Pyridoxamine as a potential scavenger of neurotoxic dopamine quinone: Inhibition of dopamine-induced α-synuclein oligomerization

Pyridoxamine as a potential scavenger of neurotoxic dopamine quinone: Inhibition of dopamine-induced α-synuclein oligomerization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Pyridoxamine as a potential scavenger of neurotoxic dopamine quinone: Inhibition of dopamine-induced α-synuclein oligomerization Seon Hwa Lee, Naoya Matsumoto, Akane Terada, Yusuke Hatakawa, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6478169/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Parkinson’s disease (PD) is a neurodegenerative disease characterized by loss of dopaminergic neurons causing reduced levels of dopamine (DA), and the presence of Lewy bodies, whose main component is fibrillar α-synuclein (α-Syn). DA is easily oxidized to DA o -quinone (DAQ), which is a key mechanism for neuronal cell death and α-Syn accumulation. Therefore, a DAQ-quenching molecule should prevent DA-induced pathogenicity in PD. Pyridoxamine (PM) is a promising drug candidate for various chronic diseases, including diabetes, because it scavenges reactive carbonyl species. In this study, we found that PM traps DAQ through stable adduct formation. The initial reaction of PM occurred at the DAQ carbonyl carbon to yield pyridoxal (PL) after hydrolysis. DA then reacted with the PL aldehyde, followed by intramolecular cyclization to produce a PL–DA adduct. The adduct structure was shown by LC-MS and NMR analyses to be (1-[3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol). The PL–DA adduct was also detected under intracellular-like conditions. DA caused α-Syn oligomerization via oxidation of methionine residues, which was inhibited by PM in a dose-dependent manner. Therefore, PM could prevent DA-induced adverse effects in the brains of PD patients by forming the PL–DA adduct, suggesting a possible therapeutic use of PM for scavenging DAQ. Biological sciences/Biochemistry Biological sciences/Chemical biology Biological sciences/Neuroscience Health sciences/Diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Parkinson’s disease (PD) is an age-related neurodegenerative disease that is the fastest growing neurological disorder in terms of disability and death 1 , 2 . PD is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting in reduced dopamine (DA) levels 1 . Additionally, the neurons contain cytosolic filamentous inclusions known as Lewy bodies, whose main component is fibrillar α-synuclein (α-Syn) 3 . Although the precise molecular mechanisms underlying PD are not fully understood, intensive studies have uncovered several pathways and mechanisms involved in pathophysiology of PD, such as oxidative stress 4 , mitochondrial dysfunction 5 , calcium homeostasis 6 , and α-Syn aggregation 7 . Despite ongoing research, there is currently no cure for PD. l -DOPA, a precursor of DA, is the first choice of treatment for PD because it replenishes DA. However, long-term use of l -DOPA causes adverse effects, such as motor fluctuations, dyskinesia, and psychiatric symptoms 8 , which are attributed partly to the formation of dopamine o -quinone (DAQ). DA is the most abundant neurotransmitter and is important in multiple physiological functions, including motor control, emotional modulation, and reward mechanisms 9 . However, DA can induce oxidative stress through various redox reactions. One-electron oxidation of DA to the semiquinone radical (DASQ) can be mediated by free heme, peroxidases, or cyclooxygenase via H 2 O 2 activation. DASQ couples with other radicals, scavenges cellular thiols, such as glutathione (GSH), undergoes further oxidation by O 2 to form DAQ and O 2 − , or disproportionates to DA and DAQ 10 , 11 . Reactive oxygen species (ROS) derived from DA oxidation can damage lipids, proteins, and DNA in the cells 12 . DA can also be oxidized to DAQ in a two-electron process promoted by transition metals or tyrosinase 13 – 15 . The electron-deficient DAQ reacts with cellular thiols and nucleophilic amino acid residues, leading to further cytotoxicity. Upon reaction with GSH, DAQ forms 5- S -glutathionyl- and 5- S -cysteinyl-DA 16 . 5- S -Cysteinyl-DA is neurotoxic 17 . DAQ binds covalently to Cys residues in the DA transporter 18 , superoxide dismutase 19 , and glucocerebrosidase 20 , decreasing enzyme activity and causing blocked DA uptake, mitochondrial dysfunction, and lysosomal dysfunction, respectively. The interaction between DA and α-Syn also causes selective neuronal cell death and the accumulation of misfolded α-Syn 21 , 22 . Although the exact mechanism is not fully defined, DA oxidation is a key mechanism. Therefore, a DAQ-quenching molecule should prevent DA-induced pathogenicity in PD, such as the death of dopaminergic neurons and aggregation of α-Syn (Fig. 1 ). Pyridoxamine (PM) is a vitamin B6 vitamer and functions as a coenzyme in enzymatic transaminations in vivo 23 . PM is also a promising pharmacological agent for the treatment of diabetic complications and other chronic conditions 23 . This is based on its multiple inhibitory effects. For example, inhibition of advanced glycation end product (AGE) formation by chelation of metal ions with the phenol and aminomethyl groups, inhibition of advanced lipoxidation end product (ALE) formation by scavenging of reactive carbonyl species (RCS), and trapping of ROS with phenol group 24 , 25 . PM is a potent scavenger of 1,2-, 1,3-, and 1,4-dicarbonyl compounds, which are RCSs. PM forms a dimer with methylglyoxal (MGO; 1,2-dicarbonyl), which blocked production of the MGO-Lys dimer and lowered the levels of MGO in red blood cells/plasma of diabetic rats 26 . PM reacts readily with glyoxal (1,2-dicarbonyl) to form a five-ring compound with a central piperazine ring. PM inhibited formation of AGE/ALE N ε -carboxymethyl Lys during incubation of bovine serum albumin with glyoxal 27 . PM traps malondialdehyde (1,3-dicarbonyl), a DNA-reactive aldehyde derived from lipid peroxidation, thereby inhibiting lipofuscin-like fluorescence induced by malondialdehyde in the reaction with bovine serum albumin 28 . PM forms a pyrrole and a lactam adduct with 4-oxopentanal and 15-E 2 -isoketal (both 1,4-dicarbonyls), respectively 29 , 30 . Recent studies 31 , 32 have demonstrated that PM inhibits lipid hydroperoxide-derived damage to proteins by trapping 4-oxo-2( E )-nonenal (ONE; 1,4-dicarbonyl), the most abundant and reactive lipid-derived aldehyde 33 , 34 . PM also attenuated ONE-derived insulin resistance by scavenging ONE 35 . PM–ONE adducts were then detected in cell culture and increased in a PM dose-dependent manner, which suggests that PM–ONE adducts could function as biomarkers of oxidative stress. Therefore, PM could inhibit DA-induced neurotoxicity by scavenging DAQ and its adduct could reflect the extent of oxidative stress and DA oxidation (Fig. 1 ). In the present study, we characterized the adduct generated from the reaction between PM and DA using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) analyses. The adduct formation was investigated in the presence of the endogenous enzyme and/or thiol to mimic the intracellular conditions. Finally, we confirmed the inhibition by PM of DA-induced oxidation/oligomerization of α-Syn. Results and Discussion Analysis of the reaction between PM and DA. LC/ESI-MS analysis after 48 h incubation at 37°C revealed the presence of two major products and residual DA ([M + H] + , m/z 154.1; retention time [ t R ], 6.8 min) and PM ([M + H] + , m/z 169.1; t R , 7.5 min) (Fig. 2 A). The MS spectrum of the most polar product eluting at 5.3 min showed [M + H] + at m/z 168.1, corresponding to a loss of 1 Da from PM. This product was identified as pyridoxal (PL), a transamination product of PM, because the LC/ESI-MS, MS/MS, and UV characteristics were identical to those for an authentic PL standard (Fig. S1 ). The product that eluted at 14.6 min (PL–DA adduct) had [M + H] + at m/z 303.2, corresponding to a 1:1 reaction of PL with DA ([M + H] + , m/z 321.3) followed by the loss of water (− 18 Da). Time course experiments were performed to clarify the formation of the PL–DA adduct further. The reaction between PM and DA in Chelex-treated phosphate buffer (pH 7.4) was monitored by LC/ESI-MS for 3 days (Fig. 2 B). DA decreased gradually and was not detectable after 72 h, whereas PL increased concomitantly. The PL–DA adduct formed after incubation for 12 h and increased to its maximum level at 72 h. The PM level did not change much throughout the reaction. DA undergoes autoxidation to form DAQ at physiological pH. DAQ reacts with nucleophilic amino acids, such as Cys, His, and Lys, yielding Michael addition products at the quinone ring 36 , 37 . The preferential positions of Cys and His addition are C-5 and C-6, respectively 36 . Therefore, it was expected that the PM–DA adduct would be produced by the nucleophilic addition of PM to the DAQ quinone core. However, the only adduct that formed was the PL–DA adduct after PL was generated. This result suggests that the initial reaction of the PM primary amino group occurs at the DAQ carbonyl carbon and yields a Schiff base (ketimine). The intermediate ketimine undergoes tautomerization to form aldimine, which is subsequently hydrolyzed to yield PL (Fig. 3 A). DA then reacts with the PL aldehyde and forms a Schiff base intermediate. The following intramolecular cyclization produces the PL–DA adduct as a 1,2,3,4-tetrahydroisoquinoline derivative (Fig. 3 B) through a Pictet–Spengler reaction 38 . The proposed mechanism and structure were confirmed by LC-MS and NMR analyses. Analysis of the reaction between PM and DA derivatives. To confirm the proposed mechanism (Fig. 3 ), PM was reacted with two DA derivatives, 3,4-dimethoxyphenethylamine (DPA) and isoproterenol (IPT). DPA is an analog of DA in which the hydroxy groups have been replaced with methoxy groups, which prevents autoxidation to the o -quinone. LC/ESI-MS analysis of the reaction between PM and DPA at 37°C for 48 h revealed the presence of DPA ([M + H] + , m/z 182.1; t R , 29.8 min) and PM ( m/z 169.1; t R , 7.4 min) (Fig. 4 A). No other products were detected. This result suggests that the o -quinone is necessary to produce PL from PM by transamination (Fig. 4 B). IPT is the isopropyl amine epinephrine analog, which can undergo autoxidation but has a bulky isopropyl group substituent on the amine. PL ( m/z 168.1; t R , 5.7 min) was detected as the only product of the reaction between PM and IPT together with residual IPT ( m/z 212.2; t R , 13.0 min) and PM (Fig. 4 C). PM was converted to PL by the reaction with IPTQ but PL failed to react with IPT to produce the Schiff base intermediate due to the steric hinderance of the isopropyl amine moiety of IPT (Fig. 4 D). These results support the proposed mechanism that involves DAQ-derived transamination of PM to PL, which subsequently reacts with the primary amino group of DA. Analysis of the reaction between PL and DA. LC/ESI-MS analysis after 48 h incubation at 37°C revealed the presence of the PL–DA adduct ([M + H] + , m/z 303.2; t R , 15.3 min) and residual DA ( m/z 154.1; t R , 7.3 min) and PL ( m/z 168.1; t R , 5.6 min) (Fig. 5 A). MS/MS analysis of the PL–DA adduct at m/z 303.2 showed the formation of product ions at m/z 285.2 (− 18 Da, − H 2 O), m/z 164.3 (− 139 Da, − C 7 H 8 NO 2 − H), m/z 152.1 (− 151 Da, − C 8 H 9 NO 2 ) and m/z 140.2 (− 163 Da, − C 9 H 10 NO 2 + H) (Fig. 5 B), which are identical to those of the PL–DA adduct formed from the reaction between PM and DA. In time course experiments for 2 days (Fig. 5 C), DA