Synthesis, structural studies and stability of the model, cysteine containing DNA-protein cross-links

Electronic Supplementary Material (ESI) for New Journal of Chemistry. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2017 ELECTRONIC SUPPLEMENTARY INFORMATION Synthesis, structural studies and stability of the model, cysteine containing DNA-protein cross-links Kinga Salus, Marcin Hoffmann, Tomasz Siodła, Bożena Wyrzykiewicz, Donata Pluskota-Karwatka* Adam Mickiewicz University in Poznań, Faculty of Chemistry, Umultowska 89b, 61-614 Poznań, Poland e-mail: donatap@amu.edu.pl 1. Chromatograms of reaction mixtures and of solutions subjected to stability studies 2 2. NMR data of M 1 MGx-Ade-NAC 5 3. Mass spectra of studied compounds 6 4. VT 1 H NMR spectra of M 1 MGx-A-NAC 17 5. Temperature calibration data 18 6. Quantum-chemical calculation results for M 1 MGx-A-NAC 20 7. Transition state structures of M 1 MGx-Ade-NAC 22 8. NMR spectra 23 NMR spectra of M 1 Gx-A-NAC (D 2 O) 24 NMR spectra of M 1 MGx-Ade-NAC (D 2 O) 30 NMR spectra of M 1 MGx-A-NAC (D 2 O) 35 1

Chromatograms of reaction mixtures and of solutions subjected to stability studies Absorbance M 1 MGx-A M 1 MGx-A-NAC 0 5 10 15 [min] Fig. S1. C18 analytical column LC-DAD chromatogram (recorded at 254 nm) of the reaction mixture of M 1 MGx-A with NAC in 0.1 M PB held at 37 C for 24 h. Absorbance M 1 MGx-A M 1 MGx-A-NH 2 M 1 MGx-A-NAC 0 5 10 15 [min] Fig. S2. C18 analytical column LC-DAD chromatogram (recorded at 254 nm) of the M 1 MGx-A-NAC solution in 0.1 M PB held at 37 C for 9 days. 2

Absorbance M 1 MGx-Ade M 1 MGx-Ade-NH 2 M 1 MGx-Ade-NAC 0 5 10 15 [min] Fig. S3. C18 analytical column LC-DAD chromatogram (recorded at 254 nm) of the M 1 MGx-Ade-NAC solution in 0.1 M PB held at 37 C for 12 days. Absorbance M 1 Gx-A-( N α -AcLys) 2 M 1 Gx-A- N α -AcLys M 1 Gx-A-NAC 0 5 10 15 [min] Fig. S4. C18 analytical column LC-DAD chromatogram (recorded at 254 nm) of the M 1 Gx-A-NAC mixture with N α - acetyllysine in 0.1 M PB solution held at 37 C for 15 min. 3

Absorbance M 1 Gx-A M 1 Gx-A-NAC 0 5 10 15 [min] Fig. S5. C18 analytical column LC-DAD chromatogram (recorded at 254 nm) of the reaction mixture of M 1 Gx-A with NAC and N α -acetyllysine in 0.1 M PB held at 37 C for 3 h. Absorbance M 1 Gx-A M 1 Gx-A- N α -AcLys M 1 Gx-A-NAC 0 5 10 15 [min] Fig. S6. C18 analytical column LC-DAD chromatogram (recorded at 254 nm) of the reaction mixture of M 1 Gx-A with NAC and N α AcLys in 0.1 M PB held at 37 C for 26 h. 4

NMR data of M 1 MGx-Ade-NAC Table S1. NMR data of M 1 MGx-Ade-NAC (D 2 O, standard NMR TSP-d 4 ) δ(h) [ppm] Multiplicity J H.H [Hz] δ(c) [ppm] HMBC CHO 9.56 s 194.13 C7, C8, C1, C2 C1 127.50 C2 8.72 171.44 C7, CHO, Cβ CH 3 2.31 s (split) 15.07 C7, C8, C1, C2, CHO H-C2 8.29 s 143.45 C3a, C9a, C9b C3a 141.98 H-C5 8.52 s (split) 138.11 C3a, C7, C9a C7 115.41 C8 145.27 C9a 146.07 C9b 118.43 CH 3 (Ac) 2.05 s (split) 24.88 C=O (Ac) C=O (Ac) 176.54 H-Cα 4.56 td 4.25; 8.02 57.74 Cβ, C=O (Ac) Ha-Cβ 3.39 dd (split) 8.36; 14.24 40.49 Cα, C2, COOH Hb-Cβ 3.64 dd (split) 4.30; 13.79 Cα, C2, COOH COOH 178.31 5

