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T Wieland

Publications and source records attributed to T Wieland.

At least 91 records · Page 5Linked to original sources
PubMed1983

Unexpected similarity of the structures of the weakly toxic amanitin (S)-sulfoxide and the highly toxic (R)-sulfoxide and sulfone as revealed by proton nuclear magnetic resonance and X-ray analysis.

The three-dimensional structures of the slightly toxic diastereomeric (S)-sulfoxide of 6'-O-methyl-alpha-amanitin [6'-O-Me-alpha-ama (S)-sulfoxide, 4] and of the corresponding highly toxic sulfone 5 have been determined by X-ray diffraction analysis. The same derivatives along with 6'-O-methyl-alpha-amanitin [O-Me-alpha-ama (R)-sulfoxide, 3] and the corresponding thioether (O-Me-alpha-ama sulfide, 6] have been investigated in dimethyl sulfoxide solutions by 360-MHz 1H NMR spectroscopy including nuclear Overhauser effects (NOE). In addition alpha-amanitin (2) has been reinvestigated by this high-resolution method involving the identification of the ABMX systems of the tryptophan, cysteine, and asparagine and discrimination between the glycine residues. The structures of compounds 2-6 are compared with the structure of beta-amanitin which was solved previously by X-ray structure analysis. The results are (1) the structures in the crystalline state of the (S)-sulfoxide 4 and sulfone 5 are practically identical and (2) in dimethyl sulfoxide solution the structures of compounds 4 and 5 are likewise identical with each other and with those of the (R)-sulfoxide 3 and the thioether 6. The general structure of the peptide backbone of the alpha-amanitin derivatives investigated here almost corresponds to that of beta-amanitin (1), the main difference being a rotated plane of the peptide bond between the asparagine and cysteine residue. In order to explain the lack of high toxicity in the (S)-sulfoxide 4 we tentatively suggest alternative hydrogen bonding of a donor from the protein, or displacement of the R oxygen to the S oxygen of a hydrogen bond donor. This alternative bonding or displacement might not occur in the sulfoxide 4. Other explanations which include local conformational changes in the inhibitors or a difference between the SO and SO2 local dipoles are also possible.

Amanitins↗
PubMed1983

The toxic peptides from Amanita mushrooms.

The results of 50 years of effort in the chemistry of Amanita toxins are reviewed. The phallotoxins, fast acting components, but not responsible for fatal intoxications after ingestion, are bicyclic heptapeptides. They combine with F-actin, stabilizing this protein against several destabilizing influences. The virotoxins likewise fast acting are monocyclic heptapeptides. The amatoxins which are the real toxins lead to death within several days by inhibiting the enzymatic synthesis of m-RNA. They are bicyclic octapeptides. The structures of all of these compounds are described, as well as conformations, chemical reactions and modification, syntheses and correlations between structures and biological activities.

Amanita↗
PubMed1983

Analogs of phalloidin. D-Abu2-Lys7-phalloin, an F-actin binding analog, its rhodamine conjugate (RLP) a novel fluorescent F-actin-probe, and D-Ala2-Leu7-phalloin, an inert peptide.

The analogs [D-Abu2-Lys7]-phalloin (1) and [D-Ala2-Leu7]-phalloin (2) are obtained by cyclization of the monocyclic thioether peptides 5 and 7 with DCCI and diphenylphosphoro-azidate respectively. For the synthesis of 5 and 7 an intramolecular Savige-Fontana reaction of the linear heptapeptide tert.-butylesters 4 and 6 is applied. On treatment in dilute solution with TFA the classically synthesized peptides 4 and 6 lose their N-terminal Boc groups thus giving rise to the reaction of the Hpi residues with the released cysteine SH-groups. The lysine analog 1 binds to F-actin with an association constant of 1.3 X 10(-6) M whereas analog 2 exhibits practically no affinity. By reaction of 1 with tetramethyl-rhodaminyl-isothiocyanate a fluorescent derivative, rhodaminyl-lysine-phallotoxin (RLP), is obtained as a novel fluorescent probe for the visualization of F-actin in cell preparations.

Actins↗
PubMed1982

Formation of a single phosphodiester bond by RNA polymerase B from calf thymus is not inhibited by alpha-amanitin.

