Sensor Enzyme, UDP-Glc: Glycoprotein. Glucosyltransferase

Supporting Information The Recognition Motif of the Glycoprotein-Folding Sensor Enzyme, UDP-Glc: Glycoprotein Glucosyltransferase Kiichiro Totani, * Yoshito Ihara, Takashi Tsujimoto, Ichiro Matsuo, and Yukishige Ito * 1) General Procedure for Synthesis 2) Synthesis of CHO-MTX conjugates as inhibitors of UGGT 3) Synthesis of substrates for UGGT 4) UGGT activities of M9-BODIPY conjugates having alkyl-type linkers 1

1) General Procedure for Synthesis Reagents and solvents were purchased from standard suppliers and used without further purification. Reactions were monitored with TLC plates precoated with Merck silica gel 60 F254. Merck silica gel-60 was used for silica gel flash chromatography. Preparative thin layer chromatography (PTLC) was developed on Merck silica gel 60 F254 plates (0.5 mm thickness). 1 H and 13 C NMR spectra were recorded on JEOL AL-400 spectrometer in CDCl 3 or D 2 O solution (JEOL AL-400 spectrometer). MALDI-TOF MS spectra were recorded in the positive ion mode on an AXIMA-CFR Kompact MALDI (Shimadzu/KRATOS). HPLC was performed on a Waters 2695 (separation module), Waters 996 (photodiode array detector) and Waters 2475 (fluorescence detector) with a TSK-GEL Amide-80 column (TOSOH) or a Mightsil RP-18 column (Kanto Chemical Co.). α-mannosidase (from Jack Beans) was obtained from SIGMA-ALDRICH Co. 1,2-α-Mannosidase, recombinant (from Aspelgillus saitoi) was purchased from Seikagaku Co. 2

2) Synthesis of CHO-MTX conjugates as inhibitors of UGGT 3

Scheme S1. Synthesis of M7A-G-MTX as an inhibitor of UGGT. Synthesis of S3. A mixture of AgOTf (153 mg, 0.595 mmol), Cp 2 HfCl 2 (115.3 mg, 0.2977 mmol) and molecular sieves 4 Å (5g) in dry toluene (50 ml) was stirred at room temperature for 30 min, then cooled at -40 C. A solution of donor S2 (1) (714.2 mg, 0.4466 mmol) and acceptor S1 (2) (500 mg, 0.298 mmol) in dry toluene (10 ml) was added dropwise over 5 min. The mixture was stirred at -40 C for 30 min, -20 C for 30 min and -10 C for 4 h. The reaction was quenched with Et 3 N (1mL). The reaction mixture was diluted with EtOAc and filtered through a pad of Celite. The filtrate was washed with saturated aqueous NaHCO 3 and brine, successively. The Organic layer was dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOAc/toluene, 1:1) to give 504.2 mg (52%) of S3 (α-isomer) and 184.2 mg (19%) of S3 (β-isomer): TLC, R f 0.39 (α-isomer), 0.44 (β-isomer) (EtOAc/toluene, 1:2, 2); 1 H NMR (400 MHz, D 2 O, α-isomer) δ 1.93 (2 s, 3H x 2), 1.97 (3 s, 3H x 3), 1.98 (s, 3 H), 2.00 (s, 3 H), 2.01 (2 s, 3 H x 2), 2.03 (s, 3 H), 2.04 (s, 3 H), 2.06 (2 s, 3 H x 2), 2.08 (s, 3 H), 2.11 (s, 3 H), 2.13 (s, 3 H), 3.18-4.19 (m, 45 H), 4.33-5.00 (m, 25 H), 5.18-5.59 (m, 15 H), 5.59 (m, 1 H), 6.72-7.78 (m, 53 H); 13 C NMR (100 MHz, CDCl 3 ) δ 20.64 x 2, 20.68 x 4, 20.72 x 4, 20.79 x 2, 20.90 x 2, 21.02, 21.13, 56.55, 61.67, 61.99, 62.25 x 2, 65.64, 66.27 x 2, 66.33 x 2, 68.48 x 3, 68.55 x 2, 68.83 x 2, 68.90 x 2, 69.20 x 2, 69.44 x 2, 69.62 x 2, 70.05 x 2, 71.79, 72.03 x 2, 72.69 x 2, 72.95 x 2, 73.35 x 2, 74.01 x 2, 74.34 x 2, 74.47, 74.63 x 2, 75.85, 77.21 x 4, 77.50 x 2, 77.68 x 2, 77.83, 78.30, 79.54, 96.92, 97.00 x 3, 98.95, 99.26 x 2, 99.57, 99.73, 117.05, 123.04, 126.74, 126.96, 127.04, 127.32, 127.37 x 3, 127.45 x 2, 127.51 x 2, 127.55 x 4, 127.64 x 4, 127.67 x 5, 127.83 x 2, 127.88 x 5, 127.98 x 2, 128.11 x 4, 128.15 x 4, 128.24 x 4, 128.25 x 5, 128.34 x 2, 128.40 x 2, 128.46 x 2, 131.65, 133.37, 133.65, 137.63, 137.90, 137.96, 138.11, 138.18, 138.51, 138.61, 169.04 x 2, 169.24 x 2, 169.28 x 2, 169.55 x 4, 169.65 x 2, 170.10, 170.16 x 2, 170.26, 170.47, 170.67, 170.66, 170.77; MS (MALDI-TOF) calcd for C 168 H 188 N 2 O 64 Na (M+Na) + m/z 3280.1, found 3280.2. 4

