Connecting metabolome and phenotype: recent advances in functional metabolomics tools for the identification of bioactive natural products.

Covering: 1995 to 2023Advances in bioanalytical methods, particularly mass spectrometry, have provided valuable molecular insights into the mechanisms of life. Non-targeted metabolomics aims to detect and (relatively) quantify all observable small molecules present in a biological system. By comparing small molecule abundances between different conditions or timepoints in a biological system, researchers can generate new hypotheses and begin to understand causes of observed phenotypes. Functional metabolomics aims to investigate the functional roles of metabolites at the scale of the metabolome. However, most functional metabolomics studies rely on indirect measurements and correlation analyses, which leads to ambiguity in the precise definition of functional metabolomics. In contrast, the field of natural products has a history of identifying the structures and bioactivities of primary and specialized metabolites. Here, we propose to expand and reframe functional metabolomics by integrating concepts from the fields of natural products and chemical biology. We highlight emerging functional metabolomics approaches that shift the focus from correlation to physical interactions, and we discuss how this allows researchers to uncover causal relationships between molecules and phenotypes.

Design and Optimization of Novel Benzimidazole- and Imidazo[4,5-b]pyridine-Based ATM Kinase Inhibitors with Subnanomolar Activities.

The ATM kinase is a promising target in cancer treatment as an important regulator of the cellular response to DNA double-strand breaks. In this work, we present a new class of specific benzimidazole-based ATM inhibitors with picomolar potency against the isolated enzyme and favorable selectivity within relative PIKK and PI3K kinases. We could identify two promising inhibitor subgroups with significantly different physicochemical properties, which we developed simultaneously. These efforts lead to numerous highly active inhibitors with picomolar enzymatic activities. Furthermore, initial low cellular activities on A549 cells could be increased significantly in numerous examples resulting in cellular IC50 values in the subnanomolar range. Further characterization of the highly potent inhibitors 90 und 93 revealed promising pharmacokinetic properties and strong activities in organoids in combination with etoposide. Additionally, 93 showed no off-target activities within a kinome-representative mini kinase panel, with favorable selectivities within the PIKK- and PI3K-families.

Branched medium-chain fatty acid profiling and enantiomer separation of anteiso-forms of teicoplanin fatty acyl side chain RS3 using UHPLC-MS/MS with polysaccharide columns.

This work reports on targeted UHPLC-tandem mass spectrometry methods for the chiral separation of anteiso-methyl branched fatty acids (aiFAs). The methods involve precolumn derivatization with 1-naphthylamine and chiral separation on Chiralpak IG-U. anteiso-Methyl branched fatty acids with up to eight carbons can be separated. A method was used for the assignment of the absolute configuration of an aiFA present as fatty acyl residue of the teicoplanin mixture, namely teicoplanin RS3. Furthermore, the excellent methylene selectivity and improved selectivity for constitutional isomers of the polysaccharide columns was exploited for the elucidation and structural confirmation of previously unknown fatty acyl residues in teicoplanin. This shows the versatility and practical applicability of polysaccharide columns as orthogonal stationary phases to reversed-phase for structural elucidation of natural compounds. The developed methods are useful tools for related subdisciplines such as targeted metabolomics and lipidomics.

Development and chromatographic exploration of stable-bonded cross-linked amino silica against classical amino phases.

The present work reports on a novel stable-bonded amino silica stationary phase obtained by crosslinking of surface aminopropyl moieties using triglycidyl isocyanurate. The obtained cross-linked amido-amino network silica material exhibited superior hydrolytic stability compared to classical 3-aminopropyl phases and showed, inter alia, excellent separation of nine therapeutically effective sulfonamides in hydrophilic interaction/weak anion exchange chromatography elution mode. Additionally, the separation of carbohydrates was investigated under classical hydrophilic interaction chromatography conditions as well proving the suitability of the novel phase for such applications. For the evaluation of the hydrolytic stability the prepared material, as well as two commercially available benchmark columns and a set of in-house synthesized amino-modified materials, were exposed to harsh aqueous mobile phase conditions for in total of 50 h at elevated temperature. In this context, the materials were examined by elemental analysis, (13 C and 29 Si cross-polarization/magic angle spinning) solid-state nuclear magnetic resonance, and a chromatographic test before and subsequent to the exposure to these stress conditions. Lastly, the new stationary phase was classified in comparison to a set of commercially available stationary phases by principal component analysis of resultant retention factors gained from chromatographic standard tests.

