The methaemoglobin forming and GSH depleting effects of dapsone and monoacetyl dapsone hydroxylamines in human diabetic and non-diabetic erythrocytes in vitro

The respective methaemoglobin forming and GSH depleting capabilities of monoacetyl dapsone hydroxylamine (MADDS-NHOH) and dapsone hydroxylamine (DDS-NHOH) were compared in human diabetic and non-diabetic erythrocytes in vitro with a view to select the most potent agent for future oxidative stress and antioxidant evaluation studies. Administration of both metabolites to non-diabetic erythrocytes over the 20 min period of the study resulted in significantly more methaemoglobin formation at all four time points compared with the diabetic erythrocytes (P<0.0001). At all four time points, significantly more methaemoglobin was formed in response to MADDS-NHOH in non-diabetic cells compared with the effects of DDS-NHOH on diabetic erythrocytes (P<0.0001). At the 5 and 10 min time points, significantly more methaemglobin was formed in non-diabetic cells in the presence of MADDS-NHOH compared with DDS-NHOH (P<0.05). At the 5 min time point only, significantly more methaemoglobin was formed in the presence of MADDS-NHOH in diabetic cells compared with that of DDS-NHOH (P<0.01). However, compared with diabetic control GSH levels, the presence of DDS-NHOH caused a significant depletion in GSH at 5, 10 and 20 min time points in diabetic cells (P<0.001). In addition, the presence of DDS-NHOH caused a significant reduction in GSH levels in diabetic cells in comparison with those of non-diabetics at the 5, 10 and 20 min, (P<0.005). DDS-NHOH was also associated with a significant depletion of GSH levels in diabetic cells compared with those of non-diabetic control erythrocytes (P<0.0001). The presence of MADDS-NHOH in diabetic erythrocytes led to a significant reduction in GSH levels at the 20 min time point compared with those of non-diabetics (P<0.001), but there were no significant differences at the 5, 10 and 15 min points. Due to its greater GSH-depleting action, DDS-NHOH will be selected for future use in the oxidative stress assessment in diabetic erythrocytes. © 2004 Elsevier B.V. All rights reserved.

A dramatic effect of double bond configuration in N-oxy-3-aza Cope rearrangements: a simple synthesis of functionalised allenes

The first examples of low temperature N-oxy-3-aza Cope rearrangements, leading to functionalised allenes are described, where the Z-configuration of the enaminic double bond in the rearranging system proves critical.

Angiotensin-converting enzyme inhibition by hydroxamic zinc-binding idrapril in humans.

The new angiotensin-converting enzyme (ACE) inhibitor idrapril acts by binding the catalytically important zinc ion to a hydroxamic group. We investigated its pharmacodynamic and pharmacokinetic properties in 8 healthy men: Increasing doses of 1, 5, and 25 mg idrapril as well as placebo or 5 mg captopril were administered intravenously (i.v.) at 1-week intervals. Six of the subjects received 100 mg idrapril orally (p.o.) last, and two ingested oral placebo as a double-blind control. Blood pressure (BP) and heart rate (HR) remained unchanged. No serious side effects were observed. ACE inhibition in vivo was evaluated by changes in the ratio of specifically measured plasma angiotensin II (AngII) and AngI concentrations determined by high-performance liquid chromatography/radioimmunoassay (HPLC/RIA) techniques. Plasma ACE activity in vitro was estimated by radioenzymatic assay; it was suppressed by > or = 93% at 15 min after injection of 25 mg idrapril or 5 mg captopril and by 96% 2 h after idrapril intake. Mean AngII levels were decreased dose dependently at 15 min after idrapril injections. At the same time, plasma renin activity (PRA) and AngI increased according to the doses. The AngII/AngI ratio was clearly related to plasma idrapril levels (r = -0.88, n = 60). Oral idrapril inhibited ACE maximally at 1-4 h after dosing, when < 7% of initial ACE activity was observed in vitro and in vivo. Idrapril is a safe and efficient ACE inhibitor in human subjects. It is well absorbed orally. Besides having a slightly slower onset of action, idrapril has pharmacodynamic effects comparable to those of captopril.

