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Home , Amanita albocreata, Antamanide, Phalloidin, Phallotoxin, Virotoxins

INVITED REVIEW In?. J. Peptideprotein Res. 22,1983,251-216

The toxic from mushrooms

THEODOR WIELAND Max-Planck-Institute for Medical Research, Heidelberg, West Germany

Received 13 December 1982, accepted for publication 15 January 1983

The results of 50 years of effort in the chemistry of Amanita are reviewed. The , fast acting components, but not responsible for fatal intoxi- cations after ingestion, are bicyclic heptapeptides. They combine with F-, stabilizing this protein against several destabilizing influences. The likewise fast acting are monocyclic heptapeptides. The 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 com- pounds are described, as well as conformations, chemical reactions and modifi- cations, syntheses and correlations between structures and biological activities. Key words: amatoxins; cyclic peptides; thioethers; phallotoxins; tryptathionine; virotoxins

Heinrich Wieland (photograph), born in 1877, was one of the most productive and versatile chemists during the first part of this century. In the thirties, at his institute the Chemical Laboratory of the Bavarian Academy of Science at Munich, Germany, students and post doctorals worked on such different themes as steroids, pigments of butterflies, tocd and snake , radical reacfions, strychnos alkaloids, alkaloids from _calabash curare and mechanism of biological oxidation. Natural substances that had any striking properties were of interest.

50 years of Amanita research In the early thirties, H. Wieland suggested to Hans A. Raab that he attempt the isolation of the poisonous principal of the most danger- ous mushroom , “der griine Knollenblatterpilz”, whose erroneous ingestion yearly accounted for numerous victims. This meant resumption of work which had begun Theodor and Heinrich Wieland, early 1950s T. Wieland more than 100 years earlier in French, American grew with the discovery of yamanitin (9), E- and German laboratories, but had not led to a amanitin (lo), (1 l), amanin amide (1 2), homogeneous substance. It was found that the (lo), amanullinic acid (1 1) and pro- aqueous extract contained a hemolytic principal amanullin (1 1). (phallin) that was destroyed by acids or by was found to be one member of a heating, and, therefore, could not be the real whole family of related cyclic peptides called which was found to be heat-stable. Raab “phulloroxins”, which included phalloin (1 3), met with considerable difficulties (1,2) as did phallisin (1 0), phallacidin (1 4), phallacin and his successor J. Renz (3), but they paved the phallisacin (1 5). The final compound isolated way for a successful continuation of the effort. was prophalloin (16). A third group of peptidic In 1937, Feodor Lynen and Ulrich Wieland toxins was detected when a number of Amunifa were able to obtain the first crystalline toxic virosa mushrooms were found and collected in component which they named phalloidin (4). the forests of West Virginia. The virotoxins are Phalloidin, after intraperitoneal injection, led to clearly separated from phallotoxins present in death of the experimental animal (white mouse) A. virosa by chromatography on Sephadex within 2-5 h. This was much faster than death LH-20 (17,18). In their mode of action, they from A. phalloides which typically takes 4-8 are closely related to the phallotoxins (LDso days. A slow acting “amanitin” was crystallized 2 mg/kg white mouse, death within 2-5 h). As by H. Wieland, R. Hallermayer and W. Zilg in it will be shown, a biochemical relationship 1941 (5). For a detailed history see one of the exists between the phallotoxins and virotoxins. early reviews (1,6). World War I1 saw an end to On looking for minor phallotoxic compo- the efforts to determine the chemical structures nents in the lipophilic part of extracts of A. of the toxins. With phalloidin, B. Witkop skill- phulloides, a fraction was obtained which, fully confirmed the suggested peptide nature of although containing phallotoxins, proved non- phalloidin by isolating from an acid hydrolysate toxic in the animal test. The suspected anti- L -, L , L axindolyalanine (“Oxy- toxic principle was purified, crystallized and ”) and L-allohydroxyproline, two characterized as a cyclic decapeptide called amino acids hitherto unknown as building anfumanide (19-21). One mg of blocks of natural peptides (7). per kg body weight when injected before, and With new methods of paper chromatography at the latest a few minutes after, a lethal dose and paper electrophoresis available after the of phalloidin, prevented certain death in the War, work was resumed in the author’s labora- mouse. Several additional but biologically tory. A sensible test reaction was developed inactive lipophilic cyclic peptides have been with cinnamaldehyde, which in a strong HCI- crystallized from A. phulloides and summarized atmosphere yields a deep violet color with as cycloamanides (CyA) (22). The three- amanitin and, less sensitive, a blue one with dimensional structure of CyA A has recently phalloidin (8). A drop of a very dilute solution been published in this Journal (23). of amanitin, dried on a free space on a news- paper upon moistening with conc. hydrochloric acid yields a blue colored spot. Neither of these reactions, cinnamaldehyde or newspaper, are yet understood. The latter depends on lignin, set free by the strong acid aldehydes, which, like cinnamaldehyde, can undergo condensation reactions forming conjugated double bond systems. By these analytical tools, a new acidic amanitin was detected and named 0-amanit in. This byproduct accompanied the original neutral amanitin, now called a-amanitin (8). In the following years, the family of the FIGURE 1 amanitin, now summarized as “amatoxins”, Formula of antamanide.

258 Amanita toxic peptides: Review

Pro- Gly -Val Pro- Ser -Phe-Phe tryptophan with L-cysteine. Acidic hydrolysis I I 1 of 2 yields 0-oxindolylalanine, 3, and L-cysteine. Ala- Phe - Phe Llle-Pro-Phe CyA A CyA B

0 0 CH, H I I &N-I!-CO# MS-CH,?-C&M Pro-kt-Leu- Gly Pro-kt-Leu-Gly H m. I 4 I 1 2 Leu- VoI- Leu --he Leu-Pro -Leu-Phe

