Structural Basis of Polydopamine Film Formation: Probing 5,6

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The structural basis of polydopamine film formation: probing 5,6dihydroxyindole-based eumelanin type units and the porphyrin issue Maria Laura Alfieri, Raffaella Micillo, Lucia Panzella, Orlando Crescenzi, Stefano Luigi Luigi Oscurato, Pasqualino Maddalena, Alessandra Napolitano, Vincent Ball, and Marco d'Ischia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09662 • Publication Date (Web): 22 Sep 2017 Downloaded from http://pubs.acs.org on September 22, 2017

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ACS Applied Materials & Interfaces

The structural basis of polydopamine film formation: probing 5,6-dihydroxyindole-based eumelanin type units and the porphyrin issue Maria L. Alfieri†, Raffaella Micillo†, Lucia Panzella†, Orlando Crescenzi†, Stefano L. Oscurato‡, Pasqualino Maddalena‡, Alessandra Napolitano†, Vincent Ball§,∥ , Marco d’Ischia†* †Department of Chemical Sciences, University of Naples Federico II, I-80126 Naples, Italy. ‡Department of Physics "Ettore Pancini", University of Naples Federico II, I-80126 Naples, Italy. § Université de Strasbourg, Faculté de Chirurgie Dentaire, 8 rue Sainte Elisabeth, 67000 Strasbourg, France. ∥

Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 1121, 11 rue Humann, 67085 Strasbourg Cedex, France.

ACS Paragon Plus Environment

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KEYWORDS: eumelanin, polydopamine, 5,6-dihydroxyindole, polymerization, film, porphyrin, tetramer, MALDI-MS.

ABSTRACT

The role of 5,6-dihydroxyindole (DHI)-based oligomers, including porphyrin-like tetramers, in polydopamine (PDA) film formation was addressed by a comparative structural investigation against model polymers from DHI and its 2,7’-dimer. MALDI-MS data showed that: a) PDA is structurally different from DHI melanin and does not contain species compatible with DHI-based oligomers as primary building blocks; b) PDA films and precipitate display a single main peak at m/z 402 in common; c) no species matching the range of m/z values expected for cyclic porphyrin-type tetramers was detected in DHI melanin produced in the presence or in the absence of folic acid (FA) as templating agent, nor by oxidation of the 2,7’-dimer of DHI as putative precursor.

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N NMR resonances and Raman spectra predicted by extensive DFT

calculations on porphyrin-type structures at various oxidation levels didn’t match spectral data for PDA or DHI melanin. Notably, unlike PDA, which gave structurally homogeneous films on quartz on atomic force microscopy (AFM) and micro-Raman spectroscopy, DHI melanin did not form any adhesive deposit after as long as 24 h. It is concluded that PDA film deposition involves structural components unrelated to DHI-based oligomers or porphyrin-type tetramers, which, on mechanism-based analysis, may arise by quinone-amine conjugation leading to polycyclic systems with extensive chain breakdown.


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Introduction Polydopamine (PDA), a black insoluble and structurally disordered eumelanin-like material produced by the oxidative polymerization of dopamine under alkaline conditions, stands to-date as the state-of-the-art for surface functionalization and coating and for various nanotechnological and biomedical applications.1 Inspired by the robust adhesion properties of catechol- and aminerich mussel adhesive proteins such as Mytilus edulis foot protein-5 (Mefp-5), PDA provides technologically versatile coatings susceptible to functionalization and chemical manipulations. PDA thin films can be deposited at the interface of different materials, including metals, oxides, inorganic semiconductors, ceramics, and polymers, can bind cells, biomolecules and metal ions, and can be used to control or modify the hydrophobicity of a variety of interfaces. PDA film properties, including hydrophilicity and thickness, can be finely tuned by a variety of experimental parameters including dopamine concentration2, nature of the buffer3, oxidant4–6 and pH.2 Because of the growing importance of PDA in materials science and technology, defining structure-property-function relationships is a major goal for optimizing its performances for tailored applications. Until 2012 two different speculative structural models were commonly assumed: the “open-chain polycatechol/quinone” model, based on linear sequences of catecholamine units linked through biphenyl-type bonds, and the “eumelanin” model, which envisaged a 5,6-dihydroxyindole (DHI) polymer arising by cyclization of dopaminequinone (Scheme 1).7,8


