US 8,685,672 B2 Page 2

(56) References Cited Kusano, R.; Tanaka, T.; Matsuo, Y.; Kouno, 1., Structures of epicatechin gallate trimer and tetramer produced by enzymatic oxi OTHER PUBLICATIONS dation. Chem Pharm Bull (2007), 55:1768-1772. Lopez, S.; Murison, S.D.; Travis, A.J.; Chesson, A.; Degradability of Grabber, J .H.; Hat?eld, R.D.; Ralph, J ., Diferulate cross-links parenchyma and sclerenchyrna cell walls isolated at different devel impede the enzymatic degradation of nonligni?ed maize walls. J Sci. opmental stages from a newly extended maize internode. Acta Bot Neerl(1993), 42:165-174. FoodAgr. (1998), 77(2), 193-200. Lu, F.; Ralph, J., Derivatization followed by reductive cleavage Grabber, J .H.; Ralph, J; Hat?eld, R.D.; Cross-Linking of maize walls (DFRC method), a new method for lignin analysis: protocol for by ferulate dimerization and incorporation into lignin. J Agric Food analysis of DFRC monomers. J Agr. Food Chem. (1997), 45(7), Chem (2000), 48:6106-6113. 2590-2592. Grabber, J .H.; Hat?eld, R.D.; Ralph, J ., Apoplastic pH and Lu, F.; Ralph, J ., Facile synthesis of 4-hydroxycinnamyl monolignol addition rate effects on lignin formation and cell wall p-coumarates. JAgric Food Chem (1998), 46:2911-2913. degradability in maize. JAg Food Chem (2003), 51:4984-4989. Lu, F.; Ralph, J ., Preliminary evidence for sinapyl acetate as a lignin Grabber, J .H.; Hat?eld, R.D., Methyl esteri?cation divergently monomer in kenaf. Chem. Commun. (2002), (1), 90-91. affects the degradability of pectic urono syls in nonligni?ed and ligni Lu, F.; Ralph, J., Non-degradative dissolution and acetylation of ?ed maize cell walls. JAg Food Chem (2005), 53:1546-1549. ball-milled plant cell walls; high-resolution solution-state NMR. Grabber, J .H., How do lignin composition, structure, and cross-link PlantJ. (2003), 35(4), 535-544. ing affect degradability? A review of cell wall model studies. Crop Sci Lu, F.; Ralph, J ., Novel B-B-structures in natural lignins incorporating (2005), 45:820-831. acylated monolignols, Thirteenth International Symposium on Wood, Grabber J .H.; Lu, F., Formation of syringyl-rich lignins in maize as Fiber, and Pulping Chemistry, Auckland, New Zealand, (2005); in?uenced by feruloylated xylans and p-coumaroylated monolignols. APPITA, Australia: pp. 233-237. Planta (2007), 226:741-751. Lu, F.; Ralph, J ., Novel tetrahydrofuran structures derived from [5-[5 Grabber, J .H.; Hat?eld, R.D.; Lu, F.; Ralph, J ., Coniferyl ferulate coupling reactions involving sinapyl acetate in kenaf lignins. Org incorporation into lignin enhances the alkaline deligni?cation and Biomol Chem (2008), 6:3681-3694. enzymatic degradation of cell walls. Biomacromolecules (2008), Marita, J .; Ralph, J .; Hat?eld, R.D.; Chapple, C., NMR characteriza 9:2510-2516. tion of lignins in Arabidopsis altered in the activity of ferulate-5 Grabber, J.H.; Mertens, D.R.; Kim, H.; Funk, C.; Lu, F.; Ralph, J., hydroxylase. Proc. Natl. Acad Sci. (1999), 96(22), 12328-12332. Cell wall fermentation kinetics are impacted more by lignin content Ohlsson, A.B.; Djerbi, S.; Winzell, A.; Bessueille, L.; Staldal, V.; Li, and ferulate cross-linking than by lignin composition. J Sci Food X.G.; Blomqvist, K.; Bulone, V.; Teeri, T.T.; Berglund, T., Cell sus Agric (2009), 89:122-129. pension cultures of Populus tremula x P-tremuloides exhibit a high Guyot, S.; Vercauteren, J .; Cheynier, V., Structural determination of level of cellulose synthase gene expression that coincides with colourless and yellow dimers resulting from (+)-catechin coupling increased in vitro cellulose synthase activity. Protoplasma (2006), catalysed by grape polyphenoloxidase. Phytochemistry (1996), 228(4), 221-229. 42:1279-1288. Oosterveld, A.; Grabber, J .H.; Beldman, G.; Ralph, J .; Voragen, A.G. Hat?eld, R.D.; Jung, H.G.; Ralph, J.; Buxton, D.R.; Weimer, P.J., A J ., Formation of ferulic acid dehydrodimers through oxidative cross comparison of the insoluble residues produced by the Klason lignin linking of sugar beet pectin. Carbohydr Res (1997), 300: 179-181. and acid detergent lignin procedures. JSci FoodAgric (1994), 65:51 Pan, X.J.; Arato, C.; Gilkes, N.; Gregg, D.; Mabee, W.; Pye, K.; Xiao, 58. Z.Z.; Zhang, X.; Saddler, J., Biore?ning of softwoods using ethanol Hat?eld, R.D.; Ralph, J.; Grabber, J .H., A potential role for sinapyl organosolv pulping: Preliminary evaluation of process streams for p-coumarate as a radical transfer mechanism in grass lignin forma manufacture of fuel-grade ethanol and coproducts. Biotechnol. tion. Planta (2008), 228:919-928. Bioeng. (2005), 90(4), 473-481. M. Hedenstrom, S. Wiklund-Lindstrom, T. Oman, F. Lu, L. Gerbner, Quideau, S.; Ralph, J., Facile large-scale synthesis of coniferyl, P. F. Schatz, B. Sundberg and J. Ralph. Identi?cation of Lignin and sinapyl, and p-coumaryl alcohol. J Agric Food Chem (1992), Polysaccharide Modi?cations in Populus Wood by Chemometric 40:1108-1110. Analysis of 2D NMR Spectra from Dissolved Cell Walls. Molecular Ralph, J .; Helm, R.F.; Quideau, S.; Hat?eld, R.D., Lignin-feruloyl Plant, (2009), 2, 933-942. ester cross-links in grasses. Part 1. Incorporation of feruloyl esters Hemmerle, H.; Burger, H.J.; Below, P.; Schubert, G.; Rippel, R.; into coniferyl alcohol dehydrogenation polymers. J Chem. Soc., Schindler, P.W.; Paulus, E.; Herling, A.W., Chlorogenic acid and Perkin Trans. 