Mycotoxin Research

, Volume 34, Issue 2, pp 141–150 | Cite as

Large-scale total synthesis of 13C3-labeled citrinin and its metabolite dihydrocitrinone

  • Dominik Bergmann
  • Florian Hübner
  • Birgit Wibbeling
  • Constantin Daniliuc
  • Benedikt Cramer
  • Hans-Ulrich Humpf
Original Article


The analysis of the nephrotoxic mycotoxin citrinin in food, feed, and physiological samples is still challenging. Nowadays, liquid chromatography coupled with mass spectrometry is the method of choice for achieving low limits of detection. But matrix effects can present impairments for this method. Stable isotope dilution analysis can prevent some of these problems. Therefore, a stable isotopically labeled standard of citrinin for use in stable isotope dilution analysis was synthesized on large scale. The improved diastereoselective total synthetic strategy offered the possibility to introduce three 13C-labels in two steps by ortho-toluate anion chemistry. This led to a mass difference of 3 Da, sufficient for preventing spectral overlap. Additionally, a stable isotopically labeled form of dihydrocitrinone, the main urinary metabolite of citrinin, was synthesized with the same mass difference. This was achieved by a sequence of cyclisation, oxidation, deprotection, and carboxylation reactions starting from a protected intermediate of the labeled citrinin synthesis. Thus, this method also offers a complete way to synthesize dihydrocitrinone from citrinin on large scale.


Mycotoxin Citrinin Metabolite Dihydrocitrinone Total synthesis Stable isotope 



We thank the NMR department of the Organisch-Chemisches Institut, Westfälische Wilhelms-Universität Münster, for measurement of some 300 MHz NMR spectra. We also thank the Deutsche Forschungsgemeinschaft (DFG) for funding (GRK1143, IRTG Münster-Nagoya).

Compliance with ethical standards

Conflict of interest


Supplementary material

12550_2018_308_MOESM1_ESM.docx (2.7 mb)
ESM 1 (DOCX 2.70 mb)
12550_2018_308_MOESM2_ESM.docx (54 kb)
ESM 2 (DOCX 53.7 kb)
12550_2018_308_MOESM3_ESM.docx (2.3 mb)
ESM 3 (DOCX 2.27 mb)


