Supporting Information for

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Supporting Information for

An efficient Strategy for the Glycosylation of Total Bufadienolides in

Venenum Bufonis

Zhi-Hao Fu

†§

, Chao Wen

†§

, Qing-Mei Ye

‡§

, Wei Huang

†

, Xuan-Ming

Liu

†

and Ren-Wang Jiang

†

*

†

Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, and International Cooperation Laboratory of TCM modernization and Innovation drug Development, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China.

‡_{Department of Pharmacy, Hainan General Hospital, Haikou 570311, P. R. China.}

§

These authors contributed equally to this work.

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S2

Supporting Figures ... S5 Figure S1. Effect of enzyme amount on conversion rate of arenobufagin in Venenum Bufonis. ... S5 Figure S2. SDS-PAGE gel of recombinant His-UGT74AN1, ASP and YjiC1 purified by affinity chromatography. Lane M: Protein Marker; Lane 1: His6-tagged UGT74AN1; Lane 2: His6-tagged ASP; Lane 3: His6-tagged YjiC1. ... S5 Figure S3. Effect of PH (A), temperature (B), metal ion (C) reaction time (D) on enzymatic activity of YjiC1. ... S6 Figure S4. 1_{H spectrum of compound 3a in MeOD(400 MHz). ... S6}

Figure S5. 13_{C spectrum of compound 3a in MeOD(400 MHz). ... S7}

Figure S6. HSQC spectrum of compound 3a in MeOD(400 MHz). ... S7 Figure S7. HRESIMS spectrum of 3a. ... S8 Figure S8. 1_{H spectrum of compound 9a in MeOD(400 MHz). ... S8}

Figure S9. 13_{C spectrum of compound 9a in MeOD(400 MHz). ... S9}

Figure S10. HRESIMS spectrum of 9a. ... S9 Figure S11. 1_{H spectrum of compound 5b in MeOD(300MHz). ... S10}

Figure S12. 13_{C spectrum of compound 5b in MeOD(300MHz). ... S10}

Figure S13. HSQC spectrum of compound 5b in MeOD(300MHz). ... S11 Figure S14. HMBC spectrum of compound 5b in MeOD(300MHz). ... S11 Figure S15. HRESIMS spectrum of 5b. ... S12 Figure S16. HPLC trace of two control experiment.……….….S12 Supporting Tables ... S13 Table S1 Tentative characterization of bufadienolides in VB by HPLC-MS/MS... S13 Table S2 Tentative characterization of YjiC1 glycosylated bufadienolides by HPLC-MS/MS. ... S13 Table S3 Tentative characterization of UGT74AN1 glycosylated bufadienolides by HPLC-MS/MS. ... S14 Table S4 Tentative characterization of OleD ASP glycosylated bufadienolides by HPLC-MS/MS. ... S14 References ... S15

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MS,

1

_{H and}

13

_{C of NMR data of the glucosylated products}

The glycosylated products catalysed by YjiC1 were identified as compound 3a: arenobufagin 3-O-β-D-glucoside and compound 9a: Cinobufagin 3-O-β-D-glucoside compound 5b: Desacetylcinobufagin 3,16-di-O-β-D-glucoside, respectively.

Compound 3a: arenobufagin 3-O-β-D-glucoside1

HR-ESI-MS: m/z=579.2650 [M+H]+ calcd. for C30H42O11: 579.3751;

1_{H NMR (400MHz, MeOD): 7.93 (1H, dd, J = 9.7, 2.2 Hz, H-22), 7.54 (1H, s, H-21),}

6.33 (1H, d, J = 9.7 Hz, H-23), 4.38 (1H, d, J = 11.1 Hz, H-11), 4.08 (1H, s, H-3), 1.19 (3H, s, H3-19), 0.92 (3H, s, H3-18);

13_{C NMR (100MHz, MeOD) δC: 215.0 (C-12), 164.5 (C-24), 151.6 (C-21), 149.1}

(C-22), 123.2 (C-20), 115.8 (C-23), 102.6 (sugar C-1), 86.2 (C-14), 78.2 (sugar C-5), 77.8 (sugar C-3), 75.5 (sugar C-4), 75.2 (sugar C-2), 75.0 (C-11), 71.7 (C-3), 63.8 (C-13), 62.8 (sugar C-6), 42.0 (C-9), 41.7 (C-17), 40.8 (C-8), 39.2 (C-5), 38.0 (C-10), 33.5 (C-4), 33.2 (C-15), 31.2 (C-1), 29.1 (C-16), 28.2 (C-2), 27.8 (C-6), 23.8 (C-19), 22.8 (C-7), 18.0 (C-18).