1 (1992), (21), 2961-2969. synthetic chlorogenic acid derivatives: Novel inhibitors of hepatic Ralph, J.; Mackay, J.J.; Hat?eld, R.D.; O’Malley, D.M.; Whetten, glucose-6-phosphate translocase. JMed Chem (1997), 40:137-145. R.W.; Sederoff, R.R., Abnormal lignin in a loblolly pine mutant. Holtzapple, M.T.; Lundeen, J.E.; Sturgis, R.; Lewis, J.E.; Dale, B.E., Science (1997), 277, 235-239. Pretreatment of lignocellulosic municipal solid waste by ammonia Ralph, J.; Hat?eld, R.D.; Piquemal, J.; Yahiaoui, N.; Pean, M.; ?ber explosion (AFEX). Appl. Biochem. Biotechnol (1992), 0273 Lapierre, C.; Boudet, A. -M., NMR characterization of altered lignins 2289. extracted from tobacco plants down-regulated for ligni?cation Hosny, M.; Rosazza, J.P.N., Novel oxidations of (+)-catechin by enzymes cinnamyl-alcohol dehydrogenase and cinnamoyl-CoA horseradish peroxidase and laccase. J Ag Food Chem (2002), reductase. Proc. Natl. Acad. Sci. (1998), 95(22), 12803-12808. 50:5539-5545. Ralph, J.; Kim, H.; Peng, J.; Lu, F., Arylpropane-1,3-diols in lignins Huang, L.S.; Colas, C.; De Montellano, P.R.O., Oxidation of from normal and CAD-de?cient pines. Org. Lett. (1999), 1(2), 323 carboxylic acids by horseradish peroxidase results in prosthetic heme 326. modi?cation and inactivation. J Am. Chem. Soc. (2004), 126(40), Ralph, J.; Lapierre, C.; Marita, J.; Kim, H.; Lu, F.; Hat?eld, R.D.; 12865-12873. Ralph, S.A.; Chapple, C.; Franke, R.; Hemm, M.R.; Van Doors Huang, Q.; Huang, Q.; Pinto, R.A.; Griebenow, K.; Schweitzer selaere, J.; Sederoff, R.R.; O’Malley, D.M.; Scott, J.T.; Mackay, J.J.; Stenner, R.; Weber, Jr W.J., Inactivation of horseradi sh peroxidase by Yahiaoui, N.; Boudet, A.-M.; Pean, M.; Pilate, G.; Jouanin, L.; phenoxyl radical attack. JAm Chem Soc (2005), 127:1431-1437. Boerjan, W., Elucidation of new structures in lignins of CAD- and Kim, H.; Ralph, J ., Simpli?ed preparation of coniferyl and sinapyl COMT-de?cient plants by NMR. Phytochem. (2001), 57(6), 993 alcohols. JAg Food Chem (2005), 55:3693-3695. 1003. Kim, H.; Ralph, J .; Akiyama, T., Solution-state 2D NMR of ball Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, PF.; milled plant cell wall gels in DMSO-d6. Bioenerg Res (2008), 1:56 Marita, J.M.; Hat?eld, R.D.; Ralph, S.A.; Christensen, J.H.; Boerjan, 66. W., Lignins: natural polymers from oxidative coupling of Kim, H.; Ralph, J ., Solution-state 2D NMR of ball-milled plant cell 4-hydroxyphenylpropanoids. Phytochem. Revs. (2004),3(1), 29-60. wall gels in DMSOd6/pyridine-d5. Org Biomol Chem (2010), 8: Ralph, J.; Bunzel, M.; Marita, J.M.; Hat?eld, R.D.; Lu, F.; Kim, H.; 576-591. Schatz, P.F.; Grabber, J .H.; Steinhart, H., Peroxidase-dependent US 8,685,672 B2 Page 3

(56) References Cited Tobimatsu,Y.; Takano, T.; Kamitakahara, H.; Nakatsubo, F., Studies on the dehydrogenative polyrnerizations of monolignol beta OTHER PUBLICATIONS glycosides. Part 2: Horseradish peroxidase catalyzed dehydrogena tive polymerization of isoconiferin. Holzforschung (2006), 60(5), cross-linking reactions of p-hydroxycinnamates in plant cell walls. 5 13 -5 18. Phytochem. Revs. (2004), 3(1), 79-96. Ulibarri, G.; Nadler, W.; Skrydstrup, T.; Audrain, H.; Chiaroni, A.; Ralph, S.A.; Landucci, L.L.; Ralph, 1., NMR Database ofLignin and Riche, C.; Grierson, D.S., Construction of the bicyclic core structure Cell Wall Model Compounds. Available over Internet at http://ars. of the enediyne antibiotic Esperamicin-a(1) in either enantiomeric usda.gov/Services/docs.htm?docid:10429, updated at least annually form from (-)-quinic acid. J Org Chem (1995), 60:2753-2761. since 1993. (2005). Ralph, J ., What makes a good monolignol substitute? In The Science Van Soest, P.J.; Van Amburgh, M.E.; Robertson, J.B.; Knaus, W.F.; and Lore ofthePlant Cell Wall Biosynthesis, Structure and Function, Validation of the 2.4 times lignin factor for ultimate extent of NDF Hayashi, T., Ed. Universal Publishers (BrownWalker Press): Boca digestion, and curve peeling rate of fermentation curves into pools. Raton, FL, (2006); pp. 285-293. In: Cornell Nutrition Conference for Feed Manufacturers; Syracuse, Ralph, J.; Akiyama, T.; Kim, H.; Lu, F.; Schatz, P.F.; Marita, J.M.; NewYork: Cornell University, Ithaca, NewYork; (2005): 139-149. Ralph, S.A.; Reddy, M.S.S.; Chen, F.; Dixon, R.A., Effects of Vanholme, R.; Morreel, K.; Ralph, J .; Boerjan, W., Lignin engineer coumarate-3-hydroxylase downregulation on lignin structure. J. ing. Curr Opin Plant Biol (2008), 11:1-8. Biol. Chem. (2006), 281(13), 8843-8853. Wagner, A.; Ralph, J.; Akiyama, T.; Flint, H.; Phillips, L.; Torr, K.M.; Ralph, J.; Brunow, G.; Boerjan, W., Lignins. In Encyclopedia ofLife Nanayakkara, B.; TE Kiri, L., Modifying lignin in conifers: The role Sciences, John Wiley & Sons, Ltd.: Chichester, UK, (2007); in press. of HCT during tracheary element formation in Pinus radiata Proc. Ralph, J.; Brunow, G.; Harris, PJ.; Dixon, R.A.; Boerjan, W., Natl. Acad. Sci. (2007), 104(28), 11856-11861. Ligni?cation: Are lignins biosynthesized via simple combinatorial Ward, G.; Hadar,Y.; Dosoretz, C.G., Inactivation of lignin peroxidase chemistry or via proteinaceous control and template replication? In during oxidation of the highly reactive substrate ferulic acid. Enzyme Advances in PolyphenolsResearch, Daayf, F.; El Hadrami, A.; Adam, Microb Tech (2001), 29(1), 34-41. L.; Ballance, G.M., Eds. Blackwell Publishing: Oxford, UK, (2007); Weimer, P.J.; Mertens, D.R.; Ponnampalam, E.; Severin, B.F.; Dale, in press. B.E., Fibex-treated rice straw as a feed ingredient for lactating dairy Ralph, J.; Kim, H.; Lu, E; Grabber, J.H.; Boerjan, W.; Leplé, J.