In situ enzymatic production of bioactive compounds from their non-toxic precursors is a relatively new strategy in drug design. If used correctly, the enzyme-prodrug binary system allows to obtain the necessary therapeutic concentrations of drugs locally and under controlled release. In nature alliinase uses S-substituted L-cysteine sulfoxide as the key part of the defense mechanisms in plants of the Allium genus, and the resulting biologically active thiosulfinates have various health benefits. In our study we describe the recombinant enzyme cystathionine beta-lyase from Klebsiella pneumoniae (kCBL) and its capability to catalyze the similar process, transforming a wide range of S-alk(en)yl- and arylalkyl-derivatives of L-cysteine sulfoxide into the corresponding thiosulfinates. The reaction proceeds with high catalytic efficiency and reaches up to 100 % conversion of the substrate. Besides a comprehensive biochemical analysis, the 3D structure of the holoenzyme and its complex with S-methyl-L-cysteine sulfoxide have been obtained at 1.9 and 2.0 & Aring; resolution, accordingly. The binding pocket of the enzyme provides suitable environment to catalyze beta-elimination reaction of the substituted Lcysteine sulfoxides of different size. To our knowledge, kCBL is the only described bacterial enzyme with such a wide substrate specificity, which makes it promising to be used as a component of the enzyme-prodrug binary system.

The use of peptides labeled with radioactive iodine isotopes enables rapid and highly sensitive assessment of their dynamic distribution in the body, as well as effective visualization and quantification of their metabolites in biological fluids and tissues down to femtomolar concentrations. In this work, we present the laboratory protocol for radioiodination of peptides containing oxidation-labile amino acids. Products of iodination of the histidine- and tyrosine-containing peptides were analyzed by NMR and high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) spectra. The proposed protocol describes a simple laboratory method for monitoring the extent of iodine isotope incorporation and a technique for isolating labeled peptides (3-10 amino acids) of high chemical and radiochemical purity without using HPLC. Using HPLC with fluorescence detection, we demonstrate the absence of significant oxidation of labile amino acids (Met, Trp, Tyr) during the radiolabeling process. The peptides labeled according to the protocol are obtained as lyophilizates with minimal inorganic salt content, allowing their direct use in cellular and animal model studies.

Bacterial methionine biosynthesis is an attractive target for research due to its central role in cellular metabolism, as most steps of this pathway are missing in mammals. Up to now little is known about sulfur metabolism in pathogenic Clostridia species, making the study of the enzymes of Cys/Met metabolism in Clostridium tetani particularly relevant. Analysis of the C. tetani genome has shown that the bacterium is capable of synthesizing methionine by direct sulfhydration. In this study, we describe purification of recombinant O-acetylhomoserine sulfhydrylase, a member of the Cys/Met metabolism pyridoxal 5'-phosphate-dependent enzyme family, from C. tetani for the first time. The gene encoding O-acetylhomoserine sulfhydrylase was cloned into the pET-28a(+) vector and expressed in Escherichia coli. The expression product was purified and identified as a 462-amino acid protein with a molecular mass of ∼50 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the C. tetani enzyme showed a high degree of similarity to O-acetylhomoserine sulfhydrylases from other bacterial sources. We confirmed the O-acetylhomoserine sulfhydrylase activity, and found the enzyme to be optimally active at pH 7.5 and 50 °C. The native enzyme assembles into a homotetramer of approx. 200 kDa as revealed by gel filtration. The obtained enzyme is capable of l-methionine formation using methanethiol as a sulfur source, that has been revealed by 1H NMR spectral data. These findings broaden the understanding of the role of O-acetylhomoserine sulfhydrylase in C. tetani Cys/Met metabolism and provide a basis for its future investigations and research.

The direct C-H activation of inert C(sp3)-H bonds in a hydrocarbon chain has been a very attractive target in organic synthesis for many decades. Among all the variety of processes, those driven by vinyl carbocations are quite scarce thus far, and it is hard to control for unstabilized vinyl cations. In this study, we designed a double C(sp3)-H functionalization of unactivated alkyl CH2 groups to produce a totally substituted quaternary carbon stereocenter via insertion of vinyl carbocations. These processes represent complicated reaction cascades with high molecular complexity controlled by the cooperative action of Ga(III) salts & GaHal4- anions and allow one-step deep poly-functionalization of simple CH substrates to be performed. In practice, this concept was initially implemented with simple starting compounds such as alkyl acetylenes and activated cyclopropanes, alkenes, or cyclobutanes to construct norbornane, cyclopentatetralin, and other important skeletons.

