Metabolism of Diazirine-Modified N-Acetylmannosamine Analogues to Photo-Cross-Linking Sialosides

Abstract

Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photocrosslinking approach in which a diazirine photocrosslinker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogs in which the length of the linker between the pyranose ring and the diazirine was varied. These analogs were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogs could be incorporated into both α2–3 and α2–6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently crosslinked to their interaction partners. We demonstrate that all three diazirine-modified analogs were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogs were less efficiently metabolized, yet could still function as effective crosslinkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogs to glycans other than sialosides, providing fidelity to selectively incorporate the crosslinker into sialylated molecules. These compounds are non-toxic and display only minimal growth inhibition at the concentrations required for crosslinking studies. This report provides essential information for the deployment of photocrosslinking analogs to capture and study ephemeral, yet essential, sialic acid-mediated interactions.

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

“…First, we prepared the diazirine-modified nucleotide-sugar donor UDP-GlcNDAz ( Fig. 1) (16) and incubated it with recombinant human OGT and a biotinylated substrate peptide derived from casein kinase II (CKII) (17). The product mixture was analyzed by MALDI mass spectrometry, revealing a mass consistent with transfer of the GlcNDAz residue to the CKII peptide (Fig.…”

“…Synthesis of Ac 4 GlcNDAz and UDP-GlcNDAz have been described (16,38). Ac 3 GlcNDAz-1-OH was produced by selective deprotection of Ac 4 GlcNDAzAc 3 GlcNDAz-1-PðAc-SATEÞ 2 was synthesized by phosphitylation of Ac 3 GlcNDAz-1-OH with bis(S-acetyl-2-thioethyl) N,Ndiisopropylphosphoramidite (20) and subsequent oxidation with mCPBA.…”

Photoaffinity labeling studies of the carbohydrate-binding proteins with different affinities

“…5,5-Azo-hexanoic acid (120 mg, 0.845 mmol) 11,15 , and N-hydroxysuccinimide (NHS; 102 mg, 0.887 mmol) was dissolved in dry CH 2 Cl 2 (2.56 mL), and added EDCI (138 mg, 0.887 mmol) at 0°C, which was stirred at 0°C for 10 min, then at room temperature for 1 h. The reaction mixture was diluted with CH 2 Cl 2 and washed with 10% NH 4 Cl three times. The organic layer was dried over Na 2 SO 4 then concentrated in vacuo to give 9 as white solid.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…"MALDI" used when instrument not specified. To form modified sialylated glycoconjugates for photo-crosslinking to interacting molecules (Bond et al, 2011b) Galacturonatecontaining disaccharides MALDI Synthesis of carbohydrates and conjugation to protein and fatty acids (Pourcelot et al, 2011) Gal 1 -Glc 1 -Man 9 -GlcNAc 2 RNase TOF/TOF (DHB)…”