Study of microheterogeneity of silatrane-based silica surface bonding chemistry and its optimization for the synthesis of chiral stationary phases for enantioselective liquid chromatography.

The present work systematically investigates the chemical microheterogeneity as part of the optimization of a single-step surface bonding chemistry of 3-mercaptopropylsilatrane (MPS) on mesoporous silica gel in comparison to the state-of-the-art silane chemistry with 3-mercaptopropyltrimethoxysilane (MPTMS). MPS functionalization turns out to be a favourable chemistry for the further use in thiol-ene click reactions such as the immobilization of chiral selectors, herein tert-butylcarbamoylquinine (tBuCQN), for the synthesis of chiral stationary phases (CSPs). MPS has higher reactivity than MPTMS and prefers the formation of trifunctional siloxane bondings unlike MPTMS which favours difunctional siloxane bonds to silica, as investigated by solid-state cross-polarization/magic angle spinning (CP/MAS) NMR (29Si and 13C nuclei). Reaction conditions (ternary mixtures of methanol, water and toluene; with and without acid; prewetting of silica; HCl pretreatment of silica) were evaluated with the aim to find conditions which promote the formation of a horizontal siloxane polymer layer on top of the silica surface. Silanization reaction times could be reduced to 2 h. The 29Si NMR signal corresponding to trifunctional siloxane bonding could be increased to 60% with no T1 signal that refers to monofunctional siloxane bonding in spite of water in the ternary reaction mixture. Furthermore, no significant disulfide bridges were formed in this approach, leading to high selector loadings. The thiol and selector coverage reached up to 4.6 and 1.4 µmol/m2, respectively. With the preferred CSP, the enantioselectivity could be increased for a chiral probe (FMOC-Phe) and the mass transfer resistance (C-term) bisected compared to the corresponding CSP prepared from benchmark MPTMS-modified silica (2.54 vs 5.72 ms). It is demonstrated that the fine-tuning of the microstructure on the silica surface can have a significant influence on enantioselectivity and mass transfer kinetics of the resultant CSPs.

Development of novel urea-based ATM kinase inhibitors with subnanomolar cellular potency and high kinome selectivity.

The ATM kinase is a key molecule regulating DNA damage response and can be targeted resulting in efficient radio- or chemosensitization. Due to the enormous size of this protein and the associated difficulties in obtaining high-quality crystal structures, we sought to develop an accurate in silico model to identify new targeting possibilities. We identified a urea group as the most beneficial chemical anchor point, which could undergo multiple interactions in the aspartate-rich hydrophobic region I of the atypical ATM kinase domain. Based on in silico data, we designed and synthesized a comprehensive set of novel urea-based inhibitors and characterized them in diverse biochemical assays. Using this strategy, we identified inhibitors with subnanomolar potency, which were further evaluated in cellular models, selectivity and early DMPK properties. Finally, the two lead compounds 34 and 39 exhibited subnanomolar cellular activity along with an excellent selectivity profile and favorable metabolic stability.

High Plasticity of the Amicetin Biosynthetic Pathway in Streptomyces sp. SHP 22-7 Led to the Discovery of Streptcytosine P and Cytosaminomycins F and G and Facilitated the Production of 12F-Plicacetin.

A chemical reinvestigation of the Indonesian strain Streptomyces sp. SHP 22-7 led to the isolation of three new pyrimidine nucleosides, along with six known analogues and zincphyrin. The structures of the new compounds (6, 7, 10) were elucidated by employing spectroscopic techniques (NMR, MS, CD, and IR) as well as enantioselective analyses of methyl branched side chain configurations. Application of the precursor-directed feeding approach led to the production and partial isolation of nine further pyrimidine analogues. The new compounds 6, 7, and 11 and three of the known compounds (2-4) were found to possess antimycobacterial and cytotoxic properties.

