Fig. 2: Synthesis of MASTER-NAADP and derivatives II A) Reagents and conditions.

[a] Thionylchloride, kat. DMF, 2 h, 80 °C, then 2.0 eq. amino-2-methyl-1-propanol, CH₂Cl₂, 2 h, 0 °C → rt, thionylchloride, 16 h, rt, 87 %; [b] 1.5 eq. 17, 1.6 eq. n-BuLi (1.6 M in hexane), THF, 1 h, –78 °C, then 1.0 eq. 2,3,5-tri-O-benzyl-ribonolactone, THF, 2 h, –78 °C → –30 °C, 3.0 eq. triethylsilane, 2.5 eq. BF₃*Et₂O, CH₂Cl₂, 16 h,–78 °C → rt, 41 %; [c] CH₃NO₂/MeI (2:1) 16 h, reflux, then MeOH/KOH 20 %, 1:1, 16 h, reflux, quantitatively; [d] 0.33 eq. triphosgene, 1.0 eq. pyridine, CH₂Cl₂, 15 h, rt, 93 %; [e] 1.1 eq. 4-hydroxyl benzyl alcohol 10, 1.0 eq. Et₃N, 1.0 eq. 21, CH₂Cl₂, 15 min, 0 °C, then 3 h, rt, 74 %; [f] 1.0 eq. 19, 1.1 eq. Yamaguchi-reagent, 1.2 eq. Et₃N, 1.2 eq. 22, 0.3 eq. DMAP, THF, 3 h 70 °C, 16 h, rt, 58 %; [g] 3.5 eq. BCl₃ (1 M, CH₂Cl₂), 2 h, –78 °C, 67 %; [h] 2.0 eq. POCl₃, 2.5 eq. Bu₃N, trimethylphosphate, 16 h, 0 °C, 42 %. [i] 10.0 eq. TFAA, 16 eq. Et₃N, 1.0 eq. monophosphate 25, CH₃CN, 10 min, 0 °C → Rt, then 6.0 eq. NMI, 10 eq. Et3N, CH₃CN, 10 min, 0 °C → rt, then 1.1 eq. 15, CH₃CN, 3 h, rt, then TEAB-buffer 1 M, 15 min, rt, 57%. B) Release of MASTER-NAADP by porcine liver extract and mass spec characterization of main product. B MASTER-NAADP was analyzed by RP-HPLC for purity (black chromatogram). A representative out of 6 experiments is shown. Then, reaction products obtained upon incubation of MASTER-NAADP with porcine liver extract were separated by RP-HPLC (blue chromatogram). The main product was then identified by ESI-mass spectrum measured in positive ionization mode as benzoic acid C-nucleoside, 2’-phospho-3’F-adenosine-diphosphate. C Molecular modeling: (a) NAADP, (b) overlay of NAADP and benzoic acid C-nucleoside, 2’-phospho-3’F-adenosine-diphosphate, (c) benzoic acid C-nucleoside, 2’-phospho-3’F-adenosine-diphosphate.