The novel hydroxylamine derivative NG-094 suppresses polyglutamine protein toxicity in Caenorhabditis elegans.

Aggregation-prone polyglutamine (polyQ) expansion proteins cause several neurodegenerative disorders, including Huntington disease. The pharmacological activation of cellular stress responses could be a new strategy to combat protein conformational diseases. Hydroxylamine derivatives act as co-inducers of heat-shock proteins (HSPs) and can enhance HSP expression in diseased cells, without significant adverse effects. Here, we used Caenorhabditis elegans expressing polyQ expansions with 35 glutamines fused to the yellow fluorescent protein (Q35-YFP) in body wall muscle cells as a model system to investigate the effects of treatment with a novel hydroxylamine derivative, NG-094, on the progression of polyQ diseases. NG-094 significantly ameliorated polyQ-mediated animal paralysis, reduced the number of Q35-YFP aggregates and delayed polyQ-dependent acceleration of aging. Micromolar concentrations of NG-094 in animal tissues with only marginal effects on the nematode fitness sufficed to confer protection against polyQ proteotoxicity, even when the drug was administered after disease onset. NG-094 did not reduce insulin/insulin-like growth factor 1-like signaling, but conferred cytoprotection by a mechanism involving the heat-shock transcription factor HSF-1 that potentiated the expression of stress-inducible HSPs. NG-094 is thus a promising candidate for tests on mammalian models of polyQ and other protein conformational diseases.

Role of gene methylation in antitumor immune response: Implication for tumor progression

Cancer immunosurveillance theory has emphasized the role of escape mechanisms in tumor growth. In this respect, a very important factor is the molecular characterization of the mechanisms by which tumor cells evade immune recognition and destruction. Among the many escape mechanisms identified, alterations in classical and non-classical HLA (Human Leucocyte Antigens) class I and class II expression by tumor cells are of particular interest. In addition to the importance of HLA molecules, tumor-associated antigens and accessory/co-stimulatory molecules are also involved in immune recognition. The loss of HLA class I antigen expression and of co-stimulatory molecules can occur at genetic, transcriptional and post-transcriptional levels. Epigenetic defects are involved in at least some mechanisms that preclude mounting a successful host-antitumor response involving the HLA system, tumor-associated antigens, and accessory/co-stimulatory molecules. This review summarizes our current understanding of the role of methylation in the regulation of molecules involved in the tumor immune response.

Biochemical analysis of the major vaccinia virus envelope antigen.

The major envelope antigen of vaccinia virus is an acylated protein of M(r) 37,000 (p37K) which is required for the formation of extracellular enveloped virions (EEV). Despite its important role in the wrapping process, p37K has not been studied in much detail. In order to better characterize this protein we have undertaken a detailed biochemical analysis. Sodium carbonate treatment showed that p37K is tightly bound to the viral envelope. Its resistance to proteinase K digestion indicates that it is not exposed on the surface of EEV but lines the inner side of the envelope. Since p37K does not contain a signal peptide characteristic of most membrane proteins, we examined the possibility that the protein acquires its membrane affinity through the addition of fatty acids. Indeed, Triton X-114 phase partitioning experiments demonstrated that p37K is hydrophobic when acylated, but hydrophilic in the absence of fatty acids. Three other viral proteins have been shown to be required for virus envelopment and release from the host cell and we therefore tested whether p37K interacts with viral proteins. In EEV and in absence of reducing agents, an 80-kDa complex reacting with an anti-37K antiserum was found. Analysis of this complex showed that it most likely consists of a p37K homodimer. Interestingly, only a small amount of p37K occurs as a complex, most of it is present in the viral envelope as monomers.