Towards enantioselective ultrahigh performance liquid chromatography-mass spectrometry-based metabolomics of branched-chain fatty acids and anteiso-fatty acids under reversed-phase conditions using sub-2-µm amylose- and cellulose-derived chiral stationary phases.

Branched-chain fatty acids (BCFAs) are mostly saturated fatty acids with one or more methyl, seldom ethyl, branches in the alkyl chain. They are derived from branched-chain amino acids, ruminant-derived food, or biosynthetic side products of acetyl-CoA carboxylase. They possess iso- (branching at penultimate carbon) and anteiso-fatty acid structure (branching at antepenultimate carbon) or are branched at any other position of the carbon chain. Except for iso-fatty acids, BCFAs are chiral. They are commonly analyzed by GC-MS, while there is a lack of enantioselective LC-MS methods. In this work, we present a methodology for targeted enantioselective UHPLC-ESI-MS/MS metabolomics of BCFAs. It makes use of precolumn derivatization with 1-naphthylamine and reversed-phase elution conditions. A homologous series of short BCFA analytes with distinct chain lengths (having up to eight carbon atoms), branching type (methyl or ethyl), and position of branching (2, 3, and 4, anteiso and iso) has been systematically studied on six commercially available polysaccharide UHPLC columns. Chiralpak IB-U exhibited the highest and broadest enantioselectivity while IH-U maintained enantioselectivity also for BCFAs with chirality distant from the carboxylic function (i.e., with other branching than in 2-position). The method was used to assign the absolute configuration of a 4-methylhexanoic acid side chain of a natural product from Streptomyces sp. SHP 22-7. The potential of the corresponding UHPLC-ESI-QTOF-MS/MS assay for analyzing stereoselectively BCFAs and other short organic acids by untargeted analysis in human urine was further elucidated in a preliminary proof-of-principle test.

Addressing a Trapped High-Energy Water: Design and Synthesis of Highly Potent Pyrimidoindole-Based Glycogen Synthase Kinase-3ß Inhibitors.

In small molecule binding, water is not a passive bystander but rather takes an active role in the binding site, which may be decisive for the potency of the inhibitor. Here, by addressing a high-energy water, we improved the IC50 value of our co-crystallized glycogen synthase kinase-3ß (GSK-3ß) inhibitor by nearly two orders of magnitude. Surprisingly, our results demonstrate that this high-energy water was not displaced by our potent inhibitor (S)-3-(3-((7-ethynyl-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile ((S)-15, IC50 value of 6 nM). Instead, only a subtle shift in the location of this water molecule resulted in a dramatic decrease in the energy of this high-energy hydration site, as shown by the WaterMap analysis combined with microsecond timescale molecular dynamics simulations. (S)-15 demonstrated both a favorable kinome selectivity profile and target engagement in a cellular environment and reduced GSK-3 autophosphorylation in neuronal SH-SY5Y cells. Overall, our findings highlight that even a slight adjustment in the location of a high-energy water can be decisive for ligand binding.

DoE Optimization Empowers the Automated Preparation of Enantiomerically Pure [18F]Talazoparib and its In Vivo Evaluation as a PARP Radiotracer.

Given the clinical potential of poly(ADP-ribose) polymerases (PARP) imaging for the detection and stratification of various cancers, the development of novel PARP imaging probes with improved pharmacological profiles over established PARP imaging agents is warranted. Here, we present a novel 18F-labeled PARP radiotracer based on the clinically superior PARP inhibitor talazoparib. An automated radiosynthesis of [18F]talazoparib (RCY: 13 ± 3.4%; n = 4) was achieved using a "design of experiments" (DoE) optimized copper-mediated radiofluorination reaction. The chiral product was isolated from the reaction mixture using 2D reversed-phase/chiral radio-HPLC (>99% ee). (8S,9R)-[18F]Talazoparib demonstrated PARP binding in HCC1937 cells in vitro and showed an excellent tumor-to-blood ratio in xenograft-bearing mice (10.2 ± 1.5). Additionally, a favorable pharmacological profile in terms of excretion, metabolism, and target engagement was observed. This synthesis of [18F]talazoparib exemplifies how DoE can enable the radiosyntheses of synthetically challenging radiolabeled compounds of high interest to the imaging community.

