3 pages, 1 figures, 2 tables, 14 references.The 13C resonance spectra of fungal melanins from Aspergillus niger, Eurotium echinolatum, and Stachybotrys chartarum are reported. The spectra were taken in 5% W/W solution of the substances in 0.1 N NaOD in D2O using the Fourier-transform-technique. The spectra and possible assignments of the bands are discussed. The similarities of these spectra to those of soil humic acids are unexpectedly small.Peer reviewe

González-Vila, Francisco Javier

Lentz, Harro

Lüdemann, H.-D.

Sáiz-Jiménez, Cesáreo

Zeitschrift für Naturforschung C

Digital.CSIC

13C Nuclear Magnetic Resonance Spectra of Fungal Melanins F. J. Gonzalez·Vila, C. Saiz-Jimenez Centro de Edafología y Biología Aplicade del Cuarto, eS.I.C., Sevilla H. Lentz lnstitut íür Physikalische Chemie und ElektrodlCmie, Karlsruhe and H.·D. Lüdemann Institut für Biophysik und Physikalische Biochemie, Regensburg Z. Naturforsdl. 33c, 291-293 (1978); receivcd Novembcr 21, 1977/January 19, 1978 Melanins, 13C Nuclear Magnetic Resona.nce The 13C resonance spectra oí íungal melanins from Aspcrgillus 1!igcr, Eurotium edti1!olalunt, and Stadtybotrys dtartarum are reported. The spectra were taken in 5% W/W solution oí the substances in 0.1 N NaOD in D20 using the Fourier-trnnsform-technique. The spectra and possible as-signments oí the bands are discussed. The similaritics oí these spectra to those oí soil humic acids ore unexpectedly small. Introduction Plant and fungal melaníns usuaIly have struc~ tures based on eatemol as opposed to the índole melanins of animal origino However, it appears that sorne fungal melanins sum as -those from Asper~ gillus niger, Stachybotrys chartarum and Eurotium echinulatum do not belong 10 these 1wo types. These pigments are eomplex quinonoid polymers formed by oxidative polymerization and linked to eell wall materials. Information about the memical structure of fungal melanins has been obtained so far by cbemieal degradation tecbniques [1- 3], whicb yield limited information only or tend in sorne cases to produce artifacts. On the other hand, spee~ trometric methods have failed to provide detailed iníormation on ,the structure of these biopolymers [4,5]. Fairly well resolved 13C resonance spectra have been obtained for humie aeids [6]. In the present paper, the first applieation of 13C NMR to fungal melanins is given. Requests for .reprints should be sent to Dr. H. Lentz. Insti-tut für Physikalische Chemie und Elektrochemie der Uni-versitat Karlsruhe, Kaiserstr. 12, D-7500 Karlsruhe 1. Material and Methods The fungi Aspergillus niger nnd Eurotiurn edzi-nulatum were isolated from a Spanish vertisol. Methods of cultivation and isolation of melanin, as well as sorne of its physieal and cllemical cllarac-teristics were reported previously [7,8]. The pig-ments of Aspergillus niger were eX'traeted from spores and the pigments of Eurotium edzinulatum from mycelium and also from n culture medium. Mycelial Stad~ybotrys chartarum melanin was pro~ vided by Dr. K. Haider, Braunsmweig, Germany. Elementary compositions oí the melanins are given in Table 1. The 13C speetra were taken of 5% W /W solutions of melanins in 0.1 N NaOD as deseribed for soíl humie substanees [6]. A Varian XL·IOO·15 speetrometer operating at 25.2 MHz was used to obtain the spectra by the Fourier-transform-tech~ ni que. The protons are noise deeoupled. 