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Protein modification by strain-promoted alkyne-nitrone cycloaddition.

Ning X, Temming RP, Dommerholt J, Guo J, Ania DB, Debets MF, Wolfert MA, Boons GJ, van Delft FL - Angew. Chem. Int. Ed. Engl. (2010)

View Article: PubMed Central - PubMed

Affiliation: Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA.

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The bioorthogonal chemical reporter strategy is emerging as a versatile method for the labeling of biomolecules, such as nucleic acids, lipids, carbohydrates, and proteins... For example, reactions of difluorinated cyclooctynes, such as (Figure 1), with azides proceed approximately 60 times faster than the corresponding reactions of unsubstituted derivatives... We have found that derivatives of the 4-dibenzocyclooctynol react fast with azido-containing saccharides and amino acids and can be employed for the visualization of metabolically labeled glycans of living cells... We report herein a novel bioorthogonal reaction pair based on strain-promoted alkyne–nitrone cycloaddition (SPANC) to give N-alkylated isoxazolines with exceptionally fast reaction kinetics... Cycloaddition reactions of – with cyclooctynol in a mixture of acetonitrile and water gave the corresponding stable isoxazolines, in most cases in high yield (Table 1)... We measured the rate constants of the cycloaddition reactions by H NMR or UV spectroscopy at 25 °C and found that the substituents on the nitrone greatly influenced the reaction kinetics... Exceptionally high reaction rates were measured for the cycloaddition of with α-carboxynitrones and... These reactions proceeded 18 and 32 times as fast, respectively, as the cycloaddition of with benzyl azide (0.12 m s)... Also, we found that a high water content increased the reaction rate constants (e.g. 12.8 m s for a derivative of in acetonitrile/water (1:9); see the Supporting Information)... Thus, the N-terminal serine residue of model peptide was oxidized with sodium periodate (1.1 equiv) to rapidly generate aldehyde, which was first treated with p-methoxybenzenethiol (6.6 equiv, 30 min), and then with N-methylhydroxylamine (2.2 equiv), p-anisidine (5 equiv), and (2.2 equiv) to give the desired isoxazoline via nitrone (Scheme 1)... We found that treatment with p-MeOC6H4SH was essential to avoid the conversion of N-methylhydroxylamine into nitrosomethane dimer ((MeNO)2) by oxidation with iodate (IO3) formed in the previous step... We have shown that 1,3-dipolar cycloadditions of cyclooctynes with nitrones that contain ester or amide α substituents exhibit much faster kinetics than similar reactions with azides... Besides serine or threonine oxidation, a variety of methods have been described for the installment of carbonyl groups in proteins, and it is to be expected that SPANC is compatible with these approaches... Furthermore, metal-free click reactions have found entry into materials science... Finally, we anticipate that SPANC will offer an attractive alternative to the well-established oxime ligation because the synthesis of nitrones is simple, the isoxazoline products are stable, and the combination of a functionalized nitrone (R is a functional group, Table 1) with a cyclooctyne conjugate (such as ) will make it possible to introduce two different functionalities in a single process.

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Ring-strained cyclooctynes for bioorthogonal cycloaddition reactions with azides.
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fig01: Ring-strained cyclooctynes for bioorthogonal cycloaddition reactions with azides.

Mentions: The first generation of cyclooctynes suffered from relatively slow reaction rates; however, it has been found that the rate of strain-promoted cycloaddition can be increased by appending electron-withdrawing groups adjacent to the triple bond. For example, reactions of difluorinated cyclooctynes, such as 1 (Figure 1), with azides proceed approximately 60 times faster than the corresponding reactions of unsubstituted derivatives.5a We have found that derivatives of the 4-dibenzocyclooctynol 2 react fast with azido-containing saccharides and amino acids and can be employed for the visualization of metabolically labeled glycans of living cells.12 Attractive features of dibenzocyclooctynols include easy synthetic access, nontoxicity, and the straightforward attachment of a variety of probes. Recently, we introduced the more polar azacyclooctyne 3,13 which exhibits a higher rate of reaction. Despite these advances, there is an urgent need for new and faster bioorthogonal reactions for labeling at low concentration.1b


Protein modification by strain-promoted alkyne-nitrone cycloaddition.

