Development and evaluation of new cyclooctynes for cell surface glycan imaging in cancer cells

Stöckmann, Henning; Neves, André; Stairs, Shaun; Ireland-Zecchini, Heather; Brindle, Kevin M.; Leeper, Finian J.

doi:10.1039/c0sc00631a

Cited by 76 publications

(67 citation statements)

References 20 publications

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“…37,38 These structures vary in terms of ring strain and electron deficiency, which in turn influence reactivity toward azides and endogenous cellular molecules, such as thiols. 26 In addition, more hydrophilic cyclooctyne structures have been developed 32 to reduce the extent of nonspecific hydrophobic binding to cells.…”

Section: Resultsmentioning

confidence: 99%

Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition

et al. 2012

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Methods for fluorophore targeting to cellular proteins can allow imaging with dyes that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the E. coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 minutes. Second, we evaluated a panel of five different cyclooctyne structures, and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.

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Section: Resultsmentioning

confidence: 99%

Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition

et al. 2012

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“…The azido sugar, on the other hand, was detected using a far-red fluorescently-labelled strained cyclooctyne, tetramethoxydibenzocyclooctyne (TMDIBO-647). 30 Our previous work showed the bioorthogonal nature of the isonitrile group as well as the orthogonality of the isonitrile/tetrazine reaction to the commonly used alkyne-azide chemistry under physiological conditions. 22 Fig.…”

Section: Resultsmentioning

confidence: 99%

Dual-sugar imaging using isonitrile and azido-based click chemistries

et al. 2013

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“…11 Several analogs of DIBO have also been reported that exhibit even higher rates of cycloaddition with azides. 5,12 …”

Section: Introductionmentioning

confidence: 99%

Polar Dibenzocyclooctynes for Selective Labeling of Extracellular Glycoconjugates of Living Cells

et al. 2012

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Although strain-promoted alkyne-azide cycloadditions (SPAAC) have found wide utility in biological and material sciences, the low polarity and limited water solubility of commonly used cyclooctynes represents a serious shortcoming. To address this problem, an efficient synthetic route has been developed for highly polar sulfated dibenzocyclooctynylamides (S-DIBO) by a Friedel-Crafts alkylation of 1,2-bis(3-methoxyphenyl)ethylamides with trichlorocyclopropenium cation followed by a controlled hydrolysis of the resulting dichlorocyclopropenes to give bis(3-methoxyphenyl)cyclooctacyclopropenones, which were subjected to methoxy group removal of the phenols, O-sulfation, and photochemical unmasking of the cyclopropenone moiety. Accurate rate measurements of the reaction of benzyl azide with various dibenzylcyclooctyne derivatives demonstrated that aromatic substitution and the presence of the amide function had only a marginal impact on the rate constants. Biotinylated S-DIBO 8 was successfully used for labeling azido-containing glycoconjugates of living cells. Furthermore, it was found that the substitution pattern of the dibenzylcyclooctynes influences subcellular location and in particular it has been shown that DIBO derivative 4 can enter cells thereby labeling intra- and extracellular azido-modified glycoconjugates, whereas S-DIBO 8 cannot pass the cell membrane and therefore is ideally suited for selective labeling of cell surface molecules. The ability to selectively label cell surface molecules will yield unique opportunities for glycomic analysis and the study of glycoprotein trafficking.

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Development and evaluation of new cyclooctynes for cell surface glycan imaging in cancer cells

Cited by 76 publications

References 20 publications

Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition

Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition

Dual-sugar imaging using isonitrile and azido-based click chemistries

Polar Dibenzocyclooctynes for Selective Labeling of Extracellular Glycoconjugates of Living Cells

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