-C.; cows. Anim Feed Sci Technol (2003), 103:41-50. Berrio Sierra, J .; Mir Derikvand, M.; Jouanin, L.; Lapierre, C., Iden Weimer, P.J.; Dien, .BS.; Springer, T.L.; Vogel, K.P., In vitro gas ti?cation of the structure and origin of a thioacidolysis marker com production as a surrogate measure of the fermentability of cellulosic pound for ferulic acid incorporation into angiosperm lignins (and a biomass to ethanol. Appl Microbiol Biotechnol (2005), 67:52-58. pseudo-marker compound for cinnamoyl-CoA reductase de? Yelle, D.L.; Ralph, J. and Frihart, C.R.; Characterization of non ciency). Plant]. (2007), submitted. derivatized plant cell walls using high-resolution solution-state NMR Ralph, J.; Schatz, P.F.; Lu, E; Kim, H.; Akiyama, T.; Nelsen, S.F., spectroscopy, Magnetic Resonance in Chemistry, (2008), 46:508 Quinone methides in ligni?cation. In: Quinone Methides. Edited by 5 17. Rokita S. Hoboken, NJ: Wiley-Blackwell; (2009). Zhu, N.; Huang, T.C.; Yu, Y.; Lavoie, E.J.; Yang, C.S.; Ho, C.T., Russell, W.R.; Burkitt, M.J.; Scobbie, L.; Chesson, A., Radical for Identi?cation of oxidation products of (-)-epigallocatechin gallate mation and coupling of hydroxycinnamic acids containing 1,2 and (-)-epigallocatechin with H202. JAgFood Chem (2000), 48:979 dihydroxy substituents. Bioorganic Chem (2003), 31:206-215. 981. Steel, R.G.D.; Torrie, J.H., Principles and procedures of statistics, Zhu, N.; Wang, M.; Wei, G.J.; Lin, J.K.;Yang, C.S.; Ho, C.T., Iden 2nd edition. New York: McGraw-Hill Publishing Co.; (1980). ti?cation of reaction products of (-)-epigallochatechin, (-)-epigal Stewart, J.J.; Akiyama, T.; Chapple, C.C.S.; Ralph, 1.; Mans?eld, lochatechin gallate and pyrogallol with 2,2-diphenyl-1 S.D., Lignins with Extreme Syringyl Levels: The Effects of Over picrylhydrazyl radical. Food Chem (2001), 73:345-349. Expression of Ferulate 5-Hydroxylase on Lignin Structure in Hybrid Poplar. J. Biol. Chem. (2007), submitted. * cited by examiner

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EH 91mm m apicatamin da?vaiivs US 8,685,672 B2 1 2 INCORPORATION OF FLAVAN-3-OLS AND “lignin” polymer. Novel (non-monolignol) monomers avail GALLIC ACID DERIVATIVES INTO LIGNIN able to the plant, discovered in lignins from studies on down TO IMPROVE BIOMASS UTILIZATION regulating genes in the monolignol pathway, include products of incomplete monolignol biosynthesis such as 5-hydroxyco CROSS-REFERENCE TO RELATED niferyl alcohol (COMT-de?ciency), and coniferaldehyde and APPLICATION sinapaldehyde (CAD-de?ciency).5 These compounds couple integrally (via a radical route) into the polymer in Priority is hereby claimed to provisional application Ser. angiosperms. The list of other compounds found integrated No. 61/325,695, ?led Apr. 19, 2010, which is incorporated into lignins in normal and/ or transgenic plants is growing.2’6’7 herein. Many of the monomers currently implicated in ligni?cation are shown in FIG. 1. REFERENCES Observations to date have allowed the present inventors to detail the ideal properties of monolignol substitutes.8 When Full citations to the references referenced herein are such compounds are introduced into lignins, even at signi? included at the end of the Detailed Description and also at the cant levels, the plants show no obvious growth/development end of the Examples. All of the referenced cited herein are phenotype. Monomers that have accessible conjugation into incorporated herein by reference. the sidechain allowing for so-called “endwise” [3-O-4-cou pling seem to fare the best. Examples are: 5-hydroxyconiferyl INTRODUCTION alcohol, the hydroxycinnamaldehydes, hydroxycinnamate 20 esters, and acylated hydroxycinnamyl alcohols. See FIGS. Lignin is a highly complex, heterogeneous polymer found 2a, 2b, 2c, and 2d, respectively. Due to incompatibilities in in all vascular plants. It rigidi?es plants and plays a crucial radical coupling reactions, p-hydroxyphenyl moieties fare role in water transport. Lignin is notable for its complex less well than guaiacyl or syringyl moieties, at least when structure. It is comprised predominately from three mono incorporating into guaiacyl-syringyl lignins, but other phe mers, p-coumaryl alcohol, coniferyl alcohol, and sinapyl nolics have not been well studied. alcohol, and a host of other structurally related monomers. 25 Replacing the entire monomer component of ligni?cation See FIG. 1, which illustrates the typical monomers found in natural lignins. Hydroxycinnamaldehydes and their corre with a novel monomer is unlikely to be an effective strategy sponding hydroxybenzaldehydes are found in all lignins. that is “acceptable” to the growing plant. Introducing strate Hydroxycinnamyl acetates are found in mo st hardwoods and gic monomers into the normal monolignol pool is, however, a viable proposition as shown by the Examples described present in high levels in kenaf and palms. Hydroxycinnamyl 30 p-hydroxybenzoates are found in willows, palms, poplars, herein. Incorporation of up to 30% novel monomer as and aspens. Hydroxycinnamyl p-coumarates are found in all described herein has produced plants with no pleiotropic grasses. These monomers are polymerized into polymeric effects or obvious growth phenotypes. Incorporation of up to lignin by combinatorial radical coupling reactions. The lig 60% novel monomer has been accomplished. A range of ni?cation of cell walls is also notable because it is likely the alternative monomers are shown herein to be consistent with single most important factor limiting the digestion of forage 35 maintaining the plant’s structural and functional integrity. by ruminants, the ‘sacchari?cation’ of structural polysaccha Thus, the crux of the present invention is a method of manu rides for conversion into biofuels and chemicals and the pro facturing modi?ed lignin using monomer substitution (as duction of cellulose-containing