Biocatalytic Polymerization of p-Cresol:  An in-Situ NMR Approach To Understand the Coupling Mechanism

The nature of the initial coupling mechanism in the enzymatic polymerization of p-cresol was investigated by in-situ 1H NMR spectroscopy in an aqueous−organic media. Two types of in-situ experiments were performed, in which the course of polymerization was monitored with (i) incremental additions of H2O2 to the reaction mixture in the NMR tube at a regular interval and (ii) a function of reaction time after a known amount of addition of H2O2. These experiments were used to understand the coupling mechanism at the very early stage of reaction and during the course of polymerization reaction. In the early stage of the polymerization, the reaction mixture contains monomer as a dominant fraction and a small concentration of low molecular weight oligomers such as dimer, trimers, etc. The analysis of the reaction mixture by 1D and 2D in-situ NMR methods suggests the ortho−ortho C−C coupling as the dominant coupling mechanism during the initial stage of the polymerization. The quantitative NMR analysis indicates that the relative consumptions of monomer and dimers are different at the early stage of polymerization; the consumption of dimer accelerates only after complete conversion of the monomer from the reaction mixture. The in-situ NMR analysis as a function of reaction time after addition of a known amount of H2O2 suggests the conversion of a quinonoid intermediate formed at the beginning from ortho−para C−C coupling, to a stable ketonic product (Pummerer's ketone). A gradual reduction of quinonoid structures and increase of Pummerer's ketone structures in the reaction mixture are monitored with time by in-situ NMR. Structural characterization of bulk polymer by multinuclei NMR spectroscopy and MALDI−TOF mass spectrometry indicates the formation of polymers (M w 1230) and the dominance of ortho−ortho and ortho−para C−C coupled products. The polymers obtained from the reaction products contained a very low concentration of Pummerer's ketones, which may be chain ends.