Synthesis of S4. To a solution of S3 (α-isomer) (254.6 mg, 78.12 µmol) in n-buoh (4 ml) was added ethylenediamine (1 ml). After being stirred at 80 C for 24 h, the solvent was removed by concentration in vacuo and co-evaporated with toluene. The residue was dissolved in pyridine (6 ml), then Ac 2 O (3 ml) and DMAP (1.0 mg, 8.2 µmol) were added at 0 C. After being stirred at room temperature for 17 h, the mixture was quenched with MeOH at 0 C and concentrated in vacuo. The residue was diluted with EtOAc and washed with aqueous CuSO 4, brine, saturated aqueous NaHCO 3 and brine. The organic layer was dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (EtOH/toluene, 1:10) to give N-acetylated compound. [Ir(COD)(PMePh 2 ) 2 ]PF 6 (52.7 mg, 60.5 µmol) was dissolved in degassed THF (5 ml) and stirred under H 2 atmosphere for 15 min then stirred under N 2 atmosphere for 1 min. A solution of the resulting N- acetylated compound in THF (10 ml) was added at 0 C and stirred under N 2 atmosphere at room temperature for 1.5 h. Then water (1.5 ml), NaHCO 3 (203.2 mg, 2.419 mmol) and iodine (30.7 mg, 0.121 mmol) were added at 0 C. After being stirred at room temperature for 15 min, the mixture was diluted with EtOAc and washed with saturated aqueous Na 2 S 2 O 3 5H 2 O and brine. The organic layer was dried over Na 2 SO 4 and concentrated in vacuo. To the residue in MeOH (10 ml) was added Pd(OH) 2 (20% on carbon, 450 mg). The mixture was stirred under H 2 atmosphere for 72 h then filtered through a pad of celite. The filtrate and washings were concentrated in vacuo. The residue was dissolved in MeOH (100 ml), and NaOMe (28% in MeOH, 1 ml) was added at 0 C. After being stirred at room temperature for 24 h, the mixture was neutralized with Amberlyst 15E (H + ). The mixture was filtered and concentrated. The residue was purified by reverse phase chromatography (Waters Sep-Pak C 18, water) to give 39.6 mg (42%, 6 steps) of S4 (α/β, 2:1): TLC, R f 0.40 (water/i-proh, 1:2); 1 H NMR (400 MHz, D 2 O, α-isomer) δ 1.90 (s, 3 H), 1.93 (s, 3 H), 3.42-3.95 (m, 52 H), 4.01 (br s, 1 H), 4.11 (br s, 1 H), 4.45 (d, J = 7.4 Hz 1H), 4.56 (d, J = 3.1 Hz, 1 H), 4.65 (s, 1 H), 4.68 (s, 1 H), 4.72 (s, 1 H), 4.89 (s, 5

1 H), 4.91 (s, 1 H), 4.94 (s, 1 H), 5.00 (s, 1 H), 5.26 (s, 1 H); MS (MALDI-TOF) calcd for C 58 H 98 N 2 O 46 Na (M+Na) + m/z 1581.5, found 1581.9. Synthesis of S5. Man 7 (A)GlcNAc 2 (S4) (22.7 mg, 14.56 µmol) was dissolved in saturated aqueous NH 4 HCO 3 (2 ml) and stirred at 40 C for 48 h then the mixture was concentrated and co-evaporated with H 2 O in vacuo. The residue was dissolved in dioxane-h 2 O (1:1)(2mL) then NaHCO 3 (5.9 mg, 69.89 µmol) and FmocGlyCl (11.0 mg, 34.94 µmol) were added at 0 C. After being stirred at 0 C for 1 h, the mixture was diluted with water and washed with CHCl 3. The aqueous layer was concentrated, and the residue was purified by reverse phase chromatography (Waters Sep-Pak C 18, water for elution of recovered S4 then water/meoh, 1:1 for elution of FmocGly-linked saccharide) to give mixture of FmocGly-linked saccharide and exess FmocGly-derivatives. The mixture was dissolved in DMF (2 ml), and piperidine (0.4 ml) was added at 0 C. After being stirred at room temperature for 4 h, the mixture was concentrated in vacuo and co-evaporated with toluene. The residue was diluted with water and washed with CHCl 3. The aqueous layer was concentrated in vacuo, and the residue was purified by gel filteration (Sephadex G15, 20% MeOH) to give 16.1 mg (69%) of S5: TLC, R f 0.13 (water/i-proh, 1:2); 1 H NMR (400 MHz, D 2 O) δ 1.86 (s, 3 H), 1.92 (s, 3 H), 3.38-3.95 (m, 54 H), 4.01 (br s, 1 H), 4.10 (br s, 1 H), 4.46 (d, J = 6.8 Hz, 1 H), 4.65 (s, 1 H), 4.72 (s, 1 H), 4.89 (s, 1 H), 4.91 (s, 1 H), 4.92 (d, J = 9.5 Hz, 1 H), 4.94 (s, 1 H), 4.99 (s, 1 H), 5.26 (s, 1 H); MS (MALDI-TOF) calcd for C 60 H 102 N 4 O 46 Na (M+Na) + m/z 1637.6.7, found 1637.3. 6

