Glutathione Mediates Control of Dual Differential Bio‐orthogonal Labelling of Biomolecules

Peschke, Frederik; Taladriz‐Sender, Andrea; Andrews, Matthew J.; Watson, Allan J. B.; Burley, Glenn A.

doi:10.1002/anie.202313063

Cited by 5 publications

(1 citation statement)

References 84 publications

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“…The CuAAC ligation between 19 and 21 proceeded cleanly in a mixture of pH 8 HEPES buffer and hexafluoroisopropanol 64 in presence of a catalytic system composed of CuSO4, the Cu(I) ligand THPTA, and sodium ascorbate as a copper(II) ion reductant. 65 Incubation at 37 °C during 4 h resulted in the complete conversion of 19 into the corresponding bis-Hmb SUMOylated peptide 22.…”

Section: Resultsmentioning

confidence: 99%

A straightforward method to prevent the under-estimated problem of aspartimide formation during chemical ligation-mediated protein synthesis

Cisse,

Aucagne

2024

Preprint

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Formation of a five-membered ring aspartimide through the attack of a backbone amide to the side chain of aspartate and asparagine residues is a long-known side-reaction in solid phase peptide synthesis, and is also associated with in vivo protein ageing and instability of purified proteins. Conversely, its possible occurrence during chemical ligation-based protein synthesis, in particular when using the gold-standard reaction NCL (native chemical ligation), is dubious. We herein report a systematic study which demonstrates that the prevalence of this side-reaction may have been overlooked, due to the difficulty to identify it through standard HPLC analytical methods, but also the in situ conversion of aspar-timide into other byproducts, having the same molecular mass as the parent aspartate residue. We show that the for-mation of aspartimide and related byproducts can be limited by adopting "good NCL practices", which involve restricting the ligation temperature and reaction times, as well as replacing the commonly used phosphate buffer with HEPES. How-ever, the efficiency of such precautions is expected to vary considerably depending on the sequence of the target protein, and the amount of byproducts is expected to grow with the length of the target protein, as a result of the number of NCL reactions and potential aspartimide hotspots. To overcome such limitations, we developed a novel straightforward and potentially generally applicable methodology based on the temporary protection of the backbone nitrogen by a 2-(4-aminobutanoyloxy)-4-methoxybenzyl (GABA-Hmb) group. This strategy was validated by the byproduct-free synthesis of SUMO-2 and a SUMOylated peptide mimic.

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Section: Resultsmentioning

confidence: 99%

A straightforward method to prevent the under-estimated problem of aspartimide formation during chemical ligation-mediated protein synthesis

Cisse,

Aucagne

2024

Preprint

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Microneedle‐Loaded Gene‐Editable Nanohybrids Engineer Cancer Cells by Telomere Stress Induction and Metabolic Interference for Enhanced Cuproptosis and Immunotherapy

Luan,

Du,

Liu

et al. 2024

Adv Funct Materials

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Cuproptosis, a novel copper‐driven cell death, offers potential for cancer therapy, yet challenges persist in targeted delivery of copper ions and therapeutic resistance management. Here, CuTG‐Cas9@PLL, a cuproptosis nano‐inducer for efficient delivery of Cu ions, 6‐Thio‐dG (6‐thio‐2′‐deoxyguanosine), and CRISPR/Cas9 to enhance cuproptosis and immunotherapy via telomere stress and metabolic interference is developed. This system, combined with microneedle patches for transdermal delivery in melanoma treatment, targets the “Warburg effect,” a key resistance mechanism, through CRISPR/Cas9‐mediated LDHA knockout, thereby sensitizing cancer cells to cuproptosis and reversing immune suppression. Moreover, 6‐Thio‐dG‐induced telomere stress not only amplifies cuproptosis via autophagy dysfunction but also boosts anti‐tumor immunity by increased immunogenicity of senescent cancer cells. This work presents a novel approach for developing efficient cuproptosis nano‐inducers and reports on the feasibility of enhancing cancer cuproptosis and anti‐tumor immunotherapy through telomere stress induction and CRISPR/Cas9‐mediated metabolic interference.

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Reactivity Profiling for High-Yielding Ynamine-Tagged Oligonucleotide Click Chemistry Bioconjugations

Peschke,

Taladriz-Sender,

Watson

et al. 2024

Bioconjugate Chem.

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The Cu-catalyzed azide–alkyne cycloaddition (CuAAC) reaction is a key ligation tool used to prepare bioconjugates. Despite the widespread utility of CuAAC to produce discrete 1,4-triazole products, the requirement of a Cu catalyst can result in oxidative damage to these products. Ynamines are superior reactive groups in CuAAC reactions and require lower Cu loadings to produce 1,4-triazole products. This study discloses a strategy to identify optimal reaction conditions for the formation of oligodeoxyribonucleotide (ODN) bioconjugates. First, the surveying of reaction conditions identified that the ratio of Cu to the choice of reductant (i.e., either sodium ascorbate or glutathione) influences the reaction kinetics and the rate of degradation of bioconjugate products. Second, optimized conditions were used to prepare a variety of ODN-tagged products and ODN-protein conjugates and compared to conventional CuAAC and Cu-free azide–alkyne (3 + 2)cycloadditions (SPAAC), with ynamine-based examples being faster in all cases. The reaction optimization platform established in this study provides the basis for its wider utility to prepare CuAAC-based bioconjugates with lower Cu loadings while maintaining fast reaction kinetics.

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Glutathione Mediates Control of Dual Differential Bio‐orthogonal Labelling of Biomolecules

Cited by 5 publications

References 84 publications

A straightforward method to prevent the under-estimated problem of aspartimide formation during chemical ligation-mediated protein synthesis

A straightforward method to prevent the under-estimated problem of aspartimide formation during chemical ligation-mediated protein synthesis

Microneedle‐Loaded Gene‐Editable Nanohybrids Engineer Cancer Cells by Telomere Stress Induction and Metabolic Interference for Enhanced Cuproptosis and Immunotherapy

Reactivity Profiling for High-Yielding Ynamine-Tagged Oligonucleotide Click Chemistry Bioconjugations

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