CyA C CyA D Tryptathionine is the chromophoric system of the phallotoxins responsible for the u.v.- maxima around 290 nm (Fig. 3). The existence FIGURE 2 of a thioether moiety in phalloidin hypotheti- cally suggested by Cornforth et al. (33) was The biologically inert cyclic peptides, cycloamanides proved i) by replacement of the sulfur bridge (CYN(23). by hydrogen atoms with the help of Raney nickel (34), forming the u.v.-spectrum of tryptophan (Amu 280 nm), and ii) by synthesis THE PHALLOTOXINS of 2-thioethers, e.g. L-methylthio-indolylacetic acid, 4, from indolylacetic acid and methyl- sulfenyl chloride (35,36). The u.v.-spectrum of The phallotoxins, with the exception of 4 coincided with that of phalloidin (Fig. 3) prophalloin, have the property of specifically proving the correctness of its thioether struc- binding to F-actin (24); the affinity constant K ture. being 10-7-10-8 M (25). By this conjugation F-actin obtains high stability against chemical C H2- CO,H C Hz-CO2H and physical influences like depolymerization + CI SCH, SCH, by chaotropic ions (0.6M KI) (26,27), degra- H C" dation by proteases (28), (29), or DNAse I (30), ultrasonic vibration (27) or denaturation by heat (31). Since the equilibrium C OaH COSH nG-actin * F-actin by phalloidin is shifted I I about 30-fold to the right (25), cells may be H-C-NH~ H-C-N H depleted from G-actin to an intolerable extent. For possible connections between this event I I and the pathological effect on the liver (swell- HO-C-H H-C-OH ing by uptake of blood) see reference 32. I I COaH CH, Chemistry of the phallotoxins 6 The building blocks. The phallotoxins are 5 bicyclic heptapeptides. Common features of all In addition to phalloidin and three neutral but one are: the constituent amino acids L- phallotoxins [phalloin (13, 37, 38), phallisin alanine, L-allohydroxyproline (4cis-hydroxy-~- (39), and prophalloin (16)], we isolated a , 1) (prophalloin has L-proline instead) group of acidic toxins from A. phalloides and the bisfunctional , L,L-tryp- characterized by the term -aci- in their names: tathionine, 2. The latter may be considered phallacidin (1 4), phallacin, and phallisacin (1 5). a product of an oxidative condensation of L- In these peptides 0-hydroxy-D-aspartic acid 259 T. Wieland

lo-'

I I NHI NHI *y, M-CH, HC-CH* HC-CH, I I I SCHEME 1 Peptide bond cleavage by lactone formation.

a-Amino-7-lactones from the different phallotoxins and amatoxins have been studied in extenso in the author's laboratory (40-44). For syntheses, photochlorination of L-leucine and L-isoleucine is a convenient method (45).

Structural and three-dimensional formulae of phallotoxins. By removing the sulfur crosslink of tryptathionine from phalloidin and by acid- olysis of the preferentially cleavable peptide FIGURE 3 bond in dethiophalloidin a linear heptapep- U.V.spectrum and CD spectrum of phalloidin in water. tide was obtained. Its stepwise degradation Broken line: CD of secophalloidin. allowed the formulation of a sequence (46), which after a slight modification (38) led to the structural formula of phalloidin and all the (erythro form, 5) replaces D-threonine (6) at phallotoxins (Fig. 4, Table 1). the same position as in the neutral phallotoxins. The LDzo values after i.p. adminstration to A second difference is the occurrence of L- the white mouse (1 5-3.0 mg/kg body weight) in the acidic instead of L-alanine in the are all in the same order of magnitude. This also neutral ones. The individual phallotoxins differ applies to their affinity to F-actin, suggesting by their number of hydroxyl-groups in the side distinct molecular sites common to all phallo- chain of L-leucine: phalloin and phallacin con- toxins being responsible for strong interaction tain 4-hydroxy-~-leucine ([2 S] -2-amino-4- with the protein. Supporting this view are hydroxyisocaproic acid, 7), phalloidin and structure-activity relations of chemically modi- phallacidin 7,6-dihydroxy-L-leucine ( [2 S, 4 R] - fied or synthesized variants and an inspection 2-amino-4, 5-dihydroxyisocaproic acid, 8) (40) of the three-dimensional formula. Such a phallisin and phallisacin 7,6,6'-trihydroxy-L- formula has been constructed according to 'H- leucine ([2 S] -2-amino-4,5,5'-trihydroxyiso- caproic acid, 9). H~C-VH-CO-NH-CH-CO-NH-CH-R'5 6 7

COlH COaH GOzH I I I I I HN HIN-C-H HzN- G- H HaN-C-H I 1 I I CHI CHI CH a I I no- c-cn, HO- c-cn.on HO-LCH, I I ins nmn H&OH 7 8 9 H I' The yhydroxylated amino acids allow a mild and very specific cleavage by acids of an adjacent peptide bond, which is split in favor FIGURE4 of lactone formation. Structural formula of phallotoxins.

260 Amanita toxic peptides: Review

TABLE 1 The naturally occurring phallotoxins. Positions of residues R' -R4 as in Fig. 4

Name R' R' R' R4

Prophalloin" CH 3 CH (OH)CH, H CH, (CH 1, OH Phalloin CH 3 CH(OH)CH3 OH CH2 (CH3 12 OH Phalloidin CH 3 CH (OH)CH, OH CH, (CH , CH ,0H)OH Phallisin CH 3 CH(OH)CH, OH CH ,(CH, OH), OH Phallacin CH (CH3)' CH (OH)CO,H OH CH,(CH,),OH Phallacidin CH (CH 3) 1 CH(OH)CO,H OH CH,(CH,, CH,OH)OH Phallisacin CH (CH3)i CH (OH)CO,H OH CH ,(CH, OH), OH

"Non-toxic with doses up to 30mg/kg (white mouse). All other phallotoxins show LD,, values of 1.5-3.0 mg/ kg.

n.m.r. measurements and minimal energy calcu- glycyl-L-cysteine cyclic sulfide (1 -+ 3), 10, lation (47). Still undecided was the helical sense exhibited a CD spectrum mirror imaged to that of the indolyl-thioether moiety. of the phallotoxins (Fig. 6) and could serve as a probe for elucidating by X-ray the absolute Helicity of phallotoxins. The structural element configuration of the thioether moiety, if indole-S-CH2- of the phallotoxins is an inherent- ly dissymmetrical chromophore. It is helical according to the strong positive Cotton effects exhibited in CD around 300nm and 240nm (Fig. 3). Helicity of the thioether can be positive or negative (Fig. 5). There is no evi- Phalloin dence (I priori as to which helical orientation is responsible for either positive or negative Cotton effects. An unambiguous method for solving this problem would be X-ray structure analysis of a , but, unfortunately, these pep- tides form very thin, long fiber-like crystals which are not suitable for X-ray analysis. There- fore several cyclic peptides having the phallo- toxin chromophoric system were synthesized (48). One of them, 2-mercapto-L-tryptophyl-

positive I plus = P -~ "1 FIGURE 5 FIGURE 6 M- and P-helicity of an indolylthioether. CD spectra of phalloin and model thioether 10.