decreased gradually and was not detectable after 48 h. The PL–DA adduct increased concomitantly to its maximum level at 18 h. The PL level did not change greatly throughout the reaction. The reaction of PL and DA yielded 11.8 times more PL–DA adduct than the reaction of PM and DA. NMR analysis of the PL–DA adduct. Assignments were made based on the chemical shifts, proton-proton couplings, and 1 H- 13 C HMQC and 1 H- 1 H COSY correlations (Fig. S2 , Table S1 ). NMR analysis revealed the presence of the isoquinoline and pyridine rings. Thus, the proton assignments were as follows: (600 MHz, DMSO) δ 7.91 (H-1, s, 1H), 2-position of pyridine; 6.44 (H-2, s, 1H), 8-position of isoquinoline; 6.01 (H-3, s, 1H), 5-position of isoquinoline; 5.20 (H-4, m, 2H), 1-position of isoquinoline; 2.48 (H-8, s, 3H), 2.59 (H-7, t, 2H, J = 7.7 Hz), 4.37 (H-6, s, 2H), 5.19 (H-5, s, 1H), 1-position of isoquinoline; 4.62 (H-5, dd, 1H), 4.54 (H-5′, dd, 1H), 3-position of pyridine hydroxymethyl; 3.19–3.21 (H-6, m, 1H), 2.81–2.85 (H-6′, m, 2H), 3-position of isoquinoline; 2.81–2.85 (H-7, m, 2H), 2.81–2.85 (H-7′, m, 1H), 4-position of isoquinoline; and 2.18 (H-8, s, 3H), 6-position of pyridine methyl. Although the 3- and 4-positions of the isoquinoline (H-6′ and H-7) overlapped in the 1 H-NMR spectrum (Fig. S2 A), they were distinguished by HMQC analysis (Fig. S2 B). The NMR, LC-MS, and MS/MS data were consistent with the structure of l-(2-methyl-3-hydroxy-5-hydroxymethyl-4-pyridyl)-6,7-dihydroxy-l,2,3,4-tetrahydroisoquinoline. Analysis of the reaction between PM and DA in the presence of tyrosinase and/or GSH. When not confined to the acidic synaptic vesicles, DA is susceptible to oxidation to DAQ by various processes. In addition to autoxidation, DAQ is generated in vivo enzymatically by tyrosinase 13 , cyclooxygenase 39 , xanthinoxidase 40 , and lipoxygenase 41 . Transition metals, such as Cu and Fe, also induce or accelerate the oxidation of DA 14 , 15 . To mimic the intracellular conditions, the present study used tyrosinase, which catalyzes the oxidation of mono- and diphenols to the corresponding quinones with the concomitant reduction of molecular oxygen to water. DA (0.1 mM) was incubated with PM (0.1 mM) in the presence of tyrosinase (0, 0.01, and 0.1 µg/mL) for 72 h. LC/ESI-MS analysis showed the tyrosinase concentration-dependent decrease in DA level (Fig. 6 A) and concomitant increase in the formation of PL (Fig. 6 B) and the PL–DA adduct (Fig. 6 C). Thus, tyrosinase-induced efficient oxidation of DA to DAQ increased the formation of the PL–DA adduct, which supports the proposed mechanism for the reaction between PM and DA (Fig. 3 ). GSH is the main intracellular non-protein thiol, and it protects cells by scavenging free radicals and reactive oxygen/nitrogen species 42 . GSH is a co-factor in the reduction of hydrogen peroxide, lipid hydroperoxides, and peroxynitrite by GSH peroxidases and GSH S -transferases (GSTs) 42 , 43 . GSH also detoxifies reactive metabolites derived from exogenous and endogenous chemicals by forming GSH adducts 43 . GSH traps DAQ through GST-mediated adduct formation occurring at C-5 to give 5- S -glutathionyl dopamine (GSH-DA adduct) 16 . In the reaction between DA (1 mM) and PM (0, 1, 2, and 5 mM) in the presence of GSH (1 mM), the formation of GSH disulfide ([M + H] + , m/z 613.2; t R , 20.7 min on LC system 4), PL ( m/z 168.1; t R , 25.1 min), the GSH-DA adduct ( m/z 459.2; t R , 32.6 min), and the PL–DA adduct ( m/z 303.1; t R , 42.0 min) were detected with residual GSH ( m/z 308.1; t R , 10.3 min), DA ( m/z 154.1; t R , 29.4 min), and PM ( m/z 169.1; t R , 45.3 min). The GSH-DA adduct level was not affected by the changes in PM concentration. However, the amount of PL and PL–DA adduct increased with the PM concentration (Fig. 6 D). Similar patterns of product formation were observed from the reaction between DA and PM in the presence of both tyrosinase (1 µg/mL) and GSH (2 mM), except for the increased GSH-DA adduct concentration due to the tyrosinase-induced increase in DAQ (Fig. 6 E). These results indicate that PM scavenges DAQ in vivo through the formation of the PL–DA adduct. PM may trap DAQ more efficiently in the brains of PD patients where tyrosinase is important in the production of DAQ but GSH is substantially depleted because of the high levels of oxidative stress 44 , 45 . DA-induced oxidation of α-Syn. Human α-Syn is composed of 140 amino acids and is mainly expressed at presynaptic sites in the nervous system. Although the function of α-Syn is not well understood, it is thought to have diverse roles in synaptic maintenance, neurotransmitter release/homeostasis, and the regulation of synaptic vesicle pools and trafficking 7 . α-Syn exists in a dynamic balance between monomeric and oligomeric states, which allows it to adopt various conformations depending on the environments and interactions 7 . In PD, α-Syn assembles into β-sheet-rich amyloid-like fibrils, generating intermediate oligomers and causing further aggregation to large, insoluble fibrils, forming Lewy bodies 21 . DA promotes oligomerization through several types of interactions with α-Syn. DAQ reacts with Lys residues of α-Syn, generating DAQ-Lys adducts followed by intra-/inter-molecular cross linking to form complicated α-Syn-DA oligomers 46 . However, Bisaglia et al . demonstrated that α-Syn-DAQ adducts retain an unfolded conformation and the main modification occurs through non-covalent interactions 47 . In addition, DA-mediated Met oxidation in α-Syn has been proposed as the dominant mechanism of cytotoxicity and oligomerization 48 , 49 . Before investigating the DA-induced modification, the conditions for enzymatic digestion of α-Syn were optimized. No reduction and alkylation steps were needed because α-Syn contains no Cys residues. Two enzymes, V8 and trypsin, were used to digest α-Syn. The sequence coverage (%) based on the number of amino acids was 88.6% for V8 and 68.6% for trypsin (Tables S2 and S3). The overall coverage was 93.6%, missing only C-terminal 9 residues (G 132 YQDYEPEA 140 ) that do not contain expected modification sites, such as Lys or Met. α-Syn (2.8 µM, final concentration) was incubated with increasing concentrations of DA (0–100 molar eq. to α-Syn), followed by proteolysis using V8 or trypsin and LC/ESI-MS(/MS) analysis. A database search based on the MS/MS spectra revealed the presence of four Met-oxidized α-Syn peptides: V8 peptides, M 1 DVFM 5 KGLSKAKE, DM 116 PVDPDNE, and AYEM 127 PSEE; and tryptic peptides, M 1 DVFM 5 K, which contain all Met residues in α-Syn. The V8 peptides (Fig. S3 ) were used to monitor changes in the oxidation level of α-Syn induced by DA. The MS intensity of each peptide peak increased with the amount of DA (Fig. 7 A–C). The relative oxidation (%) was also calculated as relative oxidation (%) = (MS intensity of the Met-oxidized peptide/sum of MS intensity of the Met-oxidized peptide and corresponding intact peptide) × 100. The relative oxidations of Met 1/5 , Met 116 , and Met 127 increased in a DA dose-dependent manner up to 90.7%, 94.9%, and 86.9%, respectively (Fig. 7 D–F). For Met 116 , 18.7% relative oxidation was observed even after incubation without DA (Fig. 7 E). Relationship between DA-induced oxidation and oligomerization of α-Syn. α-Syn (2.8 µM, final concentration) was treated with DA (0–100 molar eq. to α-Syn) at 37°C for 24 h and analyzed by SDS-PAGE. Without DA, the α-Syn monomer and dimer were detected. With DA (20 molar eq.), oligomers formed and the monomer and dimer levels decreased (Fig. 8 A left). The oligomerization of α-Syn increased in a DA dose-dependent manner. However, excess DA (100 molar eq.) induced the formation of insoluble aggregates of α-Syn, and decreased the amounts of the soluble oligomer, dimer, and monomer (Fig. 8 A right). Each band was cut out and subjected to in-gel digestion with V8, and the peptides were analyzed by LC/ESI-MS(/MS). The relative oxidations of Met-containing peptides were compared among the monomer, dimer, and oligomer. The oxidation levels of Met 1/5 were not significantly different among the three polymeric species (Fig. 8 B). In contrast, the oxidation levels of Met 116 and Met 127 were higher in higher-order species in the order monomer < dimer < oligomer (Fig. 8 C and D). The relative oxidation in the oligomer was 83.1% for Met 116 and 97.3% for Met 127 . These results support a previous study that suggested Met 127 is the main target for oxidative modification by DA 48 . Inhibition effect of PM on DA-induced oxidation/oligomerization of α-Syn. α-Syn (2.8 µM, final concentration) was treated with DA (100 molar eq. to α-Syn) in the presence of PM (0–20 molar eq. to DA) at 37°C for 24 h and analyzed by SDS-PAGE. A portion of reaction mixture was subjected to proteolysis using V8 and analyzed by LC/ESI-MS(/MS). In the absence of PM, all four Met residues were completely oxidized by DA. As the amount of PM increased, the relative oxidations of Met 1/5 , Met 116 , and Met 127 decreased in a dose-dependent manner to 69.9%, 45.4%, and 80.8%, respectively (Fig. 9 A). A PM dose-dependent decrease in oligomerization of α-Syn was also observed in SDS-PAGE analysis (Fig. 9 B). These results indicate that PM can inhibit DA-induced oxidation and oligomerization of α-Syn by scavenging DAQ through the formation of the PL–DA adduct. Conclusion We demonstrated that PM reacts with DA to produce a stable PL–DA adduct. Thus, the initial reaction of the PM amino group at the DAQ carbonyl carbon yields a Schiff base intermediate, which is hydrolyzed to form PL. DA then reacts with the PL aldehyde, followed by intramolecular cyclization to produce the PL–DA adduct as a 1,2,3,4-tetrahydroisoquinoline derivative. The proposed mechanism was verified by using DA derivatives in the reactions, and the structures were confirmed by LC/ESI-MS(/MS) and NMR analyses. Under intracellular-like conditions in the presence of tyrosinase and/or GSH, the PL–DA adduct was formed in tyrosinase and PM dose-dependent manners. DA induced the oligomerization of α-Syn via the oxidation of its Met residues, which was inhibited by PM in a dose-dependent manner. Therefore, PM could scavenge DAQ efficiently in the brains of PD patients, in which tyrosinase is overexpressed to produce more DAQ but GSH is substantially depleted by high levels of oxidative stress. PM could also inhibit DA-induced oxidation/oligomerization of α-Syn through the formation of the PL–DA adduct. Our ongoing studies are focusing on developing analytical methodology for the PL–DA adduct to use it as a biomarker of oxidative stress and DA oxidation. Materials and Methods Materials. HPLC-grade acetonitrile (MeCN), methanol (MeOH), ammonium bicarbonate (NH 4 HCO 3 ), formic acid (FA), ethanol, diethyl ether, 0.1 mol/L hydrochloric acid (HCl), sodium hydroxide, disodium hydrogenphosphate dodecahydrate (Na 2 HPO 4 •12H 2 O), sodium dihydrogenphosphate dihydrate (NaH 2 PO 4 •2H 2 O), glutathione (GSH), hydrochloric acid, dimethyl sulfoxide-d 6 (DMSO-d 6 ), CBB stain One Super, running buffer solution (10x) for SDS-PAGE, and Extra PAGE One Precast Gel 10–20% were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Pyridoxamine (PM) dihydrochloride, pyridoxal (PL) hydrochloride, endoproteinase Glu-C sequencing grade (V8), and tyrosinase from mushroom were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI). Dopamine (DA) hydrochloride and isoproterenol (IPT) hydrochloride were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). α-Synuclein (α-Syn, human, recombinant) and 3,4-dimethoxyphenethylamine (DPA) were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). Calcium