The Analgesic Acetaminophen and the Antipsychotic Clozapine Can

Subscriber access provided by PEPPERDINE UNIV

Article

The Analgesic Acetaminophen and the Antipsychotic Clozapine can each Redox-Cycle with Melanin Zülfikar Temo#in, Eunkyoung Kim, Jinyang Li, Lucia Panzella, Maria Laura Alfieri, Alessandra Napolitano, Deanna L. Kelly, William E Bentley, and Gregory F. Payne ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00310 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

The Analgesic Acetaminophen and the Antipsychotic Clozapine can each Redox-Cycle with Melanin

Zülfikar Temoҫin1,2, Eunkyoung Kim2,3, Jinyang Li2,3, Lucia Panzella4, Maria Laura Alfieri4, Alessandra Napolitano4, Deanna L. Kelly5, William E. Bentley2,3 and Gregory F. Payne2,3* 1. Department of Chemistry, Science and Arts Faculty, Kırıkkale University, Yahȿihan, 71450 Kırıkkale, Turkey 2. Institute for Bioscience and Biotechnology Research, University of Maryland 5115 Plant Sciences Building College Park, MD 20742, USA 3. Fischell Department of Bioengineering University of Maryland, College Park, MD 20742, USA 4. Department of Chemical Sciences, University of Naples Federico II Via Cintia 4, I-80126 Naples (Italy) 5. Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, MD 21228, United States. * Corresponding author Email: [email protected] Phone: 301-405-8389 FAX: 301-314-9075

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Melanins are ubiquitous but their complexity and insolubility has hindered characterization of their structures and functions. We are developing electrochemical reverse engineering methodologies that focus on properties and especially on redox properties. Previous studies have shown that melanins: (i) are redox-active and can rapidly and repeatedly exchange electrons with diffusible oxidants and reductants; and (ii) have redox potentials in mid-region of the physiological range. These properties suggest the functional activities of melanins will depend on their redox context. The brain has a complex redox context with steep local gradients in O2 that can promote redox-cycling between melanin and diffusible redox-active chemical species. Here, we performed in vitro reverse engineering studies and report that melanins can redox-cycle with two common redox-active drugs. Experimentally, we used two melanin models: a convenient natural melanin derived from cuttlefish (Sepia melanin) and a synthetic cysteinyldopamine-dopamine core-shell model of neuromelanin. One drug, acetaminophen (APAP) has been used clinically for over a century and recent studies suggest that low doses of APAP can protect the brain from oxidative stress-induced toxicity and neurodegeneration, while higher doses can have toxic effects in the brain. The second drug, clozapine (CLZ), is a second generation antipsychotic with polypharmacological activities that remain incompletely understood. These in vitro observations suggest that the redox activities of drugs may be relevant to their modes-of-action, and that melanins may interact with drugs in ways that affect their activities, metabolism and toxicities.

KEY WORDS Melanin, Neuromelanin, Redox-cycling, Acetaminophen, Clozapine, Drug, Reverse engineering

INTRODUCTION The brain is often cited as being susceptible to oxidative damage because of its high aerobic metabolism and the extensive perfusion with blood. As illustrated in Scheme 1a, these conditions also lead to steep spatial gradients in oxygen concentration which can provide a physicochemical context that promotes redox-cycling.1-2 Specifically, diffusible redox-active molecules only need to transverse short distances in a steep gradient to be switched between exposure to reducing and oxidizing conditions. Understanding the role of redox and redox-cycling reactions in biology can be complex as illustrated by the contrast between electron-transfer and proton-transfer reactions. Proton transfer reactions are rapidly reversible such that an equilibrium equation (i.e., the Henderson-Hasselbach equation) allows calculation of the protonation state of a specific chemical moiety from knowledge of environmental context (i.e., pH) and its chemical properties (i.e., pKa). For redox reactions, thermodynamic descriptions are generally insufficient for determining redox state because there can be significant kinetic barriers associated with electron transfer. Thus, a reductant (e.g., NADPH) can be kinetically stable in presence of an oxidant (e.g., O2) and enzymes may be required to catalyze electron transfer (e.g., NAD(P)H oxidases). A requirement for enzymes can make redox reactions specific and it has been suggested that redox-active drugs may be suitable agents for polypharmacology if they can be designed to redox cycle between one target where they accept electrons and a second target where they donate


Page 2 of 28

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


electrons.3-4 However, not all redox reactions have large kinetic barriers or require enzymatic catalysis as illustrated by the redox-cycling mechanism proposed to explain the linkage between some agricultural chemicals and Parkinson’s disease.5-7 For instance, paraquat is proposed to redox-cycle by accepting electrons from cells (potentially disrupting cellular redox balance) and then transferring these electrons to O2 to generate reactive oxygen species (ROS).8-9 The challenge of unraveling the roles of redox-cycling, ROS, and oxidative stress is illustrated by two emerging areas in redox biology. Several years ago it was reported that ROS generation was a common mechanism in bactericidal antibiotics,10-12 and this report generated considerable excitement and controvery.13-15 Efforts to resolve the apparent contradictions demonstrated the experimental challenges in measuring ROS and characterizing its impacts16-17 even for this comparatively simple biological system (i.e., bacteria).18 A second emerging area is redox signaling where it appears that biology uses redox as a modality for cellular communication.19 Specifically, information is exchanged among cells using diffusible redox signals (e.g., H2O2)20 and these signals are recognized by atomically specific cell receptors (e.g., sulfur switches)21 that can engage multiple intracellular signaling pathways to generate pleiotropic effects.22 These examples illustrate that redox biology is undergoing exciting changes: ROS (e.g., H2O2) is no longer viewed as entirely damaging but is emerging as an integral component of intercellular communication;23 recent results suggest redox-based signaling may impact biological systems at a global level through multiple cell signaling pathways;24 and the concept of oxidative stress is being expanded from an exclusive focus on free radical damage to a broader view that it represents a disruption in redox homeostasis.25 With these changes come the opportunity for new experimental tools.26 Electrochemistry provides unique measurement capabilities,27-28 and we are developing electrochemical methods as in vitro tools for redox biology. Scheme 1b shows that electrode voltages can be precisely set to provide high driving forces for electron transfer reactions and the resulting high localized reaction rates can generate steep gradients. Importantly, as illustrated in Scheme 1b, these imposed voltages can be switched to reverse reactions and thereby reverse the gradients. Further, these reversals can be imposed repeatedly by cycling the voltage between oxidative and reductive values. Thus, electrochemistry provides a simple experimental approach to create redox-based spatial gradients to mimic the spatial variations illustrated in Scheme 1a, and in principle allows features of a complex spatial gradient (“slices in space”) to be recapitulated by temporal variations (“slices in time”). Such an experimental “transformation” from a spatial to a temporal domain is especially useful for studying redox-cycling. Here we performed in vitro experiments to test the hypothesis that melanin can redox-cycle with redoxactive drugs. Neuromelanins are present in the brain although their functions are incompletely understood.29-30 Conventional approaches to study the redox properties of (neuro)melanins are limited for three reasons. First, only small amounts of neuromelanins can be isolated from the human brain and to overcome this limitation most experiments are conducted with natural models (e.g., melanin from cuttlefish; Sepia melanin) or synthetic models of melanin. These model melanins cannot completely recapitulate features of natural neuromelanin in brain which is associated with protein, lipid, and inorganic iron components.31-34 Second, melanins are insoluble particles and conventional