pulp for use in papermaking. well as the resulting modi?ed lignin polymer itself). The Practically speaking, lignin is indigestible in the digestive resulting modi?ed lignin drastically eases cell wall sacchari tract of ruminants. The interfering presence of indigestible 40 ?cation and fermentability, both prior to and following rela lignins limits the ability of ruminants to utilize otherwise tively mild chemical pretreatments. digestible carbohydrates present in the forage they eat. Thus, there remains a long-felt and unmet need to alter lignins in SUMMARY OF THE INVENTION such a way that improves the digestibility/fennentability of the cell wall polysaccharides to increase the nutritional con Disclosed and claimed herein is a method of manufactur tent of forages. Although cost-prohibitive for feedstuffs, 45 ing modi?ed lignin. The method comprises conducting a harsh chemical pretreatments are commonly used to break lignin-producing polymerization reaction in the presence of lignin-structural polysaccharide interactions for the indus one or more polymerizable monomers selected from the trial conversion of ?brous crops into biofuels, chemical, and group consisting of: R8 paper. Thus approaches for rendering lignin more susceptible to mild pretreatments are greatly desired for reducing produc 50 tion costs and the environmental impact of converting ?brous biomass crops into biofuels, chemicals, and paper products. Over the past decade it has become apparent that the meta bolic malleability of ligni?cation, the process of polymeriza tion of phenolic monomers to produce lignin polymers, pro 55 vides enormous potential for engineering the troublesome polymer to be less inhibitory to structural polysaccharide utilization. Massive compositional changes can be realized by perturbing single genes in the monolignol pathway, par ticularly the hydroxylases. 1'4 More strikingly, monomer sub 60 wherein R1 and R2 are independently selected from the stitution in the process of ligni?cation is now well authenti cated,l’2 particularly in cases where a plant’s ability to group consisting of hydrogen, hydroxy, alkyloxy, alkanoyl, biosynthesize the usual complement of monolignols is com alkanoyloxy, hydroxy-substituted alkoxy or alkanoyl or promised. The chemical nature of ligni?cation, involving alkanoyloxy, benzoyloxy, and mono-, di- and tri-hydroxy combinatorial radical coupling of monomers (primarily with substituted benzoyloxy. In one version of the method, R1 and the growing polymer) without direct enzymatic control, 65 R2 are not simultaneously hydrogen. allows compatible phenolic compounds present in the cell R3-R6 are independently selected from the group consist wall (CW) during ligni?cation to be incorporated into the ing of hydrogen, hydroxy, alkyl, alkyloxy, hydroxy-substi US 8,685,672 B2 3 tuted alkyl, and hydroxy-substituted alkoxy, provided that at least one of R3-R6 is hydroxy; R5 o R1 R7-Rl 1 are independently selected from the group consist ing of hydrogen, hydroxy, alkyl, alkyloxy, hydroxy-substi tuted alkyl, and hydroxy-substituted alkoxy, provided that at R4 R2 least one of R7-Rll is hydroxy. R3 In a preferred version of the method, the polymerization reaction is conducted in the presence of a polymerizable monomer wherein R2 is hydrogen, and wherein Rl-R5 are independently selected from the group R1 is: consisting of hydrogen, hydroxy, :0, hydroxy, alkyloxy, alkanoyl, alkanoyloxy, hydroxy-substituted alkoxy or alkanoyl or alkanoyloxy, benzoyloxy, and mono-, di- and tri-hydroxy- sub stituted benzoyloxy, provided that at least one of Rl-R5 is hydroxyl, and Rl-R5 are not simultaneously R13 hydrogen. See FIG. 6. The method may be conducted in the present of, or the speci?c absence of certain other polymerizable monomers, R16 R14 such as the polymerizable monolignols as depicted in FIG. 1. R15 20 It is generally preferred that from about 10% by wt to about 60% by wt of the polymerizable monomers are reacted in the polymerization reaction, although ranges above and below wherein Rlz-Rl6 are independently selected from the the stated range are explicitly within the scope of the method group consisting of hydrogen, hydroxy, alkyloxy, alkanoyl, (e.g., from 1 wt % to 100 wt %). alkanoyloxy, hydroxy-substituted alkoxy or alkanoyl or 25 The polymerization reaction may be conducted in vitro or alkanoyloxy, provided that Rlz-Rl6 are not simultaneously in vivo. hydrogen. Explicitly included within the scope of the invention are The polymerization reaction may also be carried out using modi?ed lignins produced by the method disclosed herein. gallate esters, such as those selected from the group consist Also disclosed herein are isolated ligni?ed cell walls con ing of: 30 taining a compound as recited above, wherein the compound is incorporated into the lignin of the cell wall. Likewise dis closed herein are isolated plant cells containing a compound as recited above, wherein the compound is incorporated into lignin in cell walls of the isolated plant cells. 