## 4 Discussion
### The Chemical Structure of Ochroconis Melanins
Solid-state \({}^{15}\)N NMR spectra of the melanins showed an intense signal in the peptide and amide region revealing that a major part of their organic N is bound in peptide structures, and the presence of aromatic N heterocyclic signals is negligible, if any. In addition, there were no bands assigned to pyrrole and indole ring vibrations in the SERS spectra. These data were also supported by the tests of melanin inhibition. Neither kojic acid nor tricyclazole inhibit the synthesis of _Ochroconis_ melanin, which indicates that it is not a DOPA or DHN melanin. Therefore, according to [PERSON] et al. [19] the chemical structures of these melanins are not related to DOPA, 5,6-dihydroxyindole or DHN precursors. The analytical pyrolysis of _Ochroconis_ melanins also supports this assumption because indole or naphthalene derivatives were not the major pyrolysis products. Natural DOPA melanins (e.g., from _Sepia officinalis_ and _C. neoformans_) and synthetic eumelanins share a common indole-based aromatic core, as demonstrated using \({}^{13}\)C and \({}^{15}\)N NMR [100; 101]. The same NMR approach revealed that an indole-based aromatic core could not be found in _Ochroconis_ melanins [19], but a high peptide content. Relative high peptide contents (>70% of the total intensity in the \({}^{15}\)N NMR spectra) were also found in other fungal melanins (_Aspergillus glaucus_, _Hendersonula toruloidaea_, _Ullocadium atmu_, _Trichoderma harzianun_, _E. echinultatum_, etc.). This indicates that most, if not all, of the organic nitrogen in these fungal melanins were bound in peptide-like structures [98]. The pyrolysis of DOPA melanins yield as major products those derived from an indole core: pyrroles, pyridines, toluene, phenols, and indoles [102; 103]. Although the pyrolysis of a few fungal melanins (e.g., _Aspergillus niger_, _Aspergillus sydowii_, _Penicillium funculosum_, _Coprimus micaceus_, _Stachybortys charatum_) produces the same compounds [37], they are found in comparatively lower amounts, which easily can be ascribed to pyrolysis of proteins, co-extracted or linked to the melanin [37; 94; 104]. This is also the case of the _Ochroconis_ melanins, as shown by NMR [19]. What other possibilities exist for the chemical structure of _Ochroconis_ melanins? Some authors reported fungal melanins based on DHN (e.g., _Aspergillus fumigatus_, _Neurospora crassa_) [105; 106]. A structure based on the polymerization of 1,8-dihydroxymorphthalene should yield upon pyrolysis major products derived from the naphthalene core. No conclusive pyrolysis data were found on this type of melanin. However, [PERSON] et al. [107] carried out a structural analysis of the melanin of _Mycosphaerella fijiensis_ using linear-MALDI-TOF mass spectrometry revealing a molecular mass of around 8000 Da,corresponding to 50 units of 1,8-DHN, and its spectrum showed a well-defined spacing of 161.8 Da between peaks, associated with 1,8-DHN units. To the light of the pyrolysis data of the acid hydrolyzed melanin, a core of 1,8-DHN units for _Octhroonis_ melanins should be disregarded, because the intensity of peaks related with naphthalene derivatives are similar to those obtained in the pyrolysis of proteins. It can be speculated that the structural units of _Ochroonis_ melanins would be based on the polymerization of phenols and quinones, as suggests the identification of a few pyrolysis products (phenol, methylphenols, ethylphenol, methoxyphenol, benzaldehyde, benzophenone, fluorenone, phenanthrenol) found in the acid hydrolyzed melanin (Figure 3B and Table 3). This could be indicative of a possible pyomelanin formation. In this melanin type, the process starts when homogentisic acid is oxidized to benzoquinone acetate and polymerizes to form pyomelanin. This is similar to the pathway described for the fungal melanins of _Aspergillus sidowii_[17] and _Hendersoluna toruloidea_[36], in which 2,5-dihydroxybenzoic acid (gentistic acid), among other phenols, is oxidized by a phenoloxidase to benzoquinone with the formation of melanin.