Supporting information

Supporting information The L-rhamnose Antigen: a Promising Alternative to α-gal for Cancer Immunotherapies Wenlan Chen,, Li Gu,#, Wenpeng Zhang, Edwin Motari, Li Cai, Thomas J. Styslinger, and Peng George Wang,* The Department of Chemistry and Biochemistry, The hio State University, 484 West 12th Avenue, Columbus, H 43210, USA # National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China These authors contributed equally to this work Contents 1. Supplementary figures S2 S6 2. Experimental section S7 S11 3. 1 H and 13 C NMR spectra S12 S20 S1

Figure S1. Characterization of synthetic Rha-conjugated BSA under different conditions. a) SDS-PAGE result of R1 conjugated BSA. b) MALDI spectra of R1 conjugated BSA (BSA, red; 1x PBS, green; 2x PBS, pink; sx PBS, yellow; 4x PBS brown; 5x PBS gray; dd H 2, orange). Initial model reactions employed bovine serum albumin (BSA) as a carrier protein in order for convenient characterization by SDS-PAGE and mass spectrometry. The conjugations between R1 and BSA were examined in PBS buffer with different salt concentrations (1x through 5x PBS, and dd H 2 ). It appeared that all the conjugations were successful under different medium in 1 h at RT, while slightly different installation ratios were present. Both MALDI and SDS-PAGE result confirmed that Rha installation ratio increased from 1x through 3x PBS buffer and reached maximum at 3x PBS. S2

Figure S2. SDS-PAGE evaluation of synthetic BSA and VA glycoconjugates. The glycoproteins were suspended in 12 μl sample buffer, and loaded on different lanes of a 1.5 mm-thick, 12% SDS-PAGE gel. The developed gels were visualized by Coomassie Brilliant Blue R-250 staining. It was observed that the order of the molecular weights should be BSA-Rha > BSA-linker > BSA, and VA-Rha > VA-linker > VA. The results confirmed the successful synthesis of the desired glycoconjugates. S3

Figure S3. ELISA assay of anti-rha antibody from the 1 st failed immunization experiment. Plates were coated with VA-R1 or VA-Linker-1 (10 µg/ml). Sera taken from the mice (n=5) immunized with BSA-R2 three times were added to the plates in two-fold dilutions. Anti-mouse IgG antibody and enzyme substrate tetramethylbenzidine (TMB) were added subsequently. Considering the interference of anti-linker-2 antibody, Linker-1 was used to synthesize the coating conjugates. But the absorbance of the two groups were at the same level, which might be resulted from the interference of the glycan moity on the native VA. Since VA is a glycosylated protein, some non-specific antibodies produced by Freund s complete adjuvant (FCA) interfered with the ELISA assay. S4

Figure S4. Detailed ELISA assay results for anti-rha antibody of each individual mouse from 4 different groups. Plates were coated with BSA-R1 (10 µg/ml). Sera were taken from the mice after three times of immunization with different immunogens. 34 female Balb/c mice were divided into the following 4 groups. a) Group I (No.1 19), mice immunized with VA-R2 + adjuvants (n=19). b) Group II (No.21 25), mice immunized with VA-linker-2 + adjuvants (n=5); Group III (No.26 30), mice immunized with PBS + adjuvants (n=5); Group IV (No.31 35), mice without any treatment (n=5). The absorbance was read at wavelength of 450 nm. The titers were calculated to the highest dilution that gave the D value beyond 0.1. Individual differences can be observed within the groups. Since absorbance decreases with the serum dilution folds at different extent for the individual mouse, large standard deviation appears especially at medium dilution folds in the first group. But S5

the overall absorbance of the Group I is remarkably higher than the other three groups at each dilution level, which indicated that only VA-R2 + adjuvants can effectively elicit the anti-rha antibody. S6