35 The plant cells themselves may be derived from any spe cies of the Plantae kingdom that is now known to make lignin naturally, is discovered in the future to make lignin naturally, or does not make lignin naturally, but has been genetically modi?ed to make lignin, without limitation. The plant cells 40 may also be derived from plants that have been genetically modi?ed for other purposes. This includes vascular plants of all description, monocots and dicots, hardwood and softwood trees, shrubs, grasses, grains, fruits, vegetables, etc. The term “alkyl,” by itself or as part of another substituent, 45 means, unless otherwise stated, a fully saturated, straight, R13 branched chain, or cyclic hydrocarbon radical, or combina tion thereof, and can include di- and multi-valent radicals, having the number of carbon atoms designated (e. g., C l-C 10 means from one to ten carbon atoms, inclusive). Examples of wherein RZ-Rl 1 as are de?ned in Claim 1, and 50 alkyl groups include, without limitation, methyl, ethyl, n-pro R12-Rl 6 are independently selected from the group consist pyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclo ing of hydrogen and hydroxy, provided that Rlz-Rl6 are not hexyl, (cyclohexyl)ethyl, cyclopropylmethyl, and homologs, simultaneously hydrogen. It is preferred that at least one of, at and isomers thereof, for example, n-pentyl, n-hexyl, n-heptyl, least two of, or all three of R13, R14, and R15 are hydroxy. n-octyl, and the like. The term “alkyl,” unless otherwise Preferred polymerizable monomers for use in the method 55 noted, also includes those derivatives of alkyl de?ned in more include catechin, epicatechin, gallocatechin, epigallocat detail below as “heteroalkyl” and “cycloalkyl.” echin, gallocatechin gallate, epigallocatechin gallate, and The term “alkenyl” means an alkyl group as de?ned above optical isomers thereof. containing one or more double bonds. Examples of alkenyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, Other preferred polymerizable monomers including those 60 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), etc., wherein R1, R3, and R5 are hydroxyl, as well as monomers and higher homolo gs and isomers. wherein at least one of, at least two of, or all three of R8, R9, The term “alkynyl” means an alkyl or alkenyl group as and R10 are hydroxy. de?ned above containing one or more triple bonds. Examples The polymerizable monomers may also be selected from of alkynyl groups include ethynyl, l- and 3-propynyl, 3-bu the group consisting of gallic acid and its esters, e.g., alkyl 65 tynyl, and the like, including higher homologs and isomers. gallate, as well as polymerizable monomers selected from the The terms “alkylene,” “alkenylene,” and “alkynylene,” group consisting of: alone or as part of another sub stituent means a divalent radical US 8,685,672 B2 5 6 derived from an alkyl, alkenyl, or alkynyl group, respectively, range, whether speci?cally disclosed or not. Further, these as exempli?ed by iCHZCHZCHzCHzi. numerical ranges should be construed as providing support “Substituted” refers to a chemical group as described for a claim directed to any number or sub set of numbers in that herein that further includes one or more sub stituents, such as range. For example, a disclosure of from 1 to 10 should be lower alkyl, aryl, acyl, halogen (e.g., alkylhalo such as CF3), construed as supporting a range of from 2 to 8, from 3 to 7, 5, hydroxy, amino, alkoxy, alkylamino, acylamino, thioamido, 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth. acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, both All references to singular characteristics or limitations of saturated and unsaturated cyclic hydrocarbons, heterocycles the present invention shall include the corresponding plural and the like. These groups may be attached to any carbon or characteristic or limitation, and vice-versa, unless otherwise substituent of the alkyl, alkenyl, alkynyl, alkylene, alk speci?ed or clearly implied to the contrary by the context in enylene, and alkynylene moieties. Additionally, these groups which the reference is made. may be pendent from, or integral to, the carbon chain itself. All combinations of method or process steps as used herein The term “aryl” is used herein to refer to an aromatic can be performed in any order, unless otherwise speci?ed or substituent, which may be a single aromatic ring or multiple clearly implied to the contrary by the context in which the aromatic rings which are fused together, linked covalently, or referenced combination is made. linked to a common group such as a diazo, methylene or The methods of the present invention can comprise, consist ethylene moiety. The common linking group may also be a of, or consist essentially of the essential elements and limita carbonyl as in benzophenone. The aromatic ring(s) may tions of the method described herein, as well as any additional include, for example phenyl, naphthyl, biphenyl, diphenylm or optional ingredients, components, or limitations described ethyl and benzophenone, among others. The term “aryl” 20 herein or otherwise useful in synthetic organic chemistry. encompasses “arylalkyl” and “substituted aryl.” For phenyl groups, the aryl ring may be mono-, di-, tri-, tetra-, or penta BRIEF DESCRIPTION OF THE DRAWINGS substituted. Larger rings may be unsubstituted or bear one or more substituents. FIG. 1. Natural lignin monomers. Bracketed monomers “Substituted aryl” refers to aryl as just described including 25 have not been ?rmly established as authentic monomers, one or more functional groups such as lower alkyl, acyl, although all have been found incorporated into lignin or lig halogen, alkylhalo (e. g., CF3), hydroxy, amino, alkoxy, alky nin-like plant polymers. lamino, acylamino, acyloxy, phenoxy, mercapto, and both FIGS. 2a, 2b, 2c, and 2d. Exemplary monolignol substi saturated and unsaturated cyclic hydrocarbons which are tutes. Cross-coupling and post-coupling reactions for alter fused to the aromatic ring(s), linked covalently or linked to a 30 native known monomers. FIG. 2a: Normal hydroxycinnamyl common group such as a diazo, methylene, or ethylene moi alcohol radicals