Nicotinamide riboside-amino acid conjugates that are stable to … · 2020. 4. 2. · 1...
Transcript of Nicotinamide riboside-amino acid conjugates that are stable to … · 2020. 4. 2. · 1...
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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
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Compound 4. 400 MHz 1H NMR spectrum in Acetone-d6
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Compound 4. 100 MHz 13C NMR spectrum in Acetone-d6
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Compound 7. 400 MHz 1H NMR spectrum in D2O
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Compound 7. 100 MHz 13C NMR spectrum in D2O
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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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123.05448R=118611
z=1
295.12793R=77190
z=1
173.08005R=102998
z=1
141.95781R=113872
z=1
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Compound 8. 400 MHz 1H NMR spectrum in D2O
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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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e297.14359R=77139
z=1
123.05456R=119611
z=1
141.95786R=114498
z=1
173.08007R=103770
z=1
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Compound 10a. 400 MHz 1H NMR spectrum in MeOD
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Compound 10a. 100 MHz 13C NMR spectrum in MeOD
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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
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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
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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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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
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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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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
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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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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
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
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
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
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
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
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
0
10
20
30
40
50
60
70
80
90
100R
elat
ive
Abu
ndan
ce
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.