nicotinamide riboside-amino acid conjugates that are stable to … · 2020. 4. 2. · 1...

1

Nicotinamide riboside-amino acid conjugates that are stable to purine nucleoside phosphorylase Faisal Hayat and Marie E. Migaud*

Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Ave., Mobile, AL 36604, USA

Supplementary

1. Copies of 1H NMR, 13C NMR, 19F-NMR and HRMS Spectra…………………………………..…………………....2-58 2. Enzymatic assays by 1H NMR…………………………………………………………………………………… …59-61 3. Table of Vorbrüggen reaction outcomes…………………………………………………………………………………62

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2020

2

Compound 4. 400 MHz 1H NMR spectrum in Acetone-d6

3

Compound 4. 100 MHz 13C NMR spectrum in Acetone-d6

4

Compound 7. 400 MHz 1H NMR spectrum in D2O

5

Compound 7. 100 MHz 13C NMR spectrum in D2O

6

Compound 7. HRMS spectra

mm_072018_fh_01_20_1 #242-260 RT: 3.83-4.11 AV: 19 NL: 5.55E6F: FTMS + p ESI Full ms [120.00-800.00]

130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300m/z

0

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80

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90

95

100R

elat

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Abu

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123.05448R=118611

z=1

295.12793R=77190

z=1

173.08005R=102998

z=1

141.95781R=113872

z=1

7

Compound 8. 400 MHz 1H NMR spectrum in D2O

8

Compound 8. 100 MHz 13C NMR spectrum in D2O

9

Compound 8. HRMS spectra

mm_072018_fh_01_22_1 #244-267 RT: 3.86-4.21 AV: 24 NL: 5.74E6F: FTMS + p ESI Full ms [120.00-800.00]

130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300m/z

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80

85

90

95

100R

elat

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Abun

danc

e297.14359R=77139

z=1

123.05456R=119611

z=1

141.95786R=114498

z=1

173.08007R=103770

z=1

10

Compound 10a. 400 MHz 1H NMR spectrum in MeOD

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Compound 10a. 100 MHz 13C NMR spectrum in MeOD

12

Compound 10a. HRMS spectra

mm_101618_nrh_glycine_01 #17-31 RT: 0.25-0.46 AV: 15 SB: 81 5.79-7.06 NL: 1.49E7F: FTMS + p ESI Full ms [200.00-1050.00]

450 451 452 453 454 455 456 457 458m/z

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Rel

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bund

ance

454.2186R=60354

z=1C 28 H30 N4 S-0.0052 ppm

C 21 H32 O8 N30.3863 ppm

452.2033R=59378

z=1C 14 H30 O6 N9 S

-0.3233 ppmC 15 H36 O11 N2 S

-0.3348 ppm

455.2218R=61699

z=1C 15 H35 O4 N8 S 2

0.1158 ppmC 30 H31 O40.1895 ppm

13

Compound 10b. 400 MHz 1H NMR spectrum in MeOD

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Compound 10b. 100 MHz 13C NMR spectrum in MeOD

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Compound 10b. HRMS spectra

mm_101618_nrh_leucine_01 #17-32 RT: 0.25-0.47 AV: 16 SB: 81 5.78-7.05 NL: 2.10E7F: FTMS + p ESI Full ms [200.00-1050.00]

507 508 509 510 511 512 513m/z

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510.2817R=57440

z=1C 26 H36 O4 N7-0.6538 mmuC 27 H42 O9-0.6590 mmu

C 25 H40 O8 N30.6837 mmu

508.2663R=56874

z=1C 26 H34 O4 N7-0.4031 mmuC 27 H40 O9-0.4082 mmu

C 25 H38 O8 N30.9344 mmu

511.2848R=57516

z=1C 20 H41 O10 N5

0.0095 mmuC 21 H37 O6 N9-1.3280 mmu

C 22 H43 O11 N2-1.3331 mmu

509.2696R=56261

z=?C 20 H39 O10 N5

0.4321 mmuC 21 H35 O6 N9-0.9054 mmu

C 22 H41 O11 N2-0.9106 mmu

16

Compound 10c. 400 MHz 1H NMR spectrum in MeOD

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Compound 10c. 100 MHz 13C NMR spectrum in MeOD

