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Laccase-induced C–N coupling of substituted p-hydroquinones with p-aminobenzoic acid in comparison with known chemical routes

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Fungal laccases (benzenediol:oxygen oxidoreductase, EC from Pycnoporus cinnabarinus and Myceliophthora thermophila were used as biocatalysts for enzymatic reaction of halogen-, alkyl-, alkoxy-, and carbonyl-substituted p-hydroquinones (laccase substrates) with p-aminobenzoic acid (no laccase substrate). During this reaction, the laccase substrate was oxidized to the corresponding quinones, which react with p-aminobenzoic acid by amination of the laccase substrate. The different substitutions at the hydroquinone substrates were used to prove whether the substituents influence the position of amination and product yields. The cross-coupling of methoxy-p-hydroquinone (alkoxylated) and 2,5-dihydroxybenzaldehyd (carbonyl-substituted) with p-aminobenzoic acid resulted in the formation of one monoaminated product (yield alkoxylated 52%). If monohalogen- or monoalkyl-substituted p-hydroquinones were used as laccase substrates, two monoaminated products (constitution isomers) were formed. The simultaneous formation of two different monoaminated products from the same hydroquinone substrate is the first report for laccase-mediated synthesis of aminated constitution isomers. Depending from the type of substituent of the hydroquinone, the positions of the two monoaminations are different. While the amination at the monoalkylated hydroquinone occurs at the 5- and 6-positions (yield 38%), the amination at monohalogenated hydroquinones was detectable at the 3- and 5-positions (yield 53%). The same product pattern could be achieved if instead of the biocatalyst laccase the chemical catalyst sodium iodate was used as the oxidant. However, the yields were partially much lower (0–45% of the yields with laccase).

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  1. Agematu H, Tsuchida T, Kominato K, Shibamoto N, Yoshioka T, Nishida H, Okamoto R, Shin T, Murao S (1993) Enzymatic dimerization of penicillin-X. J Antibiot 46:141–148

  2. Anyanwutaku IO, Petroski RJ, Rosazza JPN (1994) Oxidative coupling of mithramycin and hydroquinone catalysed by copper oxidases and benzoquinone. Implications for the mechanism of action of aureolic acid antibiotics. J Bioorg Med Chem 2:543–551

  3. Berka RM, Schneider P, Golightly EJ, Brown SH, Madden M, Brown KM, Halkier T, Mondorf K, Xu F (1997) Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae. Appl Environ Microbiol 63:3151–3157

  4. Bhalerao UT, Muralikrishna C, Rani BR (1994) Laccase enzyme-catalyzed efficient synthesis of 3-substituted-1,2,4-triazolo(4,3-b)(4,1,2)benzothiadiazine-8-ones. Tetrahedron 50:4019–4024

  5. Bollag JM (1992) Enzymes catalyzing oxidative coupling reactions of pollutants. In: Sigel H, Sigel A (eds) Metal ions in biological systems. vol. 28. Marcel Dekker, New York, pp 205–217

  6. Bourbonnais R, Paice MG (1990) Oxidation of non-phenolic substrates—an expanded role for laccase in lignin biodegradation. FEBS Lett 267:99–102

  7. Braun-Lüllemann A, Majcherczyk A, Hüttermann A (1997) Degradation of styrene by white-rot fungi. Appl Microbiol Biotechnol 47:150–155

  8. Brown BR (1967) Biochemical aspects of oxidative coupling of phenols. In: Taylor WJ, Battersby AR (eds) Oxidative coupling of phenols. Marcel Dekker, New York, pp 167–201

  9. Brunmark A, Cadenas E (1989) Redox and addition chemistry of quinoid compounds and its biological implications. Free Radical Bio Med 7:435–477

  10. Burton SG (2003) Laccases and phenol oxidases in organic synthesis—a review. Curr Org Chem 7:1317–1331

  11. Eggert C, Temp U, Eriksson KEL (1996) The Ligninolytic System of the White Rot Fungus Pycnoporus cinnabarinus: Purification and Characterization of the Laccase. Appl Environ Microbiol 62:1151–1158

  12. Feng X, Woonsup S, Brown SH, Wahleithner JA, Sundaram UM, Solomon EI (1996) A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability. Biochim Biophys Acta 1292:303–311