250 blocks of 1000 transients were aceumulated in the long~ term~averaging-mode. The resolutions of the speetra was ± 10 Hz (approximately 0.5 ppm). The spee· trometer was locked to an external Fluorine-19~ lock. Shift values quoted are given in the TMS scale, refereneed to a coaxial internal neat TMS eapillary. The position of the TMS signal was ob· tained in a separate run wHh accumulation of 1000 transients. No attempts to correet for magnetic susceptibility efIects were made. Table 1. Relative contents oí carbon, hydrogen o.nd nitrogen in percent by weight (Oxigen has assumed to comprise the remo.inder) and the percentage oi o.sh yielded írom fungal melonins. e H N o Ash A. niger 45.2 5.4 2.7 46.7 1.2 (spores) S. dtartarum 56.0 5.9 4.9 33.2 1.5 (mycelium) E. echinulatum 54.2 4.1 4.2 37.5 2.2 (medium) E. edtinulatum 55.2 3.8 4.7 36.3 1.8 (mycelium) Results Fig. 1 shows the 13C NMR speetra of the fungal melanins. The maximal noise oí the baseline is in~ dieated in the figure. F or S. clwrtarum a par! of the spectrum was reeorded with higher amplitudes. Then eenters oí the signals ohtained are listed in 292 Notizen ASpH~,i1US n:~c; (s¡ICresl 1\¡IV'r~tnJ" lV/\N'Ni'1A""vfJijf )r\w~0\ 2E, 200 150 100 --6Ippml so O Fig. 1. 13C NMR speetra of fungal melanins. Intensities in arbitrary units. o is the chemieal 5hift in ppm on the TMS seale. The approximate baseline noise is indieated by two horizontal bars in the seeond speetrum from the bottom. For S. chartarum a part of the speetrum was reeorded with higher amplitudes. Table 11. These signals may have more resolution; however, the low signal to noise ra-tio only allows the safe assignment af 1he bands Jisted in Table II. Plausible assignments are alsa indicated [10, 11]. These assignments could be improved by investiga-tíon oí a series oí model compounds in the same solvento The absolute chemical shift can be assumed ta be accurate ta ± (5 -10) ppm because af the high molecular weight, othe viscosity oí the solution, the neglect af magnetic susceptibility effects and dhe presence oí carbon atoms with unpaired electrons. These unpaired electrons can be deduced from ,the black colour oí the substances and moreover the Table n. l:JC chemical shifts oí fungal melanins and plausible assignrnents. ,~ -. ,~ -ci:d -cid oo-'. " .s~ :::: 8. . - .~ --o ~~ . e . E • E "'- "'- "'-230 210 r~o 2DO 198 ke~ nes 190 180 180 180 180 qmnones 170 168 168 170 COOH 160 160 150 140 140 )_."oo., 130 136 136 120 124 120 120 heteroaromatie rings llO ll5 109 100 104 102 90 80 80 79 polysaccharides 75 75 75 70 70 60 65 50 54 50 40 40 40 45 ) """.~ ",00", 30 31 30 25 lX,p,,..,-C(amino aeids) 20 18 OCH, 10 10 unstructured signals have been found in preliminary ESR-experiments at g-values drpical far carban radicals. These radicals may be also responsible for the signals in the region of positive chemical shifts. In complex substances like melanins, one has to consider another complication. The molecules might consist of rigid and mobile regions in which only the carbon-13-nucIei of Ithe flexible parts possess sufficiently long transversal relaxation times, T 2 , to be observable in a high resolution spee;trum. Con-sequently, it is hazardous to derive absolute concen-trations for a certain cIass of carbon atoms from 'lhe intensities of the signals in different samples or to deduce from the absence of a signal the absence of a specific memical structure. l3C NMR spectra may Jthus only be used for the positive assignment of certain structural eIements in a heterogenous poIy-mero The spectrum af A. niger pigments (aspergillin) shows prominent signals in ,the aliphatic zone. These resanance lines prabably belang ia palysacmarides. S. charlarum melanin shows intense signals in the aliphatic regian, whim are prabably due ta alkyl Notizen 293 chains. Cultural and mycelial E. eddnulatum mela-nins have similar 13C NMR spectra. For the mycelial pigments, the aliphatic Hnes are more resolved. The aromatic regions in both spectra of E. ed~inulatum are more pronounced than in the spectra oí other pigments. This may be due to the phenolic core and lhe peripherally linked anlhraquinones (8). Discussion The l3C NMR and also lhe pyrolysis mass speclra [9] suggest lhai aspergillin has the highest content of