Primary lignin monomers

1. Models of G subunits: Proposed mechanism UV/Vis reveals that the sterically hindered base does not coordinate to the catalyst. We envision deprotonation of the phenol substrate: Sterics: -Addition of Im or N-MeIm – 10% DMBQ -No added ligand, 30% DMBQ -Addition of 2-MeIm and 1,2-diMeIm - >75% DMBQ promote high yields of DMBQ formation. -Bulky ligands distort salen, prevent addition of a second ligand, and may raise the dz2 energy List of lignin models to test for biotransformation Electronics: -Co(salen)/2-MeIm is high spin, placing the unpaired electron in the dx2-y2 orbital -Co is displaced from the salen plane to reduce the ligand field strength -The presence of an electron in the highest energy orbital facilitates its redox transfer from CoII to the oxygen ligand, forming the CoIII-superoxo complex IMPACT We have used RHDs to transform lignin models to cis-dihydroxylated species. In addition, we have successfully oxidized models of the S and G subunits in lignin in the presence a variety of axial ligands and hindered bases. This is a starting point for the conversion of lignin into a single material. References and Notes 1. Endoma, M. A., Bui, V. P., Hansen, J., and Hudlicky, T. Org. Process Res. Dev. 2002, 6, 525. 2. Bozell, J. J.; Hames, B. R. J. Org. Chem.1995, 60, 2398. 3. Superimposed 2D HMQC spectra before (red) and after (gray) oxidation. Isolated lignin was reacted in the presence of methanol, 10% cobalt catalyst, O2 (60psi), 24 hrs. Chemical and Biochemical Catalysis for the Oxidation of p-Substituted Phenols: Models for Conversion of Lignin as a Renewable Raw Material Joseph J. Bozell,1 Diana Cedeno,1 James Daleiden,1 Alison Buchan,2 Ashley Frank,2 Mary Hadden2 and Thomas Elder3 1Center for Renewable Carbon, University of Tennessee, Knoxville, TN 37996 2Department of Microbiology, University of Tennessee, Knoxville, TN  37996 3USDA-Forest Service Southern Research Station, Pineville, LA 71360 Center for Direct Catalytic Conversion of Biomass to Biofuels CHEMICAL APPROACH Lignin comprises as much as 25% of terrestrial biomass, but remains one of the most underused carbon sources in the biosphere.1 The main obstacle in utilizing lignin is its irregular, heterogeneous structure, resulting from biosynthetic, free radical polymerization of a small group of monolignols. Thus, developing processes that convert it into a single material in high yieldwould greatly increase its value and utility within the biorefinery. This approach is focused on the single unifying structural feature of lignin - its network of aromatic rings. We examine processes that can cause selective oxidation of this aromatic network in models of S and G subunits and in lignin itself. When using these oxidizing conditions on lignin itself we observe formation ofquinones among other unidentified products.3 Model of lignin in poplar Models of S subunits Oxidized in the presence of a pyridine or imidazole coordinating base. CHEMICAL APPROACH Primary lignin monomers BIOCHEMICAL APPROACH Models of G subunits More difficult to oxidize2 but yields are improved in the presence of a non-coordinating base. Alpha spin density – primarily on O2 Beta spin density – primarily on Co Currently working to locate single electron BIOCHEMICAL APPROACH Preliminary DFT calculations, Gaussian03 B3LYP functional/6-31g basis set Preliminary calculations reproduce structure of Co(salen)/pyr complex Cellular Intermediates LARGE VOLUME RHD-mediated catabolism of aromatic compounds. In aerobic microbial metabolism, enzyme systems known as Ring Hydroxylating Dioxygenases(RHD) can catalyzetheconversion of aromatic compounds to activated cis-dihydroxylated species. The genes encoding the RHD proteins can be inserted into a bacterial expression vector and produced in a compatible host, such as E. coli for accumulation of the dihydrodiol. Small Volume Optimization Approach Combined use of recombinant microbial strains and chemical catalysis to form hydrocarbon products.1 Screening Approach Several Reactions A Two-Pronged, Parallel Strategy • Optimize conditions for maximum yield • Scale up reactions to produce more compound • Isolate and characterize compound • Screen combinations of four RHD-producing organisms and a list of lignin model compounds for chemical transformations MANY REACTIONS Initial Observations Table : UV assay results for biotransformations performed with 4 different RHDs and 12 lignin models. (+) indicates the presence of a distinct product peak in the UV range; (+/-) indicates a potential product peak in the UV range; (-) indicates no unique peak; and (ND) indicates that the given enzyme/substrate combination had not yet been tested. • UV assays suggest that, collectively, our 4 RHDs can transform 10 of 12 tested lignin models to a product that is distinct from the starting material. • Preliminary results indicate that performing the biotransformation step in minimal salts broth (MSB) significantly improves yield • Addition of substrate approximately 4h after induction with IPTG is optimal • Product formation has been seen after as long as 43h after substrate introduction • Substrate concentrations of up to ~3uL/mL medium were tolerated • Addition of smaller aliquots of substrate (50uL every 4h) gave higher product concentration The Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio) is an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0000997.