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A hydrophilic azacyclooctyne for Cu-free click chemistry.

Sletten EM, Bertozzi CR - Org. Lett. (2008)

Bottom Line: Here, we report the synthesis and evaluation of a novel azacyclooctyne, 6,7-dimethoxyazacyclooct-4-yne (DIMAC).Generated in nine steps from a glucose analogue, DIMAC reacted with azide-labeled proteins and cells similarly to cyclooctynes.However, its superior polarity and water solubility reduced nonspecific binding, thereby improving the sensitivity of azide detection.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, California, USA.

ABSTRACT
Biomolecules labeled with azides can be detected through Cu-free click chemistry with cyclooctyne probes, but their intrinsic hydrophobicity can compromise bioavailability. Here, we report the synthesis and evaluation of a novel azacyclooctyne, 6,7-dimethoxyazacyclooct-4-yne (DIMAC). Generated in nine steps from a glucose analogue, DIMAC reacted with azide-labeled proteins and cells similarly to cyclooctynes. However, its superior polarity and water solubility reduced nonspecific binding, thereby improving the sensitivity of azide detection.

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(A) Biotin conjugates of DIMAC (7) and a parent cyclooctyne (8).(5a) (B) Western blot of Jurkat cell lysates. Cells were treated with (+Az) or without (−Az) 25 μM Ac4ManNAz for 3 d, and lysates (35 μg total protein) were reacted with 250 μM 7 or 8 overnight at 25 °C. Equal protein loading was confirmed by Ponceau S staining (Figure S2, Supporting Information). The blot was probed with HRP-conjugated α-biotin. (C) Flow cytometry analysis of Jurkat cells. Cells metabolically labeled as in (B) were reacted with 250 μM 7 or 8 for 1 h at 25 °C and then stained with FITC−avidin. Similar data were obtained in three replicate experiments.
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fig2: (A) Biotin conjugates of DIMAC (7) and a parent cyclooctyne (8).(5a) (B) Western blot of Jurkat cell lysates. Cells were treated with (+Az) or without (−Az) 25 μM Ac4ManNAz for 3 d, and lysates (35 μg total protein) were reacted with 250 μM 7 or 8 overnight at 25 °C. Equal protein loading was confirmed by Ponceau S staining (Figure S2, Supporting Information). The blot was probed with HRP-conjugated α-biotin. (C) Flow cytometry analysis of Jurkat cells. Cells metabolically labeled as in (B) were reacted with 250 μM 7 or 8 for 1 h at 25 °C and then stained with FITC−avidin. Similar data were obtained in three replicate experiments.

Mentions: Next, we tested the ability of DIMAC to label glycan-associated azides within cell lysates and on the surface of live cells. DIMAC was first conjugated to biotin (Figure 2A; Scheme S3, Supporting Information), providing the means to detect its cycloadducts. For comparative purposes, we performed parallel experiments with previously reported cyclooctyne−biotin conjugate 8.(5a) The difference in water solubility of these two reagents was apparent during the preparation of 2.5 mM stock solutions. DIMAC−biotin 7 was readily soluble in aqueous buffer, whereas cyclooctyne−biotin 8 required an organic cosolvent (30% DMF).


A hydrophilic azacyclooctyne for Cu-free click chemistry.

Sletten EM, Bertozzi CR - Org. Lett. (2008)

(A) Biotin conjugates of DIMAC (7) and a parent cyclooctyne (8).(5a) (B) Western blot of Jurkat cell lysates. Cells were treated with (+Az) or without (−Az) 25 μM Ac4ManNAz for 3 d, and lysates (35 μg total protein) were reacted with 250 μM 7 or 8 overnight at 25 °C. Equal protein loading was confirmed by Ponceau S staining (Figure S2, Supporting Information). The blot was probed with HRP-conjugated α-biotin. (C) Flow cytometry analysis of Jurkat cells. Cells metabolically labeled as in (B) were reacted with 250 μM 7 or 8 for 1 h at 25 °C and then stained with FITC−avidin. Similar data were obtained in three replicate experiments.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664610&req=5

fig2: (A) Biotin conjugates of DIMAC (7) and a parent cyclooctyne (8).(5a) (B) Western blot of Jurkat cell lysates. Cells were treated with (+Az) or without (−Az) 25 μM Ac4ManNAz for 3 d, and lysates (35 μg total protein) were reacted with 250 μM 7 or 8 overnight at 25 °C. Equal protein loading was confirmed by Ponceau S staining (Figure S2, Supporting Information). The blot was probed with HRP-conjugated α-biotin. (C) Flow cytometry analysis of Jurkat cells. Cells metabolically labeled as in (B) were reacted with 250 μM 7 or 8 for 1 h at 25 °C and then stained with FITC−avidin. Similar data were obtained in three replicate experiments.
Mentions: Next, we tested the ability of DIMAC to label glycan-associated azides within cell lysates and on the surface of live cells. DIMAC was first conjugated to biotin (Figure 2A; Scheme S3, Supporting Information), providing the means to detect its cycloadducts. For comparative purposes, we performed parallel experiments with previously reported cyclooctyne−biotin conjugate 8.(5a) The difference in water solubility of these two reagents was apparent during the preparation of 2.5 mM stock solutions. DIMAC−biotin 7 was readily soluble in aqueous buffer, whereas cyclooctyne−biotin 8 required an organic cosolvent (30% DMF).

Bottom Line: Here, we report the synthesis and evaluation of a novel azacyclooctyne, 6,7-dimethoxyazacyclooct-4-yne (DIMAC).Generated in nine steps from a glucose analogue, DIMAC reacted with azide-labeled proteins and cells similarly to cyclooctynes.However, its superior polarity and water solubility reduced nonspecific binding, thereby improving the sensitivity of azide detection.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, California, USA.

ABSTRACT
Biomolecules labeled with azides can be detected through Cu-free click chemistry with cyclooctyne probes, but their intrinsic hydrophobicity can compromise bioavailability. Here, we report the synthesis and evaluation of a novel azacyclooctyne, 6,7-dimethoxyazacyclooct-4-yne (DIMAC). Generated in nine steps from a glucose analogue, DIMAC reacted with azide-labeled proteins and cells similarly to cyclooctynes. However, its superior polarity and water solubility reduced nonspecific binding, thereby improving the sensitivity of azide detection.

Show MeSH