Compound 9a: cinobufagin 3-O-β-D-glucoside2

HR-ESI-MS: m/z=605.3808 [M+H]+ calcd. for C32H44O11: 605.2992;

1_{H NMR (400MHz, MeOD): 8.05 (1H, dd, J = 9.8, 2.4 Hz, H-22), 7.39 (1H, s, H-21),} 6.26 (1H, d, J = 9.8 Hz, H-23), 4.34 (1H, d, J = 7.8 Hz, sugar H-1), 4.10 (1H, s, H-3), 3.87 (1H, dd, J = 11.8, 1.9 Hz), 3.76 (1H, s, H-15), 3.70 (1H, d, J = 5.3 Hz), 2.94 (1H, d, J = 9.3 Hz, H-17), 2.07 (1H, d, J = 11.9 Hz), 1.88 (3H, s, COCH3), 1.01 (3H, s, H3-19), 0.83 (3H, s, H3-18); 13_{C NMR (100MHz, MeOD) δ} C: 171.4 (COCH3), 163.8 (C-24), 153.2 (C-22), 150.6

(C-21), 118.1 (C-20), 113.8 (C-23), 102.4 (sugar C-1), 77.9 (sugar C-5), 77.6 (sugar C-2), 76.4 (C-16), 75.2 (sugar C-4), 74.9 (sugar C-3), 73.2 (C-14), 71.5 (C-3), 62.5 (sugar C-6), 60.5 (C-15), 51.2 (C-17), 46.1 (C-13), 40.4 (C-5), 40.7 (C-9), 37.1 (C-12), 36.1 (C-4), 34.3 (C-1), 30.8 (C-10), 30.4 (C-8), 27.2 (C-2), 26.6 (C-6), 23.7 (C-19), 21.8 (C-7), 21.4 (C-11), 20.1 (COCH3), 17.2 (C-18).

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S4

Compound 5b: desacetylcinobufagin 3,16-di-O-β-D-glucoside3

HR-ESI-MS: m/z=747.3202 [M+Na]+_{calcd. for C}

36H52O15: 747.2982;

1_{H NMR (300MHz, MeOD): 8.08 (1H, d, J = 10.3 Hz, H-22), 7.44 (1H, s, H-21), 6.21}

(1H, d, J = 9.7 Hz, H-23), 4.33 (1H, d, J = 7.7 Hz), 4.16 (1H, d, J = 7.7 Hz), 4.09 (s, 1H), 2.87 (d, J = 9.2 Hz, 1H), 1.00 (3H, s, H3-19), 0.81 (3H, s, H3-18);

13_{C NMR (75MHz, MeOD): 165.0 (C-24), 153.0 (C-21), 151.9 (C-22), 119.4 (C-20),}

113.9 (C-23), 103.4 (sugar C-1’), 102.7 (sugar C-1), 79.2 (sugar C-5), 78.2 (sugar C-5’), 78.0 (sugar C-3), 78.0 (sugar C-3’), 77.9 (C-16), 75.4 (sugar C-2), 75.2 (C-14), 74.9 (C-3), 73.5 (sugar C-2’), 71.74 (sugar C-4), 71.71 (sugar C-4’), 63.0 (C-15), 62.8 (sugar C-6), 62.8 (sugar C-6’), 50.8 (C-17), 46.1 (C-13), 40.6 (C-12), 40.5 (C-9), 37.4 (C-5), 36.3 (C-10), 34.6 (C-8), 31.1 (C-4), 30.8 (C-1) ,27.5 (C-2), 26.9 (C-6), 24.0 (C-19), 22.2 (C-7), 21.7 (C-11), 17.6 (C-18).

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Supporting Figures

Figure S1. Effect of enzyme amount on conversion rate of arenobufagin in Venenum Bufonis.

Figure S2. SDS-PAGE gel of recombinant His-UGT74AN1, ASP and YjiC1 purified by affinity chromatography. Lane M: Protein Marker; Lane 1: His6-tagged UGT74AN1; Lane 2: His6-tagged ASP; Lane 3: His6-tagged YjiC1.

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S6

Figure S3. Effect of PH (A), temperature (B), metal ion (C) reaction time (D) on enzymatic activity of YjiC1.

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Figure S5. 13C spectrum of compound 3a in MeOD(400 MHz).

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S8 Figure S7. HRESIMS spectrum of 3a.

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Figure S9. 13C spectrum of compound 9a in MeOD(400 MHz).

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S10

Figure S11. 1H spectrum of compound 5b in MeOD(300MHz).

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Figure S13. HSQC spectrum of compound 5b in MeOD(300MHz).

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S12 Figure S15. HRESIMS spectrum of 5b.

Figure S16. HPLC traces of two control experiments. A: arenobufagin 3-O--D-glucoside was incubated with the enzyme YjiC1 for one hour under the same conditions as the bioconversion reaction, and the hydrolyzed peak arenobufagin was observed; B: arenobufagin 3-O--D-glucoside was incubated for one hour under the same conditions as the bioconversion reaction without the enzyme YjiC1, and the peak for arenobufagin was not observed.