Synthesis of M7A-G-MTX. To a cold (-0 C) solution of S5 (14.1 mg, 8.73 µmol) and MTX(α t Bu) (2) (5.3 mg, 10.5 µmol) in water-meoh (1:1) (1 ml) were added DMT-MM [4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chloride](3) (2.9 mg, 10.5 µmol) and N-methylmorpholine (1.0 µl, 8.7 µmol). The mixture was stirred at room temperature for 15 h, then concentrated in vacuo. The residue was purified by reverse phase chromatography (Waters Sep-Pak C 18, water/meoh, 1:0 to 1:1) to give Man 7 (A)GlcNAc 2 -Gly-MTX(α t Bu). Man 7 (A)GlcNAc 2 -Gly-MTX(α t Bu) was dissolved in CF 3 COOH (1 ml) at 0 C. After being stirred at room temperature for 1 h, the mixture was concentrated and coevaporated with toluene in vacuo. The residue was purified by HPLC (TSKgel Amide-80, 7.6 mmφ x 30 cm, 40 C, 3% AcOH-Et 3 N aq.(ph 7.3)/CH 3 CN, 35:65 to 50:50, 80min, 3 ml/min) to give 11.5 mg (64%) of M7A-G-MTX: TLC, R f 0.38 (water/i-proh, 1:2); 1 H NMR (400 MHz, D 2 O) δ 1.78 (s, 3 H), 1.82-2.00 (m, 1 H), 1.92 (s, 3 H), 2.04-2.15 (m, 1 H), 2.20-2.36 (m, 2 H), 3.05 (s, 3 H), 3.46-3.96 (m, 57 H), 4.02 (br s, 1 H), 4.12 (br s, 1 H), 4.22 (m, 1 H), 4.43 (d, J = 7.8 Hz, 1 H), 4.53 (s, 1 H), 4.73 (s, 1 H), 4.87 (d, J = 9.5 Hz, 1 H), 4.90 (s, 1 H), 4.92 (s, 1 H), 4.96 (s, 1 H), 5.01 (s, 1 H), 5.27 (s, 1 H), 6.58 (d, J = 8.5 Hz, 2 H), 7.46 (d, J = 8.3 Hz, 2 H), 8.35 (s, 1 H); MS (MALDI-TOF) calcd for C 80 H 123 N 12 O 50 Na (M+Na) + m/z 2074.7, found 2074.7. Synthesis of GN2-G-MTX. Chitobiose (50.0 mg, 0.118 mmol) was subjected to a series of reactions in a manner as described for M7A-G-MTX (from S4). The mixture was purified by HPLC as described to give 48.7 mg (45%, 5steps) of GN2-G-MTX: TLC, R f 0.38 (water/i-proh, 1:2); 1 H NMR (400 MHz, D 2 O) δ 1.79 (s, 3 H), 1.82-1.99 (m, 1 H), 1.92 (s, 3 H), 2.04-2.14 (m, 1 H), 2.21-2.38 (m, 2 H), 2.91 (s, 3 H), 3.22-3.82 (m, 17 H), 4.22 (m, 1 H), 4.63 (d, J = 8.0 Hz, 1 H), 4.87 (d, J = 9.5 Hz, 1 H), 6.51 (d, J = 8.5 Hz, 2 H), 7.43 (d, J = 8.5 Hz, 2 H), 8.28 (s, 1 H); MS (MALDI-TOF) calcd for C 38 H 53 N 12 O 15 (M) + m/z 917.4, found 916.9. 7