The template-directed synthesis of a single phosphodiester bond by highly purified calf thymus RNA polymerase B is not inhibited by high concentrations of alpha-amanitin (10(-6) M). However, a subsequent internucleotide bond is not synthesized in the presence of alpha-amanitin. These results suggest that translocation of the nascent RNA and RNA polymerase B along the DNA template is the enzymatic process inhibited by alpha-amanitin. It is also shown that the formation of a single phosphodiester bond by RNA polymerase B results in a stable ternary transcription complex, i.e., between the enzyme, the DNA, and the nascent RNA. Under reaction conditions which normally favor the elongation of RNA, the transcriptional process is arrested at initiation by alpha-amanitin. Such ternary initiation complexes have been isolated by agarose gel electrophoresis.

Alkaline Phosphatase↗
PubMed1982

Unusual intramolecular hydrogen bonding in cycloamanide A, cyclic (LPro-LVal-LPhe-LPhe-LAla-Gly). A crystal structure analysis.

The naturally occurring cyclic hexapeptide, cycloamanide A, has only one intramolecular hydrogen bond. It is a 4 leads to 1 type that encompasses the L Phe-L Ala sequence in which the experimentally determined phi, psi values are +54 degrees, -118 degrees and -88 degrees, -4 degrees, respectively. Even though the chirality is L, L, the phi, psi values are characteristic for a D, L beta-bend, Type II'. The conformation of the molecule was established by a crystal structure determination using X-ray diffraction analysis. Cycloamanide A (C33H42N6O6 . 4H2O) crystallizes in space group P2(1)2(1)2(1) with cell parameters a = 13.307(2) A, b = 24.820(4)A and c = 11.231(1)A.

Hydrogen Bonding↗
PubMed1982

[Isolation and characterization of an amatoxin-binding protein from wheat germ (author's transl)].

The preparation and characterization of a protein from wheat germ showing strong affinity to amatoxins (ABP) but differing from RNA polymerase B (II) is described. The purification, traced by [3H]amatoxin (O-methyldehydroxymethyl-alpha-amanitin), comprises 4 chromatographic steps, on Biogel A 1.5, DEAE-Sephadex, phosphocellulose, and again Biogel A 1.5. The protein exhibited in dodecyl sulfate polyacrylamide gel electrophoresis one single band with a molecular mass of 29,000 Da. Its isoelectric point is 4.9. The dissociation constant of the complex ABP-[3H]amatoxin is KD20 = 5 X 10(-7)M as determined by equilibrium dialysis against alpha-amanitin. By cyanogen bromide the protein is split into two fragments with molecular masses of 22,000 and 7,000 Da, respectively whose amino acid analyses, on summation, give the amino acid composition of ABP.

Amanitins↗
PubMed1981

Ether derivatives of alpha-amanitin. Introduction of spacer moieties, lipophilic residues, and radioactive labels.

Etherification of alpha-amanitin with tritiated methyl iodide yielded a radioactively labeled amatoxin of high specific activity (similar to or approximately 4 Ci/mmol) which, in its inhibition capacity for RNA polymerase II, was very similar to alpha-amanitin. The labeled toxin was used successfully in binding assays with RNA polymerases II and in radioimmunological determinations of amatoxins. If long-chained alkyl bromides were reacted with alpha-amanitin, lipophilic ether derivatives were obtained with a facilitated penetration capacity into cells. As a consequence of the improved permeability, two derivatives, O-hexyl- and O-decyl-alpha-amanitin, were more toxic in vivo than alpha-amanitin, although their affinity to RNA polymerases II was much reduce. By reaction of N-tert-butyloxy-carbonyl-N'-(6-bromocaproyl)ethylenediamine with alpha-amanitin, a ten-atom spacer with a terminal amino group could be introduced into the toxin, which allowed the attachment of alpha-amanitin to proteins, solid-phase supports, or reporter groups. For example, by reaction with fluoresceinyl isothiocyanate, a fluorescent amatoxin was prepared for visualizing amatoxin-binding structures in cells. After succinylation of the spacer moiety, alpha-amanitin could be attached to proteins, e.g., fetuin, yielding a derivative with good antigenic properties. When an alpha-amanitin derivative was coupled to Sepharose 6B, an adsorbent for affinity chromatography was obtained suitable for a one-step purification of amatoxin-binding immunoglobulins from the sera of immunized rabbits.