B cross-couple with the phenolic end of the ety. The linking group may also be a carbonyl such as in growing polymerA, mainly by [3-0-4 coupling, to produce an cyclohexyl phenyl ketone. The term “substituted aryl” intermediate quinone methide which rearomatizes by nucleo encompasses “substituted arylalkyl.” philic water addition to produce the elongated lignin chain The term “acyl” is used to describe a ketone substituent, 35 A-B. The subsequent chain elongation via a further monoli iC(O)R, where R is substituted or unsubstituted alkyl, alk gnol radical C etheri?es the unit created by the prior monomer enyl, alkynyl, or aryl as de?ned herein. The term “carbonyl” B addition, producing the 2-unit-elongated polymer unit is used to describe an aldehyde substituent. The term “car A-B-C. FIG. 2b: Various y-acylated monolignols cross boxy” refers to an ester substituent or carboxylic acid, i.e., couple producing analogous products but with the [3-ether iC(O)Oi or 4C(O)4OH. 40 unit B y-acylated in the polymer unit A-B-C. FIG. 20: The term “halogen” or “halo” is used herein to refer to Hydroxycinnamaldehydes B may also cross-couple with the ?uorine, bromine, chlorine and iodine atoms. phenolic end of the growing polymer A, again mainly by The term “hydroxy” is used herein to refer to the group [3-0-4 coupling, to produce an intermediate quinone methide, iOH. but one which rearomatizes by loss of the acidic [3-proton, The term “amino” is used to designate NRR', wherein R 45 producing an unsaturated cinnamaldehyde-[3-O-4-linked B and R' are independently H, alkyl, alkenyl, alkynyl, aryl or end-unit. Incorporation further into the polymer by etheri? substituted analogs thereof. “Amino” encompasses “alky cation is analogous to FIG. 2a. FIG. 2d: 5-Hydroxyconiferyl lamino,” denoting secondary and tertiary amines, and “acy alcohol monomerA also cross-couples with the phenolic end lamino” describing the group RC(O)NR'. of the growing polymer A, mainly by [3-O-4-coupling, to The term “alkoxy” is used herein to refer to the 4OR 50 produce an intermediate quinone methide which rearoma group, where R is alkyl, alkenyl, or alkynyl, or a substituted tizes by nucleophilic water addition to produce the elongated analog thereof. Suitable alkoxy radicals include, for example, lignin chainA-B bearing a novel 5-hydroxyguaiacyl phenolic methoxy, ethoxy, t-butoxy, etc. The term “alkoxyalkyl” refers end-unit. The subsequent chain elongation is via a further to ether substituents, monovalent or divalent, e.g. 4CH2i monolignol radical C coupling [3-0-4 to the new phenolic end OiCH3 and 4CH24OiCH2i. 55 of A-B, but this time rearomatization of the quinone methide The chemical structures shown in the ?gures and drawings (not shown) is via internal attack of the 5-OH, producing purposefully do not designate stereochemistry and thus novel benzodioxane units B-C in the 2-unit-elongated poly encompass any and all stereoisomers, e.g., racemates, dias mer unit A-B-C. 5-Hydroxyconiferyl alcohol incorporation tereomers, epimers, etc., either isolated, enriched, or mixed in produces lignin with a structure that deviates signi?cantly any combination. Trivial names are used herein to denote all 60 from naturally occurring lignin. Bolded bonds are formed in stereoisomers of the stated compound unless explicitly stated the radical coupling steps. to the contrary. Thus, for example, as used herein, the terms FIGS. 3a and 3b. Biomimetically ligni?ed cell walls. FIG. “epicatechin,” “epigallocatechin,” and “epigallocatechin gal 3a: before ligni?cation. FIG. 3b: after ligni?cation. Impor late” explicitly encompass catechin, gallocatechin, and gal tantly, the ligni?cation is shown to occur within the cell wall. locatechin gallate. 65 FIGS. 4a, 4b, and 40. Examples of using NMR for identi Numerical ranges as used herein are intended to include fying monolignol substitutes incorporated into lignin. every number and subset of numbers contained within that COMT-de?cient alfalfa substitutes 5-hydroxyconiferyl alco US 8,685,672 B2 7 8 hol for sinapyl alcohol in the lignin polymerization reaction. Delineate Monomer Compatibility: FIG. 4a: Partial 2D l3C-lH correlative HMQC NMR spectra Determining the compatibility of the chosen monomers of lignins from (i) the wild-type alfalfa; and (ii) the COMT with ligni?cation via in vitro model coupling reactions is de?cient transgenic alfalfa. Dashed ovals in (i) delineate the essential to determine as any selected monomer that does not areas in which benzodioxane units H would correlate if they couple integrally into lignins is unlikely to be valuable. Cou were present. FIG. 4b: The mechanism by which 5-hydroxy pling and cross-coupling propensities are best tested empiri coniferyl alcohol incorporates into the lignin to produce novel cally as there do not appear to be any systematic rules that benzodioxane structures H. FIG. 40 Gradient-selected 2D predict whether a monomer will couple and cross-couple HMBC sub-spectra showing (x-proton correlations to carbons appropriately. We have used such methods to de?ne how within three bonds in [3-aryl ether units A, [3-5 units B, and ferulates couple into lignins, for example.19 The models and model polymers will also provide the NMR database required benzodioxane units H. These spectra demonstrate that all to identify how monomers incorporate into the more complex types of lignin monomers undergo [3-O-4 coupling to produce cell wall models and in transformed plants. [3-ethers A (AG, AS, and ASH) and also glycerol units GSH. Biomimetically Lignify the Selected Monomers into Cell Additionally, coniferyl alcohol and 5-hydroxyconiferyl alco Walls: hol (and sinapyl alcohol at lower contour levels) all add to the Selected monomers, at varying levels relative to the normal new 5-hydroxyguaiayl units formed after coupling of 5-co monolignols, are incorporated into cell walls. Strategically niferyl alcohol to form benzodioxanes HG, HSH, HS. 5-Hy 13 C-labeled monomers are used as appropriate. droxyconiferyl alcohol is clearly acting as a surrogate lignin Delineate the Resultant Cell Wall Lignin Structure: monomer in this polymerization. 