Synthesis of C-Mannosylated Glycopeptides Enabled by Ni-Catalyzed Photoreductive Cross-Coupling Reactions

Abstract

The biological functions of tryptophan C-mannosylation are poorly understood, in part, due to a dearth of methods for preparing pure glycopeptides and glycoproteins with this modification. To address this issue, efficient and scalable methods are required for installing this protein modification. Here, we describe unique Ni-catalyzed cross-coupling conditions that utilize photocatalysis or a Hantzsch ester photoreductant to couple glycosyl halides with (hetero)aryl bromides, thereby enabling the α-C-mannosylation of 2-bromo-tryptophan, peptides thereof, and (hetero)aryl bromides more generally. We also report that 2-(α-Dmannopyranosyl)-L-tryptophan undergoes facile anomerization in the presence of acid: something that must be considered when preparing and handling peptides with this modification. These developments enabled the first automated solid-phase peptide syntheses of C-mannosylated glycopeptides, which we used to map the epitope of an antibody, as well as providing the first verified synthesis of Carmo-HrTH-I, a C-mannosylated insect hormone. To complement this approach, we also performed late-stage tryptophan C-mannosylation on a diverse array of peptides, demonstrating the broad scope and utility of this methodology for preparing glycopeptides.

Tryptophan C-mannosylation is critical for Plasmodium falciparum transmission

“…3a ). Immunoblotting these proteins with the monoclonal antibody 5G12, which is specific for tryptophan C-mannosylation and recognises the H/F/L/Q/EW(Man) epitopes 8 , 39 , confirmed the C-mannosylation of rMTRAP when co-expressed with CeDPY19 (Fig. 3a ).…”

“…The rMTRAP protein expressed in the presence and absence of the C-mannosyltransferase was purified from the culture supernatant and the FLAG-SUMO* tag removed. The purified TSR domains were analysed by intact ESI-MS: 8 , 39 comparisons of deconvoluted mass spectra revealed that the C-glycosylated sample possessed +162 Da and +324 Da proteoforms, commensurate with the addition of 1-2 hexoses (Fig. 3b ).…”

Tryptophan C-mannosylation is critical for Plasmodium falciparum transmission

“…Conversely, we did obtain rMTRAP from this system with or without the co-expression of a C-mannosyltransferase. Immunoblotting these samples with the monoclonal antibody 5G12, which is speci c for tryptophan C-mannosylation and recognises the H/F/L/Q/EW(Man) epitopes 8,40 , con rmed the C-mannosylation of rMTRAP only when co-expressed with CeDPY19 (Fig. 3a).…”

“…Separation on the analytical column was undertaken using buffer A (2% DMSO, 0.1% FA) and buffer B (78% ACN, 2% DMSO and 0.1% FA) by altering the buffer composition from 3% buffer B to 23% buffer B over 29 min, 23% buffer B to 40% buffer B over 10 min, 40% buffer B to 80% buffer B over 5 min, then the composition was held at 80% buffer B for 5 min, and then dropped to 3% buffer B over 1 min and held at 3% buffer B for another 9 min. The Eclipse™ Mass Spectrometer was operated in a data-dependent mode switching between the acquisition of a single Orbitrap MS scan (120k resolution) every 3 seconds followed by Orbitrap HCD scans (maximum ll time 100 ms, AGC 2 x 105 with a resolution of 50k for the Orbitrap MS-MS scans using stepped NCE with 25,30,40).…”

Visible‐Light‐Promoted Stereoselective C(sp3)−H Glycosylation for the Synthesis of C‐Glycoamino Acids and C‐Glycopeptides

“…As shown in Table 2, glycosyl NHP esters derived from various monosaccharides, including ribose, xylose, mannose, galactose, glucose, and fructose all could participate in this reaction smoothly and afforded desired products in 65-85 % yields and > 20 : 1 d.r. (3)(4)(5)(6)(7)(8)(9)(10)(11). The bioactive sugar derivatives, such as isopropyl-β-D-thiogalactopyranoside (IPTG) and arbutin were also competent substrates (12 and 13).…”