SUPPLEMENTARY INFORMATION

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1 doi: /nature16966 Supplementary Synthetic Protocols General Solvents and chemicals were of analytical grade and were purchased from Sigma- Aldrich. Inorganic pyrophosphatase from baker's yeast and uridine-5 - diphosphoglucose pyrophosphorylase (EC ) from bovine liver were purchased from Sigma-Aldrich. Analytical thin- layer chromatography (t.l.c.) was performed on MerckSilica Gel 60F254 (0.2mm thickness on aluminum. T.l.c. plates were visualized using UV light (254 or 365 nm) and by immersion in 10% ammonium molybdate in 2 M H 2 SO 4, 5% H 2 SO 4. Size exclusion chromatography was performed using Bio-Rad Bio- Gel P-2 Gel (column size: 70 cm x 1.5 cm) at 4 C eluting with deionised water. 1 H and 13 C NMR spectra were recorded on a BrukerAvance 400inv Fourier Transform spectrometer. All spectra were recorded using an internal lock (deuterium) and are referenced internally to a residual solvent peak. 1 H and 13 C chemical shifts are quoted in parts per million (ppm) downfield of tetramethylsilane, and chemical shifts (δ) are rounded to the nearest 0.1 ppm unless increased precision was required to distinguish resonances. Coupling constants (J) are given in Hertz (Hz) and are quoted to the nearest 0.1 Hz. Proton and carbon spectral assignments were made based on COSY, TOCSY, APT, 1 H- 13 C- HSQC, and 1 H- 13 C- HMBC experiments as required. Mass spectra were acquired from sample dissolved in aqueous methanol on a Waters/Micromass instrument (electrospray ionization) and recorded using an ion-trap. Synthesis of Uridine-5'-phosphoric-α-(D-glucopyranosyl)methylphosphonic anhydride: See Supplementary Figure 1a for reaction scheme. The reaction mixture included glucose-1-phosphonate 39 (8.8 mg, 32 µmol), UTP (18.6 mg, 33.6 µmol), 25 mm TRIS buffer (ph = 7.4, 6 ml reaction volume), 4 mm MgCl 2, inorganic pyrophosphatase (5 U) and uridine-5 -diphosphoglucose pyrophosphorylase (15 U). The reaction was incubated at 37 C until TLC showed full consumption of starting materials (TLC eluent: EtOAc:H 2 O:CH 3 OH:NH 3 = 4:2:2:0.1). Subsequently the mixture was filtered through a 30 kda cut off spin filter and freeze dried. The residue was dissolved in a minimum amount of water (200 µl), loaded on a Biogel P2 column and was eluted with water (flow: 13.5 ml/h) at 4 C. Fractions containing pure product as judged by TLC were 1

