Students studying in our group are PhD candidates Mai Nguyen, Le Rae Graham, and NilminiGedevini; MS candidates Raju Kovur and Venkata Makkena; and BS candidates Matt McCarthy and Adam St Hilaire. An FBRI-REU student, Andru O'Farrill is working with us in the summer of 2007.

Overall Themes of Our Projects: the ultimate goal of our research is to develop the fundamental chemistry needed to produce new value-added products from wood. To this end, we are participants in the Maine Forest Bioproducts Research Institute, funded by the National Science Foundation. We also want to facilitate the "greening" of the processing of wood into these products and the traditional use: paper. This means we seek to use environmentally benign reagents and processes.

First a brief description of wood.

The Composition and Structure of Wood
Graphic from the B.S. thesis of Renee Whippee, UMaine, 2006

Currently in Maine the principal processing of wood is pulping: removal of the lignin and hemicelluloses, and conversion of the cellulose to paper products. The residual lignin and hemicellulose are burned to provide energy for the processing. If the hemicelluloses could be recovered efficiently, and converted to their component sugars, they could be a new source of raw materials for plastics, ethanol, and other useful products.

The hemicelluloses are covalently connected to lignin in wood cell walls. Therefore one major focus of our current efforts is the selective breaking of that connection by oxidative and enzymatic methodologies (work supported by NSF and USDA). Another focus is the efficient hydrolysis of the hemicelluloses to their constituent sugars (work supported by EPA).

Some Models for the Lignin-Hemicellulose Connection

Some examples of our specific projects follow.

Selective Chemical Oxidation of Lignin-Carbohydrate Compounds

• Synthesis of several of the above compounds by literature procedures, or improvements on literature procedures.

• Study oxidation with hydroxyl radical (OH·) and superoxide (O2·-).
  • Will superoxide oxidize the lignin component, leaving carbohydrate untouched ?

  • Will hydroxyl radical cleave these compounds at the anomeric carbon?

  • What will happen with O2 under pressure?

  • Can reactions be found that will hydroxylate the aromatic components cleanly, thus making them more susceptible to selective oxidation? [(Pertrifluoroacetic acid? Perhaps an enzyme, p-hydroxybenzoate hydroxylase, that oxidizes phenols to catechols?]

Work in this area is being carried out by PhD candidate Mai Nguyen, MS candidate Venkata Makkena, and two BS students, Jodi Wyman and Renee Whippie. We have prepared a number of compounds like the upper left example above, and shown that hydroxyl radical photochemically generated from H₂O₂ cleaves then selectively by attack at the anomeric carbon. The peroxide and hydroxyl radical then further oxidize the sugar component.

Enzymatic Oxidation of Lignin-Carbohydrate Compounds

Laccases (Chem. Rev., 1996, 96, 2563) are four-copper enzymes used by fungi to depolymerize lignin, and by some species of trees to build new lignin at sites of injury. They have two catalytic sites, one containing three coppers that reduces O2 to H2O, and a one copper center that oxidizes phenolics.

• We are examining laccase reactions with lignin-carbohydrate models, both experimentally and computationally. The experimental work includes determination of oxidation products using gc-ms or hplc-ms and comparison with products of chemical oxidation.

• Computational work examines docking of substrates to the one-copper site, and examination of mutations that might improve substrate binding. Docking of simple lignin models is underway, with Mai Nguyen working in collaboration with Ken Overly at Providence College.

Coniferyl Alcohol Docked to Laccase	Closeup of Catalytic Site

The docking results, presented at the 2005 ISWFPC, indicate that small lignin fragments bind to the enzyme as effectively as do the synthetic substrates often used as mediators.

The catechol dioxygenases (Chem. Rev., 1996, 96, 2607) are a family of bacterial enzymes that cleave dioxygenated aromatic rings to muconic acids.

• They are largely extra-cellular, non-heme iron-containing enzymes related to the peroxidases.

• Will these cut the lignin pieces off our models ?

• What will be the extent of oxidation of the aromatic portions?

A Catechol Dioxygenase

• Again, computational docking experiments will be conducted.

• We also would like to compute the redox potential of the catalytic sites of these enzymes (and laccases)
  • We will model mutations that might improve selectivity or oxidizing power

Fenton Chemistry

So-called brown rot fungi apparently use Fenton chemistry to degrade lignin and cellulose:

We are exploring the possible selectivity of the Fenton system toward lignin-carbohydrate compounds.

