Ation Reactions Research Articles

Frontispiece: Remote Fluorination and Fluoroalkyl(thiol)ation Reactions

Frontispiece: Remote Fluorination and Fluoroalkyl(thiol)ation Reactions

Remote Fluorination and Fluoroalkyl(thiol)ation Reactions.

Remote functionalization reactions have the power to transform a C-H (or C-C) bond at a distant position from a functional group. This Review summarizes recent advances and key breakthroughs in remote fluorination, trifluoromethylation, difluoromethylation, trifluoromethylthiolation, and fluoroalkenylation reactions. Several powerful strategies have emerged to control the reactivity and distal selectivity such as the undirected radical approach, the 1,5-hydrogen atom transfer, the metal migration, the use of distant directing groups, and the ring-opening reactions. These unconventional and predictable C-H (and C-C) functionalization transformations should allow for the preparation of a wide range of otherwise-difficult-to-access alkyl, aromatic, heteroaromatic, and structurally complex fluorides.

S‐Nitrosation and denitrosation of proteins by variation of superoxide/nitric oxide ratio – implications for prevention of sulfoxidation‐dependent enzyme inactivation during ischemia/reperfusion

Oxidative stress plays a key role for the development of cardiovascular, metabolic and neurodegenerative disease. This concept was never widely proven by the therapeutic use of classical antioxidants in large scale clinical trials. However, we know from numerous (pre)clinical studies that the formation of reactive oxygen and nitrogen species (RONS) as well as stable oxidative stress markers are a hallmark of most forms of diseases and higher oxidative stress markers are frequently associated with higher disease risk and mortality. Besides the detrimental role of RONS, they are also involved in cellular functions via redox signalling. We have previously identified an efficient mechanism of S‐nitrosation by low levels of nitric oxide and superoxide (3:1 ratio) with potential formation of N2O2 [Daiber A, Schildknecht S, Müller J, Kamuf J, Bachschmid MM, Ullrich V. Chemical model systems for cellular nitros(yl)ation reactions. Free Radic Biol Med. 2009 Aug 15;47(4):458–67.]. Here we, elucidated whether S‐nitrosation in the presence of higher nitric oxide than superoxide concentrations (as observed under hypoxic conditions) could prevent sulfoxidation and thereby oxidative inactivation of enzymes in the presence of higher superoxide than nitric oxide concentrations (as observed during reoxygenation). We found that increasing concentrations of xanthine oxidase/hypoxanthine caused conversion of S‐nitrosoglutathione (GSNO) to reduced glutathione (GSH) up to a certain concentration of xanthine oxidase, indicating that superoxide can induce denitrosation of GSNO. This finding was unexpected since in the presence of excess superoxide one would not expect regeneration of reduced GSH from GSNO. This was even more surprising since we observed substantial dihydrorhodamine oxidation that was prevented by uric acid and tyrosine nitration of albumin during denitrosation of GSNO by superoxide, all of which points to intermediary formation of peroxynitrite. In summary, we propose that S‐nitrosation of (mitochondrial) proteins during ischemia represents a protective mechanism to prevent irreversible overoxidation of thiols during the reperfusion phase and to re‐establish reduced thiol state in (mitochondrial) key enzymes of energy metabolism and cell survival. Wide‐spread mitochondrial protein S‐nitrosation may represent a central feature of the protective preconditioning effects of nitric oxide.Support or Funding InformationThe present work was supported by the European Cooperation in Science and Technology (COST Action BM1203/EU‐ROS and COST Action CA16225/EU‐CARDIOPROTECTION).

The Balance 2∙f (O) – f (H) as a Cornerstone in Formulation of Electrolytic Systems

The Generalized Electron Balance (GEB) concept, related to electrolytic redox systems, is considered according to principles of Generalized Approach to Electrolytic Systems (GATES). Two equivalent Approaches (I and II) to GEB are presented. The Approach I, when perceived in convention of the card game, is based on the common pool of electrons as money, introduced by electron-active elements, named as players; electron-non-active elements are called there as fans. The GEB obtained according to Approach II results from the linear combination f12 = 2∙f(O) – f(H) of elemental balances: f1 = f(H) for H, and f2 = f(O) for O. Very important properties of f12 are presented here in details, and illustrated by a redox system where comproportion- ation reactions occur

ARTD1 (PARP1) activation and NAD(+) in DNA repair and cell death.

