Supporting Information. Chemo-enzymatic Synthesis of Isotopically Labeled Nicotinamide Ribose
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1 Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry 2018 Supporting Information Chemo-enzymatic Synthesis of Isotopically Labeled Nicotinamide Ribose Ai Tran, Ryota Yokose, Yana Cen* Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, 261 Mountain View Drive, Colchester, VT * Correspondence: Yana.Cen@acphs.edu, phone:
2 Figure S1. SDS-PAGE gel of purified nicotinic acid mononucleotide adenyltransferase (NMNAT). The gene NMA1 was cloned from Saccharomyces cerevisiae gdna into the protein expression vector, Pet28a (Novagen). The PetNMA1 vector was transfected into BL21-CodonPlus(DE3)- RIPL competent cells (Agilent) and protein expression induced by 0.8 mm isopropyl- -Dthiogalactopyranoside (IPTG) when the cells reached OD 600 of 0.6~0.7 in LB media. The culture was grown for another 16 h at 37 C before the cells were pelleted and lysed with 3 freeze-thaw cycles. The protein was purified using Ni-NTA resin (Thermo Fisher) affinity column and eluted with increasing concentrations of imidazole. The protein was aliquoted in 20% glycerol plus 2 mm DTT, flash frozen and stored at -80 C. Protein concentration was determined using Bradford assay. The protein was >95% pure as determined with SDS-PAGE gel electrophoresis.
3 Figure S2. HPLC chromatograms showing that NaAD can be generated via ten enzymes coupled one-pot reaction. Traces b and c are chemical standards of NA and NaAD. Trace a is the enzymatic synthesis of 13 C-NaAD starting from U- 13 C6-glucose as described in Methods and Materials. The production of 13 C-NaAD was confirmed by the formation of a new peak on the chromatogram with identical retention time as the chemical standard of NaAD.
4 Figure S3. MS spectrum of 13 C-NaAD.
5 Figure S4. HPLC profile of enzymatic degradation of 13 C-NaAD. Traces a, b and c are chemical standards of 13 C-NaAD, AMP and NaMN. Trace d is the enzymatic degradation of 13 C-NaAD by phosphodiesterase I. Briefly, 13 C-NaAD was dissolved in 100 mm phosphate buffer ph 7.5. To the reaction was added phosphodiesterase I. The reaction was incubated at 37 C before it was injected on an HPLC fitted to a Macherey-Nagel Nucleosil C18 column. AMP, NaMN and NaAD were resolved using a gradient of 0 to 20% methanol in 20 mm ammonium acetate with a flow rate of 1 ml/min. Chromatograms were analyzed at 260 nm. 13 C-NaAD can be degraded to AMP and 13 C-NaMN as evidenced by the formation of two new peaks on the chromatogram with identical retention times as the chemical standards.
6 Figure S5. SDS-PAGE gel of purified S. pneumoniae NAD + synthetase. The gene for NAD + Synthetase, NadE, was amplified from Streptococcus pneumoniae gdna and cloned into the Pet28a vector (Novagen) at the NdeI-BamHI sites. The PetSPNadE vector was transfected into BL21-CodonPlus(DE3)-RIPL competent cells (Agilent) and protein expression induced by 0.8 mm IPTG when the cells reached OD 600 of 0.6~0.7 in LB media. The culture was grown for another 16 h at 37 C before the cells were pelleted and lysed with 3 freeze-thaw cycles. The protein was purified using a Ni-NTA resin affinity column and eluted with increasing concentrations of imidazole. The protein was aliquoted in 20% glycerol plus 2 mm DTT, flash frozen and stored at -80 C. Protein concentration was determined using Bradford assay. The protein was >95% pure as determined with SDS-PAGE gel electrophoresis.
7 Figure S6. HPLC chromatograms showing NAD + synthetase catalyzed conversion of NaAD to NAD +. Traces b and c are chemical standards of NAD + and NaAD. Trace a is the enzymatic synthesis of 13 C-NAD + starting from 13 C-NaAD as described in Methods and Materials. The production of 13 C-NAD + was confirmed by the formation of a new peak on the chromatogram with identical retention time as the chemical standard of NAD +.
8 Figure S7. HPLC profile of enzymatic degradation of 13 C-NAD +. Traces b, c and d are chemical standards of AMP, NMN and 13 C-NAD +. Trace a is the enzymatic degradation of 13 C-NAD + by phosphodiesterase I. Briefly, 13 C-NAD + was dissolved in 100 mm phosphate buffer ph 7.5. To the reaction was added phosphodiesterase I. The reaction was incubated at 37 C before it was injected on an HPLC fitted to a Macherey-Nagel Nucleosil C18 column. AMP, NMN and NAD + were resolved using a gradient of 0 to 20% methanol in 20 mm ammonium acetate with a flow rate of 1 ml/min. Chromatograms were analyzed at 260 nm. 13 C-NAD + can be degraded to AMP and 13 C-NMN as evidenced by the formation of two new peaks on the chromatogram with identical retention times as the chemical standards.