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Supporting Tables

Table S1 Tentative characterization of bufadienolides in VB by HPLC-MS/MS.

a_{Identification by comparing with standard compounds.}

Table S2 Tentative characterization of YjiC1 glycosylated bufadienolides by HPLC-MS/MS. No. tR MS MS/MS of [M+H]+ Identification [M+H]+ [2M+H]+ 1 9.18 417 399 335 Bufarenogin 2 9.93 403 805 367 349 aGamabufotalin 3 12.44 417 833 399 371 335 aArenobufagin 4 13.26 403 805 385 367 aTelocinobufagin 5 15.08 401 aDesacetylcinobufagin 6 17.11 445 889 409 367 349 Bufotalin 7 18.29 459 917 Cinobufotalin 8 20.46 387 773 369 351 333 255 aBufalin 9 24.14 443 885 401 365 347 aCinobufagin 10 24.68 367 349 339 253 aResibufogenin No. t'R MS MS/MS of [M+H]+ Predicted products [M+H]+ 1a 17.83 579 417 399 381 363 Bufarenogin 3-O-glucoside 2a 19.16 565 403 385 367 349 Gamabufotalin 3-O-glucoside 3a 23.71 579 417 399 381 Arenobufagin 3-O-glucoside 5b 24.86 725 401 Desacetylcinobufagin 3,16-di-O-glucoside 4a 33.75 565 403 367 349 Telocinobufagin 3-O-glucoside 6a 34.95 607 445 427 367 349 Bufotalin 3-O-glucoside 9b 36.8 767 443 365 Cinobufagin 3-di-O-glucoside 8a 38.77 549 387 369 351 Bufalin 3-O-glucoside 3 38.93 417 335 Arenobufagin

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S14

Table S3 Tentative characterization of UGT74AN1 glycosylated bufadienolides by HPLC-MS/MS. No. t'R MS MS/MS of [M+H]+ Predicted products [M+H]+ 2a 19.97 565 403 367 349 321 Gamabufotalin 3-O-glucoside 3a 23.37 579 417 399 381 363 335 Arenobufagin 3-O-glucoside 4a 33.29 565 403 385 367 349 339 Telocinobufagin 3-O-glucoside 6a 34.52 607 445 385 367 349 Bufotalin 3-O-glucoside 3 37.55 417 399 371 Arenobufagin 7a 38.1 621 543 441 423 381 363 345 Cinobufotalin 3-O-glucoside 8a 38.71 549 387 369 351 333 269 255 Bufalin 3-O-glucoside

10a 41.47 547 385 367 349 339 321 271 Resibufogenin 3-O-glucoside

9a 41.97 605 443 425 383 365 347 337 Cinobufagin 3-O-glucoside

4 43.92 403 385 367 349 339 Telocinobufagin

Table S4 Tentative characterization of OleD ASP glycosylated bufadienolides by HPLC-MS/MS. No. t'R MS MS/MS of [M+H]+ Predicted products [M+H]+ 2a 20.11 565 403 385 367 349 321 Gamabufotalin 3-O-glucoside 3a 23.09 579 417 399 381 363 Arenobufagin 3-O-glucoside 6a 34.57 607 445 427 385 367 349 339 Bufotalin 3-O-glucoside 9b 36.45 767 605 443 425 383 365 347 Cinobufagin 3-di-O-glucoside 3 37.53 417 399 371 Arenobufagin 8a 38.71 549 387 369 351 333 269 Bufalin 3-O-glucoside

10a 41.42 547 385 367 349 339 321 Resibufogenin 3-O-glucoside

9a 41.94 605 443 425 383 365 347 319 Cinobufagin 3-O-glucoside

4 43.92 403 385 367 349 331 271 253 Telocinobufagin

6 45.18 445 409 385 367 349 331 Bufotalin

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References

1. Wen, C.; Huang, W.; Zhu, X. L.; Li, X. S.; Zhang, F.; Jiang, R. W. UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation. Org. Lett., 2018, 20, 534-537.

2. Zhu, X. L.; Wen, C.; Ye, Q. M.; Xu, W.; Zou, D. L.; Liang, G. P.; Zhang, F.; Chen, W. N.; Jiang, R. W. Probing the stereoselectivity of OleD-catalyzed glycosylation of cardiotonic steroids. RSC Adv. 2018, 8, 5071-5078.

3. He, X.; Tang, J.; Qiao, A.; Wang, G.; Jiang, M.; Liu, R. H.; Yao, X. Cytotoxic biotransformed products from cinobufagin by Mucor spinosus and Aspergillus

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