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13C NMR spectrum of quercetin-3′-O-sulfate (150.93 MHz, DMSO-d6, 30 oC). .............................. 4 .... -78.0±16.2 -47.7±16.4. -30.3±7.7 quercetin 3´-O-sulfate.
Sulfated Quercetin Metabolites

Supporting information

Chemoenzymatic Preparation and Biophysical Properties of Sulfated Quercetin Metabolites Kateřina Valentová1,*, Kristýna Káňová1, Florent Di Meo2,*, Helena Pelantová1, Christopher Steven Chambers1, Lenka Rydlová1, Lucie Petrásková1, Alena Křenková1, Josef Cvačka3, Patrick Trouillas2,4, Vladimír Křen1 Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20 Prague, Czech Republic; [email protected] (K.K.); [email protected] (H.P.); [email protected] (C.S.C.); [email protected] (L.P.); [email protected] (A.K.); [email protected] (V.K.). 2INSERM U850, Univ. Limoges, School of Pharmacy, 2 rue du Docteur Marcland, 87025 Limoges, France; [email protected] (P.T.). 3Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, CZ 16610 Prague, Czech Republic; [email protected]. 4Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacký University, tř. 17. listopadu 12, 771 46 Olomouc, Czech Republic 1

Table of Content: Figure S1.

HPLC chromatograms of quercetin sulfation reaction mixture at (a) 1 h, (b) 5 h, (c) 24 h. ............ 2

Figure S2.

Distance of quercetin derivatives to lipid bilayer center over MD simulations. ............................... 3

Table S1.

Average non-covalent interactions between solute and (a) lipids or (b) lipid tails. ......................... 3

Figure S3.

1H

Figure S4.

13C

Figure S5.

Mass spectrum (ESI−) of quercetin-3′-O-sulfate. ........................................................................... 5

Figure S6.

HPLC chromatogram of quercetin-3′-O-sulfate. ............................................................................. 5

Figure S7.

1H

Figure S8.

13C

Figure S9.

Mass spectrum (ESI−) of quercetin-4′-O-sulfate. ........................................................................... 7

Figure S10.

HPLC chromatogram of quercetin-4′-O-sulfate. ............................................................................. 7

Figure S11.

1H

Figure S12.

13C

Figure S13.

Mass spectrum (ESI−) of quercetin-3-O-sulfate. ............................................................................ 9

Figure S14.

HPLC chromatogram of quercetin-3-O-sulfate. .............................................................................. 9

Figure S15.

Expanded 1H NMR spectrum of quercetin-di-O-sulfates mixture (600.23 MHz, DMSO-d6, 30 oC). ..... 10

Figure S16.

Expanded 13C NMR spectrum of quercetin-di-O-sulfates mixture (150.93 MHz, DMSO-d6, 30 oC). ....... 10

Figure S17.

Mass spectrum (ESI−) of quercetin-di-O-sulfates. ....................................................................... 11

Figure S18.

HPLC chromatogram of quercetin-di-O-sulfates mixture.............................................................. 11

NMR spectrum of quercetin-3′-O-sulfate (600.23 MHz, DMSO-d6, 30 oC). ............................... 4 NMR spectrum of quercetin-3′-O-sulfate (150.93 MHz, DMSO-d6, 30 oC). .............................. 4

NMR spectrum of quercetin-4′-O-sulfate (600.23 MHz, DMSO-d6, 30 oC). ............................... 6 NMR spectrum of quercetin-4′-O-sulfate (150.93 MHz, DMSO-d6, 30 oC). .............................. 6

NMR spectrum of quercetin-3-O-sulfate (399.87 MHz, DMSO-d6, 30 oC). ................................ 8 NMR spectrum of quercetin-3-O-sulfate (100.55 MHz, DMSO-d6, 30 oC). ............................... 8

1

Sulfated Quercetin Metabolites (a)

Supporting information

70

5 6

Intensity (AU)

60 50

3

40 30

2

1

20

4

10 0 0

Intensity (AU)

(b)

5

45 40 35 30 25 20 15 10 5 0

10 Retention time (min)

15

3

4

6

2 1 0

5

10 Retention Time (min)

15

4

140

(c)

5

Intensity (AU)

120 100

3

80 60

5

6

2

40

1

20 0 0

5

10

15

Retention Time (min)

Figure S1. HPLC chromatograms of quercetin sulfation reaction mixture at (a) 1 h, (b) 5 h, (c) 24 h. Peak assignment: 1 – p-NP, 2 – quercetin disulfates, 3 – p-NPS, 4 – quercetin-3′-O-sulfate, 5 quercetin-4′-O-sulfate, 6 – quercetin; detection at 370 nm.