26 1 T. Wieland group of alanine, and the aromatic ring in proper arrangement. More insight into the role of the other side chains and of some structural features was gained from chemically modified phallotoxins and synthetic analogs.

Chemical modifications of phallotoxins Chemical means of reacting with phallotoxins in a definite way are acids, reductants and oxidants. Acids preferentially cleave the peptide bond between the carboxyl of the y-hydroxylated amino acid No. 7 and alanine (resp. valine) No. I, in favor of formation of a ylactone (cf. Scheme 1). The seco-compounds formed are entirely non-toxic, serving as compounds for studying the conditions for syntheses of phallo- toxins and analogs as described. The CD spectra of the seco-compounds are quite different from the bicyclic system, with positive Cotton effects around 280nm and a strong negative one at 235 nm (Fig. 3) pointing to a distortion of the chromophoric and binding moiety (Fig. 3). Reduction with Raney nickel removes the sulfur crosslink by replacing it by hydrogen atoms. The dethio compounds so formed (34) are non-toxic and exhibit CD spectra without any characteristic features. This points to a loss of stable conformations in solution of the dethio-compounds, the spatial arrangement of the groups responsible for binding to F-actin FIGURE 7 being disordered. Three-dimensional structures (a) of thioether peptide Oxidants affect the molecules in various 10 (M-helical) and (b) phalloin (P-helical). ways. Peroxy acids attack the sulfur atom yielding the diastereomeric (R) and (9 sulf- oxides and the sulfone (50). The assignment to obtained in suitable crystals. An N-p-bromoben- the (R)- and (a-configuration of the sulfoxides zene-sulfonyl derivative of 10 was obtained in has been made by comparison of the ORD crystals that revealed M-helicity of the chromo- curves with that of a synthetic (R)-sulfoxide of phoric system by X-ray diffraction. P-helicity 2-methylmercapto-~-tryptophan(1 1). must be ascribed to the phallotoxins with their opposite Cotton effect (49). n TTT The synthetic thioether peptides of ref. 48, including 10, are biologically inactive. They do not bind to F-actin because they lack the struc- u sulfoxides sulfone tural elements important for interaction with the 11 protein and visible at the left side of the three- dimensional formula (Fig. 7b): the cis-standing The absolute configuration of 11 was solved OH-group of (prophalloin, by X-ray diffraction analysis (5 1). Whereas the without OH, does not bind at all), the methyl (R)-sulfoxide of phalloidin and the sulfone are

262 Amanita toxic peptides: Review nearly as toxic as the parent substance, and 7 CHI -NH-cH-cH,-C-CH~OH -cH-cH,-CHI“‘DH) bind to F-actin by comparable affinities, the I I (S)-sulfoxide is not toxic with doses up to F0 OH I bH 20mg/kg and does not bind. Since the ORD- Phalloidin De-methylph~flO~ curves of (R)- and (S)-sulfoxides virtually behave like image and mirror image, indicating -CH-CH,-C=O a pair of enantiomeric sulfoxide moieties, it Krtopholloidin does not seem probable that a fundamental +HzC-CH-CH,J L An/ difference exists in the conformations of the q-g molecules. As will be shown, the amatoxins which are (R)-sulfoxides per se, reveal an $HI - CH-C Ha- amazing similarity of the three-dimensional ‘i) structure to that of the much less toxic (S)- sulfoxide. The difference in the biological ICH.COCI activities may rest upon the opposite or FL-WC=S IRH-N-Z=S I directions of the strong S-0 dipoles.

Oxidation of a side chain. The glycolic part of -CH-CHa-CH1-~~, the side chain of -y,6-dihydroxyleucine (No. 7) w-NHCOCI+,I W-NH- 5-NH-FLIRHI Norphalloin can be oxidized by periodate (52). The “keto- S phalloidin” (u-aminolevulinic acid-7-phalloidin) FL-fluoreueinyl. RH-Rhodaminyl formed is as toxic as phalloidin, demonstrating that the nature of this side chain does not SCHEME 2 influence the binding strength to actin. Accord- Chemical reactions at side chain No. 7 of phalloidin. ingly, further transformations (53) do not influence the activity: reduction of the keto group by NaBH4 leads to an equally toxic suffers cleavage to secophalloidin under such demethyl-phalloin. Radioactivity (3H) can be mild conditions as with aqueous at introduced (54) by this reaction. The keto room temperature (53). group could be reacted with bis-thioglycol to yield a dithiolane which on mild treatment The carboxyl group of the acidic phallotoxins. with Raney nickel (without destroying of the The second side chain modified by chemical tryptathionine moiety) was converted to a means was that of 0-hydroxyaspartic acid in methylene of norphalloin (norvaline-7-phal- phallacidin (PHC). We synthesized the amide, loidin) (53). a similarly toxic compound. With 1-amino-2, ‘ 3-dithiopropane an aminomethyl- dithiolane was formed whose amino group served as a function for linking to iodoacetyl (for affinity labeling) or to fluorescein- or rhodamine-isothiocyanate (5 6,57). The fluorescent derivatives still bind strongly to F-actin, thus providing sensitive stains for visualization of F-actin in biological objects (58) (Fig. 8). Fluorescent labels are also intro- duced in 6-amino-phalloin. This primary is obtained from 6-tosylphalloidin (53) by ammonolysis (59). Other reactions performed with 6-tosylphal- loidin were substitution of OTos by methyl- FIGURE^ mine and hydrogensulfide, respectively. With F-Actin cables in a fibroblast cell made visible by alkali an epoxide is formed which, interestingly, staining with a fluorescent phallotoxin.