Mass spectra of studied compounds * 507.1283 * 508.1333 * 509.1345 510.1334 507 507.5 508 508.5 509 509.5 510 510.5 Fig. S7. Positive ions MS spectrum of M 1 Gx-A-NAC 246.0445 375.0865 158.0579 202.0719 333.0758 507.1296 288.0541 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 Fig. S8. Positive ions MS/MS spectrum of M 1 Gx-A-NAC; collision energy 30 ev 505.1122 (M-H)- 506.1154 (M-H)- 507.1156 (M-H)- 505 505.2 505.4 505.6 505.8 506 506.2 506.4 506.6 506.8 507 507.2 507.4 507.6 Fig. S9. Negative ions MS spectrum of M 1 Gx-A-NAC 6

* 389.1021 * 390.1051 * 391.1026 392.1034 393.1038 388.5 389 389.5 390 390.5 391 391.5 392 392.5 393 393.5 Fig. S10. Positive ions MS spectrum of M 1 MGx-Ade-NAC 389.1030 260.0601 232.0652 347.0912 302.0696 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 Fig. S11. Positive ions MS/MS spectrum of M 1 MGx-Ade-NAC; collision energy 10 ev * 387.0858 (M-H)- 388.0886 (M-H)- 389.0847 (M-H)- 390.0852 (M-H)- 386.5 387 387.5 388 388.5 389 389.5 390 Fig. S12. Negative ions MS spectrum of M 1 MGx-Ade-NAC 7

258.0443 117.0194 162.0217 196.0614 330.9745 387.0853 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Fig. S13. Negative ions MS/MS spectrum of M 1 MGx-Ade-NAC; collision energy 10 ev * 505.1494 * 506.1523 * 507.1513 508.1532 509.1515 505 505.5 506 506.5 507 507.5 508 508.5 509 509.5 Fig. S14. Positive ions MS spectrum of M 1 MGx-dA-NAC 389.1024 505.1495 260.0598 232.0648 281.1464 347.0919 453.3425 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 Fig. S15. Positive ions MS/MS spectrum of M 1 MGx-dA-NAC; collision energy 15 ev 8

503.1325 (M-H)- 504.1351 (M-H)- 505.1341 (M-H)- 502.5 503 503.5 504 504.5 505 505.5 506 Fig. S16. Negative ions MS spectrum of M 1 MGx-dA-NAC 374.0922 503.1348 162.0235 215.9772 258.0424 303.0050 347.0322 431.1972 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 Fig. S17. Negative ions MS spectrum of M 1 MGx-dA-NAC; collision energy 10 ev * 521.1448 * 522.1475 * 523.1469 524.1480 520.5 521 521.5 522 522.5 523 523.5 524 524.5 525 525.5 526 526.5 527 Fig. S18. Positive ions MS spectrum of M 1 MGx-A-NAC 9

521.1446 Intensywność 389.1025 216.0890 260.0597 345.1118 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 Fig. S19. Positive ions MS/MS spectrum of M 1 MGx-A-NAC; collision energy 10 ev * 362.1101 363.1145 364.1165 363.3594 365.1195 362 362.5 363 363.5 364 364.5 365 Fig. S20. Positive ions MS spectrum of M 1 Gx-A 230.0668 362.1090 84.0442 120.0185 321.3146 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 Fig. S21. Positive ions MS/MS spectrum of M 1 Gx-A; collision energy 10 ev 10

361.1257 362.1301 363.1332 364.1362 361 361.5 362 362.5 363 363.5 364 364.5 Fig. S22. Positive ions MS spectrum of M 1 Gx-A-NH 2 229.0828 361.1248 184.0606 212.0564 147.0669 302.8024 120 140 160 180 200 220 240 260 280 300 320 340 360 Fig. S23. Positive ions MS/MS spectrum of M 1 Gx-A-NH 2 ; collision energy 10 ev * 244.0834 * 245.0870 246.0900 243.5 244 244.5 245 245.5 246 246.5 247 Fig. S24. Positive ions MS spectrum of M 1 MGx-Ade 11