Nicotinamide adenine dinucleotide, NAD(+), is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD(+) and NADH). NAD(+) is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalyzed by the sirtuins (SIRTs) which use NAD(+) as a substrate. In this review, we highlight the significance of NAD(+) catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by ARTD1 activation and energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked, are dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischaemia/reperfusion.

7th International Symposium on Enabling Technologies for Life Sciences (ETP)

7^th International Symposium on Enabling Technologies for Life Sciences (ETP)

Poly(ADP‐ribose) Polymerase 1 (PARP1) in Atherosclerosis: From Molecular Mechanisms to Therapeutic Implications

Poly(ADP-ribosyl)ation reactions, carried out by poly(ADP-ribose) polymerases (PARPs/ARTDs), are reversible posttranslational modifications impacting on numerous cellular processes (e.g., DNA repair, transcription, metabolism, or immune functions). PARP1 (EC 2.4.2.30), the founding member of PARPs, is particularly important for drug development for its role in DNA repair, cell death, and transcription of proinflammatory genes. Recent studies have established a novel concept that PARP1 is critically involved in the formation and destabilization of atherosclerotic plaques in experimental animal models and in humans. Reduction of PARP1 activity by pharmacological or molecular approaches attenuates atherosclerotic plaque development and enhances plaque stability as well as promotes the regression of pre-established atherosclerotic plaques. Mechanistically, PARP1 inhibition significantly reduces monocyte differentiation, macrophage recruitment, Sirtuin 1 (SIRT1) inactivation, endothelial dysfunction, neointima formation, foam cell death, and inflammatory responses within plaques, all of which are central to the pathogenesis of atherosclerosis. This article presents an overview of the multiple roles and underlying mechanisms of PARP1 activation (poly(ADP-ribose) accumulation) in atherosclerosis and emphasizes the therapeutic potential of PARP1 inhibition in preventing or reversing atherosclerosis and its cardiovascular clinical sequalae.

Spectral and photophysical properties of zinc(II) complexes with alkylsubstituted derivatives of dipyrrolylmethene and their resistance to protolytic dissociation and photochemical destruction

The spectral and photophysical properties of zinc(II) complexes with alkylsubstituted derivatives of dipyrrolylmethene, along with the kinetics of their protolytic dissociation and photochemical degradation in organic solvents of different natures, are studied by means of electronic absorption and fluorescence spec� troscopy. It is found that protic solvents have the strongest effect on absorption spectra, while electron donors affect fluorescence spectra due to differences in the mechanisms of solvation for the ground and excited states of molecules. It is shown that the reaction product of the protolytic dissociation of complexes in benzene solutions of acetic acid is a protonated form of ligand. The observable and genuine rate constants of dissoci� ation reactions are determined and activation parameters are calculated. A kinetic model of the process is proposed, and the regularities of the effect the nature of ligand has on the kinetics of dissociation are estab� lished. The photochemical degradation of compounds proceeds on monopyrrole products, the photolysis rate falls as the a degree of alkylation increases, and the nature of the substituent in the β�position of the ligand's pyrrole ring has a greater effect on the stability of a compound.

Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

Chromatin repair after oxidative stress: Role of PARP-mediated proteasome activation

Oxidative stress is an inevitable process in the nucleus, especially in antitumor chemotherapy, and adaptation by defense mechanisms seems to be one element in the development of long-term resistance to many chemotherapeutic drugs. In this study, a potential chromatin repair mechanism during oxidative stress was investigated in HT22 cells. The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified histone proteins in the nucleus. Poly(ADP-ribosyl)ation reactions also play an important role in DNA repair as a consequence of oxidative damage and single-strand breaks. Such a reaction may occur also with the 20S proteasome—with a known increase in enzymatic activity—and also with histones—reducing their proteolytic susceptibility as shown for the first time here. After hydrogen peroxide treatment of HT22 cells, degradation of the model peptide substrate suc-LLVY-MCA and degradation of oxidized histones by nuclear proteasome increased. During the removal of protein carbonyls, single-strand breaks and 8-hydroxy-2′-deoxyguanosine, proteasome, and poly(ADP-ribose) polymerase-1 enzymes were shown to play tightly interacting roles. Our results following the repair of oxidative damage show the proteolytic activation of proteasome concerning poly(ADP-ribosyl)ation together with a decline in poly(ADP-ribosyl)ation of oxidized histones, leading to a selective recognition of oxidatively modified histones.