Amino acid analogues with a phosphorus-containing moiety replacing the carboxylic group are promising sources of biologically active compounds. The H-phosphinic group, with hydrogen-phosphorus-carbon (H-P-C) bonds and a flattened tetrahedral configuration, is a bioisostere of the carboxylic group. Consequently, amino-H-phosphinic acids undergo substrate-like enzymatic transformations, leading to new biologically active metabolites. Previous studies employing NMR-based metabolomic and proteomic analyses show that in Escherichia coli, α-KG-γ-PH (the distal H-phosphinic analogue of α-ketoglutarate) can be converted into L-Glu-γ-PH. Notably, α-KG-γ-PH and L-Glu-γ-PH are antibacterial compounds, but their intracellular targets only partially overlap. L-Glu-γ-PH is known to be a substrate of aspartate transaminase and glutamate decarboxylase, but its substrate properties with NAD+-dependent glutamate dehydrogenase (GDH) have never been investigated. Compounds containing P-H bonds are strong reducing agents; therefore, enzymatic NAD+-dependent oxidation is not self-evident. Herein, we demonstrate that L-Glu-γ-PH is a substrate of eukaryotic GDH and that the pH optimum of L-Glu-γ-PH NAD+-dependent oxidative deamination is shifted to a slightly alkaline pH range compared to L-glutamate. By 31P NMR, we observe that α-KG-γ-PH exists in a pH-dependent equilibrium of keto and germinal diol forms. Furthermore, the stereospecific enzymatic synthesis of α-KG-γ-PH from L-Glu-γ-PH using GDH is a possible route for its bio-based synthesis.

Candida albicans and non-albicans Candida species are a common cause of human mucosal infections, as well as bloodstream infections and deep mycoses. The emergence of resistance of Candida spp. to antifungal drugs used in practice requires the search for new antimycotics. The present study unravels the antifungal potential of the synthetic dialk(en)ylthiosulfinates in comparison with an enzymatic in situ methionine γ-lyase-based thiosulfinate generation system (TGS). The kinetics of the TGS reaction, namely, the methionine γ-lyase-catalyzed β-elimination of S-alk(en)yl-L-cysteine sulfoxides, was investigated via 1H NMR spectroscopy for the first time, revealing fast conversion rates and the efficient production of anticandidal dialk(en)ylthiosulfinates. The anticandidal potential of this system vs. synthetic thiosulfinates was investigated through an in vitro assay. TGS proved to be more effective (MIC range 0.36–1.1 μg/mL) than individual substances (MIC range 0.69–3.31 μg/mL). The tested preparations had an additive effect with the commercial antimycotics fluconazole, amphotericin B and 5-flucytosine demonstrating a fractional inhibitory coefficient index in the range of 0.5–2 μg/mL. TGS can be regarded as an attractive candidate for the targeted delivery of antimycotic thiosulfinates and for further implementation onto medically implanted devices.

Five new bifunctional conjugates of pyropheophorbide a with 17-substituted testosterone, dihydrotestosterone and epitestosterone differing in the length of linker (1 � 5) and two new complex conjugates 6 and 7 (containing three functional units: pyropheophorbide a, 17?-substituted testosterone, and lipophylic hexadecyl chain, connected with L-lysine joining block) were synthesized. Mutual influence of steroidal and macrocyclic fragments in conjugates (1 � 7) was established by analysis of 1H NMR spectra and molecular models of conjugates. Studies of interaction of conjugates 1 � 5 with prostate carcinoma cells revealed that their uptake and internalization were dependent on the structure of conjugates, particularly on the stereochemical configuration of 17-hydroxyl group in steroidal moiety, and the length of linker connecting pyropheophorbide a with steroid fragments. Conjugates 1 � 5 significantly decreased the growth and proliferation of LNCaP and PC-3 cells. The highest anti-proliferative activity demonstrated by epitestosterone derivative 3, comprising short linker. Irradiation of labeled cells with light (? = 660 nm) was significantly increased cytotoxicity. Trifunctional conjugates 6 and 7 easily formed mixed micells with phosphatidyl choline and pluronic F68; these mixed micelles efficiently internalized by human hepatocarcinoma Hep G2 cells. The binding of conjugates 6 and 7 in the form of mixed micelles to Hep G2 cells depended on the conjugate structure, rather than on the method of solubilization.

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown.This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.

This study presents a methodology for the synthesis of the ionic cyclopropenium-derived triplatinum cluster complex [(Ph3C3)(2)Pt-3(MeCN)(4)](2+)(BF4-)(2) and its analogue with the replacement of one of the BF4- anions with a Cl- anion, which represents a chain coordination polymer in crystalline form. The structures of the complexes were studied by several physicochemical methods, primarily X-ray diffraction using synchrotron radiation and Pt-195 NMR spectroscopy in solution with measurement and analysis of the Pt-195-Pt-195 and C-13-Pt-195 spin-spin coupling constants. In addition, a high catalytic activity of the synthesized Pt-3 complex was observed in hydrosilylation reactions. The catalytic efficiency of the complex was found to be comparable to that of Karstedt's catalyst and in some cases even higher.

