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Readily accessible bicyclononynes for bioorthogonal labeling and three-dimensional imaging of living cells.

Dommerholt J, Schmidt S, Temming R, Hendriks LJ, Rutjes FP, van Hest JC, Lefeber DJ, Friedl P, van Delft FL - Angew. Chem. Int. Ed. Engl. (2010)

View Article: PubMed Central - PubMed

Affiliation: Radboud University Nijmegen, Institute for Molecules and Materials, Heijendaalseweg 135, 6525 AJ, Nijmegen, The Netherlands.

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The advent of chemical biology tools for imaging and tracking of biomolecules (proteins, lipids, glycans) in their native environment is providing unique insights into cellular processes that are not achievable with traditional biochemical or molecular biology tools... Bioorthogonal labeling of biomolecules has proven particularly useful for the detection and study of glycans and lipids, based on a highly selective reaction between an abiotic functional tag and a designed chemical probe... Most prominently, azides were find to react with cyclooctynes with high reaction rates in a so-called strain-promoted alkyne–azide cycloaddition (SPAAC)... The toolbox of metal-free bioorthogonal reactions was most recently further expanded by our research group and others, by demonstrating that cyclooctynes undergo even more rapid strain-promoted cycloaddition with nitrones (SPANC), a procedure that was found suitable for dual, irreversible, and site-specific N-terminal modification of proteins... The desired bicyclo[6.1.0]non-4-yn-9-ol (endo-) was thus obtained in 61 % overall yield after purification (the diastereomeric exo-isomer of was prepared in 53 % yield form exo-)... Both exo- or endo- were found sufficiently stable for prolonged storage at −20 °C and did not undergo structural change upon stirring in the presence of 5 mm glutathione for 48 hours in CD3CN/D2O (1:2)... Reaction rates increased, as anticipated, in a more polar mixture (CD3CN/D2O (1:2)), measuring 0.29 and 0.19 m s for endo- and exo-, respectively, values similar to or better than other cyclooctyne systems... Strain-promoted acetylene–nitrone cycloaddition (SPANC) with nitrone (Scheme 2 b) instead of an azide was found to be significantly faster, measuring 1.66 and 1.32 m s for endo- and exo-, respectively... The usefulness of BCN for bioorthogonal functionalization of biomolecules was next investigated in the one-pot SPANC functionalization of a model peptide with an N-terminal serine... In all cases, cells retained morphological integrity and cell surface fluorescence, with consistently higher labeling for BCN than for DIBO, as detected by confocal microscopy (Figure 2 a) and flow cytometry (Figure 2 b)... By using flow cytometry, high monophasic intensities and excellent signal-to-noise ratio (SNR) were found for both DIBO-biotin (SNR=116) and BCN-biotin (SNR=221)... Thereby, submicron resolution reveals fine surface distribution of sialic acids at leading edge filopodia, focal clusters at actin-rich contact sites to collagen fibers, and substantial glycan-rich deposits into the tissue matrix from the trailing edge (see the Supporting Information)... In conclusion, in view of the non-toxic labeling procedure, the tunable fluorescent properties by choice of dye and the high signal-to-noise ratio, BCN will be useful for addressing molecular glycan function studies in live-cell and other systems.

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Surface and total fluorescence intensity of MV3 melanoma, cultured in the absence or presence of Ac4ManNAz (50 μm), followed by labeling with a cyclooctyne-biotin conjugate and secondary labeling with streptavidin-Alexa Fluor 488. a) Representative confocal images of unlabeled cells (top), cells labeled with DIBO-biotin (middle) or BCN-biotin 9 (bottom). Bar: 10 μm. b) Label intensity assessed by flow cytometry, indicated as mean fluorescence intensity (MFI). Numbers denote the average of green fluorescent cells for that particular experiment. Black trace: untreated; red trace: Ac4ManNAz + SA-AF488; blue trace: w/o Ac4ManNAz + DIBO + SA-AF488; dark red trace: Ac4ManNAz + DIBO + SA-AF488; green trace: w/o Ac4ManNAz + BCN + SA-AF488; magenta trace: Ac4ManNAz + BCN + SA-AF488.
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fig02: Surface and total fluorescence intensity of MV3 melanoma, cultured in the absence or presence of Ac4ManNAz (50 μm), followed by labeling with a cyclooctyne-biotin conjugate and secondary labeling with streptavidin-Alexa Fluor 488. a) Representative confocal images of unlabeled cells (top), cells labeled with DIBO-biotin (middle) or BCN-biotin 9 (bottom). Bar: 10 μm. b) Label intensity assessed by flow cytometry, indicated as mean fluorescence intensity (MFI). Numbers denote the average of green fluorescent cells for that particular experiment. Black trace: untreated; red trace: Ac4ManNAz + SA-AF488; blue trace: w/o Ac4ManNAz + DIBO + SA-AF488; dark red trace: Ac4ManNAz + DIBO + SA-AF488; green trace: w/o Ac4ManNAz + BCN + SA-AF488; magenta trace: Ac4ManNAz + BCN + SA-AF488.

