, Volume 21, Issue 3, pp 1395–1407 | Cite as

Towards lignin-protein crosslinking: amino acid adducts of a lignin model quinone methide

  • Brett G. Diehl
  • Heath D. Watts
  • James D. Kubicki
  • Matthew R. Regner
  • John Ralph
  • Nicole R. Brown
Original Paper


The polyaromatic structure of lignin has long been recognized as a key contributor to the rigidity of plant vascular tissues. Although lignin structure was once conceptualized as a highly networked, heterogeneous, high molecular weight polymer, recent studies have suggested a very different configuration may exist in planta. These findings, coupled with the increasing attention and interest in efficiently utilizing lignocellulosic materials for green materials and energy applications, have renewed interest in lignin chemistry. Here we focus on quinone methides (QMs)—key intermediates in lignin polymerization—that are quenched via reaction with cell-wall-available nucleophiles. Reactions with alcohol and uronic acid groups of hemicelluloses, for example, can lead to lignin-carbohydrate crosslinks. Our work is a first step toward exploring potential QM reactions with nucleophilic groups in cell wall proteins. We conducted a model compound study wherein the lignin model compound guaiacylglycerol-β-guaiacyl ether 1, was converted to its QM 2, then reacted with amino acids bearing nucleophilic side-groups. Yields for the QM-amino acid adducts ranged from quantitative in the case of QM-lysine 3, to zero (no reaction) in the cases of QM-threonine (Thr) 10 and QM-hydroxyproline (Hyp) 11. The structures of the QM-amino acid adducts were confirmed via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and density functional theory (DFT) calculations, thereby extending the lignin NMR database to include amino acid crosslinks. Some of the QM-amino acid adducts formed both syn- and anti-isomers, whereas others favored only one isomer. Because the QM-Thr 10 and QM-Hyp 11 compounds could not be experimentally prepared under conditions described here but could potentially form in vivo, we used DFT to calculate their NMR shifts. Characterization of these model adducts extends the lignin NMR database to aid in the identification of lignin-protein linkages in more complex in vitro and in vivo systems, and may allow for the identification of such linkages in planta.


Nuclear magnetic resonance spectroscopy Lignin Protein Quinone methide Amino acid Crosslinking Density functional theory 



This research was supported as part of The Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001090, and the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). The authors would like to thank and acknowledge the Center for Lignocellulose Structure and Formation (CLSF) and the members thereof. Student fellowships were provided by the USDA National Needs Program and the National Science Foundation. The authors would like to thank Dr. Alan Benesi and Dr. Wenbin Luo for assistance in acquiring NMR spectra of the lignin model compounds, Dr. James Miller for acquiring mass spec data, and Dr. Josh Stapleton for providing assistance with UV/Vis. The primary author would also like to acknowledge Paul Munson and Curtis Frantz for valuable discussion, and valuable interactions with Dan Gall and other members of the Wisconsin lab.

Supplementary material

10570_2014_181_MOESM1_ESM.doc (8.1 mb)
The online version of this article contains supplementary material, which is available to authorized users. (DOC 8251 kb)