Cinchona alkaloids and BINOL derivatives as privileged catalysts or ligands in asymmetric synthesis

During the last fifteen years organocatalysis emerged as a powerful tool for the enantioselective functionalization of the most different organic molecules. Both C-C and C-heteroatom bonds can be formed in an enantioselective fashion using many types of catalyst and the field is always growing. Many kind of chiral catalysts have emerged as privileged, but among them Proline, cinchona alkaloids, BINOL, and their derivatives showed to be particularly useful chiral scaffolds. This thesis, after a short presentation of many organocatalysts and activation modes, focuses mainly on cinchona alkaloid derived primary amines and BINOL derived chiral Brønsted acids, describing their properties and applications. Then, in the experimental part, these compounds are used for the catalysis of new transformations. The enantioselective Friedel-Crafts alkylation of cyclic enones with naphthols using cinchona alkaloid derived primary amines as catalysts is presented and discussed. The results of this work were very good and this resulted also in a publication. The same catalysts are then used to accomplish the enantioselective addition of indoles to cyclic enones. Many catalysts in combination with many acids as co-catalysts were tried and the reaction was fully studied. Selective N-alkylation was obtained in many cases, in combination with quite good to good enantioselectivities. Also other kind of catalysis were tried for this reaction, with interesting results. Another aza-Michael reaction between OH-free hydroxylamines and nitrostyrene using cinchona alkaloid derived thioureas is briefly discussed. Then our attention focused on Brønsted acid catalyzed transformations. With this regard, the Prins cyclization, a reaction never accomplished in an enantioselective fashion until now, is presented and developed. The results obtained are promising. In the last part of this thesis the work carried out abroad is presented. In Prof. Rueping laboratories, an enantioselective Nazarov cyclization using cooperative catalysis and the enantioselective desymmetrization of meso-hydrobenzoin catalyzed by Brønsted acid were studied.

Synthetic routes toward functional block copolymers and bioconjugates via RAFT polymerization