20 Structural characterization of the cell walls reveal whether FIG. 5. Histogram depicting alkali extractability of modi the monomers integrate into wall lignins and also provide ?ed lignin according to the present invention as compared to materials for conversion testing. Structures are examined by natural lignin. See the Detailed Description for experimental degradative methods and, most importantly, via the whole details. As can be seen from the ?gure, an increase in the cell-wall dissolution and NMR procedures28 (where the stra number of phenolic hydroxy groups in the replacement 25 tegic labeling helps reveal the bonding patterns). monomer positively correlates with increased alkaline Test Biomass Processing Impacts: extractability. This is a highly desirable outcome because Monomers are selected for their potential to improve bio lignin extractability in alkali solutions depends on the ability mass processing ef?ciency. Arti?cially ligni?ed cell walls are to cleave the lignin polymer into smaller pieces and the solu tested under a variety of biomass conversion methods to bility of the resulting fragments in the extraction solution. 30 delineate how much improvement might be expected in The increased number of phenolic hydroxyl groups in the planta from utilization of the monomer substitutes are various modi?ed lignin presumably enables larger fragments of the levels. modi?ed lignin to be solubilized and separated from the other These steps are described more fully below. components of the cells wall. Delineating Monomer Compatibility: FIG. 6. Other monomers that may be used to make the 35 Synthetic in vitro coupling reactions, although they do not modi?ed lignin described herein. Included are gallate esters provide materials suitable for testing the effects of lignin and closely related compounds such as ethyl gallate, epigal modi?cation, play a valuable role in the initial selection of locatechin gallate, gallocatechin gallate, epigallocatechin-3, potential monomers. The reasoning is simple. All the cou 5-di-O-gallate, hyperoside, 2"-O-gallohyperin, l,2,3,4,6-O pling reactions evidenced in lignins in vivo are also produced, pentagalloylglucose, and corilagin. 40 admittedly at different relative levels, in vitro. If coupling and FIG. 7. A graph depicting the relationship of ?nal gas cross-coupling compatibility is not observed in synthetic cou production (fermentability) to the number of OH groups on pling reactions, failure in planta is almost certainly assured the catechin derivatives. because the in vivo reaction is also purely chemical. Because the monomer-substitutes are envisioned to incorporate into DETAILED DESCRIPTION 45 ligni?cation with the normal monolignols, it is important that they be compatible with coupling and, more importantly, Novel monomers that appear to be well suited for ligni? cross-coupling reactions with the growing polymer derived in cation can be found throughout the plant kingdom. Several part from those monolignols. groups of compounds, as described below, are suitable for It is for these reasons that some suggestions, seemingly producing modi?ed lignins. As a general proposition, the 50 logical on paper, will simply not work. For example, it has most suitable monomer for producing modi?ed lignins fall already been established that non-methoxylated phenolic into ?ve (5) classes: (1) bifunctional monomers or monomer entities such a tyramine and p-coumarate do not become conjugates linked via cleavable ester or amide bonds; (2) integrated into the polymer by coupling reactions. They are monomers that produce novel cleavable functionalities in the found in lignin polymers, but only as “appendages” or end lignin polymer; (3) hydrophilic monomers; (4) monomers 55 units. For example, p-coumarates are exclusively found as that minimize lignin-polysaccharide cross-linking; and (5) acylating groups in lignin sidechain y-positions. They are monomers that produce simpler lignins. Each of these classes free-phenolic (non-etheri?ed), meaning that they do not of monomers will be described below. In each instance, suit undergo radical coupling reactions. On their own, in vitro, able monomers are polymerized into a modi?ed lignin. The p-coumarates will couple, but what happens during ligni?ca modi?ed lignins are then assessed to see whether and how the 60 tion in the presence of normal monolignols and lignin guai modi?ed lignins impact biomass processing in biomimetic acyl/syringyl units is that radical transfer from these less cell wall systems. stable radicals occurs before they will enter into radical Many of the experimental procedures referenced herein are coupling.9 Thus, lignifying with coniferyl- or sinapyl p-cou described only brie?y. Full-text references describing the marate is known not to work. The coniferyl and sinapyl alco known procedures can be found at: http://www.dfrc.ars.us 65 hol moieties incorporate as usual, but the p-coumarate end, da.gov/DFRCWebPDFs/pdendex.html. See also the refer despite being phenolic and potentially capable of radical cou ences cited