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Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions.

ABSTRACT: Antibody-drug conjugates hold considerable promise as anticancer agents, however, producing them remains a challenge and there is a need for mild, broadly applicable, site-specific conjugation methods that yield homogenous products. It was envisaged that enzymatic remodeling of the oligosaccharides of an antibody would enable the introduction of reactive groups that can be exploited for the site-specific attachment of cytotoxic drugs. This is based on the observation that glycosyltransferases often tolerate chemical modifications in their sugar nucleotide substrates, thus allowing the installation of reactive functionalities. An azide was incorporated because this functional group is virtually absent in biological systems and can be reacted by strain-promoted alkyne-azide cycloaddition. This method, which does not require genetic engineering, was used to produce an anti-CD22 antibody modified with doxorubicin to selectively target and kill lymphoma cells.

SUBMITTER: Li X

PROVIDER: S-EPMC4128391 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions.

Li Xiuru X Fang Tao T Boons Geert-Jan GJ

Angewandte Chemie (International ed. in English) 20140523 28

Antibody-drug conjugates hold considerable promise as anticancer agents, however, producing them remains a challenge and there is a need for mild, broadly applicable, site-specific conjugation methods that yield homogenous products. It was envisaged that enzymatic remodeling of the oligosaccharides of an antibody would enable the introduction of reactive groups that can be exploited for the site-specific attachment of cytotoxic drugs. This is based on the observation that glycosyltransferases of ...[more]

PMID: 24862406

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Project description:O-linked β-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

Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions.

Publications

Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions.

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