Figure 1

Schematic diagram of the Pyo_ENZ process (in blue) that uses a laccase and showing the associated decarboxylation mechanism and the two resulting products identified in the polymer. Comparison to the Pyo_CHEM synthesis (abiotic autoxidation, in red). The common precursor 2,5-DMPA could also be synthesized from 2,5-dimethoxyacetophenone by a Willgerodt-Kindler reaction type²⁵. Step 4 is the final HCl precipitation followed by washing and drying. Doubling the amount of HBr (step 1) led to incomplete demethylation and the extra formation of 2,5-dihydroxyphenylacetaldehyde (~ 6%) identified from the HPLC-DAD spectrum (λ_max 292 nm) and the EI-MS profile (molecular ion [M + 2TMS] at m/z 294, characteristic fragments [M – CHO] at m/z 265, and [M – CH₂CHO] at m/z 251), similarly to the NIST data bank and previous data³⁴. In step 2, the addition of sulfite (Na₂SO₃)²⁵ was unnecessary because the solution was immediately buffered to 6.8 and the polymerization by the laccase followed (step 3). The alkaline opening of the lactone was essential, indeed the rMt laccase was unable to open the lactone nor demethylate 2,5-DMPA at pH 6.8, even after several days of agitation. BQA, 1,4-benzoquinone acetic acid; gentisaldehyde, 2,5-dihydroxybenzaldehyde; gentisyl alcohol, 2,5-dihydroxybenzyl alcohol; 2,5-DMPA, 2,5-dimethoxyphenylacetic acid; 2,5-DMAPO, 2,5-dimethoxyacetophenone.