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

Abstract

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.

Improved synthesis and purification of methylated amanitins using diazomethane*

“…In contrast, we have been unable to selectively and quantitatively achieve monoO-alkylation using the method of Faulstich et al (12), since unaltered AMA, N-alkyl-AMA, and N,O-dialkyl-AMA are recovered from the reaction as well (unpublished); these results appear to be consonant with those of Faulstich e l al. Our modification of the original method of Wieland & Fahrmeir (8) can quantitatively yield meAMA, and the conditions can be changed if dimeAMA is desired.…”

“…Positions A, B, and C correspond to AMA, meAMA and dimeAMA, respectively. In addition, the dimeAMA shows a bathochromic shift of approximately 3 nm, relative to meAMA, suggesting an N-methyl substitution previously observed for dimeAMA (12). 3.…”

“…derivative (12). This reaction has proven effective for the direct introduction of tritium via C3H31, although the specific radioactivities that may be obtained with this approach are lower than those that may routinely be obtained with the NaB3H4 reduction of the aldoamanitin, and the yields of the meAMA via reaction with CH3I appear to be limited to 30% (12).…”

Production of 15N-labeled α-amanitin in Galerina marginata

“…To the best of our knowledge, α-amanitin has never been chemically synthesized, which thwarts this route to producing α-amanitin labeled with either radioactive or stable isotopes. Chemical radiolabeling with 125 I led to a chemically distinct compound with reduced activity (Morris et al., 1978; Faulstich et al, 1981). In vivo labeling is hindered by the difficulty of culturing most species of Amanita .…”

Sensitivity of Carrot Cell Cultures and RNA Polymerase II to Amatoxins

“…Data from Figure 3 relating the 50% inhibition doses for suspension cells given by AMA, meAMA, and deAMA are tabulated (Table II) Drosophila embryos seen with derivatives of AMA modified to be more lipophilic (14). Given the above, the use of meAMA or deAMA for mutant selection or RNA synthesis studies would be better choices than AMA itself.…”