214.0721 186.0771 Intensywność 119.0352 244.0824 198.0771 242.0669 80.0495 98.0600 171.0662 136.0615 160.0728 226.0719 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 Fig. S25. Positive ions MS/MS spectrum of M 1 MGx-Ade; collision energy 30 ev * 243.0989 * 265.0807 (M+Na)+ * 281.0545 (M+K)+ 245 250 255 260 265 270 275 280 285 Fig. S26. Positive ions MS spectrum of M 1 MGx-Ade-NH 2 80.0495 198.0771 97.0761 172.0614 119.0351 136.0615 186.0772 226.0719 243.0985 157.0507 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 Fig. S27. Positive ions MS/MS spectrum of M 1 MGx-Ade-NH 2 ; collision energy 30 ev 12

* 376.1247 * 377.1282 Fig. S28. Positive ions MS spectrum of M 1 MGx-A 378.1306 376 376.25 376.5 376.75 377 377.25 377.5 377.75 378 378.25 378.5 378.75 244.0828 376.1246 216.0873 262.9798 341.3043 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 Fig. S29. Positive ions MS/MS spectrum of M 1 MGx-A; collision energy 15 ev * 375.1408 * 376.1434 377.1463 378.1487 374.5 375 375.5 376 376.5 377 377.5 378 378.5 379 379.5 380 380.5 381 Fig. S30. Positive ions MS spectrum of M 1 MGx-A-NH 2 13

375.1407 243.0987 158.1526 226.0710 267.2650 305.1721 339.1364 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Fig. S31. Positive ions MS/MS spectrum of M 1 MGx-A-NH 2 ; collision energy 10 ev * 532.2139 * 533.2174 Fig. S32. Positive ions MS spectrum of M 1 Gx-A-N α AcLys * 534.2198 535.2229 536.2235 532 532.5 533 533.5 534 534.5 535 535.5 536 536.5 537 400.1723 158.9650 267.1354 372.1772 212.0575 445.7024 532.2146 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 Fig. S33. Positive ions MS/MS spectrum of M 1 Gx-A-N α AcLys; collision energy 20 ev 14

* 702.3199 * 703.3231 704.3264 702 702.5 703 703.5 704 704.5 705 Fig. S34. Positive ions MS spectrum of M 1 Gx-A-(N α AcLys) 2 702.3202 570.2776 267.1330 382.1598 465.2330 528.2666 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 Fig. S35. Positive ions MS/MS spectrum of M 1 Gx-A-(N α AcLys) 2 ; collision energy 20 ev * 546.2299 547.2328 Fig. S36. Positive ions MS spectrum of M 1 MGx-A-N α AcLys 548.2350 549.2357 546 546.5 547 547.5 548 548.5 549 549.5 15

546.2297 414.1875 226.0712 281.1488 326.1689 386.1921 510.7546 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 Fig. S37. Positive ions MS/MS spectrum of M 1 MGx-A-N α AcLys; collision energy 10 ev 716.3354 717.3379 718.3397 716 716.5 717 717.5 718 718.5 719 Fig. S38. Positive ions MS spectrum of M 1 MGx-A-(N α AcLys) 2 716.3345 163.6157 280.1604 584.2911 386.1878 672.3416 100 150 200 250 300 350 400 450 500 550 600 650 700 750 Fig. S39. Positive ions MS spectrum of M 1 MGx-A-(N α AcLys) 2 ; collision energy 10 ev 16

VT 1 H NMR spectra of M 1 MGx-A-NAC 376 K 373 K 365 K 364 K 342 K 297 K 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 Fig. S40. Fragments of 1 H NMR spectra of M 1 MGx-A-NAC (D 2 O) recorded at 297 K, 342 K, 364, 365 K and 376. The split acetyl proton signals at 1.96 coalesced to singlet at Tc = 376 K, methyl protons signals at 2.23 ppm coalesced at lower temperature, but artefacts may be misleading. High temperature caused partial degradation of M 1 MGx-A-NAC leading to the formation of products which gave rise to additional signals in 1 H NMR spectra of the studied compound. This process interfered with the variable-temperature studies. 17

