A facile preparation of functional cycloalkynes <i>via</i> an azide-to-cycloalkyne switching approach

Yoshida, Suguru; Kuribara, Tomoko; Ito, Hiromitsu; Meguro, Tomohiro; Nishiyama, Yoshitake; Karaki, Fumika; Hatakeyama, Yasutomo; Koike, Y.; Kii, Isao; Hosoya, Takamitsu

doi:10.1039/c9cc01113g

“…We then fluorescently-labelled azide-JVZ007 via a methodology that utilised both a copper-catalysed Huisgen cycloaddition reaction between an azide and a terminal alkyne and a copper-free strain-promoted azide-alkyne Huisgen cycloaddition between an azide and a dibenzocyclooctyne (DBCO) motif (SI, Figure S31). 51 As expected, SDS-PAGE gel analysis demonstrated only azide-JVZ007 was fluorescently labelled, whereas α-oxo aldehyde JVZ007 remained unlabelled (Figure 5).…”

Section: Resultssupporting

confidence: 74%

Catalyst-free site-selective cross-aldol bioconjugations

Yates

¹

,

Akkad

²

,

Noble

³

et al. 2022

Preprint

View full text Add to dashboard Cite

The bioconjugation of proteins to small molecules serves as an invaluable tool for probing biological mechanisms and creating biomaterials. The most powerful bioconjugation stratagems are those which have rapid kinetics, are site-selective, can be conducted in aqueous solvent under mild conditions and do not require the use of additional potentially toxic catalysts. Herein we present spontaneous coupling via aldol ligation of proteins (SCALP) a catalyst-free “green” site-selective protein ligation between α-oxo aldehyde functionalised proteins and enolisable aldehyde probes at neutral pH, and demonstrate the utility of this system in the targeting of prostate cancer cells with functionalised nanobodies.

show abstract

“…Liberation of copper from the DBCO peptides could be accomplished with aqueous ammonia, EDTA, or metal scavengers, as reported by the Hosoya group. [88][89] As other metal salts have been demonstrated to complex with cycloalkyne rings, 90,106 these may also be able to protect against acid-mediated degradation.…”

Section: Discussionmentioning

confidence: 99%

“…The Hosoya group previously reported that a copper(I) salt, tetrakis(acetonitrile)copper(I) tetrafluoroborate or (MeCN)4CuBF4, could reversibly protect cyclooctynes from reacting with azides. [88][89] This strategy builds on the finding that various metals can form complexes with cycloalkyne rings, 90 which led us to examine copper as an additive to prevent the acid-mediated 5-endo-dig cycloisomerization of DBCO. Guided by the methodology reported by the Hosoya group, initial attempts at protecting peptides 1a and 2a were performed by treating peptide resins with ~3 equiv.…”

Section: Synthesis Of Dbco Peptides Via Fmoc-sppsmentioning

confidence: 99%

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Erickson¹,

Fulcher²,

Kay

³

2020

Preprint

View full text Add to dashboard Cite

<div><div><div><p>Chemoselective ligation reactions, such as native chemical ligation (NCL), enable the assembly of synthetic peptides into proteins. However, the scope of proteins accessible to total chemical synthesis is limited by ligation efficiency. Sterically hindered thioesters and poorly soluble peptides can undergo incomplete ligations, leading to challenging purifications with low yields. This work describes a new method, ClickAssisted NCL (CAN), which overcomes these barriers. In CAN, peptides are modified with traceless “helping hand” lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles for strain-promoted alkyne-azide cycloaddition (SPAAC) reactions. This cycloaddition templates the peptides to increase their effective concentration and greatly accelerate ligation kinetics. After ligation, mild hydroxylamine treatment tracelessly removes the linkers to afford the native ligated peptide. Although DBCO is incompatible with standard Fmoc solid-phase peptide synthesis (SPPS) due to an acid-mediated rearrangement that occurs during peptide cleavage, we demonstrate that copper(I) protects DBCO from this side reaction, enabling direct production of DBCO-containing synthetic peptides. Excitingly, low concentrations of triazole-linked model peptides reacted ~1,200-fold faster than predicted for non-templated control ligations, which also accumulated many side products due to the long reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that the SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in a one-pot fashion. CAN will be useful for overcoming ligation challenges to expand the reach of chemical protein synthesis.</p></div></div></div>

show abstract

“…The Hosoya group previously reported that a copper(I) salt, tetrakis(acetonitrile)copper(I) tetrafluoroborate or (MeCN)4CuBF4, could reversibly protect cyclooctynes from reacting with azides. [88][89] This strategy builds on the finding that various metals can form complexes with cycloalkyne rings, 90 To address this issue, we next tested the direct addition of ~1.5 equiv.…”

Section: Synthesis Of Dbco Peptides Via Fmoc-sppsmentioning

confidence: 99%

“…Liberation of copper from the DBCO peptides could be accomplished with aqueous ammonia, EDTA, or metal scavengers, as reported by the Hosoya group. [88][89] As other metal salts have been demonstrated to complex with cycloalkyne rings, 90,106 these may also be able to protect against acid-mediated degradation.…”

Section: Click-assisted Ncl Of the E Coli 50s Ribosomal Subunit L32mentioning

confidence: 99%

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Erickson¹,

Fulcher²,

Kay³

2020

Preprint

View full text Add to dashboard Cite

<div><div><div><p>Chemoselective ligation reactions, such as native chemical ligation (NCL), enable the assembly of synthetic peptides into proteins. However, the scope of proteins accessible to total chemical synthesis is limited by ligation efficiency. Sterically hindered thioesters and poorly soluble peptides can undergo incomplete ligations, leading to challenging purifications with low yields. This work describes a new method, ClickAssisted NCL (CAN), which overcomes these barriers. In CAN, peptides are modified with traceless “helping hand” lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles for strain-promoted alkyne-azide cycloaddition (SPAAC) reactions. This cycloaddition templates the peptides to increase their effective concentration and greatly accelerate ligation kinetics. After ligation, mild hydroxylamine treatment tracelessly removes the linkers to afford the native ligated peptide. Although DBCO is incompatible with standard Fmoc solid-phase peptide synthesis (SPPS) due to an acid-mediated rearrangement that occurs during peptide cleavage, we demonstrate that copper(I) protects DBCO from this side reaction, enabling direct production of DBCO-containing synthetic peptides. Excitingly, low concentrations of triazole-linked model peptides reacted ~1,200-fold faster than predicted for non-templated control ligations, which also accumulated many side products due to the long reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that the SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in a one-pot fashion. CAN will be useful for overcoming ligation challenges to expand the reach of chemical protein synthesis.</p></div></div></div>

show abstract

A facile preparation of functional cycloalkynes via an azide-to-cycloalkyne switching approach

Cited by 23 publications

References 60 publications

Catalyst-free site-selective cross-aldol bioconjugations

Catalyst-free site-selective cross-aldol bioconjugations

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Contact Info

Product

Resources

About