Synthesis of M1-G-MTX. The reaction mixture contained, in a total volume of 1 ml, α-mannosidase (Jack bean) (62.5 µg, 1 U), 50 mm Tris HCl buffer, ph 5.86, and M1-G-MTX (2) (3.33 mg, 2.37 µmol). After 48 h at 37 C, the reactions were stopped by adding excess CH 3 CN. The reaction mixture was purified by HPLC (TSKgel Amide-80, 7.6 mmφ x 30 cm, 40 C, 3% AcOH-Et 3 N aq.(ph 7.3)/CH 3 CN, 35:65 to 50:50, 80 min, 3 ml/min) to give 1.64 mg (64%) of M1-G-MTX: 1 H NMR (400 MHz, D 2 O) δ 1.80 (s, 3 H), 1.84-1.98 (m, 1 H), 1.94 (s, 3 H), 2.03-2.14 (m, 1 H), 2.24-2.38 (m, 2 H), 3.04 (s, 3 H), 3.26-4.00 (m, 25 H), 4.25 (m, 1 H), 4.47 (d, J = 7.8 Hz, 1 H), 4.88 (d, J = 9.8 Hz, 1 H), 5.05 (s, 1 H), 6.72 (d, J = 7.8 Hz, 2 H), 7.54 (d, J = 8.8 Hz, 2 H), 8.43 (s, 1 H); MS (MALDI-TOF) calcd for C 44 H 62 N 12 O 20 (M) + m/z 1078.4, found 1078.9. Synthesis of M5A-G-MTX. The reaction mixture contained, in a total volume of 1 ml, 1,2-α-mannosidase, recombinant (from Aspergilllus saitoi) (5 µg, 0.05 U), 100 mm AcOH/Et 3 N buffer, ph 5.5, and M7A-G-MTX (4 mg, 1.95 µmol). After 5 h at 37 C, the reactions were stopped by adding excess CH 3 CN. The reaction mixture was purified by HPLC (TSKgel Amide-80, 7.6 mmφ x 30 cm, 40 C, 3% AcOH-Et 3 N aq.(ph 7.3)/CH 3 CN, 35:65 to 50:50, 80min, 3 ml/min) to give 2.1 mg (61%) of M5A-G-MTX: 1 H NMR (400 MHz, D 2 O) δ 1.79 (s, 3 H), 1.82-1.98 (m, 1 H), 1.93 (s, 3 H), 2.02-2.15 (m, 1 H), 2.20-2.38 (m, 2 H), 3.06 (s, 3 H), 3.22-3.95 (m, 44 H), 4.02 (br s, 1 H), 4.13 (br s, 1 H), 4.24 (m, 1 H), 4.47 (d, J = 7.3 Hz, 1 H), 4.56 (s, 1 H), 4.70 (s, 1 H), 4.74 (s, 1 H), 4.78 (s, 1 H), 4.87 (d, J = 9.8 Hz, 1 H), 4.97 (s, 1 H), 6.67 (d, J = 8.5 Hz, 2 H), 7.51 (d, J = 8.5 Hz, 2 H), 8.41 (s, 1 H); MS (MALDI-TOF) calcd for C 68 H 102 N 12 O 40 (M) + m/z 1726.6, found 1726.9. 8

3) Synthesis of substrates for UGGT. Synthesis of M9-G-E-Fmoc. To a solution of M9-G (4) (29.4 mg, 15.2 µmol) and FmocGlu(α t Bu) (10.3 mg, 24.3 µmol) in water-meoh (1:1) (1 ml) were added DMT-MM [4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chrolide](3) (6.7 mg, 24.3 µmol) and N-methylmorpholine (1.6 µl, 15.2 µmol). The mixture was stirred at room temperature for 20 h, then concentrated in vacuo. The residue was purified by reverse phase chromatography (Waters Sep-Pak C 18, water/meoh, 1:0 to 1:1) and following gel filtration (Sephadex G15, 20% MeOH) to give 27.5 mg (77%) of M9-G-E-Fmoc: TLC, R f 0.68 (water/i-proh, 1:2); 1 H NMR (400 MHz, D 2 O) δ 1.35 (s, 9 H), 1.83 (s, 3 H), 1.93 (s, 3 H), 1.62-1.82 (m, 1 H), 1.82-2.10 (m, 1 H), 2.10-2.40 (m, 2 H), 3.40-4.00 (m, 73 H), 4.03 (br s, 1 H), 4.09 (br s, 1 H), 4.46 (br s, 1 H), 4.65 (s, 1 H), 4.67 (s, 1 H), 4.74 (s, 1 H), 4.92 (m, 1 H x 3), 5.01 (s, 1 H), 5.18 (s, 1 H), 5.20 (s, 1 H), 5.28 (s, 1 H), 6.82 (m, 4 H), 7.06 (m, 4 H); MS (MALDI-TOF) calcd for C 96 H 147 N 5 O 61 Na (M+Na) + m/z 2368.8, found 2369.2. Synthesis of M9-G-E. Compound M9-G-E-Fmoc (35.7 mg, 27.5 µmol) was dissolved in DMF (4 ml), and piperidine (0.8 ml) was added at 0 C. After being stirred at room temperature for 24 h, the mixture was concentrated in vacuo and co-evaporated with toluene. The residue was purified by gel filtration (Sephadex G15, 20% MeOH) to give 23.1 mg (92%) of M9-G-E: TLC, R f 0.14 (water/i-proh, 1:2); 1 H NMR (400 MHz, D 2 O) δ 1.38 (s, 9 H), 1.88 (s, 3 H), 1.94 (s, 3 H), 1.93-2.15 (m, 1 H), 2.17-2.25 (m, 1 H), 2.25-2.42 (m, 2 H), 3.42-3.98 (m, 67 H), 4.03 (br s, 1 H), 4.10 (br s, 1 H), 4.48 (d, J = 7.1 Hz, 1 H), 4.65 (s, 1 H), 4.67 (s, 1 H), 4.74 (s, 1 H), 4.92 (m, 1 H x 3), 5.02 (s, 1 H), 5.18 (s, 1 H), 5.21 (s, 1 H), 5.28 (s, 1 H); MS (MALDI-TOF) calcd for C 81 H 137 N 5 O 59 Na (M+Na) + m/z 2146.8, found 2147.1. 9

