Design, Synthesis, and Biochemical Evaluation of Alpha‐Amanitin Derivatives Containing Analogs of the<i>trans</i>‐Hydroxyproline Residue for Potential Use in Antibody‐Drug Conjugates

Matinkhoo, Kaveh; Wong, Antonio A. W. L.; Hambira, Chido M.; Kato, Brandon; Wei, Charlie; Müller, Christoph; Hechler, Torsten; Braun, Alexandra; Gallo, Francesca; Pahl, Andreas; Perrin, David M.

doi:10.1002/chem.202101373

Cited by 11 publications

(6 citation statements)

References 77 publications

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“…To date, most antecedent reports have been limited to amatoxins obtained by modifying or degrading the natural product or constructing analogues from natural amino acids. A recent SAR study of hydroxyproline analogues showed significant intolerance to OH replacement by various bio-isosteres, highlighting challenges in the rational design . As we were preparing this manuscript, Süssmuth et al.…”

Section: Introductionsupporting

confidence: 91%

“…A recent SAR study of hydroxyproline analogues showed significant intoler-ance to OH replacement by various bio-isosteres, highlighting challenges in the rational design. 32 As we were preparing this manuscript, Sussmuth et al disclosed an array of 25 analogues of dideoxyamanitin (S-deoxy-amaninamide) of which none exceeds 20% of the cytotoxicity of α-amanitin or its dideoxy analogue, further underscoring the challenges in designing potent amatoxins (see Discussion and Conclusions). 33 These recent reports, together with a vast literature spanning more than 5 decades, underscore the implasticity of α-amanitin, which is consistent with a highly rigid structure as well as with the extensive noncovalent interactions within Pol II.…”

Section: ■ Introductionmentioning

confidence: 89%

“…The majority of this work is adapted from previous protocols established in this lab ,, and detailed further below.…”

Section: Methodsmentioning

confidence: 99%

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Rationally Designed Amanitins Achieve Enhanced Cytotoxicity

et al. 2022

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For 70 years, α-amanitin, the most cytotoxic peptide in its class, has been without a synthetic rival; through synthesis, we address the structure–activity relationships to inform the design of new amatoxins and disclose analogues that are more cytotoxic than the natural product when evaluated on CHO, HEK293, and HeLa cells, whereas on liver-derived HepG2 cells, the same toxins show diminished cytotoxicity.

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Section: Introductionsupporting

confidence: 91%

Section: ■ Introductionmentioning

confidence: 89%

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Rationally Designed Amanitins Achieve Enhanced Cytotoxicity

et al. 2022

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“…For NCL with an N-terminal mercaptoproline, the 4 R stereochemistry at proline is required for proline NCL to proceed, due to a lack of functional group proximity that prevents S → N acyl transfer with 4 S -mercaptoproline. ,, 4 R -Mercaptoproline has been applied in NCL reactions to synthesize cyclic peptides; a polyglycosylated 88-amino acid fragment of erythropoietin; and O -GlcNAcylated heat shock proteins, in work that demonstrated that O -GlcNAcylation inhibits amyloidosis of Aβ and α-synuclein. − Mercaptoproline stereoisomers have also been incorporated in conformationally constrained cyclic peptides that function as protein inhibitors or as α-helical capping motifs, as well as in other applications in medicinal chemistry, polymers, and materials science. − …”

Section: Introductionmentioning

confidence: 99%

N-Terminal Proline Editing for the Synthesis of Peptides with Mercaptoproline and Selenoproline: Mechanistic Insights Lead to Greater Efficiency in Proline Native Chemical Ligation

Ludwig,

Forbes,

Zondlo

2024

ACS Chem. Biol.

