Synthesis of Glycopeptides from Type II Collagen‐Incorporating Galactosylated Hydroxylysine Mimetics and Their Use in Studying the Fine Specificity of Arthritogenic T Cells

Abstract

Five analogues of the bovine type II collagen (bCII) immunodominant glycopeptide [beta-D-Gal-(5R)-5-Hyl264]CII(256-270) (1) carrying diverse modifications at the critical hydroxylysine (Hyl) 264 side chain were designed and synthesised, to explore the fine specificity of bCII-reactive T cells involved in the initiation and/or regulation of collagen-induced arthritis (CIA), a mouse model for rheumatoid arthritis (RA). Beta-D-galactosyl-(5R)-5-hydroxy-L-lysine (19) and corresponding mimetics (22-25), conveniently protected for solid-phase synthesis, were all obtained by a divergent route involving enantiopure 5-hydroxylated 6-oxo-1,2-piperidinedicarboxylates as the key intermediates. All three bCII-specific T hybridomas used, as well as a recurrent pathogenic CD4+ T-cell clone isolated from bCII-immunised DBA/1 mice, recognised the galactosylated form 1 of the immunodominant bCII (256-270) epitope. These cells were extremely sensitive to changes at the epsilon-amino group of Hyl264, but differed in their pattern of recognition of analogues with a Hyl264 side chain modified at C-5 (i.e. inversion of stereochemistry, methylation). These data further document the importance of collagen post-translational modifications in autoimmunity and in the CIA model in particular, and provide a new insight into the molecular interaction between glycopeptide 1 and the TCR of pathogenic T cells.

Synthesis of glycosylated 5-hydroxylysine, an important amino acid present in collagen-like proteins such as adiponectin

“…By using this more sterically demanding galactosyl donor excellent yields (89%) of galactosylated 5-HL were obtained, using similar conditions to Kihlberg et al 51 (Guichard: 1.5 eq bulkier tetra-pivaloylated galactosyl bromide, silver silicate, CH 2 Cl 2 , rt, 8 h). Guichard et al 52 later reported use of their galactosylated-HL in SPPS to afford the glycopeptide corresponding to residues 256-270 of bovine type II collagen.…”

Insights into spatial configuration of a galactosylated epitope required to trigger arthritogenic T-cell receptors specific for the sugar moiety

“…The panel of modifications incorporated at the Gal-Hyl 264 side chain in the sequence of the bovine or mouse immunodominant CII(256–270) glycopeptide is shown in Figure 1 . The synthesis of N -Fmoc-protected Gal-Hyl residue and glycosylated building blocks with modifications at the Gal-Hyl side chain (specifically, GalPiv-Hyl, Gln-Hyl, Gal-Hyl[N 3 ], Gal-Hyl [OH], Gal [5 S ]-Hyl and Gal [5Me]-Hyl and Gal[6]-Hnl-[5S]-NH 2 ), as well as corresponding glycopeptides, were previously reported in detail [ 18 , 19 ]. The synthesis of the Gal [4R]-Hyl building block and corresponding glycopeptide will be described elsewhere.…”

Synthesis of a Galactosylated 4‐Hydroxylysine Building Block and Its Incorporation into a Collagen Immunodominant Glycopeptide

“…[9] To further investigate the basis of epitope recognition by CII-reactive T cells, we became interested in synthesizing and evaluating the [Gal-5-Hyl264]CII(256-270) variant 2 obtained by substituting a galactosylated 4-hydroxylysyl residue for the galactosylated 5-hydroxylysyl residue at position 264. We have shown previously that enantiopure 5-hydroxy-and 4-hydroxy-6-oxo-1,2-piperidinedicarboxylates are versatile building blocks for the synthesis of 5-and 4-hydroxylysine derivatives, respectively.…”