Synthesis of M9-G-Fmoc. M9 (1) (5.0 mg, 2.7 µmol) was dissolved in saturated aqueous NH 4 HCO 3 (1 ml) and stirred at 40 C for 24 h then the mixture was concentrated and co-evaporated with H 2 O in vacuo. The residue was dissolved in dioxane-h 2 O (1:1)(2mL) then NaHCO 3 (0.5 mg, 6.4 µmol) and FmocGlyCl (1.0 mg, 3.2 µmol) were added at 0 C. The mixture was stirred at 0 C for 1 h then NaHCO 3 (0.5 mg, 6.4 µmol) and FmocGlyCl (1.0 mg, 3.2 µmol) were added. After being stirred at 0 C for 1 h, the mixture was diluted with water (10 ml) and washed with EtOAc (5 ml x 5). The aqueous layer was concentrated, and the residue was purified by reverse phase chromatography chromatography (Waters Sep-Pak C 18, water/meoh, 1:0 to 1:1) and following gel filtration (Sephadex G15, 20% MeOH) to give 4.8 mg (82%) of M9-G-Fmoc: TLC, R f 0.63 (water/i-proh, 1:2); 1 H NMR (400 MHz, D 2 O) δ 1.88 (s, 3 H), 1.95 (s, 3 H), 3.42-3.99 (m, 69 H), 4.05 (s, 1 H), 4.09 (s, 1 H), 4.48 (d, J = 7.1 Hz, 1 H), 4.68 (s, 1 H), 4.75 (s, 1 H), 4.91 (2s, 1 H x 2), 4.95 (s, 1 H), 4.99 (d, J = 9.5 Hz, 1 H), 5.02 (s, 1 H), 5.19 (s, 1 H), 5.22 (s, 1 H), 5.28 (s, 1 H), 6.84 (m, 4 H), 7.04 (m, 4 H); MS (MALDI-TOF) calcd for C 87 H 132 N 4 O 58 Na (M+Na) + m/z 2183.7, found 2183.4. Synthesis of M9-G-E-5TAMRA. To a solution of M9-G-E (0.5 mg, 0.24 µmol) and 5-TAMRA-SE (5-carboxytetramethylrhodamin, succinimidyl ester) (0.25 mg, 0.45 µmol) in DMF (1 ml) was added N,N-diisopropylethylamine (0.13 µl, 0.72 µmol). After being stirred at room temperature for 24h, the mixture was concentrated in vacuo. The residue was purified by PTLC (water/i-proh, 1:2) and then HPLC (TSKgel Amide-80, 7.6 mmφ x 30 cm, 40 C, 3% AcOH-Et 3 N aq.(ph 7.3)/CH 3 CN, 35:65 to 50:50, 80min, 3mL/min) to give 0.4 mg 10