Glucan linked to proteins is a natural mega-glycoconjugate (mGC) playing the central role as a structural component of a yeast cell wall (CW). Regulation of functioning of non-covalently bound glucanosyltransglycosylases (ncGTGs) that have to remodel mGC to provide CW extension is poorly understood. We demonstrate that the main ncGTGs Bgl2 and Scw4 have phosphorylated and glutathionylated residues and are represented in CW as different pools of molecules having various firmness of attachment. Identified pools contain Bgl2 molecules with unmodified peptides, but differ from each other in the presence and combination of modified ones, as well as in the presence or absence of other CW proteins. Correlation of Bgl2 distribution among pools and its N-glycosylation was not found. Glutathione affects Bgl2 conformation, probably resulting in the mode of its attachment and enzymatic activity. Bgl2 from the pool of unmodified and monophosphorylated molecules demonstrates the ability to fibrillate after isolation from CW. Revealing of Bgl2 microcompartments and their mosaic arrangement summarized with the results obtained give the evidence that the functioning of ncGTGs in CW can be controlled by reversible post-translational modifications and facilitated due to their compact localization. The hypothetical scheme of distribution of Bgl2 inside CW is represented.

The novel class of gallium catalysts based on cationic gallium phthalocyanines (RPcGa+) with SbF6– and Sb2F11– weakly coordinating anions was developed in order to using in the catalytic version for gallium-specific chemistry. Two cycloaddition/annulation processes of styrylmalonates with aromatic aldehydes and styrene were implemented in the catalytic version using this approach. The synthetic strategy to gallium phthalocyanines and their structural features were studied in details, including the use of specially adapted 71Ga NMR spectroscopy in combination with 19F and 121Sb ones, as well as the mechanistic aspects.

An aptamer is a synthetic oligonucleotide with a unique spatial structure that provides specific binding to a target. To date, several aptamers to hemagglutinin of the influenza A virus have been described, which vary in affinity and strain specificity. Among them, the DNA aptamer RHA0385 is able to recognize influenza hemagglutinins with highly variable sequences. In this paper, the structure of RHA0385 was studied by circular dichroism spectroscopy, nuclear magnetic resonance, and size-exclusion chromatography, demonstrating the formation of a parallel G-quadruplex structure. Three derivatives of RHA0385 were designed in order to determine the contribution of the major loop to affinity. Shortening of the major loop from seven to three nucleotides led to stabilization of the scaffold. The affinities of the derivatives were studied by surface plasmon resonance and an enzyme-linked aptamer assay on recombinant hemagglutinins and viral particles, respectively. The alterations in the loop affected the binding to influenza hemagglutinin, but did not abolish it. Contrary to aptamer RHA0385, two of the designed aptamers were shown to be conformationally homogeneous, retaining high affinities and broad binding abilities for both recombinant hemagglutinins and whole influenza A viruses.

A combination of 19F and 1H NMR with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was used to examine the mobility of liquids in the interplane space of graphite oxide (GO) for the first time. The proposed approach allows for the reduction of NMR signals from immobile hydrogen-containing groups on the surface of GO and for monitoring of the molecular mobility of intercalated liquids. The mobile fractions of H2O, CH3CN and CF3CH2OH were detected inside the corresponding swollen GO samples. For H2O, the amount of mobile liquid showed a peculiar temperature dependence where a certain portion survived well below 273 K. The sensitivity of the proposed 1H NMR + CPMG procedure is also compared to the sensitivity of the EPR nitroxide spin probe method.

Human cardiac myosin has two isoforms, kinetically fast α and slow β. There is high sequence similarity between isoforms. Extensive molecular dynamics simulations reveal stable isoform-specific salt bridges in three regions of myosin head, the loop1 region, the loop switch 1 region, and the force-generating region. We hypothesized that isoform-specific inter-residue electrostatic interactions affect kinetics of actomyosin. We designed four mutants of human cardiac myosin, I303V:I313V, D208Q:K450L, E45Q, and R694N in β-isoform background, to remove β-isoform-specific salt bridges, potentially affecting actomyosin kinetics. We expressed and purified recombinant human cardiac myosin β-isoform, wild type and four mutants, using adenovirus mediated C2C12 murine myoblast expression system. ATP-induced actomyosin dissociation and the competitive inhibition of actomyosin dissociation by ADP were studied in detail. All mutants and the wild type myosin exhibit high affinity of ADP to actomyosin. The rate of ADP dissociation from actomyosin depends on the introduced mutation. We found that destabilization of the electrostatic interactions within the force-generating region increases the rate of ADP dissociation from actomyosin. This reduces the time of myosin strongly bound state and decreases myosin duty ratio. In myosin head, the relay helix spans from the loop switch II of the active site to the force-generating region. We speculate that the relay helix translates the increased dynamics of the force-generating region to the active site, which results in the changed equilibrium of the open and close state of myosin active site.

