The Polymerization of Homogentisic Acid In Vitro as a Model for Pyomelanin Formation

Abstract

Melanins are a class of biopolymers that are widespread in nature, with diverse origins, compositions, and functions, and their chemical and optoelectronic properties render them potentially useful for application in materials science for various biotechnological applications. For patients with alkaptonuria, the accumulation of homogentisic acid (HGA) in their bodies is associated with the concomitant deposition of pyomelanin, which is a pigment that contains significant amounts of polymerized HGA (polyHGA) in the bodily tissues of the patients. The polymerization of HGA under various different conditions in vitro is investigated using a selection of different analytical chemistry techniques to understand if there may be a correlation between the conditions and pigment deposition in vivo, and their potential for application as green/sustainable and components of electronic devices.

Phenolic Polymers as Model Melanins

“…Indeed, in a previous study we investigated the polymerization of HGA under various conditions in vitro, specifically in the absence (control) or presence of enzymes (a laccase, peroxidase, or tyrosinase) at either pH 5 or 7.4 with a view to understand the potential role of enzymes and pH on the formation of ochronotic pigment: we observed interesting trends in the yields of polyHGAs produced with subtle differences in their properties; notably, conductive tip atomic force microscopy data for polyHGAs showed that those generated in the presence of LACC at pH 7.4, or HRP at pH 7.4 or 5.0, respectively, displayed measurable conductivity owing to the formation of polymers connected via CC bonds. [ 47 ]…”

“…Various methods of polymerization of aromatics (e.g., phenols) exist, including enzymatic, [42][43][44][45][46][47] electrochemical, [48][49][50][51][52][53][54][55][56][57] oxidative, [58,59] etc. and the polymerization of phenols is the subject of an excellent review from Kobayashi and co-workers, [58,59] which examined the propensity for C-C or C-O-C bond formation which would be expected to play a role in the conductivity of the resulting polymers (polymers connected via C-C bonds akin to graphene derivatives [60] are expected to have higher electrical conductivity than polymers connected via C-O-C bonds).…”

“…[58] Indeed, in a previous study we investigated the polymerization of HGA under various conditions in vitro, specifically in the absence (control) or presence of enzymes (a laccase, peroxidase, or tyrosinase) at either pH 5 or 7.4 with a view to understand the potential role of enzymes and pH on the formation of ochronotic pigment: we observed interesting trends in the yields of poly-HGAs produced with subtle differences in their properties; notably, conductive tip atomic force microscopy data for polyHGAs showed that those generated in the presence of LACC at pH 7.4, or HRP at pH 7.4 or 5.0, respectively, displayed measurable conductivity owing to the formation of polymers connected via C-C bonds. [47] In this study, a green approach was applied using a weakly basic aqueous solution of the commercially available monomers CAT, DOPA, and HGA (Figure 1) to polymerize the monomers via autooxidation with a view to understand the potential role of subtle differences in monomer structure and the properties of the resulting conjugated polymers, polyCAT, poly-DOPA, and polyHGA, respectively (Schemes S1-S3, Supporting Information).…”

“…[6,70] SEM images of lyophilized solutions of the polymers deposited on SEM stubs shows the particles/precipitates formed were irregularly shaped with sizes between tens to hundreds of micrometers characteristic of melanins (Figure 4); while the HS particles are generally smaller than the LS precipitates, there is no meaningful correlation between particle/precipitate shapes/sizes as the polymers are produced under unconstrained conditions. [47] By comparison, the shapes/sizes of melanin particles/precipitates in vivo would be constrained by the intracellular/extracellular environment in which they are produced. EDX data suggests that all samples are mainly composed of C and O for all polymers with a trace of N present in the polyDOPA samples (similar to the XPS data), with additional traces of Ca, K, and Na from the buffer, Au (sputter coating) and Si (substrate) (Figure 4 and Figure S11, Supporting Information).…”

“…This work follows on from our earlier work that was the first detailed chemical analysis of polyHGA. [ 47 ] Herein polymerization of a variety of phenolic monomers (CAT, DOPA, and HGA), yielded conjugated polymers polyCAT‐HS, polyCAT‐LS, polyDOPA‐HS, polyDOPA‐LS, or polyHGA‐HS, respectively. A variety of analytical techniques (UV–Vis, NMR, XRD, DSC, XPS, zeta potential measurements, TEM, SEM, EDX, FTIR, EPR, CV, and conductivity measurements) were employed to examine the polymers produced.…”