8 8 8 2
DOI: 10.1039/b411428k 4-hydroxy-3-methoxycinnamyl, and 3,5-dimethoxy-4-hydroxy-4-hydroxy-3-methoxycinnamyl, and (4-hydroxycinnamyl, alcohols sinapyl monolignols, and coniferyl, coumaryl, three the are monomers lignin primary The sinapyl implicating hydroxybenzoate asaligninprecursor. tissues, also poplar is lignifying that in product detected 8-8-cross-coupled an produces alcohol sinapyl of Cross-coupling evidence (to be moreevidencebe fullyyear,(to this documented Ralphand Lu itself. polymer lignin the on occurs acylation whether or reactions, coupling free-radical ventional con the by lignin into cross-coupling and coupling byporated incor then conjugatesarewhichproduce ester areacylated to first monolignols whether resolve to important become has it resources, plant of utilization the improve to order in sought are acylation such of functions the and processes various ling control genes As unknown. are enzymes transferase involved mains unresolved and the genes associated with the presumably or or 9- so-called (the alcohol primary sidechain their have plants woody and crops important agriculturally many of Lignins alcohols). cinnamyl First publishedasanAdvanceArticleontheweb28thSeptember2004 Received 27thJuly2004,Accepted10thSeptember2004 c b a Fachuang Lu, sinapyl alcoholandsinapyl Preparation across-coupling andrelevance product between of lignifying xylem tissue in poplar. Intermediate 1 Scheme
Ghent University, B-9000Gent,Belgium E-mail: [email protected]; Fax: +16082645147;Tel: +16082645407 WI 53706,USA Dept. PlantSystemsBiology, Flanders Interuniversity Institutefor Biotechnology, Dept. Forestry, Wisconsin-Madison,Madison,WI53706,USA. University of US Dairy Forage Research Center, USDA-ARS, 1925LindenDrive West, Madison, p . m e h C . l o m o i B . g r O -coumaroylated. following Coupling reactions of sinapyl alcohol alcohol sinapyl of reactions Coupling a,b the monolignol radical coupling reactions or or reactions coupling radical monolignol the John Ralph,* 1–3 c O) acetylated, -OH) The biochemistry of such acylation re acylation such of biochemistry The , 4 0 0 2 , 4,5 p -hydroxybenzoate and sinapyl sinapyl and -hydroxybenzoate We have provided preliminary preliminary provided have We a,b KrisMorreel, 2 0 9 8 2 – 8 8 8 2 , p QMbb p -hydroxybenzoylated -hydroxybenzoate 1a is a surprisingly stable, isolable product in homo-coupling reactions of and sinapyl and c EricMessens s i l a n r u o j s i h T p p p -hydroxybenzoate ------c andWout Boerjan independent preparation of the independent compound preparationhere. of productis crucial the of proof structural The (COMT). acid caffeic by down-regulated heavily plants normal but decreased to below detectable levels in plants substituent. benzoate coveredproductnovel8-8-coupling a pathway,biosynthetic lignin the on genes/enzymes in deficient transgenics and plants poplar normal between ences evidence for monolignol kenaf sinapyl alcohol, in pre-acetylated from part, 9-acetylation significant in of least at arises, level lignins high the that unpublished) intermediate intermediate reactions were insensitive the to 9-OH. the acylation of 2aa the cross-coupled product of yield isolated 25% satisfactory a produced alcohol sinapyl sinapyl of amounts equimolar of tion achieve. to difficult be can reactions cross-coupling homo-dehydrodimerization; opig ecin ivlig ooinl srnl favor strongly monolignols involving reactions Coupling differ metabolite phenolic profiling studies recent During Sinapyl alcohol alcohol Sinapyl : 2ab © : 4 0 0 2 y r t s i m e h C f o y t e i c o S l a y o R e h T 2bb 1b . Cross-coupled product Cross-coupled . was essentially 1 : 2 : 1 suggesting that the coupling bis 6 and have provided at least good circumstantial -quinone methide methide -quinone c 2,11 1a 9 This product was readily detected in in detected readily was product This eetees peroxidase–H Nevertheless, radical 8-8-coupling produces an an produces 8-8-coupling radical p -coumarates in grasses. 2ab , Scheme 1.† The ratio productsof C O M M U N I C A T I O N 2ab QMaa 2ab has been found in actively in found been has p -hydroxybenzoate and bearing a bearing 1b O , Scheme 1, which which 1, Scheme , . -methyltransferase
