Diversity-oriented Enzymatic Modular Assembly of ABO Histo-blood Group Antigens

Supporting Information for Diversity-oriented Enzymatic Modular Assembly of ABO Histo-blood Group Antigens Jinfeng Ye,, Xian-wei Liu,, Peng Peng, Wen Yi, Xi Chen, Fengshan Wang,, Hongzhi Cao*,, National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan 250100, China Institute of Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA Key Laboratory of Chemical Biology, Ministry of Education, and School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China State Key Laboratory of Microbiology, Shandong University, Jinan 250100, China. J.Y. and X.L. contributed equally to this work. * To whom correspondence should be addressed: Email: hzcao@sdu.edu.cn (H. Cao). Table of Contents General Methods...S2 Experimental procedures...s2-s10 1 H and 13 C NMR spectra of compound 9...S11-S12 1 H and 13 C NMR spectra of compound 10...S13-S14 1 H and 13 C NMR spectra of compound 11......S15-S16 1 H and 13 C NMR spectra of compound 12......S17-S18 1 H and 13 C NMR spectra of compound 13...S19-S20 1 H and 13 C NMR spectra of compound 14...S21-S22 1 H and 13 C NMR spectra of compound 15...S23-S24 1 H and 13 C NMR spectra of compound 16......S25-S26 1 H and 13 C NMR spectra of compound 17......S27-S28 1 H and 13 C NMR spectra of compound 18...S29-S30 1 H and 13 C NMR spectra of compound 19...S31-S32 1 H and 13 C NMR spectra of compound 20......S33-S34 1 H and 13 C NMR spectra of compound 21......S35-S36 1 H and 13 C NMR spectra of compound 22...S37-S38 1 H and 13 C NMR spectra of compound 23...S39-S40 S1

General method All chemicals were obtained from commercial suppliers and used without further purification unless noted. Thin layer chromatography (TLC) was performed on silica gel plates 60 F 254 (Merck, Billerica MA). Plates were visualized under UV light and/or by treatment with 5% sulfuric acid in ethanol or p-anisaldehyde sugar stain followed by heating. Silica gel 60 (300-400 mesh, Haiyang, Qingdao, China) was used for flash column chromatography. Gel filtration chromatography was performed using a column (100 cm 2.5 cm) packed with BioGel P-2 Fine resins (Bio-Rad, Hercules, CA). 1 H NMR (600 MHz or 400 MHz) and 13 C NMR (151 MHz) spectra were recorded on Bruker AVANCE-600 spectrometer, Bruker AVANCE-400 spectrometer, or Agilent VNMRS-600 spectrometer at 25 C. NMR spectra were calibrated using solvent signals ( 1 H: 3.34 for CD 3 OD, 13 C: 77.0 for CDCl 3 ). High resolution electrospray ionization (ESI) mass spectra were obtained at the National Glycoengineering Research Center and Drug Testing and Analysis Center in Shandong University. BlNahK-EcGlmU 1, a fusion enzyme from Bifidobacterium longum N-acetylhexosamine-1-kinase (BlNahK) and Escherichia coli N-acetylglucosamine uridyltransferase (EcGlmU), Escherichia coli K-12 galactokinase (EcGalK) 2, Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP) 3, Neisseria meningitides 1 4-galactosyltransferase (NmLgtB) 4, Bifidobacterium infantis D-galactosyl- 1 3-N-acetyl-D-hexosamine phosphorylase (BiGalHexNAcP) 2, Bacteroides fragilis bifunctional L-fucokinase/GDP-L-fucose pyrophosphorylase (BfFKP) 5, Helicobacter mustelae 1 3-N-acetylgalactosaminyltransferase (BgtA) 6, Helicobacter pylori 1 2-fucosyltransferase (FutC) 7 were expressed and purified as described previously. The genes encoding the 1 2-fucosyltransferase (WbgL) 8 from Escherichia coli O126 fusing with the propeptide sequence of the lipase of Staphylococcus hyicus at N-terminal and human 1 3-galactosyltransferase (GTB) 9 were synthesized by GENEWIZ (Suzhou, China) according to the reported sequences. The synthetic genes were then constructed into expressing vector, expressed and purified as previously reported 8,9. Experimental Procedures Gal 1 3(Fuc 1-2)GlcNAc ProN 3 (9) Gal 1 3GlcNAc ProN 3 (4) 2 (160 mg, 0.34 mmol), L-fucose (85 mg, 0.52 mmol), adenosine 5'-triphosphate (ATP) (284 mg, 0.52 mmol) and guanosine 5'-triphosphate (GTP) (311 mg, 0.52 mmol) were dissolved in water in a 50 ml centrifuge tube containing Tris-HCl buffer (100 mmol, ph 7.5) and MnCl 2 (10 mmol). After the addition of appropriate amount of recombinant FKP 5 (2 mg) and FutC 7 (2 mg), water was added to bring the volume of the reaction mixture to 10 ml. The reaction mixture S2

was incubated in a shaking incubator at 37 C for 4 h with agitation at 100 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group H type I trisaccharide 9 (168 mg, 80%) as a white solid. 1 H NMR (400 MHz, D 2 O) δ 5.12 (d, J = 4.0 Hz, 1H), 4.57 (d, J = 7.6 Hz, 1H), 4.35 (d, J = 8.4 Hz, 1H), 4.22 (q, J = 6.5 Hz, 1H), 3.93 3.34 (m, 17 H), 3.28 (m, 2H), 2.00 (s, 3H), 1.76 (m, 2H), 1.15 (d, J = 6.6 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 173.56, 101.67, 100.07, 99.38, 77.11, 76.51, 75.30, 74.94, 73.33, 71.66, 69.29, 69.01, 68.62, 67.91, 66.97, 66.38, 61.03, 60.55, 54.74, 47.61, 28.04, 22.06, 15.07; HRMS (ESI) m/z calcd for C 23 H 41 N 4 O 15 [M+H] + 613.2568, found 613.2570. Gal 1 4(Fuc 1 2)GlcNAc ProN 3 (10) Gal 1 4GlcNAc ProN 3 (5) 4 (100 mg, 0.22 mmol), L-fucose (53 mg, 0.32 mmol), adenosine 5'-triphosphate (ATP) (175 mg, 0.32 mmol) and guanosine 5'-triphosphate (GTP) (190 mg, 0.32 mmol), MnCl 2 (10 mmol) were dissolved in water in a 50 ml centrifuge tube containing Tris-HCl buffer (100 mmol, ph 7.5). After the addition of appropriate amount of recombinant FKP 5 (2 mg) and WbgL 8 (3 mg), water was added to bring the volume of the reaction mixture to 10 ml. The reaction mixture was incubated in a shaking incubator at 27 C for 36 h. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group H type II trisaccharide 10 (95 mg, 72%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.32 (s, 1H), 4.55 (d, J = 7.7 Hz, 1H), 4.53 (d, J = 8.4 Hz, 1H), 4.23 (q, J = 6.5 Hz, 1H), 4.00 3.66 (m, 16 H), 3.48 (dd, J = 5.8, 7.7 Hz, 1H), 3.39 (t, J = 6.5 Hz, 2H), 2.07 (s, 3H), 1.86 (p, J = 6.2 Hz, 2H), 1.24 (d, J = 6.7 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 174.51, 101.17, 100.22, 99.38, 76.35, 76.03, 75.25, 73.53, 72.28, 71.66, 69.60, 69.14, 68.18, 67.22, 66.91, 61.12, 60.16, 55.29, 47.78, 28.10, 22.22, 15.33; HRMS (ESI) m/z calcd for C 23 H 41 N 4 O 15 [M+H] + 613.2568, found 613.2563. Gal 1 3(Fuc 1 2)GalNAc ProN 3 (11) The enzymatic fucosylation of disaccharide 6 2 (200 mg, 0.43 mmol) was performed following the same procedure as described above for 9. The product formation was S3

monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group H type III trisaccharide 11 (277 mg, 95%) as a white solid. 1 H NMR (400 MHz, D 2 O) δ 5.16 (d, J = 4.0 Hz, 1H), 4.56 (d, J = 7.7 Hz, 1H), 4.16 (q, J = 6.3 Hz, 1H), 4.11 4.07 (m, 3H), 3.95 (t, J = 4.5 Hz, 1H), 3.82 (d, J = 3.2 Hz, 1H), 3.77 (dd, J = 3.4, 9.7 Hz, 1H), 3.74 3.54 (m, 11H), 3.40 (m, 3H), 1.98 (s, 3H), 1.82 (p, J = 6.1 Hz, 2H), 1.12 (d, J = 6.5 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 173.50, 101.90, 99.12, 96.62, 76.05, 74.89, 73.88, 73.39, 71.69, 70.42, 69.45, 68.96, 68.94, 67.91, 66.67, 64.62, 61.07, 60.81, 49.40, 48.00, 27.91, 21.76, 15.25; HRMS (ESI) m/z calcd for C 23 H 44 N 5 O 15 [M+NH 4 ] + 630.2834, found 630.2836. Gal 1 3(Fuc 1 2)GalNAc ProN 3 (12) The enzymatic fucosylation of disaccharide 7 2 (200 mg, 0.43 mmol) was performed following the same procedure as described above for 9. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group H type IV trisaccharide 12 (233 mg, 89%) as a white solid. 1 H NMR (400 MHz, D 2 O) δ 5.16 (d, J = 4.0 Hz, 1H), 4.54 (d, J = 7.7 Hz, 1H), 4.26 (d, J = 7.5 Hz, 1H), 4.16 (q, J = 6.4 Hz, 1H), 4.04 (d, J = 1.8 Hz, 1H), 3.94 3.46 (m, 16H), 3.29 (m, 2H), 1.99 (s, 3H), 1.76 (m, 2H), 1.14 (d, J = 6.6 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 173.59, 102.55, 101.96, 99.09, 76.48, 75.88, 74.95, 74.67, 73.44, 71.67, 69.39, 69.00, 68.38, 67.90, 66.87, 66.68, 60.86, 60.82, 51.30, 47.61, 28.08, 22.14, 15.14; HRMS (ESI) m/z calcd for C 23 H 41 N 4 O 15 [M+H] + 613.2568, found 613.2577. Gal 1 4(Fuc 1 2)Glc ProN 3 (13) The enzymatic fucosylation of disaccharide 8 10 (100 mg, 0.24 mmol) was performed following the same procedure as described above for 10. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group H type VI trisaccharide 13 (66 mg, 49%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.30 (d, J = 2.2 Hz, 1H), 4.51 (d, J = 7.7 Hz, 1H), 4.44 (d, J = 8.0 Hz, 1H), 4.22 (q, J = 6.5 Hz, 1H), 4.00 3.64 (m, S4

14H), 3.58 (t, J = 9.2 Hz, 1H), 3.45 (t, J = 6.7 Hz, 3H), 3.32 (t, J = 8.6 Hz, 1H), 1.90 (p, J = 6.4 Hz, 2H), 1.22 (d, J = 6.5 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 102.20, 100.18, 99.27, 76.23, 75.80, 75.25, 75.13, 74.20, 73.49, 72.83, 71.59, 69.53, 69.05, 68.08, 67.33, 66.82, 61.02, 60.10, 47.79, 28.15, 15.21; HRMS (ESI) m/z calcd for C 21 H 37 N 3 NaO 15 [M+Na] + 594.2122, found 594.2120. GalNAc 1 3Gal 1 3(Fuc 1 2)GlcNAc ProN 3 (14) Trisaccharide 9 (73 mg, 0.12 mmol), N-acetylgalactosamine (GalNAc, 32 mg, 0.14 mmol), adenosine 5'-triphosphate (ATP, 79 mg, 0.14 mmol) and uridine 5'-triphosphate (UTP, 69 mg, 0.14 mmol) were dissolved in water in a 50 ml centrifuge tube containing Tris-HCl buffer (100 mmol, ph 8.0) and MgCl 2 (20 mmol). After the addition of appropriate amount of BlNahK/EcGlmU 1 (1.5 mg) and BgtA 6 (1.0 mg), water was added to bring the volume of the reaction mixture to 10 ml. The reaction mixture was incubated in a shaking incubator at 37 C for 12 hrs with agitation at 100 rpm. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 4:2:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group A type I tetrasaccharide 14 (92 mg, 95%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.22 (d, J = 4.1 Hz, 1H), 5.15 (d, J = 3.7 Hz, 1H), 4.68 (d, J = 7.6 Hz, 1H), 4.40 (d, J = 8.5 Hz, 1H), 4.31 (q, J = 6.5 Hz, 1H), 4.27 (t, J = 6.1 Hz, 1H), 4.19 (m, 2H), 3.99 3.90 (m, 6H), 3.79 3.70 (m, 9H), 3.64 (dd, J = 3.6, 8.2 Hz, 1H), 3.62 3.58 (m, 2H), 3.50 3.45 (m, 2H) 3.33 (m, 2H), 2.05 (s, 3H) 2.01 (s, 3H), 1.80 (m, 2H), 1.21 (d, J = 6. Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 174.72, 173.69, 101.72, 99.85, 99.03, 91.05, 77.17, 75.29, 75.27, 74.64, 73.79, 71.70, 70.82, 69.60, 68.73, 68.41, 67.55, 67.47, 67.01, 66.54, 62.77, 61.25, 61.08, 60.54, 54.67, 49.51, 47.63, 28.06, 22.16, 21.83, 15.08; HRMS (ESI) m/z calcd for C 31 H 53 N 5 NaO 20 [M+Na]+ 838.3182, found 838.3193. GalNAc 1 3Gal 1 4(Fuc 1 2)GlcNAc ProN 3 (15) The enzymatic fucosylation of trisaccharide 10 (25 mg, 0.04 mmol) was performed following the same procedure as described above for 14. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 4:2:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After S5

centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group A type II tetrasaccharide 15 (35 mg, 95%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.34 (d, J = 4.0 Hz, 1H), 5.17 (d, J = 3.6 Hz, 1H), 4.59 (d, J = 7.7 Hz, 1H), 4.50 (d, J = 8.4 Hz, 1H), 4.31 (q, J = 6.5 Hz, 1H), 4.24 4.20 (m, 3H), 4.00 3.64 (m, 19H), 3.45 (m, 1H), 3.38 (m, 2H), 2.05 (s, 3H), 2.03 (s, 3H), 1.84 (p, J = 6.2 Hz, 2H), 1.24 (d, J = 6.6 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 174.72, 174.44, 101.09, 99.96, 98.55, 91.21, 76.07, 75.57, 75.23, 75.07, 72.32, 72.26, 71.59, 70.98, 69.85, 68.39, 67.67, 67.57, 67.14, 66.78, 62.95, 61.22, 61.11, 60.06, 55.27, 49.41, 47.69, 28.01, 22.14, 21.87, 15.09; HRMS (ESI) m/z calcd for C 31 H 53 N 5 NaO 20 [M+Na] + 838.3182, found 838.3196. GalNAc 1 3Gal 1 3(Fuc 2)GalNAc ProN 3 (16) The enzymatic fucosylation of trisaccharide 11 (97 mg, 0.16 mmol) was performed following the same procedure as described above for 14. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 4:2:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group A type III tetrasaccharide 16 (116 mg, 90%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.25 (d, J = 4.1 Hz, 1H), 5.14 (d, J = 3.7 Hz, 1H), 4.84 (d, J = 2.0 Hz, 1H), 4.66 (d, J = 7.6 Hz, 1H), 4.29 4.09 (m, 7H), 3.99 (t, J = 6.1 Hz, 1H), 3.95 (d, J = 2.6 Hz, 1H), 3.91 (m, 2H), 3.86 (d, J = 2.6 Hz, 1H), 3.85 (m, 1H), 3.78 3.69 (m, 7H), 3.63 (d, J = 2.9 Hz, 1H), 3.60 (dd, J = 4.2, 7.7 Hz, 1H), 3.55 (dd, J = 3.1, 10.3 Hz, 1H), 3.48 3.39 (m, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.85 (p, J = 6.1 Hz, 2H), 1.17 (d, J = 6.5 