2 freeze dried to give uridine-5'-phosphoric-α-(d-glucopyranosyl)methylphosphonic anhydride (Yield: 8.5 mg, 45%). Impure fraction were further purified by another P2 column to give more uridine-5'-phosphoric-α-(d-glucopyranosyl)methylphosphonic anhydride (Yield: 7.8 mg, 42%). 1 H-NMR (D 2 O at 10 C) δ = 7.83 (d, J=8.12 Hz, 1H, H- 6 ), (m, 1H, H-1 ), 5.80 (d, J=8.16 Hz, 1H, H-5 ), (m, 3H, H-1, H- 2, H-3 ), (m, 3H, H-4, H-5 a, H-5 b), 3.68 (dd, J=2.10, Hz, 1H, H-6a), (m, 2H, H-5, H-6b), 3.45 (pseudo t, J=9.38 Hz, 1H, H-3), 3.23 (pseudo t, J=9.38 Hz, 1H, H-4), 2.09 (ddd, J=11.29, 16.05, Hz, 1H, H-1 a), 1.96 (ddd, J=3.42, 15.75, Hz, 1H, H-1 b); 13 C-NMR (D 2 O at 10 C) d = (C-4 ), (C-2 ), (C-6 ), (C-5 ), 88.7 (C-1 ), (d, J=9.08 Hz, C-4 ), 74.1 (C-3 ), 73.4 (C-3), 72.9 (C-5), (d, J=4.94 Hz, C-1), (d, J=12.63 Hz, C-2), 70.3 (C-4), 69.8 (C-2 ), (d, J=4.60 Hz, C-5 ), 60.9 (C-6), (1C, d, J= Hz, C-1 ). 31 P-NMR (D 2 O) δ = 15.3 (d, J=26.73 Hz, 1P, CH 2 P), (d, J=26.73 Hz, 1P), see Supplementary Figure 2a and b; LRMS (ESI negative mode): [M-H] -. Synthesis of UDP-2-deoxy-2-fluoro-α-D-glucopyranose: See Supplementary Figure 1b for reaction scheme. The reaction mixture included disodium 2-deoxy-2-fluoro-α-D-glucopyranose 40 (3.1 mg, 10 µmol), UTP (5.8 mg, 10 µmol), 25 mm TRIS buffer (ph = 7.4, 2 ml reaction volume), 4 mm MgCl 2, inorganic pyrophosphatase (1.5 U) and uridine-5 -diphosphoglucose pyrophosphorylase (3.5 U). The reaction was incubated at 37 C until TLC showed full consumption of starting materials (~3 hours) (TLC eluent: EtOAc:H 2 O:CH 3 OH:NH 3 = 4:2:2:0.1). Subsequently the mixture was filtered through a 30 kda cut off spin filter and freeze dried. The residue was dissolved in a minimum amount of water (100 µl), loaded on a Biogel P2 column and was eluted with water (flow: 13.5 ml/h) at 4 C. Fractions containing pure product as judged by TLC were freeze dried to give UDP-2-deoxy-2-fluoro-α-D-glucopyranose (Yield: 2.9 mg, 49%). All spectroscopic data were in accordance with previous literature 41. Previously not reported: 19 F-NMR (D 2 O) δ = (1F, dd, J= 49.1, 12.9 Hz); 31 P-NMR (D 2 O) δ = (d, J= 20.0 Hz, 1P), (d, J= 20.0 Hz, 1P), see Supplementary Figure 2c; LRMS (ESI negative mode): [M-H]

3 Synthesis of Octyl UDP-6-thio-α-D-galactopyranose disulfide: See Supplementary Figure 1c for the reaction scheme. 3-Methoxy-2-pyridyl 2,3,4-tri-O-acetyl-6-thioacetyl-β-D-galactopyranoside (3) To a mixture of 1 (1.82 g, 4.5 mmol) in anhydrous dichloromethane (DCM) (25 ml), anhydrous ethyl acetate (2.5 ml) and acetic anhydride (0.2 ml), stirred at rt under N 2, was added TiBr 4 (3.3 g, 2.0 equiv) as one portion. After stirring overnight, more TiBr 4 (0.5 g) was added, and the mixture was stirred for another 6 h. The reaction mixture was diluted with DCM (150 ml), washed with cold water (100 ml) and cold brine (100 ml), dried over MgSO 4, filtered and evaporated, co-evaporated with dry toluene twice to afford a residue. To the residue were added dry toluene (15 ml) and silver 3-methoxy-2- pyridoxide 42, and the suspension was stirred vigorously for 1 h at 110 C under N 2. After completion, the reaction mixture was filtered through Celite, and washed with ethyl acetate (200 ml). The combined filtrate was washed with saturated NaHCO 3 (100 ml) and brine (100 ml), dried over MgSO 4, filtered and concentrated. The residue was purified by flash column chromatography (2:1 petroleum ether-ethyl acetate) to afford the product as a white foam 3 (1.016 g, 48 %). 1 H NMR (CDCl 3, 300 MHz): δ 7.70 (dd, 1 H, J 1.3 Hz, J 4.9 Hz, Ar-H), 7.10 (dd, 1 H, Ar-H), 6.93 (dd, 1 H, J 7.8 Hz, Ar-H), 6.18 (d, 1 H, J 1,2 8.3 Hz, H-1), 5.52 (dd, 1 H, J 2, Hz, H-2), 5.45 (brd, 1 H, H-4), 5.12 (dd, 1 H, J 3,4 3.4 Hz, H-3), 3.90 (brt, 1 H, H-5), 3.81 (s, 3 H, OCH 3 ), 3.14 (dd, 1 H, J 5,6a, 7.8 Hz, J 6a,6b 13.8 Hz, H-6a), 3.02 (dd, 1 H, J 5,6b, 6.5 Hz, H-6b), 2.28 (s, 3 H, CH 3 CO), 2.15 (s, 3 H, CH 3 CO), 1.97 (s, 3 H, CH 3 CO), 1.93 (s, 3 H, CH 3 CO). 13 C NMR (CDCl 3, 75 MHz): δ 194.5, 170.5, 170.2, 169.5, 151.8, 144.3, 136.9, 119.4, 119.1, 94.0, 72.8, 71.5, 68.5, 67.8, 56.1, 30.5, 28.3, 20.83, 20.78, 20.7,; EIS-MS: Calcd for [C 20 H 25 NO 10 S + H] + : Found m/z: Octyl 3-Methoxy-2-pyridyl 2,3,4-tri-O-acetyl-6-thio-β-D-galactopyranoside Disulfide (4) A solution of 3 (0.424 g, 0.9 mmol) in dry DMF (10 ml) was degassed with N 2 for 15 minutes, and then hydrazine acetate (162 mg, 2 equiv) was added as one portion. The reaction mixture was stirred for 0.5 h under N 2, diluted with ethyl acetate (100 ml), washed with saturated NaHCO 3 (50 ml) and brine (50 ml), dried over MgSO 4, filtered and concentrated, dried under vacuum for 1 h to give a residue. To the residue in dry 3