1,2,3,4-tetra--acetyl-α-L-rhamnopyranose (1) L-rhamnose (5.0 g, 30 mmol) in pyridine (20 ml) was added acetic anhydride (17 ml, 183 mmol) followed by addition of DMAP (0.1 g). After stirring overnight, the solvent was removed. The residue was diluted with EtAc and washed with 1N HCl (aq), sat. NaHC 3 (aq) and brine. The organic layer was concentrated and dried under vacuum to give crude product 1 for direct use in the next step. 1 H NMR (500 MHz, CDCl 3 ) δ 6.00 (d, J = 1.8 Hz, 1H), 5.29 (dd, J = 10.0, 3.3 Hz, 1H), 5.24 (dd, J = 3.3, 1.9 Hz, 1H), 5.11 (t, J = 10.0 Hz, 1H), 3.96-3.90 (m 2H), 2.15 (s, 3H), 2.14 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H), 1.22 (d, J = 6.4 Hz, 3H). Rha-linker 3 Peracetate α-l-rhamnose 1 (1.1 g, 3.31 mmol) and azido acceptor 2 (0.70 g, 3.97 mmol) in anhydrous CH 2 Cl 2 (10 ml) was cooled to 0 C, followed by addition of BF 3 -Et 2 (1 ml, 8.26 mmol) dropwise. The reaction mixture was allowed to warm up to RT slowly. After stirring overnight, the reaction was quenched by addition of sat. NaHC 3 (aq) and diluted by EtAc. The organic phase was washed sat. NaHC 3 (aq) and brine. The combined organic layers were dried over anhydrous Na 2 S 4. The residue after concentration was purified by flash silica gel column chromatography (3:1 hexanes/etac) to give product 3 (1.0 g, 68 %). 1 H NMR (500 MHz, CDCl 3 ) δ 5.30 (dd, J = 10.0, 3.5 Hz, 1H), 5.25 (dd, J = 3.5, 1.8 Hz, 1H), 5.06 (t, J = 9.9 Hz, 1H), 4.77 (d, J = 1.7 Hz, 1H), 3.95-3.89 (m, 1H), 3.82-3.78 (m, 1H), 3.69-3.65 (m, 9H), 3.39 (t, J = 5.1 Hz, 2H), 2.13 (s, 3H), 2.04 (s, 3H), 1.97 (s, 3H), 1.21 (d, J = 6.3 Hz, 3H); 13 C NMR (125.69 MHz, CDCl 3 ) δ 170.3, 170.2, 170.1, 97.8, 71.4, 71.1, 70.9, 70.4, 70.3, 70.1, 69.3, 67.4, 66.5, 50.9, 21.1, 21.0, 20.9, 17.6; HRMS calcd. for C 18 H 29 N 3 10 Na ([M + Na] + ) 4470.1745; found 470.1747. Rha-linker 4 Triacetyl azido compound 3 (0.25 g, 0.56 mmol) in anhydrous MeH (5 ml) was added NaMe (0.2 ml, 0.5 M in MeH). The reaction mixture was allowed to stir at RT for overnight. The acidic resin was added to neutralize the reaction. The resulting S7

solution was filtered and filtrate was concentrated to give product 4 (0.17 g, 95 %). 1 H NMR (500 MHz, CDCl 3 ) δ 4.69 (d, J = 1.6 Hz, 1H), 3.79-3.74 (m, 2H), 3.66-3.55 (m, 11H), 3.36-3.34 (m, 2H), 3.28 (m, 1H), 1.23 (d, J = 6.2 Hz, 3H); 13 C NMR (125.69 MHz, CDCl 3 ) δ 101.9, 74.2, 72.5, 72.4, 71.9, 71.7, 71.6, 71.4, 69.9, 67.9, 51.9, 18.2; HRMS calcd. for C 12 H 23 N 3 7 Na ([M + Na] + ) 344.1428; found 344.1435. Rha-linker 6 To a solution of azide 4 (0.17 g, 0.53 mmol), 5-hexynoic acid 5 (0.12 ml, 1.06 mmol) in EtH/H 2 (4 ml, 1:1) was added (+)-sodium L-ascorbate (0.26 g, 1.33 mmol) followed by CuS 4-5H 2 (67 mg). After being stirred at RT overnight, the reaction mixture was extracted with CHCl 3 to remove excess organic reagents. The resulting aqueous solution was concentrated and purified by flash silica gel column chromatography (9:3:1 EtAc/iPrH/H 2 ) to give product 6 (0.20 g, 87 %). 1 H NMR (500 MHz, CDCl 3 ) δ 7.52 (s, 1H), 4.56 (s, 2H), 4.43 (d, J = 1.5 Hz, 1H), 4.26 (t, J = 5.09 Hz, 2H), 3.59 (t, J = 5.2 Hz, 2H), 3.52 (dd, J = 3.4, 1.7 Hz, 1H), 3.48-3.44 (m, 1H), 3.35 (dd, J = 9.6, 3.4 Hz, 1H), 3.33-3.27 (m, 6H), 3.08 (t, J = 9.5 Hz, 1H), 2.45 (t, J = 7.7 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.67 (m, 2H), 0.96 (d, J = 6.2 Hz, 3H); 13 C NMR (125.69 MHz, CDCl 3 ) δ 176.0, 146.9, 122.8, 100.4, 72.6, 70.9, 70.8, 70.2, 70.1, 70.0, 69.1, 68.4, 66.3, 49.9, 33.1, 24.6, 24.3, 16.7; HRMS calcd. for C 18 H 31 N 3 9 Na ([M + Na] + ) 456.1953; found 456.1966. Rha-Linker-1 (R1) TSTU (83 mg, 0.28 mmol) and Et 3 N (40 μl, 0.28 mmol) was added to a solution of acid linker 6 (100 mg, 0.23 mmol) in 2 ml anhydrous DMF. The reaction was monitored by LC-MS. After been stirred at RT for 1 h, the free acid completely disappeared. The reaction mixture was then concentrated and dried under vacuum to give crude product R1 for direct use in the future. HRMS calcd. for C 22 H 34 N 4 11 Na ([M + Na] + ) 553.2116; found 553.2111. S8