Temperature calibration data Table S2. Temperature values calculated based on 1 H NMR chemical shift values of hydroxyl and methylene protons of ethylene glycol Thermostat temperature (T t ) δ ( OH) [ppm] δ (-CH 2 ) [ppm] Δδ [ppm] T calc * 298 4.87 3.22 1.65 298.20 308 4.73 3.17 1.56 307.38 318 4.91 3.48 1.43 320.64 323 4.00 2.59 1.41 322.68 332 3.90 2.61 1.29 334.92 337 4.44 3.21 1.23 341.04 340 5.07 3.87 1.20 344.10 342 3.96 2.78 1.18 346.14 *Temperature values calculated based on T calc = 466.5 102.00 * Δδ 1,70 1,60 y = -0.0109x + 4.8972 R² = 0.9959 1,50 δ[ppm] 1,40 1,30 1,20 1,10 290 300 310 320 330 340 350 Thermostat temperature, T t [K] Chart S1. The relationship between 1 H NMR chemical shift difference values ( δ) of ethylene glycol protons and temperature values showed by thermostat (T t ) 18

350 Actual temperature, T [K] 340 330 320 310 300 y = 1.1082x - 33.009 R² = 0.9959 290 290 300 310 320 330 340 350 Thermostat temperature, T t [K] Chart S2. Calibration curve showing the relationship between thermostat temperature values (T t ) and calculated temperature (T calc ). Equation T = 1.1082 * T t 33.009 allows to determine the actual temperature in thermostat. 19

Quantum-chemical calculation results for M 1 MGx-A-NAC Relative energy [kcal/mol] 14 12 10 8 6 4 2 0-180 -150-120 -90-60 -30 0 30 60 90 120 150 180 Dihedral angle N6-C7-C1"-C2" value s-cis s-trans Chart S3. The relative energy of s-cis and s-trans conformers of M 1 MGx-A-NAC dependent on the N6-C7-C1 -C2 dihedral angle value calculated at M06/6-31G(d) level of theory in vacuo Fig. S41. Energy minima structures of M 1 MGx-A-NAC: s-cis/m -50 (A) and s-cis/p (B); arrows show correlations observed in NOESY spectrum of M 1 MGx-A-NAC (D 2 O) 20

Table S3. Comparison of the relative energies ( E) and Gibbs free energies ( G) of M 1 MGx-A-NAC s-cis and s-trans conformers calculated at M06/6-31G(d) level of theory in vacuo, and using PCM for water Dihedral angle* In vacuo E [kcal/mol] G [kcal/mol] Dihedral angle* PCM (water) E [kcal/mol] G [kcal/mol] s-cis/m -50-52.8º 0 0-54.4º 0 0 s-cis/m -120-121.4º 0.62 0.61-116.1º 1.06 1.68 s-cis/p 130.0º 2.20 1.98 129.5º 1.80 1.44 s-cis/ts_0 4.5º 10.12 12.01 2.7º 11.05 12.66 s-cis/ts _180-176.3º 12.95 13.90-174.1º 12.58 13.67 s-trans/m -135.2º 1.57 1.68-128.9º 1.72 2.42 s-trans/p 130.5º 3.93 2.94 128.8º 2.48 3.36 s-trans/ts_0-9.3º 13.00 12.03-6.4º 16.36 16.90 s-trans/ts _180 179.3º 11.21 12.07 178.0º 11.47 12.87 * N6-C7-C1 -C2 dihedral angle value Table S4. The relative energy values ( E) of s-cis and s-trans conformers of M 1 MGx-A-NAC calculated using M06/6-31++G(d,p), B3LYP/6-31++G(d,p), ωb97x-d/6-31g(d) and MP2/6-31G(d) in vacuo. For comparison the E values calculated at initial M06/6-31G(d) level were given. Dihedral angle* M06/ 6-31G(d) M06/ 6-31++G(d,p) B3LYP/ 6-31++G(d,p) ωb97x-d/ 6-31G(d) MP2/ 6-31G(d) s-cis/m -50-52.8º 0.00 0.00 0.00 0.00 0.04 s-cis/p 130.0º 2.20 1.74 1.53 2.81 3.06 s-cis/ts_0 4.5º 10.12 9.72 9.41 11.35 13.73 s-cis/ts _180-176.3º 12.95 12.42 13.04 14.83 20.11 s-trans/m -135.2º 1.57 0.83 1.71 1.41 0.00 s-trans/p 130.5º 3.93 3.42 3.89 4.15 2.82 s-trans/ts_0-9.3º 13.00 12.01 12.09 13.04 11.42 s-trans/ts _180 179.3º 11.21 10.91 11.49 12.27 14.51 * N6-C7-C1 -C2 dihedral angle value 21