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Compound 10c. HRMS spectra

mm_101618_nrh_valine_01 #16-30 RT: 0.23-0.44 AV: 15 SB: 81 5.78-7.06 NL: 1.51E7F: FTMS + p ESI Full ms [200.00-1050.00]

493 494 495 496 497 498m/z

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bund

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496.2657R=58296

z=1C 24 H38 O8 N3

0.3563 mmu

494.2505R=58700

z=1C 25 H32 O4 N7-0.5448 mmu

497.2691R=58231

z=1C 19 H39 O10 N5

-0.0212 mmu

495.2542R=58360

z=?C 20 H33 O6 N9-0.6596 mmu

19

Compound 10d. 400 MHz 1H NMR spectrum in MeOD

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Compound 10d. 100 MHz 13C NMR spectrum in MeOD

21

Compound 10d. HRMS spectra

mm_101618_nrh_tryptophan_01 #17-41 RT: 0.25-0.61 AV: 25 SB: 81 5.78-7.06 NL: 2.03E7F: FTMS + p ESI Full ms [200.00-1050.00]

680 681 682 683 684 685 686 687 688m/z

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bund

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683.3276R=50853

z=1

684.3305R=50849

z=1

681.3123R=50150

z=1685.3333R=51048

z=1682.3158R=50741

z=?

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Compound 10e. 400 MHz 1H NMR spectrum in MeOD

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Compound 10e. 100 MHz 13C NMR spectrum in MeOD

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Compound 10e. HRMS spectra

mm_072219_nrh_phenylalanine_1 #5-28 RT: 0.07-0.26 AV: 8 NL: 2.55E7F: FTMS + p ESI Full ms [100.00-600.00]

540 541 542 543 544 545 546 547 548 549m/z

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544.26593R=56245

z=1

545.26919R=55336

z=1

542.25069R=56007

z=1

543.25400R=56282

z=1

546.27201R=57569

z=1

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Compound 10f. 400 MHz 1H NMR spectrum in MeOD

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Compound 10f. 100 MHz 13C NMR spectrum in MeOD

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Compound 10f. HRMS spectra

mm_101618_nrh_isoleucine_01 #17-33 RT: 0.25-0.49 AV: 17 SB: 81 5.78-7.06 NL: 2.47E7F: FTMS + p ESI Full ms [200.00-1050.00]

507 508 509 510 511 512 513 514m/z

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510.2821R=56884

z=1C 26 H36 O4 N7

-0.4491 ppmC 27 H42 O9-0.4593 ppm

C 25 H40 O8 N32.1720 ppm

511.2851R=56955

z=1C 20 H41 O10 N5

0.6638 ppmC 21 H37 O6 N9

-1.9522 ppmC 22 H43 O11 N2

-1.9623 ppm

508.2667R=57549

z=1C 27 H40 O90.0774 ppm

C 26 H34 O4 N70.0876 ppm

C 28 H36 O5 N4-2.5540 ppm

509.2700R=57198

z=?C 21 H35 O6 N9

-0.9270 ppmC 22 H41 O11 N2

-0.9372 ppmC 20 H39 O10 N5

1.6993 ppm

28

Compound 10g. 400 MHz 1H NMR spectrum in MeOD

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Compound 10g. 100 MHz 13C NMR spectrum in MeOD

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Compound 10g. HRMS spectra

mm_101618_nrh_methionine_01 #18-32 RT: 0.26-0.47 AV: 15 SB: 81 5.78-7.06 NL: 2.46E7F: FTMS + p ESI Full ms [200.00-1050.00]