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 174.70, 173.53, 102.03, 98.66, 96.54, 91.11, 75.25, 74.70, 74.38, 72.71, 71.70, 70.84, 70.37, 69.85, 69.04, 68.42, 67.59, 67.54, 66.78, 64.57, 62.86, 61.29, 61.11, 60.87, 49.46, 49.34, 47.99, 27.94, 21.86, 21.85, 15.24; HRMS (ESI) m/z calcd for C 31 H 53 N 5 NaO 20 [M+Na] + 838.3182, found 838.3206. GalNAc 1 3Gal 1 3(Fuc 1 2)GalNAc ProN 3 (17) The enzymatic fucosylation of trisaccharide 12 (75 mg, 0.12 mmol) was performed following the same procedure as described above for 14. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 4:2:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After S6

centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group A type IV tetrasaccharide 17 (94 mg, 94%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.24 (d, J = 4.1 Hz, 1H), 5.14 (d, J = 3.8 Hz, 1H), 4.64 (d, J = 7.6 Hz, 1H), 4.29 (d, J = 7.6 Hz, 1H), 4.25 (m, 2H), 4.21 4.19 (m, 2H), 4.11 (d, J = 1.9 Hz, 1H), 3.96 3.68 (m, 16H), 3.60 3.56 (m, 2H), 3.53 (dd, J = 3.2, 10.3 Hz, 1H), 3.33 (m, 2H), 2.03 (s, 3H), 2.01 (s, 3H), 1.77 (m, 2H), 1.19 (d, J = 6.6 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 174.70, 173.64, 102.64, 102.01, 98.66, 91.10, 76.96, 75.32, 74.72, 74.65, 72.74, 71.66, 70.84, 69.75, 68.44, 68.39, 67.57, 67.53, 66.90, 66.82, 62.85, 61.27, 60.90, 60.85, 51.18, 49.45, 47.63, 28.10, 22.23, 21.85, 15.14; HRMS (ESI) m/z calcd for C 31 H 53 N 5 NaO 20 [M+Na] + 838.3182, found 838.3209. GalNAc 1 3Gal 1 4(Fuc 1 2)Glc ProN 3 (18) The enzymatic fucosylation of trisaccharide 13 (20 mg, 0.04 mmol) was performed following the same procedure as described above for 14. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 4:2:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group A type VI tetrasaccharide 18 (30 mg, 92%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.35 (d, J = 3.3 Hz, 1H), 5.20 (d, J = 3.6 Hz, 1H), 4.58 (d, J = 7.6 Hz, 1H), 4.44 (d, J = 7.9 Hz, 1H), 4.32 (q, J = 6.4 Hz, 1H), 4.24 4.20 (m, 3H), 4.02 3.71 (m, 16H), 3.66 (d, J = 4.8 Hz, 1H), 3.58 (t, J = 9.3 Hz, 1H), 3.47 (m, 3H), 3.33 (t, J = 9.0 Hz, 1H), 2.04 (s, 3H), 1.92 (p, J = 6.2 Hz, 2H), 1.25 (d, J = 6.3 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 174.75, 102.23, 100.03, 98.54, 91.24, 75.92, 75.62, 75.29, 75.05, 74.29, 72.87, 72.31, 71.61, 71.00, 69.86, 68.41, 67.68, 67.58, 67.36, 66.81, 62.97, 61.24, 61.11, 60.08, 49.43, 47.80, 28.17, 21.91, 15.09; HRMS (ESI) m/z calcd for C 29 H 50 N 4 NaO 20 [M+Na] + 797.2916, found 797.2933. Gal 1 3Gal 1 3(Fuc 1 2)GlcNAc ProN 3 (19) Trisaccharide 9 (35 mg, 0.06 mmol), galactose (12 mg, 0.07 mmol) adenosine 5'- triphosphate (ATP, 38 mg, 0.07 mmol) and uridine 5'-triphosphate (UTP, 33 mg, 0.07 mmol) were dissolved in water in a 50 ml centrifuge tube containing Tris-HCl buffer (100 mmol, ph 7.5) and MgCl 2 (20 mmol). After the addition of appropriate amount of EcGalK 2 (2 mg), BLUSP 3 (1.5 mg) and GTB 9 (2.0 mg), water was added to bring S7