Transition state structures of M 1 MGx-Ade-NAC s-cis/ts_00 s-cis/ts_180 s-trans/ts 0 s-trans/ts_180 M/TS_-900 P/TS_90 M/TS_90 P/TS -90 Fig. S42. Transition state structures of M 1 MGx-Ade-NAC calculated at M06/6-31G(d) level of theory in vacuo 22

NMR spectra 23

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 Fig. S43. 1 H NMR spectrum of M 1 Gx-A-NAC (D 2 O) 24 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1000 900 800 700 600 500 400 300 200 100 0 1.00 1.00 0.95 1.02 0.93 1.07 7.69 6.21 8.49 8.63 8.66 9.52 1.16 0.87 4.86 4.93 1.16 1.20 1.12 1.11 1.09 0.95 0.95 4.30 4.50 4.57 3.61 3.42 3.88 3.94 3.10 2.05 B (dd) 4.57 J(4.33, 7.79) C (dd) 4.30 J(4.12, 7.34) E (dd) 3.88 J(4.30, 12.72) D (dd) 3.95 J(3.04, 12.73) F (dd) 3.61 J(4.33, 14.19) G (dd) 3.42 J(7.83, 14.18) 5.1 4.9 4.7 4.5 4.3 4.1 3.9 3.7 3.5 3.3 600 500 400 300 200 100 0-100 4.29 4.30 4.30 4.31 4.49 4.50 4.50 4.56 4.57 3.87 3.88 3.89 3.94 3.94 3.96 3.40 3.42 3.43 3.44 3.60 3.61 3.62 4.86 4.93

200 190 180 170 160 Fig. S44. 13 C NMR spectrum of M 1 Gx-A-NAC (D 2 O) 150 140 130 120 110 25 100 90 80 70 60 50 40 30 20 30000 28000 26000 24000 22000 20000 40.65 24.86 18000 16000 14000 12000 10000 8000 6000 4000 2000 0-2000 -4000-6000 178.30 176.58 194.12 170.62 144.45 141.67 143.95 136.57 139.51 125.60 127.63 119.23 91.75 88.29 73.25 76.84 64.25 57.68

2.0 2.5 {4.57,3.43} {3.61,3.44} {4.58,3.62} {3.43,3.62} {4.30,3.86} 3.0 3.5 {4.88,4.49} {6.22,4.88} {4.50,4.88} {4.49,4.34} {3.95,4.30} {3.61,4.58} {3.44,4.57} {4.31,4.48} 4.0 4.5 5.0 5.5 {4.88,6.22} 6.0 6.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 Fig. S45. COSY spectrum of M 1 Gx-A-NAC (D 2 O) 26

{2.02,24.59} 10 20 {3.61,40.35} {3.42,40.34} 30 40 {4.57,57.32} {3.94,63.96} 50 60 {4.88,76.57} {3.88,63.95} 70 {6.22,91.52} {4.30,88.10} 80 90 100 110 120 {8.64,139.41} {7.70,136.45} 130 140 {8.49,143.83} 150 {8.66,169.53} 160 170 {9.52,194.23} 180 190 200 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 Fig. S46. HSQC spectrum of M 1 Gx-A-NAC (D 2 O) 27