Enantioselective multiple heart cutting online two-dimensional liquid chromatography-mass spectrometry of all proteinogenic amino acids with second dimension chiral separations in one-minute time scales on a chiral tandem column.

In this work, we present a unique, robust and fully automated analytical platform technology for the enantioselective amino acid analysis using a multiple heart cutting RPLC-enantio/stereoselective HPLC-ESI-QTOF-MS method. This 2D-LC method allows the full enantioselective separation of 20 proteinogenic AAs plus 5 isobaric analogues, namely allo-Threonine (aThr), homoserine (Hse), allo-isoleucine (aIle), tert-Leucine (Tle) and Norleucine (Nle), after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ). This N-terminal AA-derivatization method introduces on the one hand beneficial chromatographic properties for 1D RP-LC (stronger retention) and 2D chiral separation (better chiral recognition), and on the other hand favorable detection properties with its chromophoric, fluorophoric, and easily ionizable quinoline mass tag. The entire separation occurs within a total 2DLC run time of 45 min, which includes the 1D-RP run and the 68 s 2D chiral separations of 30 heart-cuts (from the 1D-RP-run) on a chiral quinine carbamate (core-shell QNAX/fully porous ZWIX) tandem column. This relatively short overall run time was only possible by utilizing the highly efficient "smart peak parking" algorithm for the heart cuts and the resulting optimized analysis order thereof. 1D retention time precisions of <0.21% RSD were a requirement for the time-based sampling mode and finally led to a robust, fully automated enantioselective amino acid analysis platform. This achiral-chiral 2DLC method was applied for the amino acid stereoconfiguration assignment of three peptides (aureobasidin A, a lipopeptide research sample, and octreotide) using an L-[u-13C15N] labelled internal AA standard mix spiked to each sample. The isotopically labelled L-AA standard allowed an easy and straightforward identification and configuration assignment, as well as the relative quantification of amino acids within the investigated peptides, allowing the direct determination of the number of respective amino acids and their chirality within a peptide.

Controllable organosilane monolayer density of surface bonding using silatranes for thiol functionalization of silica particles for liquid chromatography and validation of microanalytical method for elemental composition determination.

The present work systematically investigates a new strategy for the functionalization of silica gel using alkyl silatrane chemistry instead of alkylsilanes for synthesis of chromatographic stationary phases. In this work, silica was chemically modified for further functionalization by a thiol-ene click reaction. Thus, 3-mercaptopropylsilatrane (MPS) was used which is capable to form self-assembled monolayers (SAM) on top of silanol surfaces in a controlled manner as previously shown for silicon wafers. The utility of this chemistry for stationary phase synthesis in liquid chromatography was not evaluated yet. Hence, silica surface modifications using MPS were studied in comparison to established 3-mercaptopropyltrimethoxysilane (MPTMS) chemistry. First, the employed elemental analysis method was validated and it showed excellent intra-day and inter-day precisions (typically less than 5% RSD). It could be shown that the reaction kinetics of MPS was roughly 35-times faster than with MPTMS. After 30 min reaction time with MPS, the thiol content reached 74% of the maximal coverage. Due to controlled chemistry with MPS, which does not lead to oligomeric siloxane network at the silica surface, the ligand coverage was lower. However, multiple silanization cycles with MPS led to a dense surface coverage (around 4 µmol m-2). 29Si cross polarization/magic angle spinning (CP/MAS) solid-state NMR revealed distinct T1/T2/T3 ratios for MPS and MPTMS materials with up to 80% T3 (indicative for trifunctional siloxane linkage) for MPS and around 20% T3 for MPTMS. This indicates a more homogeneous, thinner monolayer film of MPS on the silica surface, as compared to an irregular thick oligomeric siloxane network with MPTMS. Bonding of quinine carbamate as chiral selector afforded an efficient chiral stationary phase (CSP) for chromatographic enantiomer separation. Separation factors were comparable to MPTMS-bonded CSP, however, chromatographic efficiency was much better for the MPS-bonded CSP. H/u curves indicated a reduced mass transfer resistance by roughly factor 3 for MPS- compared to MPTMS-bonded CSP. This confirms better chromatographic performance of surfaces with homogeneous monolayer compared to network structures on the silica surface which suffer from poor stationary phase mass transfer.