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

electrochemical methods are not well-adapted to studying the redox properties of insoluble particulates. Third, non-electrochemical methods generally can set conditions to be oxidizing or reducing, and thus allow redox reactions to be studied only in one direction. We are developing an alternative electrochemical reverse engineering approach to study the redox properties of insoluble materials (e.g., melanins) as illustrated in Scheme 1c.35-37 There are several features of this reverse engineering approach as illustrated in Scheme 1c. First, we entrap the insoluble melanin adjacent to the electrode in a thin film of the aminopolysaccharide chitosan. This film is permeable to diffusible species and is non-conducting. Second, soluble redoxactive chemical species are purposely added that are capable of permeating throughout the film and shuttling electrons between the melanin sample and the underlying electrode. As illustrated in Scheme 1c, one of these electron shuttles is the drug being tested while the second electron shuttle is Ru(NH3)6Cl3 (Ru3+). As will be shown, both drugs being tested can engage the entrapped melanin in oxidative redox-cycling reactions that serve to transfer electrons from melanin to the electrode when an oxidative electrode voltage is imposed. Since the capacity of the melanin to supply electrons is finite and can be depleted by this oxidative redox-cycling, we add the Ru3+ mediator to provide a mechanism to replenish the melanin with electrons through reductive redox-cycling that occurs under reductive electrode voltages. Third, a sequence of voltage inputs is imposed at the electrode (e.g., cyclic inputs are illustrated in Scheme 1c) to control these redox-cycling reactions. Finally, the electron transfer reactions at the electrode result in electrochemical currents and these response characteristics are analyzed to detect and characterize the drug-melanin redox-cycling. In this study, we tested two drugs acetaminophen (paracetamol) and clozapine. Acetaminophen (APAP) has been used clinically for over a century and remains one of the most widely used drugs38 although the mechanism(s) of action appears to still be under investigation.39 Toxicity studies of acetaminophen have generally focused on the liver while recent studies have suggested toxicity can also occur in the brain.40 Interestingly recent reports indicate that acetaminophen has underappreciated antioxidant properties and at low doses can have protective effects against oxidative stress-induced brain toxicity41 and neurodegeneration.42 The second drug, clozapine (CLZ), is a second generation antipsychotic and considered to be a preeminent example of serendipitous polypharmacology in that it appears to be a broad spectrum ligand43 capable of interacting with multiple target receptors.44 However, there remains considerable controversy regarding clozapine’s unique efficacy and profile.43

RESULTS AND DISCUSSION Characterization of Natural and Synthetic Melanins We investgated the ability of drugs to redox-cycle using both the natural melanin from Sepia and a synthetic cysteinyldopamine-dopamine core-shell model of neuromelanin as described previously.32-33, 45 Figure 1a shows the natural Sepia melanin has a granular morphology with 2-10 µm particles composed of smaller granules of 100-200 nm. Figure 1b shows the synthetic melanin is more homogeneous in structure but with larger size distribution of granules in the range of 0.5-1 µm. Figure 1c shows the


Page 4 of 28

Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


elemental analysis of natural and synthetic melanins using Energy Dispersive X-ray Spectroscopy (EDX). In the table of Figure 1c, the content of nitrogen is a little higher than the value (∼10%) reported in other studies.46-47 The difference might be due to the limitation of EDX spectroscopy to accuarately quantify nitrogen content (EDX spectra of two melanins are shown in Figure S1 of Supporting Information). However, as expected, the synthetic melanin has a compartively higher sulfur content consistent with the incorporation of cysteine into the structure33, 45, 48-49. Initial Evidence for Drug-Melanin Redox Interactions (Natural Melanin) The hypothesis in this work is that acetaminophen (APAP) and clozapine (CLZ) can individually engage melanin in oxidative redox-cycling. One prerequisite for such an oxidative redox-cycling is illustrated in the thermodynamic plot of Figure 2a: the redox potentials of these drugs must be more oxidative than the redox potential of the entrapped melanin. Previous experimental measurements suggest that natural melanin has a redox potential in the range from -0.25 V to +0.25V (vs Ag/AgCl).36-37 To measure the redox potential of APAP and CLZ we used cyclic voltammetry (CV) with a gold electrode immersed in phosphate buffered (0.1 M, pH 7.0) solutions. The CV scan in Figure 2b for APAP (50 µM) shows an oxidative peak at +0.39 V and a reductive peak at +0.29 V which suggests APAP can undergo reversible redox reactions. The CV for CLZ (50 µM) also shows an oxidative peak at +0.38 V but only two small reductive peaks at +0.31 V and +0.13 V. This result suggests CLZ’s oxidation is less reversible which is consistent with the previous studies.50 In summary, Figure 2b shows that APAP and CLZ are redox active and have redox potentials that are more oxidative than the redox potential reported for natural melanin. To provide initial evidence for melanin’s redox-cycling, we prepared electrodes coated with melanincontaining chitosan films and performed CV measurements with these coated-electrodes in solutions containing drug (50 µM) plus Ru3+ (50 µM). One control in Figure 2c is an electrode coated with a melanin-chitosan film immersed in a solution lacking both APAP and Ru3+: no CV peaks are observed for this control. A second control in Figure 2c is an electrode coated with a chitosan film (without melanin) and incubated with APAP and Ru3+: small CV peak currents are observed in this control for APAPoxidation and Ru3+-reduction. When an electrode coated with the melanin-containing film was incubated with both APAP and Ru3+, Figure 2c shows large peak currents for both APAP-oxidation and Ru3+-reduction. Amplification of currents is consistent with an ability of melanin to engage in redoxcycling while the paired amplifications indicate the melanin is accepting electrons from one species (i.e., Ru3+) and donating electrons to a second species (i.e., APAP). Using the same experimental approach we tested the ability of CLZ to engage melanin in oxidative redox-cycling. The CV results in Figure 2d show that the melanin-chitosan coated electrode have amplified currents for both CLZ-oxidation and Ru3+-reduction. This paired amplification in oxidation and reduction currents is an electrochemical signature indicating that the melanin-containing film is redox active and can undergo both oxidative and reductive redox-cycling. We should note that it is often reported that melanins can bind drugs 51-53 and metal ions54-56. We first checked the possibility of melanin-drug binding and the results presented in Figure S2 of Supporting Information indicate that APAP is not adsorbed by either melanins while CLZ can bind to both melanins (29 µg (0.89 nmol) CLZ



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

/mg Sepia melanin, and 8 µg (0.25 nmol) CLZ /mg synthetic melanin). In addition, we checked for melanin-metal (Fe, Ru) binding and results presented in Figure S3 of Supporting Information indicate that neither ruthenium nor iron are bound to our melanins, which supports the assumption that the redox cycling reactions with drugs occurred through the catechol/quinone moieties of melanin rather than through bound metal ions. In summary, the results in Figure 2 provide initial evidence that both APAP and CLZ can undergo redox interactions with melanin. Evidence for Repeated Redox-cycling (Natural Melanin) To provide more definitive evidence for drug-melanin redox-cycling, we subjected the melanincontaining films to repeated cyclic potential inputs and observed the stability of the redox-cycling signatures. Experimentally, we prepared electrodes coated with melanin-chitosan and chitosan (control) films, and immersed these film-coated electrodes in buffered solutions containing drug (50 μM) plus Ru3+ (50 μM), and imposed voltage inputs that were cycled between +0.7 V and -0.4 V for 20 cycles (scan rate 2 mV/s). Figure 3a shows the CVs of APAP and Ru3+ for these two film-coated electrodes. The CVs for the control chitosan film shows small peaks for APAP oxidation at +0.6 V, and the reduction peak for Ru3+ at -0.25 V. The CV for the film with entrapped natural melanin shows amplification both for APAP’s oxidation and Ru3+’s reduction. After the first cycle, there were no noticeable changes in the output current peaks for the CVs with this melanin-containing film. The results in Figure 3a are re-plotted as input-output curves in Figure 3b, and this representation further illustrates that the amplified APAP oxidation and Ru3+ reduction for the melanin-containing film were “steady” over multiple cycles with little attenuation in peak currents. Steady amplification of output currents is a signature of repeated redox-cycling which indicates that the melanin can repeatedly be oxidized by the oxidative redox cycling reaction of APAP and reduced by the reductive redox cycling reaction of Ru3+. Figure 3c shows CV results for the two film-coated electrodes tested in solutions of CLZ and Ru3+. The CVs for the control chitosan film shows small peaks for CLZ oxidation between +0.4 and +0.6 V, and the reduction peak for Ru3+ at -0.26 V. The CV for the film with entrapped natural melanin shows considerable amplification of both CLZ’s oxidation and Ru3+’s reduction and again these output currents were steady after the first cycle. This amplified and steady response is further illustrated by the inputoutput representation shown in Figure 3d. Quantitative analysis of the CV “signals” in Figure 3 was performed as illustrated in Figure 4a for the case of APAP. Specifically, the CVs are divided into 4 quadrants based on whether the currents are oxidizing or reducing, and whether the currents are assigned to APAP or Ru3+. The assignment of each quadrant to a single electrochemical reaction (e.g., APAP oxidation or Ru3+ reduction) is an approximation of the underlying electrochemistry and is used for data analysis. The charge transfer (Q; μC) associated with each quadrant is calculated by integration of the current. Figure 4b shows the values of Q calculated for the APAP oxidation quadrant for the melanin-chitosan film and the control chitosan film. Similarly, Figure 4c shows the Q values for the Ru3+ reduction quadrant (Note: Figure S4 of Supporting Information shows the Q values calculated for all quadrants). For both APAP oxidation and Ru3+ reduction, the melanin-chitosan film shows considerably larger charge transfer consistent with a redox-cycling with the entrapped melanin. Figure 4a also shows the “excess” electron transfer between


