title: Synthesis of a Cytotoxic Amanitin for Biorthogonal Conjugation.
abstract: Alpha-amanitin is an exceedingly toxic, naturally occurring, bicyclic octapeptide that inhibits RNA polymerase and results in cellular and organismal death. Here we report the straightforward synthesis of an amanitin analogue that exhibited near-native toxicity. A pendant alkyne was readily installed to enable copper-catalyzed alkyne-azide cycloaddition (CuAAC) to azido-rhodamine and two azide-bearing versions of the RGD peptide. The fluorescent toxin analogue entered cells and provoked morphological changes consistent with cell death. The latter two conjugates are as toxic as the parent alkyne precursor, which demonstrates that conjugation does not diminish toxicity. In addition, we showed that toxicity depends on a single diastereomer of the unnatural amino acid, dihydroxyisoleucine (DHIle), at position 3. The convenient synthesis of a heptapeptide precursor now provides access to bioactive amanitin analogues that may be readily conjugated to biomolecules of interest.
authors.affiliation: Chemistry Department, UBC, 2036 Main Mall, Vancouver, B.C. V6T 1Z1 (Canada).
authors.forename: Liang
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Chemistry Department, UBC, 2036 Main Mall, Vancouver, B.C. V6T 1Z1 (Canada).
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: May
authors.affiliation: Chemistry Department, UBC, 2036 Main Mall, Vancouver, B.C. V6T 1Z1 (Canada).
authors.forename: Antoine
authors.initials: A
authors.lastname: Blanc
authors.affiliation: Chemistry Department, UBC, 2036 Main Mall, Vancouver, B.C. V6T 1Z1 (Canada).
authors.forename: David J
authors.initials: DJ
authors.lastname: Dietrich
authors.affiliation: Chemistry Department, UBC, 2036 Main Mall, Vancouver, B.C. V6T 1Z1 (Canada).
authors.forename: Anastak
authors.initials: A
authors.lastname: Loonchanta
authors.affiliation: Chemistry Department, UBC, 2036 Main Mall, Vancouver, B.C. V6T 1Z1 (Canada).
authors.forename: Kaveh
authors.initials: K
authors.lastname: Matinkhoo
authors.affiliation: Chemistry Department, UBC, 2036 Main Mall, Vancouver, B.C. V6T 1Z1 (Canada).
authors.forename: Alla
authors.initials: A
authors.lastname: Pryyma
authors.affiliation: Chemistry Department, UBC, 2036 Main Mall, Vancouver, B.C. V6T 1Z1 (Canada). dperrin@chem.ubc.ca.
authors.forename: David M
authors.initials: DM
authors.lastname: Perrin
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D000546:Amanitins / Q000138:chemical synthesis* / Q000737:chemistry / Q000633:toxicity; D000818:Animals; D001386:Azides / Q000737:chemistry*; D016466:CHO Cells; D045744:Cell Line, Tumor; D057930:Click Chemistry / Q000379:methods; D003412:Cricetulus; D061565:Cycloaddition Reaction; D003603:Cytotoxins / Q000138:chemical synthesis* / Q000737:chemistry / Q000633:toxicity; D006367:HeLa Cells; D006801:Humans; D009842:Oligopeptides / Q000737:chemistry; D010455:Peptides; D011042:Poisons / Q000138:chemical synthesis / Q000737:chemistry / Q000633:toxicity; D012235:Rhodamines / Q000737:chemistry
chemical_list: D000480:Alkynes; D000546:Amanitins; D001386:Azides; D003603:Cytotoxins; D009842:Oligopeptides; D010455:Peptides; D011042:Poisons; D012235:Rhodamines; C047981:arginyl-glycyl-aspartic acid
keywords: amanitin; bioconjugates; click chemistry; natural products; peptides