13 research outputs found
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 authorâs 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
Peptide Analysis: Zwitterionic Chiral Ion-Exchangers as Complementary Option to HILIC and to Reversed-Phase Chromatography
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. GCâMS, 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-3ÎČ,16ÎČ,20-triol esters) in the n-hexane extract of C. officinalis with high mass resolution and good mass accuracy.Struktur- und Innovationsfonds Baden-WĂŒrttemberg/[INST 37/821-1 FUGG]/SI-BW/AlemaniaGerman Research Foundation/[INST 37/821-1 FUGG]/DFG/AlemaniaUCR::VicerrectorĂa de InvestigaciĂłn::Unidades de InvestigaciĂłn::Ciencias BĂĄsicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::VicerrectorĂa de Docencia::Ciencias BĂĄsicas::Facultad de Ciencias::Escuela de QuĂmic
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
IC<sub>50</sub> values in the low nanomolar range. Furthermore, we
developed a kinome selective irreversible inhibitor <b>45a</b> with an IC<sub>50</sub> 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
UHPLC-ESI-MS/MS assay for quantification of endocannabinoids in cerebrospinal fluid using surrogate calibrant and surrogate matrix approaches
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
structureâactivity 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)Âpyridin-2-yl)Âamino)Âphenyl)Âbenzamide (<b>7</b>) inhibits the JNK3 in the subnanomolar range (IC<sub>50</sub> =
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 Ï-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<sub>Δ</sub> 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 I<sup>1</sup>/<sub>2</sub> p38α MAP Kinase Inhibitors with Excellent Selectivity, High Potency, and Prolonged Target Residence Time by Interfering with the RâSpine
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 kinaseâs 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 I<sup>1</sup>/<sub>2</sub> binding. Moreover,
microsomal studies showed convenient metabolic stability of the most
potent, herein reported representatives