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NH 2

HO HO dopamine [O] O

HO

NH 2 early cyclization

O

[O]

quinone

N HO H 5,6-dihydroxyindole (DHI) (a)

HO HO

NH 2

dimerization [O] HO

OH

OH

HO HO

NH 2

HN

HN H N

HO HO (b)

HO

HO HO

N H

2,4'-dimer

OH OH

2,7'-dimer

N H

HO HO

N H

late cy cli zat ion

polymerization

polydopamine

Scheme 1. Simplified view of the possible oxidative pathways leading to eumelanin building blocks emphasizing the generation of amine-containing open chain oligomers versus cyclized indole-type species. Highlighted are: a) the oxidative coupling of 5,6-dihydroxyindole (DHI) leading to two main dimers, the 2,4’- and the 2,7’-biindolyl; b) the possible occurrence of late coupling cyclization pathways as an alternate route to indolic oligomers from early cyclization.

Since 2012, several groups have begun to address in detail the chemical nature of PDA, its basic scaffolds and functional groups. The following structural models have been proposed in order of time:


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a)

A supramolecular aggregate consisting primarily of 5,6-dihydroxyindoline and

dopaminochrome held together through a combination of charge transfer, π-π stacking, and hydrogen-bonding interactions.9 b)

A physical trimer of (dopamine)2/DHI, derived from a self-assembly mechanism.10

c)

A trimer of DHI and other MALDI-MS-detectable species with m/z 401 and 448 on

PDA-coated glass.11 d)

A three-component structure of polydopamine, comprising uncyclized (catecholamine)

and cyclized (indole) units, as well as pyrrolecarboxylic acid moieties, with partial incorporation of tris(hydroxymethyl)aminomethane (Tris buffer).12,13 e)

Mixtures of different oligomers containing indole units with various degrees of

(un)saturation and open-chain dopamine units.14 f)

Dopamine and C=N-containing tautomers of quinone and indole species in growing films

deposited on gold surfaces over an interval of time from 2 to 60 min.15 g)

A (DHI)2/PCA (pyrrolecarboxylic acid) trimer complex (m/z 402) as primary component

to build up the supramolecular structure of PDA.16,17 Very recently, a set of computational methods applied to investigate nearly 3000 probable molecular structures of PDA and eumelanin indicated that i) more planar oligomers are more stable; ii) there is a group of tetramers relatively more stable than the others, implying repetitive structural motifs despite inherent chemical diversity; iii) larger oligomers are less likely to form.18


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Parallel to these structural studies, a number of papers over the past few years interpreted PDA properties in terms of a simplified structural model first proposed for eumelanins by Kaxiras et al. in 200619 and involving porphyrin-type building blocks derived from oxidative cyclization of DHI tetramers built via 2,7’- bondings (Scheme 2). HO

HO

[O]

N HO H 5,6-dihydroxyindole (DHI)

NH

DHI

HO

OH x2

HO

OH

HO

HO N H

HO NH

N H

HO H N

NH OH

DHI

OH HO

N H

HO

dimerization (2,7'-coupling)

OH

HO

HN H N

OH

OH OH porphyrin-type tetramer

Scheme 2. Putative pathways of porphyrin tetramer formation during PDA synthesis. Time-dependent density functional theory (DFT) calculations suggested that the structural model reproduces fairly well the main features of eumelanin, including broadband absorption spectrum, X-ray scattering data, the ability to capture and release metal ions, and the characteristic size of eumelanin protomolecules. Subsequent DFT calculations showed that such tetramers can not only associate with an interlayer distance of 3.0-3.3 Å but can also bind in a covalent manner through interlayer C-C bonds.20 Additional indirect support to the porphyrin model came from combined molecular dynamics simulations and high resolution transmission electron microscopy (TEM) data suggesting that most of eumelanin/PDA properties could be explained by self assembly of porphyrin-like indole