Page 6 of 28

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the melanin-chitosan film and the control chitosan film is quantified by the redox capacity (NOx and NRed). Figure 4d shows the calculated values of NOx and NRed are relatively constant over the 20 cycles with average values calculated as 76 ± 8 nmole/cm2 for APAP oxidation and 69 ± 2 nmole/cm2 for Ru3+ reduction. Similar calculations were performed for analysis of the CLZ results. Figure 4e shows the calculated Q values for the CLZ oxidation quadrant and Figure 4f shows the values for the Ru3+ reduction quadrant (Note: Figure S4 of Supporting Information shows the Q values calculated for all quadrants). Comparison of these calculated Q values indicate a nearly steady amplification of CLZ-oxidation and Ru3+–reduction for the melanin-chitosan film consistent with redox-cycling with the melanin. Figure 4g shows the excess electron transfer (NOx and NRed) associated with melanin to be 61 ± 4 nmole/cm2 for CLZ oxidation and 65 ± 2 nmole/cm2 for Ru3+ reduction. In summary, the results in Figure 3 and Figure 4 provide more rigorous evidence that both APAP and CLZ can undergo repeated oxidative redox-cycling with melanin. [Note: we believe the gradual declines in Q and N values observed over multiple cycles may be due to the limited oxidative stability of the drugs over this 8 hour experiment.] Amplified Current Response to Step Changes in Input Voltages (Natural Melanin) In the next set of experiments, film-coated electrodes were probed in the presence of drug (50 μM) plus Ru3+ (50 μM) using step inputs in voltage as illustrated in Figure 5a. An initial charging step was used to transfer electrons from the electrode to the entrapped melanin by imposing reducing conditions (-0.4 V for 30 min) sufficient to engage Ru3+ in reductive redox-cycling. After charging the melanin with electrons, the potential was stepped to an oxidative voltage (+0.7 V for 30 min) and redox-cycling based discharging was monitored. Figure 5b shows that for the case of APAP, considerably larger oxidative charge transfer (Q) was observed for the melanin-containing film compared to the control film. Figure 5b shows this difference was quantified in terms of the excess electron transfer (NOx) which for APAP was calculated to be 310 ± 20 nmole/cm2. Next, Figure 5a shows a reducing voltage was imposed (-0.4 V for 30 min) to re-charge the melanin with electrons through the Ru3+-mediated reductive redox-cycling. Again, Figure 5c shows that for the case of APAP, a considerably larger reductive charge transfer is observed for the melanin-containing film compared to the control film, and the excess electron transfer (NRed) was calculated to be 240 ± 30 nmole/cm2. The excess electron transfer values observed during these step change experiments were 3 to 4 fold larger than values observed in Figure 4d when cyclic voltage inputs were used: presumably this difference reflects the use of longer times to more completely discharge and charge the melanin. Figure 5d and Figure 5e show similar results for the case of CLZ. Specifically, the melanin-containing film showed amplified oxidative charge transfer associated with CLZ’s redox-cycling and amplified reductive charge transfer associated with Ru3+-mediated reductive redox-cycling. The excess electron transfer values calculated from the CLZ results were NOx =250 ± 20 nmole/cm2 and NRed =250 ± 50 nmole/cm2, which were also 4 fold higher than the values calculated from experiments with cyclic imposed voltages (Figure 4g).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In sum, the responses observed in Figure 5 further support the conclusion that both APAP and CLZ can underdo oxidative redox-cycling with natural melanin. Response to Complex Sequence of Input Voltages (Natural Melanin) In a final experimental test for redox-cycling, we immersed the film-coated electrodes in solutions containing drug (50 μM) plus Ru3+ (50 μM), and imposed a sequence of cyclic voltage inputs of differing amplitudes. The initial input amplitude in Figure 6a (designated “Steady 1”) was applied between +0.7 and -0.4 V which, as shown in Figure 3a, allows both oxidative APAP-mediated redox-cycling and Ru3+– mediated reductive redox-cycling. As expected, the output curve in Figure 6a and sequence of CVs in Figure 6b show time-invarying steady current outputs for this initial input sequence. As indicated by the input curve in Figure 6a, the second voltage amplitude was varied between 0 and 0.4 V which is sufficiently reductive for Ru3+-reduction but insufficiently oxidative for APAP-oxidation (this amplitude is designated Unsteady-Reduction; UR). As expected, the output curve in Figure 6a and the sequence of CVs in Figure 6b shows no APAP oxidation peaks and a progressive decay in the Ru3+reduction peak currents. This time-varying (i.e., unsteady) output response is consistent with a progressive charging of the melanin with electrons by Ru3+ redox-cycling but this reductive redox-cycling cannot continue indefinitely in the absence of a discharging mechanism (i.e., the melanin becomes fully reduced or “saturated” with electrons). The third input amplitude was adjusted back to the initial amplitude. The output curve in Figure 6a and CVs in Figure 6b show that output currents return to a time-invarying steady response. The fourth input amplitude (designated Unsteady-Oxidation; UO) was varied between 0 and +0.7 V which is sufficiently oxidative for APAP oxidation but insufficiently reductive for Ru3+-reduction. As expected, the output curve in Figure 6a and the sequence of CVs in Figure 6b show no Ru3+-reduction while the APAP oxidation peaks were observed to progressively decay with each cycle. This time-varying (i.e., unsteady) output response is consistent with a progressive discharging of electrons from melanin by APAP redoxcycling but this oxidation cannot continue indefinitely in the absence of a mechanism to replenish the melanin with electrons (i.e., the melanin becomes fully depleted of electrons). The final input amplitude was adjusted back to the initial amplitude, and the output curve in Figure 6a and CVs in Figure 6b show the output currents return to a time-invarying steady response. We used the same methodology described in Figure 4a to quantitatively analyze the CV signals in Figure 6b. Figure 6c shows the calculated Q values in the APAP oxidation region, Figure 6d shows the calculated Q values in the Ru3+ reduction region, and Figure 6e shows the excess electron transfer (NOx and NRed). In essence, Figure 6e provides a quantitative summary of the experiment and indicates that incorporation of melanin in the film leads to large excess electron transfer for both APAP oxidation and Ru3+ reduction which is consistent with the redox-cycling. In the absence of balanced redox-cycling, the NOx and NRed values are observed to decay consistent with the requirement that both oxidative and reductive redox-cycling mechanisms must be operative to achieve steady paired amplifications. [Note:


Page 8 of 28

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


we believe the gradual decline in Q and N values observed for APAP oxidation over this 13 hour experiment is due to the limited oxidative stability of APAP.] The same experimental approach and signal analysis was performed for the case of CLZ. Figure 7a shows the input-output curves and Figure 7b shows the summary of results for this experiment (Figure S5 of the Supporting Information shows the complete data set for this experiment). The results for CLZ are similar to those with APAP and further support the conclusion that these drugs can each undergo oxidative redox-cycling with natural melanin. Confirmatory Studies with Synthetic Cysteinyldopamine-Dopamine Core-Shell Melanin Finally, we tested the synthetic cysteinyldopamine-dopamine core-shell model of neuromelanin for redox-cycling using the same experimental approach as in Figure 6. Previously, this synthetic melanin was observed to be redox-active and could undergo both oxidation and reduction32-33. Figure 8a shows the input-output curves for the case of APAP, while Figure 8b shows the corresponding CVs. Figure 8 also shows the calculated values for Q and N. Consistent with results in Figure 6 for natural melanin, the results in Figure 8 show: both APAP and Ru3+ currents are amplified in the presence of the synthetic melanin; amplifications are nearly steady and paired when an input voltage amplitude is sufficient to allow both oxidative and reductive redox-cycling; and the amplified currents are unbalanced if the input voltage amplitude only allows one redox-cycling mechanism to occur. Finally, we used the same experimental approach to test redox interactions between CLZ and the synthetic cysteinyldopamine-dopamine core-shell model. The results in Figure 9 for CLZ are similar to those observed for APAP (Figure S6 of the Supporting Information shows the complete data set for this experiment). These observations are all consistent with the conclusion that both APAP and CLZ can undergo oxidative redox-cycling with the cysteinyldopamine-dopamine core-shell model melanin.