  1. Ali N, Blaszkewicz M, Mohanto NC, Rahman M, Alim A, Hossain K, Degen GH (2015) First results on citrinin biomarkers in urines from rural and urban cohorts in Bangladesh. Mycotoxin Res 31:9–16. Google Scholar
  2. Asam S, Rychlik M (2015) Recent developments in stable isotope dilution assays in mycotoxin analysis with special regard to Alternaria toxins. Anal Bioanal Chem 407(25):7563–7577. CrossRefPubMedGoogle Scholar
  3. Barber JA, Staunton J (1979) Protium as a tracer in polyketide biosynthesis: incorporation of 13CH3 13CO2H into citrinin produced on a medium based on D2O. J Chem Soc Chem Commun (23):1098–1099.
  4. Barber JA, Staunton J (1981) The total synthesis of some deuterium labelled pentaketide derivatives of orsellinic acid. J Chem Soc Perkin Trans 1:1685–1689. Google Scholar
  5. Barber JA, Carter RH, Garson MJ, Staunton J (1981) The biosynthesis of citrinin by Penicillium citrinum. J Chem Soc Perkin Trans 1:2577–2583. Scholar
  6. Barber JA, Staunton J, Wilkinson MR (1986) A diastereoselective synthesis of the polyketide antibiotic citrinin using toluate anion chemistry. J Chem Soc, Perkin Trans 1:2101–2109. Scholar
  7. Barber JA, Chapman AC, Howard TD, Tebb G (1988) Recycling of polyketides by fungi: the degradation of citrinin by Penicillium citrinum. Appl Microbiol Biotechnol 29(4):387–391. CrossRefGoogle Scholar
  8. Barluenga S, Moulin E, Lopez P, Winssinger N (2005) Solution- and solid-phase synthesis of radicicol (monorden) and pochonin C. Chem Eur J 11(17):4935–4952. CrossRefPubMedGoogle Scholar
  9. Birch AJ, Fitton P, Pride E, Ryan AJ, Smith H, Whalley WB (1958) Studies in relation to biosynthesis. Part XVII. Sclerotiorin, citrinin, and citromycetin. J Chem Soc: 4576–4581. Scholar
  10. Blanc PJ, Laussac JP, Le Bars J, Le Bars P, Loret MO, Pareilleux A, Prome D, Prome JC, Santerre AL, Goma G (1995) Characterization of monascidin A from Monascus as citrinin. Int J Food Microbiol 27(2-3):201–213.
  11. Blaszkewicz M, Muñoz K, Degen GH (2013) Methods for analysis of citrinin in human blood and urine. Arch Toxicol 87:1087–1094. Scholar
  12. Bowden K, Heilbron IM, Jones ERH, Weedon BCL (1946) Researches on acetylenic compounds. Part I. The preparation of acetylenic ketones by oxidation of acetylenic carbinols and glycols. J Chem Soc:39–45.
  13. Brown JP, Cartwright NJ, Robertson A, Whalley WB (1948) Structure of citrinin. Nature 162:72–73. Scholar
  14. Brown JP, Cartwright NJ, Robertson A, Whalley WB (1949a) The chemistry of fungi. Part IV. The constitution of the phenol, C11H16O3, from citrinin. J Chem Soc: 859–867. Scholar
  15. Brown JP, Robertson A, Whalley WB, Cartwright NJ (1949b) The chemistry of fungi. Part V. The constitution of citrinin J Chem Soc 867–879. Scholar
  16. Carpenter TA, Evans GE, Leeper FJ, Staunton J, Wilkinson MR (1984) Reactions of the carbanion from an orsellinate derivative with electrophiles. J Chem Soc Perkin Trans 1:1043–1051. Scholar
  17. Cartwright NJ, Robertson A, Whalley WB (1949a) A synthesis of citrinin. Nature 163:94–95. Scholar
  18. Cartwright NJ, Robertson A, Whalley WB (1949b) The chemistry of fungi. Part VII. Syntheses of citrinin and dihydrocitrinin. J Chem Soc:1563–1567. Scholar
  19. Chang H, Li X, Fu J, Cheng K, Ye Y-H, Guo J-H (2009) Crystal structure of (3R,4S)-6,8-dihydroxy-3,4,5-trimethyl-1-oxoisochroman-7-carboxylic acid, C13H14O6. Z Kristallogr-New Cryst Struct 224:425–427. Scholar
  20. Chien MM, Schiff PL, Slatkin DJ, Knapp JE (1977) Metabolites of aspergilli. III. Isolation of citrinin, dihydrocitrinone and sclerin from Aspergillus carneus. Lloydia 40(3):301–302PubMedGoogle Scholar
  21. Cram DJ (1948) Mold metabolites. III. The structure of citrinin. J Am Chem Soc 70(12):4244–4247. CrossRefPubMedGoogle Scholar
  22. Cram DJ (1950) Mold metabolites. V. The stereochemistry and ultraviolet absorption spectrum of citrinin. J Am Chem Soc 72(2):1001–1002. CrossRefGoogle Scholar
