Electron diffraction and solid-state NMR reveal the structure and exciton coupling in a eumelanin precursor

Abstract

Eumelanin, a versatile biomaterial found throughout the animal kingdom, performs essential functions like photoprotection and radical scavenging. The diverse properties of eumelanin are attributed to its elusive and heterogenous structure with DHI (5,6-dihydroxyindole) and DHICA (5,6-dihydroxyindole-2-carboxylic acid) precursors as the main constituents. Despite DHICA being recognized as the key eumelanin precursor, its crystal structure and functional role in the assembled state remain unknown. Herein, we employ a synthesis-driven, bottom-up approach to elucidate the structure and assembly-specifics of DHICA, a critical building block of eumelanin. We introduce an interdisciplinary methodology to analyse the nanocrystalline assembly of DHICA, employing three-dimensional electron diffraction (3D ED), solid-state NMR and density functional theory (DFT), while correlating the structural aspects with the electronic spectroscopic features. The results underscore charge-transfer exciton delocalization as the predominant energy transfer mechanism within the π-π stacked and hydrogen-bonded crystal network of DHICA. Additionally, extending the investigation to the ¹³C-labelled DHICA-based polymer improves our understanding of the chemical heterogeneity across the eumelanin pigment, providing crucial insights into the structure of eumelanin.

Scheme 1. Schematic diagram representing the spontaneous polymerization of DHICA, the monomeric unit of eumelanin into the black polymeric DHICA-melanin.

Fig. 1. UV-visible-NIR diffuse-reflectance transformed absorption spectra of DHICA and DHICA-melanin in the solid state.

Fig. 2. Selected TEM images and electron diffraction patterns obtained for DHICA.

Fig. 3. (A) The 2D ¹³C{¹H} cross-polarization heteronuclear correlation (CP HETCOR) NMR spectrum of ¹³C labelled DHICA obtained at a spinning rate of 60 kHz and a magnetic field of B_o = 14.1 T, utilizing natural isotopic abundance with a contact time of 5 ms; (B) the 2D CP INADEQUATE spectrum of ¹³C labelled DHICA obtained at 60 kHz spinning speed; (C) the ¹H–¹H DQ-SQ spectrum of the sample collected at 60 kHz with a recoupling time corresponding to two rotor cycles; (D) the ¹H{¹⁴N} DHMQC spectrum of the DHICA monomer acquired at a spinning speed of 50 kHz; (E) the one-dimensional slice extracted from the ¹⁴N dimension of the ¹H{¹⁴N} DHMQC spectrum.

Fig. 4. (A) The crystal packing of DHICA showing the near-perpendicular orientation of the π–π stacks; (B) π–π stacked dimers connected to the adjacent stack through hydrogen bonding.

Fig. 5. (A) The 2D CP INADEQUATE spectrum of ¹³C selectively labelled DHICA-melanin showing the correlation between the adjacent carbon atoms. (B) The 2D ¹³C{¹H} CP HETCOR NMR spectrum of DHICA-melanin at a spinning speed of 60 kHz and a magnetic field strength of B_o = 14.1 T; (C) the ¹H{¹⁴N} DHMQC spectrum of DHICA-melanin collected at 50 kHz spinning speed.