Heinrich Trischmann

Über Verdazyle, eine neue Klasse cyclischer N-haltiger Radikale

Triphenylformazan last sich mit Dimethylsulfat bzw. Methyljodid, aber auch mit Formaldehyd bzw. Methylenjodid, in ein tief grunes freies Radikal verwandeln. Mit weiteren Alkylhalogeniden bzw. Aldehyden sowie mit anderen Formazanen sind eine ganze Reihe von Radikalen dieses Typs (Verdazyle) dargestellt worden. Ihre chemischen und physikalischen Eigenschaften werden beschrieben. Im Zusammenhang mit den ESR-Spektren wird die Frage der Formulierung erortert. Mit Halogenen entsteht aus dem grunen Radikal 1,3,5-Triphenyl-verdazyl (V) das entsprechende violette Kation, das diamagnetisch ist. Durch Reduktionsmittel werden die Verdazyle unter Aufnahme von 1 H-Atom in farblose Verbindungen verwandelt, aus denen die tieffarbigen Radikale, meist schon durch Luftsauerstoff, zuruckgebildet werden. Die Ruckverwandlung in Formazane gelang auf photochemischem Wege. Beim thermischen Abbauwurden unter Ringverengung Triazole erhalten.

Preparation of tetramethylrhodaminyl-phalloidin and uptake of the toxin into short-term cultured hepatocytes by endocytosis

A fluorescent phallotoxin with high photostability, tetramethylrhodaminyl-phalloidin (Rh-phalloidin), has been prepared. The affinity of this compound to rabbit muscle actin has been determined to be about 6 times lower than that of phalloidin. In freshly isolated hepatocytes the internalized fluorescent toxin stains the cellular actin. In contrary, there is no actin staining visible in cultured hepatocytes. Short-term cultured hepatocytes (5 h of culturing) incorporate the toxin by endocytosis; it is kept sealed in the endocytotic vesicles, which are usually found accumulated at the sites where cells touch after reaggregation.

A rapid radioimmunoassay, using a nylon support, for amatoxins from Amanita mushrooms

A fetuin derivative of alpha-amanitin was prepared and used as an antigen in rabbits. The antigen was superior to previous bovine serum albumin derivatives of beta-amanitin by its lower toxicity and high immunogenicity. On the other hand, the antibodies raised with the alpha-amanitin derivative did not show full crossreactivity with the other amatoxins, as did the immunoglobulins induced by protein derivatives of beta-amanitin. The procedure for activating nylon surfaces and coupling proteins onto them was improved with respect to surface charge and homogeneity. A partially purified IgG-fraction derived from the sera of rabbits immunized against amatoxins was covalently attached to the activated nylon surfaces. The covalently coupled immunoglobulins were complexed with a tritiated amatoxin. Then small pieces of the nylon sheet were punched out and incubated with the amatoxin solution to be analyzed. This procedure represents a method for dosing, in one step and without pipetting, the immunoglobulins and the labelled hapten. Determination of amatoxin concentrations was achieved by counting the radioactivity in the incubation fluid. The limit of detection was about 3 ng of amatoxins per ml. The radioimmunoassay was used to measure amatoxin concentrations in serum, urine, duodenal fluid, and gastric juice of patients with Amanita poisoning. Since such assays can be performed in 2-3 hr, the results can be used to determine the therapeutic protocol. The assay was likewise used to determine the concentration of amatoxins in mushroom tissue. For Amanita phalloides, for example, we found that the amatoxin concentration (mg/g dry weight) is 4.5 times higher in the gills than in the bulb.