263 T. Wieland

7 FHJ NH, CH, peptide synthesis between the carboxyl func- -NH-CH-CH,-C-CH.-OT~~ or NH,cH, -CH,--t--CH,-NH, tion of the y-hydroxy acid and the amino group I LO bH - OH -NHCH, of alanine-1. As a relais reaction, the recyc- I NH lization of secophalloidin (or secophalloin) was I d-hno covds attempted without success. Under the con- I-10s-pholloidtn ditions of all methods tried, elimination of water took place exclusively by lactone forma- -CU-C--CH,P, tion. As a result, we turned our attention to I1 d,l"l, -FH---CH1 co cn, OCld seco-ketophalloidin whose C-terminal amino 20' * NH acid cannot lactonize. Seco-ketophalloidin was I -y, prepared from secophalloidin by oxidation with periodate (52). By the mixed anhydride method using ethoxycarbonylchloride clearly SCHEME 3 toxic ketophalloidin was obtained and it was Some reactions of 6-toxyl phalloidin. demonstrated that, in the monocyclic secopep- tide, no odd geometry will prevent a second cyclization (66). When it was recognized that methylamide and dimethylamide which, in norphalloin with its simple propyl side chain this sequence, were progressively less toxic is likewise toxic, its total synthesis was started (15). PHC covalently bound by its carboxylic by F. Fahrenholz (67). He ended up with a group to bovine serum albumin using a water toxic preparation identical in every respect to soluble carbodiimide proved non-toxic in doses the substance obtained from natural phalloidin as high as 200mg/kg white mouse (67). The via ketophalloidin (Scheme 2). carboxyl function was also used to introduce A breakthrough in the total synthesis of the the fluorescing 2-nitrobenz-2-oxa-l , 3-diazole natural phallotoxin phalloin was achieved by residue via tnL 'hylene diamide -CO-NH- E. Munekata. Although the yields were small, CH2-CH2-NH2 by Barak et al. (61,62). Munekata showed that the recyclization of At the indole nitrogen atom alkylations were secophalloidin is feasible. The lactone ring was carried out in order to study the influence of opened by hydrolysis occurring on passage of their bulkiness on the toxicity of the derivatives the substance through a column of Sephadex (63) with the following results: N-CH3 deriva- LH-20 in 0.004M ammonia. The zwitterion so tive showed an LDSo (per kg body weight of formed was subjected to a rapid mixed anhy- the white mouse) like that of phalloidin dride procedure at low temperature, and the (2.5 mg); the N-ethyl derivative had 10 mg; the recyclized toxin was isolated by very careful N-propyl compound was non-toxic at doses up chromatographic separation of the product to 20 mg/kg. Consequently, N-methylation can mixture (68). be used for introducing radioactive atoms into A facile racemization occurs with N-acylated, the molecule without loss of its toxic properties. even urethane-type protected, a-amino-y-lac- tones on alkaline ring opening (69). Since, Partial and total syntheses additionally, some racemization occurs in frag- The synthetic chemistry of natural phallotoxin ment peptide condensations, a diastereomeric has a long history beginning in 1958 with the phalloidin containing y,6-dihydroxy-D-leucine thioether formation by the sulfenylchloride in pos. 7 was also formed and isolated. It was method of appropriate cysteine-peptides with non-toxic with a dose of up to 30 mg/kg (70). tryptophan-containing counterparts (64). A Secophalloidin, easily prepared from phal- main obstacle was the y-hydroxylated amino loidin by mild acid treatment, served as a acid in position 7, which, due to lactone forma- parent compound for preparing a series of tion, as a component inside of a peptide easily analogs: After removal of the N-terminal gives rise to a cleaving of the carboxyl-standing alanine-1 by Edman degradation, other amino peptide bond (65). Therefore as the last step acids were introduced (Scheme 4). Recyclization it was intended to close the second ring by a was also carried out at the de-Ala-secopeptide

264 Amanita toxic peptides: Review

-NH- !1( -NH-Fr 01-0 oc-0 Edman- H,N*I deQradL tic-cn, -NH-iO --NH2 seco-laclon nc-cn,tcn,c,n,i coupling with BocF'hr UNHdO I Phalloidin -NH-t/r Ikrokgur: benzyl inrtmd of methyl1 1 -Boc -NH-boc- HNBoc -* Yp' HC--a(zwI SCHEME 4 (HA1 n$-cnIhY I -NH-CO -M-CO Exchange of Ma' by Phe scco-zwitterton in phalloidin.

and after introduction of two yielding Two appropriate peptides were joined by the a bicyclic hexapeptide, de-A1a'-phalloidin and classical thioether synthesis via S-chloride (3.9, an octapeptide, endo-Ala'-phalloidin, respect- and the thioether treated as shown in Scheme 5. ively. The latter analogs showed no toxicity Final ring closure yielded phalloin indistinguish- and, correspondingly, no affinity to F-actin. able from the natural substance. Analogously, The difference of their molecular shapes from the toxic analog Leu7-phalloin and the natural the normal phallotoxins was also evident from non-toxic prophalloin (16) were obtained by their differing CD curves (71). total synthesis. A more detailed review of this After the conditions for a final ring closure synthetic work has been given by Munekata had been elaborated, the first total synthesis of (75). a natural phallotoxin, phalloin, was started A second successful approach to the phallo- (72). All previous syntheses of analogs were toxins was made possible by Fontana's obser- with norvaline in pos. 7, avoiding the difficulties vation that Savige's oxidation product of L- caused by lactonization (13,74). Following a tryptophan, L-1 ,2,3,3a,8,8a-hexahydropyrrolo similar pathway secophalloin was synthesized. (2,3b) indole-3-carboxylic acid, 12, abbrev. Hpi

a KCl (1) CF3COOH Boc-Trp-OH +Hl*-Lac. HCI Boc-Trp-Hle-Lac 907. (2) Boc-*la-ONSO

CF3CoCm Boc 41.- D - Thr-Cr(S-Cl)pnyp- Boc-h-irp-nie-LN H- Ala-Trp-Hb-Lac CF3 CKJH D - in CHSOOH 332

Ab-Trp-Lw (OH)

aHyp-Ala-D-Thr SCHEME 5 Total synthesis of phalloin.

