A spontaneous gold(i)-azide alkyne cycloaddition reaction yields gold-peptide bioconjugates which overcome cisplatin resistance in a p53-mutant cancer cell line

Abstract

Solid-phase peptide synthesis (SPPS) is a versatile technique for the assembly of small to medium size peptides, that can help in the delivery of bound metal complexes to certain cellular compartments, for example in cancer cells. This work shows a new route to gold-peptide bioconjugates via a non-catalyzed [3 + 2] cycloaddition reaction of gold azides with alkynyl peptides. Gold(I) tetrapeptide conjugates with a mitochondria-targeting sequence were synthesized and display prolonged stability in the presence of thiol-containing biological media. Their antiproliferative potency against selected cancer cells (2-50 mM) corresponds to the lipophilicity of the conjugates. The cellular uptake of Au, determined by atomic absorption spectroscopy (AAS), shows that high initial uptake equals strong cytotoxicity. Respiration and acidification rates react immediately upon treatment with the Au-peptide conjugates, and a terminal breakdown of essential cellular functions is complete within ca. 12 h at most, as observed by online monitoring of the cancer cell metabolism in a microfluidic biosensor device (Bionas sensorchip system). The mode of action of these Au-peptide bioconjugates was elucidated by a variety of biochemical and cell biological experiments. First, a strong selective inhibition of the enzyme thioredoxin reductase (TrxR), a regulator of cellular redox processes, was found. In this context, elevated levels of reactive oxygen species (ROS) and strong effects on the respiration of isolated mouse liver mitochondria were found. These finally lead to cell death via apoptotic pathways, as indicated by flow cytometry, low mitochondrial membrane potential (MMP) and DNA fragmentation. Intriguingly, cisplatin-resistance in p53-mutant MDA-MB231 breast cancer cells could be overcome by the Au-peptide conjugates presented herein.Scheme 2 Fmoc solid-phase peptide synthesis of (phosphine)gold(I) tetrapeptide triazoles 8a-c. Cleavage was effected with TFA-phenol-TIS (75 : 20 : 5) for 1 h in a Schlenk flask under nitrogen. Phenol was added to avoid oxidation of the gold(I) centre under the strongly acidic conditions. Synthesis of (phosphine)gold(I) dipeptides 6a-cTo a flame-dried Schlenk flask 1a (51 mg, 0.1 mmol), alternatively 1b (45 mg, 0.11 mmol) or 1c (43 mg, 0.12 mmol) were added under nitrogen flow together with 1.4 molar equivalents of 3. The mixture was suspended in 10 ml absolute THF and stirred for 4 d under a nitrogen atmosphere protected from light. After completion of the reaction, as indicated by TLC, the solution was evaporated to dryness. The crude product was purified by column chromatography on silica (0.062-0.2 mm) using petroleum ether-ethyl acetate mixtures.

Au-iClick mirrors the mechanism of copper catalyzed azide–alkyne cycloaddition (CuAAC)

“…[2][3][4] Others [5][6][7] extended iClick to include the already prevalent [8][9] cycloaddition of an organic substrate to either a metal-azide or metal-acetylide. 7,[10][11][12][13][14][15][16][17][18][19][20][21] Important to this work, Gray et al 22 demonstrated that gold-acetylides and gold-azides will undergo cycloaddition to their organic counterparts. Despite the fact that several transition metals other than Cu(I) catalyze the azide-alkyne cycloaddition, including ruthenium, silver, and iridium, [23][24][25][26] it became apparent that not all metal-acetylide/azide species will participate in the iClick cycloaddition.…”

Azide Chemistry – An Inorganic Perspective, Part II[‡] [3+2]‐Cycloaddition Reactions of Metal Azides and Related Systems

“…[447,449] Gold-peptide bioconjugates overcome cisplatin resistance in a cancer cell line. [449] An allylpalladium azide which undergoes 1,3-dipolar cycloaddition with alkynes is proposed as key intermediate in the www.zaac.wiley-vch.de [458] three component-coupling reaction of activated alkynes, allyl-methyl carbonate and trimethylsilyl azide for the regiospecific synthesis of 2-allyl-1,2,3-triazoles. [330,444] Alkylation (at N 3 ) and subsequent deprotonation (at C 5 -H) of 1,2,3-triazoles lead to "abnormal" or mesoionic carbenes (aNHC) with excellent ligand properties.…”

“…[427] Recently, a series of interesting gold complexes with carbon-bound triazolate ligands has been obtained by Gray and by Metzler-Nolte and co-workers from reactions of phosphinegold(I) azides with terminal organic alkynes (Equation (13)). [446][447][448][449] In the place of phosphine ligands, also N-heterocyclic carbenes have been used, and a series of functionalized alkynes has been introduced in these reactions. [448] Until now the mechanism of formation of the C-bound triazolate has not been established.…”

Transition metal bioconjugates with an organometallic link between the metal and the biomolecular scaffold

“…Reaction with a (phosphine)gold azide complex induces a [2+3] cycloaddition and affords a triazolyl gold complex covalently attached to the peptide scaffold (Scheme 11) [88]. Dissociation of the organogold peptide conjugate from the solid support yields a gold oligopeptide conjugate with activity against some cisplatin-resistant breast cancer cell lines.…”

“…Dissociation of the organogold peptide conjugate from the solid support yields a gold oligopeptide conjugate with activity against some cisplatin-resistant breast cancer cell lines. Scheme 11 Synthesis of a tetrapeptide triazolyl (phosphine)gold(I) complex with antitumor activity [88].…”