Synthetic Routes toward Functional Block Copolymers and Bioconjugates via RAFT PolymerizationrnSynthesewege für funktionelle Blockcopolymere und Biohybride über RAFT PolymerisationrnDissertation von Dipl.-Chem. Kerstin T. WissrnIm Rahmen dieser Arbeit wurden effiziente Methoden für die Funktionalisierung beider Polymerkettenenden für Polymer- und Bioanbindung von Polymeren entwickelt, die mittels „Reversible Addition-Fragmentation Chain Transfer“ (RAFT) Polymerisation hergestellt wurden. Zu diesem Zweck wurde ein Dithioester-basiertes Kettentransferagens (CTA) mit einer Aktivestereinheit in der R-Gruppe (Pentafluorphenyl-4-phenylthiocarbonylthio-4-cyanovaleriansäureester, kurz PFP-CTA) synthetisiert und seine Anwendung als universelles Werkzeug für die Funktionalisierung der -Endgruppe demonstriert. Zum Einen wurde gezeigt, wie dieser PFP-CTA als Vorläufer für die Synthese anderer funktioneller CTAs durch einfache Aminolyse des Aktivesters genutzt werden kann und somit den synthetischen Aufwand, der üblicherweise mit der Entwicklung neuer CTAs verbunden ist, reduzieren kann. Zum Anderen konnte der PFP-CTA für die Synthese verschiedener Poly(methacrylate) mit enger Molekulargewichtsverteilung und wohl definierter reaktiver -Endgruppe verwendet werden. Dieses Kettenende konnte dann erfolgreich mit verschiedenen primären Aminen wie Propargylamin, 1-Azido-3-aminopropan und Ethylendiamin oder direkt mit den Amin-Endgruppen verschiedener Peptide umgesetzt werden.rnAus der Reaktion des PFP-CTAs mit Propargylamin wurde ein Alkin-CTA erhalten, der sich als effizientes Werkzeug für die RAFT Polymerisation verschiedener Methacrylate erwiesen hat. Der Einbau der Alkin-Funktion am -Kettenende wurde mittels 1H und 13C NMR Spektroskopie sowie MALDI TOF Massenspektroskopie bestätigt. Als Modelreaktion wurde die Kopplung eines solchen alkin-terminierten Poly(di(ethylenglykol)methylethermethacrylates) (PDEGMEMA) mit azid-terminiertem Poly(tert-butylmethacrylat), das mittels Umsetzung einer Aktivester-Endgruppe erhalten wurde, als kupferkatalysierte Azid-Alkin-Cycloaddition (CuAAC) durchgeführt. Die Aufarbeitung des resultierenden Diblockcopolymers durch Fällen ermöglichte die vollständige Abtrennung des Polymerblocks 1, der im Überschuss eingesetzt wurde. Darüber hinaus blieb nur ein sehr kleiner Anteil (< 2 Gew.-%) nicht umgesetzten Polymerblocks 2, was eine erfolgreiche Polymeranbindung und die Effizienz der Endgruppen-Funktionalisierung ausgehend von der Aktivester--Endgruppe belegt.rnDie direkte Reaktion von stimuli-responsiven Polymeren mit Pentafluorphenyl(PFP)ester-Endgruppen, namentlich PDEGMEMA und Poly(oligo(ethylenglykol)methylethermethacrylat), mit kollagen-ähnlichen Peptiden ergab wohl definierte Polymer-Peptid-Diblockcopolymere und Polymer-Peptid-Polymer-Triblockcopolymer unter nahezu quantitativer Umsetzung der Endgruppen. Alle Produkte konnten vollständig von nicht umgesetztem Überschuss des Homopolymers befreit werden. In Analogie zu natürlichem Kollagen und dem nicht funktionalisierten kollagen-ähnlichen Peptid bilden die PDEGMEMA-basierten, entschützten Hybridcopolymere Trimere mit kollagen-ähnlichen Triple-Helices in kalter wässriger Lösung, was mittels Zirkular-Dichroismus-Spektroskopie (CD) nachgewiesen werden konnte. Temperaturabhängige CD-Spektroskopie, Trübungsmessungen und dynamische Lichtstreuung deuteten darauf hin, dass sie bei höheren Temperaturen doppelt stimuli-responsive Überstrukturen bilden, die mindestens zwei konformative Übergänge beim Aufheizen durchlaufen. Einer dieser Übergänge wird durch den hydrophoben Kollaps des Polymerblocks induziert, der andere durch Entfalten der kollagen-ähnlichen Triple-Helices.rnAls Ausweitung dieser synthetischen Strategie wurde homotelecheles PDEGMEMA mit zwei PFP-Esterendgruppen dargestellt, wozu der PFP-CTA für die Funktionalisierung der -Endgruppe und die radikalische Substitution des Dithioesters durch Behandlung mit einem Überschuss eines funktionellen AIBN-Derivates für die Funktionalisierung der -Endgruppe ausgenutzt wurde. Die Umsetzung der beiden reaktiven Kettenenden mit dem N-Terminus eines Peptidblocks ergab ein Peptid-Polymer-Peptid Triblockcopolymer.rnSchließlich konnten die anorganisch-organischen Hybridmaterialien PMSSQ-Poly(2,2-diethoxyethylacrylat) (PMSSQ-PDEEA) und PMSSQ-Poly(1,3-dioxolan-2-ylmethylacrylat) (PMSSQ-PDMA) für die Herstellung robuster, peptid-reaktiver Oberflächen durch Spin Coaten und thermisch induziertes Vernetzen angewendet werden. Nach saurem Entschützen der Acetalgruppen in diesen Filmen konnten die resultierenden Aldehydgruppen durch einfaches Eintauchen in eine Lösung mit einer Auswahl von Aminen und Hydroxylaminen umgesetzt werden, wodurch die Oberflächenhydrophilie modifiziert werden konnte. Darüber hinaus konnten auf Basis der unterschiedlichen Stabilität der zwei hier verglichenen Acetalgruppen Entschützungsprotokolle für die exklusive Entschützung der Diethylacetale in PMSSQ-PDEEA und deren Umsetzung ohne Entschützung der zyklischen Ethylenacetale in PMSSQ-PDMA entwickelt werden, die die Herstellung multifunktioneller Oberflächenbeschichtungen z.B. für die Proteinimmobilisierung ermöglichen.

Dapsone hydroxylamine induces premature removal of human erythrocytes by membrane reorganization and antibody binding

N-hydroxylation of dapsone leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with dapsone therapy. Dapsone has been associated with decreased lifespan of erythrocytes, with consequences such as anaemia and morbidity in patients treated with dapsone for malaria. Here, we investigated how dapsone and/or its hydroxylamine derivative (DDS-NHOH) induced erythrocyte membrane alterations that could lead to premature cell removal.