4 DCM (40 ml), stirred at rt under N 2, were added diethyl-n- (octylsulfanyl)hydrazodicarboxylate, prepared by reaction of diethylazodicarboxylate with octanethiol in methylene chloride 43, (0.92 g, 2.7 mmol, 3 equiv) and trimethylamine (30 µl). The reaction mixture was stirred for 0.5 h under the same conditions, diluted with DCM (100 ml), washed with 1M HCl (60 ml), saturated NaHCO 3 (60 ml) and brine (60 ml), dried over MgSO 4, filtered and evaporated. The resulting residue was purified by flash column chromatography (4:1 petroleum ether-ethyl acetate) to afford the product 4 as a syrup (0.428 g, 83 %). 1 H NMR (CDCl 3, 400 MHz): δ 7.68 (dd, 1 H, J 1.2 Hz, J 4.8 Hz, Ar-H), 7.08 (dd, 1 H, Ar-H), 6.91 (dd, 1 H, J 8.0 Hz, Ar-H), 6.23 (d, 1 H, J 1,2 8.4 Hz, H-1), 5.53 (m, 2 H, H-2 & H-4), 5.18 (dd, 1 H, J 2, Hz, J 3,4 2.4 Hz, H-3), 4.09 (brt, 1 H, H-5), 3.79 (s, 3 H, OCH 3 ), 2.88 (dd, 1 H, J 5,6a, 7.2 Hz, J 6a,6b 14.0 Hz, H- 6a), 2.71 (dd, 1 H, J 5,6b, 6.4 Hz, H-6b), 2.63 (t, 1 H, J 7.4 Hz, CH 2 S), 2.12 (s, 3 H, CH 3 CO), 1.97 (s, 3 H, CH 3 CO), 1.92 (s, 3 H, CH 3 CO), 1.57 (m, 2 H, CH 2 ), 1.26 (m, 10 H, 5XCH 2 ), 0.84 (t, 3 H, J 6.4 Hz, CH 3 ). 13 C NMR (CDCl 3, 100 MHz): δ 170.3, 170.2, 169.5, 151.9, 144.3, 136.9, 119.4, 119.1, 94.1, 72.8, 71.6, 70.0, 68.7, 68.4, 56.1, 39.2, 38.9, 31.9, 29.3, 29.2, 29.1, 28.5, 22.7, 20.84, 20.79, 20.73, 14.2; EIS-MS: Calcd for [C 26 H 39 NO 9 S 2 + H] + : Found m/z: Octyl 3-Methoxy-2-pyridyl 6-thio-β-D-galactopyranoside Disulfide (5) Ammonia was bubbled into a cooled (0 C) solution of 4 (0.4 g, 0.7 mmol) in dry methanol (50 ml) for 10 minutes, and then the reaction mixture was stirred for 48 h under the same conditions. After this time the solvent was evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (50:1 & 20:1 DCM-MeOH) to afford the product as a white foam (0.285 g, 91 %). 1 H NMR (CD 3 OD, 400 MHz): δ 7.73 (dd, 1 H, J 1.2 Hz, J 4.8 Hz, Ar-H), 7.33 (dd, 1 H, Ar-H), 7.02 (dd, 1 H, J 8.0 Hz, Ar-H), 5.96 (d, 1 H, J 1,2 8.0 Hz, H-1), 4.03 (brd, 1 H, H-4), 3.93 (m, 2 H, H-2 & H-5), 3.89 (s, 3 H, OCH 3 ), 3.71 (dd, 1 H, J 3,4 3.6 Hz, J 2,3 9.6 Hz, H-3), 3.03 (dd, 1 H, J 5,6a, 6.8 Hz, J 6a,6b 13.6 Hz, H-6a), 2.98 (dd, 1 H, J 5,6b, 6.8 Hz, H-6b), 2.74 (t, 1 H, J 7.2 Hz, CH 2 S), 1.65 (m, 2 H, CH 2 ), 1.37 (m, 10 H, 5XCH 2 ), 0.95 (t, 3 H, J 6.8 Hz, CH 3 ). 13 C NMR (CD 3 OD, 100 MHz): δ 152.6, 144.5, 136.6, 119.3, 118.6, 96.4, 74.1, 4

