# Ochronotic Deposition in Alkaptonuria: Semiquinone-Mediated Oxidative Coupling and Metabolic Drivers of Homogentisic Acid Accumulation

## TL;DR

This study explores how a rare metabolic disorder causes pigment buildup in the body and identifies molecular structures that could help develop new treatments.

## Contribution

The study reveals the oxidative coupling mechanism and structural motifs of pigment formation in alkaptonuria, offering targets for future therapies.

## Key findings

- Semiquinone radical intermediates are involved in homogentisic acid polymerization under physiological conditions.
- Phenolic ether and biphenyl linkages are key structural features of the pigment formed in alkaptonuria.
- Alkaline conditions accelerate pigment formation while preserving the aromatic scaffold of homogentisic acid.

## Abstract

Alkaptonuria (AKU) is a rare metabolic disorder caused by homogentisate 1,2-dioxygenase (HGD) deficiency, leading to homogentisic acid (HGA) accumulation and ochronotic pigment deposition, which drug therapy cannot reverse. The process of pigment formation and deposition is still unclear. This study offers molecular insights into the polymeric structure, with the goal of developing future adjuvant strategies that can inhibit or reverse pigment formation, thereby complementing drug therapy in AKU. HGA polymerisation was examined under physiological, acidic, and alkaline conditions using liquid and solid phase nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and polyacrylamide gel electrophoresis. At physiological pH, HGA polymerised slowly, while alkaline catalysis accelerated pigment formation while retaining the HGA aromatic scaffold. During the process, EPR detected a semiquinone radical intermediate, consistent with an oxidative coupling mechanism. Reactivity profiling showed the diphenol ring was essential for polymerisation, while –CH2COOH modifications did not impair reactivity. Pigments displayed a polydisperse molecular weight range (11–50 kDa) and a strong negative charge. Solid-state NMR has revealed the presence of phenolic ether and biphenyl linkages. Collectively, these identified structural motifs can serve as a foundation for future molecular targeting related to pigment formation.