Thio-Linked UDP–Peptide Conjugates as O-GlcNAc Transferase Inhibitors

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA (Ki = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay.


List of Contents
Instrumentation and general methods 2 General procedure for Fmoc SP peptide synthesis 3 Photo thiol-ene conjugation reactions, general remarks 4 Synthetic procedures and spectral data for selected compounds 4 Supplementary Table1 16 Copies of NMR spectra for selected compounds 17 Crystallography and structure solution 22 hOGT activity measurement 23 typically 5% to 30% in 5 min, flow rate 20 mL min −1 . Detection was at double wavelength 214 and 220 nm. Appropriate fractions were pooled and freeze-dried to provide the target compounds as fluffy powders. Integrity and the purity of the peptides was confirmed by LC_MS ESI(+) analysis.
L-ribose (5.0 g, 33.33 mmol) was mixed with 0.2 M HCl-MeOH solution at 0 °C. The reaction was stirred at RT for 4 h and the clear solution was neutralized with Amberlite IRA-400 (OH ˗ ) anion exchange resin. The resin was filtered off and the filtrate was concentrated. The residue was briefly dried in vacuum, dissolved in pyridine (25 mL) and solution was cooled down to 0 °C (ice-bath). Then, acetic anhydride (Ac2O; 10 mL, 105 mmol) was added dropwise and the reaction was stirred at RT for 16 h. The reaction was quenched by addition of MeOH (1 mL) and stirred for 30 min. The reaction was concentrated and the residue was partitioned between EA (40 mL) and 1M HCl (40 mL). The layers were separated. The organic layer was successively washed with water and a mixture of NaHCO3 saturated aqueous solution and brine. The aqueous layers were back extracted with EA (40 mL). The combined organic layer was dried and concentrated to give 9.5 g of the crude intermediate product as a syrup. This was dissolved in a mixture of glacial acetic acid (30 mL) and Ac2O (7.5 mL) and the solution was cooled down to 0 °C (ice-bath). Concentrated H2SO4 (2.1 mL) was then added dropwise and the reaction was stirred at room temperature for 16 h and poured on crushed ice (50 g). After ice melted, water (50 mL) and EA (50 mL) was added and the layers were separated. The organic layer was successively washed with water and a mixture of NaHCO3 saturated aqueous solution and brine.
The aqueous layers were back extracted with EA (40 mL). The combined organic layer was dried and concentrated to give 9.5 g of the crude product as a syrup. This was co-evaporated with toluene (2×20 mL) and dissolved in ethyl ether (20 mL). The solution was kept in a freezer at -18 °C for 16 h. The crystalline deposit was filtered off, washed with hexane˗ether (2:1, 50 mL) and dried to give 5.5 g (17 mmol, 52%) of the target product S1. m.p. 81-83 °C.   Step 3 (see below) without purification.
Step The layers were separated by centrifugation for 10 min at 4 °C; the aqueous layer was collected.
The extraction was repeated two times more. The combined aqueous layer was purified by size exclusion chromatography (Bio-Gel P2 fine; column 2.6×100 cm; flow rate 1 mL/min; elution with 0.25 M NH4HCO3). Appropriate fractions were pooled and freeze dried to give 0.22 g (0.46 mmol, 60%) of the crude product as amorphous solid.
The crude product was purified by HPLC on Waters Peptide Separation Technology C18 column     Figure S5. HSQC spectrum for compound 6.

Crystallography and structure solution
Human OGT (312-1031) was recombinantly expressed and purified as a cleavable GST-fusion protein, as described previously 1  as a search model. The structure was fully refined using iterative cycles of Refmac5 6 and COOT 7 . Ligand topology was generated using PRODRG 8 . Data collection and refinement statistics can be found in Supplementary Table 2.

hOGT activity measurement
Human OGT (312-1031) was recombinantly expressed as a cleavable His6-fusion protein and was purified as described previously 9   Binding constants for ligands in a displacement experiment were determined using GraphPad Prism7 and the equation reported by Nikolovska-Coleska et al. 11 .

OGT inhibition in cell extracts
HeLa cells were grown in DMEM medium supplemented with 10% FBS, 2 mM L-Glutamine and 1% Penicillin/Streptomycin (100 U/ml and 100 μg/ml respectively) at 37 °C and 5% CO2. Cells were lysed for 10 min on ice in a buffer containing 50 mM Tris-HCl, 0.1 mM EGTA, 1 mM EDTA,