Solid‐Phase‐Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides

• Published in: Angewandte Chemie International Edition 2024-08, Vol.63 (34), p.e202405671-n/a
• Main Authors: Lin, Po‐han, Xu, Yongmei, Bali, Semiha Kevser, Kim, Jandi, Gimeno, Ana, Roberts, Elijah T., James, Deepak, Almeida, Nuno M. S., Loganathan, Narasimhan, Fan, Fei, Wilson, Angela K., Jonathan Amster, I., Moremen, Kelley W., Liu, Jian, Jiménez‐Barbero, Jesús, Huang, Xuefei
• Format: Article
• Language: English
• Subjects: Cathepsin G; Chemical reactions; Chemical synthesis; Chondroitin sulfate; chondroitin sulfate glycopeptide; Core protein; Dodecasaccharides; Elongated structure; Enzymatic synthesis; enzymes; Glycan; Glycopeptides; Glycosaminoglycans; Mass spectrometry; Mass spectroscopy; NMR; Nuclear magnetic resonance; Peptides; Protein biosynthesis; proteoglycan; Proteoglycans; solid phase; Solid phases; Sulfates; Sulfation; synthesis
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202405671

Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80 %. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs. Chemoenzymatic synthesis has been successfully performed producing the longest homogeneous chondroitin sulfate (CS) glycopeptides to date. Compared to more traditional chemical synthesis, this new strategy was much more efficient, which could reduce the total number of synthetic steps by 80 %. The availability of well‐defined CS glycopeptides enabled the study of the impacts of sulfation on glycopeptide structures and functions.