the volume of the reaction mixture to 10 ml. The reaction mixture was incubated in a shaking incubator at 37 C for 36 hrs. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). To stop the reaction, the reaction mixture was added with same volume of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group B type I tetrasaccharide 19 (40 mg, 90%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.18 (d, J = 3.7 Hz, 1H), 5.17 (d, J = 4.3 Hz, 1H), 4.66 (d, J = 7.6 Hz, 1H), 4.37 (d, J = 8.4 Hz, 1H), 4.29 (q, J = 6.5 Hz, 1H), 4.22 (m, 2H), 3.96 3.43 (m, 21 H), 3.30 (m, 2H), 2.03 (s, 3H), 1.78 (m, 2H), 1.18 (d, J = 6.5 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 173.69, 101.71, 99.92, 99.06, 92.89, 77.21, 75.91, 75.23, 74.44, 73.73, 71.68, 70.92, 69.60, 69.24, 69.15, 68.69, 67.97, 67.54, 66.97, 66.49, 63.26, 61.16, 61.03, 60.53, 54.63, 47.61, 28.05, 22.14, 15.04; HRMS (ESI) m/z calcd for C 29 H 51 N 4 O 20 [M+H] + 775.3097, found 775.3098. Gal 1 3Gal 1 4(Fuc 1 2)GlcNAc ProN 3 (20) The enzymatic fucosylation of trisaccharide 10 (40 mg, 0.06 mmol) was performed following the same procedure as described above for 19. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group B type II tetrasaccharide 20 (48 mg, 95%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.29 (d, J = 4.1 Hz, 1H), 5.21 (d, J = 2.9 Hz, 1H), 4.58 (d, J = 7.6 Hz, 1H), 4.46 (d, J = 8.4 Hz, 1H), 4.27 (q, J = 6.6 Hz, 1H), 4.25 (d, J = 2.6 Hz, 1H), 4.17 (t, J = 6.1 Hz, 1H), 3.97 3.61 (m, 20 H), 3.42 3.31 (m, 3 H), 2.02 (s, 3H), 1.81 (p, J = 6.4 Hz, 2H), 1.20 (d, J = 6.6 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ174.40, 101.10, 99.99, 98.67, 92.88, 76.05, 76.03, 75.20, 74.82, 72.43, 72.23, 71.57, 71.01, 69.87, 69.37, 69.14, 67.93, 67.56, 67.09, 66.68, 63.35, 61.15, 61.03, 60.04, 55.20, 47.64, 27.98, 22.08, 15.03; HRMS (ESI) m/z calcd for C 29 H 51 N 4 O 20 [M+H] + 775.3097, found 775.3095 Gal 1 3Gal 1 3(Fuc 1 2)GalNAc ProN 3 (21) The enzymatic fucosylation of trisaccharide 11 (40 mg, 0.08 mmol) was performed following the same procedure as described above for 19. The product formation was S8

monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group B type III tetrasaccharide 20 (30 mg, 60%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.23 (d, J = 4.1 Hz, 1H), 5.21 (d, J = 3.4 Hz, 1H), 4.84 (s, 1H), 4.68 (d, J = 7.6 Hz, 1H), 4.28 4.22 (m, 3H), 4.19 (s, 1H), 4.11 (s, 2H), 4.00 (t, J = 6.2 Hz, 1H), 3.95 3.70 (m, 13H), 3.64 (dd, J = 4.4, 7.7 Hz, 1H), 3.62 (d, J = 2.8 Hz, 1H), 3.54 (dd, J = 3.1, 10.0 Hz, 1H), 3.43 (m, 3H), 2.03 (s, 3H), 1.86 (p, J = 6.2 Hz, 2H), 1.16 (d, J = 6.5 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 173.53, 102.11, 98.72, 96.53, 92.78, 75.79, 74.50, 74.42, 72.71, 71.69, 70.92, 70.36, 69.90, 69.34, 69.18, 69.07, 67.98, 67.55, 66.75, 64.57, 63.29, 61.25, 61.10, 60.84, 49.34, 47.99, 27.95, 21.86, 15.23; HRMS (ESI) m/z calcd for C 29 H 50 N 4 NaO 20 [M+Na] + 797.2916, found 797.2925. Gal 1 3Gal 1 3(Fuc 1 2)GalNAc ProN 3 (22) The enzymatic fucosylation of trisaccharide 12 (20 mg, 0.10 mmol) was performed following the same procedure as described above for 19. The product formation was monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group