Rha-linker 8 The solution of peracetate α-l-rhamnose 1 (1.0 g, 3.0 mmol) and acceptor 7 (0.85 g, 3.6 mmol) in anhydrous CH 2 Cl 2 (8 ml) was cooled to 0 C, followed by addition of BF 3 -Et 2 (0.93 ml, 7.5 mmol) dropwise. The reaction mixture was allowed to warm up to RT slowly. After stirring overnight, the reaction was quenched by sat. NaHC 3 (aq) and diluted with EtAc. The organic phase was washed sat. NaHC 3 (aq) and brine. The combined organic layers were dried over anhydrous Na 2 S 4. The residue after concentration was purified by flash silica gel column chromatography (2:1 hexanes/etac) to give product 8 (1.1 g, 73 %). 1 H NMR (500 MHz, CDCl 3 ) δ 7.84 (dd, J = 5.4, 3.1 Hz, 2H), 7.70 (dd, J = 5.4, 3.1 Hz, 2H), 5.27 (dd, J = 9.9, 3.5 Hz, 1H), 5.24 (dd, J = 3.5, 1.7 Hz, 1H), 5.03 (t, J = 9.9 Hz, 1H), 4.76 (d, J = 1.7 Hz, 1H), 3.90-3.87 (m, 3H), 3.75-3.72 (m, 3H), 3.66-3.64 (m, 2H), 3.62-3.58 (m, 1H), 2.14 (s, 3H), 2.04 (s, 3H), 1.96 (s, 3H), 1.18 (d, J = 6.2 Hz, 3H); 13 C NMR (125.69 MHz, CDCl 3 ) δ 170.3, 170.2, 170.1, 168.4 (2), 134.1 (2), 132.4 (2), 123.5 (2), 97.8, 71.4, 70.2, 69.7, 69.3, 68.3, 67.2, 66.5, 37.4, 21.1, 21.0, 20.9, 17.6; HRMS calcd. for C 24 H 29 N 11 Na ([M + Na] + ) 530.1633; found 530.1617. Rha-linker 9 The solution of peracetate α-l-rhamnose phthalamide 8 (0.4 g, 0.79 mmol) in anhydrous MeH (30 ml) was added hydrazine (0.40 ml, 12.7 mmol) dropwise. After stirring overnight, the reaction mixture was filtered and the filtrate was concentrated. The residue was purified by flash silica gel column chromatography (5:1 MeH/NH 3 -H 2 ) to give amine 9 (0.19 g, 96 %). 1 H NMR (500 MHz, CD 3 D) δ 4.68 (d, J = 1.5 Hz, 1H), 3.77 (dd, J = 3.5, 1.6 Hz, 1H), 3.75 (dd, J = 5.1, 2.0 Hz, 1H), 3.62-3.56 (m, 4H), 3.49 (t, J = 5.3 Hz, 2H), 3.33 (t, J = 9.4 Hz, 1H), 3.28 (m, 1H), 2.75 (t, J = 5.3 Hz, 2H), 1.22 (d, J = 6.2 Hz, 3H); 13 C NMR (125 MHz, CD 3 D) δ 101.9, 74.1, 73.7, 72.5, 72.4, 71.4, 69.9, 67.9, 42.3, 18.2; HRMS calcd. for C 10 H 22 N 6 ([M + H] + ) 252.1442; found 252.1443. S9