  23. Cram DJ (1952) Studies in stereochemistry. V. Phenonium sulfonate ion-pairs as intermediates in the intramolecular rearrangements and solvolysis reactions that occur in the 3-phenyl-2-butanol system. J Am Chem Soc 74(9):2129–2137. CrossRefGoogle Scholar
  24. Cram DJ, Singer LA (1963) Macro rings. XXVI. [2.2]Paracyclophanyl as a neighboring group. J Am Chem Soc 85(8):1075–1079. CrossRefGoogle Scholar
  25. Cramer B, Beyer M, Humpf H-U (2009) Stable isotope labeled mycotoxins as standards for HPLC-MS/MS analysis. In: Appell M, Kendra DF, Trucksess MW (eds) mycotoxin prevention and control in agriculture, ACS Symp Ser 1031, American Chemical Society, Washington, DC, pp 265–276. Scholar
  26. Cui X, Zhu G, Liu H, Jiang G, Wang Y, Zhu W (2016) Diversity and function of the Antarctic krill microorganisms from Euphausia superba. Sci Rep 6:36496. Scholar
  27. Curtis RF, Hassall CH, Nazar M (1968) The biosynthesis of phenols. Part XV. Some metabolites of Penicillium citrinum related to citrinin. J Chem Soc C:85–93.
  28. Destro R, Marsh RE (1984) Temperature dependence of tautomeric equilibria in the solid state: the case of citrinin. J Am Chem Soc 106(23):7269–7271. CrossRefGoogle Scholar
  29. Donner CD, Gill M (2002) Pigments of fungi. Part 68. Synthesis and absolute configuration of thysanone. J Chem Soc Perkin Trans 1:938–948. Scholar
  30. Dunn BB, Stack ME, Park DL, Joshi A, Friedman L, King RL (1983) Isolation and identification of dihydrocitrinone, a urinary metabolite of citrinin in rats. J Toxicol Environ Health 12(2-3):283–289. CrossRefPubMedGoogle Scholar
  31. EC (2014) Commission regulation (EU) no 212/2014 of 6 March 2014 amending regulation (EC) no 1881/2006 as regards maximum levels of the contaminant citrinin in food supplements based on rice fermented with red yeast Monascus purpureus. Off J Eur Union 57:3–4Google Scholar
  32. EFSA (2012) Scientific opinion on the risks for public and animal health related to the presence of citrinin in food and feed. EFSA J 10(3):2605. Scholar
  33. Fang B, Xie X, Zhao C, Jing P, Li H, Wang Z, Gu J, She X (2013) Asymmetric total synthesis of fusarentin 6-methyl ether and its biomimetic transformation into fusarentin 6,7-dimethyl ether, 7-O-demethylmonocerin, and (+)-monocerin. J Org Chem 78(12):6338–6343. CrossRefPubMedGoogle Scholar
  34. Flajs D, Peraica M (2009) Toxicological properties of citrinin. Arch Ind Hyg Toxicol 60(4):457–464. Google Scholar
  35. Föllmann W, Behm C, Degen GH (2014) Toxicity of the mycotoxin citrinin and its metabolite dihydrocitrinone and of mixtures of citrinin and ochratoxin A in vitro. Arch Toxicol 88:1097–1107. Scholar
  36. Gerding J, Cramer B, Humpf H-U (2014) Determination of mycotoxin exposure in Germany using an LC-MS/MS multibiomarker approach. Mol Nutr Food Res 58:2358–2368. Scholar
  37. Hassall CH, Jones DW (1962) The biosynthesis of phenols. Part IV. A new metabolic product of Aspergillus terreus Thom. J Chem Soc 4189–4191. Scholar
  38. He Y, Cox RJ (2016) The molecular steps of citrinin biosynthesis in fungi. Chem Sci 7(3):2119–2127. CrossRefGoogle Scholar
  39. Hetherington AC, Raistrick H (1931) Studies in the biochemistry of micro-organisms. Part XIV. On the production and chemical constitution of a new yellow colouring matter, citrinin, produced from glucose by Penicillium citrinum Thom. Phil Trans R Soc Lond B 220(468-473):269–295. CrossRefGoogle Scholar
  40. Hill RK, Gardella LA (1964) The absolute configuration of citrinin. J Org Chem 29(3):766–767. CrossRefGoogle Scholar
  41. IARC (1986) Citrinin. In: Some naturally occurring and synthetic food components, furocoumarins and ultraviolet radiation, vol 40. IARC, Lyon, France, pp 67–87Google Scholar
  42. Jenny EF, Winstein S (1958) 14C-Umlagerung, Salzeffekte und Ionenpaar-Rückkehr in der Solvolyse von [2-(p-Anisyl)-äthyl]-p-toluolsulfonat. Helv Chim Acta 41(3):807–823. CrossRefGoogle Scholar
  43. Larghi EL, Kaufman TS (2006) The oxa-Pictet-Spengler cyclization: synthesis of isochromans and related pyran-type heterocycles. Synthesis:187–220. Google Scholar