9 Figure S8. MS spectrum of 18 O-NAM.
10 Figure S9. MS spectrum of 13 C, 18 O-NAD +.
11 Figure S10. HPLC chromatograms showing enzymatic conversion of 13 C, 18 O-NAD + to 13 C, 18 O- NR. Traces b, c and d are chemical standards of NAD +, NR and NMN. Trace a is the enzymatic synthesis of 13 C, 18 O-NR starting from 13 C, 18 O-NAD + as described in Methods and Materials. The production of 13 C, 18 O-NR was confirmed by the formation of a new peak on the chromatogram with identical retention time as the chemical standard of NR.
12 Figure S11. MS spectrum of 13 C, 18 O-NR.
13 Figure S12. 1 H NMR spectrum of 13 C, 18 O-NR. 1 H NMR (D 2 O, 500 MHz) (ppm): 3.81 (m, 0.5H), 3.92 (m, 0.5H), 4.10 (m, 1H), 4.22 (m, 1H), 4.31 (m, 0.5H), 4.42 (m, 0.5H), 4.50 (m, 0.5H), 4.61 (m, 0.5H), 6.11 (m, 0.5H), 6.45 (m, 0.5H), 8.31 (dd, J = 6.4, 7.9 Hz, 1H), 9.02 (dt, J = 1.3, 8.1 Hz, 1H), 9.31 (d, J = 6.3 Hz, 1H), 9.64 (s, 1H).
14 Figure S C NMR spectrum of 13 C, 18 O-NR. 13 C NMR (D 2 O, 125 MHz) (ppm): 60.2 (d, J = 41.8 Hz), 70.2 (dt, J = 3.6, 38.3 Hz), 77.4 (dd, J = 37.6, 42.4 Hz), 88.7 (dd, J = 39.1, 41.4 Hz), 99.9 (dd, J = 3.1, 42.5 Hz), 128.4, 134.0, 140.4, 142.6, 145.6, The inset highlights the carbons on the nicotinamide ring.
15 Figure S14. Characterization of 14 C-NR. A B A. HPLC chromatograms showing that 14 C-NR can be phosphorylated by human NRK1 to 14 C- NMN. Traces a and b are chemical standards of NMN and NR. Trace c is the enzymatic reaction containing 14 C-NR, ATP and recombinant human NRK1. The formation of 14 C-NMN was observed and the radioactivity was confirmed by scintillation counting. B. HPLC chromatograms showing that 14 C-NR can be degraded by PNP to 14 C-NAM. Traces a and c are chemical standards of NR and NAM. Trace b is the enzymatic reaction containing 14 C-NR and human PNP. The formation of 14 C-NAM was observed and the radioactivity was detected by scintillation counting.
16 Figure S C incorporation into NR metabolites. Cells were incubated with 200,000 cpm 14 C-NR for 2 to 6 h. Cells were harvested, rinsed and re-suspended in 1 ml of fresh medium. Cell number was determined using a hemocytometer. The cell suspension was then re-pelleted for NAD + metabolites measurements. To the cell pellet was added 30 L of ice-cold 7% perchloric acid. The sample was then vortexed for 30 s and sonicated on ice for 5 min. The vortex-sonication cycle was repeated three times. The sample was centrifuged at 14,000 x g for 3 min at RT. Clear supernatant was removed and neutralized to ph 7 with 3 M NaOH and 1 M phosphate buffer (ph 9). The sample was then injected on an HPLC fitted to a Macherey-Nagel Nucleosil C18 column. NMN, NR, NAM and NAD + were resolved using a gradient of 0 to 20 methanol in 20 mm ammonium acetate with a flow rate of 1 ml/min. Chromatograms were analyzed at 260 nm. Fractions containing each of the NAD + metabolites were collected and radioactivity was determined by scintillation counting.
17 Figure S16. Phosphorolysis of NR by human PNP. Reactions were performed in 100 mm phosphate buffer ph 7.5 containing increasing concentrations of NR. The reactions were initiated by the addition of 5 M recombinant human PNP and were incubated at 37 C for 10 min before being quenched by 10% TFA. The samples were injected on an HPLC fitted to a Macherey-Nagel Nucleosil C18 column. NR and NAM were resolved using a gradient of 0 to 20% methanol in 20 mm ammonium acetate with a flow rate of 1 ml/min. Chromatograms were analyzed at 260 nm. Reactions were quantified by integrating areas of peaks corresponding to NR and NAM. Rates were plotted as a function of NR concentration and best fits of points to the Michaelis-Menten equation were performed with Kaleigagraph.
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