Karel D. Klika

A comprehensive examination of the self-disproportionation of enantiomers (SDE) of chiral amides via achiral, laboratory-routine, gravity-driven column chromatography

This work explores the self-disproportionation of enantiomers (SDE) of chiral amides via achiral, gravity-driven column chromatography as typically used in laboratory settings. The major findings of this work are: (1) the remarkable persistence and high magnitude of the SDE for the analytes under a variety of conditions, including polar solvents and different achiral stationary phases and (2) the notable generality of the SDE phenomenon as it occurs for a wide range of chiral amide substrates and even for a broad range of starting ee. This last aspect is unusual and not commonly observed. The key conclusion of this work is that it judiciously conveys the predictability of the SDE for chiral amides under the routine conditions of achiral chromatography. These results are consequently of concern for practitioners in the area of catalytic asymmetric synthesis involving chiral amides as intermediates or products and the inferents need to be taken extremely seriously by workers in the field.

Linker Modification Strategies To Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors

Since prostate-specific membrane antigen (PSMA) is up-regulated in nearly all stages of prostate cancer (PCa), PSMA can be considered as a viable diagnostic biomarker and treatment target in PCa. This project is focused on the development and evaluation of a series of compounds directed against PSMA. The modifications to the linker are designed to improve the binding potential and pharmacokinetics for theranostic application. In addition, the results help to further elucidate the structure-activity relationships (SAR) of the resulting PSMA inhibitors. Both in vitro and in vivo experiments of 18 synthesized PSMA inhibitor variants showed that systematic chemical modification of the linker has a significant impact on the tumor-targeting and pharmacokinetic properties. This approach can lead to an improved management of patients suffering from recurrent prostate cancer by the use of one radiolabeling precursor, which can be radiolabeled either with (68)Ga for diagnosis or with (177)Lu or (225)Ac for therapy.

Signature of protein unfolding in chemical exchange saturation transfer imaging.

Chemical exchange saturation transfer (CEST) allows the detection of metabolites of low concentration in tissue with nearly the sensitivity of MRI with water protons. With this spectroscopic imaging approach, several tissue‐specific CEST effects have been observed in vivo. Some of these originate from exchanging sites of proteins, such as backbone amide protons, or from aliphatic protons within the hydrophobic protein core. In this work, we employed CEST experiments to detect global protein unfolding. Spectral evaluation revealed exchange‐ and NOE‐mediated CEST effects that varied in a highly characteristic manner with protein unfolding tracked by fluorescence spectroscopy. We suggest the use of this comprehensive spectral signature for the detection of protein unfolding by CEST, as it relies on several spectral hallmarks. As proof of principle, we demonstrate that the presented signature is readily detectable using a whole‐body MR tomograph (B0 = 7 T), not only in denatured aqueous protein solutions, but also in heat‐shocked yeast cells. A CEST imaging contrast with the potential to detect global protein unfolding would be of particular interest regarding protein unfolding as a marker for stress, ageing, and disease. Copyright

Quantitative pulsed CEST-MRI using Ω-plots.

Chemical exchange saturation transfer (CEST) allows the indirect detection of dilute metabolites in living tissue via MRI of the tissue water signal. Selective radio frequency (RF) with amplitude B1 is used to saturate the magnetization of protons of exchanging groups, which transfer the saturation to the abundant water pool. In a clinical setup, the saturation scheme is limited to a series of short pulses to follow regulation of the specific absorption rate (SAR). Pulsed saturation is difficult to describe theoretically, thus rendering quantitative CEST a challenging task. In this study, we propose a new analytical treatment of pulsed CEST by extending a former interleaved saturation–relaxation approach. Analytical integration of the continuous wave (cw) eigenvalue as a function of the RF pulse shape leads to a formula for pulsed CEST that has the same structure as that for cw CEST, but incorporates two form factors that are determined by the pulse shape. This enables analytical Z‐spectrum calculations and permits deeper insight into pulsed CEST. Furthermore, it extends Dixons Ω‐plot method to the case of pulsed saturation, yielding separately, and independently, the exchange rate and the relative proton concentration. Consequently, knowledge of the form factors allows a direct comparison of the effect of the strength and B1 dispersion of pulsed CEST experiments with the ideal case of cw saturation. The extended pulsed CEST quantification approach was verified using creatine phantoms measured on a 7 T whole‐body MR tomograph, and its range of validity was assessed by simulations. Copyright

Aggregation-induced changes in the chemical exchange saturation transfer (CEST) signals of proteins

Chemical exchange saturation transfer (CEST) is an MRI technique that allows mapping of biomolecules (small metabolites, proteins) with nearly the sensitivity of conventional water proton MRI. In living organisms, several tissue‐specific CEST effects have been observed and successfully applied to diagnostic imaging. In these studies, particularly the signals of proteins showed a distinct correlation with pathological changes. However, as CEST effects depend on various properties that determine and affect the chemical exchange processes, the origins of the observed signal changes remain to be understood. In this study, protein aggregation was identified as an additional process that is encoded in the CEST signals of proteins. Investigation of distinct proteins that are involved in pathological disorders, namely amyloid beta and huntingtin, revealed a significant decrease of all protein CEST signals upon controlled aggregation. This finding is of particular interest with regard to diagnostic imaging of patients with neurodegenerative diseases that involve amyloidogenesis, such as Alzheimers or Huntingtons disease. To investigate whether the observed CEST signal decrease also occurs in heterogeneous mixtures of aggregated cellular proteins, and thus prospectively in tissue, heat‐shocked yeast cell lysates were employed. Additionally, investigation of different cell compartments verified the assignment of the protein CEST signals to the soluble part of the proteome. The results of in vitro experiments demonstrate that aggregation affects the CEST signals of proteins. This observation can enable hypotheses for CEST imaging as a non‐invasive diagnostic tool for monitoring pathological alterations of the proteome in vivo.