CONCLUSIONS Melanins are ubiquitous in nature but their functions are poorly understood. Previous studies have shown that melanins have redox activities and can accept electrons from common biological reductants (e.g., NADPH and glutathione)57-58 and donate electrons to O2 to generate ROS.36, 59-63 Using our electrochemical reverse engineering approach, we extended these studies to show that melanin’s redox activity is reversible in the sense that melanins can rapidly and repeatedly exchange electrons. Thus melanins can serve (at least in some circumstances) as a kind of redox catalyst for electron transfer.37 Further, melanins have redox potentials in physiologically-intermediate range and can both donate and accept electrons37 analogous to ubiquinones in the respiratory electron transport chain. These observations indicate that melanins may be important participants in redox-biology and may perform functions such as redox-catalysis, redox buffering and/or radical scavenging. In addition, these observations indicate that melanin’s pathophysiological behavior will be highly dependent on redox context. In the brain, the redox context is quite complex with steep gradients that allow soluble redoxactive chemical species to be exposed to oxidizing and reducing regions by diffusing only short distances. Thus, redox-cycling could be an important but under-appreciated activity of neuromelanins.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 28

The specific conclusion of this in vitro study is that two drugs (acetaminophen and clozapine) with somewhat reversible redox-activities can oxidatively redox-cycle with melanin. It is known that some drugs can bind to melanin51-53 but to our knowledge, this is the first report that drugs can actually exchange electrons with melanin. Both drugs in our study are relatively old, neither were designed for redox-cycling, and there remain questions of their mechanisms of action. One implication of these in vitro observations is that the redox activities of these drugs may be relevant to their mode of action (e.g., potentially through redox-responsive receptors) as suggested for an even older drug methylene blue.64 A second broad implication is that melanins may interact with drugs in ways that affect their activities, metabolism and toxicities. The obvious question for any in vitro or reverse engineering study is if/how the in vitro observations relate to in vivo biological activities. Despite these inherent limitations, these observations may provide important clues of the polypharmacological, antioxidant and pro-oxidant activities of drugs which could be important for off-target effects.

EXPERIMENTAL SECTION Chemicals. The following were purchased from Sigma-Aldrich: chitosan, Ru(NH3)6Cl3 (Ru3+), acetaminophen (APAP), clozapine (CLZ), and melanin from Sepia officinalis. The water (>18 MΩ) used in this study was obtained from a Super Q water system (Millipore). Chitosan solutions (1%, pH 5.5) were prepared by dissolving chitosan flakes in HCl to achieve a final pH of 5-6. The solutions of acetaminophen, clozapine and Ru3+ were prepared in phosphate buffer (0.1 M, pH 7.0). Synthetic melanin preparation. 5-S-Cysteinlydopamine was prepared on gram scale by the method previously described.65 Cysteinyldopamine melanin was prepared by peroxide/H2O2 oxidation as previously described.66 The core-shell melanin was prepared by cysteinyldopamine melanin induced oxidation of dopamine (dopamine/cysteinyldopamine melanin 1:1 w/w) as previously described.48 Preparation of Film-Coated Electrode. For the fabrication of the melanin-chitosan film, melanin (5 mg/mL) was suspended in the chitosan solution (0.5%, pH 5.5), and 20 µL of this suspension was spread onto the standard gold electrode (r =1 mm). The film was vacuum-dried at room temperature for 20 min and then immersed in phosphate buffer (pH 7.0; 30 min) to neutralize the chitosan and thus form an insoluble film. Electrochemical Instruments. Electrochemical measurements (cyclic voltammetry and i-t amperometry) were performed using a three electrode system with Ag/AgCl as a reference electrode and Pt wire as a counter electrode (CHI Instruments 6273C electrochemical analyzer). Most electrochemical experiments were performed in a mixed solution containing both 50 µM Ru3+ and 50 µM APAP or 50 µM CLZ in phosphate buffer (0.1 M, pH 7.0). Air was excluded during electrochemical measurements by purging the solution with N2 during the experiment. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDX). Natural Sepia melanin or synthetic melanin powders were suspended in water and then spread onto gold substrates after which the substrates were dried. For the elemental analysis of Sepia melanin or synthetic melanin,


Page 11 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Energy Dispersive X-ray (SEM-EDX) spectroscopy was performed using a Bruker SOL-XE detector (Bruker AXS Inc.). The elemental analysis was obtained by averaging the analyzed values at three different points of each sample (melanin spread on the gold substrate). For image analysis, all samples were coated with Au under argon plasma using a Hummer X sputter coater (Anatech, Springfield, VA) and the images were obtained using a scanning electron microscope (SEM, SU-70, Hitachi, Pleasanton, CA).

SUPPORTING INFORMATION Additional Information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. EDX spectra for element analysis, UV-VIS spectra for acetaminophen and clozapine with/without melanin, Quantitative analysis of redox-cycling signatures including controls, Supporting data for clozapine redox-cycling reaction. (PDF)

AUTHOR INFORMATION Corresponding author Gregory F. Payne, E-mail: [email protected]. Tel: 301-405-8389

Fax: 301-314-9075.

ORCID Gregory F. Payne: 0000-0001-6638-9459 Eunkyoung Kim: 0000-0003-2566-4041 Author Contributions Z.T., E.K. and G.F.P. conceived the project and designed the experiments. Z.T. and E.K. performed the experiments. L.P, M.L.A. and A.N synthesized the cysteinyldopamine melanin. A.N, D.K. W.E.B. and G.F.P. supervised the work. Z.T., E.K, J.L., A.N. and G.F.P. wrote and edited the manuscript. Notes The authors declare no competing financial interest.

Funding The authors gratefully acknowledge financial support from the United State’s National Science Foundation (CBET-1435957) and the Department of Defense (Defense Threat Reduction Agency; HDTRA1-13-1-0037).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACKNOWLEDGMENT We acknowledge the support of the Maryland NanoCenter and its AIMLab. Zulfikar Temocin thanks the Scientific and Technological Research Council of Turkey (TUBITAK) for their support to allow him to conduct this research at the University of Maryland, College Park in USA.


Page 12 of 28

Page 13 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURE LEGENDS Scheme 1. (a) The redox-context of the brain is complex with steep gradients between oxidizing and reducing conditions that provide the opportunity for redox-active molecules to engage in redox-cycling over short distances. (b) Electrochemistry allows spatial gradients to be generated, while cyclically imposed voltages allows redox-cycling to be probed over short times. (c) Electrochemical reverse engineering to evaluate redox-cycling interactions between melanin and the redox-active drugs acetaminophen (APAP) and clozapine (CLZ). Figure 1. Morphological and chemical characterization. (a) SEM images of natural Sepia melanin show granular structure. (b) SEM images of synthetic melanin show homogeneous structure. (c) Chemical analysis of natural Sepia melanin and synthetic melanin using Energy Dispersive X-ray Spectroscopy (EDX). Figure 2. Initial evidence for drug-melanin redox-cycling (natural Sepia melanin). (a) Redox-cycling interactions with melanin: reductive redox-cycling with Ru(NH3)6Cl3 (Ru3+) transfers electrons from the electrode to melanin and oxidative redox-cycling with either APAP or CLZ transfers electrons from melanin to the electrode. (b) Control cyclic voltammograms (CVs) with uncoated gold electrodes show APAP and CLZ can each undergo somewhat reversible redox reactions. Cyclic voltammogram (CV) of melanin-chitosan film shows the paired amplifications for the reduction of Ru3+ and the oxidation of either (c) APAP or (d) CLZ (compared to the control chitosan film). All CVs were for 3rd cycle at a scan rate of 2 mV/s. Figure 3. Evidence for repeatable redox-cycling (natural Sepia melanin). (a) Cyclic voltammograms (CVs) and (b) input-output curves show that melanin-containing films have paired amplifications for Ru3+-reduction and APAP-oxidation that appear “steady” over 20 cycles. (c) CVs and (d) input-output curves also show paired and steady amplifications for Ru3+-reduction and CLZ-oxidation. CV scan rate of 2 mV/s. Figure 4. Quantitative analysis of redox-cycling signatures (natural Sepia melanin). (a) Schematic illustrating quantification of output response “signal” in terms of charge transfer (Q) and excess electron transfer (N) for drug-oxidation and Ru3+-reduction. Near steady responses are observed for (b) APAPoxidation, (c) Ru3+-reduction, and (d) their excess electron transfers. Similarly, near steady responses are observed for (e) CLZ-oxidation, (f) Ru3+-reduction, and (g) their excess electron transfers. Figure 5. Evidence for redox-cycling using step changes in input voltages (natural Sepia melanin). (a) Three step input potentials are imposed in the presence of Ru3+ and drug to probe for melanin’s oxidative discharging and reductive charging. (b) APAP-mediated discharging of electrons (oxidation) and (c) subsequent Ru3+-mediated charging of electrons (reduction). (d) CLZ-mediated discharging of electrons (oxidation) and (e) subsequent Ru3+-mediated charging of electrons (reduction). Figure 6. Evidence for APAP’s redox-cycling using a complex sequence of input voltages (natural Sepia melanin). (a) Input-output curves for cyclic potential inputs of differing amplitudes. (b) CV curves show steady (time-invarying) output currents when large input amplitudes are imposed to allow both