polysaccharides of aU the investigated melanins. Nicolaus [5] reported that polysacdlarides are bound to this melanin. AIso nigeran, a polysac-dIaride, is produced by A. niger. However, other authors [1] have reported a very low amount of aliphatic compounds and a high amount of benzene-carboxylic and phenolic acids. This contradiotion and the others reported below may be explained by the variability oí difIerent strains from the same fungi. The S. chartarum melanin seems 'to be constitll'ted by alkylbenzenes and alkylphenols as major como ponents. For a similar melanin, Sdmitzer and Ney-roud [12] found phenolic, benzenecarboxylic and aIiphatic acids as major components from the alka. line cupric oxide oxidation. The IR spectrum of this particular melanin reveals high concentrations of aliphatic and aromatic constituents [14], ",hereas the 13C spectrum indicates ihat rthis melanin has a {airly homogeneous polymeric structure corrobo-[1] M. Schnitzer, M. 1. Ortiz, and K. Ivarson, Soil ScL Soco Amer. Proc. 37, 229 (1973). [2] J. P. Martin, K Haider, and C. Sáiz-Jiménez, Soil Sci. Soco Amer. Proc. 38, 760 (1974). [3] C. Sáiz-Jiménez and F. Martin, Izvest. Akad. Nauk SSSR, Ser. Biol. (in press) (1976). [4] M. Schnitzer and S. U. Khan, Rumie Substanees in the Environment (Maree1 Dekker, New York 1972). [5] R. A. Nicolaus, Melanins (Hermann, Paris 1968). [6] F. J. Gonza1ez-Vila, H. Lentz, and H.-D. Lüdeman, Biophys. Biochem. Res. Commun. 72, 1063 (1976). [7] C. Sáiz-Jiméncz and F. Martin, An. Edaf. Agrobiol. 31. 133 (1972). [8] C. Sáiz-Jiménez, K. Haider, and J. P. Martin, Soil Sci. Soe. Amer. Proc. 39, 649 (1975). rating the pyrolysis mass results by Meuzelaar et al. [9]. The E. ed~inu1atum pigments show stronger sig-naIs in the aromatic region than the other pig-ments discussed here. These lines appear al a slightly smaller, o value than most C-aromatic com· pounds. Most probably these resonances can be al-tributed to phenols and anthraquinones, though other hetera-aroma tic compounds cannot be ex-cluded. Phenols and anthraquinones were found in extracts from culture media after 14 - 18 days of incubation. These compounds are incorporated later ¡nto the poIymer and recovered among the pro-ducts of dIemical degradation [8]. The 13C NMR spectra from fungal melanins can be compared with the spectra from soil humic acids obtained under similar conditions [6]. The simi-larilies of the spectra froro both groups of sub-stances are much smaller than would be expected from the similarities of the anaIytical data reported in the literature, amI one shouId be very cautious in transferring structural information from fungal pigments to huroic acids and vice versa. At this stage of the investigation, 1he signal to noise ratio is poor and the interpretation of the spectra obtained must remain qualitative. Thus it appears unjustified 10 interprete the broad lines with further iníorrnation drawn from mernical de-gradation studies. A better signal to noise ratio may be obtained in the future with apparatus employing higher magnetic fields and/or larger sample volumes. [9] H. L. C. Meuzelaar, K. Haider, B. R. Nagar, and J. P. Martín, Geodcrma 17. 239 (1977). [10] E. Brcitmaier and W. Voe1ter, l:1C NMR Spectroseopy (Verlag Chcmic, Weinheim 1974). lU] G. C. Levy and G. L. Nc1son, Carbon-13 Nuclear Magnetic Resonanee for Organie Chemist (Wiley, Ncw York 1972). [12] K. Haider, B. R. Nagar, H. L. C. Meuzelaar, C. Sáiz-Jiménez, and J. P. Martin, Intern. Symp. on Soil Org. Matter Studies, Brunswick (Germany) (in press) (1976) . [13] M. Sch.nitzer and J. A. Neyroud, Soil Biol. Biocnem. 7, 365 (1975). [14] Z. Filip, K. Haidcr, H. Beutelspadter, snd J. P. Mar-tin, Geoderma n. 37 (1974). 

13C nuclear magnetic resonance spectra of fungal melanins

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