265 T. Wieland (76), on proton catalysis will react smoothly The phallotoxin-like bicyclic peptide 15 with thiols yielding 2-thioethers of tryptophan shows a conformation similar to phalloidin as (78,79) (Hpi by photo-oxidation see ref. 77). revealed by CD spectroscopy, but has no bio- logical activity. An F-actin binding analog was also synthesized following the novel route. In order to have available an amino functional phal- lotoxin independently from natural sources, we 12 synthesized an analog with L-lysine in the “tole- rant” 7-position. Since D-cr-aminobutyric acid In our laboratories this reaction was soon instead of D-threonine in pos. 2 does not influ- applied to Boc-Hpi-(STrt)cysteine methylester ence binding to F-actin (74), D-Abu’ -LYs(Z)~- affording cyclic thioether 13 which on heating phalloin was synthesized via the heptapeptide was transformed to the “miniphallotoxin” 14 Boc -Hpi - Lys (Z)- Ala - D- Abu -Cys (STrt)-a-Hyp- (Scheme 5). The heptapeptide Boc-Hpi-Gly- Ala-tert.-butylester. Removal of Boc, Trt and t- Gly-Gly-Cys(STrt)-Gly-Gly-OHon subjection to butyl by trifluoroacetic acid along with intra- trifluoroacetic acid loosing the Boc- and Trt- molecular thioether formation yielded a mono- groups was cyclized to form a thioether which cyclic heterodetic peptide, which after second by an internal peptide synthesis (mixed anhy- cyclization with DCC and hydrogenolytical dride) yielded the simplest all-Gly-phallotoxin removal of the 7-residue afforded the desired cyclic (~-cysteinylglycylglycyl-2-mercapto-~-D-Abu2 -Lys7-phallotoxin. It exhibited an tryptophylglycylglycylglycy1)cyclic (1 -4) sul- affinity for F-actin of approximately 25% of fide 15 (48). that of phalloidin and, therefore, was conju- gated with rhodamine isothiocyanate to form an excellent fluorescent probe for F-actin in cell preparations (81). H N-CH -CO-Gly Structure-toxicity relations r I I Ample information has been collected on the I dependence of toxic behavior (affinity to F- actin) on structural details of the phallotoxins. In Table 2 all of the analogs have been listed together with their toxicity in the white mouse. For the contribution of each side chain, the following conclusions can be drawn: the cis- standing hydroxyl group in hydroxyproline-4, the methyl group of alanine-5 and the indole moiety are the chief binding sites. Proof of the participation of the aromatic system is an -CH- NH-Gly increase of the respective u.v.-absorption on binding to F-actin as shown by difference- spectroscopic measurements (82). The D-side 15 chain of residue 2 belongs to the binding

cy-c-n/HZ

SCHEME 6 co.tn. 0c-L- Synthesis of the smallest 13 14 bicyclic thioether peptide.

266 Arnanita toxic peptides: Review

TABLE 2 Analogs of phalloidin and their toxicity

Norphalloin (NPN)b CH, CH (OH)CH, cis OH CH, CH,CH,CH, ++ D-Abul-NPN CH, CH,CH, cis OH CH1cH2CH3 ++ D-Abu’-Lys’-PHN CH, CH,CH, cis OH CH3 (CHa),-NHa -c D-Ala’-Leu’PHN CH, (D-) CH, cis OH CH, CH,CH(CH,), - - Pro‘ -NPN CH, CH(OH)CH, H cH1CH1CH3 Gly5-NPN CH, CH (OH)CH, cis OH H CH,CH,CH, - Leu’-PHN CH, CH(OH)CH, transOH CH, CH,CH(CH,), ++ Glyl-PHD H CH(OH)CH, cis OH CH, + Val’-PHD CH (CH 3 11 CH (OH)CH, cis OH CH, ++ LeuI-PHD CH,CH(CH,), CH(OH)CH, cis OH CH, ++ Phe’-PHD 5 CH (OH)CH, cis OH CH, H,COH f D-Alal-PHD DCH, CH(OH)CH, cis OH CH, CH, -C-CH, - D-(OH),Leu-PHD CH, CH(OH)CH, cis OH CH, OH - PHC-amide CH (CH 3)’ CH(OH)CONH, cis OH CH ++ PHC-methylamide CH(CH,), -CONHCH, cis OH CH, + PHC-dimethylamide CH (CH,), -CON(CH,), cis OH CH, + des-AlaI-PHD cyclic hexapeptide residue no. 1 missing - endo-Ala’.-PHD cyclic octapeptide additional Ala between 1 and 7 -

.Toxicity LD,, (mg/kg body weight) at white mouse after i.p. injection LD,, 1-4 + +, 4-10 +, 10-30 f, 30-. bNPN, norphalloin; PHD, phalloidin; PHC, phallacidin. ODoes not, as a cation, enter the liver cell but has about 20% of PHDaffnity to Factin.

elements, for it has to have at least two carbon different virotoxins as well as their proportions atoms. Minor contribution makes side chain are given in Table 3. No. 1, which can vary considerably in structure As shown in Fig. 9, there exist in the amino or even be absent. Least important for binding acid compositions some features common with to F-actin is the nature of side chain No. 7, which, consequently, can be modified to a TABLE 3 handle for introducing biochemically The naturally occurring virotoxins. For meaning of X, functional groups (56). R‘ and R’ see Fig. 9

THE VIROTOXINS Name X R’ R’ % of total

The virotoxins discovered as components of the Vioidin so, CH(CH,), a, 18 white mushroom Amanifa uirosa produce the Desoxo- SO CH(CH,), CH, 4 same toxicological symptoms as the phallo- viroidin toxins. Surprisingly, the elucidation of the Alal-viroidin SO, CH, (33, 10 structure of viroidin led to the conclusion that fi’-desoxo- so CH3 CH, the virotoxins are not bicyclic peptides like the virOidin Viroisin phallotoxins but monocyclic heptapeptides Desoxo- SO, CH(CH,), CH,OH 49 (18). The general formula is shown in Fig. 9. SO CH(CH,), CH,OH 19 viroisin The variations of side chains leading to the six