  44. Larghi EL, Kaufman TS (2011) Synthesis of oxacycles employing the oxa-Pictet-Spengler reaction: recent developments and new prospects. Eur J Org Chem 2011:5195–5231. Scholar
  45. Lee CC, Slater GP, Spinks JWT (1957) Rearrangement studies with C14. V. The solvolysis of 2-phenylethyl-1-C14 p-toluenesulphonate. Can J Chem 35(12):1417–1422. CrossRefGoogle Scholar
  46. López P, de Nijs M, Spanjer M, Pietri A, Bertuzzi T, Starski A et al (2017) Generation of occurrence data on citrinin in food. EFSA Support Publ 2017:EN-1177. Scholar
  47. Mechoulam R, Ben-Zvi Z (1969) Carboxylation of resorcinols with methylmagnesium carbonate. Synthesis of cannabinoid acids. J Chem Soc D (7):343–344.
  48. Mehta PP, Whalley WB (1963) The chemistry of fungi. Part XLII. The absolute configuration of citrinin J Chem Soc:3777–3779. Scholar
  49. Osteresch B, Viegas S, Cramer B, Humpf H-U (2017) Multi-mycotoxin analysis using dried blood spots and dried serum spots. Anal Bioanal Chem 409(13):3369–3382. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Ostry V, Malir F, Ruprich J (2013) Producers and important dietary sources of ochratoxin A and citrinin. Toxins 5:1574–1586. Scholar
  51. Raistrick H, Smith G (1935) Studies in the biochemistry of micro-organisms. XLII. The metabolic products of Aspergillus terreus Thom. A new mould metabolic product – terrein. Biochem J 29:606–611. Scholar
  52. Regan AC, Staunton J (1987) Asymmetric synthesis of (+)-citrinin using an ortho-toluate carbanion generated by a chiral base. J Chem Soc Chem Commun (7):520–521.
  53. Rödel T, Gerlach H (1995) Enantioselective synthesis of the polyketide antibiotic (3R,4S)-(−)-citrinin. Liebigs Ann 1995(5):885–888. CrossRefGoogle Scholar
  54. Santesson J (1970) Syntheses of orsellinic acid and related compounds. Acta Chem Scand 24:3373–3378. CrossRefGoogle Scholar
  55. Saunders WH, Ašperger S, Edison DH (1958) Rates of solvolysis of some deuterated 2-phenylethyl p-toluenesulfonates. J Am Chem Soc 80:2421–2424. Scholar
  56. Schwenk E, Schubert M, Stahl E (1949) New reactions of citrinin. Arch Biochem 20:220–226PubMedGoogle Scholar
  57. Schwenk E, Alexander GJ, Gold AM, Stevens DF (1958) Biogenesis of citrinin. J Biol Chem 233(5):1211–1213PubMedGoogle Scholar
  58. Stiles M, Finkbeiner HL (1959) Chelation as a driving force in synthesis. A new route to α-nitro acids and α-amino acids. J Am Chem Soc 81(2):505–506. CrossRefGoogle Scholar
  59. Timonin MI, Rouatt JW (1944) Bacteriostatic activity of citrinin in vitro. Can J Public Health 35:396–406Google Scholar
  60. Ting SZY, Baird LJ, Dunn E, Hanna R, Leahy D, Chan A, Miller JH, Teesdale-Spittle PH, Harvey JE (2013) Synthesis of diastereomeric, deoxy and ring-expanded sulfone analogues of aigialomycin D. Tetrahedron 69(49):10581–10592. CrossRefGoogle Scholar
  61. Wang P, Zhang Z, Yu B (2005) Total synthesis of CRM646-A and -B, two fungal glucuronides with potent heparinase inhibition activities. J Org Chem 70(22):8884–8889.
  62. Wünsch B, Zott M (1992) Chirale 2-Benzopyran-3-carbonsäure-Derivate durch Oxa-Pictet-Spengler-Reaktion von (S)-3-Phenylmilchsäure-Derivaten. Liebigs Ann Chem 1992(1):39–45. CrossRefGoogle Scholar
  63. Xin Z-H, Tian L, Zhu T-J, Wang W-L, Du L, Fang Y-C et al (2007) Isocoumarin derivatives from the sea squirt-derived fungus Penicillium stoloniferum QY2-10 and the halotolerant fungus Penicillium notatum B-52. Arch Pharm Res 30(7):816–819. CrossRefPubMedGoogle Scholar
  64. Xu B-J, Jia X-Q, Gu L-J, Sung C-K (2006) Review on the qualitative and quantitative analysis of the mycotoxin citrinin. Food Control 17(4):271–285. CrossRefGoogle Scholar
  65. Zheng H, Zhao C, Fang B, Jing P, Yang J, Xie X, She X (2012) Asymmetric total synthesis of cladosporin and isocladosporin. J Org Chem 77:5656–5663. Scholar

Copyright information

© Society for Mycotoxin Research and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Food ChemistryMünsterGermany
  2. 2.Organisch-Chemisches InstitutMünsterGermany

Personalised recommendations