  13. Heinzkill M, Bech L, Halkier T, Schneider P, Anke T (1998) Characterization of laccases and peroxidases from wood-rotting fungi (family Coprinaceae). Appl Environ Microbiol 64:1601–1606

  14. Hosny M, Rosazza JPN (2002) Novel oxidations of (+)-catechin by horseradish peroxidase and laccase. J Agr Food Chem 50:5539–5545

  15. Ikeda R, Uyama H, Kobayashi S (1996) Novel synthetic pathway to a poly(phenylene oxide). Laccase-catalyzed oxidative polymerization of syringic acid. Macromolecules 29:3053–3054

  16. Ikeda R, Tanaka H, Uyama H, Kobayashi S (2001) A new crosslinking method of vinyl polymers having a phenol moiety via oxidative coupling. Polym J 33:959–961

  17. Jonas U (1998) Biotransformation von Biarylverbindungen durch Weißfäulepilze unter besonderer Berücksichtigung des ligninolytischen Enzymsystems von Pycnoporus cinnabarinus und Trametes versicolor. Ph.D. thesis, Greifswald, Germany

  18. Jonas U, Hammer E, Schauer F, Bollag J-M (1998) Transformation of 2-hydroxy dibenzofuran by laccases of the white rot fungi Trametes versicolor and Pycnoporus cinnabarinus and characterization of oligomerisation products. Biodegradation 8:321–328

  19. Kallmayer HJ, Tappe C (1986) Quinone-amine reactions. 18. 2-Methyl-1,4-benzoquinone derivatives from psychopharmacological agents with secondary amine structure. Pharmazie 41:29–33

  20. Leontievsky AA, Myasoedova NM, Baskunov BP, Golovleva LA, Bucke C, Evans CS (2001) Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase. Appl Microbiol Biotechnol 57:85–91

  21. Liu SY, Minard RD, Bollag JM (1981) Coupling reactions of 2,4-dichlorophenol with various anilines. J Agric Food Chem 29:253–257

  22. Majcherczyk A, Johannes C, Hüttermann A (1999) Oxidation of aromatic alcohols by laccase from Trametes versicolor mediated by the 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) cation radical and dication. Appl Microbiol Biotechnol 51:267–276

  23. Manda K, Hammer E, Mikolasch A, Niedermeyer THJ, Dec J, Jones AD, Benesi AJ, Schauer F, Bollag JM (2005) Laccase-induced cross-coupling of 4-aminobenzoic acid with para-dihydroxylated compounds 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide and 2,5-dihydroxybenzoic acid methyl ester. J Mol Catal B Enzym 35:86–92

  24. Manda K, Hammer E, Mikolasch A, Gördes D, Thurow K, Schauer F (2006) Laccase-induced derivatization of unprotected amino acid L-tryptophan by coupling with p-hydroquinone 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide. Amino Acids 31:409–419

  25. Mayer A, Staples R (2002) Laccase: new functions for an old enzyme. Phytochemistry 60:551–565

  26. Mickel M, Hee-Cheol K, Hampp N (2003) Origin of the mediator losses in electrochemical delignification processes: primary and secondary reactions of violuric acid and N,N-dimethylvioluric acid radicals with lignin model compounds. Green Chem 5:8–14

  27. Mikolasch A, Schauer F (2003) Biotransformation of N-(2-alkylamino-4-phenylimidazol-1-yl)-acetamides and kinetic studies by using cells and laccase from Trametes versicolor. J Basic Microb 43:508–521

  28. Mikolasch A, Niedermeyer THJ, Lalk M, Witt S, Seefeldt S, Hammer E, Schauer F, Gesell M, Hessel S, Julich WD, Lindequist U (2006) Novel penicillins synthesized by biotransformation using laccase from Trametes spec. Chem Pharm Bull 54:632–638

  29. Mikolasch A, Niedermeyer THJ, Lalk M, Witt S, Seefeldt S, Hammer E, Schauer F, Gesell M, Hessel S, Julich WD, Lindequist U (2007) Novel cephalosporins synthesized by amination of 2,5-dihydroxybenzoic acid derivatives using fungal laccases. Chem Pharm Bull 55:412–416

  30. Milligan C, Ghindilis A (2002) Laccase based sandwich scheme immunosensor employing mediatorless electrocatalysis. Electroanalysis 14:415–419