Amanitins↗
PubMed1981

Phalloidin uptake by the liver of cholestatic rats in vivo, in isolated perfused liver and isolated hepatocytes.

Rats made cholestatic by bile duct ligation survive phalloidin poisoning. This protection against phalloidin poisoning is correlated with the rate of the toxin uptake by the liver. [3H]demethylphalloin was used as a tracer for uptake studies. This is justified because there is no significant difference in the rate of uptake of the phallotoxins used. Within 4 h after bile duct ligation phalloidin uptake is reduced to about 25% and after 24 h to about 15% of controls. Isolated perfused livers and isolated hepatocytes from cholestatic rats bind less phalloidin than normal controls. Besides morphological changes serum factors may account for the decreased rate of phallotoxin uptake in cholestatic cells.

Alkaloids↗
PubMed1981

The effect of the chemical nature of the side chains of amatoxins in the inhibition of eukaryotic RNA polymerase B.

The inhibition constants (Ki) of DNA-dependent RNA polymerase B (or II) from calf thymus were measured for eight synthetically obtained bicyclic amanitin-like thioethers, two R-sulfoxides, and two S-sulfoxides. These Ki values were compared with those of alpha-amanitin, its 6'-O-methylether Ia (an R-sulfoxide), the S-sulfoxide, the sulfone, the S-deoxo derivative (Id) of Ia, and several previously described amatoxins. The necessity of a beta-methyl side chain in position 3 and a hydroxy group in proline-2 was confirmed. Additionally, the presence of an isoleucine side chain in position 6 and the absence of a side chain in position 5 was recognized as important for binding to the enzyme. In the three sulfoxide samples examined, the R-diastereomer was found to be a stronger inhibitor than the S-form. The contribution of every structural element to biological activity has been discussed.

Amanitins↗
PubMed1981

Interaction of long-chain acyl-CoA analogs with pig kidney general acyl-CoA dehydrogenase.

The interaction of two long-chain acyl-CoA analogs with pig kidney general acyl-CoA dehydrogenase (EC 1.3.99,3) was examined. The effect of S-heptadecyl-CoA and heptadecan-2-onyl-dethio-CoA on the flavo-protein was observed spectrophotometrically using the flavin as an active-site probe. The S-heptadecyl thioether analog bound strongly to the enzyme (Kd = 17 nM) and was a powerful competitive inhibitor (Ki less than 40 nM). In contrast to the thioether analog, the dethiocarba derivative, heptadecan-2-onyl-dethio-CoA, was a substrate inthe standard assay system being dehydrogenated at about 60% of the rate shown by palmitoyl-CoA. These results support the proposal that alpha-carbanion formation is an early event in the dehydrogenation of acyl-CoA substrates.

Acyl Coenzyme A↗
PubMed1981

Analogs of amanin. Synthesis of Ile3-amaninamide and its diastereoisomeric (S)-sulfoxide.

Ile3-amaninamide (3-R) and its diastereomeric sulfoxide (3-S) are obtained by oxidation of the bicyclic thioether peptide 2 by hydrogen peroxide in acetic acid. 2 was prepared by an intramolecular Savige-Fontana reaction of the linear octapeptide tert.-butylester 4 whose N-terminal Boc-Hpi residue on treatment with TFA loses the Boc group and reacts under thioether formation with the released cysteine-SH. The concomitantly deprotected carboxyl terminus is coupled intramolecularly with the free amino group of the secocompound 5 using the MA or DCCI method, thus forming the homodetic peptide ring. Compounds 3-R and 3-S agree very well with analog samples in chiroptical behavior. Thioether 2 and sulfoxide 3-R exert 50% inhibition of RNA polymerase II (or B) from Drosophila melanogaster in 10(-6) M solution whereas Ki of 3-S is about five times higher.

Amanitins↗
PubMed1980

Virotoxins: actin-binding cyclic peptides of Amanita virosa mushrooms.