(74%) of M9-G-E-5TAMRA: TLC, R f 0.22 (water/i-proh, 1:2); MS (MALDI-TOF) calcd for C 106 H 157 N 7 O 63 Na (M+Na) + m/z 2558.9, found 2559.2; HPLC chromatogram see below. TSKgel Amide-80, 4.6 mmφ x 25 cm, 40 C, 3% AcOH-Et 3 N aq.(ph 7.3)/CH 3 CN, 35:65 to 50:50, 50min,1mL/min Detection; λ Max 580 nm (Em. 555 nm) Synthesis of M9-G-E-6TAMRA. To a solution of M9-G-E (0.5 mg, 0.24 µmol) and 6-TAMRA-SE (6-carboxytetramethylrhodamin, succinimidyl ester) (0.25 mg, 0.45 µmol) in DMF (1 ml) was added N,N-diisopropylethylamine (0.13 µl, 0.72 µmol). After being stirred at room temperature for 24h, the mixture was concentrated in vacuo. The residue was purified by PTLC (water/i-proh, 1:2) and then HPLC (TSKgel Amide-80, 7.6 mmφ x 30 cm, 40 C, 3% AcOH-Et 3 N aq.(ph 7.3)/CH 3 CN, 35:65 to 50:50, 80 min, 3mL/min) to give 0.4 mg (70%) of M9-G-E-6TAMRA: TLC, R f 0.20 (water/i-proh, 1:2); MS (MALDI-TOF) calcd for C 106 H 157 N 7 O 63 (M) + m/z 2535.9, found 2536.3; HPLC chromatogram see below. 11

Synthesis of M9-G-E-BODIPY. To a solution of M9-G-E (0.50 mg, 0.24 µmol) and BODIPY(FL)-SE (4,4-difluoro-5,7-dimethyl-4- bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester) (0.2 mg, 0.5 µmol) in DMF (1 ml) was added N,N-diisopropylethylamine (0.13 µl, 0.72 µmol). After being stirred at room temperature for 24h, the mixture was concentrated in vacuo. The residue was purified by PTLC (water/i-proh, 1:2) and then HPLC (Mightsil RP-18, 10 mmφ x 25 cm, 40 C, water/ch 3 CN, 100:0 to 40:60, 55 min, 3mL/min) to give 0.4 mg (76%) of M9-G-E-BODIPY: TLC, R f 0.50 (water/i-proh, 1:2); MS (MALDI-TOF) calcd for C 95 H 150 BF 2 N 7 O 60 Na (M+Na) + m/z 2420.9, found 2421.2; HPLC chromatogram see below. TSKgel Amide-80, 4.6 mmφ x 25 cm, 40 C, TSKgel Amide-80, 4.6 mmφ x 25 cm, 40 C, water/ch 3% AcOH-Et 3 CN, 35:65 to 50:50, 50 min, 1 ml/min 3 N aq.(ph 7.3)/CH 3 CN, Detection; λ 35:65 to 50:50, Max 513 nm (Em. 504 nm) 50 min, 1mL/min Detection; λ Max 580 nm (Em. 555 nm) 12

Synthesis of M9-G-E-BODIPY(493). To a solution of M9-G-E (0.50 mg, 0.24 µmol) and BODIPY(493/503)-SE (4,4-difluoro-1,3,5,7- TSKgel Amide-80, 4.6 mmφ x 25 cm, 40 C, water/ch 3 CN, 35:65 to 50:50, 50 min, 1 ml/min Detection; λ Max 503 nm (Em. 493 nm) tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid, succinimidyl ester) (0.2 mg, 0.5 µmol) in DMF (1 ml) was added N,N-diisopropylethylamine (0.13 µl, 0.72 µmol). After being stirred at room temperature for 24h, the mixture was concentrated in vacuo. The residue was purified by PTLC (water/i- PrOH, 1:2) and then HPLC (Mightsil RP-18, 10 mmφ x 25 cm, 40 C, water/ch 3 CN, 100:0 to 40:60, 55 min, 3 ml/min) to give 0.4 mg (77%) of M9-G-E-BODIPY(493): TLC, R f 0.48 (water/i-proh, 1:2); MS (MALDI-TOF) calcd for C 97 H 155 BF 2 N 7 O 60 Na (M+Na) + m/z 2449.9, found 2449.5; HPLC chromatogram see below. Synthesis of M9-G-BODIPY. 13

To a TSKgel Amide-80, 4.6 mmφ x 25 cm, 40 C, water/ch 3 CN, 35:65 to 50:50, 50 min, 1mL/min Detection; λ Max 513 nm (Em. 504 nm) solution of M9-G (0.50 mg, 0.24 µmol) and BODIPY( FL)-SE (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester) (0.2 mg, 0.5 µmol) in DMF (1 ml) was added N,N-diisopropylethylamine (0.13 µl, 0.72 µmol). After being stirred at room temperature for 24h, the mixture was concentrated in vacuo. The residue was purified by PTLC (water/i-proh, 1:2) and then HPLC (Mightsil RP-18, 10 mmφ x 25 cm, 40 C, water/ch 3 CN, 100:0 to 40:60, 55 min, 3 ml/min) to give 0.4 mg (70%) of M9-G-BODIPY: TLC, R f 0.50 (water/i-proh, 1:2); MS (MALDI-TOF) calcd for C 86 H 137 BF 2 N 6 O 57 Na (M+Na) + m/z 2237.8, found 22237.4; HPLC chromatogram see below. 14