525 526 527 528 529 530 531m/z

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528.2387R=56309

z=1

529.2419R=56244

z=1

526.2235R=55989

z=1

31

Compound 12a. 400 MHz 1H NMR spectrum in D2O

32

Compound 12a. 100 MHz 13C NMR spectrum in D2O

33

Compound 12a. 377 MHz 19F NMR spectrum in MeOD

34

Compound 12a. HRMS spectra

mm_101618_nr_glycine_01 #13-21 RT: 0.18-0.31 AV: 9 SB: 82 5.78-7.07 NL: 2.79E6F: FTMS + p ESI Full ms [200.00-1050.00]

305 310 315 320m/z

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312.1197R=73560

z=1C 14 H14 O 2 N7

-1.9392 ppmC 15 H20 O 7-1.9558 ppm

C 13 H18 O 6 N32.3460 ppm

313.1230R=74186

z=1C 8 H19 O 8 N5

0.7378 ppmC 22 H17 O 22.3591 ppm

C 9 H15 O 4 N9-3.5336 ppm

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Compound 12b. 400 MHz 1H NMR spectrum in D2O

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Compound 12b. 100 MHz 13C NMR spectrum in D2O

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Compound 12b. 377 MHz 19F NMR spectrum in MeOD

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Compound 12b. HRMS spectra

mm_101618_nr_leucine_01 #13-22 RT: 0.18-0.32 AV: 10 SB: 82 5.78-7.06 NL: 4.94E6F: FTMS + p ESI Full ms [200.00-1050.00]

366 367 368 369 370 371 372m/z

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368.1816R=67557

z=1C 16 H20 O N10

-0.1071 ppmC 17 H26 O 6 N3

-0.1212 ppm

369.1849R=66613

z=1C 26 H25 O 2

0.0990 ppmC 12 H27 O 8 N5-1.2761 ppm

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Compound 12c. 400 MHz 1H NMR spectrum in D2O

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Compound 12c. 100 MHz 13C NMR spectrum in D2O

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Compound 12c. 377 MHz 19F NMR spectrum in MeOD

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Compound 12c. HRMS spectra

mm_101618_nr_valine_01 #13-44 RT: 0.18-0.65 AV: 32 SB: 82 5.77-7.06 NL: 1.32E6F: FTMS + p ESI Full ms [200.00-1050.00]

352 353 354 355 356 357m/z

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100R

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danc

e

354.1661R=69575

z=1C 16 H24 O 6 N3

0.3722 ppm

355.1695R=67522

z=1C 25 H23 O 2

0.6424 ppm

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Compound 12d. 400 MHz 1H NMR spectrum in D2O

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Compound 12d. 100 MHz 13C NMR spectrum in D2O

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Compound 12d. 377 MHz 19F NMR spectrum in MeOD

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Compound 12d. HRMS spectra

mm_101618_nr_tryptophan_01 #13-21 RT: 0.18-0.31 AV: 9 SB: 82 5.78-7.07 NL: 7.47E5F: FTMS + p ESI Full ms [200.00-1050.00]

438 439 440 441 442 443 444 445 446m/z

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441.1773R=61755

z=1C 8 H27 O 12 N9

-0.2166 ppmC 22 H25 O 6 N4

0.9341 ppm

442.1805R=61373

z=1C 17 H26 O 8 N6

-0.3455 ppmC 16 H30 O 12 N2

2.6793 ppm

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Compound 12e. 400 MHz 1H NMR spectrum in D2O

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Compound 12e. 100 MHz 13C NMR spectrum in D2O

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Compound 12e. 377 MHz 19F NMR spectrum in MeOD

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Compound 12e. HRMS spectra

mm_101618_nr_phenylalanine_01 #13-23 RT: 0.18-0.33 AV: 11 SB: 82 5.77-7.06 NL: 4.94E6F: FTMS + p ESI Full ms [200.00-1050.00]

399 400 401 402 403 404 405 406 407m/z

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402.1663R=64928

z=1C 20 H24 O 6 N3

0.8076 ppm

403.1696R=63979

z=1C 15 H25 O 8 N5

-0.4827 ppm

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Compound 12f. 400 MHz 1H NMR spectrum in D2O