7,8
www.rsc.org/obc
p 2 9,10 O -hydroxy- OBC 2 we dis we oxida via the - - - m e h C . l o m o i B . g r O , 4 0 0 2 , . 2 , 0 9 8 2 – 8 8 8 2 9 8 8 2 8 8 8 2
DOI: 10.1039/b411428k . m e h C . l o m o i B . g r O , 4 0 0 2 , 2 0 9 8 2 – 8 8 8 2 , s i l a n r u o j s i h T © 4 0 0 2 y r t s i m e h C f o y t e i c o S l a y o R e h T
C O M M U N I C A T I O N
www.rsc.org/obc OBC Finding the cross-couplingtheproduct Finding product of produces and Rearomatization addition, water requires 9-OH. other the is single moiety the by such trapped one internally but methide, bis-quinone intermediary between reaction cross-coupling but dehydrodimer, a product not the therefore is product moiety. methide resulting quinone The other the trapping following of seen capable OH typically is as 8- addition, water rearomatizes external methide by quinone quinone one the Consequently, trapping methide. of incapable obviously is sinapyl it however, of “dimerization” the in as via afford syringaresinol to rearomatizes Utilization of WoodUtilization of and Wood Fiber #2001-02176). and (Improved Initiatives Research (#DE-AI02-00ER15067) National USDA-CSREES the program Biosciences Energy DOE the through funding partial acknowledge gratefullyWe Acknowledgements plants. these in their and transferases genes, the and to attempt the acylation lignin to role understand of find to be will goals ultimate The resulting structures of incorporation of from levels low if determining at aimed be of types three the natural for lignin acylation observed in one nature. Future research general will a be to appears therefore p fiber bast lignification kenaf in implicated is acetate sinapyl that finding 1b from this quinone methide intermediate. naphthalene compound mainly produces washingand saturatedwith EtOAcNH aqueous into product reaction entire the Extracting structure. possible to obtain sufficiently good crystals for an X-ray crystal after over 8 months in a freezer. Regrettably, it has not yet been known. long been have analogs bromo crystalline although unstable, considered NMR spectra to be recorded in 1983, allow to solution in stable sufficiently were methides quinone organic common methide in DMSO-d dissolve in to NMR solvents. difficult was that formed QMbb compound 1b thehomo-coupling of reaction usingsinapyl sachalinensis. Salix 2ab contains lignin substrate formonolignol the implicated acylating sinapyl tion that demonstrates it p importantly, lignin the More for tissue destined polymer. lignifying compound a actively be may it in that suggests detection Its co-injection). by (including time retention HPLC and data, spectral MS-MS) (including mass and UV its by identical be to shown was here poplar. old month 3 of tissue xylem alcohol, sinapyl pathway; of dehydrodimer the from lignan) havearisen to (or unlikely is it lignin the in “monomer” authentic an sinapyl that evidence -coumarate in maize (and other grasses). other (and maize in -coumarate hdoyezae s h “ooe” n h culn reac coupling the in “monomer” the is -hydroxybenzoate O In In conclusion, demonstration that sinapyl aspects unexpectedchemical and moreintriguing the of One product Indeed n peroxidase–H in is an authentic precursor of lignification augments the the augments lignification of precursor authentic an is the two the groups.9-OH has also been isolated previously, without comment, from from comment, previously,without isolated been also has -4-dehydrodimerization. n planta. in a ioal ad tbe§ pl ylo precipitate yellow pale A stable.§ and isolable was 2aa QMbb 1b 2bb into the lignification process in poplar, where the the poplar,where in process lignification the into 6 . 2bb and the evidence for sinapyl and coniferyl coniferyl and sinapyl for evidence the and 2 bb A A was that the intermediary quinone methide methide quinone intermediary the that was , with a molecular mass 16 units higher. The The higher. units 16 mass molecular a with , . Although certain syringyl and guaiacyl guaiacyl and syringyl certain Although . p ) demonstrating that compound compound that demonstrating ) 18 hdoyezyae units. -hydroxybenzoylated 2ab 16 p
