Chemoselective Solution- and Solid-Phase Synthesis of Disulfide-Linked Glycopeptides

Abstract

Glycosylation of peptides and proteins is a widely employed strategy to mimic important post-translational modifications or to modulate the physicochemical properties of peptides to enhance their delivery. Furthermore, glycosylation via a sulfur atom imparts increased chemical and metabolic stability to the resulting glycoconjugates. Herein, we report a simple and chemoselective procedure to prepare disulfide-linked glycopeptides. Acetate-protected glycosylsulfenyl hydrazines are shown to be highly reactive with the thiol group of cysteine residues within peptides, both in solution and as part of conventional solid-phase peptide synthesis protocols. The efficiency of this glycosylation methodology with unprotected carbohydrates is also demonstrated, which avoids the need for deprotection steps and further extends its utility, with disulfide-linked glycopeptides produced in excellent yields. Given the importance of glycosylated peptides in structural glycobiology, pharmacology, and therapeutics, the methodology outlined provides easy access to disulfide-linked glycopeptides as molecules with multiple biological applications.

Scheme 1. One-Pot Protocol to Prepare Disulfide-Linked Glycopeptides

Scheme 2. Two-Pot Protocol to Access Disulfide-Linked Glycopeptide

Scheme 3. Solid-Phase Disulfide-Linked-Glycosylation of Peptides

Scheme 4. Solid-Phase Glycosylation of Fmoc-Met-Pro-Ala-Cys-Gly-Ser-Ser-OH (Yielding Compounds 3g and 8g) and H₂N-Tyr-Thr-Gly-Phe-Leu-Cys-Leu-OH (Yielding Compounds 3m and 8m)

Synthetic conditions: (i) CH₂Cl₂/TFA/TIS 95:2:3, 3 cycles of 10 min; (ii) 5 or 7 (2.5 equiv), N,N-diisopropylethylamine (DIPEA) (1 equiv), dimethylformamide (DMF) 2 cycles × 30 min; (iii) TFA/TIS/water 95:2.5:2.5, 4 h.

Figure 1

¹³C NMR spectra recorded in CD₃OD. (A) ¹³C NMR spectrum of d-thioglucose sodium salt; (B) ¹³C NMR spectrum of reaction of d-thioglucose sodium salt with dilute deuterated methanolic DEAD (2.0 equiv) (instant reaction); (C) ¹³C NMR spectrum after addition of PhSH (2.0 equiv) (1 h).

Scheme 5. Schematic Representation of Synthesis of Unprotected Glycosyl Disulfide 16

Scheme 6. Synthesis of Symmetrical Dithiosaccharides

Figure 2

High-performance liquid chromatography (HPLC) chromatogram (λ = 254 nm) of peptide 2g (green), and crude reaction mixtures after reaction with symmetrical glycosyl disulfide 17 (yielding compound 23g, purple), and 19 (yielding compound 24g, black), respectively.