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Compound 12f. 100 MHz 13C NMR spectrum in D2O

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Compound 12f. 377 MHz 19F NMR spectrum in MeOD

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Compound 12f. HRMS spectra

mm_101618_nr_isoleucine_01 #13-24 RT: 0.18-0.35 AV: 12 SB: 82 5.78-7.07 NL: 4.58E6F: FTMS + p ESI Full ms [200.00-1050.00]

366 367 368 369 370 371m/z

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368.1820R=68181

z=1C 17 H26 O 6 N3

0.9686 ppm

369.1854R=65044

z=1C 12 H27 O 8 N5

-0.1098 ppm

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Compound 12g. 400 MHz 1H NMR spectrum in D2O

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Compound 12g. 100 MHz 13C NMR spectrum in D2O

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Compound 12g. 377 MHz 19F NMR spectrum in MeOD

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Compound 12g. HRMS spectra

mm_101618_nr_methionine_01 #12-22 RT: 0.17-0.32 AV: 11 SB: 81 5.78-7.05 NL: 2.48E6F: FTMS + p ESI Full ms [200.00-1050.00]

384 385 386 387 388 389 390m/z

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386.1376R=65949

z=1

387.1410R=64324

z=1

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* Nicotinamide riboside ∆ Nicotinamide ● D-Ribose-1-phosphate

** **

*

** **

*

* * *

** * **

**

** *

* * **

60

† NR-glycine * Nicotinamide riboside

61

OH OH

N +

N H 2O

OOH N H 3

+

O

O–

NH2

O

NO HOH

O H

O –O

POO

OHO HOH

O NH2

N+OO

NH2

O

GlycineNR

D-Ribose-1-phosphateNAM

0-6 hoursPNP, HEPES H2PO4- ,

NR-Glycine

+

+

† NR-glycine ∆ Nicotinamide ● D-Ribose-1-phosphate Detailed PNP enzyme activity assay. The phosphorolysis of nicotinamide riboside chloride (NR-Cl) and glycine nicotinamide riboside conjugate (Gly-NR) catalyzed by PNP (Purine Nucleoside Phosphorylase) was performed at room temperature and monitored by 1H NMR at t = 0, 20 min and 6 h. All one dimensional spectra were obtained at 300 K on a Bruker AscendTM 400 MHz ultrashielded spectrometer (Bruker Biospin) operating at 400.13 MHz for protons (9.39 Tesla). TopSpin 3.2 (Bruker BioSpin) was used for all NMR spectral acquisition (ns=128) and pre-processing, and the automation of sample submission was performed using ICON-NMR (Bruker BioSpin). All samples were automatically shimmed, and the spectra acquisition time was 10 minutes 08 seconds (ns=128). Each NMR tube (7inch x 5mm) contained 450 µl HEPES buffer (100.0 mM, pH 7.0) which included 100 mM KH2PO4, 5 µl PNP (1mg dissolved in 50µl HEPES buffer), 50.0 µl NR-Cl and NR glycine conjugate (50.0 mM in 1 mL D2O). For each independent experiment used freshly prepared solutions of NR-Cl and NR glycine conjugate in HEPES buffer, containing 100 mM KH2PO4 (13.6 mg/mL) and 50 µl D2O.

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TABLE 1: exploratory conditions for the synthesis of NR amino acid conjugate 5 by ball-milling.

Entry

TMSOTf

Solvent

Reaction outcomes a,b

1 1 eqv 2 eqv DCE (1 eqv) trace 2 1 eqv 2 eqv DCE (1eqv) trace 3 1 eqv 0.5 eqv DCM (1 eqv) trace 4 1 eqv 1 eqv CH3CN (1 eqv) trace 5 1 eqv 2 eqv CH3CN (3 eqv) trace 6 1 eqv 3 eqv CH3CN (5 eqv) trace

a ball-milling (30Hz), 30 minutes; b crude 1H-NMR indicated decomposition of the riboside and formation of trace quantities of the expected product, product formation was confirmed by

MS.