  1. Adamo C, Barone V (1998) Exchange functionals with improved long-range behavior and adiabatic connection methods without adjustable parameters: the mPW and mPW1PW models. J Chem Phys 108:664–675CrossRefGoogle Scholar
  2. Albersheim P, Darvill A, Roberts K, Sederoff R, Staehelin A (2010) Principles of cell wall architecture and assembly. In: Plant cell walls. Garland Science, New York, New York, pp 227–272 Google Scholar
  3. Awad HM, Boersma MG, Vervoort J, Rietjens IMCM (2000) Peroxidase-catalyzed formation of quercetin quinone methide-glutathione adducts. Arch Biochem Biophys 378:224–233CrossRefGoogle Scholar
  4. Balakshin M, Capanema E, Gracz H, Chang H, Jameel H (2011) Quantification of lignin-carbohydrate linkages with high-resolution NMR spectroscopy. Planta 233:1097–1110CrossRefGoogle Scholar
  5. Barone G, Duca D, Silvestri A, Gomez-Paloma L, Riccio R, Bifulco G (2002) Determination of the relative stereochemistry of flexible organic compounds by ab initio methods: conformational analysis and Boltzmann-averaged GIAO 13C NMR chemical shifts. Chem Eur J 8(14):3240–3245CrossRefGoogle Scholar
  6. Beat K, Templeton MD, Lamb CJ (1989) Specific localization of a plant cell wall glycine-rich protein in protoxylem cells of the vascular system. Proc Natl Acad Sci USA 86:1529–1533CrossRefGoogle Scholar
  7. Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Ann Rev Plant Biol 54:519–546CrossRefGoogle Scholar
  8. Bolton JL, Turnipseed SB, Thompson JA (1997) Influence of quinone methide reactivity on the alkylation of thiol and amino groups in proteins: studies utilizing amino acid and peptide models. Chem Biol Interact 107:185–200CrossRefGoogle Scholar
  9. Buhl M, Kaupp M, Malkina OL, Malkin VG (1999) The DFT route to NMR chemical shifts. J Comput Chem 20:91–105CrossRefGoogle Scholar
  10. Cances E, Mennucci B, Tomasi J (1997) A new integral equation formalism for the polarizable continuum model: theoretical background and applications to isotropic and anisotropic dielectrics. J Chem Phys 107(8):3032–3041CrossRefGoogle Scholar
  11. Capanema EA, Balakshin MY, Kadla JF (2004) A comprehensive approach for quantitative lignin characterization by NMR spectroscopy. J Agric Food Chem 52:1850–1860CrossRefGoogle Scholar
  12. Cassab IG, Varner JE (1988) Cell wall proteins. Ann Rev Plant Physiol Plant Mol Biol 39:321–353CrossRefGoogle Scholar
  13. Chapple C, Ladisch M, Meilan R (2007) Loosening lignin’s grip on biofuel production. Nat Biotechnol 25:746–748CrossRefGoogle Scholar
  14. Cheeseman JR, Trucks GW, Keith TA, Frisch MJ (1996) A comparison of models for calculating nuclear magnetic resonance shielding tensors. J Chem Phys 104:5497–5509CrossRefGoogle Scholar
  15. Chen F, Dixon RA (2008) Genetic manipulation of lignin biosynthesis to improve biomass characteristics for agro-industrial processes. In Vitro Cell Dev Biol Anim 44:S28–S29Google Scholar
  16. Clark T, Chandrasekhar J, Spitznagel GW, Schleyer PVR (1983) Efficient diffuse function-augmented basis sets for anion calculations. III. The 3-21 + G basis set for first-row elements, Li–F. J Comput Chem 4(3):294–301CrossRefGoogle Scholar
  17. Cosgrove D (2005) Growth of the plant cell wall. J Nat Rev Mol Cell Biol 6:850–861CrossRefGoogle Scholar
  18. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Montgomery JA (2009) Gaussian 09, revision B01. Gaussian, Inc, WallingfordGoogle Scholar
  19. Gogonea V (1998) Self-consistent reaction field methods: cavities. In: Schleyer PVR, Schreiner PR, Allinger NL, Clark T, Gasteiger J, Kollman P, Schaefer HF III (eds) Encyclopedia of computational chemistry. Wiley, New York, pp 2560–2574Google Scholar
  20. Gottlieb HE, Kotlyar V, Nudelman A (1997) NMR chemical shifts of common laboratory solvents as trace impurities. J Org Chem 62(21):7512–7515CrossRefGoogle Scholar
  21. Harrak H, Chamberland H, Plante M, Bellemare G, Lafontaine JG, Tabaeizadeh Z (1991) A proline-, threonine-, and glycine-rich protein down-regulated by drought is localized in the cell wall of xylem elements. Plant Phys 121:557–564CrossRefGoogle Scholar
  22. Hohenberg P, Kohn W (1964) Inhomgeneous electron gas. Phys Rev 136(3b):B864–B871CrossRefGoogle Scholar
  23. Jose M, Puigdomenech P (1993) Structure and expression of genes encoding for structural proteins of the plant cell wall. New Phytol 125:259–282CrossRefGoogle Scholar
