Hexose phosphorylation for a non-enzymatic glycolysis and pentose phosphate pathway on early Earth

Glycolysis and pentose phosphate pathways play essential roles in cellular processes and are assumed to be among the most ancient metabolic pathways. Non-enzymatic metabolism-like reactions might have occurred on the prebiotic Earth and been inherited by the biological reactions. Previous research has identified a part of the non-enzymatic glycolysis and the non-enzymatic pentose phosphate pathway from glucose 6-phosphate and 6-phosphogluconate, which are intermediates of these reactions. However, how these phosphorylated molecules were formed on the prebiotic Earth remains unclear. Herein, we demonstrate the synthesis of glucose and gluconate from simple aldehydes in alkaline solutions and the formation of glucose 6-phosphate and 6-phosphogluconate with borate using thermal evaporation. These results imply that the initial stages of glycolysis-like and pentose phosphate pathway-like reactions were achieved in borate-rich evaporative environments on prebiotic Earth, suggesting that non-enzymatic metabolism provided biomolecules and their precursors on prebiotic Earth.


Fig. S2 .
Fig. S2.LC-MS/MS analysis of glucose phosphate in the phosphorylation experiment at °C (m/z: 259>97).(a) MRM chromatogram of the standard of glucose 6-phosphate 3. (b) Fragment pattern spectrum of the glucose 6-phosphate 3. (c) MRM chromatogram of the experimental product at 95 °C.(d) Fragment pattern spectrum of the experimental product at °C.

Fig. S3 .
Fig. S3.LC-MS/MS analysis of phosphogluconate in the phosphorylation experiment at °C (m/z: 275>97).(a) MRM chromatogram of the standard of 6-phosphogluconate 4. (b) Fragment pattern spectrum of the 6-phosphogluconate 4. (c) MRM chromatogram of the experimental product at 95 °C.(d) Fragment pattern spectrum of the experimental product at °C.

Fig. S5 .
Fig. S5.Fragmentation pattern of glucose 6-phosphate.The molecular ion mass is 259.Fragments from phosphate moieties (79 and 97) show intense signals.The signals of 199, 169, and 139 are created by the fragmentation in glucose.The signal 139 is characteristic of the 6-phosphate and 5-phosphate.Phosphates combined at 2, 3, or 4-hydroxyl do not form the fragment 139.

Fig. S11 .
Fig. S11. 13C-NMR spectra of product ureido-glucose phosphate.(a) Full spectrum of the experimental product.(b) Enlarged spectrum view of chemical shifts from glucose of the experimental product (green-shaded area in a).

Fig
Fig. S12.LC-MS/MS analysis of residual glucose (m/z: 179>59).(a) Residual glucose in the experiment with borate.The yields represent the average of triplicate experiments.(b) Residual glucose in the experiment without borate.The yields represent the average of triplicate experiments.(c) The standard of glucose.

Fig
Fig. S13.LC-MS/MS analysis of residual gluconic acid in the phosphorylation experiments (m/z: 195>75).(a) Residual gluconic acid in the experiment with borate.The yields represent the average of triplicate experiments.(b) Residual gluconic acid in the experiment without borate.The yields represent the average of triplicate experiments.(c) The standard of gluconic acid.

Fig. S14 .
Fig. S14. 1 H-NMR spectra of the condensation products of glucose and urea.(a) Experimental product in the presence of borate.(b) Experimental product in the absence of borate.

Fig. S15 .
Fig. S15. 13C-NMR of the condensation products of glucose and urea.(a) Experimental product in the presence of borate.(b) Experimental product in the absence of borate.

Fig. S16 .
Fig. S16.Full 13 C-NMR spectra of condensation products of glucose and urea.(a) Experimental product in the presence of borate.(b) Experimental product in the absence of borate.

Fig. S17 .
Fig. S17.MS spectra of the direct infusion analysis of glucose and urea condensation products in the presence of borate.(a) Negative ESI-MS spectrum showing the formation of ureido-glucose combined with one or two borates.The m/z signal at 243 Da corresponds to ureidoglucose combining one boric acid.The m/z signal at 291 Da corresponds to ureido-glucose combining one boric acid and one borate.Other major peaks are attributed to polyborates.(b) The fragmentation spectrum of the precursor ion 291 Da that corresponds to the mass of ureido-glucose combined with two borate molecules.The m/z signals of 43 and 59 are attributed to fragments from urea.

Fig
Fig. S19 LC-MS/MS analysis of phosphogluconate in the phosphorylation experiment in the presence of glucose, gluconate, and Ca 2+ .(a) MRM chromatogram of the standard of 6phosphogluconate 4 (m/z: 275>97).(b) Fragment pattern spectrum of the 6-phosphogluconate 4. (c) MRM chromatogram of the experimental product (m/z: 275>97).(d) Fragment pattern spectrum of the experimental product.

Fig. S24 .
Fig. S24.Possible reaction pathways from glucose.Combining with borate at 2-hydroxyl, the isomerization from imine to ketone was prevented, which contributes to the stabilization of ureidoglucose.The stabilized molecules are subjected to the phosphorylation.