267 T. Wieland H

I c H, I OH NH I CO CH3X H NH HOCH,I N-CO-C H OH OH HOHOE I IH H NH-CO-CH-NH-CO 16 17

It is highly probable that the virotoxins are derived from the phallotoxins or from a 9 FIGURE common precursor molecule. A reasonable bio- Structural formula of virotoxins. chemical transformation of phalloidin to viroidin is formulated in Scheme 7. The toxicity of the virotoxins corresponds the phallotoxins: alanine (resp. valine) in to that of the phallotoxins (LDSO 2,5 mg/kg, position 1, D-threonine (pos. 2), L-alanine (pos. white mouse). They also bind strongly to F- 5), and L-leucine, containing a varying number actin. This is surprising since the virotoxin mol- of hydroxy groups, in position 7. Amino acids ecule is not preformed a priori for binding, as different from those of phallotoxins are: D- its uncharacteristic CD spectrum shows. A serine instead of the L-cysteine-part of tryp- similar monocyclic structure in the class of the tathionine in pos. 3; 2.3-trans-3,4-trans-3,4-phallotoxins as a dethiocompound does not dihydroxyc-proline, 16, instead of 2,4-cis-4- exhibit any affinity to the protein. Features of hydroxy-L-proline in pos. 4 and 2'-(methyl- virotoxins not present in dethiophalloidin su1finyl)-L-tryptophan or 2'-(methyl-sulfonyl)- which could be responsible for such a strong L-tryptophan instead of the tryptophan part of interaction with F-actin by induced fit are the tryptathionine in pos. 6. The 3,4-dihydroxy- hydroxyl group of D-serine instead of L-alanine, proline (1 6) was unknown before detection as the additional trans-hydroxyl group in 3'- a component of the virotoxins (83), while the position of 4cis-4-hydroxy-L-proline and the diastereomeric 2,3-cis-3,4-trans-3,4-dihydroxy-methylsufonyl group at the tryptophan moiety. L-proline (1 7) was recognized as a component A methyl sulfonyl group has been introduced of a protein in diatoms several years ago (84). into dethiophalloidin by A. Buku and J.U. Kahl Both of these diastereomers have been obtained (personal communication); the product, how- by synthesis in the author's laboratory (85). ever, exhibited no affinity for F-actin.

SCHEME 7 Suggested biochemical path from phalloidin to viroidin [from (18)].

268 Amanita toxic peptides: Review The virotoxins, like the phallotoxins, most For a recent survey on toxicological and clinical probably are not involved in the fatal intoxi- research see reference 87. cation by A. virosa, but it contains the in a concentration comparable to Chemistry of amatoxins that of a-and 0-amanitin in A. phaffoides(1 2). The amatoxins, one family of bicyclic octapep- The white mushroom A. vernu does not tides, are derived from the same amino acid contain virotoxins. Apparently it is a white backbone but differ in the number of hydroxyl variety of the green A. phalfoides as it yields groups. a chromatographic pattern of its components The amatoxins consist exclusively of L- very similar to that of the green mushroom amino acids and glycine. Uncommon building (1 7). The yellow Amanita mushroom A. citrina blocks are the crosslinking 6’-hydroxytrypta- has no toxic peptides but a rather high content thionine-(R)sulfoxide (No. 4-8), which is of bufotenine (86). responsible for the u.v.-absorption around 300nm and for the positive Cotton effects in THE AMATOXINS the same region of circular dichroism (Fig. 11). On addition of alkali the u.v.-maximum is The amatoxins are the toxic components of shifted by 30nm to long wavelengths as a Amanita species, which after ingestion can consequence of phenolate-ion formation at the cause death to patients (87, 89). Not every 6‘hydroxyl group. This is expressed by the CD animal species will be intoxicated by peroral spectrum. The OH group is responsible for the administration like man, the dog and guinea very intensive color reaction with cinnamal- pig. These species resorb the toxins from the dehyde plus hydrochloric vapors (deep violet), intestinal tract, whereas rodents don’t resorb it and for the blue staining on newspaper with at all or only by such a slow rate that the 10 N hydrochloric acid (p. 258). symptoms of intoxication will not occur. The On strong irradiation at 314nm, i.e. longer symptoms are: after 8-10 h, nausea, vomiting, than the absorption maxima of proteins, a- diarrhea, and, after a deceptive intercept of 2- amanitin is decomposed, thus leaving the bind- 3 days, necrosis of the liver and kidneys, and ing site of RNA polymerase B (92). Side chain death by coma on the 5th to the 7th day. The No. 1 is that of aspartic acid in 0-amanitin and lethal dose for an adult person is about lOmg of asparagine in a-amanitin. For proof the latter amatoxins, the contents of one average green was obtained from 0-amanitin by amidation via death cap mushroom. mixed anhydride plus ammonia (93). In pos. 3 The molecular mechanism of action of is an L-isoleucine which is monohydroxylated amatoxins is their strong inhibtion of one of in y-position (y-amanitin, eamanitin) or bis- the eukaryotic DNA-dependent RNA poly- hydroxylated in y- and 6-position (a-amanitin, merases (form I1 or B) which is responsible for O-amanitin). The exact stereochemistry of this the transcription of the precursor of m-RNA (89). Half inhibition occurs in a concentration as low as lo-’ M. The toxins bind to the sub- unit of 140 KD in a ratio of 1 :1 (90). In its presence, one step of elongation, formation of HN- CH-I CO-NH-CH-CO-NH-CH,-CO4 6 a new phosphodiester bond, still goes on but IJ I I the next step, translocation of the growing RNA chain to the catalytic site of the enzyme, is blocked. The toxin forms a ternary complex of enzyme, DNA template and nascent RNA which can be isolated by gel chromatography OC- CH-NH-CO-CH-NH -co-~,-NH (91). Depletion from m-RNA will lead to a d-COA’ a 7 diminished synthesis and eventually arrest of protein formation in the liver and kidneys, FIGURE 10 leading to necrosis of the parenchymal cells. Structural formula of amatoxins.

