iterative approach of docking, compound preparation, biological testing, and SAR interpretation, robust and flexible synthetic routes were established. As a result, we report two reversible inhibitors <b>11d</b> and <b>11e</b> of the clinically challenging triple mutant L858R/T790M/C797S with IC₅₀ values in the low nanomolar range. Furthermore, we developed a kinome selective irreversible inhibitor <b>45a</b> with an IC₅₀ value of 1 nM against the EGFR L858R/T790M double mutant. Target binding kinetics and metabolic stability data are included. These potent mutant EGFR inhibitors may serve as a basis for the development of structurally novel EGFR probes, tools, or candidates

Methods for the comprehensive structural elucidation of constitution and stereochemistry of lipopeptides

A panel of methods of general suitability for complete structural elucidation of the stereochemistry of cyclopeptides, depsipeptides and lipopeptides is presented and described in detail. The suitability of the proposed methods was exemplified on the lipopeptide poaeamide from Pseudomonas poae. Amino acid configurations have been assigned by direct LC enantiomer separation with Chiralpak ZWIX(+) and were confirmed by GC enantiomer separation on Chirasil L-Val. 3-Hydroxydecanoic acid absolute configuration was analyzed on Chiralpak ZWIX(+) and confirmed by injection on ZWIX(-) which showed opposite elution order. Plenty of D-amino acids have been found in this lipopeptide. It contained in total 5 Leu residues of which one had n-configuration. The position of the D-Leu in the peptide sequence was determined by pepsin and chemical digestions in combination with isolation of diagnostic peptide-fragments and subsequent identification of absolute configurations of the Leu residues. This allowed pinpointing the position of the D-amino acid. The complementarity of the peptide retention profiles on Chiralpak ZWIX column as compared to both RPLC and HILIC suggests its great utility as an alternative peptide separation tool. (C) 2015 Elsevier B.V. All rights reserved

Tri- and Tetrasubstituted Pyridinylimidazoles as Covalent Inhibitors of c-Jun N-Terminal Kinase 3

The concept of covalent inhibition of c-Jun N-terminal kinase 3 (JNK3) was successfully transferred to our well validated pyridinylimidazole scaffold varying several structural features in order to deduce crucial structure–activity relationships. Joint targeting of the hydrophobic region I and methylation of imidazole-N1 position increased the activity and reduced the number of off-targets. The most promising covalent inhibitor, the tetrasubstituted imidazole 3-acrylamido-<i>N</i>-(4-((4-(4-(4-fluorophenyl)-1-methyl-2-(methylthio)-1<i>H</i>-imidazol-5-yl)­pyridin-2-yl)­amino)­phenyl)­benzamide (<b>7</b>) inhibits the JNK3 in the subnanomolar range (IC₅₀ = 0.3 nM), shows high metabolic stability in human liver microsomes, and displays excellent selectivity in a screening against a panel of 410 kinases. Covalent bond formation to Cys-154 was confirmed by incubation of the inhibitors with wild-type JNK3 and JNK3-C154A mutant followed by mass spectrometry

Targeting the Gatekeeper MET146 of C-Jun N-Terminal Kinase 3 Induces a Bivalent Halogen/Chalcogen Bond

We target the gatekeeper MET146 of c-Jun N-terminal kinase 3 (JNK3) to exemplify the applicability of X···S halogen bonds in molecular design using computational, synthetic, structural and biophysical techniques. In a designed series of aminopyrimidine-based inhibitors, we unexpectedly encounter a plateau of affinity. Compared to their QM-calculated interaction energies, particularly bromine and iodine fail to reach the full potential according to the size of their σ-hole. Instead, mutation of the gatekeeper residue into leucine, alanine, or threonine reveals that the heavier halides can significantly influence selectivity in the human kinome. Thus, we demonstrate that, although the choice of halogen may not always increase affinity, it can still be relevant for inducing selectivity. Determining the crystal structure of the iodine derivative in complex with JNK3 (4X21) reveals an unusual bivalent halogen/chalcogen bond donated by the ligand and the back-pocket residue MET115. Incipient repulsion from the too short halogen bond increases the flexibility of C_ε of MET146, whereas the rest of the residue fails to adapt being fixed by the chalcogen bond. This effect can be useful to induce selectivity, as the necessary combination of methionine residues only occurs in 9.3% of human kinases, while methionine is the predominant gatekeeper (39%)

Design, Synthesis, and Biological Evaluation of Novel Type I¹/₂ p38α MAP Kinase Inhibitors with Excellent Selectivity, High Potency, and Prolonged Target Residence Time by Interfering with the R‑Spine

We recently reported <b>1a</b> (skepinone-L) as a type I p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, as a type I inhibitor, it is entirely ATP-competitive and shows just a moderate residence time. Thus, the scope was to develop a new class of advanced compounds maintaining the structural binding features of skepinone-L scaffold like inducing a glycine flip at the hinge region and occupying both hydrophobic regions I and II. Extending this scaffold with suitable residues resulted in an interference with the kinase’s R-Spine. By synthesizing 69 compounds, we could significantly prolong the target residence time with one example to 3663 s, along with an excellent selectivity score of 0.006 and an outstanding potency of 1.0 nM. This new binding mode was validated by cocrystallization, showing all binding interactions typifying type I¹/₂ binding. Moreover, microsomal studies showed convenient metabolic stability of the most potent, herein reported representatives