5 73.9, 70.4, 69.3, 55.2, 39.1, 38.7, 31.8, 29.1 (2 C), 28.9, 28.2, 22.5, EIS-MS: Calcd for [C 20 H 33 NO 6 S 2 + H] + : Found m/z: Octyl 6-thio-α-D-galactopyranosyl phosphate Disulfide (6) The 3-methoxy-2-pyridyloxy glycoside 5 (0.105 g; mol) was concentrated twice from toluene (2 x 10 ml) before being dissolved in dry N,N-dimethylformamide (2 ml) under an argon atmosphere. This solution was transferred by canula to a flask containing dry H 3 PO 4 (0.125 g; 1.27 mmol; 5.4 eq.). The original flask was rinsed with an additional 2 ml of the same solvent, which was then also transferred to the phosphoric acid containing flask by canula. The reaction was stirred under Argon at room temperature for approximately one hour before neutralization by addition of a slight excess of sodium hydroxide in water (600 ul). The solution was then partially concentrated by rotary evaporator under reduced pressure and purified by tc18 SepPak (Waters) to give 6 (0.033 g, 31 %) and recovered 5 (67 mg, 64%). 1 H NMR (D 2 O, 400 MHz): δ 5.51 (ddd, 1 H, J 1,2 3.1 Hz, J 1,P 7.6 Hz, H-1; in 31 P decoupled 1 H NMR signal collapses to d, J 1,2 3.1 Hz), 4.37 (dd, 1 H, H-5), 4.17 (brd, 1 H, J ~2 Hz, H-4), 3.99 (dd, 1 H, J 3,4 3.2 Hz, J 3, Hz, H-3), 3.79 (dd, 1 H, J 2, Hz, J 2,1 3.1 Hz, H-2), 3.09 (dd, 1 H, J 6a,6b 13.6 Hz, J 6a,5 6.4 Hz, H-6a), 3.00 (dd, 1 H, J 6b,5 8.0 Hz, H-6b), (m, 2 H, SCH 2 ), (m, 2 H, SCH 2 CH 2 ), (m, 10 H), 0.91 (m, 3H, CH 3 ). 13 C NMR (D 2 O, 100 MHz): δ 94.0 (d, 3 J 1,P = 5.0 Hz, C-1), (C-5), (C-3), 69.6 (C-4), 69.3 (d, 4 J 2,P = 6.0 Hz C-2), 38.4 (C-6), 38.3 (S-CH 2 ), 31.2, 28.5, 28.4, 28.4, 27.7, 22.2, 13.6 see Supplementary Figure P NMR (D 2 O, 162 MHz) δ 2.5. ESI-HRMS: Calcd for [C 14 H 28 O 8 PS 2 ]: Found m/z: Octyl UDP-6-thio-α-D-galactopyranose Disulfide (7): The glycosyl phosphate 6 (9.1 mg; 20.5 umol) was suspended in methanol (1.0 ml) and cooled to 4 C. Cold Amberlyst IR 120H (~ 20 mg) was added and the mixture was stirred for 5 minutes in the fridge, during which time the remaining sugar solids appeared to dissolve. The mixture was filtered through cotton in a Pasteur pipette into cold pyridine (2 ml), rinsing with cold methanol (2 ml). The solution was concentrated on a rotary evaporator under reduced pressure (< 30 C), redissolved in dry pyridine and concentrated again (2 x 4 ml). The galactosyl phosphate pyridinium salt was then 5

