Toxic Dopamine Metabolite DOPAL Forms an Unexpected Dicatechol Pyrrole Adduct with Lysines of α‐Synuclein

Abstract

Parkinson’s disease has long been known to involve the loss of dopaminergic neurons in the substantia nigra and the coincidental appearance of Lewy bodies containing oligomerized forms of α-synuclein. The ‘catecholaldehyde hypothesis’ posits a causal link between these two central pathologies mediated by 3,4-dihydroxyphenylacetaldehyde (DOPAL), the most toxic dopamine metabolite. Here we determine the structure of the dominant product in reactions between DOPAL and α-synuclein, a dicatechol pyrrole lysine adduct. This novel modification results from the addition of two DOPAL molecules to the Lys sidechain amine through their aldehyde moieties and the formation of a new carbon-carbon bond between their alkyl chains to generate a pyrrole ring. The product is detectable at low concentrations of DOPAL and its discovery should provide a valuable chemical basis for future studies of DOPAL-induced crosslinking of α-synuclein.

Isoindole Linkages Provide a Pathway for DOPAL-Mediated Cross-Linking of α-Synuclein

“…DCPL was prepared and purified as described previously. 37 After elution from the reverse phase column, each DCPL preparation was divided evenly into several aliquots, which were flash frozen in liquid nitrogen and lyophilized to dryness in the dark. A single aliquot was resuspended in 600 μL of the PBS buffer with ascorbate described above, and the amount of DCPL was calculated via the integrated intensity of its acetyl methyl signal in a 1D 1 H NMR spectrum, again with N α -acetylated lysine used as a reference.…”

“…40 DOPAL was purchased from VDM Biochemicals and stocks were prepared in methanol and quantified as described previously. 37 …”

Pharmacokinetics, metabolism and safety of deuterated L‐DOPA (SD‐1077)/carbidopa compared to L‐DOPA/carbidopa following single oral dose administration in healthy subjects

“…To confirm central effects of the drug in humans, the efficacy and safety of SD‐1077 needs to be investigated in patients. Additionally, a lower exposure to the transient toxic metabolites DOPAL 25 and a lower catecholamine load in the brain could be expected, both with the potential to reduce neurotoxic effects 26. The peripheral effects of higher DA plasma peak levels after SD‐1077 are deemed to be acceptable in Parkinson's disease patients considering reported exposure to DA in this population (e.g.…”

Covalent modification of biological targets with natural products through Paal–Knorr pyrrole formation

“…In a recent study, a team led by Levine and Bax reacted DOPAL with Ac-α-synuclein and characterized the reaction products with LC-MS. 47 To the investigators’ surprise, instead of the expected increase in mass of +134 Da due to the Schiff base formation between DOPAL and a lysine residue, 48 the modified protein showed a clear and homogeneous molecular ion peak corresponding to an increase in mass of +266. Because α-synuclein contains 15 lysine residues, they continued their study of this unexpected covalent modification in a simplified system with N -Ac-Lys.…”

“…Reaction monitoring using NMR showed that the concentration of DCPL increased linearly over the first 2.5 h and reached a maximum value after 5 h. This was followed by a plateau in DCPL formation, presumably resulting from its further reactions, such as oxidative degradation and further reactions with DOPAL, among other possibilities. 47 …”