In this work, general approaches for preparing p-tolylsilanes as promising precursors for the synthesis of functionalized organosilicon compounds are discussed. Various synthetic techniques, including new and specially developed ones, were used to obtained p-tolylsiloxanes of different structures. Our findings resulted in advanced methods over the previously reported procedures, since they do not imply the use of expensive metal-containing catalysts. All the products were obtained and isolated in gram amounts (up to 225 g) in high yields (63-90%) and were characterized by NMR, ESI-HRMS and X-Ray data. (c) 2020 Elsevier B.V. All rights reserved.

Human cardiac myosin has two isoforms, alpha and beta, sharing significant sequence similarity, but different in kinetics: ADP release from actomyosin is an order of magnitude faster in the alpha myosin isoform. Apparently, small differences in the sequence are responsible for distinct local inter-residue interactions within alpha and beta isoforms, leading to such a dramatic difference in the rate of ADP release. Our analysis of structural kinetics of alpha and beta isoforms using molecular dynamics simulations revealed distinct dynamics of SH1:SH2 helix within the force-generation region of myosin head. The simulations showed that the residue R694 of the helix forms two permanent salt bridges in the beta isoform, which are not present in the alpha isoform. We hypothesized that the isoform-specific electrostatic interactions play a role in the difference of kinetic properties of myosin isoforms. We prepared R694N mutant in the beta isoform background to destabilize electrostatic interactions in the force-generating region of the myosin head. Our experimental data confirm faster ADP release from R694N actomyosin mutant, but is not as dramatic as the difference of kinetics of ADP release in the alpha and beta isoforms.

The first selective process of formal [2+2]‐cycloaddition of donor–acceptor cyclopropanes (DAC) to multiple C–C bonds to form a four‐membered ring in the product has been developed. Bicyclobutylidene is used do demonstrate this reaction. The process is promoted by GaCl3 and occurs through generation of 1,2‐zwitterionic intermediates. A carbocationic rearrangement occurs in the bicyclobutylidene moiety. It results in expansion of both cyclobutane rings into five‐membered ones and formation of a [2.3.3]propellane skeleton in the final product.

We have studied the kinetics of ATP induced actomyosin dissociation and ADP release in two mutants of beta isoform of the human cardiac myosin S1. The mutations were introduced in the Loop 1 (D208Q: K450L) and in the force-generating region (R694N) of the myosin head. Both mutants support myosin ATPase activity. We introduced these mutations to remove electrostatic interactions between charged residues in the specified domains of myosin S1. Our computational analysis showed that in the wild-type myosin, permanent salt bridges are present in the specified regions of the beta isoform. Those salt bridges are absent in alpha isoform. Since the kinetics of alpha isoform is an order of magnitude faster than the kinetics of beta isoform, we hypothesize that electrostatic interactions in the specified regions play a crucial role in myosin kinetics regulation. Obtained experimental data were analyzed using two models, assuming either rapid equilibrium between actomyosin and nucleotide or slow dissociation of ADP from actomyosin. We discuss the effect of mutations on the retention of the nucleotide by actomyosin and the potential role of electrostatic interactions in the Loop1 and in the force-generating region of human cardiac myosin on its regulation. We conclude that these salt bridges are essential for the proper functioning of beta isoform despite their absence in the alpha isoform.

Seven new oxazoline, benzoxazole and benzimidazole derivatives were synthesized from 3β-acetoxyandrosta-5,16-dien-17-carboxylic, 3β-acetoxyandrost-5-en-17β-carboxylic and 3β-acetoxypregn-5-en-21-oic acids. Docking to active site of human 17α-hydroxylase/17,20-lyase revealed that all oxazolines, as well as benzoxazoles and benzimidazoles comprising Δ16 could form stable complexes with enzyme, in which steroid moiety is positioned similarly to that of abiraterone and galeterone, and nitrogen atom coordinates heme iron, while 16,17-saturated benzoxazoles and benzimidazoles could only bind in a position where heterocycle is located nearly parallel to heme plane. Modeling of the interaction of new benzoxazole and benzimidazole derivatives with androgen receptor revealed the destabilization of helix 12, constituting activation function 2 (AF2) site, by mentioned compounds, similar to one induced by known antagonist galeterone. The synthesized compounds inhibited growth of prostate carcinoma LNCaP and PC-3 cells at 96 h incubation; the potency of 2'-(3β-hydroxyandrosta-5,16-dien-17-yl)-4',5'-dihydro-1',3'-oxazole and 2'-(3β-hydroxyandrosta-5,16-dien-17-yl)-benzimidazole was superior and could inspire further investigations of these compounds as potential anti-cancer agents.