Multicomponent reactions, especially the Ugi-four component reaction (U-4CR), provide powerful protocols to efficiently access compounds having potent biological and pharmacological effects. Thus, a diverse library of betulinic acid (BA), fusidic acid (FA), cholic acid (CA) conjugates with TEMPO (nitroxide) have been prepared using this approach, which also makes them applicable in electron paramagnetic resonance (EPR) spectroscopy. Moreover, convertible amide modified spin-labelled fusidic acid derivatives were selected for post-Ugi modification utilizing a wide range of reaction conditions which kept the paramagnetic center intact. The nitroxide labelled betulinic acid analogue 6 possesses cytotoxic effects towards two investigated cell lines: prostate cancer PC3 (IC₅₀ 7.4 ± 0.7 μM) and colon cancer HT29 (IC₅₀ 9.0 ± 0.4 μM). Notably, spin-labelled fusidic acid derivative 8 acts strongly against these two cancer cell lines (PC3: IC₅₀ 6.0 ± 1.1 μM; HT29: IC₅₀ 7.4 ± 0.6 μM). Additionally, another fusidic acid analogue 9 was also found to be active towards HT29 with IC₅₀ 7.0 ± 0.3 μM (CV). Studies on the mode of action revealed that compound 8 increased the level of caspase-3 significantly which clearly indicates induction of apoptosis by activation of the caspase pathway. Furthermore, the exclusive mitochondria targeting of compound 18 was successfully achieved, since mitochondria are the major source of ROS generation.

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor.

Incorporating metal ions into synthetic polymer hydrogels results in toughening of these hydrogels. Herein, it is demonstrated that addition of small volumes of a 0.1maq. solution of Cu²⁺‐salts to poly(acrylic acid)‐based hydrogels (physically cross‐linked by Ca²⁺) increases their mechanical/rheological properties by several order of magnitude. Continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopic techniques reveal that the origin of the observed boost in mechanical properties is due to parts of the hydrogel network that interact with and through hydrated copper ions. With EPR spectroscopy, it is found that these complexes mainly have a sixfold (octahedral) H₂O‐coordination shell. Hence, in copper‐containing hydrogels, in addition to direct ionic and dipolar (H‐bonding) interactions between calcium and carboxylic anions of the side chains of polymer, a second interaction arises from the noncovalent, likely electrostatic interactions of the copper hexaaqua complex and PAA carboxylic acids that are screened through the lower charge density in the hydrated complexes. Also, short‐ranged hydrogen‐bonded networks of Cu²⁺with aqueous solvent molecules inside the water‐filled regions of the polymeric hydrogel may play a pivotal role. The synergy of these interactions explains the improved elastic properties of copper containing hydrogels.

The quinone compound 2,6-dimethoxy-1,4-benzoquinone is hydroxylated in alkaline aqueous solution with pH above 12. Electron paramagnetic resonance experiments showed that two transient radicals are formed in this reaction. The radical appearing first is assigned to a one electron reduced 2,6-dimethoxy-1,4-benzoquinone, receiving the electron from an intermediate anionic hydroxylated species. For this primary radical, all proton couplings were determined (quinoid ring protons: 1.453 G, methyl protons: 0.795 G). The density functional theory method was applied to obtain electronic and structural information of the primary radical and a solution structure is suggested. For approaching the experimental hyperfine couplings in theoretical models, it was necessary to consider effects of external polarisation arising from water molecules near one carbonyl group, and the orientation of methoxy groups towards the quinone ring. With this approach, the secondary radical formed in the hydroxylation reaction, and the transient radicals found for other biologically important quinones (including coenzymes Q) and their hydroxylated species may become accessible.

Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d⁵ electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe²⁺. Without added Fe²⁺, most high spin features of bound Fe³⁺ were preserved, while different g’-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe³⁺/Fe² in samples with supplemented Fe²⁺. The absence of any EPR signal related to Fe³⁺ or Fe²⁺ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.

This work focuses on the reaction mechanism of poly(sulfur nitride) ((SN)x, (S15N)x), with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate [EMIm][OAc]. We compare this with the reaction of the IL with S4N4 or its 15N labeled form S415N4, a precursor for the synthesis of (SN)x and (S15N)x. After purification of the S4N4-IL- and S415N4-system via column chromatography, we characterize the reaction products with 1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy and with electron spray ionization time-of-flight mass spectrometry (ESI-ToF-MS). Furthermore, time-resolved electron paramagnetic resonance (EPR) spectroscopy as well as time-resolved ultraviolet-visible (UV/Vis) spectroscopy are carried out. Thus, radical intermediates are detected which are consumed with reaction progress. Finally, we postulate a reaction mechanism for the S4N4-IL- and S415N4-IL system and compare this with the respective data for the (SN)x-IL- and (S15N)x-IL-system.