Chemical model systems for cellular nitros(yl)ation reactions

S-nitros(yl)ation belongs to the redox-based posttranslational modifications of proteins but the underlying chemistry is controversial. In contrast to current concepts involving the autoxidation of nitric oxide ( NO, nitrogen monoxide), we and others have proposed the formation of peroxynitrite (oxoperoxonitrate (1−)) as an essential intermediate. This requires low cellular fluxes of NO and superoxide ( O 2 −), for which model systems have been introduced. We here propose two new systems for nitros(yl)ation that avoid the shortcomings of previous models. Based on the thermal decomposition of 3-morpholinosydnonimine, equal fluxes of NO and O 2 − were generated and modulated by the addition of NO donors or Cu,Zn-superoxide dismutase. As reactants for S-nitros(yl)ation, NADP +-dependent isocitrate dehydrogenase and glutathione were employed, for which optimal S-nitros(yl)ation was observed at nanomolar fluxes of NO and O 2 − at a ratio of about 3:1. The previously used reactants phenol and diaminonaphthalene (C- and N-nitrosation) demonstrated potential participation of multiple pathways for nitros(yl)ation. According to our data, neither peroxynitrite nor autoxidation of NO was as efficient as the 3 NO/1 O 2 − system in mediating S-nitros(yl)ation. In theory this could lead to an elusive nitrosonium (nitrosyl cation)-like species in the first step and to N 2O 3 in the subsequent reaction. Which of these two species or whether both together will participate in biological S-nitros(yl)ation remains to be elucidated. Finally, we developed several hypothetical scenarios to which the described NO/ O 2 − flux model could apply, providing conditions that allow either direct electrophilic substitution at a thiolate or S-nitros(yl)ation via transnitrosation from S-nitrosoglutathione.

The expanding field of poly(ADP‐ribosyl)ation reactions. ‘Protein Modifications: Beyond the Usual Suspects’ Review Series

Poly(ADP-ribosyl)ation is a post-translational modification of proteins that is mediated by poly(ADP-ribose) polymerases (PARPs). Although the existence and nature of the nucleic acid-like molecule poly(ADP-ribose) (PAR) has been known for 40 years, understanding its biological functions--originally thought to be only the regulation of chromatin superstructure when DNA is broken--is still the subject of intense research. Here, we review the mechanisms controlling the biosynthesis of this complex macromolecule and some of its main biological functions, with an emphasis on the most recent advances and hypotheses that have developed in this rapidly growing field.

Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli.

The expanding field of poly(ADP‐ribosyl)ation reactions

Poly(ADP-ribosyl)ation is a post-translational modification of proteins that is mediated by poly(ADP-ribose) polymerases (PARPs). Although the existence and nature of the nucleic acid-like molecule poly(ADP-ribose) (PAR) has been known for 40 years, understanding its biological functions—originally thought to be only the regulation of chromatin superstructure when DNA is broken—is still the subject of intense research. Here, we review the mechanisms controlling the biosynthesis of this complex macromolecule and some of its main biological functions, with an emphasis on the most recent advances and hypotheses that have developed in this rapidly growing field.