Fig. S47. HMBC spectrum of M 1 Gx-A-NAC (D 2 O) 28

M1GxA_NAC_1 KS_M1MGx_NAC_1 spin off temp. 298K 1 2 {8.66,3.60} {8.64,3.40} {8.66,3.41} {3.62,3.42} {8.49,3.90} {4.88,3.87} {8.66,4.56} {8.49,4.49} {6.22,4.46} {8.65,4.91} {8.49,4.87} {6.21,4.91} {4.88,4.51} {3.89,4.30} {6.22,4.29} {3.94,4.29} {3.42,3.61} {3.43,4.57} {3.60,4.56} 3 4 5 {8.49,6.21} 6 {2.05,7.64} 7 {6.21,8.49} {9.53,8.66} {3.60,8.65} {3.44,8.65} {8.66,9.52} 8 9 10 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Fig. S48. NOESY spectrum of M 1 Gx-A-NAC (D 2 O) 29

C (d) 2.31 J(4.94) B (d) 2.05 J(8.16) 1500 1000 1500 1400 500 1300 1200 3.19 3.22 0 1100 2.3 2.2 2.1 2.0 1000 9.56 D (d) 8.52 J(13.10) A (td) 4.56 J(4.25, 8.02) F (ddd) 3.64 J(4.30, 9.37, 13.79) E (ddd) 3.39 J(8.36, 10.40, 14.24) C (d) 2.31 J(4.94) B (d) 2.05 J(8.16) 900 800 700 600 8.29 500 8.72 400 300 200 4.56 100 0 1.00 0.95 1.00 0.98 1.08 1.12 1.09 3.19 3.22-100 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Fig. S49. 1 H NMR spectrum of M 1 MGx-Ade-NAC (D 2 O) 30

200 190 180 170 160 150 140 Fig. S50. 13 C NMR spectrum of M 1 MGx-Ade-NAC (D 2 O) 130 120 110 31 100 90 80 70 60 50 40 30 20 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 40.49 15.64 24.82 12000 10000 8000 6000 4000 2000 0-2000 -4000-6000 194.54 171.59 176.54 178.31 138.51 140.21 142.34 143.82 145.27 146.07 127.50 115.41 118.43 57.65 57.81 180 176 172 20000 10000 0 60 55 50 45 40 30000 20000 10000 0 178.31 176.54 171.59 57.65 57.81 40.49

2 {4.55,3.40} {4.56,3.64} {3.62,3.43} 3 {3.40,3.61} {3.64,4.57} {3.39,4.56} 4 5 6 7 8 9 10 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 Fig. S51. COSY spectrum of M 1 MGx-Ade-NAC (D 2 O) 32

{4.56,57.31} {2.27,15.07} {3.62,40.13} {3.37,40.03} {2.03,24.88} 0 20 40 60 80 100 {8.52,138.11} {8.29,143.45} 120 140 {9.56,194.13} {8.72,171.44} 160 180 200 220 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Fig. S52. HSQC spectrum of M 1 MGx-Ade-NAC (D 2 O) 33

{8.72,39.96} {4.57,39.70} 30 40 {3.37,57.36} {3.63,57.31} 50 60 70 80 90 {9.56,114.83} {8.29,118.50} {9.53,127.07} {8.51,115.43} {8.52,145.44} {8.28,145.82} {9.56,144.87} {8.29,142.49} {9.56,171.64} {8.72,194.26} 100 {2.30,114.85} 110 {2.30,127.37} 120 130 {2.30,145.00} 140 150 160 {3.63,171.34} {3.38,171.49}{2.30,171.53} {3.63,178.04} {3.39,177.94} 170 {2.04,176.25} 180 {4.55,178.35} {2.30,193.72} 190 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 200 Fig. S53. HMBC spectrum of M 1 MGx-Ade-NAC (D 2 O) 34

4000 4000 3800 G (dd) 4.87 J(5.14, 10.20) F (ddd) 4.58 J(4.34, 8.36, 9.88) E (m) 4.50 D (dd) 4.30 J(3.67, 7.51) C (ddd) B (m) 3.92 3.65 J(3.73, 12.77, 17.04) 1000 800 A (ddd) 600 3.41 J(8.32, 14.22, 15.59) 400 200 0 B (d) 2.33 J(6.98) 3000 A (d) 2000 2.07 J(9.66) 1000 0 2.32 2.08 3600 3400 3200 3000 2800 2600 2400 2200 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 2.3 2.2 2.1 2000 9.57 1800 1600 8.55 8.74 1400 1200 8.49 6.20 4.87 4.59 4.50 4.30 3.94 3.90 3.66 3.41 1000 800 600 400 200 0 1.00 1.03 1.12 1.00 1.08 1.25 1.07 1.14 1.16 1.16 1.17 1.05 1.06 3.10 3.10-200 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 Fig. S54. 1 H NMR spectrum of M 1 MGx-A-NAC (D 2 O) 35