chloride (CaCl 2 ) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Ammonium formate (HCOONH 4 ) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Chelex® 100 chelating resin was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Ultrapure water was obtained from a Milli-Q Integral 10 (MilliporeSigma, Burlington, MA) equipped with a 0.22-µm membrane cartridge. Liquid chromatography. For LC systems 1–4, an Agilent 1100 LC system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a 1100 G1312A binary pump, 1100 G1379A degasser, 1100 G1367A autosampler, 1100 G1316A column heater, and a 1100 G1315B photodiode array detector was used. LC systems 1–3 used a scherzo SW-C18 column (150 × 2.0 mm i.d., 3 µm, 13 nm; Imtakt Corporation, Kyoto, Japan) with a flow rate of 0.2 mL/min. Solvent A was H 2 O containing 0.3% (v/v) FA, and solvent B was100 mM HCOONH 4 /MeCN = 7/3 (v/v). Gradient elution was performed as follows: LC system 1, 2% B at 0 min, 22% B at 40 min, 90% B at 41 min, 90% B at 51 min, 2% B at 52 min, and 2% B at 82 min; LC system 2, 2% B at 0 min, 62% B at 120 min, 90% B at 121 min, 90% B at 131 min, 2% B at 132 min, and 2% B at 162 min; LC system 3, 2% B at 0 min, 17% B at 30 min, 90% B at 31 min, 90% B at 41 min, 2% B at 42 min, and 2% B at 72 min. LC system 4 used a scherzo SS-C18 column (150 × 2.0 mm i.d., 3 µm, 13 nm; Imtakt Corporation) with a flow rate of 0.2 mL/min. Solvent A was H 2 O containing 0.1% (v/v) FA, and solvent B was100 mM HCOONH 4 /MeCN = 6/4 (v/v). Gradient elution was performed as follows: 0% B at 0 min, 0% B at 3 min, 45% B at 48 min, 100% B at 49 min, 100% B at 64 min, and 0% B at 65 min. For LC system 5, an Ultimate 3000 LC system (Thermo Fisher Scientific, Inc.) equipped with an SRD-3600 degasser, DGP-3600MB pump, FLM-3100B (nano, 2X2P-10P) flow manager, and WPS-3000TBPL (nano, CAP) autosampler was used. A Jupiter C18 column (150 × 2.0 mm i.d., 5 µm, 300 Å; Phenomenex, Torrance, CA, USA) with a flow rate of 0.2 mL/min and a column oven temperature of 40°C. Solvent A was H 2 O containing 0.1% (v/v) FA, and solvent B was MeCN containing 0.1% (v/v) FA. Gradient elution was performed as follows: 0% B at 0 min, 0% B at 3 min, 32.5% B at 70 min, 90% B at 71 min, 90% B at 80 min, 0% B at 81 min, and 0% B at 100 min. Mass spectrometry. The LCQ-DECA ion trap mass spectrometer (Thermo Fisher Scientific Inc.) equipped with an ESI source was used in positive ion mode for LC systems 1–4. Data was processed using an Xcalibur (version 2.0 SR2, Thermo Fisher Scientific Inc.). The operating conditions were as follows: heated capillary, 300°C; ion spray voltage, 4.5 kV; sheath and auxiliary gas (nitrogen) pressures, 85 and 15 arbitrary units (arb), respectively; mass range, m/z 100–1000; isolation width, 2; normalized collision energy (CE), 40%; activation Q, 0.25; and activation time, 30 ms. The LTQ Orbitrap Velos hybrid ion trap-orbitrap mass spectrometer (Thermo Fisher Scientific, Inc.) equipped with an ESI source was used in the positive ion mode LC system 5. Data were processed using an Xcalibur (version 2.2.SR2). The operating conditions were as follows: analyzer, ion trap; heated capillary, 275°C; spray voltage, 3.0 kV; scan rate, normal (33,000 amu/s); sheath and auxiliary gas (nitrogen) pressures, 50 and 15 arb, respectively. Full scanning analyses were performed in the range of m/z 300–2000. Tandem mass spectrometry (MS/MS) was performed with data dependent scan and its settings were as follows: precursor, top 10 ions; default charge state, 2; isolation width, 4; normalized CE, 35%; activation Q, 0.25; and activation time, 10 ms. Database search. Peptide sequences and modifications were identified with Proteome Discoverer 1.3 (Thermo Fisher Scientific, Inc.). The peak list was searched by Sequest (University of Washington, Seattle, WA) against National Center for Biotechnology Information (alpha-synuclein isoform NACP140 [Homo sapiens], NP_000336.1). Search settings were as follows: enzyme, V8 or trypsin; maximum missed cleavage, 3; dynamic modification, oxidation (Met); precursor mass tolerance, 2 Da; fragment mass tolerance, 0.8 Da; target false discovery rate (FDR), 0.01%. DA (or DAQ) (Lys), [DA (or DAQ) − H 2 O] (Lys), [DA (or DAQ) − 2H] (Lys) were added to the dynamic modification list. NMR. The NMR spectrum was recorded on a JNM-ECA600 spectrometer (JEOL Ltd., Tokyo, Japan) at 25°C. Data was processed using a Delta NMR software (version 5.2, JEOL Ltd.) an Xcalibur (version 2.0 SR2, Thermo Fisher Scientific Inc.). The sample was dissolved in DMSO-d 6 . Chemical shifts were reported on the δ scale (ppm) by assigning the residual solvent peak for DMSO as internal reference to 2.49 and 39.5 for 1 H and 13 C, respectively. Acquisition conditions for 1 H NMR were as follows: Domain, proton; offset, 5 ppm; sweep, 15 ppm; points, 16384; prescans, 1; scans, 256. Acquisition conditions for 1 H- 1 H COSY were as follows: Domain, proton (x and y); offset, 5 ppm (x and y); sweep, 15 ppm; points, 1280 (x) and 256 (y); prescans, 4 (x) and 0 (y); scans, 1. Acquisition conditions for 1 H- 13 C HMQC were as follows: Domain, proton (x) and carbon-13 (y); offset, 5 ppm (x) and 85 ppm (y); sweep, 15 ppm (x) and 170 ppm (y); points, 1024 (x) and 256 (y); prescans, 4 (x) and 0 (y); scans, 4. Preparation of PL-DA adduct for NMR analysis. The water (8 mL) solution containing DA hydrochloride (100 mg) and PL hydrochloride (100 mg) were neutralized with 5 M sodium chloride (1 µL), followed by incubation at room temperature for 4 h. The white precipitate (PL-DA adduct) formed was dissolved in DMSO (60°C, 1.1 mL) and recrystallized by adding a cold water (1.1 mL) dropwise. After centrifugation, the supernatant was removed and the solid was dried under vacuum. PL-DA adduct was obtained as a white solid (6.6 mg isolated). Reaction of PM or PL with DA. A solution of 1 mM DA hydrochloride in 50 mM Chelex-treated sodium phosphate buffer (PB, pH 7.4, 100 µL) and 1 mM PM dihydrochloride or PL hydrochloride in PB (100 µL) were added to PB (800 µL). The reaction mixture was incubated at 37°C for 96 h. A portion of the reaction mixture (50 µL) was withdrawn at various time points and filtered through a syringe filter, followed by LC/ESI-MS(/MS) analysis using LC system 1. Reaction of PM with DPA or IPT. A solution of 1 mM DPA or IPT in PB (100 µL) and 1 mM PM dihydrochloride in PB (100 µL) were added to PB (800 µL). The reaction mixture was incubated at 37°C for 48 h and analyzed by LC/ESI-MS(/MS) analysis using LC system 2. Reaction of PM with DA in the presence of tyrosinase. DA hydrochloride in PB (1 mM, 100 µL) and PM dihydrochloride in PB (1 mM, 100 µL) were added to a solution of tyrosinase (0, 10, or 100 ng) in PB (800 µL). The reaction mixture was incubated at 37°C for 72 h. A portion of the reaction mixture (50 µL) was withdrawn at various time points and filtered through a syringe filter, followed by LC/ESI-MS(/MS) analysis using LC system 3. Reaction of PM with DA in the presence of GSH. DA hydrochloride in PB (10 mM, 100 µL) and GSH in PB (10 mM, 100 µL) were added to a solution of PM dihydrochloride (0, 1, 2, 5, or 10 µmol) in PB (800 µL). The reaction mixture was incubated at 37°C for 48 h and filtered through a syringe filter, followed by LC/ESI-MS(/MS) analysis using LC system 4. Reaction of PM with DA in the presence of tyrosinase and GSH. DA hydrochloride in PB (10 mM, 100 µL), tyrosinase in PB (10 µg/mL, 100 µL), and GSH in PB (20 mM, 100 µL) were added to a solution of PM dihydrochloride (0, 1, 2, 5, or 10 µmol) in PB (700 µL). The reaction mixture was incubated at 37°C for 48 h and filtered through a syringe filter, followed by LC/ESI-MS(/MS) analysis using LC system 4. In-solution digestion of α-Syn. α-Syn (0.1 µg/µL in H 2 O, 20 µL) was added to ammonium bicarbonate buffer (12.5 mM, 80 µL), followed by incubation with sequencing-grade modified trypsin or V8 (0.002 µg/µL, 20 µL) at 37°C for 24 h. A portion of sample (100 µL) was then analyzed by LC/ESI-MS(/MS) using LC system 5. Reaction of α-Syn with DA. α-Syn (0.1 µg/µL in H 2 O, 20 µL) was incubated with DA (0, 0.14, 1.4, 2.8, 7.0, or 14 nmol) in PB (30 µL) at 37°C for 24 h (final concentration was as follows: α-Syn, 2.8 µM; DA, 0, 2.8, 28, 56, 140, 280 µM). The solutions were digested with trypsin or V8 as described above. A portion of samples (100 µL) were then analyzed by LC/ESI-MS(/MS) using LC system 5. For SDS-PAGE, samples after the 24-h incubation were evaporated to dryness and redissolved in 10 µL of H 2 O. Reaction of α-Syn with DA in the presence of PM. α-Syn (0.1 µg/µL in H 2 O, 20 µL) and DA in PB (14 nmol, 10 µL) was added to PM (0, 14, 70, 140, or 280 nmol) in PB (20 µL) at 37°C for 24 h (final concentration was as follows: α-Syn, 2.8 µM; DA, 280 µM; PM, 0, 0.28, 1.4, 2.8, 5.6 mM). The solution was digested with trypsin or V8 as described above. A portion of sample (100 µL) was then analyzed by LC/ESI-MS(/MS) using LC system 5. For SDS-PAGE, samples after the 24-h incubation were evaporated to dryness and redissolved in 10 µL of H 2 O. SDS-PAGE. The sample (10 µL) was mixed with Laemmli sample buffer (3.3 µL) and boiled at 95°C for 5 min. The sample was loaded onto 10–20% polyacrylamide gel and run in the running buffer (24 mM Tris, 192 mM glycine, 0.1% (v/v) SDS, pH 8.3) at 300 V for 210 min. The sample was then stained by CBB R-250. In-gel digestion of α-Syn. α-Syn band was excised from the gel and washed in water (200 µL) for 30 s and then washed two times in 200 µL of 50 mM NH 4 HCO 3 /MeOH (1:1, v/v) for 1 min. The band pieces were dehydrated in 200 µL of 50 mM NH 4 HCO 3 /MeCN (1:1, v/v) for 5 min, followed by 100% MeCN (200 µL) for 30 s while vortex mixing. The supernatant was removed, and band pieces were dried in the centrifugal evaporator. V8 (0.04 µg/µL) suspended in 50 mM NH 4 HCO 3 was added, and band pieces were rehydrated for 10 min on ice and digested at 37°C for 24 h. The samples were then centrifuged and the supernatant was transferred to a fresh tube, followed by evaporation to dryness. The samples were redissolved in 30 µL of water and analyzed by LC/ESI-MS(/MS) using LC system 5. Declarations Acknowledgements We are grateful to the Central Analytical Center at Graduate School of Pharmaceutical Sciences, Tohoku University for the use of their LTQ Orbitrap Velos. This work was supported in part by a Grants-in-Aid for Scientific Research (C) (to S.H.L., 19K07187 for 2019−2021) and for Scientific Research (B) (to T.O., 16H05078 for 2016–2018) from the Japan Society for the Promotion of Science (JSPS). Author contributions S.H.L.: Writing – original draft, Project administration, Methodology, Funding acquisition, Conceptualization. N.M. and A.T.: Visualization, Investigation, Data curation. Y.H.: Writing – review & editing. T.O.: Writing – review & editing, Supervision, Resources, Funding acquisition. 