Preparation and characterization of poly(3-mercaptopropyl)methylsiloxane functionalized silica particles and their further modification for silver ion chromatography and enantioselective high-performance liquid chromatography.

The present work reports on the preparation of polythiol-functionalized silica particles by thermally and photo-initiated radical addition reactions using poly(3-mercaptopropyl)methylsiloxane (PMPMS) as sulfhydryl group-rich surface modification reagent. Prior to surface modification with PMPMS, the silica was vinylized with vinyl trimethoxysilane. Finally, the usefulness of the thiolated silica particles was demonstrated by their further modification for various HPLC applications such as argentation chromatography and chiral separations. Aiming at a sulfhydryl group-rich, thin PMPMS layer on the surface of the silica several factors such as quantity of PMPMS, radical starter and reaction time were investigated by a design of experiment (DoE) approach. In thermally induced polymerization reactions 2,2'-azobis(isobutyronitrile) (AIBN) was used as radical starter, in photo-induced reactions 2,2-dimethoxy-2-phenylacetophenone (DMPA) was used instead. The incorporation of PMPMS was evaluated by elemental analysis and reactive and accessible sulfhydryl groups were determined by performing a thiol-disulfide exchange reaction with 2,2'-dipyridyl disulfide (DPDS). Consequently, thiol-functionalized silica particles (200 Å, 5 µm) with 1.81 ± 0.07 µmol sulfhydryl groups per m2 were prepared and further functionalized for silver ion chromatography and chiral separation chromatography clearly proving its utility as platform for further silica functionalization. The fabricated stationary phase for silver ion chromatography showed promising separation abilities for fatty acid methyl esters (FAME) according to the amount of double bonds within the fatty acid residue and cis- and trans-stilbene as model molecule for cis-trans isomerism. After the successful incorporation of O-tert-butylcarbamoyl quinine (tBuCQN) as chiral selector via thiol-ene click chemistry onto the PMPMS layer, the obtained chiral stationary phases (CSP) showed good separation of derivatized amino acids in polar organic elution mode comparable with a column based on commercially available CHIRALPAK QN-AX silica particles (120 Å, 5 µm).

Discovery and Evaluation of Enantiopure 9H-pyrimido[4,5-b]indoles as Nanomolar GSK-3ß Inhibitors with Improved Metabolic Stability.

Glycogen synthase kinase-3ß (GSK-3ß) is a potential target in the field of Alzheimer's disease drug discovery. We recently reported a new class of 9H-pyrimido[4,5-b]indole-based GSK-3ß inhibitors, of which 3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propanenitrile (1) demonstrated promising inhibitory potency. However, this compound underwent rapid degradation by human liver microsomes. Starting from 1, we prepared a series of amide-based derivatives and studied their structure-activity relationships against GSK-3ß supported by 1 µs molecular dynamics simulations. The biological potency of this series was substantially enhanced by identifying the eutomer configuration at the stereocenter. Moreover, the introduction of an amide bond proved to be an effective strategy to eliminate the metabolic hotspot. The most potent compounds, (R)-3-(3-((7-chloro-9H-pyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)-3-oxopropanenitrile ((R)-2) and (R)-1-(3-((7-bromo-9Hpyrimido[4,5-b]indol-4-yl)(methyl)amino)piperidin-1-yl)propan-1-one ((R)-28), exhibited IC50 values of 480 nM and 360 nM, respectively, and displayed improved metabolic stability. Their favorable biological profile is complemented by minimal cytotoxicity and neuroprotective properties.