  24. Jung HG (1989) Forage lignins and their effects on fiber digestibility. Agron J 81:33–38CrossRefGoogle Scholar
  25. Jung HG, Allen MS (1995) Characteristics of plant cell walls affecting intake and digestibility of forages by ruminants. J Anim Sci 73:2774–2790Google Scholar
  26. Karadakov PB (2008) Ab initio calculation of NMR shielding constants. In: Webb GA (ed) Modern magnetic resonance. Springer, New York, pp 63–70Google Scholar
  27. Kawai S, Okita K, Sugishita K, Tanaka A, Ohashi H (1999) Simple method for synthesizing phenolic β-O-4 dilignols. J Wood Sci 45:440–443CrossRefGoogle Scholar
  28. Kieliszewski M, Lamport DTA, Tan L, Cannon MC (2011) Hydroxyproline-rich glycoproteins: form and function. Ann Plant Rev 41:321–342Google Scholar
  29. Kim H, Ralph J (2010) Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d 6/pyridine-d 5. Org Biomol Chem 8:576–591CrossRefGoogle Scholar
  30. Kohn W, Sham LJ (1965) Self-consistent equations including exchange and correlation effects. Phys Rev 140(4A):A1133–A1138CrossRefGoogle Scholar
  31. Krishnan RBJS, Binkley JS, Seeger R, Pople JA (1980) Self-consistent molecular orbital methods. XX. A basis set for correlated wave functions. J Chem Phys 72:650CrossRefGoogle Scholar
  32. Landucci LL, Geddes SA, Kirk TK (1981) Synthesis of 14C labeled 3-methoxy-4-hydroxy-α-(2-methoxy-phenoxy)-β-hydroxypropiophenone, a lignin model compound. Holzforschung 35:66–69CrossRefGoogle Scholar
  33. Leary GJ (1980) Quinone methides and the structure of lignin. Wood Sci Technol 14:21–34CrossRefGoogle Scholar
  34. Leary G, Miller IJ, Thomas W, Woolhouse AD (1977) The chemistry of reactive lignin intermediates. Part 5. Rates of reactions of quinone methides with water, alcohols, phenols, and carboxylic acids. J Chem Soc, Perkin Trans 2 13:1737–1739CrossRefGoogle Scholar
  35. Li X, Weng JK, Chapple C (2008) Improvement of biomass through lignin modification. Plant J 54:569–581CrossRefGoogle Scholar
  36. Liang H, Frost CJ, Wei X, Brown NR, Carlson JE, Tien M (2008) Improved sugar release from lignocellulosic material by introducing a tyrosine-rich cell wall peptide gene in poplar. Clean 36(8):662–668Google Scholar
  37. Lodewyk MW, Siebert MR, Tantillo DJ (2012) Computational prediction of 1H and 13C chemical shifts: a useful tool for natural product, mechanistic, and synthetic organic chemistry. Chem Rev 112(3):1839–1862CrossRefGoogle Scholar
  38. Mansfield SD, Kim H, Lu F, Ralph J (2012) Whole plant cell wall characterization using solution-state 2D NMR. Nat Protoc 7(9):1579–1589CrossRefGoogle Scholar
  39. McQueen-Mason S, Cosgrove D (1994) Disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension. J Proc Natl Acad Sci USA 91:6574–6578CrossRefGoogle Scholar
  40. Miyagawa Y, Takemoto O, Takano T, Kamitakahara H, Nakatsubo F (2012) Fractionation and characterization of lignin carbohydrate complexes (LCCs) of Eucalyptus globulus in residues left after MWL isolation. Part I: analyses of hemicellulose-lignin fractionation (HC-L). Holzforschung 66:459–465CrossRefGoogle Scholar
  41. Mostaghni F, Abbas T, Seyed AM (2013) Synthesis, spectroscopic characterization and DFT calculations of β-O-4 type lignin model compounds. Spectrochimica Acta Part A Mol Biomol Spec 110:430–436CrossRefGoogle Scholar
  42. Nagy PI, Tejada FR, Messer WS (2005) Theoretical studies of the tautomeric equilibria for five-member N-heterocycles in the gas phase and in solution. J Phys Chem 109:22588–22602CrossRefGoogle Scholar
  43. Papajak E, Zheng J, Xu X, Leverentz HR, Truhlar DG (2011) Perspectives on basis sets beautiful: seasonal plantings of diffuse basis functions. J Chem Theory Comput 7(10):3027–3034CrossRefGoogle Scholar
  44. Ralph J, Young RA (1983) Stereochemical aspects of addition reactions involving lignin model quinone methides. J Wood Chem Technol 3(2):161–181CrossRefGoogle Scholar
  45. Ralph J, Lundquist K, Brunow G, Lu F, Kim H, Schatz PF, Marita JM, Hatfield RD, Ralph SA, Christensen JH (2004a) Lignins: natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem Rev 3:29–60CrossRefGoogle Scholar
  46. Ralph SA, Ralph J, Landucci LL (2004) NMR database of lignin and cell wall model compounds. Accessed 27 Sept 2013