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

oxidative and reductive redox-cycling, while un-steady (time-varying) output currents are observed when the input amplitude is limited to allow only reductive redox-cycling or only oxidative redox-cycling. Quantification of output response in terms of charge transfer (Q) for (c) APAP- oxidation and (d) Ru3+reduction, and (e) excess electron transfers for APAP-oxidation (NOx) and Ru3+-reduction (NRed). Figure 7. Evidence for CLZ’s redox-cycling using a complex sequence of input voltages (natural Sepia melanin). (a) Input-output curves for cyclic potential inputs of differing amplitudes. (b) Summary of output response for excess electron transfers for CLZ-oxidation (NOx) and Ru3+-reduction (NRed). Figure 8. Evidence for APAP’s redox-cycling using a complex sequence of input voltages (synthetic melanin). (a) Input-output curves for cyclic potential inputs of differing amplitudes. (b) CV curves show steady (time-invarying) output currents when large input amplitudes are imposed to allow both oxidative and reductive redox-cycling, while un-steady (time-varying) output currents are observed when the input amplitude is limited to allow only reductive redox-cycling or only oxidative redox-cycling. Quantification of output response in terms of charge transfer (Q) for (c) APAP- oxidation and (d) Ru3+reduction, and (e) excess electron transfers for APAP-oxidation (NOx) and Ru3+-reduction (NRed). Figure 9. Evidence for CLZ’s redox-cycling using a complex sequence of input voltages (synthetic melanin). (a) Input-output curves for cyclic potential inputs of differing amplitudes. (b) Summary of output response for excess electron transfers for CLZ-oxidation (NOx) and Ru3+-reduction (NRed).

REFERENCES 1. Mehta, N. J.; Asmaro, K.; Hermiz, D. J.; Njus, M. M.; Saleh, A. H.; Beningo, K. A.; Njus, D., Hypochlorite converts cysteinyl-dopamine into a cytotoxic product: A possible factor in Parkinson's Disease. Free Radical Biol. Med. 2016, 101, 44-52. 2. Herrera, A.; Munoz, P.; Steinbusch, H. W. M.; Segura-Aguilar, J., Are Dopamine Oxidation Metabolites Involved in the Loss of Dopaminergic Neurons in the Nigrostriatal System in Parkinson's Disease? ACS Chem. Neurosci. 2017, 8 (4), 702-711. 3. Sidorov, P.; Desta, I.; Chesse, M.; Horvath, D.; Marcou, G.; Varnek, A.; Davioud-Charvet, E.; Elhabiri, M., Redox Polypharmacology as an Emerging Strategy to Combat Malarial Parasites. Chemmedchem 2016, 11 (12), 1339-1351. 4. Ehrhardt, K.; Deregnaucourt, C.; Goetz, A. A.; Tzanova, T.; Gallo, V.; Arese, P.; Pradines, B.; Adjalley, S. H.; Bagrel, D.; Blandin, S.; Lanzer, M.; Davioud-Charvet, E., The Redox Cycler Plasmodione Is a FastActing Antimalarial Lead Compound with Pronounced Activity against Sexual and Early Asexual BloodStage Parasites. Antimicrob. Agents Chemother. 2016, 60 (9), 5146-5158. 5. Kamel, F., Paths from Pesticides to Parkinson's. Science 2013, 341 (6147), 722-723. 6. Goldman, S. M., Environmental toxins and Parkinson's disease. Annu. Rev. Pharmacol. Toxicol. 2014, 54, 141-64. 7. Baltazar, M. T.; Dinis-Oliveira, R. J.; de Lourdes Bastos, M.; Tsatsakis, A. M.; Duarte, J. A.; Carvalho, F., Pesticides exposure as etiological factors of Parkinson's disease and other neurodegenerative diseases--a mechanistic approach. Toxicol. Lett. 2014, 230 (2), 85-103.


Page 14 of 28

Page 15 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8. Drechsel, D. A.; Patel, M., Differential Contribution of the Mitochondrial Respiratory Chain Complexes to Reactive Oxygen Species Production by Redox Cycling Agents Implicated in Parkinsonism. Toxicol. Sci. 2009, 112 (2), 427-434. 9. Rodriguez-Rocha, H.; Garcia-Garcia, A.; Pickett, C.; Li, S. M.; Jones, J.; Chen, H.; Webb, B.; Choi, J.; Zhou, Y.; Zimmerman, M. C.; Franco, R., Compartmentalized oxidative stress in dopaminergic cell death induced by pesticides and complex I inhibitors: Distinct roles of superoxide anion and superoxide dismutases. Free Radical Biol. Med. 2013, 61, 370-383. 10. Kohanski, M. A.; Dwyer, D. J.; Hayete, B.; Lawrence, C. A.; Collins, J. J., A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007, 130 (5), 797-810. 11. Dwyer, D. J.; Collins, J. J.; Walker, G. C., Unraveling the Physiological Complexities of Antibiotic Lethality. In Annual Review of Pharmacology and Toxicology, Vol 55, Insel, P. A., Ed. Annual Reviews: Palo Alto, 2015; Vol. 55, pp 313-332. 12. Dwyer, D. J.; Belenky, P. A.; Yang, J. H.; MacDonald, I. C.; Martell, J. D.; Takahashi, N.; Chan, C. T. Y.; Lobritz, M. A.; Braff, D.; Schwarz, E. G.; Ye, J. D.; Pati, M.; Vercruysse, M.; Ralifo, P. S.; Allison, K. R.; Khalil, A. S.; Ting, A. Y.; Walker, G. C.; Collins, J. J., Antibiotics induce redox-related physiological alterations as part of their lethality. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (20), E2100-E2109. 13. Liu, Y. Y.; Imlay, J. A., Cell Death from Antibiotics Without the Involvement of Reactive Oxygen Species. Science 2013, 339 (6124), 1210-1213. 14. Keren, I.; Wu, Y. X.; Inocencio, J.; Mulcahy, L. R.; Lewis, K., Killing by Bactericidal Antibiotics Does Not Depend on Reactive Oxygen Species. Science 2013, 339 (6124), 1213-1216. 15. Mahoney, T. F.; Silhavy, T. J., The Cpx Stress Response Confers Resistance to Some, but Not All, Bactericidal Antibiotics. J. Bacteriol. 2013, 195 (9), 1869-1874. 16. Van Acker, H.; Gielis, J.; Acke, M.; Cools, F.; Cos, P.; Coenye, T., The Role of Reactive Oxygen Species in Antibiotic-Induced Cell Death in Burkholderia cepacia Complex Bacteria. PLoS One 2016, 11 (7), 20. 17. Renggli, S.; Keck, W.; Jenal, U.; Ritz, D., Role of Autofluorescence in Flow Cytometric Analysis of Escherichia coli Treated with Bactericidal Antibiotics. J. Bacteriol. 2013, 195 (18), 4067-4073. 18. Imlay, J. A., Diagnosing oxidative stress in bacteria: not as easy as you might think. Curr. Opin. Microbiol. 2015, 24, 124-131. 19. Janssen-Heininger, Y. M. W.; Mossman, B. T.; Heintz, N. H.; Forman, H. J.; Kalyanaraman, B.; Finkel, T.; Stamler, J. S.; Rhee, S. G.; van der Vliet, A., Redox-based regulation of signal transduction: Principles, pitfalls, and promises. Free Radical Biol. Med. 2008, 45 (1), 1-17. 20. D'Autreaux, B.; Toledano, M. B., ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat. Rev. Mol. Cell Biol. 2007, 8 (10), 813-824. 21. Go, Y.-M.; Chandler, J. D.; Jones, D. P., The cysteine proteome. Free Radical Biol. Med. 2015, 84, 227-245. 22. García-Santamarina, S.; Boronat, S.; Hidalgo, E., Reversible Cysteine Oxidation in Hydrogen Peroxide Sensing and Signal Transduction. Biochemistry 2014, 53 (16), 2560-2580. 23. Bartosz, G., Reactive oxygen species: Destroyers or messengers? Biochem. Pharmacol. 2009, 77 (8), 1303-1315. 24. Go, Y.-M.; Jones, D. P., Redox biology: Interface of the exposome with the proteome, epigenome and genome. Redox Biology 2014, 2, 358-360. 25. Jones, D. P., Redefining Oxidative Stress. Antioxidants & Redox Signaling 2006, 8 (9-10), 1865-1879. 26. Li, J.; Liu, Y.; Kim, E.; March, J. C.; Bentley, W. E.; Payne, G. F., Electrochemical reverse engineering: A systems-level tool to probe the redox-based molecular communication of biology. Free Radical Biol. Med. 2017, 105, 110-131. 27. Rodeberg, N. T.; Sandberg, S. G.; Johnson, J. A.; Phillips, P. E. M.; Wightman, R. M., Hitchhiker's Guide to Voltammetry: Acute and Chronic Electrodes for in Vivo Fast-Scan Cyclic Voltammetry. ACS Chem. Neurosci. 2017, 8 (2), 221-234.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

