Phosphorylation of prebiotically important organic compounds after a wet-to-dry transition

Here, 24.2 mg (0.13 mmol, 1 eq.) of calcium imidazole phosphate (see SI Section ^S3 for the synthesis of calcium imidazole phosphate) and 98.5 mg (0.65 mmol, 5 eq.) of d-ribose were dissolved in 2.0 mL of MilliQ water to give a 65 mM calcium imidazole phosphate and a 325 mM d-ribose solution. The solution was added to a petri dish and left with the lid off to dry at 22 °C for 24 h. The resulting paste was scraped from the petri dish and placed into an Eppendorf. The Eppendorf was spun on a centrifuge for 5 min at 5590 × g in order to move all paste to the bottom of the Eppendorf and compact it together in order to remove air pockets introduced during the transfer of the paste to the Eppendorf. An ~10 mg sample of the paste was taken to record the extent of phosphorylation after the wet-to-dry transition to a paste. The Eppendorf was placed in a thermoshaker at 50 °C and 600 r.p.m. for up to 168 h. The reaction was followed by periodically removing samples (~10 mg), dissolving them in 0.5 mL of 0.5 M citric acid buffer at pH 6.85 and 9:1 H₂O:D₂O, and analysing them with ³¹P-NMR and ¹H-NMR, and ¹H ³¹P HMBC (Heteronuclear Multiple Bond Correlation) NMR spectroscopy. The citric acid buffer chelated calcium ions enabling full solubilization of all calcium phosphate salts. Reactions with other nucleophiles were conducted using an identical procedure unless otherwise specified in the SI Section ^S7.