Virotoxins are toxic peptides singularly found in Amanita virosa mushrooms. After purification and resolution by high-pressure liquid chromatography, the main component, viroisin, was selectively cleaved and submitted to Edman degradation. The structure could be completely elucidated and was in part found to be the same as in phallotoxins. Differing from the phallotoxins, however, virotoxins are monocyclic peptides and contain D-serine instead of L-cysteine. In addition, two amino acids were detected in virotoxins which thus far have not been found in nature: 2,3-trans-3,4-dihydroxy-L-proline and 2'-(methylsulfonyl)-L-tryptophan. The biological activity of viroisin is comparable to that of the phallotoxins: e.g., with 2.5 mg of viroisin per kg (white mouse), 50% of the animals die within 2-5 h by hemorrhagia of the liver. Also, on the molecular level, the virotoxins behave similar to the phallotoxins. Thus, viroisin binds to rabbit muscle actin as proved by difference UV spectroscopy. With an apparent equilibrium dissociation constant KD approximately 2 x 10(-8) M, the affinity of viroisin is very similar to that of phalloidin. However, the flexibility of the monocyclic structure and the presence of two additional hydroxy groups in the virotoxins suggest a different mode of interaction with actin. While there is proof that the bicyclic phallotoxins possess a rigid binding site, the virotoxins may adopt the biologically active conformation by an induced-fit mechanism upon contact with actin.

Actins↗
PubMed1980

Concentration-dependent influence of various cytochalasins and chaetoglobosins on the phalloidin-induced polymerization of G-actin in 0.6 M potassium iodide.

Phalloidin, a bicyclic peptide from the poisonous Amanita phalloides mushroom, stimulates the viscosimetrically determined polymerization of G-actin to F-actin in 0.6 m potassium iodide, a medium in which spontaneous polymerization does not occur. The cytochalasins B, D, E, and G (CB, CD, etc.) and the chaetoglobosins A, B, C, E, F, and J (Ch-A, Ch-B, etc.) have been found to influence the rat of polymerization in different ways, depending on their chemical structure and concentrations applied. Class I cytotoxins, e.g., CB, CG, Ch-C, Ch-E, and Ch-F, which exert on F-actin a weak degradative power (DP, "Spudich effect"), increase the polymerization rate when present in a ratio of 4 mol to 1 mol of actin. Polymerization is slightly enhanced or retarded at molar ratios ranging from 0.04:1 to 0.4:1. Class III cytotoxins of strong degradative power, i.e., high affinity for actin, such as CE, Ch-B, and Ch-J, decrease the polymerization rate at a molar ratio of 4:1 during the first 30-40 min; however, they increase it at a molar ratio of 0.4:1. Members of class II (CD, Ch-A) exert an effect that can be interpreted as a combination of the effects of class I and class III cytotoxins. An explanation of this difference in behavior is offered on the basis of experiments on F-actin degradation by Ch-J and its reconstitution by phalloidin in the presence of Ch-J.

Actins↗
PubMed1980

Amaninamide, a new toxin of Amanita virosa mushrooms.

Amaninamide, a toxin closely related to the family of amatoxins, was found exclusively in Amanita virosa mushrooms. It differs from the well known toxin alpha-amanitin in that it lacks the 6'-hydroxyl group of the tryptophan unit, and from the toxin amanin found in Amanita phalloides by the presence of a carboxamide group instead of a carboxylic acid groups.

Agaricales↗
PubMed1980

2,3-trans-3,4-trans-3,4-Dihydroxy-L-proline: An amino acid in toxic peptides of Amanita virosa mushrooms.

Among the four possible stereoisomers of 3,4-dihydroxy-L-proline,2,3-trans-3,4-trans-3,4-dihydroxy-L-proline (IV) had not been found in nature previously. It has now been detected as a component of virotoxins, toxic peptides of Amanita virosa mushrooms. Because periodate failed to effect an oxidative glycol splitting reaction, the two hydroxyl groups in positions 3 and 4 were expected to be in a trans configuration. Furthermore, the formation of a 4-lactone on treatment with acids pointed to the carboxyl group and the hydroxyl group at position 4 being in a cis configuration. These results are in agreement with structure IV only. Final proof for structure IV was given by NMR spectroscopy and direct comparison with the 2,3-cis-3,4-trans-3,4-dihydroxy-L-proline isomer.

Journal Article↗