-hydroxybenzoyl transferase is therefore therefore is transferase -hydroxybenzoyl QMbb 2 O was identified in the actively lignifying lignifying actively the in identified was 2 2ab p t H t idpnety generate independently to 5 pH at -hydroxybenzoate must therefore be be therefore must -hydroxybenzoate 13 ‡ When the 9-OH group is acylated, ‡ When group is 9-OH the acylated, and and in the solid state did not degrade degrade not did state solid the in 13 6 2bb At this point there is an internal is an there internal point At this revealed it to be the quinone quinone the be to it revealed 2bb aog ih h tetrahydro the with (along 1a can be detected in lignins. in detected be can 2ab 9 and and 17 The product synthesized synthesized product The 2aa they have generally been p in plants is compellingis plants in -hydroxybenzoate -hydroxybenzoate 1a 4 Cl acidified with HCl with acidified Cl by internal trapping trapping internal by 1b to feed the acylated acylated the to feed p 7,8 p also produces an an produces also -hydroxybenzoate -hydroxybenzoate The mechanism mechanism The 15 The product product The 2bb derives 2ab 1b - - . . , 140.4 (S7), 152.2 (S5), 153.7 (S3), 163.3 (P4), 166.2 (P7), 175.29 (S4). 104.8 (S6), 112.9 (S2), 115.9 (P3/5), 121.1 (P1), 132.3 (P2/6), 133.5 (S1), (2H, m, P2/6); d, (1H, OMe), 4.02 (1H, m, S8), 4.42 (2H, m, S9), 6.56 (1H, d, § also produced but typically8- at less than 9% yield. the alone, alcohol sinapyl with reactions In ‡ 8- aryltetralin as 8-8-products were not further examined. such products other of amounts Small 64.0 (B9); 122.4 (C1), 132.5 (C2/6), 116.0 (C3/5), 162.6 (C4), 166.4 (C7). 134.5 (B1), 104.45 (B2/6), 148.5 (B3/5), 136.0 (B4), 85.3 (B7), 49.8 (B8), 104.53 (A2/6), 148.5 (A3/5), 135.9 (A4), 72.6 (A7), 48.4 (A8), 70.0 (A9); 6.87 (2H, m, C3/5) (* assignments may be interchanged); C2/6), m, (2H, 7.77 s. B2/6); (2H, 6.66 s,B3/5-OMe), (6H, 3.74* B9b), d, (1H, 4.90 A2/6); s, (2H, 6.72 A3/5-OMe), s, separately.(6H, 3.78* A9s), m, reported (2H, been have data (acetone-d MS and UV solvent. 1 chromatographycyclohexane–EtOAc, gel with eluting : silica byas 1 compound cross-coupled The organicthe phase.white foam)(pale evaporation after wasobtained of MgSO over dried was phase organic HCl (3%) to pH around 3–4 and extracted with EtOAc NH (300 ml × 2). with The The saturated was disappearance. mixture material starting complete indicated TLC when h 1 for stirred was mixture water. This ml 10 in dissolved mmol) 2.13 mg, (200 hydrogencomplex the ureaperoxide mg of addition followedby units 150–250 Type II, (Sigma, peroxidaseHorseradish flask. ml 500 in ml) 400 4.5, pH phosphate,dihydrogen sodium mM (20 buffer phosphate into added and ml) (100 acetone in alcohol sinapyl and mmol) 2.12 and alcohol sinapyl from product sinapyl cross-coupled the of Synthesis † Notes andreferences 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 QMbb
J
J. Ralph, C. Chen, B. Burggraeve, M. Van Montagu, E. Messens Messens E. Montagu, Van M. B. Burggraeve, C. Chen, J. Ralph, R. Busson,P. Bruyn, Herdewijn, De A. B. Devreese, B. Pollet, J. C. Lapierre, VanBeeumen, Morreel, K. J. M. Meyermans,Marita, H. DehydrogenativeRes. 3,5-disubstituted T. Higuchi, of polymerization and Takeuchi H. Tanahashi,M. Lignin C.A. Neish. and FreudenbergK. and coniferyl of sinapyl alcohols, syntheses selective Highly J. Ralph, and F. Lu Perkin Trans. 