Synthesis of M9-G-P4-BODIPY. To a solution of M9-G (1.0 mg, 0.52 µmol) and N-Fmoc-amido-dPEG 4 acid [15-amino-N-(9- fluorenylmethoxycarbonyl)-4,7,10,13-tetraoxapentadecanoic acid] (0.64 mg, 1.2 µmol) in water-meoh (1:1) (1 ml) were added DMT-MM [4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chrolide] 3 (0.34 mg, 1.2 µmol) and N-methylmorpholine (0.057 µl, 0.52 µmol). The mixture was stirred at room temperature for 48 h, then concentrated in vacuo. The residue was dissolved in DMF (1 ml), and piperidine (0.2 ml) was added at 0 C. After being stirred at room temperature for 24 h, the mixture was concentrated in vacuo and co-evaporated with toluene. The residue was purified by gel filteration (Sephadex G15, 20% MeOH) to give 0.6 mg (57%, 2 steps) of M9-Gly-PEG 4 -acid: 1 H NMR (400 MHz, D 2 O) δ 1.85 (s, 3 H), 1.91 (s, 3 H), 3.02-4.07 (m, 88 H), 4.45 (d, J = 7.0 Hz, 1 H), 4.68 (s, 1 H), 4.71 (s, 1 H), 4.73 (s, 1 H), 4.89 (m, 1 H x 3), 5.00 (s, 1 H), 5.15 (s, 1 H), 5.18 (s, 1 H), 5.25 (s, 1 H); MS (MALDI-TOF) calcd for C 83 H 143 N 5 O 61 Na (M+Na) + m/z 2208.8, found 2208.8. To a solution of M9-Gly-PEG 4 -acid (0.60 mg, 0.27 µmol) and BODIPY(FL)-SE (4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester) (0.2 mg, 0.5 µmol) in DMF (1 ml) was added N,N-diisopropylethylamine (0.14 µl, 0.81 µmol). After being stirred at room temperature for 24h, the mixture was concentrated in vacuo. The residue was purified by PTLC (water/i- PrOH, 1:2) and then HPLC (Mightsil RP-18 10 mmφ x 25 cm, 40 C, water/ch 3 CN, 100:0 to 40:60, 55 min, 3 ml/min) to give 0.4 mg (64%) of M9-G-P4-BODIPY: TLC, R f 0.36 (water/i-proh, 1:2); MS (MALDI-TOF) calcd for C 97 H 156 BF 2 N 7 O 62 Na (M+Na) + m/z 2482.9, found 2482.9; HPLC chromatogram see below. 15

TSKgel Amide-80, 4.6 mmf x 25 cm, 40 C, water/ch 3 CN, 35:65 to 50:50, 50 min, 1 ml/min Detection; λ Max 513 nm (Em. 504 nm) Synthesis of M9-G-P8-BODIPY. To a solution of M9-G (1.0 mg, 0.52 µmol) and N-Fmoc-amido-dPEG 8 acid [27-amino-N-(9- fluorenylmethoxycarbonyl)-4,7,10,13,16,19,22,25-octaoxapentadecanoic acid] (0.84 mg, 1.2 µmol) in water-meoh (1:1) (1 ml) were added DMT-MM [4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chrolide](3) (0.34 mg, 1.2 µmol) and N-methylmorpholine (0.057 µl, 0.52 µmol). The mixture was stirred at room temperature for 48 h, then concentrated in vacuo. The residue was dissolved in DMF (1 ml), and piperidine (0.2 ml) was added at 0 C. After being stirred at room temperature for 24 h, the mixture was concentrated in vacuo and co-evaporated with toluene. The residue was purified by gel filteration (Sephadex G15, 20% MeOH) to give 0.7 mg (59%, 2 steps) of M9-Gly-PEG 8 -acid: 1 H NMR (400 MHz, D 2 O) δ 1.85 (s, 3 H), 1.91 (s, 3 H), 3.00-4.00 (m, 104 H), 4.44 (d, J = 7.1 Hz, 1 H), 4.63 (s, 1 H), 4.68 (s, 1 H), 4.71 (s, 1 H), 4.88 (m, 1 H x 3), 4.98 (s, 1 H), 5.15 (s, 1 H), 5.18 (s, 1 H), 5.25 (s, 1 H); MS (MALDI-TOF) calcd for C 91 H 159 N 5 O 65 Na (M+Na) + m/z 2384.9, found 2385.4. To a solution of M9-Gly-PEG 8 -acid (0.70 mg, 0.30 µmol) and BODIPY(FL)-SE (4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester) (0.2 mg, 0.6 µmol) in 16

