4-Deoxy-4-fluoro-GalNAz (4FGalNAz) Is a Metabolic Chemical Reporter of O-GlcNAc Modifications, Highlighting the Notable Substrate Flexibility of O-GlcNAc Transferase

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.


Table of contents:
. Synthesis of Ac34FGalNAz Page S2 Figure S2. 4FGalNAz is not particularly toxic to mammalian cells.
Page S2 Figure S3. Characterization of 4FGalNAz in Jurkat cells.
Page S3 Figure S4. 4FGalNAz is a substrate for GalK2 and AGX1.
Page S3

Experimental Methods Page S4
NMR characterization of synthetic compounds Page S7

References
Page S11! S2 Figure S1. Synthesis of Ac34FGalNAz. Figure S2. 4FGalNAz is not particularly toxic to mammalian cells. CHO cells were incubated with MCR or DMSO vehicle under the indicated conditions before cell viability was measured using an MTT assay.  . 4FGalNAz is a substrate for GalK2 and AGX1. a) 4FGalNAz is accepted by GALK2. Michaelis-Menten enzyme curves were measured using recombinant GalK2 and the indicated concentrations of either GalNAc or 4FGalNAz. Enzyme constants were determined using line fitting in Graphpad Prism 9. b) 4FGalNAz-1-phosphate is accepted by AGX1. Michaelis-Menten enzyme curves were measured using recombinant AGX1 and the indicated concentrations of either GalNAc or 4FGalNAz. Enzyme constants were determined using line fitting in Graphpad Prism 9.!

Chemical synthesis
General information All reagents used for chemical synthesis were purchased from Sigma-Aldrich, Alfa Aesar or EMD Millipore unless otherwise specified and used without further purification. All anhydrous reactions were performed under argon or nitrogen atmosphere. Analytical thin-layer chromatography (TLC) was conducted on EMD Silica Gel 60 F254 plates with detection by ceric ammonium molybdate (CAM), anisaldehyde or UV. For flash chromatography, 60 Å silica gel (EMD) was utilized. 1 H spectra were obtained at 400, 500, or 600 MHz on Varian spectrometers Mercury 400, VNMRS-500, or -600. Chemical shifts are recorded in ppm (δ) relative to solvent. Coupling constants (J) are reported in Hz. 13 C spectra were obtained at 100, 125, or 150 MHz on the same instruments.

1-O-Benzyl-N-acetyl-α-D-glucosamine (2) 1
To a stirred suspension of N-acetylglucosamine 1 (10.0 g, 45.2 mmol) in benzyl alcohol (125 mL) at 0 °C, acetyl chloride (10.9 mL, 12.0 g, 152 mmol) was added dropwise. The reaction mixture was stirred for 30 min at rt and further stirred for 24 h at 65 °C. The benzyl glycoside was precipitated using cold Et2O and the liquid phase was discarded. The resulting syrup corresponding to the benzyl glycoside was washed with cold Et2O, solubilized in MeOH and neutralized with NaHCO3 (solid) until pH 7 was achieved. The suspension was filtered through a short pad of Celite TM , further washed with MeOH, and the solvent was removed under reduced pressure. Recrystallization from EtOH yielded 2 (11.0 g, 78%) as a white solid.
Benzaldehyde (3.46 ml, 23.0 mmol) and p-toluenesulfonic acid (2 g, 11.5 mmol) were added to a suspension of the starting material 2 (6.5 g, 20.88 mmol) in anhydrous DMF (20 ml). The mixture was stirred at 65°C for 20 h. After this time the solvent was evaporated under reduced pressure, then the solid residue washed and triturated with hexane. The residue was then triturated with a warm NaHCO3 saturated solution. After cooling to room temperature, the solid was filtered and the solvent evaporated to afford the compound 3 (7.6 g, 91%) as a solid.