herein. pling, will not incorporateithe units remain as free-phenolic US 8,685,672 B2 10 pendant units.9 As a result, cleaving the esters will release the as the secondary wall producing systems from tobacco,23 p-coumarate but will not cause any depolymerization of the poplar,24 as well as Wagner’s pine tracheary element sys polymer. Similarly, the idea of using tyramine ferulate will tem.25 Such systems provide assorted ligni?ed plant cell not work either; tyramine units (also non-methoxylated phe walls. nolics) do not enter into coupling reactions.20 That said, if Delineating Resultant Lignin Structure: lignins are derived from higher levels of the non-methoxy An important aspect of this work is in establishing how lated monolignol, p-coumaryl alcohol, p-coumarates and well the novel monomer incorporated into lignin. With model tyramines will cross-couple into those p-hydroxyphenyl-rich data from the model coupling reactions in B.1 above, NMR polymers. Thus exploring the chemical compatibility of methods in particular, and degradative methods such as ana monomers ?rst will delineate whether it is worth introducing lytical thioacidolysis26 and the DFRC method,27 enable those monomers into C3H-de?cient plants, for examplei delineating how well incorporated a novel monomer plants in which the coniferyl and sinapyl alcohol levels are becomes, and into what types of structures it is incorporated. depleted at the expense of the potentially compatible p-cou This provides particularly important data for delineating maryl alcohol.21 whether plant alteration has been successful. Although tyramine ferulate was noted as not being a can For example, FIGS. 4a, 4b, and 4c illustrate various meth didate for introducing cleavable bonds into lignins, an analog ods to elucidate how hydroxycinnamaldehydes incorporate can be found in certain plants. 3-Methoxytyramine ferulate, into CAD-de?cient angiosperms, and how 5-hydroxyco for example, is a bifunctional molecule in which both moi niferyl alcohol incorporates into COMT-de?cient eties are entirely compatible with ligni?cation. It therefore angiosperms. In both instances, detailed NMR data showed incorporates fully, from both ends, into lignin. The cleavable 20 not only the incorporation but revealed the detailed incorpo amide functionality then introduced into the backbone of the ration pro?les. Using this date, the present inventors were polymer is exactly the kind of zipper unit that will allow such able to discover valuable marker compounds for monitoring a polymer to be more readily depolymerized. such incorporation. Biomimetic Ligni?cation into Suspension-Cultured Cell In addition to carefully evaluated individual spectra, Walls: 25 emerging cell wall 2D NMR “?ngerprint” pro?les28 and Once the monomers have been obtained/ synthesized, they chemometrics methods29’3O can be used to relate the detailed are then tested for their lignifying ability. As a general rule, it structural information available in the pro?le to various con is not preferred to make synthetic lignins by simple in vitro version parameters. polymerization of these monomers (with or without the tra Testing Biomass Processing: ditional monomers) because the in vitro materials give little 30 More straightforward but no less important is the process insight into the behavior of the cell wall during biomass ing and testing of the cell walls with modi?ed lignins to assess processing. It is much preferred to produce cell walls ligni?ed the impact of the ligni?cation changes on biomass conversion with the novel monomers (either in the presence of, or the ef?ciency. These processes are all well established and won’t absence of the normally present monolignols). A suspension be detailed herein, for example, the ethanolysis process for cultured corn system for producing cell walls amenable to 35 producing cellulose that is ideal for sacchari?cation and fer controlled ligni?cation by exogenously supplying the lignin mentation.3 l monomers has been described in reference 22, incorporated The walls from above will be subjected to various biomass herein by reference. See FIGS. 3a and 3b, which illustrate a processing conditions, and compared to controls. To develop maize cell prior to ligni?cation (FIG. 3a) and after ligni?ca a comprehensive database of conditions, at least the following tion (FIG. 3b). The cell walls already contain the polysaccha 40 processing pretreatments should be tested: ethanolysis3 1 (and ride complement, and contain their own peroxidases. Com other organosolv methods), aminolysis, including the APEX patible phenolic monomers and a supply of H202 are the only (ammonia ?ber explosion) process,32 alkaline pulping, and requirements to effect in muro ligni?cation. When normal acid hydrolysis.An example protocol for processing via alka monolignols are fed, the lignins are structurally extremely line pulping is as follows: The alkaline solubility of lignins is similar to those in the analogous growing plant.22 A repre 45 determined by incubating cell walls under N2 atmosphere sentative protocol is as follows: Primary cell walls (~1.2 g dry with 100 mL/g of 0.5 M aqueous NaOH for 22 h at 30° C. or weight) isolated from 14 d old maize cell suspensions were for 2.5 h at 100 or 160° C. Anthraquinone (0.02 mg/mL) is stirred in 120 mL of HOMOPIPES buffer (25 mM, pH 5.5 added to catalyze the hydrolysis of lignin ether inter-unit with 4 mM CaClZ) and arti?cially ligni?ed over ~24 h by linkages at 1600 C. After cooling, alkaline residues are pel adding separate solutions of lignin precursors (250 mg in 70 50 leted (5,000>