269 T. Wieland TABLE 4 The naturally occurring amatoxins. Positions of residues R1-R' as in Fig. 10

Name R' R' R3 R' RS LD,, (mg/kg white mouse) a-Amanitin CH,OH OH NHa OH OH 0.3 #-Amanitin CH,OH OH OH OH OH 0.5 r-Amanitin CH, OH NHZ OH OH 0.2 edmanitin CH3 OH OH OH OH 0.3 Amanin CH,OH OH OH H OH 0.5 Amaninamidea CH,OH OH NH, H OH 0.3 Amanullin CH3 H NHZ OH OH > 20 Amanuliinic CH, H OH OH OH > 20 acid Proamanullin CH3 H NHa OH H > 20

A. virosa only. side chain is: (25,3R, 4R)-2-amino-3-methyl-4, group at the tryptophan moiety. All of 5-dihydroxy-valeric acid (94). Amanullin has a the amatoxins are (R)-sulfoxides. A three- normal L-isoleucine side chain. Amanin and dimensional structure formula of a-amanitin in amaninamide differ from 0- resp. a-amanitin dimethylsulfoxide solution was derived in 1978 only by the lack of the phenolic 6'-hydroxyl from 'H-n.m.r. data (95) and, more recently, for 6'-O-methyl a-amanitin (96). These struc- tures are in accordance with a space formula of p-amanitin obtained by X-ray diffraction analysis (97). Chemical modificationsof amatoxins As in the phallotoxin series and the amatoxins a preferential cleavage of one peptide bond

Ii It I' b' -8.

FIGURE I1 d U.v.-spectra and CD-spectra (broken lines) of a- FIGURE 12 amanitin in water at different pH values. Three-dimensional structure of p-amanitin [from (97)j 270 Amanita toxic peptides: Review occurs under mild acidolytic conditions radioactivity, tritium, into the toxin). between the y-hydroxylated isoleucine and the The aspartic acid side chain of 0-amanitin tryptophan moiety yielding monocyclic non- was derivatized as methyl ester, thiophenylester toxic seco compounds. Hydrogenolytic removal and as amides of various structures all deriva- of the sulfoxide bridge by treatment with tives being still toxic (93). The carboxylic Raney-nickel yielded non-toxic monocyclic group was used as a handle for fixation of 0- dethio products. Edman-degradation of a linear amanitin to serum albumin by the use of a dethioseco octapeptide revealed the structure carbodiimide to yield conjugates, which were of the amatoxins (98). The oxygen atom of the even more toxic than the drug itself (101). (R)-sulfoxide bridge in 6’-O-methyl-cu-amanitin As a third point for manipulations by could be removed by treatment with Raney chemical means, the phenolic hydroxyl group nickel or K3MoC16. Re-oxidation by peroxy at the indole nucleus may be mentioned. It is acids yielded besides the original (R)-sulfoxide not essential for toxicity, since amanin and the almost non-toxic (8)-diastereomer, and, amaninamide are toxic inspite of their lacking with excessive oxidant, the sulfone which the 6‘-OH group (11, 121). An inspection of exhibits almost full toxicity (99). The assump- the three-dimensional formula of 0-amanitin tion of different conformations of the consider- (Fig. 12) shows that the indole nucleus pro- ably toxic sulfone and the much less toxic (9-trudes distinctly out from the octapeptide cycle sulfoxide was disproved by X-ray structure which, apparently, forms the binding part, the analysis which revealed a surprising similarity of OH-group being the most separate entity. both the structures (96). This was confirmed by Hence, it is reasonable to conclude that modifi- H-n.m .r. spectroscopy in dimethylsulfoxide cations at the side don’t disturb binding to the solution not only for both derivatives but also enzyme. Such modifications are 1) etherifi- for the thioether and the (R)-sulfoxide. As with cation, 2) diazo-coupling, 3) iodination and 4) an analogous situation in the phallotoxin series, replacement of OH by tritium. a reason for the differing affinities to RNA polymerase I1 (or B) of (R)- and (8)-sulfoxides Ethers. More than six alkyl ethers of growing may be the differing direction of the strong chain length and a couple of ethers with long $-0 dipoles in the diasteriomieric compounds. amide spacer moieties have been prepared In the foregoing and following oxidation which all exhibit notable binding strength to experiments, the phenolic hydroxyl group sus- RNA polymerase (102). An ether prepared with ceptible to oxidative attack had to be protected Ecaproylethylenediamine with its free amino by methylation. 6‘-0-methyl-a-amanitin was group was conjugated with fluoresceine isothio- oxidized by periodate which split the -y, &-glycol cyanate; the resulting fluorescent amatoxin grouping of side chain No. 3 (100). The resulting (FAMA) allowed the visualization of amatoxin- “aldo-amanitin” proved non-toxic and had only binding substance during mitosis in cultured rat 1% of the affinity to RNA polymerase 11. Part kangaroo (PtKl) cells (103). After succinoyl- of toxicity and binding strength to the enzyme ation of the amino group the toxin by its spacer was restored by hydrogenation of the aldehyde was coupled to diverse proteins, particularly to group by NaBH, yielding a primary alcohol fetuin. With this antigen antibodies could be function (this is one method for introducing raised in rabbits by the use of which a radio-

0-Me -or-amanitin Aldo - Dc-hydrorymethyl lo-20% SCHEME 8 Toxicity: W)OH 0 v.