DMF (1 ml) was added N,N-diisopropylethylamine (0.15 µl, 0.89 µmol). After being stirred at room temperature for 24h, the mixture was concentrated in vacuo. The residue was purified by PTLC (water/i- PrOH, 1:2) and then HPLC (Mightsil RP-18, 10 mmφ x 25 cm, 40 C, water/ch 3 CN, 100:0 to 40:60, 55 min, 3 ml/min) to give 0.5 mg (62%) of M9-G-P8-BODIPY: TLC, R f 0.28 (water/i-proh, 1:2); MS (MALDI-TOF) calcd for C 105 H 172 BF 2 N 7 O 66 Na (M+Na) + m/z 2659.0, found 2658.7; HPLC chromatogram see below. TSKgel Amide-80, 4,6 mmf x 25 cm, 40 C, water/ch 3 CN, 35:65 to 50:50, 50 min, 1 ml/min Detection; λ Max 513 nm (Em. 504 nm) Synthesis of M9-G-P12-BODIPY. To a solution of M9-G (1.0 mg, 0.52 µmol) and N-Fmoc-amido-dPEG 12 acid [39-amino-N-(9- fluorenylmethoxycarbonyl)-4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxapentadecanoic acid] (1.1 mg, 1.2 µmol) in water-meoh (1:1) (1 ml) were added DMT-MM [4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chrolide](3) (0.34 mg, 1.2 µmol) and N-methylmorpholine (0.057 µl, 0.52 µmol). The mixture was stirred at room temperature for 48 h, then concentrated in vacuo. The residue was dissolved in DMF (1 ml), and piperidine (0.2 ml) was added at 0 C. After being stirred at room temperature for 24 h, the mixture was concentrated in vacuo and co-evaporated with toluene. The residue was purified by gel filteration (Sephadex G15, 20% MeOH) to give 0.76 mg (58%, 2 steps) of M9-Gly-PEG 12 -acid: 1 H NMR (400 MHz, D 2 O) δ 1.84 (s, 3 H), 1.90 (s, 3 H), 3.06-4.07 (m, 120 H), 17

4.45 (d, J = 7.2 Hz, 1 H), 4.63 (s, 1 H), 4.66 (s, 1 H), 4.70 (s, 1 H), 4.88 (m, 1 H x 3), 4.98 (s, 1 H), 5.15 (s, 1 H), 5.17 (s, 1 H), 5.25 (s, 1 H); MS (MALDI-TOF) calcd for C 99 H 175 N 5 O 69 Na (M+Na) + m/z 2561.0, found 2561.4. To a solution of M9-Gly-PEG 8 -acid (0.76 mg, 0.30 µmol) and BODIPY(FL)-SE (4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester) (0.2 mg, 0.6 µmol) in DMF (1 ml) was added N,N-diisopropylethylamine (0.15 µl, 0.89 µmol). After being stirred at room temperature for 24h, the mixture was concentrated in vacuo. The residue was purified by PTLC (water/i- PrOH, 1:2) and then HPLC (Mightsil RP-18, 10 mmφ x 25 cm, 40 C, water/ch 3 CN, 100:0 to 40:60, 55 min, 3 ml/min) to give 0.5 mg (60%) of M9-G-P12-BODIPY: TLC, R f 0.14 (water/i-proh, 1:2)18 MS (MALDI-TOF) calcd for C 113 H 188 BF 2 N 7 O 70 Na (M+Na) + m/z 2835.1, found 2835.0; HPLC chromatogram see below. TSKgel Amide-80, 4.6 mmf x 25 cm, 40 C, water/ch 3 CN, 35:65 to 50:50, 50 min, 1 ml/min Detection; λ Max 513 nm (Em. 504 nm) 4) UGGT activities of M9-BODIPY conjugates having alkyl-type linkers 18

The experiments were carried out as described in Experimental Procedures (Glucose transfer assay, method B). References for Supporting Information. 19

(1) Matsuo, I., Totani, K., Tatami, A., and Ito, Y. (2006) Comprehensive synthesis of ER related highmannose-type sugar chains by convergent strategy. Tetrahedron, 62, 8262-8277. (2) Totani, K., Matsuo, I., and Ito, Y. (2004) Tight binding ligand approach to oligosaccharide-grafted protein. Bioorg. Med. Chem. Lett., 14, 2285-2289. (3) Kunishima, M., Kawachi, C., Hioki, K., Terao, K., and Tani, S. (2001) Formation of carboxamides by direct condensation of carboxylic acids and amines in alcohols using a new alcohol- and watersoluble condensing agent: DMT-MM. Tetrahedron, 57, 1551-1558. (4) Totani, K., Ihara, Y., Matsuo, I., and Ito, Y. (2006) High-mannose-type glycan modifications of dihydrofolate reductase using glycan-methotrexate conjugates. Bioorg. Med. Chem. 14, 5220-5229. 20