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Application of Cu(I)-catalyzed azide-alkyne cycloaddition for the design and synthesis of sequence specific probes targeting double-stranded DNA.

ABSTRACT: Efficient protocols based on Cu(I)-catalyzed azide-alkyne cycloaddition were developed for the synthesis of conjugates of pyrrole-imidazole polyamide minor groove binders (MGB) with fluorophores and with triplex-forming oligonucleotides (TFOs). Diverse bifunctional linkers were synthesized and used for the insertion of terminal azides or alkynes into TFOs and MGBs. The formation of stable triple helices by TFO-MGB conjugates was evaluated by gel-shift experiments. The presence of MGB in these conjugates did not affect the binding parameters (affinity and triplex stability) of the parent TFOs.

SUBMITTER: Vasilyeva SV

PROVIDER: S-EPMC4979877 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Application of Cu(I)-catalyzed azide-alkyne cycloaddition for the design and synthesis of sequence specific probes targeting double-stranded DNA.

Vasilyeva Svetlana V SV Filichev Vyacheslav V VV Boutorine Alexandre S AS

Beilstein journal of organic chemistry 20160630

Efficient protocols based on Cu(I)-catalyzed azide-alkyne cycloaddition were developed for the synthesis of conjugates of pyrrole-imidazole polyamide minor groove binders (MGB) with fluorophores and with triplex-forming oligonucleotides (TFOs). Diverse bifunctional linkers were synthesized and used for the insertion of terminal azides or alkynes into TFOs and MGBs. The formation of stable triple helices by TFO-MGB conjugates was evaluated by gel-shift experiments. The presence of MGB in these co ...[more]

PMID: 27559384

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Project description:O-linked ?-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein post-translational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression, and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac4 GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells. In addition, we made a labeling efficiency comparison between two modes of azide-alkyne bioorthogonal reactions in click chemistry: copper-catalyzed azide-alkyne cycloaddition (CuAAC) with Biotin-Diazo-Alkyne and stain-promoted azide-alkyne cycloaddition (SPAAC) with Biotin-DIBO-Alkyne. After conjugation with click chemistry in vitro and enrichment via streptavidin resin, proteins with O-GlcNAc modification were separated by SDS-PAGE and identified with mass spectrometry. Proteomics data analysis revealed that 229 putative O-GlcNAc modified proteins were identified with Biotin-Diazo-Alkyne conjugated sample and 188 proteins with Biotin-DIBO-Alkyne conjugated sample, among which 114 proteins were overlapping. Interestingly, 74 proteins identified from Biotin-Diazo-Alkyne conjugates and 46 verified proteins from Biotin-DIBO-Alkyne conjugates could be found in the O-GlcNAc modified proteins database dbOGAP (http://cbsb.lombardi.georgetown.edu/hulab/OGAP.html). These results suggested that CuAAC with Biotin-Diazo-Alkyne represented a more powerful method in proteomics with higher protein identification and better accuracy compared to SPAAC. The proteomics credibility was also confirmed by the molecular function and cell component gene ontology (GO). Together, the method we reported here combining metabolic labeling, click chemistry, affinity-based enrichment, SDS-PAGE separation, and mass spectrometry, would be adaptable for other post-translationally modified proteins in proteomics.

Dataset Information

Application of Cu(I)-catalyzed azide-alkyne cycloaddition for the design and synthesis of sequence specific probes targeting double-stranded DNA.

Publications

Application of Cu(I)-catalyzed azide-alkyne cycloaddition for the design and synthesis of sequence specific probes targeting double-stranded DNA.

Similar Datasets

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