Ultra-low Temperature Oxidation of 5,6-Dihydroxyindole: A Novel Approach to Study Synthetic Melanogenesis[dagger]

Photochemistry and Photobiology, May/Jun 2008 by Hatcher, Lanying Q, Simon, John D

ABSTRACT

The detailed structure of melanin remains elusive due to the complexity and insolubility of the pigment. Herein we describe a novel oxidation of 5,6-dihydroxyindole (DHI) as a means to characterize soluble intermediates formed prior to oligomerization. The approach entails the use of a biomimetic copper-peroxo oxidant, at ultra-low temperature (-787deg;C). DHI oxidized by [LCu^sup II^(O2)Cu^sup II^L]B(C^sub 6^F^sub 5^)^sub 4^(L = 2,6,10-trimethyl-2,6,10-triazaundecane) under argon produces the one electron oxidation product, semiquinone radical, which is spectroscopically observed at -78°C. MS analysis of the reaction mixture reveals the DHI dimer as well as other extensively oxidized DHI units.

INTRODUCTION

The structural characterization of synthetic or natural melanin pigment has challenged chemists for over several decades (1). Difficulty comes from the fact that the pigment is insoluble and cannot be cleanly separated from the proteins and lipids present in the melanosome (2-4). Previous approaches to determine the intact melanin molecular structure have been limited to solid state (SS) spectroscopic studies such as SS-NMR, IR and X-ray absorption spectroscopy (XAS) (5-7). These techniques provide functional group information, but do not provide detailed atomic connectivity. SS analysis of synthetic melanin and Sepia melanin by matrix-assisted laser desorption ionization MS yielded a more descriptive structural analysis, suggesting the presence of oligomers of oxidized 5,6-dihydroxyindole (DHI) units (8,9).

Melanin can be divided into the subgroups eumelanin and pheomelanin, giving rise to a brown/black and reddish color, respectively (1). The chemical difference between the two types comes from the differing monomeric starting materials whereby the latter incorporates cysteine molecules during melanogenesis.

In this study, we focus on the formation of eumelanin. The initial steps of melanogenesis for eumelanin are summarized by the Raper-Mason scheme (Fig. 1). While many of the initial steps are well characterized, much ambiguity remains about the reactions that transform the monomers DHI and 5,6-dihydroxyindole carboxylic acid (DHICA) to the final pigment.

Oligomers produced by the melanin precursors DHI and DHICA have been obtained in vitro by oxidation with the tyrosinase/O2 or peroxidase/H^sub 2^O^sub 2^ enzymatic systems (8,10-13). However, these oxidation techniques result in rapid formation of the insoluble melanin polymer because the reactions are performed at room temperature in an aqueous environment that contains excess O2. Water itself can act as a nucleophile to intermediate quinone-imines, thereby complicating the reaction mixture and accelerating oxidative oligomerization.

The initial oxidation steps are mediated by tyrosinase, an enzyme belonging to a family of proteins that has a type 3 copper active site whereby two copper centers activate dioxygen through formation of a Cu^sub 2^(µ-η^sup 2^:η^sup 2^-O^sub 2^) "side-on" structure (14,15). Side-on peroxo dicopper(II) centers in proteins and model complexes have been studied extensively with respect to structural and electronic properties, and oxidative reactivity (16,17). Inorganic model complexes of the tyrosinase active site show oxidative reactivity with various organic substrates (18) and therefore can serve as a model oxidant for DHI.

In this report, a novel, ultra-low temperature (78°C) approach to study melanogenesis is presented. A side-on peroxo dicopper(II) complex, similar in structure and function to the dioxygen-bound tyrosinase active site (Fig. 2), is used as the biomimetic oxidant in organic solvents under an inert argon atmosphere. The approach taken is to trap soluble intermediates created by this oxidation. This method simplifies the reaction by removing water and controlling the amount of O2. The ultra-low temperature slows the reaction allowing intermediates to be spectroscopically and structurally characterized.

MATERIALS AND METHODS

Materials. 5,6-Diacetoxyindole and 2,6,10-trimethyl-2,6,10-triazaundecane were purchased from TCI America. CH^sub 2^Cl^sub 2^, CH^sub 3^CN, Et^sub 2^O and Cu(MeCN)^sub 4^]PF^sub 6^ were purchased from Sigma-Aldrich. Optima® LCMS solvents were purchased from Fisher Scientific. LiB(C^sub 6^F^sub 5^)^sub 4^.Et^sub 2^O was purchased from Boulder Scientific Company. High purity O2, N^sub 2^ and Ar were purchased from National Welders.

Synthesis of DHI. 5,6-Diacetoxyindole (0.389 g) was dissolved in minimal MeOH (ca. 12 mL). Argon was bubbled through the solution for 20 min with an air-free addition funnel. The deoxygenated solution was added to solid K^sub 2^CO^sub 3^ under argon. The reaction mixture was stirred for 35 min until deemed complete by thin-layer chromatography. The solvent was evaporated under high vacuum and the remaining solid was dissolved in deoxygenated Et^sub 2^O. The product was stirred in Et^sub 2^O overnight. The solid K^sub 2^CO^sub 3^ was filtered under argon. After rotary evaporation, DHI was collected in 90% yield. ^sup 1^H NMR (DMSO): δ 6.118 (H3, d (J = 2) 6.752 (H4, s), 6.823 (H7, s), 6.980 (H2 d (J = 2.4)). ^sup 13^C NMR (CDC13): δ 97.45 (C4), 100.57 (C3), 104.79 (C7), 120.64 (C9), 122.93 (C2), 130.45 (C8), 140.82 (C5), 142.57 (C6). The NMR and mass spectral analysis indicated a purity of >99%.

Synthesis of [Cu^sup I^(MeCN)]B(C^sub 6^F^sub 5^]^sub 4^. 0.544g (1.46 mmol) [Cu^sup I^ (MeCN)]PF^sub 6^ was dissolved in 15 mL MeCN and added to an air-free addition funnel (Chemglass). The solution was deoxygenated by bubbling argon through it for 10 min. The solution was added anaerobically to a Shlenk flask containing 1.11 g (1.46 mmol) LiB(C^sub 6^F^sub 5^)^sub 4^.Et^sub 2^O and allowed to stir for 40 min. Then, deoxygenated H2O was added to precipitate the product. The white solid [Cu^sup I^MeCN)]B(C^sub 6^F^sub 5^)^sub 4^ was filtered and dried under vacuum overnight. 1.041 g (79% yield) was collected.