2

Sulfated Quercetin Metabolites

Supporting information

Figure S2. Distance of quercetin derivatives to lipid bilayer center over MD simulations. Each quercetin replica is depicted in red, green blue and cyan. Average phosphate distances to lipid center is plotted in yellow. Table S1. Average non-covalent interactions between solute and (a) lipids or (b) lipid tails. (a) quercetin quercetin 3´-O-sulfate quercetin 4´-O-sulfate quercetin 3',4´-O-disulfate quercetin-7',3´-O-disulfate quercetin-7',4´-O-disulfate

Eint

Eelec

-78.0±16.2 -134.7±27.2 -138.6±20.2 -288.7±53.8 -222.2±31.7 -216.1±30.2

-47.7±16.4 -92.6±22.7 -94.1±17.2 -245.5±47.4 -170.1±28.4 -163.1±26.0

Eint

Eelec

Evdw -30.3±7.7 -42.2±8.7 -44.5±8.0 -43.2±10.4 -52.1±7.5 -53.0±8.5

(b) Evdw

quercetin -24.1±7.0 -0.9±0.6 -23.3±6.8 quercetin 3´-O-sulfate -23.0±6.7 -0.4±1.2 -22.6±6.2 quercetin 4´-O-sulfate -29.4±7.3 -1.2± -28.2±6.6 quercetin 3',4´-O-disulfate -17.3±6.4 0.2± -17.6±6.1 quercetin-7',3´-O-disulfate -25.6±5.9 -1.2± -24.4±4.9 quercetin-7',4´-O-disulfate -30.5±6.8 -1.4± -29.1±5.9 -1 -1 total (Eint, kcal.mol ), electrostatic (Eelec, kcal.mol ) and van der Waals energies (Evdw, kcal.mol-1) over the last 200 ns 3

Sulfated Quercetin Metabolites

Figure S3.

Supporting information

H NMR spectrum of quercetin-3′-O-sulfate (600.23 MHz, DMSO-d6, 30 oC).

1

Signal assignment: 6.158 (1H, d, J = 2.0 Hz, H-6), 6.392 (1H, d, J = 2.0 Hz, H-8), 6.977 (1H, d, J = 8.6 Hz, H-5′), 7.841 (1H, dd, J = 8.6, 2.3 Hz, H-6′), 8.026 (1H, d, J = 2.3 Hz, H-2′)

Figure S4.

C NMR spectrum of quercetin-3′-O-sulfate (150.93 MHz, DMSO-d6, 30 oC).

13

Signal assignment: 93.55 (C-8), 98.45 (C-6), 102.82 (C-10), 117.25 (C-5′), 122.36 (C-1′), 122.65 (C-2′), 124.89 (C-6′), 136.05 (C-3), 140.85 (C-3′), 145.95 (C-2), 151.19 (C-4′), 156.25 (C-9), 160.74 (C-5), 164.73 (C-7), 175.87 (C-4)

4

Sulfated Quercetin Metabolites

Supporting information

051216servisHR_2- #29-36 RT: 1.62-2.02 AV: 8 NL: 7.35E5 T: FTMS - p ESI Full ms [200.00-2000.00] 381.0

100 95 90 85 80 75 70

Relative Abundance

65 60 55 50 45 40 35 30 301.0 25 20 382.0 15 10 283.3

5 0 200

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280

383.0

302.0 300

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Figure S5. Mass spectrum (ESI−) of quercetin-3′-O-sulfate. [M – H]-, m/z 381.0; [M – H – SO3]-, m/z 301.0. 70

Intensity (AU)

60 50 40 30 20 10 0 0

2

4

6

8 10 12 Retention time (min)

Figure S6. HPLC chromatogram of quercetin-3′-O-sulfate.

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14

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20

520

Sulfated Quercetin Metabolites

Figure S7.

Supporting information

H NMR spectrum of quercetin-4′-O-sulfate (600.23 MHz, DMSO-d6, 30 oC).

1

Signal assignment: 6.181 (1H, d, J = 2.1 Hz, H-6), 6.428 (1H, d, J = 2.1 Hz, H-8), 7.377 (1H, d, J = 8.6 Hz, H-5′), 7.573 (1H, dd, J = 8.6, 2.2 Hz, H-6′), 7.672 (1H, d, J = 2.2 Hz, H-2′)

Figure S8.

C NMR spectrum of quercetin-4′-O-sulfate (150.93 MHz, DMSO-d6, 30 oC).

