Semisynthesis of Biologically Active Glycoforms of the Human Cytokine Interleukin 6

Abstract

Human interleukin 6 (IL‐6) is a potent cytokine with immunomodulatory properties. As the influence of N‐glycosylation on the in vivo activities of IL‐6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL‐6 was established by sequential native chemical ligation. The four cysteines of IL‐6 are convenient for ligations and require only the short synthetic glycopeptide 43–48. The Cys‐peptide 49–183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1–42 was accessible by the simultaneous cleavage of two inteins, leading to the 1–42 thioester with the native N‐terminus. Ligation and refolding studies showed that the inherently labile AspPro bond 139–140 was detrimental for the sequential C‐ to N‐terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full‐length IL‐6 glycoproteins, which showed full bioactivity after efficient refolding and purification.

Exploring chemoselective S-to-N acyl transfer reactions in synthesis and chemical biology

“…The synthetic potential of the methodology is abundantly clear from the numerous reported total-syntheses of proteins that utilize this approach. An impressive array of complex peptides and proteins have been successfully synthesized by NCL including the sialic acid-binding lectin siglec-7 (127 AA) 92 , human interleukin-6 glyoprotein (183 AA) 93 , and the pore-forming antimicrobial protein caenopore-5 (82 AA) 94 . In a ground-breaking study, Valiyaveetil and co-workers employed a semisynthetic approach combining EPL and SPPS to prepare a K + channel containing the D-enantiomer of alanine in place of a conserved glycine residue.…”

Recent Developments in Synthetic Carbohydrate‐Based Diagnostics, Vaccines, and Therapeutics

“…In fact, glycoengineered versions of EPO and follicle‐stimulating hormone (FSH) were developed that incorporated additional sialylated N ‐glycans and exhibited increased in vivo stability and improved bioactivity 100. While the recombinant cellular biosynthetic technologies offer advantages in terms of speed, scalability and production, synthetic chemistry101 has provided access to chemically pure, homogeneously glycosylated proteins, including EPO (Figure 11),102 interferon‐β,103 interleukin‐6,104 and some members of the family of human glycoprotein hormones 105. Merging the best of both biological and chemical methods, the laboratories of Davis and others have utilized unnatural linkages to attach synthetic carbohydrates to recombinant, bio‐orthogonally tagged proteins in a site‐specific manner by chemoselective conjugation reactions to provide synthetic glycoprotein mimics that retain biological activity 22.…”

Chemical Modification of the N Termini of Unprotected Peptides for Semisynthesis of Modified Proteins by Utilizing a Hydrophilic Protecting Group

“…In order to demonstrate E. coli expression of a short peptide and its subsequent N‐terminal modification, we examined the expression of model peptide 4 using a small ubiquitin‐related modifier (SUMO) fusion tag . Recombinant expression of peptides with less than 100 amino acids is usually prone to degradation in E. coli .…”