28. Veloso, A.; Kerman, K., Advances in electrochemical detection for study of neurodegenerative disorders. Anal. Bioanal. Chem. 2013, 405 (17), 5725-5741. 29. Zucca, F. A.; Basso, E.; Cupaioli, F. A.; Ferrari, E.; Sulzer, D.; Casella, L.; Zecca, L., Neuromelanin of the Human Substantia Nigra: An Update. Neurotox. Res. 2014, 25 (1), 13-23. 30. Zucca, F. A.; Giaveri, G.; Gallorini, M.; Albertini, A.; Toscani, M.; Pezzoli, G.; Lucius, R.; Wilms, H.; Sulzer, D.; Ito, S.; Wakamatsu, K.; Zecca, L., The neuromelanin of human substantia nigra: physiological and pathogenic aspects. Pigment Cell Res. 2004, 17 (6), 610-7. 31. Ferrari, E.; Capucciati, A.; Prada, I.; Zucca, F. A.; D'Arrigo, G.; Pontiroli, D.; Bridelli, M. G.; Sturini, M.; Bubacco, L.; Monzani, E.; Verderio, C.; Zecca, L.; Casella, L., Synthesis, Structure Characterization, and Evaluation in Microglia Cultures of Neuromelanin Analogues Suitable for Modeling Parkinson's Disease. ACS Chem. Neurosci. 2017, 8 (3), 501-512. 32. Kim, E.; Leverage, W. T.; Liu, Y.; Panzella, L.; Alfieri, M. L.; Napolitano, A.; Bentley, W. E.; Payne, G. F., Paraquat-Melanin Redox-Cycling: Evidence from Electrochemical Reverse Engineering. ACS Chem. Neurosci. 2016, 7 (8), 1057-67. 33. Bush, W. D.; Garguilo, J.; Zucca, F. A.; Albertini, A.; Zecca, L.; Edwards, G. S.; Nemanich, R. J.; Simon, J. D., The surface oxidation potential of human neuromelanin reveals a spherical architecture with a pheomelanin core and a eumelanin surface. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (40), 14785-14789. 34. Ferrari, E.; Engelen, M.; Monzani, E.; Sturini, M.; Girotto, S.; Bubacco, L.; Zecca, L.; Casella, L., Synthesis and structural characterization of soluble neuromelanin analogs provides important clues to its biosynthesis. JBIC Journal of Biological Inorganic Chemistry 2013, 18 (1), 81-93. 35. Kim, E.; Gordonov, T.; Liu, Y.; Bentley, W. E.; Payne, G. F., Reverse engineering to suggest biologically relevant redox activities of phenolic materials. ACS Chem. Biol. 2013, 8 (4), 716-24. 36. Kim, E.; Liu, Y.; Leverage, W. T.; Yin, J. J.; White, I. M.; Bentley, W. E.; Payne, G. F., Contextdependent redox properties of natural phenolic materials. Biomacromolecules 2014, 15 (5), 1653-62. 37. Kim, E.; Panzella, L.; Micillo, R.; Bently, W.; Napolitano, A.; Payne, G., Reverse Engineering Applied to Red Human Hair Pheomelanin Reveals Redox-Buffering as a Pro-Oxidant Mechanism. Sci. Rep. 2015. 38. Bertolini, A.; Ferrari, A.; Ottani, A.; Guerzoni, S.; Tacchi, R.; Leone, S., Paracetamol: New vistas of an old drug. Cns Drug Reviews 2006, 12 (3-4), 250-275. 39. Anderson, B. J., Paracetamol (Acetaminophen): mechanisms of action. Pediatric Anesthesia 2008, 18 (10), 915-921. 40. Ghanem, C. I.; Perez, M. J.; Manautou, J. E.; Mottino, A. D., Acetaminophen from liver to brain: New insights into drug pharmacological action and toxicity. Pharmacol. Res. 2016, 109, 119-131. 41. Nazıroğlu, M.; Cihangir Uğuz, A.; Koçak, A.; Bal, R., Acetaminophen at Different Doses Protects Brain Microsomal Ca2+-ATPase and the Antioxidant Redox System in Rats. J. Membr. Biol. 2009, 231 (2), 5764. 42. Zhao, W. X.; Zhang, J. H.; Cao, J. B.; Wang, W.; Wang, D. X.; Zhang, X. Y.; Yu, J.; Zhang, Y. Y.; Zhang, Y. Z.; Mi, W. D., Acetaminophen attenuates lipopolysaccharide-induced cognitive impairment through antioxidant activity. J. Neuroinflammation 2017, 14, 15. 43. Wenthur, C. J.; Lindsley, C. W., Classics in Chemical Neuroscience: Clozapine. ACS Chem. Neurosci. 2013, 4 (7), 1018-1025. 44. Cardozo, T.; Shmelkov, E.; Felsovalyi, K.; Swetnam, J.; Butler, T.; Malaspina, D.; Shmelkov, S. V., Chemistry-based molecular signature underlying the atypia of clozapine. Translational Psychiatry 2017, 7, 7. 45. Ito, S., Encapsulation of a reactive core in neuromelanin. Proc. Natl. Acad. Sci. USA 2006, 103 (40), 14647-14648. 46. Eibl, O.; Schultheiss, S.; Blitgen-Heinecke, P.; Schraermeyer, U., Quantitative chemical analysis of ocular melanosomes in the TEM. Micron 2006, 37 (3), 262-276.