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Additional Declarations No competing interests reported. Supplementary Files MatsumotoSR2025Sup.pdf TableS2SynV8.xlsx TableS3SynTrypsin.xlsx Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 Jul, 2025 Reviews received at journal 30 Jun, 2025 Reviewers agreed at journal 26 Jun, 2025 Reviewers agreed at journal 26 Jun, 2025 Reviews received at journal 12 May, 2025 Reviewers agreed at journal 06 May, 2025 Reviewers agreed at journal 06 May, 2025 Reviewers invited by journal 06 May, 2025 Editor assigned by journal 28 Apr, 2025 Editor invited by journal 27 Apr, 2025 Submission checks completed at journal 25 Apr, 2025 First submitted to journal 18 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6478169","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":452610434,"identity":"bcfcd982-4ca1-48e2-819d-4818ac111ddc","order_by":0,"name":"Seon Hwa Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDACCQbGAwwMNiAmGwPDAYigAQEtIHVpIA2kaTmMqgUvkJ/dwHDgQ8X5aAb55mMPPpw5zMDffoChuACPFoM7BxgOzjhzO7eBjS3dcMaNwwwSZxIYjGfg0yKR/+EwbxtIC4+ZNM8HoAtvMDAY8+Bz2IwEhsN//51DaJEnpIXhBlALY8MBqBagwwwIaTEAajnYcyw5t40tLU1yxpl0HsMziQ14/QJ0GOODHzV2uf3Mh49JfDhmLSd3/PAxY3whBgdsUBroJMY2Y2J0oADmxyRrGQWjYBSMguEMAIEpTJAe05H7AAAAAElFTkSuQmCC","orcid":"","institution":"Tohoku University","correspondingAuthor":true,"prefix":"","firstName":"Seon","middleName":"Hwa","lastName":"Lee","suffix":""},{"id":452610435,"identity":"76c40ea5-de29-4a64-a3a8-8e895e69cba0","order_by":1,"name":"Naoya Matsumoto","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Naoya","middleName":"","lastName":"Matsumoto","suffix":""},{"id":452610436,"identity":"6c69a484-9ae7-4a7d-842b-21a6fae55e46","order_by":2,"name":"Akane Terada","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Akane","middleName":"","lastName":"Terada","suffix":""},{"id":452610437,"identity":"0a9d7323-3b82-401d-84a1-dd73cb453694","order_by":3,"name":"Yusuke Hatakawa","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Hatakawa","suffix":""},{"id":452610438,"identity":"3f6075f6-1cab-4871-80d1-54ff77d1bac3","order_by":4,"name":"Tomoyuki Oe","email":"","orcid":"","institution":"Tohoku University","correspondingAuthor":false,"prefix":"","firstName":"Tomoyuki","middleName":"","lastName":"Oe","suffix":""}],"badges":[],"createdAt":"2025-04-18 10:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6478169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6478169/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-31292-8","type":"published","date":"2025-12-26T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82562808,"identity":"316545fe-b66d-4b4f-aca8-8ee636b0dd0b","added_by":"auto","created_at":"2025-05-13 01:50:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":505453,"visible":true,"origin":"","legend":"\u003cp\u003ePyridoxamine (PM) scavenges neurotoxic dopamine quinone (DAQ) by forming the PL–DA adduct, and inhibits DA-induced oligomerization of α-synuclein (α-Syn).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/8f8068fa7ea71cb3e4c939fe.png"},{"id":82563282,"identity":"19984d84-c35b-4038-8451-5f100f20cb56","added_by":"auto","created_at":"2025-05-13 01:58:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1408560,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) LC/ESI-MS analysis of the reaction between PM and DA at 37 °C for 48 h. (\u003cstrong\u003eB\u003c/strong\u003e) Time course of the reaction between PM and DA over 72 h.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/6204b17b9d07c053761f7717.png"},{"id":82562193,"identity":"f5d4e1d2-f18a-4951-b321-ed56a2eca2a4","added_by":"auto","created_at":"2025-05-13 01:42:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1104310,"visible":true,"origin":"","legend":"\u003cp\u003eProposed\u003cstrong\u003e \u003c/strong\u003emechanisms. (\u003cstrong\u003eA\u003c/strong\u003e) Formation of PL from the reaction between PM and DA. (\u003cstrong\u003eB\u003c/strong\u003e) Formation of the PL–DA adduct from the reaction between PL and DA.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/c23457214fb04577dc3a90d7.png"},{"id":82562197,"identity":"7cc970f4-88d7-43d9-968b-266522d2ffb7","added_by":"auto","created_at":"2025-05-13 01:42:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2044255,"visible":true,"origin":"","legend":"\u003cp\u003eLC/ESI-MS analysis of the reaction between PM and (\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eDPA or (\u003cstrong\u003eC\u003c/strong\u003e) IPT at 37 °C for 48 h. Proposed mechanisms for the reaction between PM and (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eDPA or (\u003cstrong\u003eD\u003c/strong\u003e) IPT.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/f29076f38d953b3ed4f7182d.png"},{"id":82562217,"identity":"a85c5d54-6d58-4333-8273-b54ac99f2bb1","added_by":"auto","created_at":"2025-05-13 01:42:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1454073,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) LC/ESI-MS analysis of the reaction between PL and DA at 37 °C for 48 h. (\u003cstrong\u003eB\u003c/strong\u003e) MS/MS analysis of the PL–DA adduct at \u003cem\u003em/z\u003c/em\u003e 303.2 ([M + H]\u003csup\u003e+\u003c/sup\u003e). (\u003cstrong\u003eC\u003c/strong\u003e) Time course of the reaction between PL and DA over 48 h.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/bf6f32e3408648fc3847907b.png"},{"id":82562216,"identity":"fd9fe612-3276-43d4-907d-b301c17cf84f","added_by":"auto","created_at":"2025-05-13 01:42:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1502053,"visible":true,"origin":"","legend":"\u003cp\u003eFormation of (\u003cstrong\u003eA\u003c/strong\u003e) DA, (\u003cstrong\u003eB\u003c/strong\u003e) PL, and (\u003cstrong\u003eC\u003c/strong\u003e) the PL–DA adduct from the reaction between DA (0.1 mM) and PM (0.1 mM) in the presence of tyrosinase (0, 0.01, and 0.1 μg/mL) at 37 °C for 72 h. Formation of the GSH-DA adduct, PL, and the PL–DA adduct from the reaction between DA (1 mM) and PM (0, 1, 2, and 5 mM) in the presence of (\u003cstrong\u003eD\u003c/strong\u003e) GSH (1 mM) or (\u003cstrong\u003eE\u003c/strong\u003e) tyrosinase (1 μg/mL) and GSH (2 mM) at 37 °C for 48 h.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/6d95ef5612f6b815dfe8af29.png"},{"id":82562200,"identity":"217569c8-7ef3-40ef-b64e-66e599ca402e","added_by":"auto","created_at":"2025-05-13 01:42:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1565315,"visible":true,"origin":"","legend":"\u003cp\u003eLC/ESI-MS analysis of Met-oxidized α-Syn peptides (\u003cstrong\u003eA\u003c/strong\u003e) \u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sup\u003eDVF\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sup\u003eKGLSKAKE, (\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eD\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e116\u003c/strong\u003e\u003c/sup\u003ePVDPDNE, and (\u003cstrong\u003eC\u003c/strong\u003e) AYE\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e127\u003c/strong\u003e\u003c/sup\u003ePSEE formed from the reaction of α-Syn and DA (molar eq. to α-Syn: 0, top; 10, middle; 50, bottom) at 37 °C for 24 h. Relative oxidation (%) of (\u003cstrong\u003eD\u003c/strong\u003e) \u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sup\u003eDVF\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sup\u003eKGLSKAKE, (\u003cstrong\u003eE\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eD\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e116\u003c/strong\u003e\u003c/sup\u003ePVDPDNE, and (\u003cstrong\u003eF\u003c/strong\u003e) AYE\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e127\u003c/strong\u003e\u003c/sup\u003ePSEE in the reaction of α-Syn and DA (0–100 molar eq. to α-Syn).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/ebb80e02888979bcf1563f3c.png"},{"id":82562207,"identity":"fe4484e4-8919-4d64-8343-142ec7e52ba7","added_by":"auto","created_at":"2025-05-13 01:42:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1243007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e SDS-PAGE analysis of α-Syn treated with DA (0–100 molar eq. to α-Syn). Relative oxidation (%) of (\u003cstrong\u003eB\u003c/strong\u003e) \u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sup\u003eDVF\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sup\u003eKGLSKAKE, (\u003cstrong\u003eC\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eD\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e116\u003c/strong\u003e\u003c/sup\u003ePVDPDNE, and (\u003cstrong\u003eD\u003c/strong\u003e) AYE\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e127\u003c/strong\u003e\u003c/sup\u003ePSEE in monomer (M), dimer (D), and oligomer (O) bands of SDS-PAGE in \u003cstrong\u003eA\u003c/strong\u003e. Each band was cut out and subjected to in-gel digestion with V8. The peptides were analyzed by LC/ESI-MS(/MS).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/162ea613a848765a9a2c1541.png"},{"id":82563284,"identity":"23f0f0d4-7610-4372-91b3-83ff23549e34","added_by":"auto","created_at":"2025-05-13 01:58:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1166051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Relative oxidation (%) of \u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sup\u003eDVF\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sup\u003eKGLSKAKE (top), D\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e116\u003c/strong\u003e\u003c/sup\u003ePVDPDNE (middle), and AYE\u003cstrong\u003eM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e127\u003c/strong\u003e\u003c/sup\u003ePSEE (bottom) formed from the reaction of α-Syn and DA in the presence of PM (0–20 eq. to DA). \u003cstrong\u003eB.\u003c/strong\u003e SDS-PAGE analysis of α-Syn treated with DA in the absence/presence of PM (0, 5, and 10 eq. to DA).\u0026nbsp;\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/106b934f91073a37572d9c04.png"},{"id":99172472,"identity":"6943a2dc-4f4e-4d1e-9ed4-bb60218d1c42","added_by":"auto","created_at":"2025-12-29 16:10:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13054872,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/abdedf1a-e627-4eaf-8961-ee2aae275746.pdf"},{"id":82562190,"identity":"35c32302-cc95-49bb-be99-98f73f4a3e95","added_by":"auto","created_at":"2025-05-13 01:42:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":373906,"visible":true,"origin":"","legend":"","description":"","filename":"MatsumotoSR2025Sup.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/692ee214157cbae211c88911.pdf"},{"id":82562807,"identity":"deb882b0-f7cb-4338-ad0a-ec4aa7783de2","added_by":"auto","created_at":"2025-05-13 01:50:26","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11995,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2SynV8.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/8d050d83e4d3b7f4605d9de4.xlsx"},{"id":82562192,"identity":"d070616d-da7a-4102-95ff-9761d5ada555","added_by":"auto","created_at":"2025-05-13 01:42:26","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11107,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3SynTrypsin.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6478169/v1/4a1428ea59febce6b9ae45cb.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pyridoxamine as a potential scavenger of neurotoxic dopamine quinone: Inhibition of dopamine-induced α-synuclein oligomerization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is an age-related neurodegenerative disease that is the fastest growing neurological disorder in terms of disability and death\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. PD is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting in reduced dopamine (DA) levels\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Additionally, the neurons contain cytosolic filamentous inclusions known as Lewy bodies, whose main component is fibrillar α-synuclein (α-Syn)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Although the precise molecular mechanisms underlying PD are not fully understood, intensive studies have uncovered several pathways and mechanisms involved in pathophysiology of PD, such as oxidative stress\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, mitochondrial dysfunction\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, calcium homeostasis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, and α-Syn aggregation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Despite ongoing research, there is currently no cure for PD. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-DOPA, a precursor of DA, is the first choice of treatment for PD because it replenishes DA. However, long-term use of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-DOPA causes adverse effects, such as motor fluctuations, dyskinesia, and psychiatric symptoms\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, which are attributed partly to the formation of dopamine \u003cem\u003eo\u003c/em\u003e-quinone (DAQ). DA is the most abundant neurotransmitter and is important in multiple physiological functions, including motor control, emotional modulation, and reward mechanisms\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, DA can induce oxidative stress through various redox reactions. One-electron oxidation of DA to the semiquinone radical (DASQ) can be mediated by free heme, peroxidases, or cyclooxygenase via H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e activation. DASQ couples with other radicals, scavenges cellular thiols, such as glutathione (GSH), undergoes further oxidation by O\u003csub\u003e2\u003c/sub\u003e to form DAQ and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, or disproportionates to DA and DAQ\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Reactive oxygen species (ROS) derived from DA oxidation can damage lipids, proteins, and DNA in the cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. DA can also be oxidized to DAQ in a two-electron process promoted by transition metals or tyrosinase\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The electron-deficient DAQ reacts with cellular thiols and nucleophilic amino acid residues, leading to further cytotoxicity. Upon reaction with GSH, DAQ forms 5-\u003cem\u003eS\u003c/em\u003e-glutathionyl- and 5-\u003cem\u003eS\u003c/em\u003e-cysteinyl-DA\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. 5-\u003cem\u003eS\u003c/em\u003e-Cysteinyl-DA is neurotoxic\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. DAQ binds covalently to Cys residues in the DA transporter\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, superoxide dismutase\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and glucocerebrosidase\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, decreasing enzyme activity and causing blocked DA uptake, mitochondrial dysfunction, and lysosomal dysfunction, respectively. The interaction between DA and α-Syn also causes selective neuronal cell death and the accumulation of misfolded α-Syn\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Although the exact mechanism is not fully defined, DA oxidation is a key mechanism. Therefore, a DAQ-quenching molecule should prevent DA-induced pathogenicity in PD, such as the death of dopaminergic neurons and aggregation of α-Syn (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePyridoxamine (PM) is a vitamin B6 vitamer and functions as a coenzyme in enzymatic transaminations \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. PM is also a promising pharmacological agent for the treatment of diabetic complications and other chronic conditions\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This is based on its multiple inhibitory effects. For example, inhibition of advanced glycation end product (AGE) formation by chelation of metal ions with the phenol and aminomethyl groups, inhibition of advanced lipoxidation end product (ALE) formation by scavenging of reactive carbonyl species (RCS), and trapping of ROS with phenol group\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. PM is a potent scavenger of 1,2-, 1,3-, and 1,4-dicarbonyl compounds, which are RCSs. PM forms a dimer with methylglyoxal (MGO; 1,2-dicarbonyl), which blocked production of the MGO-Lys dimer and lowered the levels of MGO in red blood cells/plasma of diabetic rats\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. PM reacts readily with glyoxal (1,2-dicarbonyl) to form a five-ring compound with a central piperazine ring. PM inhibited formation of AGE/ALE \u003cem\u003eN\u003c/em\u003e\u003csup\u003eε\u003c/sup\u003e-carboxymethyl Lys during incubation of bovine serum albumin with glyoxal\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. PM traps malondialdehyde (1,3-dicarbonyl), a DNA-reactive aldehyde derived from lipid peroxidation, thereby inhibiting lipofuscin-like fluorescence induced by malondialdehyde in the reaction with bovine serum albumin\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. PM forms a pyrrole and a lactam adduct with 4-oxopentanal and 15-E\u003csub\u003e2\u003c/sub\u003e-isoketal (both 1,4-dicarbonyls), respectively\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Recent studies\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e have demonstrated that PM inhibits lipid hydroperoxide-derived damage to proteins by trapping 4-oxo-2(\u003cem\u003eE\u003c/em\u003e)-nonenal (ONE; 1,4-dicarbonyl), the most abundant and reactive lipid-derived aldehyde\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. PM also attenuated ONE-derived insulin resistance by scavenging ONE\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. PM\u0026ndash;ONE adducts were then detected in cell culture and increased in a PM dose-dependent manner, which suggests that PM\u0026ndash;ONE adducts could function as biomarkers of oxidative stress. Therefore, PM could inhibit DA-induced neurotoxicity by scavenging DAQ and its adduct could reflect the extent of oxidative stress and DA oxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we characterized the adduct generated from the reaction between PM and DA using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) analyses. The adduct formation was investigated in the presence of the endogenous enzyme and/or thiol to mimic the intracellular conditions. Finally, we confirmed the inhibition by PM of DA-induced oxidation/oligomerization of α-Syn.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eAnalysis of the reaction between PM and DA.\u003c/b\u003e LC/ESI-MS analysis after 48 h incubation at 37\u0026deg;C revealed the presence of two major products and residual DA ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, \u003cem\u003em/z\u003c/em\u003e 154.1; retention time [\u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e], 6.8 min) and PM ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, \u003cem\u003em/z\u003c/em\u003e 169.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 7.5 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The MS spectrum of the most polar product eluting at 5.3 min showed [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e at \u003cem\u003em/z\u003c/em\u003e 168.1, corresponding to a loss of 1 Da from PM. This product was identified as pyridoxal (PL), a transamination product of PM, because the LC/ESI-MS, MS/MS, and UV characteristics were identical to those for an authentic PL standard (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The product that eluted at 14.6 min (PL\u0026ndash;DA adduct) had [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e at \u003cem\u003em/z\u003c/em\u003e 303.2, corresponding to a 1:1 reaction of PL with DA ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, \u003cem\u003em/z\u003c/em\u003e 321.3) followed by the loss of water (\u0026minus;\u0026thinsp;18 Da). Time course experiments were performed to clarify the formation of the PL\u0026ndash;DA adduct further. The reaction between PM and DA in Chelex-treated phosphate buffer (pH 7.4) was monitored by LC/ESI-MS for 3 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). DA decreased gradually and was not detectable after 72 h, whereas PL increased concomitantly. The PL\u0026ndash;DA adduct formed after incubation for 12 h and increased to its maximum level at 72 h. The PM level did not change much throughout the reaction. DA undergoes autoxidation to form DAQ at physiological pH. DAQ reacts with nucleophilic amino acids, such as Cys, His, and Lys, yielding Michael addition products at the quinone ring\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The preferential positions of Cys and His addition are C-5 and C-6, respectively\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Therefore, it was expected that the PM\u0026ndash;DA adduct would be produced by the nucleophilic addition of PM to the DAQ quinone core. However, the only adduct that formed was the PL\u0026ndash;DA adduct after PL was generated. This result suggests that the initial reaction of the PM primary amino group occurs at the DAQ carbonyl carbon and yields a Schiff base (ketimine). The intermediate ketimine undergoes tautomerization to form aldimine, which is subsequently hydrolyzed to yield PL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). DA then reacts with the PL aldehyde and forms a Schiff base intermediate. The following intramolecular cyclization produces the PL\u0026ndash;DA adduct as a 1,2,3,4-tetrahydroisoquinoline derivative (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) through a Pictet\u0026ndash;Spengler reaction\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The proposed mechanism and structure were confirmed by LC-MS and NMR analyses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the reaction between PM and DA derivatives.\u003c/b\u003e To confirm the proposed mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e), PM was reacted with two DA derivatives, 3,4-dimethoxyphenethylamine (DPA) and isoproterenol (IPT). DPA is an analog of DA in which the hydroxy groups have been replaced with methoxy groups, which prevents autoxidation to the \u003cem\u003eo\u003c/em\u003e-quinone. LC/ESI-MS analysis of the reaction between PM and DPA at 37\u0026deg;C for 48 h revealed the presence of DPA ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, \u003cem\u003em/z\u003c/em\u003e 182.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 29.8 min) and PM (\u003cem\u003em/z\u003c/em\u003e 169.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 7.4 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). No other products were detected. This result suggests that the \u003cem\u003eo\u003c/em\u003e-quinone is necessary to produce PL from PM by transamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). IPT is the isopropyl amine epinephrine analog, which can undergo autoxidation but has a bulky isopropyl group substituent on the amine. PL (\u003cem\u003em/z\u003c/em\u003e 168.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 5.7 min) was detected as the only product of the reaction between PM and IPT together with residual IPT (\u003cem\u003em/z\u003c/em\u003e 212.2; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 13.0 min) and PM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). PM was converted to PL by the reaction with IPTQ but PL failed to react with IPT to produce the Schiff base intermediate due to the steric hinderance of the isopropyl amine moiety of IPT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results support the proposed mechanism that involves DAQ-derived transamination of PM to PL, which subsequently reacts with the primary amino group of DA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the reaction between PL and DA.\u003c/b\u003e LC/ESI-MS analysis after 48 h incubation at 37\u0026deg;C revealed the presence of the PL\u0026ndash;DA adduct ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, \u003cem\u003em/z\u003c/em\u003e 303.2; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 15.3 min) and residual DA (\u003cem\u003em/z\u003c/em\u003e 154.