Ning X, Temming RP, Dommerholt J, Guo J, Ania DB, Debets MF, Wolfert MA, Boons GJ, van Delft FL - Angew. Chem. Int. Ed. Engl. (2010)

Ring-strained cyclooctynes for bioorthogonal cycloaddition reactions with azides.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2871956&req=5

fig01: Ring-strained cyclooctynes for bioorthogonal cycloaddition reactions with azides.
Mentions: The first generation of cyclooctynes suffered from relatively slow reaction rates; however, it has been found that the rate of strain-promoted cycloaddition can be increased by appending electron-withdrawing groups adjacent to the triple bond. For example, reactions of difluorinated cyclooctynes, such as 1 (Figure 1), with azides proceed approximately 60 times faster than the corresponding reactions of unsubstituted derivatives.5a We have found that derivatives of the 4-dibenzocyclooctynol 2 react fast with azido-containing saccharides and amino acids and can be employed for the visualization of metabolically labeled glycans of living cells.12 Attractive features of dibenzocyclooctynols include easy synthetic access, nontoxicity, and the straightforward attachment of a variety of probes. Recently, we introduced the more polar azacyclooctyne 3,13 which exhibits a higher rate of reaction. Despite these advances, there is an urgent need for new and faster bioorthogonal reactions for labeling at low concentration.1b

View Article: PubMed Central - PubMed

Affiliation: Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

The bioorthogonal chemical reporter strategy is emerging as a versatile method for the labeling of biomolecules, such as nucleic acids, lipids, carbohydrates, and proteins... For example, reactions of difluorinated cyclooctynes, such as (Figure 1), with azides proceed approximately 60 times faster than the corresponding reactions of unsubstituted derivatives... We have found that derivatives of the 4-dibenzocyclooctynol react fast with azido-containing saccharides and amino acids and can be employed for the visualization of metabolically labeled glycans of living cells... We report herein a novel bioorthogonal reaction pair based on strain-promoted alkyne–nitrone cycloaddition (SPANC) to give N-alkylated isoxazolines with exceptionally fast reaction kinetics... Cycloaddition reactions of – with cyclooctynol in a mixture of acetonitrile and water gave the corresponding stable isoxazolines, in most cases in high yield (Table 1)... We measured the rate constants of the cycloaddition reactions by H NMR or UV spectroscopy at 25 °C and found that the substituents on the nitrone greatly influenced the reaction kinetics... Exceptionally high reaction rates were measured for the cycloaddition of with α-carboxynitrones and... These reactions proceeded 18 and 32 times as fast, respectively, as the cycloaddition of with benzyl azide (0.12 m s)... Also, we found that a high water content increased the reaction rate constants (e.g. 12.8 m s for a derivative of in acetonitrile/water (1:9); see the Supporting Information)... Thus, the N-terminal serine residue of model peptide was oxidized with sodium periodate (1.1 equiv) to rapidly generate aldehyde, which was first treated with p-methoxybenzenethiol (6.6 equiv, 30 min), and then with N-methylhydroxylamine (2.2 equiv), p-anisidine (5 equiv), and (2.2 equiv) to give the desired isoxazoline via nitrone (Scheme 1)... We found that treatment with p-MeOC6H4SH was essential to avoid the conversion of N-methylhydroxylamine into nitrosomethane dimer ((MeNO)2) by oxidation with iodate (IO3) formed in the previous step... We have shown that 1,3-dipolar cycloadditions of cyclooctynes with nitrones that contain ester or amide α substituents exhibit much faster kinetics than similar reactions with azides... Besides serine or threonine oxidation, a variety of methods have been described for the installment of carbonyl groups in proteins, and it is to be expected that SPANC is compatible with these approaches... Furthermore, metal-free click reactions have found entry into materials science... Finally, we anticipate that SPANC will offer an attractive alternative to the well-established oxime ligation because the synthesis of nitrones is simple, the isoxazoline products are stable, and the combination of a functionalized nitrone (R is a functional group, Table 1) with a cyclooctyne conjugate (such as ) will make it possible to introduce two different functionalities in a single process.

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