Page 16 of 28

Page 17 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


47. Costa, T. G.; Younger, R.; Poe, C.; Farmer, P. J.; Szpoganicz, B., Studies on Synthetic and Natural Melanin and Its Affinity for Fe(III) Ion. Bioinorg. Chem. Appl. 2012, 2012, 712840. 48. Greco, G.; Panzella, L.; Gentile, G.; Errico, M. E.; Carfagna, C.; Napolitano, A.; d'Ischia, M., A melanin-inspired pro-oxidant system for dopa(mine) polymerization: mimicking the natural casing process. Chem. Commun. 2011, 47 (37), 10308-10310. 49. Wakamatsu, K.; Murase, T.; Zucca, F. A.; Zecca, L.; Ito, S., Biosynthetic pathway to neuromelanin and its aging process. Pigment Cell & Melanoma Research 2012, 25 (6), 792-803. 50. Ben-Yoav, H.; Winkler, T. E.; Kim, E.; Chocron, S. E.; Kelly, D. L.; Payne, G. F.; Ghodssi, R., Redox cycling-based amplifying electrochemical sensor for in situ clozapine antipsychotic treatment monitoring. Electrochim. Acta 2014, 130, 497-503. 51. Karlsson, O.; Lindquist, N. G., Melanin and neuromelanin binding of drugs and chemicals: toxicological implications. Arch. Toxicol. 2016, 90 (8), 1883-1891. 52. Larsson, B. S., Interaction Between Chemicals and Melanin. Pigment Cell Res. 1993, 6 (3), 127-133. 53. Ito, S., A Chemist's View of Melanogenesis. Pigment Cell Res. 2003, 16 (3), 230-236. 54. Zecca, L.; Tampellini, D.; Gatti, A.; Crippa, R.; Eisner, M.; Sulzer, D.; Ito, S.; Fariello, R.; Gallorini, M., The neuromelanin of human substantia nigra and its interaction with metals. J. Neural Transm. 2002, 109 (5), 663-672. 55. Hong, L.; Simon, J. D., Current Understanding of the Binding Sites, Capacity, Affinity, and Biological Significance of Metals in Melanin. The Journal of Physical Chemistry B 2007, 111 (28), 7938-7947. 56. Ferrari, E.; Capucciati, A.; Prada, I.; Zucca, F. A.; D’Arrigo, G.; Pontiroli, D.; Bridelli, M. G.; Sturini, M.; Bubacco, L.; Monzani, E.; Verderio, C.; Zecca, L.; Casella, L., Synthesis, Structure Characterization, and Evaluation in Microglia Cultures of Neuromelanin Analogues Suitable for Modeling Parkinson’s Disease. ACS Chem. Neurosci. 2017, 8 (3), 501-512. 57. Panzella, L.; Leone, L.; Greco, G.; Vitiello, G.; D'Errico, G.; Napolitano, A.; d'Ischia, M., Red human hair pheomelanin is a potent pro-oxidant mediating UV-independent contributory mechanisms of melanomagenesis. Pigment Cell Melanoma Res. 2014, 27 (2), 244-252. 58. Napolitano, A.; Panzella, L.; Monfrecola, G.; d'Ischia, M., Pheomelanin-induced oxidative stress: bright and dark chemistry bridging red hair phenotype and melanoma. Pigment Cell & Melanoma Research 2014, 27 (5), 721-733. 59. Napolitano, A.; Panzella, L.; Leone, L.; D'Ischia, M., Red Hair Benzothiazines and Benzothiazoles: Mutation-Inspired Chemistry in the Quest for Functionality. Acc. Chem. Res. 2013, 46 (2), 519-528. 60. Sarna, T.; Sealy, R. C., Free radicals from eumelanins: Quantum yields and wavelength dependence. Arch. Biochem. Biophys. 1984, 232 (2), 574-578. 61. Ye, T.; Hong, L.; Garguilo, J.; Pawlak, A.; Edwards, G. S.; Nemanich, R. J.; Sarna, T.; Simon, J. D., Photoionization thresholds of melanins obtained from free electron laser-photoelectron emission microscopy, femtosecond transient absorption spectroscopy and electron paramagnetic resonance measurements of oxygen photoconsumption. Photochem. Photobiol. 2006, 82 (3), 733-737. 62. Jacobson, E. S., Pathogenic roles for fungal melanins. Clin. Microbiol. Rev. 2000, 13 (4), 708-17. 63. Jacobson, E. S.; Hong, J. D., Redox buffering by melanin and Fe(II) in Cryptococcus neoformans. J. Bacteriol. 1997, 179 (17), 5340-6. 64. Howland, R. H., Methylene Blue The Long and Winding Road From Stain to Brain: Part 2. J. Psychosoc. Nurs. Ment. Health Serv. 2016, 54 (10), 21-26. 65. Aureli, C.; Cassano, T.; Masci, A.; Francioso, A.; Martire, S.; Cocciolo, A.; Chichiarelli, S.; Romano, A.; Gaetani, S.; Mancini, P.; Fontana, M.; d'Erme, M.; Mosca, L., 5-S-cysteinyldopamine neurotoxicity: Influence on the expression of α-synuclein and ERp57 in cellular and animal models of Parkinson's disease. J. Neurosci. Res. 2014, 92 (3), 347-358. 66. Napolitano, A.; De Lucia, M.; Panzella, L.; D’Ischia, M., The “Benzothiazine” Chromophore of Pheomelanins: A Reassessment†. Photochem. Photobiol. 2008, 84 (3), 593-599.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC graphic


Page 18 of 28

Page 19 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1

Scheme 1. (a) The redox-context of the brain is complex with steep gradients between oxidizing and reducing conditions that provide the opportunity for redox-active molecules to engage in redox-cycling over short distances. (b) Electrochemistry allows spatial gradients to be generated, while cyclically imposed voltages allows redox-cycling to be probed over short times. (c) Electrochemical reverse engineering to evaluate redox-cycling interactions between melanin and the redox-active drugs acetaminophen (APAP) and clozapine (CLZ).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1

Figure 1. Morphological and chemical characterization. (a) SEM images of natural Sepia melanin show granular structure. (b) SEM images of synthetic melanin show homogeneous structure. (c) Chemical analysis of natural Sepia melanin and synthetic melanin using Energy Dispersive X-ray Spectroscopy (EDX).


Page 20 of 28

Page 21 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2

Figure 2. Initial evidence for drug-melanin redox-cycling (natural Sepia melanin). (a) Redox-cycling interactions with melanin: reductive redox-cycling with Ru(NH3)6Cl3 (Ru3+) transfers electrons from the electrode to melanin and oxidative redox-cycling with either APAP or CLZ transfers electrons from melanin to the electrode. (b) Control cyclic voltammograms (CVs) with uncoated gold electrodes show APAP and CLZ can each undergo somewhat reversible redox reactions. Cyclic voltammogram (CV) of melanin-chitosan film shows the paired amplifications for the reduction of Ru3+ and the oxidation of either (c) APAP or (d) CLZ (compared to the control chitosan film). All CVs were for 3rd cycle at a scan rate of 2 mV/s.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3

Figure 3. Evidence for repeatable redox-cycling (natural Sepia melanin). (a) Cyclic voltammograms (CVs) and (b) input-output curves show that melanin-containing films have paired amplifications for Ru3+-reduction and APAP-oxidation that appear “steady” over 20 cycles. (c) CVs and (d) input-output curves also show paired and steady amplifications for Ru3+-reduction and CLZ-oxidation. CV scan rate of 2 mV/s.


Page 22 of 28

Page 23 of 28

Figure 4 Definition of Parameters to Analyze Redox Cycling Signature

(a)

2

QRu,Red

Current (µA)

1.5

Oxidation

Reduction

1

QAPAP,Red

0.5

Qox (Charge)

Chit

0

QRu,Ox

-0.5 Mel-chit

-1

Q=

-1.5

QAPAP,Ox

-2

∫

i ⋅ dt =

∫

i⋅

1

ν

Nox

⋅ dE

0.5

0

QRed (Charge)

−Chit Q Mel − Q Chit APAP,Ox APAP,Ox

−Chit Q RMel − Q Chit u,Red Ru,Red

NRed

F⋅A

F⋅A

N – Redox capacity: excess electron transfer of film during oxidation (Nox) and reduction (NRed)

-2.5 -0.5

E(V) vs Ag/AgCl Charge Transfer for Ru3+-Reduction

Charge Transfer for APAP-Oxidation

(c)

400

300

200 Chit

QAPAP,Ox

100

0

NOx

200 150 100 Chit

QRu,Red

50 0

0

5

10

15

20

5

10

(f) Charge (µC, QRu)

250 200 Chit

QCLZ,Ox

100 50 0 5

10

Number of cycles

15

15

20

0

20

20

(g)

Mel-chit

200 150 Chit QRu,Red

100

10

15

20

Redox Capacity

QRu,Red

250

5

Number of cycles

300

Mel-chit

QCLZ,Ox

0

NRed

40

Charge Transfer for Ru3+-Reduction

350

150

60

Number of cycles

Charge Transfer for CLZ-Oxidation

300

80

0 0

Number of cycles

(e)

100

QRu,Red

250

NFilm (nmole/cm2)

Charge (µC, QRu)

Charge (µC, QAPAP)

300

(d)

Mel-chit

Mel-chit

QAPAP,Ox

Redox Capacity

100

NFilm (nmole/cm2)

(b)

Charge (µC, QCLZ)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

NRed

80 60

NOx

40 20 0

0 0

5

10

15

Number of cycles

20

0

5

10

15

Number of cycles

20

Figure 4. Quantitative analysis of redox-cycling signatures (natural Sepia melanin). (a) Schematic illustrating quantification of output response “signal” in terms of charge transfer (Q) and excess electron transfer (N) for drug-oxidation and Ru3+-reduction. Near steady responses are observed for (b) APAPoxidation, (c) Ru3+-reduction, and (d) their excess electron transfers. Similarly, near steady responses are observed for (e) CLZ-oxidation, (f) Ru3+-reduction, and (g) their excess electron transfers.