A click chemistry approach identifies target proteins of xanthohumol

SCOPE Many phytochemicals with beneficial pharmacological properties contain electrophilic sites, e.g. α,β-unsaturated carbonyl (enone) groups. There is increasing evidence that many biological effects of electrophilic compounds depend on covalent conjugation to reactive protein thiols. For example, the reaction of electrophiles with cysteinyl residues of the sensor protein Keap1 activates the cell-protective Nrf2 response. Thus it is of interest to identify more generally the proteins to which small molecule electrophiles bind covalently. METHODS AND RESULTS Here we use a Click chemistry approach to identify target proteins of the chemopreventive phytochemical xanthohumol (XN), an enone-containing chalcone from hops (Humulus lupulus L.). Using an alkynylated analog of XN (XN-alkyne), we purified covalent protein-electrophile conjugates from cell lysates. We confirm the previously described conjugation of XN to Keap1. One of the newly identified candidate target proteins is glucose-6-phosphate dehydrogenase (G6PDH). We confirm that XN attenuates intracellular G6PDH activity at low micromolar concentrations. CONCLUSION We find support for the notion that XN modulates multiple pathways and processes by covalent modification of proteins with reactive cysteines.

The self-disproportionation of enantiomers (SDE): a menace or an opportunity?

Herein we report on the well-documented, yet not widely known, phenomenon of the self-disproportionation of enantiomers (SDE): the spontaneous fractionation of scalemic material into enantioenriched and -depleted fractions when any physicochemical process is applied.

The Application of Simple and Easy to Implement Decoupling Pulse Scheme Combinations to Effect Decoupling of Large Values with Reduced Artifacts

Of the various problems in decoupling one nucleus type from another using standard decoupling pulse schemes for broadband decoupling, a particular challenge is to effect full, artifact-free decoupling when the size of the coupling constant is very large. Herein it is demonstrated that 1H decoupling of the 31P NMR spectrum of diethyl phosphonate HP(=O)(OCH2CH3)2 can be accomplished with reduced artifacts despite the large value of 693 Hz by using a combination of decoupling pulse schemes involving continuous-wave (CW) irradiation and either adiabatic-pulse decoupling (APD), MPF decoupling, or traditional composite-pulse decoupling (CPD) schemes such as WALTZ or GARP. The considered strategy is simple, efficient, and easy to implement on most instruments. The best result was attained for a combination of CW and CPD using GARP with a standard pulse width of 60 μs. Altogether, the advantages of the methodology include low power requirements, complete decoupling, tolerance of a range of large values, large bandwidth for normal-sized values, and the suppression of sidebands.

Comparison of the major polyphenols in mature Argan fruits from two regions of Morocco

Previous studies have shown that Argan fruits contain a large variety of polyphenolic compounds. Recently, another class of polyphenolic compounds, namely amino phenols have been detected and identified in immature Argan fruits. The objective of this study, was to establish whether or not, these novel compounds are also present in mature Argan fruits. To this end, a comparison was made between mature fruits from two regions of Morocco. Nineteen major compounds were identified and quantitated, including amino phenols, flavonoids, and phenolic acids by chromatographic methods in mature Argan fruits from the two regions of Morocco (Essaouira and Agadir). The phenolic acids were identified as gallic acid and 3,4-dihydroxybenzoic acid; the amino phenols as Arganimide A, and argaminolics A-C, and the flavonoids as rutin pentoside, quercetin-3-O-arabinoside, quercetin glycogallate, quercetin-3-O-rhamnogalactoside, rutin, quercetin-3-O-galactoside (hyperoside), quercetin-3-O-glucoside (quercitrin), quercetin-3-O-arabinoside, quercetin glycohydroxybenzoate, quercetin glycosinapate, quercetin glycoferulate, quercetin glycocoumarate and quercetin. n=145.

Targeting Thioredoxin-1 by dimethyl fumarate induces ripoptosome-mediated cell death

Constitutively active NFκB promotes survival of many cancers, especially T-cell lymphomas and leukemias by upregulating antiapoptotic proteins such as inhibitors of apoptosis (IAPs) and FLICE-like inhibitory proteins (cFLIPs). IAPs and cFLIPs negatively regulate the ripoptosome, which mediates cell death in an apoptotic or necroptotic manner. Here, we demonstrate for the first time, that DMF antagonizes NFκB by suppressing Thioredoxin-1 (Trx1), a major regulator of NFκB transcriptional activity. DMF-mediated inhibition of NFκB causes ripoptosome formation via downregulation of IAPs and cFLIPs. In addition, DMF promotes mitochondrial Smac release and subsequent degradation of IAPs, further enhancing cell death in tumor cells displaying constitutive NFκB activity. Significantly, CTCL patients treated with DMF display substantial ripoptosome formation and caspase-3 cleavage in T-cells. DMF induces cell death predominantly in malignant or activated T-cells. Further, we show that malignant T-cells can die by both apoptosis and necroptosis, in contrast to resting T-cells, which are restricted to apoptosis upon DMF administration. In summary, our data provide new mechanistic insight in the regulation of cell death by targeting NFκB via Trx1 in cancer. Thus, interference with Trx1 activity is a novel approach for treatment of NFκB-dependent tumors.