13

signal assignment: 93.57 (C-8), 98.39 (C-6), 103.05 (C-10), 116.30 (C-2′), 119.17 (C-6′), 122.11 (C-5′), 126.90 (C-1′), 136.61 (C-3), 142.71 (C-4′), 145.83 (C-2), 148.55 (C-3′), 156.35 (C-9), 160.76 (C-5), 164.48 (C-7), 176.10 (C-4) 6

Sulfated Quercetin Metabolites

Supporting information

051216servisHR_3- #28-34 RT: 1.56-1.91 AV: 7 NL: 1.69E6 T: FTMS - p ESI Full ms [200.00-2000.00] 381.0

100 95 90 85 80 75 70

Relative Abundance

65 60 55 50 45 40 301.0

35 30 25 20

382.0 15 10 302.0

5 0 200

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383.0

320

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380

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520

Figure S9. Mass spectrum (ESI−) of quercetin-4′-O-sulfate. [M – H]-, m/z 381.0; [M – H – SO3]-, m/z 301.0.

Intensity (AU)

250 200 150 100 50 0 0

2

4

6

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Retention time (min)

Figure S10. HPLC chromatogram of quercetin-4′-O-sulfate.

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Sulfated Quercetin Metabolites

Supporting information

Figure S11. 1H NMR spectrum of quercetin-3-O-sulfate (399.87 MHz, DMSO-d6, 30 oC). signal assignment: 1H NMR: 6.159 (1H, d, J = 2.1 Hz, H-6), 6.363 (1H, d, J = 2.1 Hz, H-8), 6.795 (1H, d, J = 8.5 Hz, H-5′), 7.560 (1H, d, J = 2.2 Hz, H-2′), 7.613 (1H, dd, J = 8.5, 2.2 Hz, H-6′)

Figure S12.

C NMR spectrum of quercetin-3-O-sulfate (100.55 MHz, DMSO-d6, 30 oC).

13

signal assignment: 93.32 (C-8), 98.41 (C-6), 104,15 (C-10), 115.16 (C-5′), 116.00 (C-2′), 121.63a (C-1′), 121.66a (C-6′), 132.35 (C-3), 144.74 (C-3′), 148.38 (C-4′), 156.12 (C-9), 156.58 (C-2), 161.42 (C-5), 164.01 a (C-7), 177.77 (C-4) … might be interchanged

8

Sulfated Quercetin Metabolites

Supporting information

020617servisHR_2- #28-35 RT: 1.56-1.97 AV: 8 NL: 1.70E6 T: FTMS - p ESI Full ms [200.00-2000.00] 381.0

100 95 90 85 80 75 70

Relative Abundance

65 301.0

60 55 50 45 40 35 30 25 20

382.0 15 300.0

10

403.0

302.0

482.9

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383.0

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Figure S13. Mass spectrum (ESI−) of quercetin-3-O-sulfate. [M – H]-, m/z 381.0; [M – H – SO3]-, m/z 301.0. Signals of a disulfate were found at m/z 482.9 ([M – 2H + Na]-), m/z 403.0 ([M – 2H + Na – SO3]-), and m/z 230.0 ([M – 2H]2-). 30000

Intensity (AU)

25000 20000 15000 10000 5000 0 0

5

10

Retention time (min)

Figure S14. HPLC chromatogram of quercetin-3-O-sulfate.

9

15

20

Sulfated Quercetin Metabolites

Supporting information

Figure S15. Expanded 1H NMR spectrum of quercetin-di-O-sulfates mixture (600.23 MHz, DMSO-d6, 30 oC). Signals of individual components are marked as A: quercetin-3’,4’-di-O-sulfate, B: quercetin-7,3’-diO-sulfate, and C: quercetin-7,4’-di-O-sulfate.

Figure S16. Expanded 13C NMR spectrum of quercetin-di-O-sulfate mixture (150.93 MHz, DMSO-d6, 30 oC).

10

Sulfated Quercetin Metabolites

Supporting information

170215servisHR_2#61-69 RT: 1.62-1.84 AV: 9 SB: 15 1.16-1.54 NL: 5.72E4 T: FTMS - p ESI Full ms [200.00-2000.00] 498.9

100 95 90 85 482.9

80 403.0 75

Relative Abundance

70 65 60 55 50

460.9

45 418.9 40

395.0 432.0

35 30

397.0

25 20

478.0

15

429.0

447.1

432.5

463.0

10

499.9

483.9

455.0

439.9 5 0 390

400

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510

m/z

Figure S17. Mass spectrum (ESI−) of quercetin-di-O-sulfates. [M – 2H + K]-, m/z 498.9; [M – 2H + Na]-, m/z 482.9; [M – 2H + NH4]-, m/z 478.0; [M – H]-, m/z 460.9; [M – 2H + K – SO3]-, m/z 418.9; [M – 2H + Na – SO3]-, m/z 403.0. 70 Intensity (AU)

60 50 40 30 20 10 0

0

2

4

6

8 10 12 Retention time (min)

14

Figure S18. HPLC chromatogram of quercetin-di-O-sulfate mixture.

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