40.37 15.63 24.90 190 180 170 160 150 140 130 120 Fig. S55. 13 C NMR spectrum of M 1 MGx-A-NAC (D 2 O) 110 36 100 90 80 70 60 50 40 30 20 194.36 171.54 171.91 176.54 178.29 138.75 138.79 141.98 143.75 144.13 145.10 124.83 127.27 127.35 115.25 115.30 91.79 88.35 73.31 76.81 138.75 138.79 64.31 57.68 194.5 194.0 145 144 143 142 141 140 139 40 35 30 25 20 15 194.36 194.40 145.10 143.75 144.13 141.98 40.37 40.60 15.63 24.90

1.5 2.0 2.5 3.0 {4.58,3.43} {4.59,3.64} {4.31,3.88} 3.5 {6.20,4.88} {4.49,4.33} {4.88,4.51} {4.50,4.87} {3.95,4.30} {3.90,4.31} {3.41,4.59} {4.31,4.48} {3.66,4.59} 4.0 4.5 5.0 5.5 {4.88,6.20} 6.0 6.5 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 Fig. S56. COSY spectrum of M 1 MGx-A-NAC (D 2 O) 37

0 {2.30,15.82} 10 20 {4.58,58.00} {4.50,73.65} {3.40,40.62} {2.07,25.02} {3.65,40.70} {3.88,64.52} {3.95,64.57} 30 40 50 60 70 {6.20,92.12} {4.88,77.01} {4.30,88.57} 80 90 100 110 120 {8.54,139.07} {8.49,144.47} 130 140 150 {8.74,171.88} 160 170 180 {9.54,194.80} 190 200 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Fig. S57. HSQC spectrum of M 1 MGx-A-NAC (D 2 O) 38

20 {8.74,40.28} {8.49,91.51} {6.19,76.73} {6.20,88.67} {4.58,40.38} {3.66,57.60} {3.41,57.55} {4.31,72.97} {4.50,64.28} {4.88,91.55} {4.50,91.68} 40 60 80 {9.57,115.27} {8.55,114.08} {9.55,126.95} {8.48,124.63} {4.30,91.98} {2.33,115.17} {3.39,127.55} {2.33,127.51} 100 120 {9.57,145.03} {8.49,141.89}{6.20,141.49} {6.20,144.80} {3.66,127.28} {2.33,145.01} {8.54,142.32} {9.57,171.89} {3.41,171.66}{2.32,171.63} {3.66,178.61} {8.74,194.30} {4.58,176.95} {3.40,180.91}{2.33,194.34} {2.08,175.58} 140 160 180 200 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Fig. S58. HMBC spectrum of M 1 MGx-A-NAC (D 2 O) 39

{9.58,2.12} {8.74,2.07} 2 {9.58,2.31} {8.74,2.33} {8.74,3.40} 3 {4.58,3.43} {3.67,3.38} {8.74,3.65} {4.50,3.89} {8.48,4.50} {8.49,3.90} {8.49,4.87} {8.74,4.58} {6.19,4.31} {3.42,3.66} {4.31,3.94} {2.32,3.65} {3.42,4.57} {4.88,4.51} {3.97,3.88} {3.94,4.30} {6.20,4.87} 4 {3.66,4.58} 5 {4.50,4.87} {8.49,6.19} {4.31,6.20} {4.87,6.20} 6 7 {6.20,8.48} {4.88,8.48} {9.57,8.74} {4.50,8.49} 8 {3.66,8.74} {4.58,8.74} {8.74,9.57} 9 {3.40,8.74} 10 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 Fig. S59. NOESY spectrum of M1MGx-A-NAC (D2O) 40 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0