A radioimmunoassay for amanitin

In the past several attempis [l-4] were made to raise antibodies against the toxins of the dangerous toadstool Amanita phalloides. These experiments used the crude extracts of the mushrooms as antigens; however, it appears most unlikely that the toxic peptides, with mol. wts of about 900, exhibit any antigenic properties. In 1959 Boehringer and Wieland [5] conjugated /3-amanitin via the thiophenylester of its carboxylic group to bovine serum albumin and found that the conjugate was too toxic to be used as an antigen in animals. In 1969 Fiume et al. [6,7] prepared the same conjugate using carbodiimides. They found that amanitin bound to albumin had a specific toxicity for protein-consuming cells like the sinusoidal cells of the liver, the macrophages [8], or the cells of the proximal tubules in the kidney [9]. Much less toxic conjugates of amanitin with bovine serum albumin and also dextranes of high mol. wt were obtained by Faulstich and Trischmann [lo], who used cy-amanitin bound to the macromolecules by a spacer containing an azo-linkage. These compounds, however, were poor antigens, either due to the azo linkage being unstable under physiologic conditions, or the toxin molecule having lost one of its predominant antigenic sites by the chemical modification at the 6-hydroxyindole moiety. In this situation Fiume et al. decided on the rat for raising antibodies. The rat is less sensitive to amatoxins [ 111 as well as to amanitin-albumin conjugates [12], than are the rabbits. They succeeded in obtaining a serum, which in a radioimmunoassay enabled.the.detectjon 500 pg of amatoxins [ 121. In otir study we made use of the N-hydroxysuccinimidester of /3-amanitin, which is better soluble in aqueous protein solutions than is the thiophenylester of the toxin. After the coupling reaction with bovine serum albumin we found 1.3 mol of /3-amanitin bound per mole of protein [ 161. The resulting amanitinalbumin conjugate was less toxic than that obtained by carbodiimide coupling, since the active ester reaction favors the formation of amide linkages over ester linkages between the toxin and the protein, and besides this, avoids side reactions and oligomerisation of albumin [ 131. Both, the presence of ester linkages and an increase in molecular weight probably contribute to the extremely high toxicity of amanitin-albumin conjugates prepared by carbodiimides. Nevertheless, the toxicity in the white mouse of the resulting derivative was still 3-4 times higher than that of /3-amanitin itself. This, however, was overcome by a covalent attachment of the peptide-protein-complex to polylysine. The amanitin-albumin-polylysine conjugate was mixed with Freund’s complete adjuvant and administered to rabbits by intradermal application. Though many of the animals died, and though the titer of amanitin-binding proteins in the sera of the surviving animals remained low, a radioimmunoassay could be established. By the use of [3H]O-methyl-demethyl-yamanitin [ 14,151 with a specific activity of 2.4 Ci/ mmol we succeeded in detecting as little as 50 pg of cy-amanitin.

Stabile N-haltige Biradikale und Triradikale

Darstellung und Eigenschaften von kristallisierten freien Radikalen, die zwei und drei Verdazyl-Ringsysteme pro Molekul enthalten, werden beschrieben. Wahrend der Paramagnetismus der drei dargestellten Triradikale 3 ungepaarten Elektronen entsprach und bis 77° K unverandert blieb, fand sich unter den neun dargestellten Biradikalen eines, das beim Abkuhlen reversible diamagnetisch wurde. Die Absorptionsspektren und ESR-Spektren werden im Zusammenhang mit Strukturfragen diskutiert.

NMR-Spektren von Verdazylen

DieNMR-Spektren von Verdazylen, deren Arylreste Methoxy-, Athyl- undtert.-Butylgruppen tragen, ergeben aus den paramagnetischen Verschiebungen der Protonenresonanz-Linien die isotropen Kopplungskonstanten mit ihren Vorzeichen fur die C-6-Methylenprotonen im Verdazylring und fur die Protonen der eingefuhrten Substituenten. Die Vorzeichen der gemessenen Kopplungskonstanten stimmen mit den Vorzeichen der berechneten Spindichten uberein.

Ringöffnung von Verdazylen zu N-Formyl-formazanen

Verdazyle mit einer Methylenbrucke in 6-Stellung werden in Gegenwart von Carboraffin durch Luftsauerstoff unter Ringoffnung zu N-Formyl-formazanen oxydiert. Mit Acetanhydrid/BF3-Atherat ergeben C-Aryl-formazane C-Aryl-N-Acetyl-formazane.

Über Tetraaza-pentenyl-Radikale

Die BF3-katalysierte Kondensation von Orthoameisensaureester mit asymmetrischen Diarylhydrazinen liefert 1,1,5,5-Tetraaryl-1,2,4,5-tetraaza-pentene. Ihre Dehydrierung mit PbO2 ergibt blaugrune Tetraaza-pentenyl-Radikale vom Typ11, die Oxydation im sauren Medium fuhrt zu Diaza-trimethin-Farbstoffen. Die thermische Zersetzung liefert u.a. Azoverbindungen (z. B.12). Die Absorptions- und ESR-Spektren werden im Zusammenhang mit der Struktur der Verbindungen diskutiert.

Aminoalkylierung von Formazanen und Ringerweiterung von Tetrazoliumsalzen zu Verdazylen

Formazane reagieren mit Formaldehyd und sekundaren Aminen zu Verdazylen, die in 6-Stellung den N-haltigen Substituenten tragen. Diese Substituenten wirken, wie auch -C≡CH und Phenyl in 6-Stellung, hypsochrom. Die unter Verwendung sekundarer Amine erhaltenen Verdazyle werden durch Saure nicht in stabile violette Kationen verwandelt, sondern unter Entfarbung zerstort. Aus Triphenyltetrazoliumchlorid wurden mit CH2N2-Losungen von 1,3,5-Triphenyl-verdazyl erhalten.