Collaborator of Stat6 (CoaSt6)-associated Poly(ADP-ribose) Polymerase Activity Modulates Stat6-dependent Gene Transcription

The transcription factor Stat6 plays a critical role in interleukin-4-dependent gene activation. To mediate this function, Stat6 recruits canonical transcriptional co-activators including the histone acetyl transferases CREB-binding protein and NCoA-1 and other proteins such as a p100 co-factor. However, much remains unknown regarding the constituents of Stat6 enhancer complexes, and the exact molecular events that modulate Stat6-dependent gene activation are not fully understood. Recently, we identified a novel co-factor, CoaSt6 (collaborator of Stat6), which associates with Stat6 and enhances its transcriptional activity. Sequence homologies place CoaSt6 in a superfamily of poly(ADP-ribosyl)polymerase (PARP)-like proteins. We have demonstrated here that PARP enzymatic activity is associated with CoaSt6, and this function of CoaSt6 can append ADP-ribose to itself and p100. Further, we show that a catalytically inactive mutant of CoaSt6 was unable to enhance Stat6-mediated transcription of a test promoter. Consistent with these findings, chemical inhibition of PARP activity blocked interleukin-4-dependent transcription from target promoters in vivo. Taken together, we have identified a CoaSt6-associated PARP activity and provided evidence for a role of poly(ADP ribosyl)ation in Stat-mediated transcriptional responses involving a novel PARP.

KIF4 Motor Regulates Activity-Dependent Neuronal Survival by Suppressing PARP-1 Enzymatic Activity

In brain development, apoptosis is a physiological process that controls the final numbers of neurons. Here, we report that the activity-dependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein 4 (KIF4), a microtubule-based molecular motor. The C-terminal domain of KIF4 is a module that suppresses the activity of poly (ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme known to maintain cell homeostasis by repairing DNA and serving as a transcriptional regulator. When neurons are stimulated by membrane depolarization, calcium signaling mediated by CaMKII induces dissociation of KIF4 from PARP-1, resulting in upregulation of PARP-1 activity, which supports neuron survival. After dissociation from PARP-1, KIF4 enters into the cytoplasm from the nucleus and moves to the distal part of neurites in a microtubule-dependent manner. We suggested that KIF4 controls the activity-dependent survival of postmitotic neurons by regulating PARP-1 activity in brain development.

Mild and Efficient Copper‐Catalyzed Cyanation of Aryl Iodides and Bromides.

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Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/β-Catenin Complex

T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by beta-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/beta-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/beta-catenin transcriptional complex by means of proteomics. A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the beta-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. In this study, we identified PARP-1 as a novel coactivator of the beta-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis.

A universal surface complexation framework for modeling proton binding onto bacterial surfaces in geologic settings

Adsorption onto bacterial cell walls can significantly affect the specia- tion and mobility of aqueous metal cations in many geologic settings. However, a unified thermodynamic framework for describing bacterial adsorption reactions does not exist. This problem originates from the numerous approaches that have been chosen for modeling bacterial surface protonation reactions. In this study, we compile all currently available potentiometric titration datasets for individual bacterial species, bacterial consortia, and bacterial cell wall components. Using a consistent, four discrete site, non-electrostatic surface complexation model, we determine total func- tional group site densities for all suitable datasets, and present an averaged set of 'universal' thermodynamic proton binding and site density parameters for modeling bacterial adsorption reactions in geologic systems. Modeling results demonstrate that the total concentrations of proton-active functional group sites for the 36 bacterial species and consortia tested are remarkably similar, averaging 3.2 1.0 (1) 10 4 moles/wet gram. Examination of the uncertainties involved in the development of proton-binding modeling parameters suggests that ignoring factors such as bacterial species, ionic strength, temperature, and growth conditions introduces relatively small error compared to the unavoidable uncertainty associated with the determination of cell abundances in realistic geologic systems. Hence, we propose that reasonable estimates of the extent of bacterial cell wall deprotonation can be made using averaged thermodynamic modeling parameters from all of the experiments that are considered in this study, regardless of bacterial species used, ionic strength, temperature, or growth condition of the experiment. The average site densities for the four discrete sites are 1.1 0.7 10 4 , 9.1 3.8 10 5 , 5.3 2.1 10 5 , and 6.6 3.0 10 5 moles/wet gram bacteria for the sites with pKa values of 3.1, 4.7, 6.6, and 9.0, respectively. It is our hope that this thermodynamic framework for modeling bacteria-proton binding reactions will also provide the basis for the development of an internally consistent set of bacteria-metal binding constants. 'Universal' constants for bacteria-metal binding reactions can then be used in conjunc- tion with equilibrium constants for other important metal adsorption and complex- ation reactions to calculate the overall distribution of metals in realistic geologic systems.