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 7.3 min) and PL (\u003cem\u003em/z\u003c/em\u003e 168.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 5.6 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). MS/MS analysis of the PL\u0026ndash;DA adduct at \u003cem\u003em/z\u003c/em\u003e 303.2 showed the formation of product ions at \u003cem\u003em/z\u003c/em\u003e 285.2 (\u0026minus;\u0026thinsp;18 Da, \u0026minus; H\u003csub\u003e2\u003c/sub\u003eO), \u003cem\u003em/z\u003c/em\u003e 164.3 (\u0026minus;\u0026thinsp;139 Da, \u0026minus; C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;H), \u003cem\u003em/z\u003c/em\u003e 152.1 (\u0026minus;\u0026thinsp;151 Da, \u0026minus; C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e9\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e) and \u003cem\u003em/z\u003c/em\u003e 140.2 (\u0026minus;\u0026thinsp;163 Da, \u0026minus; C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;H) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), which are identical to those of the PL\u0026ndash;DA adduct formed from the reaction between PM and DA. In time course experiments for 2 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), DA decreased gradually and was not detectable after 48 h. The PL\u0026ndash;DA adduct increased concomitantly to its maximum level at 18 h. The PL level did not change greatly throughout the reaction. The reaction of PL and DA yielded 11.8 times more PL\u0026ndash;DA adduct than the reaction of PM and DA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNMR analysis of the PL\u0026ndash;DA adduct.\u003c/b\u003e Assignments were made based on the chemical shifts, proton-proton couplings, and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC HMQC and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH COSY correlations (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). NMR analysis revealed the presence of the isoquinoline and pyridine rings. Thus, the proton assignments were as follows: (600 MHz, DMSO) δ 7.91 (H-1, s, 1H), 2-position of pyridine; 6.44 (H-2, s, 1H), 8-position of isoquinoline; 6.01 (H-3, s, 1H), 5-position of isoquinoline; 5.20 (H-4, m, 2H), 1-position of isoquinoline; 2.48 (H-8, s, 3H), 2.59 (H-7, t, 2H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz), 4.37 (H-6, s, 2H), 5.19 (H-5, s, 1H), 1-position of isoquinoline; 4.62 (H-5, dd, 1H), 4.54 (H-5\u0026prime;, dd, 1H), 3-position of pyridine hydroxymethyl; 3.19\u0026ndash;3.21 (H-6, m, 1H), 2.81\u0026ndash;2.85 (H-6\u0026prime;, m, 2H), 3-position of isoquinoline; 2.81\u0026ndash;2.85 (H-7, m, 2H), 2.81\u0026ndash;2.85 (H-7\u0026prime;, m, 1H), 4-position of isoquinoline; and 2.18 (H-8, s, 3H), 6-position of pyridine methyl. Although the 3- and 4-positions of the isoquinoline (H-6\u0026prime; and H-7) overlapped in the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR spectrum (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA), they were distinguished by HMQC analysis (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). The NMR, LC-MS, and MS/MS data were consistent with the structure of l-(2-methyl-3-hydroxy-5-hydroxymethyl-4-pyridyl)-6,7-dihydroxy-l,2,3,4-tetrahydroisoquinoline.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the reaction between PM and DA in the presence of tyrosinase and/or GSH.\u003c/b\u003e When not confined to the acidic synaptic vesicles, DA is susceptible to oxidation to DAQ by various processes. In addition to autoxidation, DAQ is generated \u003cem\u003ein vivo\u003c/em\u003e enzymatically by tyrosinase\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, cyclooxygenase\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, xanthinoxidase\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and lipoxygenase\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Transition metals, such as Cu and Fe, also induce or accelerate the oxidation of DA\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. To mimic the intracellular conditions, the present study used tyrosinase, which catalyzes the oxidation of mono- and diphenols to the corresponding quinones with the concomitant reduction of molecular oxygen to water. DA (0.1 mM) was incubated with PM (0.1 mM) in the presence of tyrosinase (0, 0.01, and 0.1 \u0026micro;g/mL) for 72 h. LC/ESI-MS analysis showed the tyrosinase concentration-dependent decrease in DA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and concomitant increase in the formation of PL (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and the PL\u0026ndash;DA adduct (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Thus, tyrosinase-induced efficient oxidation of DA to DAQ increased the formation of the PL\u0026ndash;DA adduct, which supports the proposed mechanism for the reaction between PM and DA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGSH is the main intracellular non-protein thiol, and it protects cells by scavenging free radicals and reactive oxygen/nitrogen species\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. GSH is a co-factor in the reduction of hydrogen peroxide, lipid hydroperoxides, and peroxynitrite by GSH peroxidases and GSH \u003cem\u003eS\u003c/em\u003e-transferases (GSTs)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. GSH also detoxifies reactive metabolites derived from exogenous and endogenous chemicals by forming GSH adducts\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. GSH traps DAQ through GST-mediated adduct formation occurring at C-5 to give 5-\u003cem\u003eS\u003c/em\u003e-glutathionyl dopamine (GSH-DA adduct)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the reaction between DA (1 mM) and PM (0, 1, 2, and 5 mM) in the presence of GSH (1 mM), the formation of GSH disulfide ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, \u003cem\u003em/z\u003c/em\u003e 613.2; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 20.7 min on LC system 4), PL (\u003cem\u003em/z\u003c/em\u003e 168.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 25.1 min), the GSH-DA adduct (\u003cem\u003em/z\u003c/em\u003e 459.2; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 32.6 min), and the PL\u0026ndash;DA adduct (\u003cem\u003em/z\u003c/em\u003e 303.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 42.0 min) were detected with residual GSH (\u003cem\u003em/z\u003c/em\u003e 308.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 10.3 min), DA (\u003cem\u003em/z\u003c/em\u003e 154.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 29.4 min), and PM (\u003cem\u003em/z\u003c/em\u003e 169.1; \u003cem\u003et\u003c/em\u003e\u003csub\u003eR\u003c/sub\u003e, 45.3 min). The GSH-DA adduct level was not affected by the changes in PM concentration. However, the amount of PL and PL\u0026ndash;DA adduct increased with the PM concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Similar patterns of product formation were observed from the reaction between DA and PM in the presence of both tyrosinase (1 \u0026micro;g/mL) and GSH (2 mM), except for the increased GSH-DA adduct concentration due to the tyrosinase-induced increase in DAQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These results indicate that PM scavenges DAQ \u003cem\u003ein vivo\u003c/em\u003e through the formation of the PL\u0026ndash;DA adduct. PM may trap DAQ more efficiently in the brains of PD patients where tyrosinase is important in the production of DAQ but GSH is substantially depleted because of the high levels of oxidative stress\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDA-induced oxidation of α-Syn.\u003c/b\u003e Human α-Syn is composed of 140 amino acids and is mainly expressed at presynaptic sites in the nervous system. Although the function of α-Syn is not well understood, it is thought to have diverse roles in synaptic maintenance, neurotransmitter release/homeostasis, and the regulation of synaptic vesicle pools and trafficking\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. α-Syn exists in a dynamic balance between monomeric and oligomeric states, which allows it to adopt various conformations depending on the environments and interactions\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In PD, α-Syn assembles into β-sheet-rich amyloid-like fibrils, generating intermediate oligomers and causing further aggregation to large, insoluble fibrils, forming Lewy bodies\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. DA promotes oligomerization through several types of interactions with α-Syn. DAQ reacts with Lys residues of α-Syn, generating DAQ-Lys adducts followed by intra-/inter-molecular cross linking to form complicated α-Syn-DA oligomers\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. However, Bisaglia \u003cem\u003eet al\u003c/em\u003e. demonstrated that α-Syn-DAQ adducts retain an unfolded conformation and the main modification occurs through non-covalent interactions\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In addition, DA-mediated Met oxidation in α-Syn has been proposed as the dominant mechanism of cytotoxicity and oligomerization\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBefore investigating the DA-induced modification, the conditions for enzymatic digestion of α-Syn were optimized. No reduction and alkylation steps were needed because α-Syn contains no Cys residues. Two enzymes, V8 and trypsin, were used to digest α-Syn. The sequence coverage (%) based on the number of amino acids was 88.6% for V8 and 68.6% for trypsin (Tables S2 and S3). The overall coverage was 93.6%, missing only C-terminal 9 residues (G\u003csup\u003e132\u003c/sup\u003eYQDYEPEA\u003csup\u003e140\u003c/sup\u003e) that do not contain expected modification sites, such as Lys or Met. α-Syn (2.8 \u0026micro;M, final concentration) was incubated with increasing concentrations of DA (0\u0026ndash;100 molar eq.\u0026nbsp;to α-Syn), followed by proteolysis using V8 or trypsin and LC/ESI-MS(/MS) analysis. A database search based on the MS/MS spectra revealed the presence of four Met-oxidized α-Syn peptides: V8 peptides, M\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eDVFM\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003eKGLSKAKE, DM\u003csup\u003e116\u003c/sup\u003ePVDPDNE, and AYEM\u003csup\u003e127\u003c/sup\u003ePSEE; and tryptic peptides, M\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eDVFM\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003eK, which contain all Met residues in α-Syn. The V8 peptides (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) were used to monitor changes in the oxidation level of α-Syn induced by DA. The MS intensity of each peptide peak increased with the amount of DA (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;C). The relative oxidation (%) was also calculated as relative oxidation (%) = (MS intensity of the Met-oxidized peptide/sum of MS intensity of the Met-oxidized peptide and corresponding intact peptide) \u0026times; 100. The relative oxidations of Met\u003csup\u003e1/5\u003c/sup\u003e, Met\u003csup\u003e116\u003c/sup\u003e, and Met\u003csup\u003e127\u003c/sup\u003e increased in a DA dose-dependent manner up to 90.7%, 94.9%, and 86.9%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u0026ndash;F). For Met\u003csup\u003e116\u003c/sup\u003e, 18.7% relative oxidation was observed even after incubation without DA (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRelationship between DA-induced oxidation and oligomerization of α-Syn.\u003c/b\u003e α-Syn (2.8 \u0026micro;M, final concentration) was treated with DA (0\u0026ndash;100 molar eq.