Thiol-ene photo-click immobilization of a chiral chromatographic ligand on silica particles.

This work reports procedures for the immobilization of vinyl ligands on silica particles by UV-initiated thiol-ene radical addition reaction (photo-click immobilization). tert­Butylcarbamoyl quinine was the functional ligand (ene component) for the synthesis of chiral stationary phases. Two distinct surface chemistries were evaluated. In one approach, the ligand was directly attached to 3-mercaptopropyl-silica triggered by radicals generated by UV irradiation from a photoinitiator. In another approach, the ligand was immobilized onto vinyl silica via poly(3-mercaptopropyl-methylsiloxane) (PMPMS) as crosslinker by a photoinitiated double click reaction in which functionalization with chiral ligand and crosslinking to vinylsilica occurred simultaneously in one synthesis step. PMPMS-bonded CSPs were prepared from suspension (slurry method) or solventless after coating of the polythiol onto the vinylsilica surface (film method). Optimization by a design of experiment approach showed that the reaction time is the prime variable to optimize the surface coverage of chiral selector which also increased with PMPMS concentration. When the film formation of the latter approach was assisted by a minute volume of toluene during photo-click immobilization, selector coverage could be significantly increased to 0.73 µmol/m2 in a 2 h synthesis compared to 0.44 µmol/m2 by the solventless film method and 0.47 µmol/m2 by the slurry method under otherwise comparable conditions. The solvent assistance improved the chromatographic efficiency of the film method and resulted equal minimal reduced plate height of 2.6 as the slurry method for a chiral probe (Fmoc-Phe). The mass transfer resistance was around factor 2 smaller with the solvent-assisted film method as compared to the film approach without toluene, presumably due to a more homogenous distribution of the thin polymer film owing to lower dynamic viscosity in presence of toluene.

Evaluation of superficially porous particle based zwitterionic chiral ion exchangers against fully porous particle benchmarks for enantioselective ultra-high performance liquid chromatography.

Zwitterionic chiral ion-exchange selectors (ZWIX) obtained by conjugation of quinine and 2-aminocyclohexanesulfonic acid via a carbamate bond were immobilized on three different silica particle types, viz. 120 Š3 µm fully porous particles (FPP), 200 Š3 µm FPP and 160 Š2.7 µm superficially porous particles (SPP). Selector densities were determined by elemental analysis and the porosities of packed columns measured by inverse size exclusion chromatography with polystyrene standards. Liquid chromatographic tests with a set of chiral zwitterionic, acidic and basic analytes showed that the surface chemistry was successfully transferred to the distinct particle morphologies. The chromatographic performance of the three columns was evaluated by acquiring van Deemter curves. The results showed that the column packed with the SPP particles gives the best performance and kinetic plots further demonstrated that they represent the most favorable compromise in terms of speed, efficiency and pressure drop. Sub-minute separations could be accomplished at much lower pressure drop on the core-shell column, e.g. 2-amino-2-phenylbutyric acid was baseline separated in less than 15 s on a 5 cm long column. The Maxwell effective medium theory with second order approximation was applied to calculate effective diffusion in the mesoporous zones of SPP and FPP, which allowed eventually to deconvolute the individual peak dispersion contributions (ha, hb, hc,m, hc,s, hc,ads). The efficiency gain of the 160 ŠSPP column compared to the 120 ŠFPP (benchmark) column was mainly due to lower eddies (ha), smaller c-term accounting for slow adsorption-desorption kinetics in enantioselective chromatography (hc,ads), and also due to lower stationary mass transfer resistance (hc,s). Enhanced effective diffusion (Deff) in the SPP column contributed to a lower longitudinal diffusion (hb). In contrast, the mobile phase mass transfer coefficient was similar in the two columns leading to comparable hc,m contributions. This study discloses some options for improvement of the efficiency of ZWIX-type chiral columns such as replacing narrow pore (120 Å) by wide pore (200 Å) particles, substituting FPP by SPP and reducing the selector density on the surface.