• MS candidate Satyavan Singh has observed cleavage of methyl b-D-glucoside and b-cellobioside in the Fenton system (without the reductase present)

• Singh also has carried out homology modeling of the quinone reductase, which was sequenced by Ken Hammel and coworkers at the FPL; docking of the FMN cofactor is underway

Model of Quinone Reductase

• We also are exploring computationally the role of the redox potential of the hydroquinone and quinone in both the Fenton chemistry and the reduction of the quinone.

• Can a more efficient chemical sequence be designed using a commercially or synthetically available quinone and reductant?

Hydrolysis of Hemicelluloses

This project addresses the hydrolysis of hemicellulose to individual sugar components.

• Acid hydrolysis (1 M sulfuric acid) can recover some of the sugars

• Further reactions of the sugars and the resistance of some of the glycosidic bonds to these conditions limit the efficiency of this simple treatment.

• Organisms that feed on wood and cell walls generally secrete a variety of glucanases, one or more for each type of glycosidic bond: xylanases, glucuronidases, arabinofuranosidases, etc.

  An Example of a Xylanase	An Arabinofuranosidase

• Working initially with birch xylan PhD candidate LeRae Graham is exploring the enzymatic hydrolyses.

• We want to determine
  • conditions for most efficient reaction

  • nature of the products

  • whether multiple enzymes can be used simultaneously to hydrolyze multiple linkage types

  • the extent to which residual phenolic lignin interferes with the hydrolysis.

• If lignin residues pose a significant problem, then we will explore the extent to which the problem can be alleviated by the methodologies developed above.

• Work then will proceed to hemicellulose fractions obtained from Maine softwood.

Modeling is a significant component of this problem as well. Glucanases are modular enzymes, with variable binding modules and relatively strongly conserved catalytic modules.

We shall conduct docking experiments to determine the fit of various substrate types, and in silico mutations that might alter selectivity towards a broader range of glycosidic bonds.

T. Elder and R. C. Fort Jr., "Computational Electrochemistry of Lignin Model Compounds", POSTER presented at the 13th International Symposium on Wood, Fiber, and Pulping Chemistry, Auckland, NZ, May, 2005; Abstracts, p. 113.

R. C. Fort Jr., B. J. W. Cole, M. Nguyen, R. M. Pedro, and K. R. Overly, "Selectivity in the Oxidation of Carbohydrate-bound Lignin. I. Docking of Lignin Models to the Laccase from Trametes versicolor", POSTER presented at the 13th International Symposium on Wood, Fiber, and Pulping Chemistry, Auckland, NZ, May, 2005; Abstracts, p. 87.

D. F. Guay, B. J. W. Cole, R. C. Fort, Jr., M. C. Hausman, J. M. Genco, and T. J. Elder, "Mechanisms of Oxidative Degradation of Carbohydrates During Oxygen Delignification. III. Reaction of Photochemically Generated Hydroxyl Radicals with 1,5-Anhydrocellobitol and Cellulose", J. Pulp Paper Sci., 2002, 28, 217.

T. Solouki, R. C. Fort, Jr., A. Alomary, and A. Fattahi, "Gas Phase Hydrogen-Deuterium Exchange Reactions of a Model Peptide: FT-ICR and Computational Analyses of Metal-Induced Conformational Mutations", J. Am. Soc. Mass Spectr., 2001, 12, 1272.

D. F. Guay, B. J. W. Cole, R. C. Fort, Jr., M. C. Hausman, J. M. Genco, T. J. Elder, and K. R. Overly, "Mechanisms of Oxidative Degradation of Carbohydrates During Oxygen Delignification. II. Reaction of Photochemically Generated Hydroxyl Radicals with Methyl b-D-cellobioside", J. Wood Chem. Technol., 2001, 21(1), 67.

D. F. Guay, B. J. W. Cole, R. C. Fort, Jr., J. M. Genco, and M. C. Hausman, "Mechanisms of Oxidative Degradation of Carbohydrates During Oxygen Delignification. I. Reaction of Photochemically Generated Hydroxyl Radicals with Methyl -D-glucoside", J. Wood Chem. Technol., 2000, 20(4), 375.

B. J. W. Cole, J. Yang, and R. C. Fort, Jr. "The Bleaching and Photostabilization of High-Yield Pulp by Sulfur Compounds. II. Reaction of Glycol Mercaptoesters with Model Quinones", J. Wood Chem. Technol., 2000, 20(1), 1.