\u0026nbsp;to α-Syn) at 37\u0026deg;C for 24 h and analyzed by SDS-PAGE. Without DA, the α-Syn monomer and dimer were detected. With DA (20 molar eq.), oligomers formed and the monomer and dimer levels decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e8\u003c/span\u003eA left). The oligomerization of α-Syn increased in a DA dose-dependent manner. However, excess DA (100 molar eq.) induced the formation of insoluble aggregates of α-Syn, and decreased the amounts of the soluble oligomer, dimer, and monomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e8\u003c/span\u003eA right). Each band was cut out and subjected to in-gel digestion with V8, and the peptides were analyzed by LC/ESI-MS(/MS). The relative oxidations of Met-containing peptides were compared among the monomer, dimer, and oligomer. The oxidation levels of Met\u003csup\u003e1/5\u003c/sup\u003e were not significantly different among the three polymeric species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). In contrast, the oxidation levels of Met\u003csup\u003e116\u003c/sup\u003e and Met\u003csup\u003e127\u003c/sup\u003e were higher in higher-order species in the order monomer\u0026thinsp;\u0026lt;\u0026thinsp;dimer\u0026thinsp;\u0026lt;\u0026thinsp;oligomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and D). The relative oxidation in the oligomer was 83.1% for Met\u003csup\u003e116\u003c/sup\u003e and 97.3% for Met\u003csup\u003e127\u003c/sup\u003e. These results support a previous study that suggested Met\u003csup\u003e127\u003c/sup\u003e is the main target for oxidative modification by DA\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibition effect of PM on DA-induced oxidation/oligomerization of α-Syn.\u003c/b\u003e α-Syn (2.8 \u0026micro;M, final concentration) was treated with DA (100 molar eq.\u0026nbsp;to α-Syn) in the presence of PM (0\u0026ndash;20 molar eq.\u0026nbsp;to DA) at 37\u0026deg;C for 24 h and analyzed by SDS-PAGE. A portion of reaction mixture was subjected to proteolysis using V8 and analyzed by LC/ESI-MS(/MS). In the absence of PM, all four Met residues were completely oxidized by DA. As the amount of PM increased, the relative oxidations of Met\u003csup\u003e1/5\u003c/sup\u003e, Met\u003csup\u003e116\u003c/sup\u003e, and Met\u003csup\u003e127\u003c/sup\u003e decreased in a dose-dependent manner to 69.9%, 45.4%, and 80.8%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). A PM dose-dependent decrease in oligomerization of α-Syn was also observed in SDS-PAGE analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). These results indicate that PM can inhibit DA-induced oxidation and oligomerization of α-Syn by scavenging DAQ through the formation of the PL\u0026ndash;DA adduct.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe demonstrated that PM reacts with DA to produce a stable PL\u0026ndash;DA adduct. Thus, the initial reaction of the PM amino group at the DAQ carbonyl carbon yields a Schiff base intermediate, which is hydrolyzed to form PL. DA then reacts with the PL aldehyde, followed by intramolecular cyclization to produce the PL\u0026ndash;DA adduct as a 1,2,3,4-tetrahydroisoquinoline derivative. The proposed mechanism was verified by using DA derivatives in the reactions, and the structures were confirmed by LC/ESI-MS(/MS) and NMR analyses. Under intracellular-like conditions in the presence of tyrosinase and/or GSH, the PL\u0026ndash;DA adduct was formed in tyrosinase and PM dose-dependent manners. DA induced the oligomerization of α-Syn via the oxidation of its Met residues, which was inhibited by PM in a dose-dependent manner. Therefore, PM could scavenge DAQ efficiently in the brains of PD patients, in which tyrosinase is overexpressed to produce more DAQ but GSH is substantially depleted by high levels of oxidative stress. PM could also inhibit DA-induced oxidation/oligomerization of α-Syn through the formation of the PL\u0026ndash;DA adduct. Our ongoing studies are focusing on developing analytical methodology for the PL\u0026ndash;DA adduct to use it as a biomarker of oxidative stress and DA oxidation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eMaterials.\u003c/b\u003e HPLC-grade acetonitrile (MeCN), methanol (MeOH), ammonium bicarbonate (NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e), formic acid (FA), ethanol, diethyl ether, 0.1 mol/L hydrochloric acid (HCl), sodium hydroxide, disodium hydrogenphosphate dodecahydrate (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u0026bull;12H\u003csub\u003e2\u003c/sub\u003eO), sodium dihydrogenphosphate dihydrate (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026bull;2H\u003csub\u003e2\u003c/sub\u003eO), glutathione (GSH), hydrochloric acid, dimethyl sulfoxide-d\u003csub\u003e6\u003c/sub\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e), CBB stain One Super, running buffer solution (10x) for SDS-PAGE, and Extra PAGE One Precast Gel 10\u0026ndash;20% were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Pyridoxamine (PM) dihydrochloride, pyridoxal (PL) hydrochloride, endoproteinase Glu-C sequencing grade (V8), and tyrosinase from mushroom were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI). Dopamine (DA) hydrochloride and isoproterenol (IPT) hydrochloride were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). α-Synuclein (α-Syn, human, recombinant) and 3,4-dimethoxyphenethylamine (DPA) were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). Calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Ammonium formate (HCOONH\u003csub\u003e4\u003c/sub\u003e) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Chelex\u0026reg; 100 chelating resin was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Ultrapure water was obtained from a Milli-Q Integral 10 (MilliporeSigma, Burlington, MA) equipped with a 0.22-\u0026micro;m membrane cartridge.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLiquid chromatography.\u003c/b\u003e For LC systems 1\u0026ndash;4, an Agilent 1100 LC system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a 1100 G1312A binary pump, 1100 G1379A degasser, 1100 G1367A autosampler, 1100 G1316A column heater, and a 1100 G1315B photodiode array detector was used. LC systems 1\u0026ndash;3 used a scherzo SW-C18 column (150 \u0026times; 2.0 mm i.d., 3 \u0026micro;m, 13 nm; Imtakt Corporation, Kyoto, Japan) with a flow rate of 0.2 mL/min. Solvent A was H\u003csub\u003e2\u003c/sub\u003eO containing 0.3% (v/v) FA, and solvent B was100 mM HCOONH\u003csub\u003e4\u003c/sub\u003e/MeCN\u0026thinsp;=\u0026thinsp;7/3 (v/v). Gradient elution was performed as follows: LC system 1, 2% B at 0 min, 22% B at 40 min, 90% B at 41 min, 90% B at 51 min, 2% B at 52 min, and 2% B at 82 min; LC system 2, 2% B at 0 min, 62% B at 120 min, 90% B at 121 min, 90% B at 131 min, 2% B at 132 min, and 2% B at 162 min; LC system 3, 2% B at 0 min, 17% B at 30 min, 90% B at 31 min, 90% B at 41 min, 2% B at 42 min, and 2% B at 72 min. LC system 4 used a scherzo SS-C18 column (150 \u0026times; 2.0 mm i.d., 3 \u0026micro;m, 13 nm; Imtakt Corporation) with a flow rate of 0.2 mL/min. Solvent A was H\u003csub\u003e2\u003c/sub\u003eO containing 0.1% (v/v) FA, and solvent B was100 mM HCOONH\u003csub\u003e4\u003c/sub\u003e/MeCN\u0026thinsp;=\u0026thinsp;6/4 (v/v). Gradient elution was performed as follows: 0% B at 0 min, 0% B at 3 min, 45% B at 48 min, 100% B at 49 min, 100% B at 64 min, and 0% B at 65 min.\u003c/p\u003e \u003cp\u003eFor LC system 5, an Ultimate 3000 LC system (Thermo Fisher Scientific, Inc.) equipped with an SRD-3600 degasser, DGP-3600MB pump, FLM-3100B (nano, 2X2P-10P) flow manager, and WPS-3000TBPL (nano, CAP) autosampler was used. A Jupiter C18 column (150 \u0026times; 2.0 mm i.d., 5 \u0026micro;m, 300 \u0026Aring;; Phenomenex, Torrance, CA, USA) with a flow rate of 0.2 mL/min and a column oven temperature of 40\u0026deg;C. Solvent A was H\u003csub\u003e2\u003c/sub\u003eO containing 0.1% (v/v) FA, and solvent B was MeCN containing 0.1% (v/v) FA. Gradient elution was performed as follows: 0% B at 0 min, 0% B at 3 min, 32.5% B at 70 min, 90% B at 71 min, 90% B at 80 min, 0% B at 81 min, and 0% B at 100 min.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMass spectrometry.\u003c/b\u003e The LCQ-DECA ion trap mass spectrometer (Thermo Fisher Scientific Inc.) equipped with an ESI source was used in positive ion mode for LC systems 1\u0026ndash;4. Data was processed using an Xcalibur (version 2.0 SR2, Thermo Fisher Scientific Inc.). The operating conditions were as follows: heated capillary, 300\u0026deg;C; ion spray voltage, 4.5 kV; sheath and auxiliary gas (nitrogen) pressures, 85 and 15 arbitrary units (arb), respectively; mass range, \u003cem\u003em/z\u003c/em\u003e 100\u0026ndash;1000; isolation width, 2; normalized collision energy (CE), 40%; activation Q, 0.25; and activation time, 30 ms.\u003c/p\u003e \u003cp\u003eThe LTQ Orbitrap Velos hybrid ion trap-orbitrap mass spectrometer (Thermo Fisher Scientific, Inc.) equipped with an ESI source was used in the positive ion mode LC system 5. Data were processed using an Xcalibur (version 2.2.SR2). The operating conditions were as follows: analyzer, ion trap; heated capillary, 275\u0026deg;C; spray voltage, 3.0 kV; scan rate, normal (33,000 amu/s); sheath and auxiliary gas (nitrogen) pressures, 50 and 15 arb, respectively. Full scanning analyses were performed in the range of \u003cem\u003em/z\u003c/em\u003e 300\u0026ndash;2000. Tandem mass spectrometry (MS/MS) was performed with data dependent scan and its settings were as follows: precursor, top 10 ions; default charge state, 2; isolation width, 4; normalized CE, 35%; activation Q, 0.25; and activation time, 10 ms.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDatabase search.\u003c/b\u003e Peptide sequences and modifications were identified with Proteome Discoverer 1.3 (Thermo Fisher Scientific, Inc.). The peak list was searched by Sequest (University of Washington, Seattle, WA) against National Center for Biotechnology Information (alpha-synuclein isoform NACP140 [Homo sapiens], NP_000336.1). Search settings were as follows: enzyme, V8 or trypsin; maximum missed cleavage, 3; dynamic modification, oxidation (Met); precursor mass tolerance, 2 Da; fragment mass tolerance, 0.8 Da; target false discovery rate (FDR), 0.01%. DA (or DAQ) (Lys), [DA (or DAQ)\u0026thinsp;\u0026minus;\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO] (Lys), [DA (or DAQ)\u0026thinsp;\u0026minus;\u0026thinsp;2H] (Lys) were added to the dynamic modification list.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNMR.\u003c/b\u003e The NMR spectrum was recorded on a JNM-ECA600 spectrometer (JEOL Ltd., Tokyo, Japan) at 25\u0026deg;C. Data was processed using a Delta NMR software (version 5.2, JEOL Ltd.) an Xcalibur (version 2.0 SR2, Thermo Fisher Scientific Inc.). The sample was dissolved in DMSO-d\u003csub\u003e6\u003c/sub\u003e. Chemical shifts were reported on the δ scale (ppm) by assigning the residual solvent peak for DMSO as internal reference to 2.49 and 39.5 for \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, respectively. Acquisition conditions for \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR were as follows: Domain, proton; offset, 5 ppm; sweep, 15 ppm; points, 16384; prescans, 1; scans, 256. Acquisition conditions for \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH COSY were as follows: Domain, proton (x and y); offset, 5 ppm (x and y); sweep, 15 ppm; points, 1280 (x) and 256 (y); prescans, 4 (x) and 0 (y); scans, 1. Acquisition conditions for \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC HMQC were as follows: Domain, proton (x) and carbon-13 (y); offset, 5 ppm (x) and 85 ppm (y); sweep, 15 ppm (x) and 170 ppm (y); points, 1024 (x) and 256 (y); prescans, 4 (x) and 0 (y); scans, 4.