Figure 5 (a) Input for Step Changes in Potential E (V) vs Ag/AgCl

-1 -0.5

Initial charging

Reductive charging

-0.4V

-0.4V

0 0.5

Oxidative discharging

+0.7V

1 0

30

60

90

Time (min)

(b) Output for Oxidative Step

(c) Output for Reductive Step (Ru3+)

(APAP) 1.2

-0.4

Charge (mC)

Charge (mC)

0 Chit

-0.8 Mel-chit NOX ≈ 310 nmole/cm2

-1.2 -1.6

0.9 0.6

NRed ≈ 240 nmole/cm2

Mel-chit

0.3 Chit

-2

0 0

10

20

0

30

Time (min)

10

20

30

Time (min)

(d) Output for Oxidative Step

(e) Output for Reductive Step

(CLZ)

(Ru3+)

0

1.2

-0.3

Charge (mC)

Charge (mC)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

Chit

-0.6

Mel-chit

-0.9

NOX ≈ 250 nmole/cm2

0.9 0.6 Mel-chit

NRed ≈ 250 nmole/cm2

0.3

-1.2

Chit

-1.5

0 0

10 20 Time (min)

30

0

10 20 Time (min)

30

Figure 5. Evidence for redox-cycling using step changes in input voltages (natural Sepia melanin). (a) Three step input potentials are imposed in the presence of Ru3+ and drug to probe for melanin’s oxidative discharging and reductive charging. (b) APAP-mediated discharging of electrons (oxidation) and (c) subsequent Ru3+-mediated charging of electrons (reduction). (d) CLZ-mediated discharging of electrons (oxidation) and (e) subsequent Ru3+-mediated charging of electrons (reduction).


Page 25 of 28

Figure 6 (a)

Input Steady 1

UR

Output

Steady 2

UO

Steady 1

Steady 1

UR

Steady 2

UO

Steady 1

Chit

1.5

-0.3

Current (µA)

E(V) vs Ag/AgCl

-0.6

0 0.3 0.6

0.5 -0.5 -1.5

Mel-chit -2.5

0.9 0

100

200

300

400

500

600

0

700

100

200

300

(b)

Steady 1

Current (µA)

2

2

Unsteady -Reduction

1 1

Chit

0 Mel-chit

-1

0

0

-1

-1

0.7 0.4 0.1 -0.2 -0.5

0.7

0.4

600

700

0

-1

-1

-2 0.7

0.1 -0.2 -0.5

Steady 3

2 Oxidation + + APAP APAP +2H +2e 1

0

-2

-2

-2

500

Unsteady-Oxidation

Steady 2

2

Reduction Ru3+ + e- Ru2+

1

1

400

Time (min)

Time (min)

0.4

-2 0.7 0.4 0.1 -0.2 -0.5

0.1 -0.2 -0.5

0.7

0.4

0.1 -0.2 -0.5

E(V) vs Ag/AgCl

(d) Charge Transfer for Ru3+-Reduction 300

300

250

Charge (µC, QRu)

350

250 200

Mel-chit QAPAP,Ox

150 100 Chit

50

QAPAP,Ox

0

200 150

0

10

20

30

Number of cycles

40

50

100

Mel-chit QRu,Red

100 50 0

(e) Redox Capacity

NFilm (nmole/cm2)

(c) Charge Transfer for APAP-Oxidation

Charge (µC, QAPAP)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60

NRed

40 20

NOx

Chit

QRu,Red 0

10

0 20

30

40

50

Number of cycles

0

10

20

30

40

50

Number of cycles

Figure 6. Evidence for APAP’s redox-cycling using a complex sequence of input voltages (natural Sepia melanin). (a) Input-output curves for cyclic potential inputs of differing amplitudes. (b) CV curves show steady (time-invarying) output currents when large input amplitudes are imposed to allow both oxidative and reductive redox-cycling, while un-steady (time-varying) output currents are observed when the input amplitude is limited to allow only reductive redox-cycling or only oxidative redox-cycling. Quantification of output response in terms of charge transfer (Q) for (c) APAP- oxidation and (d) Ru3+reduction, and (e) excess electron transfers for APAP-oxidation (NOx) and Ru3+-reduction (NRed).



Figure 7 Input

(a) Steady 1

E(V) vs Ag/AgCl

-0.6

UR

Steady 2

UO

Steady 3

-0.3 0 0.3 0.6 0.9 0

100

200

300

400

500

600

700

Time (min) Output Steady 1 1.5

UR

Steady 2

UO

Steady 3

Chit

Current (µA)

1 0.5 0 -0.5 -1

Mel-chit

-1.5 0

100

200

300

400

500

600

700

Time (min)

(b) Redox Capacity 80

NFilm (nmole/cm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

70 60

NRed

50 40 30 20 10

NOx

0 0

10

20

30

40

50

Number of cycles

Figure 7. Evidence for CLZ’s redox-cycling using a complex sequence of input voltages (natural Sepia melanin). (a) Input-output curves for cyclic potential inputs of differing amplitudes. (b) Summary of output response for excess electron transfers for CLZ-oxidation (NOx) and Ru3+-reduction (NRed).


Page 27 of 28

Figure 8 (a)

Input Steady 1

-0.6

UR

Output

Steady 2

UO

Steady 1

Steady 1

UR

Steady 2

UO

Steady 1

1.2

-0.3

Current (µA)

E(V) vs Ag/AgCl

Chit

0 0.3 0.6

0.6 0 -0.6

Synt. mel-chit

-1.2

0.9 0

100

200

300

400

500

600

0

700

100

200

300

(b)

Steady 1

Unsteady -Reduction 1

Current (µA)

1

Steady 2

Reduction Ru3+ + e- Ru2+

0.5

0.5

1

1

0.5

0.5

Chit

Synt.Mel-chit

700

Steady 3

1 Oxidation + + APAP APAP +2H +2e 0.5

0

0

0

-0.5

-0.5

-0.5

0.7 0.4 0.1 -0.2 -0.5

600

Unsteady-Oxidation

0

-1

-1

500

-0.5

0 -0.5

400

Time (min)

Time (min)

-1

-1

-1

0.7 0.4 0.1 -0.2 -0.5

0.7 0.4 0.1 -0.2 -0.5

0.7 0.4 0.1 -0.2 -0.5

0.7 0.4 0.1 -0.2 -0.5

E(V) vs Ag/AgCl

(c) Charge Transfer for APAP-Oxidation

(d) Charge Transfer for Ru3+-Reduction

180

(e) Redox Capacity

120 Syn.Mel-chit

QAPAP,Ox

120 90

Chit QAPAP,Ox

60 30 0

30

100

NFilm (nmole/cm2)

150

Charge (µC, QRu)

Charge (µC, QAPAP)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Syn. Mel-Chit

80 QRu,Red 60 40 Chit

20

QRu,Red

0 0

10

20

30

Number of cycles

40

50

25 20 15

NRed

10 5

NOx

0 0

10

20

30

40

50

Number of cycles

0

10

20

30

40

50

Number of cycles

Figure 8. Evidence for APAP’s redox-cycling using a complex sequence of input voltages (synthetic melanin). (a) Input-output curves for cyclic potential inputs of differing amplitudes. (b) CV curves show steady (time-invarying) output currents when large input amplitudes are imposed to allow both oxidative and reductive redox-cycling, while un-steady (time-varying) output currents are observed when the input amplitude is limited to allow only reductive redox-cycling or only oxidative redox-cycling. Quantification of output response in terms of charge transfer (Q) for (c) APAP- oxidation and (d) Ru3+reduction, and (e) excess electron transfers for APAP-oxidation (NOx) and Ru3+-reduction (NRed).



Figure 9 (a)

Input Steady 1

E(V) vs Ag/AgCl

-0.6

UR

Steady 2

UO

Steady 3

-0.3 0 0.3 0.6 0.9 0

100

200

300

400

500

600

700

Time (min) Output UR

Steady 1

1

UO

Steady 2

Steady 3

Current (µA)

Chit 0.5 0 -0.5

Synt. Mel-chit -1 0

100

200

300

400

500

600

700

Time (min)

(b) Redox Capacity 35

NFilm (nmole/cm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 28

30 25

NRed

20 15 10 5

NOx

0 0

10

20

30

40

50

Number of cycles

Figure 9. Evidence for CLZ’s redox-cycling using a complex sequence of input voltages (synthetic melanin). (a) Input-output curves for cyclic potential inputs of differing amplitudes. (b) Summary of output response for excess electron transfers for CLZ-oxidation (NOx) and Ru3+-reduction (NRed).


The Analgesic Acetaminophen and the Antipsychotic Clozapine Can

Recommend Documents