Elucidating the Structural Framework of Synthetic and Fungal Melanins by Solid-State NMR

Abstract

Melanins are an important class of natural pigments associated with biological functions that include protection from sunlight, camouflage, free radical scavenging, and microbial virulence. However, structural investigation of these enigmatic pigments remains a challenging task because of their insolubility and heterogeneous architecture. High-resolution solid-state NMR spectroscopy offers a unique tool to probe melanin structure, and melanogenesis in Cryptococcus neoformans (CN) requiring exogenous obligatory catecholamine precursors such as L-dopa provides unparalleled opportunities to investigate melanin assembly in fungal cells. 1D solid-state 13C NMR and EPR demonstrated the structural differences among CN melanins produced by systematic variation of catecholamine precursors and in cell-free media. Broad aromatic resonances in both fungal and synthetic melanins indicated a heterogeneous disordered aromatic domain in these pigments, whereas differences among the aliphatics suggested incorporation of glucose-derived cellular materials in the fungal materials. A mechanistic scheme was proposed based on the Mason-Raper pathway, including potential intermediates for the biosynthesis of indole-based melanin polymers and the consequent precursor preferences observed in CN melanogenesis. 13C and 15N solid-state NMR, including 2D through-space and through-bond correlation spectra of pigments produced with exogenously 13C-and 15N-enriched L-dopa and/or [U-13C6]-glucose supplied to the growth media, revealed the molecular framework formed by the pigments and in CN melanization. Findings include: (1) the major aromatic moieties are located spatially within 5-7 A of the long-chain aliphatics; (2) glucose scrambles metabolically to form oxygenated- and long-chain aliphatic structures; (3) particular aromatic, oxygenated- and long-chain aliphatic moieties are attached covalently to carboxyl groups of the pigment; (4) nitrogenous components are found primarily in the aromatic domain of both synthetic and fungal pigments. The structural framework of melanins deduced in this investigation can guide the design of new antivirulence drugs and therapeutics against melanoma tumors.