D erythrose 4 phosphate

N-terminal histidine-tagged CpgA was purified as described by Absalon and coworkers^{10 (link)}. Then, His-tagged CpgA was dialyzed in 20 mM HEPES (pH 8) buffer, and protein concentration was determined using Bradford colorimetric assay kit. In a 96 well plate, P_i release was measured using 10 μM CpgA with 1 mM of substrates: guanosine-5’-triphosphate (Roche), D-glucose-6-phosphte (Sigma), D-fructose-6-phosphate (Sigma), D-gluconate-6-phosphate (Sigma), D-erythrose-4-phosphate (Sigma), and D-erythronate-4-phosphate (Santa Cruz Biotechnology), in a 100 mM MOPS buffer (pH 7.5) containing 1 mM MgCl₂. The reactions were incubated at 37 °C for 20 min. and released phosphates were measured spectroscopically at 620 nm by adding 25 μl of malachite green working reagent (Sigma-Aldrich, MAK308) incubated for 30 min at room temperature.

Br₂-saturated H₂O (616 μl, ∼35 mg/ml, 135 μmol) was added to a stirred mixture of d-erythrose-4-phosphate (12.0 mg, 54.0 μmol; Sigma) in aqueous Na₂CO₃ (400 μl, 0.405 M, 162 μmol) at room temperature. After 1 h, excess bromine was removed by sparging for 2 h with N₂. The resultant mixture was passed down a column containing Amberlite Ag50 (acid form, 2-ml bed volume, ∼1.7 mM H⁺ per ml), which was rinsed with H₂O (1.5 ml three times). Cyclohexylamine (39 μl, 0.324 mmol) was added to the eluted product, and the volatiles were removed by rotary evaporation and further drying under an N₂ stream overnight. The crude material was purified on a Shimadzu 2020 LC-MS instrument, using an ACE Excel 5 Super C₁₈ column (150 mm by 2.1 mm) with 0.1% formic acid (solvent A) and methanol (solvent B). A linear gradient was performed from 80% to 10% across 15 min, and elution of 4-PE was determined using the theoretical exact mass of 4-PE. Separation of 4-PE from contaminants was monitored using the MS in full scan mode and with UV detection. Verification of the purified 4-PE was performed by rerunning the collected aliquot on the instrument and confirming no other masses or UV peaks were observed (above background). Expected [M – H]⁻ was 214.9962 m/z; observed was 214.9968 m/z.