Rha-linker 10 The solution of amine 9 (50 mg, 0.20 mmol) and succinic anhydride (20 mg, 0.2 mmol) in anhydrous MeH (1 ml) was stirred at RT for 3 h. Then the solvent was removed and the residue was purified by flash silica gel column chromatography (2:1 CH 2 Cl 2 /MeH) to give acid 10 (60 mg, 92 %). 1 H NMR (400 MHz, CD 3 D) δ 4.68 (d, J = 1.5 Hz, 1H), 3.78 (dd, J = 3.4, 1.6 Hz, 1H), 3.76-3.72 (m, 1H), 3.64-3.55 (m, 6H), 3.51 (t, J = 5.6 Hz, 2H), 3.36-3.32 (m, 2H), 2.51-2.42 (m, 4H), 1.22 (d, J = 6.2 Hz, 3H); 13 C NMR (100 MHz, CD 3 D) δ 176.8, 173.9, 100.3, 72.6, 70.9, 70.8, 69.8, 69.3, 68.4, 66.4, 39.0, 31.1, 30.6, 16.6; HRMS calcd. for C 14 H 25 N 9 ([M + Na] + ) 374.1422 found 374.1424. Rha-Linker-2 (R2) TSTU (62 mg, 0.2 mmol) and Et 3 N (28 μl, 0.2 mmol) was added to a solution of acid linker 10 (60 mg, 0.17 mmol) in 2 ml anhydrous DMF. The reaction was monitored by LC-MS. After been stirred at RT for 1 h, the free acid completely disappeared. The reaction mixture was then concentrated and dried under vacuum to give crude product R2 for direct use in the next step. HRMS calcd. for C 18 H 28 N 2 11 Na ([M + Na] + ) 471.1585; found 471.1581. Linker-1 The suspension of azide 2 (0.1 g, 0.57 mmol), 5-hexynoic acid 5 (65 μl, 0.63 mmol), (+)-sodium L-ascorbate (0.28 g, 1.42 mmol) and CuS 4 (15 mg, 0.06 mmol) in EtH/H 2 (4 ml; v:v = 1:1) was stirred at RT overnight. Then the reaction was concentrated and the residue was purified by flash silica gel column chromatography (5:1 CH 2 Cl 2 /MeH) to give compound 11 (0.14 g, 85%) for next step. TSTU (63 mg, 0.21 mmol) and Et 3 N (30 μl, 0.21 mmol) was added to a solution of acid linker 11 (50 mg, 0.17 mmol) in 2 ml anhydrous DMF. The reaction was monitored by LC-MS. After been stirred at RT for 1 h, the free acid completely disappeared. The reaction mixture was then concentrated and dried under vacuum to S10

give crude product Linker-1 (in quantative yield) for direct use in the next step. HRMS calcd. for C 16 H 24 N 4 7 Na ([M + Na] + ) 407.1537; found 407.1545. Linker-2 The solution of 2-(2-aminoethoxy) ethanol (0.5 ml, 5.0 mmol) and succinic anhydride (0.5 g, 5.0 mmol) in anhydrous MeH (10 ml) was stirred at RT overnight. Then the solvent was removed to give crude product 12 directly for next step. TSTU (80 mg, 0.27 mmol) and Et 3 N (50 μl, 0.36 mmol) was added to a solution of acid linker 12 (50 mg, 0.24 mmol) in 2 ml anhydrous DMF. The reaction was monitored by LC-MS. After been stirred at RT for 1 h, the free acid completely disappeared. The reaction mixture was then concentrated and dried under vacuum to give crude product Linker-2 (in quantative yield) for direct use in the next step. HRMS calcd. for C 12 H 18 N 2 7 Na ([M + Na] + ) 325.1006; found 325.1008. S11

Ac Ac Ac Ac 1 S12

H N 3 2 S13

N 3 Ac Ac Ac 3 S14

N 3 H H H 4 S15

N N N H H H H 6 S16

H N 7 S17

Ac Ac Ac 8 N S18

NH 2 H H H 9 S19

S20