  31. Minussi R, Pastore G, Duran N (2002) Potential applications of laccase in the food industry. Trends Food Sci Technol 13:205–216

  32. Murugesan K, Arulmani M, In-Hyun N, Young-Mo K, Yoon-Seok C, Kalaichelvan PT (2006) Purification and characterization of laccase produced by a white rot fungus Pleurotus sajorcaju under submerged culture condition and its potential in decolorization of azo dyes. Appl Microbiol Biotechnol 72:939–946

  33. Nagai M, Sato T, Watanabe H, Saito K, Kawata M, Enei H (2002) Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes, and decolorization of chemically different dyes. Appl Microbiol Biotechnol 60:327–335

  34. Nakamura T (1960) On the process of enzymatic oxidation of hydroquinone. Biochem Biophys Res Commun 2:111–113

  35. Niedermeyer THJ, Mikolasch A, Lalk M (2005) Nuclear amination catalyzed by fungal laccases: reaction products of p-hydroquinones and primary aromatic amines. J Org Chem 70:2002–2008

  36. Osiadacz J, Al-Adhami AJH, Bajraszewska D, Fischer P, Peczynska-Czoch W (1999) On the use of Trametes versicolor laccase for the conversion of 4-methyl-3-hydroxyanthranilic acid to actinocin chromophore. J Biotechnol 72:141–149

  37. Pardo M, Joos K, Schäfer W (1979) Über die oxidative Aminierung von 1′,4′-dihydroxy-2′-acetonaphthon. Liebigs Ann Chem 1979:503–521

  38. Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24:219–226

  39. Rochefort D, Leech D, Bourbonnais R (2004) Electron transfer mediator systems for bleaching of paper pulp. Green Chem 6:14–24

  40. Schäfer W, Aguado A (1971) Chemistry of substituted benzoquinones. 8. Oxidative amination of hydroquinones. Angew Chem Int Ed 10:405–406

  41. Schäfer W, Aguado A, Sezer U (1971) Chemistry of Substituted Benzoquinones. 9. New method of preparing heterocyclic quinones. Angew Chem Int Ed 10:406–407

  42. Schäfer A, Specht M, Hetzheim A, Francke W, Schauer F (2001) Synthesis of substituted imidazoles and dimerization products using cells and laccase from Trametes versicolor. Tetrahedron 57:7693–7699

  43. Tatsumi K, Freyer A, Minard RD, Bollag JM (1994b) Enzyme-mediated coupling of 3,4-dichloroanilin and ferulic acid: a model for pollutant binding to humic materials. Environ Sci Technol 28:210–215

  44. Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26

  45. Torres T, Eswaran SV, Schäfer W (1985) Quinone chemistry. Synthesis of 3-methoxy[2,1]benzisoxazole- and 3-methoxynaphth[2,3-c]isoxazolequinones. J Heterocycl Chem 22:697–699

  46. Uchida H, Fukuda T, Miyamoto H, Kawabata T, Suzuki M, Uwajima T (2001) Polymerization of bisphenol A by purified laccase from Trametes villosa. Biochem Biophys Res Commun 287:355–358

  47. Yaropolov AI, Skorobogatko OV, Vartanov SS, Varfolomeyev SD (1994) Laccase—properties, catalytic mechanism, and applicability. Appl Biochem Biotechnol 49:257–280

  48. Yogo M, Ito C, Furukawa H (1991) Synthesis of some carbazolequinone alkaloids and their analogs—facile palladium-assisted intramolecular ring-closure of arylamino-1,4-benzoquinones to carbazole-1,4-quinones. Chem Pharm Bull 39:328–334

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We thank K. Weisz (Institute of Biochemistry, University of Greifswald) for providing NMR data and R. Jack (Institute of Immunology, University of Greifswald) for help in preparing the manuscript.

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Correspondence to Annett Mikolasch.

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Laccase-induced C–N coupling of substituted p-hydroquinones with p-aminobenzoic acid in comparison with known chemical routes (DOC 779 KB)

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Mikolasch, A., Matthies, A., Lalk, M. et al. Laccase-induced C–N coupling of substituted p-hydroquinones with p-aminobenzoic acid in comparison with known chemical routes. Appl Microbiol Biotechnol 80, 389 (2008). https://doi.org/10.1007/s00253-008-1595-y

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  • Laccase
  • Coupling
  • Hydroquinones
  • p-aminobenzoic acid