## 4 Discussion
### The Chemical Structure of Ochroconis Melanins  
If we assume that the fungal melanins are a mixture of compounds and polymers extracted from the fungal cells and that most of the compounds released by thermal desorption or pyrolysis are not part of the real melanin moiety, what is the chemical structure of this fungal melanin? The decades from the 1960s to the 1980s of the last century were characterized by the efforts to investigate the reaction between phenols and amino acids and proteins, considering that this was the formation process of soil humic substances and fungal melanins [11; 17; 18; 94; 95; 96]. Briefly, phenols are readily oxidized to quinones. Phenols and quinones interact with proteins reversibly by hydrogen bonding, or irreversibly by covalent bonding. Phenolic compounds and amines are linked together by oxidative coupling. Products of oxidative coupling reactions result from C-C and C-O coupling of phenols and from C-N and N-N coupling of amines [95; 96] although C-S from thiols were also considered [17]. This originated the formation of black polymers from which part of the amino acids can be recovered after acid hydrolysis. The percentage of residual nitrogen after acid hydrolysis in some fungal melanins ranges between 5 and 56% and that of sulfur from 41 to 75% [12; 17; 18]. This denotes that the coupling C-N and C-S is resistant to acid hydrolysis. According to [PERSON] et al. [94] the reaction products of phenols with amino acids were stable against acid hydrolysis and it was found that all amino acids, except the N-terminal bound to oxidized phenols, could be hydrolyzed. It is highly probable that this type of units, formed by the reaction of phenols and quinones with proteins are present in the melanin. The hypothesis of a phenolic-protein polymer for some fungal melanins is supported by the data obtained from the pyrolysis of _E. echinulatum_ melanin (Supplementary Figure S1), and their \({}^{13}\)C and \({}^{15}\)N NMR spectra [19]. In addition, the pyrolysis-mass spectra of this melanin and a synthetic phenolic-protein polymer were almost identical [97]. Although the _Ochroconis_ melanins showed a relatively high aromatic character (16 % of the total \({}^{13}\)C intensity) and carboxyl and carbonyl functional groups (16%), _E. echinulatum_ reached the highest aromatic character (25%) and carboxyl and carbonyl functional groups (18%) of all the melanins investigated [98]. Therefore, the _E. echinultatum_ melanin is representative of a phenolic-protein polymer. In fact, the major pyrolysis products were phenol and benzenediol derivatives including a few anthraquinones previously identified in the culture medium [18] and linked to the polymer, as well as some pyrolysis products from proteins, polysaccharides, and lipids. Another model melanin was extracted from the fungus _Cryptococcus neoformans_. This DOPA-melanin, upon pyrolysis, should yield indole-derivatives and phenols. Pyrolysis showed an abundance of fatty acid derivatives and sterols, natural component of the cells, co-extracted with the melanin (Supplementary Figure S2). Fatty acids were represented by unmethylated fatty acids and their methyl and propyl esters, as well as fatty acids amides. The covalent binding of _C. neoformans_ melanin to aliphatic moieties (polysaccharides, chitin and chitosan, and lipids) has been shown by NMR analyses [99]. In addition, melanin-associated proteins were identified from _C. neoformans_ melanin granules [99]. The presence of alkanes, alkenes and alkylbenzenes suggest a breakdown or cyclization from fatty acids upon pyrolysis, as reported by [PERSON] [24; 25]. Phenols and nitrogen derivatives were also relatively abundant, as well as polycyclic aromatic hydrocarbons, which might be evidence of a DOPA core. Previous studies on solid-state cross polarization magic-angle spinning \({}^{13}\)C and \({}^{15}\)N NMR and SERS spectroscopy of the melanins extracted from the mycelia of _O. lascausensis_, _O. anomala_, and _O. tshawvtschae_ revealed that they have similar spectra [19].