We used glycerol as a model substrate to assess how the structure of histidyl peptides affects their catalytic performance. Phosphorylated glycerol is an amphiphile precursor required for the formation of membranes and compartmentalisation. The phosphorylations were performed by preparing aqueous solutions containing the amphiphile precursor glycerol, calcium imidazole phosphate and a histidyl peptide at pH 7.5 (Figure , SI Section S3). The solution was evaporated to a paste by leaving the solution open to the air at 22 °C. Unlike in our previous study, we did not heat the pastes to 50 °C in order to promote phosphate transfer but kept the temperature at 22 °C throughout. The reaction was followed for 62 h by the periodic removal of samples from the paste. Samples were dissolved in a 0.5 M citric acid buffer at pH 6.85 with an HMPA internal standard and analysed by 31 P NMR spectroscopy. The yield of phosphorylated glycerol at each time point was determined by summating together the yield of glycerol-1-phosphate, glycerol-2phopshate and cyclo-glycerol-1,2-phosphate.