Mentions: The bioavailability and tolerability of labeling surface glycans on living human melanoma MV3 cells was addressed using the chemical reporter strategy.[1b] MV3 melanoma cells are highly invasive and metastatic, and their abundant production of surface glycans was previously implicated in invasion processes.[24] Thus, MV3 cells were incubated with peracetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz), labeled with BCN-biotin conjugate 9, and stained with streptavidin-Alexa Fluor 488 (Figure 2). To be able to compare the efficiency of labeling of 9 to that of dibenzocycylooctyne (DIBO, 2), one of the most reactive cyclooctyne systems known to date,[18] cells were also labeled with a DIBO-biotin conjugate. In all cases, cells retained morphological integrity and cell surface fluorescence, with consistently higher labeling for BCN than for DIBO, as detected by confocal microscopy (Figure 2 a) and flow cytometry (Figure 2 b). By using flow cytometry, high monophasic intensities and excellent signal-to-noise ratio (SNR) were found for both DIBO-biotin (SNR=116) and BCN-biotin (SNR=221). No signs of label-induced cytotoxicity were detected after propidium iodide staining (see the Supporting Information).


Readily accessible bicyclononynes for bioorthogonal labeling and three-dimensional imaging of living cells.

Dommerholt J, Schmidt S, Temming R, Hendriks LJ, Rutjes FP, van Hest JC, Lefeber DJ, Friedl P, van Delft FL - Angew. Chem. Int. Ed. Engl. (2010)

Surface and total fluorescence intensity of MV3 melanoma, cultured in the absence or presence of Ac4ManNAz (50 μm), followed by labeling with a cyclooctyne-biotin conjugate and secondary labeling with streptavidin-Alexa Fluor 488. a) Representative confocal images of unlabeled cells (top), cells labeled with DIBO-biotin (middle) or BCN-biotin 9 (bottom). Bar: 10 μm. b) Label intensity assessed by flow cytometry, indicated as mean fluorescence intensity (MFI). Numbers denote the average of green fluorescent cells for that particular experiment. Black trace: untreated; red trace: Ac4ManNAz + SA-AF488; blue trace: w/o Ac4ManNAz + DIBO + SA-AF488; dark red trace: Ac4ManNAz + DIBO + SA-AF488; green trace: w/o Ac4ManNAz + BCN + SA-AF488; magenta trace: Ac4ManNAz + BCN + SA-AF488.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3021724&req=5