1 cross-coupling, and dimerization of novel influence The reveals poplar G.and K. Syrjanen Brunow,biosynthesis.Regioselectivitylignin in COMT-deficient of benzodioxane oligolignols, Messens, E. profiling and W. Phenolic Boerjan Eycken, der J. Van J. Goeman, L. K. Morreel, J. Ralph, F. reveals Lu, G. Goeminne, R. Busson, P. oligolignols Herdewijn, Physiol. xylem, of poplar lignifying in conditions Profiling coupling monolignol W. Boerjan, and Messens E. S. Ralph, A. G. Goeminne, F. Lu, Kim, H. J. Ralph, Morreel, K. units in lignins, F. Lu and J. Ralph, Detection and determination of lignin as revealed by NMR, H.-J. G. Jung,Pathway of J. Ralph, R. D. Hatfield, S. Quideau, R. F. Helm, J. H.Grabber and lignin monomer in kenaf, a as acetate sinapyl for evidence Preliminary J. Ralph, and F. Lu esters in lignin, coniferyl of coniferyl and Syntheses II. lignin. bamboo in of linkage Ester T. Higuchi, and Y. Nakamura and isolated lignins, native on regiochemistry acetate determine to method modified A 6. Partanalysis. lignin for DFRCmethod F.J. The and Ralph Lu, pyrolysis-gas Identifying Spectrom. using spectrometry, fibers T. A. Martinez, non-wood in chromatography/mass and units lignin Gutierrez A. acetylated Rio, J. C. del 4-hydroxyphenylpropanoids, of coupling oxidative from polymers natural W. and Lignins: Boerjan, J. Christensen H. S. Ralph, A. D. R. Hatfield, J. Marita, M. P. F. Schatz, Kim, H. F. G. Lu, Brunow, Lundquist, K. J. Ralph, Rev. Plant Biol. Biosynthesis, Lignin Baucher, M. W. J. and Ralph Boerjan, = 6.3 Hz, B7), 2.55 (1H, m, B8), 4.41 (1H, m, B9a), 4.68 (1H, m, m, (1H, 4.68 B9a), m, (1H, 4.41 B8), m, (1H, 2.55 B7), Hz, 6.3 = , 1976, J : NMR (DMSO-d NMR : p = 1.9, S6), 6.82 (2H, m, P3/5), 6.93 (1H, d, d, (1H, 6.93 P3/5), m, (2H, 6.82 S6), 1.9, = , Springer-Verlag, Berlin–Heidelberg–New York, 1968. hdoyezae sinapyl -hydroxybenzoate: 6 ) ) , 2004, in press. d m e h C . l o m o i B . g r O , 2004, H d 61 4.96 (1H, d, d, (1H, 4.96 C 40.7 (S8), 55.55 (S5-OMe), 55.62 (S3-OMe), 65.9 (S9), , 44. p , 2000, 183. , 2003, -coumarate as possible precursors of aromatic acid acid aromatic of precursors possible as -coumarate Cellul. Chem. Technol. J. Agric. Food Chem. J. Agric. Food Chem. 18 , 1181. J. Agric. Food Chem. 54 6 ) ) , 519. Chem. Commun. d J p H Plant Physiol. -coumaricacidincorporation intomaize = 5.0 Hz, A7), 3.99 (1H, m, A8), 4.14 4.14 A8), m, (1H, 3.99 A7), Hz, 5.0 = J. Am. Chem. Soc. 3.66 (3H, s, S3-OMe), 3.70 (3H, s,S5- (3H, 3.70 s,S3-OMe), (3H, 3.66 2ab 12 4 Phytochem. Rev. Cl and acidified with dilute aqueous aqueous dilute with acidified and Cl (460 mg, 2.19 mmol) were dissolved dissolved were mmol) 2.19 mg, (460 20m, 5 yed ws isolated was yield) 25% mg, (280 4 and filtered. The crude product crude The filtered. and Constitution and Biosynthesis of of Biosynthesis and Constitution , 4 0 0 2 , . p , 1999, -hydroxybenzoate , 1998, , 1978, p cuay alcohols, -coumaryl , 1998, , 2004, in press. Rapid Commun. Mass Mass Commun. Rapid , 2002, 90. O 47 46 14 -4-dehydrodimers and and -4-dehydrodimers 12 −1 , 2004, , 1988. , 1994, 2 O 8 g ws added was mg) 8 , , 1794. , 199. , 46 -4-dehydrodimer is is -4-dehydrodimer p -hydroxybenzoate J 0 9 8 2 – 8 8 8 2 J , 4616. p =10.3, S7), 6.56 p = 1.9, S2), 7.82 7.82 S2), 1.9, = -coumaric acid acid -coumaric -coumaroylated 3 . hm Soc., Chem. J. 116 d , in press. C 5 (700 mg, mg, (700 136.0 (A1), , 9448. 9 NMR NMR Annu. Annu. Wood Plant Plant 9 8 8 2 and W. Boerjan, Modifications in lignin and accumulation of 17 J. Ralph and B. R. Adams, Determination of the conformation phenolic glucosides in poplar xylem upon down-regulation of and isomeric composition of lignin model quinone methides by caffeoyl-coenzyme A O-methyltransferase, an enzyme involved NMR, J. Wood Chem. Technol., 1983, 3, 183. in lignin biosynthesis, J. Biol. Chem., 2000, 275, 36899. 18 T. Zinke and O. Hahn, Über die Einwirkung von Brom und von 16 H. Lee, N. Watanabe, T. Sasaya and S. Ozawa, Extractives of Chlor auf Phenole: Substitutionsprodukten, Pseudobromide and short-rotation hardwood species. I. Phenolics of the wood of Salix Pseudochloride; IX. Über die Einwirkung von Brom auf Isoeugenol, sachalinensis Fr. Schm, Mokuzai Gakkaishi, 1993, 39, 1409. Justus Liebigs Ann. Chem., 1903, 329, 1.
2 8 9 0 Org. Biomol. Chem. , 2 0 0 4 , 2 , 2888–2890