B type IV tetrasaccharide 21 (25 mg, 98%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.22 (d, J = 4.2 Hz, 1H), 5.20 (d, J = 3.5 Hz, 1H), 4.64 (d, J = 7.4 Hz, 1H), 4.28 (d, J = 7.8 Hz, 1H), 4.26 4.22 (m, 3H), 4.10 (s, 1H), 3.96 3.67 (m, 17H), 3.63 (dd, J = 4.3, 7.6 Hz, 1H), 3.57 (m, 1H), 3.52 (dd, J = 3.1, 10.3 Hz, 1H), 3.32 (m, 2H), 2.02 (s, 3H), 1.79 (m, 2H), 1.17 (d, J = 6.6 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 173.61, 102.63, 102.07, 98.72, 92.78, 76.98, 75.86, 74.62, 74.51, 72.73, 71.62, 70.91, 69.78, 69.30, 69.13, 68.43, 67.95, 67.50, 66.85, 66.79, 63.26, 61.19, 60.86, 60.81, 51.14, 47.60, 28.08, 22.20, 15.09; HRMS (ESI) m/z calcd for C 29 H 51 N 4 O 20 [M+H] + 775.3097, found 775.3102. Gal 1 3Gal 1 4(Fuc 1 2)Glc ProN 3 (23) The enzymatic fucosylation of trisaccharide 13 (35 mg, 0.06 mmol) was performed following the same procedure as described above for 19. The product formation was S9

monitored by TLC (EtOAc/MeOH/H 2 O/HOAc, 8:3:1:0.2, v/v). The reaction was stopped by adding 10 ml of ice-cold ethanol and incubated at 4 C for 30 min. After centrifugation, the supernatant containing the product was concentrated, purified by BioGel P-2 column (eluted with H 2 O) to provide group B type VI tetrasaccharide 23 (44 mg, 97%) as a white solid. 1 H NMR (600 MHz, D 2 O) δ 5.30 (d, J = 4.1 Hz, 1H), 5.22 (d, J = 2.8 Hz, 1H), 4.57 (d, J = 7.6 Hz, 1H), 4.41 (d, J = 8.0 Hz, 1H), 4.29 (q, J = 6.5 Hz, 1H), 4.26 (d, J = 2.5 Hz, 1H), 4.18 (t, J = 6.1 Hz, 1H), 3.99 3.66 (m, 18H), 3.55 (t, J = 9.3 Hz, 1H), 3.45 3.41 (m, 3H), 3.31 (t, J = 8.5 Hz, 1H), 1.89 (p, J = 6.5 Hz, 2H), 1.21 (d, J = 6.6 Hz, 3H); 13 C NMR (151 MHz, D 2 O) δ 102.24, 100.04, 98.65, 92.90, 76.09, 75.85, 75.26, 74.79, 74.26, 72.79, 72.40, 71.58, 71.02, 69.88, 69.38, 69.15, 67.95, 67.56, 67.31, 66.71, 63.36, 61.16, 61.02, 60.06, 47.75, 28.12, 15.02; HRMS (ESI) m/z calcd for C 27 H 51 N 4 O 20 [M+NH 4 ] + 751.3097, found 751.3091. References 1. Zhai, Y.; Liang, M.; Fang, J.; Wang, X.; Guan, W.; Liu, X. W.; Wang, P.; Wang, F. Biotechnol. Lett. 2012, 34, 1321-1326. 2. Yu, H.; Thon, V.; Lau, K.; Cai, L.; Chen, Y.; Mu, S.; Li, Y.; Wang, P. G.; Chen, X. Chem. Commun. 2010, 46, 7507-7509. 3. Muthana, M. M.; Qu, J.; Li, Y.; Zhang, L.; Yu, H.; Ding, L.; Malekan, H.; Chen, X. Chem. Commun. 2012, 48, 2728-2730. 4. Lau, K.; Thon, V.; Yu, H.; Ding, L.; Chen, Y.; Muthana, M. M.; Wong, D.; Huang, R.; Chen, X. Chem. Commun. 2010, 46, 6066-6068. 5. Zhao, G.; Guan, W.; Cai, L.; Wang, P. G. Nat. Protoc. 2010, 5, 636-646. 6. Yi, W.; Shen, J.; Zhou, G.; Li, J.; Wang, P. G. J. Am. Chem. Soc. 2008, 130, 14420-14421. 7. Stein, D. B.; Lin, Y.-N.; Lin, C.-H. Adv. Syn. Catal. 2008, 350, 2313-2321. 8. Engels, L.; Elling, L. Glycobiology 2014, 24, 170-178. 9. Seto, N. O. L.; Palcic, M. M.; Hindsgaul, O.; Bundle, D. R.;Narang, S. A. Eur. J. Biochem. 1995, 234, 323 328. 10. Chen, C.; Zhang, Y.; Xue, M.; Liu, X.-w.; Li, Y.; Chen, X.; Wang, P. G.; Wang, F.; Cao, H. Chem. Commun. 2015, 51, 7689-7692. S10

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