fig02: Surface and total fluorescence intensity of MV3 melanoma, cultured in the absence or presence of Ac4ManNAz (50 μm), followed by labeling with a cyclooctyne-biotin conjugate and secondary labeling with streptavidin-Alexa Fluor 488. a) Representative confocal images of unlabeled cells (top), cells labeled with DIBO-biotin (middle) or BCN-biotin 9 (bottom). Bar: 10 μm. b) Label intensity assessed by flow cytometry, indicated as mean fluorescence intensity (MFI). Numbers denote the average of green fluorescent cells for that particular experiment. Black trace: untreated; red trace: Ac4ManNAz + SA-AF488; blue trace: w/o Ac4ManNAz + DIBO + SA-AF488; dark red trace: Ac4ManNAz + DIBO + SA-AF488; green trace: w/o Ac4ManNAz + BCN + SA-AF488; magenta trace: Ac4ManNAz + BCN + SA-AF488.
Mentions: The bioavailability and tolerability of labeling surface glycans on living human melanoma MV3 cells was addressed using the chemical reporter strategy.[1b] MV3 melanoma cells are highly invasive and metastatic, and their abundant production of surface glycans was previously implicated in invasion processes.[24] Thus, MV3 cells were incubated with peracetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz), labeled with BCN-biotin conjugate 9, and stained with streptavidin-Alexa Fluor 488 (Figure 2). To be able to compare the efficiency of labeling of 9 to that of dibenzocycylooctyne (DIBO, 2), one of the most reactive cyclooctyne systems known to date,[18] cells were also labeled with a DIBO-biotin conjugate. In all cases, cells retained morphological integrity and cell surface fluorescence, with consistently higher labeling for BCN than for DIBO, as detected by confocal microscopy (Figure 2 a) and flow cytometry (Figure 2 b). By using flow cytometry, high monophasic intensities and excellent signal-to-noise ratio (SNR) were found for both DIBO-biotin (SNR=116) and BCN-biotin (SNR=221). No signs of label-induced cytotoxicity were detected after propidium iodide staining (see the Supporting Information).

View Article: PubMed Central - PubMed

Affiliation: Radboud University Nijmegen, Institute for Molecules and Materials, Heijendaalseweg 135, 6525 AJ, Nijmegen, The Netherlands.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

The advent of chemical biology tools for imaging and tracking of biomolecules (proteins, lipids, glycans) in their native environment is providing unique insights into cellular processes that are not achievable with traditional biochemical or molecular biology tools... Bioorthogonal labeling of biomolecules has proven particularly useful for the detection and study of glycans and lipids, based on a highly selective reaction between an abiotic functional tag and a designed chemical probe... Most prominently, azides were find to react with cyclooctynes with high reaction rates in a so-called strain-promoted alkyne–azide cycloaddition (SPAAC)... The toolbox of metal-free bioorthogonal reactions was most recently further expanded by our research group and others, by demonstrating that cyclooctynes undergo even more rapid strain-promoted cycloaddition with nitrones (SPANC), a procedure that was found suitable for dual, irreversible, and site-specific N-terminal modification of proteins... The desired bicyclo[6.1.0]non-4-yn-9-ol (endo-) was thus obtained in 61 % overall yield after purification (the diastereomeric exo-isomer of was prepared in 53 % yield form exo-)... Both exo- or endo- were found sufficiently stable for prolonged storage at −20 °C and did not undergo structural change upon stirring in the presence of 5 mm glutathione for 48 hours in CD3CN/D2O (1:2)... Reaction rates increased, as anticipated, in a more polar mixture (CD3CN/D2O (1:2)), measuring 0.29 and 0.19 m s for endo- and exo-, respectively, values similar to or better than other cyclooctyne systems... Strain-promoted acetylene–nitrone cycloaddition (SPANC) with nitrone (Scheme 2 b) instead of an azide was found to be significantly faster, measuring 1.66 and 1.32 m s for endo- and exo-, respectively... The usefulness of BCN for bioorthogonal functionalization of biomolecules was next investigated in the one-pot SPANC functionalization of a model peptide with an N-terminal serine... In all cases, cells retained morphological integrity and cell surface fluorescence, with consistently higher labeling for BCN than for DIBO, as detected by confocal microscopy (Figure 2 a) and flow cytometry (Figure 2 b)... By using flow cytometry, high monophasic intensities and excellent signal-to-noise ratio (SNR) were found for both DIBO-biotin (SNR=116) and BCN-biotin (SNR=221)... Thereby, submicron resolution reveals fine surface distribution of sialic acids at leading edge filopodia, focal clusters at actin-rich contact sites to collagen fibers, and substantial glycan-rich deposits into the tissue matrix from the trailing edge (see the Supporting Information)... In conclusion, in view of the non-toxic labeling procedure, the tunable fluorescent properties by choice of dye and the high signal-to-noise ratio, BCN will be useful for addressing molecular glycan function studies in live-cell and other systems.

Show MeSH
Related in: MedlinePlus