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of PL-DA adduct for NMR analysis.\u003c/b\u003e The water (8 mL) solution containing DA hydrochloride (100 mg) and PL hydrochloride (100 mg) were neutralized with 5 M sodium chloride (1 \u0026micro;L), followed by incubation at room temperature for 4 h. The white precipitate (PL-DA adduct) formed was dissolved in DMSO (60\u0026deg;C, 1.1 mL) and recrystallized by adding a cold water (1.1 mL) dropwise. After centrifugation, the supernatant was removed and the solid was dried under vacuum. PL-DA adduct was obtained as a white solid (6.6 mg isolated).\u003c/p\u003e \u003cp\u003e \u003cb\u003eReaction of PM or PL with DA.\u003c/b\u003e A solution of 1 mM DA hydrochloride in 50 mM Chelex-treated sodium phosphate buffer (PB, pH 7.4, 100 \u0026micro;L) and 1 mM PM dihydrochloride or PL hydrochloride in PB (100 \u0026micro;L) were added to PB (800 \u0026micro;L). The reaction mixture was incubated at 37\u0026deg;C for 96 h. A portion of the reaction mixture (50 \u0026micro;L) was withdrawn at various time points and filtered through a syringe filter, followed by LC/ESI-MS(/MS) analysis using LC system 1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReaction of PM with DPA or IPT.\u003c/b\u003e A solution of 1 mM DPA or IPT in PB (100 \u0026micro;L) and 1 mM PM dihydrochloride in PB (100 \u0026micro;L) were added to PB (800 \u0026micro;L). The reaction mixture was incubated at 37\u0026deg;C for 48 h and analyzed by LC/ESI-MS(/MS) analysis using LC system 2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReaction of PM with DA in the presence of tyrosinase.\u003c/b\u003e DA hydrochloride in PB (1 mM, 100 \u0026micro;L) and PM dihydrochloride in PB (1 mM, 100 \u0026micro;L) were added to a solution of tyrosinase (0, 10, or 100 ng) in PB (800 \u0026micro;L). The reaction mixture was incubated at 37\u0026deg;C for 72 h. A portion of the reaction mixture (50 \u0026micro;L) was withdrawn at various time points and filtered through a syringe filter, followed by LC/ESI-MS(/MS) analysis using LC system 3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReaction of PM with DA in the presence of GSH.\u003c/b\u003e DA hydrochloride in PB (10 mM, 100 \u0026micro;L) and GSH in PB (10 mM, 100 \u0026micro;L) were added to a solution of PM dihydrochloride (0, 1, 2, 5, or 10 \u0026micro;mol) in PB (800 \u0026micro;L). The reaction mixture was incubated at 37\u0026deg;C for 48 h and filtered through a syringe filter, followed by LC/ESI-MS(/MS) analysis using LC system 4.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReaction of PM with DA in the presence of tyrosinase and GSH.\u003c/b\u003e DA hydrochloride in PB (10 mM, 100 \u0026micro;L), tyrosinase in PB (10 \u0026micro;g/mL, 100 \u0026micro;L), and GSH in PB (20 mM, 100 \u0026micro;L) were added to a solution of PM dihydrochloride (0, 1, 2, 5, or 10 \u0026micro;mol) in PB (700 \u0026micro;L). The reaction mixture was incubated at 37\u0026deg;C for 48 h and filtered through a syringe filter, followed by LC/ESI-MS(/MS) analysis using LC system 4.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-solution digestion of α-Syn.\u003c/b\u003e α-Syn (0.1 \u0026micro;g/\u0026micro;L in H\u003csub\u003e2\u003c/sub\u003eO, 20 \u0026micro;L) was added to ammonium bicarbonate buffer (12.5 mM, 80 \u0026micro;L), followed by incubation with sequencing-grade modified trypsin or V8 (0.002 \u0026micro;g/\u0026micro;L, 20 \u0026micro;L) at 37\u0026deg;C for 24 h. A portion of sample (100 \u0026micro;L) was then analyzed by LC/ESI-MS(/MS) using LC system 5.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReaction of α-Syn with DA.\u003c/b\u003e α-Syn (0.1 \u0026micro;g/\u0026micro;L in H\u003csub\u003e2\u003c/sub\u003eO, 20 \u0026micro;L) was incubated with DA (0, 0.14, 1.4, 2.8, 7.0, or 14 nmol) in PB (30 \u0026micro;L) at 37\u0026deg;C for 24 h (final concentration was as follows: α-Syn, 2.8 \u0026micro;M; DA, 0, 2.8, 28, 56, 140, 280 \u0026micro;M). The solutions were digested with trypsin or V8 as described above. A portion of samples (100 \u0026micro;L) were then analyzed by LC/ESI-MS(/MS) using LC system 5. For SDS-PAGE, samples after the 24-h incubation were evaporated to dryness and redissolved in 10 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReaction of α-Syn with DA in the presence of PM.\u003c/b\u003e α-Syn (0.1 \u0026micro;g/\u0026micro;L in H\u003csub\u003e2\u003c/sub\u003eO, 20 \u0026micro;L) and DA in PB (14 nmol, 10 \u0026micro;L) was added to PM (0, 14, 70, 140, or 280 nmol) in PB (20 \u0026micro;L) at 37\u0026deg;C for 24 h (final concentration was as follows: α-Syn, 2.8 \u0026micro;M; DA, 280 \u0026micro;M; PM, 0, 0.28, 1.4, 2.8, 5.6 mM). The solution was digested with trypsin or V8 as described above. A portion of sample (100 \u0026micro;L) was then analyzed by LC/ESI-MS(/MS) using LC system 5. For SDS-PAGE, samples after the 24-h incubation were evaporated to dryness and redissolved in 10 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSDS-PAGE.\u003c/b\u003e The sample (10 \u0026micro;L) was mixed with Laemmli sample buffer (3.3 \u0026micro;L) and boiled at 95\u0026deg;C for 5 min. The sample was loaded onto 10\u0026ndash;20% polyacrylamide gel and run in the running buffer (24 mM Tris, 192 mM glycine, 0.1% (v/v) SDS, pH 8.3) at 300 V for 210 min. The sample was then stained by CBB R-250.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-gel digestion of α-Syn.\u003c/b\u003e α-Syn band was excised from the gel and washed in water (200 \u0026micro;L) for 30 s and then washed two times in 200 \u0026micro;L of 50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e/MeOH (1:1, v/v) for 1 min. The band pieces were dehydrated in 200 \u0026micro;L of 50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e/MeCN (1:1, v/v) for 5 min, followed by 100% MeCN (200 \u0026micro;L) for 30 s while vortex mixing. The supernatant was removed, and band pieces were dried in the centrifugal evaporator. V8 (0.04 \u0026micro;g/\u0026micro;L) suspended in 50 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e was added, and band pieces were rehydrated for 10 min on ice and digested at 37\u0026deg;C for 24 h. The samples were then centrifuged and the supernatant was transferred to a fresh tube, followed by evaporation to dryness. The samples were redissolved in 30 \u0026micro;L of water and analyzed by LC/ESI-MS(/MS) using LC system 5.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the Central Analytical Center at Graduate School of Pharmaceutical Sciences, Tohoku University for the use of their LTQ Orbitrap Velos. This work was supported in part by a Grants-in-Aid for Scientific Research (C) (to S.H.L., 19K07187 for 2019−2021) and for Scientific Research (B) (to T.O., 16H05078 for 2016–2018) from the Japan Society for the Promotion of Science (JSPS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.H.L.: Writing – original draft, Project administration, Methodology, Funding acquisition, Conceptualization. N.M. and A.T.: Visualization, Investigation, Data curation. Y.H.: Writing – review \u0026amp; editing. T.O.: Writing – review \u0026amp; editing, Supervision, Resources, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePoewe, W. et al. Parkinson disease. \u003cem\u003eNat. Rev. Dis. Prim.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 17013 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhidayasiri, R., Kalia, L. V. \u0026amp; Bloem, B. R. Tackling Parkinson\u0026rsquo;s Disease as a Global Challenge. \u003cem\u003eJ. 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Chem.\u003c/em\u003e \u003cb\u003e294\u003c/b\u003e, 5657\u0026ndash;5665 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6478169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6478169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a neurodegenerative disease characterized by loss of dopaminergic neurons causing reduced levels of dopamine (DA), and the presence of Lewy bodies, whose main component is fibrillar α-synuclein (α-Syn). DA is easily oxidized to DA \u003cem\u003eo\u003c/em\u003e-quinone (DAQ), which is a key mechanism for neuronal cell death and α-Syn accumulation. Therefore, a DAQ-quenching molecule should prevent DA-induced pathogenicity in PD. Pyridoxamine (PM) is a promising drug candidate for various chronic diseases, including diabetes, because it scavenges reactive carbonyl species. In this study, we found that PM traps DAQ through stable adduct formation. The initial reaction of PM occurred at the DAQ carbonyl carbon to yield pyridoxal (PL) after hydrolysis. DA then reacted with the PL aldehyde, followed by intramolecular cyclization to produce a PL\u0026ndash;DA adduct. The adduct structure was shown by LC-MS and NMR analyses to be (1-[3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol). The PL\u0026ndash;DA adduct was also detected under intracellular-like conditions. DA caused α-Syn oligomerization via oxidation of methionine residues, which was inhibited by PM in a dose-dependent manner. Therefore, PM could prevent DA-induced adverse effects in the brains of PD patients by forming the PL\u0026ndash;DA adduct, suggesting a possible therapeutic use of PM for scavenging DAQ.\u003c/p\u003e","manuscriptTitle":"Pyridoxamine as a potential scavenger of neurotoxic dopamine quinone: Inhibition of dopamine-induced α-synuclein oligomerization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 01:42:21","doi":"10.21203/rs.3.rs-6478169/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-24T20:30:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-30T16:19:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280341325966816982413612129651876456689","date":"2025-06-26T19:24:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47237491321236471323753180674452327794","date":"2025-06-26T13:28:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-12T14:50:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172360301471183338118179019558920137483","date":"2025-05-06T10:41:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129870072735846937256976886172444825061","date":"2025-05-06T10:14:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-06T09:55:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T08:10:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-28T03:17:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-25T04:15:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-18T10:04:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2c0aa8bd-0202-4638-a890-3eb764b5cb6f","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48133299,"name":"Biological sciences/Biochemistry"},{"id":48133300,"name":"Biological sciences/Chemical biology"},{"id":48133301,"name":"Biological sciences/Neuroscience"},{"id":48133302,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2025-12-29T16:05:41+00:00","versionOfRecord":{"articleIdentity":"rs-6478169","link":"https://doi.org/10.1038/s41598-025-31292-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-26 15:58:00","publishedOnDateReadable":"December 26th, 2025"},"versionCreatedAt":"2025-05-13 01:42:21","video":"","vorDoi":"10.1038/s41598-025-31292-8","vorDoiUrl":"https://doi.org/10.1038/s41598-025-31292-8","workflowStages":[]},"version":"v1","identity":"rs-6478169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6478169","identity":"rs-6478169","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}