The observations reported here are a continuation and expansion of observations described earlier: the typical conditions to synthesize melanin-like materials from catecholic precursors (air-oxidation in an alkaline environment) leads to the generation of a light-colored substance in addition to the typical dark substances. This light-colored substance appears to be associated with the dark-colored materials through non-covalent interactions. We employed a novel co-precipitation process that allows for a simple separation of this light-colored substance from the dark-colored materials. We studied some of the physic-chemical properties (color, fluorescence, FT-IR absorbance) of some of the fractions we obtained and discussed these properties in the context of what has been discussed for eumelanin, pheomelanin or neuromelanin. Overall, the current observations strengthen our hypothesis that synthetic melanins may be built from at least two types of substances: 1) a soluble, yellow-to-orange colored component, possibly rich in unoxidized precursors and 2) an insoluble, dark component. Our observations do suggest that when studying the chemistry or physiology of melanins one should look beyond the dark colors typically displayed by melanin-like materials.

<p>We investigated the synthesis of melanin-like materials from DOPA, dopamine, norepinephrine and epinephrine in the presence of L-cysteine. We observed that L-cysteine delayed the formation of pigment from these catecholamines and that the presence of L-cysteine yielded darker-colored reaction mixtures. No reddish pigment was observed that would indicate the synthesis of pheomelanin-like material. The reactions were performed in the presence of Na₂CO₃ and through the addition of CaCl₂ at the end of the reaction; the black, eumelanin-like material was co-precipitated with CaCO₃. The remaining supernatant solutions were observed to be light-yellow to rusty-orange in color depending on the catecholamine used in the reaction. Size exclusion chromatography (SEC) analyses indicated that the removal of the black pigment left behind an oligomeric material that exhibited a strong absorbance band around 280nm. Our experimental and analytical observations prompt us to raise a number of points of discussion or hypotheses. 1) The presence of L-cysteine during the air-mediated oxidation of catecholamines leads to darker-colored pigments; not reddish or lighter-colored pigments that would visually resemble pheomelanin-like pigments, 2) SEC analyses suggested that the black pigment generated during the air-mediated oxidation of catecholamines is not necessarily the main reaction product, 3) The pre-formed, dark-colored pigments obtained through the air-mediated oxidative melanogenesis process can readily be deposited on insoluble mineral surfaces using an <i>in situ</i> co-precipitation procedure, 4) The air-mediated oxidation of catecholamines leads to a binary product that contains an insoluble, melanin-like substance and a soluble, oligo- or polymeric substance containing unoxidized precursor units, 5) The melanogenesis process leads to a binary product involving a non-covalently bonded combination of dark-colored pigment and a lighter-colored or colorless substance; the latter being understudied or ignored in the <i>in vitro</i> or <i>in vivo</i> studies of the melanogenesis process, 6) The kinetics of the melanogenesis process may determine the balance between insoluble and soluble components of the binary product generated; the slower the reaction the more dark-colored, insoluble pigment generated, 7) One should consider the possibility of intermolecularly, N-to-C, bonded units of catecholamines when evaluating the structure of melanins, polydopamines, etc. and 8) There is a need for a systematic study of the effect of amino acids (beyond just L-cysteine) and amines in general on the melanogenesis process.</p>

<p>We investigated the synthesis of melanin-like materials from DOPA, dopamine, norepinephrine and epinephrine in the presence of L-cysteine. We observed that L-cysteine delayed the formation of pigment from these catecholamines and that the presence of L-cysteine yielded darker-colored reaction mixtures. No reddish pigment was observed that would indicate the synthesis of pheomelanin-like material. The reactions were performed in the presence of Na₂CO₃ and through the addition of CaCl₂ at the end of the reaction; the black, eumelanin-like material was co-precipitated with CaCO₃. The remaining supernatant solutions were observed to be light-yellow to rusty-orange in color depending on the catecholamine used in the reaction. Size exclusion chromatography (SEC) analyses indicated that the removal of the black pigment left behind an oligomeric material that exhibited a strong absorbance band around 280nm. Our experimental and analytical observations prompt us to raise a number of points of discussion or hypotheses. 1) The presence of L-cysteine during the air-mediated oxidation of catecholamines leads to darker-colored pigments; not reddish or lighter-colored pigments that would visually resemble pheomelanin-like pigments, 2) SEC analyses suggested that the black pigment generated during the air-mediated oxidation of catecholamines is not necessarily the main reaction product, 3) The pre-formed, dark-colored pigments obtained through the air-mediated oxidative melanogenesis process can readily be deposited on insoluble mineral surfaces using an <i>in situ</i> co-precipitation procedure, 4) The air-mediated oxidation of catecholamines leads to a binary product that contains an insoluble, melanin-like substance and a soluble, oligo- or polymeric substance containing unoxidized precursor units, 5) The melanogenesis process leads to a binary product involving a non-covalently bonded combination of dark-colored pigment and a lighter-colored or colorless substance; the latter being understudied or ignored in the <i>in vitro</i> or <i>in vivo</i> studies of the melanogenesis process, 6) The kinetics of the melanogenesis process may determine the balance between insoluble and soluble components of the binary product generated; the slower the reaction the more dark-colored, insoluble pigment generated, 7) One should consider the possibility of intermolecularly, N-to-C, bonded units of catecholamines when evaluating the structure of melanins, polydopamines, etc. and 8) There is a need for a systematic study of the effect of amino acids (beyond just L-cysteine) and amines in general on the melanogenesis process.</p>

<p>This report is a continuation of an earlier report on the effect of L-cysteine on the synthesis of melanin-like materials from catecholamines. We confirmed the pattern of results indicating that in the presence of L-cysteine the appearance of dark colors during the melanogenesis reactions is delayed, but that ultimately the presence of L-cysteine yields reaction mixtures with darker colors. The results of the current report indicate that the effect of L-cysteine is very reproducible in the cases of DOPA or dopamine, but much more variable in the cases of norepinephrine or epinephrine. The delays in color formation in the cases of norepinephrine and epinephrine were much longer compared to the cases of DOPA and dopamine. In addition to L-cysteine, we tested the effect of a few amino acids on the melanogenesis reactions. In general, we observed that the presence of the amino acids slowed, but did not delay the color formation. Given enough reaction time, the presence of amino acids did result in reaction mixtures exhibiting darker colors. In addition, some of the results obtained indicated a difference between a-amino acids and their corresponding primary amines, e.g., tyrosine vs. tyramine.</p>

This report is a combination of new and former experimental results collected over the past three years. It details our observations and spectroscopic studies of the air-mediated oxidation of a wide variety of catecholic precursors into melanin-like materials. Depending on the precursor involved and the reaction conditions employed, dark-brown to black or yellow to rusty-orange colored reaction mixtures, akin to eumelanin- or pheomelanin-like materials, can be obtained. This report expands and confirms an earlier report that behind the dark colors of eumelanin-like materials, distinct, light-colored, pheomelanin-like materials are hidden. In addition, this report expands and confirms earlier observations regarding the effect of the presence of amino acids on the color of the reaction mixture; particularly the effect of the presence of cysteine. The presence of cysteine delays the onset of color formation and leads to dark-colored reaction mixtures; turning pheomelanin-like reaction mixtures into eumelanin-like mixtures. Visible and infrared spectroscopic analyses were made to evaluate the nature, eumelanin- vs. pheomelanin-like, of the materials generated under the varying reaction conditions. Reactions involving epinephrine as the precursor presented unique results which are detailed in this report.