27 1 T. Wieland immunoassay could be developed for trace hydroxy-L-tryptophan, for the natural ama- amounts of amatoxins (104). toxins, amanin and amaninamide lack the 6-OH As phenols the amatoxins are prone to coup- group. Since the sulfoxide-oxygen may be ling with aromatic diazonium ions forming absent without diminishing the toxicity, several highly colored azocompounds. In 1940 this analogs were synthesized containing trypta- method was used to detect the phenolic nature thionine and amino acids other than y-hydroxy- of amanitin (9,and later for introducing spacer isoleucine in pos. 3. Using the “classical” thio- moieties envisioning diverse biological utiliz- ether formation via S-chlorides we prepared a ations (105). Most recently it has been extended non-toxic homoserine analog (1 10) and an iso- to several diazonium compounds and studied in leucine analog which, like amanullin, was non- detail (106). toxic but exhibited a considerable affinity to Iodination of a-amanitin occurs in 7’- RNA polymerase B (1 1 1). position, and the use of ‘’’I is a further possi- Amanullin, a component of the mushroom, bility for introducing radioactivity (107). proved non-toxic in mice but had an affinity Tritium was introduced into 0-amanitin by to RNA polymerase B comparable to that of hydrogenation of the 6’4 1-phenyl)tetrazolyl toxic amatoxins. The reasons for its ineffectivity ether. Replacement of the oxygen with hydrogen in vivo may be: 1) the rate constant of its dis- leads from the amanitin to the amanin-series sociation from the enzyme is about 10 times (108) and use of 3H-containing hydrogen greater than that of a-amanitin (1 12) and 2) allowed the synthesis of 6I-jH-amanin (109). it differs pharmacodynamically from the toxic peptides due to its more lipophilic property. Synthetic approaches A fresh impetus was given to amatoxin The total synthesis of a natural amatoxin has chemistry by adaptation of the novel indolyl not been accomplished to date. The main thioether synthesis (78,79). As with the phallo- difficulty is the tricky y-hydroxyisoleucine, toxins, the first ring can be closed advan- which, like y-hydroxyleucine in the phallotoxin tageously by an internal attack of the cysteine series, as an endo-component of a peptide gives sulfur at the Hpi-residue of appropriate octa- rise to acidolytic cleavage of the adjacent CO- peptides. Using this method, G. Zanotti synthe- NH-, and, other than with y-hydroxyleucine sized a variety of amatoxin analogs all lacking doesn’t form a trace of peptide bond with its the y-hydroxylated isoleucine building block carboxyl group but exclusively the y-lactone - (1 13, 114). Nevertheless, a comparison of their most probably because of steric hindrance. As inhibitory capacity of RNA polymerase B gave a result, it has not been possible to recyclize a much information on the role of the different seco-amatoxin. side chains for binding strength. The synthesis Less grave is the difficult accessibility of 6- of Ile3-amaninamide is indicated in Scheme 9.

_.. H CO-Gly-llr-Gly-NH-CH-CO-Asn-Hyp-lie-OtBu HI Boc cy TrtS’

ab..TFE I MA HO%I 147%)

SCHEME 9 Am-Cys-bty Synthesis of Ile3-amanin- Asn-CO- CH-NH-Gdly 112- om amide.

272 Amanita toxic peptides: Review Structure-toxicity relations Attempts have been made in several laboratories Summarizing our experiences from natural to cultivate toxic without any success. occurring, chemically modified and synthesized Amatoxins, however, occur also in fungi other amatoxins and analogs one can draw the follow- than Amanita. Some Calerina species have been ing conclusions: For strong inhibition the mol- found to contain amatoxins, and one of them, ecule must be bicyclic. Of the side chains the C. sulciceps, has been reported to grow in hydroxyl group of 4-trans-hydroxy-~-proline greenhouses (1 16). This observation may set (No. 2) is essential since proamanullin, lacking an end to the necessity of Amanita mushroom this OH, binds at least a hundred-fold weaker hunting in the forests. to the enzyme than amanullin. The chemical nature of the side chain No. 3 is decisive. For ACKNOWLEDGMENT strong binding, it must consist of four C-atoms with a methyl branch at the 0-C atom (iso- The results summarized in this review would never leucine). Methyl branching at the 7-C atom have been obtained without the excellent collaboration (leucine) reduces the affinity to about with numerous students and postdoctoral fellows. 5%, Most of them have been mentioned in the references to whereas omission of the terminal methyl of iso- this article, but the names of a few colleagues to leucine (=dine, 0-branched, methyl) has 25% whom my thanks are due for longer lasting excellent affinity. Optimal binding occurs if the isoleucine assistance should be noted here: Angeliki Buku, Heinz side chain No. 3 contains at least one hydroxyl Faulstich, Christian Birr, Eisuke Munekata and group in 7-position (yamanitin) or, as in a- Giancarlo Zanotti. The extremely valuable experi- and P-amanitin, an additional one at the 6- mental technical assistance of Annemarie Seeliger, carbon. Furthermore isoleucine in pos. 6 takes Renate Altmann and Heinrich Trischmann is grate- part in tight binding, since its replacement by fully acknowledged. My thanks are also due to the alanine in an analog increased the inhibitory pharmachemical company Boehringer Ingelheim for providing processed extracts of A. phalloides (Drs. constant for RNA polymerase I1 by about a W. Konz and W. LUttke) for many years and for thousand-fold. From these and additional data carrying out a great number of toxicological tests (109), it follows that the amatoxins owe their (Ms. A. Schmitz). fixation at the protein to hydroxyl and lipo- philic groups specifically arranged at the whole REFERENCES peptide ring, and held in the proper position by a very rigid structure. 1. Raab, H. (1932) Hoppe-Seylers Z. Physiol. Chem. 207,157-181 2. Raab, H. & Renz, J. (1933) Hoppe-Seylers Z. CONCLUSION Physiol. Chem. 216, 224-229 In the present review, the predominant aspects 3. Renz, J. (1934) Hoppe-Seylers Z. Physiol. Chem. considered have been the chemical rather than 230,245-258 the toxicological, clinical or biological; the 4. Lynen, F. & Wieland, U. (1938) Liebigs Ann. Chem. 533,93-117 story of antamanide has not been considered. A 5. Wieland, H., Hallermayer, R. & Zilg, W. (1941) more detailed treatment of these questions is Liebigs Ann. Chem. 548, 1-18 offered in the Proceedings of an International 6. Wieland, T. & Wieland, 0. (1959) Pharmacol. Symposium held at the end of 1978 (87) and in Rev. 11,87-107 a summarizing article with more than 400 7. Wieland, H. & Witkop, B. (1940) Liebigs Ann. references (1 15). However, a short botanical Chem. 543,171-183 remark shall finish this review. Phallotoxins 8. Wieland, T., Wirth, L. & Fischer, E. (1949) occur in Amanita species as A. phalloides, A. Liebigs Ann. Chem. 564,152-160 verna, A. virosa, A. bisporigera, A. tenuifolia, A. 9. Wieland, T. & Dudensing, C. (1956) Liebigs Ann. ocreata and others, compiled in reference 87. In Chem. 600,156-160 10. Wieland, T., Rempel, D., Gebert, U., Buku, A. & obtaining phallotoxins and amatoxins, which Boehringer, H. (1967) Liebigs Ann. Chem. 704, proved valuable tools in cell biology, only the 226-236 phallotoxins are accessible by synthesis; the ll.Buku, A. & Wieland, T. (1974) Liebigs Ann. others still have to be isolated from mushrooms. Chem., 1581-1595

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274 Amanita toxic peptides: Review

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