Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent

Abstract

Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)₂, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water.

Figure 1

Natural products containing phenolic O-glycosyl moieties and strategies to access phenolic O-glycosyl linkages in small and complex molecules (a) Selected examples of glycosylated natural products exhibiting varied biological activity. (b) Schematic representation of direct vs. convergent synthetic methodologies to access glycopeptides. Coloured circles represent amino acids. (c) Conditions for previously reported aromatic O-glycosylation methods, discussed and referenced in the text; those require a protecting group for the oxygen sites (OPG). (d) Previously reported protecting-group-free aqueous glycosylation of sucrose promoted by Ca²⁺ and NMe₃ (ref. 56). The OH groups required for reaction are highlighted in green, the OH reaction site is shown in blue. (e) This work: O-glycosylation in aqueous solvent.

Figure 2

Evaluating substrate scope. The reactions were carried out at a substrate concentration of 1.0 M, the yields shown refer to isolated products. Reaction conditions: 0.5 mmol substrate, α-D-fluoroglycoside (1.5 mmol, 3 equiv.), Ca(OH)₂ (1.5 mmol, 3 equiv.), H₂O (1.0 M in substrate), rt, 1 h. The products were purified via reversed-phase flash column chromatography.

Figure 3

Tyrosine-selective glycosylation of glucagon-like peptide 1. HPLC yield reported (calculated by integrating peak corresponding to monosubstituted peptide at 214 nm). Reaction conditions: 0.5 or 1.0 mg substrate, α-D-fluoroglucose (1.0 M, 1000 equiv.), Ca(OH)₂ (1.0 M, 1000 equiv.), H₂O (1.0 mM in substrate), rt, 10 min. Reactions quenched with 0.5 M EDTA solution (pH = 8.0) and purified using desalting and buffer exchange column (SpinOUT™ GT-100).

Figure 4

Tyrosine-selective glycosylation of biologically active peptides. *Isolated yield reported (Reaction conditions specified in Supplementary Page 49). **HPLC yield reported (calculated by integrating peak corresponding to monosubstitutated peptide at 214 nm), reaction conditions: 0.5 or 1.0 mg substrate, α-D-fluoroglucose (1.0 M, 1000 equiv.), Ca(OH)₂ (1.0 M, 1000 equiv.), H₂O (1.0 mM in substrate), rt, 10 min. Reactions quenched with 0.5 M EDTA solution (pH = 8.0) and purified using desalting and buffer exchange column (SpinOUT™ GT-100).

Figure 5

Reactivity of cysteine under the aqueous glycosylation conditions. (a) Glycosylation of Boc-Cys-OH (33). (b) Glycosylation of Boc-Tyr-Cys-OH (35).