6 dissolved in dry pyridine (3 ml) and transferred by canula to a flask containing UMP morpholidate (28 mg; ~ 2 eq; Sigma) that had previously been dissolved in dry pyridine and concentrated (3 x 4 ml). To this solution was next added 1H-tetrazole (5 mg) in freshly distilled pyridine. The mixture was stirred for ~72 hours after which time methanol (1 ml) was added and the solution was concentrated to dryness. The residue was dissolved in 20 mm NH 4 Cl and applied to a tc18 SepPak (Waters) preconditioned with methanol and then water (2 x column volume). The SepPak was eluted with water, 10, 20, 30, 50, and 70% methanol in 20 mm NH 4 Cl, and then 100% methanol. The desired product was obtained in the 70% methanol fractions. After concentration the UDP-sugar was further purified on a Biogel P2 column and was eluted with water (flow: 13.5 ml/h) at 4 C to obtain 7 (6 mg; 39%). 1 H NMR (D 2 O, 400 MHz): δ 7.90 (d, 1 H, J 8.4 Hz, =CH), 5.90 (d, 1 H, =CH), 5.87 (d, 1 H, J 3.2 Hz, H-1), 5.52 (dd, 1 H, J 1,2 3.6 Hz, J P,H 7.6 Hz, H-1 ), (m, 6 H, H-2, H-3, H-4, H-5a, H-5b, H-5 ), 4.02 (d, 1 H, H- 4 ), 3.84 (dd, 1 H, J 3,4 3.2 Hz, J 2, Hz, H-3 ), 3.70 (dt, 1 H, H-2 ), 2.92 (dd, 1 H, J J 5,6a, 6.4 Hz, J 6a,6b 13.6 Hz, H-6a), 2.84 (dd, 1 H, J 5,6b, 7.2 Hz, H-6b), 2.67 (t, 1 H, J 7.2 Hz, CH 2 S), 1.58 (m, 2 H, CH 2 ), 1.18 (m, 10 H, 5XCH 2 ), 0.77 (t, 3 H, J 6.6 Hz, CH 3 ). 13 C NMR (D 2 O, 150 MHz): δ 165.9, 151.3, 141.2, 102.1, 95.3 (d, J P,C 7.5 Hz), 88.1, 82.6 (d, J P,C 9.0 Hz), 73.4, 70.1, 68.9, 68.87, 68.8, 67.8 (d, J P,C 6.8 Hz ), 64.2 (d, J P,C 6.8 Hz ), 37.6, 37.5, 30.6, 27.7, 27.0, 21.5, 20.6 (2 C), P NMR (D 2 O, 162 MHz): δ (d, J 20.0 Hz), (dd, J 6.0 Hz), see Supplementary Figure P(H) NMR (D 2 O, 162 MHz): δ (d, J 20.1 Hz), (d). ESI-HRMS: Calcd for [C 23 H 39 N 2 O 16 P 2 S 2 ] - : Found m/z:

7 Supplementary Figures Supplementary Figure 1 Outline of chemical syntheses. Synthesis of (a) Uridine-5'-phosphoric-α-(Dglucopyranosyl)-methylphosphonic anhydride, (b) UDP-2-deoxy-2-fluoro-α-D-glucopyranose, and (c) Octyl UDP-6-thio-α-D-galactopyranose disulfide. 7

8 doi: /nature16966 Supplementary Figure 2 NMR product characterization. (a) 1H- and (b) 13C-NMR spectra of uridine5'-phosphoric-α-(d-glucopyranosyl)methylphosphonic anhydride. (c) 1H-NMR spectrum of UDP-2-deoxy2-fluoro-α-D-glucopyranose. 8

9 doi: /nature16966 Supplementary Figure 3 NMR product characterization.1h- and 13C-NMR spectra of Octyl 6-thio-αD-galactopyranosyl phosphate disulfide; upper and lower panels, respectively. 9

10 Supplementary Figure 4 NMR product characterization. 1 H- and 13 C-NMR spectra of uridine-5'- phosphoric-α-(d-glucopyranosyl)methylphosphonic anhydride; upper and lower panel, respectively. 10