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

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100R

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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

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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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90

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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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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

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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

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

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

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

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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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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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100R

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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

** **

*

** **

*

* * *

** * **

**

** *

* * **

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† NR-glycine * Nicotinamide riboside

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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.