  47. Ralph J, Schatz PF, Lu F, Kim H, Akiyama T, Nelsen SF (2009) Quinone methides in lignification. In: Rokita SE (ed) Quinone methides. Wiley, New Jersey, pp 385–420CrossRefGoogle Scholar
  48. Ramakrishnan K, Fisher J (1983) Nucleophilic trapping of 7,11-dideoxyanthracyclinone quinone methides. J Am Chem Soc 105:7187–7188CrossRefGoogle Scholar
  49. Ryser U, Schorderet M, Zhao G, Studer D, Ruel K, Hauf G, Keller B (1997) Structural cell-wall proteins in protoxylem development: evidence for a repair process mediated by a glycine-rich protein. Plant J 12(1):97–111CrossRefGoogle Scholar
  50. Sarotti AM, Pellegrinet SC (2009) A multi-standard approach for GIAO 13C NMR calculations. J Org Chem 74(19):7254–7260CrossRefGoogle Scholar
  51. Sarotti AM, Pellegrinet SC (2012) Application of the multi-standard methodology for calculating (1)H NMR chemical shifts. J Org Chem 77(14):6059–6065CrossRefGoogle Scholar
  52. Schreckenbach G, Ziegler T (1995) Calculation of NMR shielding tensors using gauge-including atomic orbitals and modern density functional theory. J Chem Phys 99(2):606–611CrossRefGoogle Scholar
  53. Stewart JJ, Kadla JF, Mansfield SD (2006) The influence of lignin chemistry and ultrastructure on the pulping efficiency of clonal aspen (Populus termuloides Michx). Holzforschung 60:111–122CrossRefGoogle Scholar
  54. Terashima N, Atalla RH, Ralph SA, Landucci LL, Lapierre C, Monties B (1995) New preparations of lignin polymer models under conditions that approximate cell wall lignification. Holzforschung 49:521–527CrossRefGoogle Scholar
  55. Toikka M, Jussi S, Teleman A, Brunow G (1998) Lignin-carbohydrate model compounds. Formation of lignin-methyl arabinoside and lignin-methyl galactoside benzyl ethers via quinone methide intermediates. J Chem Soc, Perkin Trans 1 1:3813–3818CrossRefGoogle Scholar
  56. Vanholme R, Morreel K, Ralph J, Boerjan W (2010) Lignin biosynthesis and structure. Plant Phys 153:895–905CrossRefGoogle Scholar
  57. Watts HD, Mohamed MNA, Kubicki JD (2011) Comparison of multistandard and TMS-standard calculated NMR shifts for coniferyl alcohol and application of the multistandard method to lignin dimers. J Phys Chem B 115(9):1958–1970CrossRefGoogle Scholar
  58. Whitmore FW (1978a) Lignin-carbohydrate complex formed in isolated cell walls of callus. Phytochem 17:421–425CrossRefGoogle Scholar
  59. Whitmore FW (1978b) Lignin-protein complex catalyzed by peroxidase. Plant Sci Lett 13:241–245CrossRefGoogle Scholar
  60. Whitmore FW (1982) Lignin-protein complex in cell walls of Pinus elliottii: amino acid constituents. Phytochem 21(2):315–318CrossRefGoogle Scholar
  61. Wolinski K, Hinton JF, Pulay P (1990) Efficient implementation of the gauge-independent atomic orbital method for NMR chemical shift calculations. J Am Chem Soc 112(23):8251–8260CrossRefGoogle Scholar
  62. Xu Y, Chen C, Thomas TP, Azadi P, Diehl B, Tsai C, Brown N, Carlson JE, Tien M, Liang H (2013) Wood chemistry analysis and expression profiling of a poplar clone expressing a tyrosine-rich peptide. Plant Cell Rep 32:1827–1841CrossRefGoogle Scholar
  63. Yuan T, Sun S, Xu F, Sun R (2011) Characterization of lignin structures and lignin-carbohydrate complex (LCC) linkages by quantitative 13C and 2D HSQC NMR spectroscopy. J Agric Food Chem 59:10604–10614CrossRefGoogle Scholar
  64. Zhao Y, Shultz NE, Truhlar DG (2006) Design of density functionals by combining the method of constraint satisfaction with parametrization for thermochemistry, thermochemical kinetics, and noncovalent interactions. J Chem Theory Comput 2(2):364–382CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Brett G. Diehl
    • 1
  • Heath D. Watts
    • 2
  • James D. Kubicki
    • 3
  • Matthew R. Regner
    • 4
  • John Ralph
    • 4
  • Nicole R. Brown
    • 5
  1. 1.Department of Agricultural and Biological EngineeringThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.Department of GeosciencesThe Pennsylvania State UniversityUniversity ParkUSA
  3. 3.Department of Geosciences and the Earth and Environmental Systems InstituteThe Pennsylvania State UniversityUniversity ParkUSA
  4. 4.Department of Biochemistry and DOE Great Lakes Bioenergy Research CenterWisconsin Energy InstituteMadisonUSA
  5. 5.Department of Agricultural and Biological EngineeringThe Pennsylvania State UniversityUniversity ParkUSA

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