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Native chemical ligation (NCL) at proline has been limited by cost and synthetic access. In addition, prior examples of NCL using mercaptoproline have exhibited stalling of the reaction after thioester exchange, due to inefficient S → N acyl transfer. Herein, we develop methods, using inexpensive Boc-4R-hydroxyproline, for the solid-phase synthesis of peptides containing Nterminal 4R-mercaptoproline and 4R-selenoproline. The synthesis proceeds via proline editing on the N-terminus of fully synthesized peptides on the solid phase, converting an N-terminal Boc-4R-hydroxyproline to the 4S-bromoproline, followed by an S N 2 reaction with potassium thioacetate or selenobenzoic acid. After cleavage from the resin and deprotection, peptides with functionalized N-terminal proline amino acids were obtained. NCL reactions with mercaptoproline proceeded slowly under standard NCL conditions, with the S-acyl transthioesterification intermediate observed as a major species. Computational investigations indicated that the bicyclic intermediates and transition states for S → N acyl transfer are sufficiently low in energy (10−15 kcal mol −1 above starting material) that ring strain cannot explain the slow S → N acyl transfer. Instead, the bicyclic zwitterionic tetrahedral intermediate has a low barrier for reversion to the S-acyl intermediate, causing reversion to the thioester (reverse reaction) to occur preferentially over elimination to generate the amide (forward reaction). We hypothesized that a buffer capable of general acid and/ or general base catalysis could promote S → N acyl transfer and thus achieve greater efficiency in proline NCL. In the presence of 2 M imidazole at pH 6.8, NCL with mercaptoproline proceeded efficiently to generate the peptide with a native amide bond. NCL with selenoproline also proceeded efficiently to generate the desired products when a thiophenol thioester was employed as a ligation partner. After desulfurization or deselenization, the products obtained were identical to those synthesized directly, confirming that the solid-phase proline editing reactions proceeded stereospecifically and without epimerization.

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“…Even though amatoxins are very promising payloads for antibody-drug conjugates, 16,18 they could not be used in cancer research until recently, because their synthesis is extremely challenging. In mushrooms, amatoxins are ribosomally synthesised, 4,19 but obtaining amatoxins in sufficient quantity and quality from mushroom farms proved to be impossible.…”

Section: Introductionmentioning

confidence: 99%

The influence of N-methylation on the ansamers of an amatoxin: Gly5Sar-amanullin

Wenz

Kosol

Yao

et al. 2022

Preprint

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Amatoxins are strong inhibitors of RNA polymerase II, and cause cell death. Because of their cytotoxicity, they are candidates for anti-cancer drugs, and understanding their structure-activity relationship is crucial. Amatoxins have a rigid bicyclic scaffold which consists of a cyclic octapeptide bridged by cysteine and tryptophan side chain forming a tryptathionine bridge. Here, we show the influence of the N-methylation on the amatoxin scaffold by studying Gly5Sar-amanullin with MD simulations and NMR experiments. Since we have shown recently that the amatoxin scaffold allows for two isomeric forms (ansamers), we studied both isomers of Gly5Sar-amanullin. We found that both isomers of Gly5Sar-amanullin form two long-living conformations, which is unusual for amatoxins, and that they are differently affected by the N-methylation. The natural Gly5Sar-amanullin forfeits the hydrogen bonds to Gly5 due to the N-methylation, which is expected from existing crystal structures for alpha-amanitin. Our results however indicate that this does not cause more flexibility due to a shift in the hydrogen bond pattern. In the unnatural isomer, we observe an interesting cis-trans-isomerisation of the backbone angles in Trp4 and Gly7, which is enabled by the N-methylation. We expect that our perspective on the effect of N-methylation in amatoxins could be a starting point for further SAR-studies which are urgently needed for the design of better anti-cancer agents.

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Design, Synthesis, and Biochemical Evaluation of Alpha‐Amanitin Derivatives Containing Analogs of thetrans‐Hydroxyproline Residue for Potential Use in Antibody‐Drug Conjugates

Cited by 11 publications

References 77 publications

Rationally Designed Amanitins Achieve Enhanced Cytotoxicity

Rationally Designed Amanitins Achieve Enhanced Cytotoxicity

N-Terminal Proline Editing for the Synthesis of Peptides with Mercaptoproline and Selenoproline: Mechanistic Insights Lead to Greater Efficiency in Proline Native Chemical Ligation

The influence of N-methylation on the ansamers of an amatoxin: Gly5Sar-amanullin

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