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tetramers.21 Moreover, a detailed ES (+)-MS analysis of PDA reported a signal at m/z 595.1841 compatible with a cyclotetramer.14 Other studies based on MALDI-MS showed that folic acid (FA) influences the morphology and nanostructure of PDA favoring formation of species giving a cluster of peaks at 584–589 Da that were fitted to a cyclic tetramer structure proposed as an important structural motif.22 More recently, an integrated approach based on an electrochemical fingerprinting technique suggested that natural eumelanin pigments contain porphyrin-like protomolecules at appreciable concentrations.23 Despite the evident importance of elucidating PDA structure for understanding and controlling its properties for technological applications, the picture emerging from these studies is still confusing and partly based on theoretical models that are not adequately supported by unambiguous experimental evidence. It is therefore mandatory for progress in PDA research that critical open questions about the structural components responsible for adhesion are definitively settled. A crucial issue of central relevance to PDA film forming properties is: Do film formation properties of PDA depend on eumelanin-type, DHI-based components ? Settling this issue is the main goal of this paper. Before discussing the results, however, a concise background of mechanistic arguments and a list of the main difficulties that need to be circumvented to achieve this goal with possible approaches is proposed to support the rationale of the study. Though PDA is often referred to as a synonymous of eumelanin because it complies to the definition24 of “nitrogenous biopolymer produced by oxidative polymerization of a tyrosine-


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derived indole precursor” and shows similar physicochemical properties, it differs from typical natural or synthetic eumelanins because of the lack of carboxylated indole units (5,6dihydroxyindole-2-carboxylic acid, DHICA). Nonetheless, PDA may contain DHI units24 or mixtures of partially degraded indole units. Thus, answering the question key aforementioned requires an understanding of whether and to what extent proportion PDA films consist of intact or partially degraded DHI oligomers, and whether such components arise from polymerization of preformed DHI or from the late cyclization of linear dopamine oligomers. A strictly related issue is the role of porphyrin-like tetramers in PDA film formation. Thus far, formation of porphyrin structures via DHI chemistry has not been unambiguously demonstrated by adequate experimental data. Chemical evidence indicates that DHI tends to couple mainly via two different pathways, leading to the 2,4’- and the 2,7’-dimers, though other modes of coupling, e.g. via the 3-position, may become significant at the dimer-dimer coupling stage.25 Assuming that each coupling step of DHI proceeds statistically via 2,4’- and 2,7’-bondings (i.e. 50% each: an oversimplification which however roughly reflects the experimental proportion of the two coupling modes in dimer formation), and considering that porphyrin formation requires four sequential 2,7’-coupling steps, including the final cyclization step, the expected final yield of tetramer would be not higher than ca. 6% based on reacted DHI. Furthermore, non-quantitative conversion of dopamine to DHI, due to other competing pathways of dopamine quinone evolution, would further decrease porphyrin contribution to PDA structure. Overall, the above arguments would make a strong mechanism-based case against a significant involvement of porphyrin tetramers in PDA and eumelanin structure. However, it’s possible that specific control mechanisms e.g. mediated by aggregation induce porphyrin assembly from DHI. This point can be addressed by revisiting the polymerization of DHI and dopamine in the presence and in the


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absence of folic acid (FA) as a templating agent, as reported.22 It is also possible that, even though produced in very low yields, porphyrin components may be primary and specific components of the very minute fraction of adhesive PDA components leading to film formation, though a recent MS analysis of PDA film did not provide evidence in support of this view.15 In any case, obtaining direct experimental evidence for porphyrin tetramers in eumelanin-type materials is a difficult task. MALDI-MS data, which support most of current suggestions, cannot be taken as unambiguous proof for bona fide porphyrins. For example, the previous suggestion14 that the pseudomolecular ion peak in the MALDI-MS spectrum of PDA at m/z 595 could be assigned to a cyclotetrameric structure bearing indoline and aminochrome units would not withstand critical analysis since indoline type leucodopaminochrome structures would be readily reoxidized to dopaminochrome and then to DHI, thus being stable enough to provide a main peak under MALDI conditions. More convincing options for the species at m/z 595 could include mixed-type tetramers involving both DHI and uncyclized units (dopamine is more stable to autoxidation than the indoline and is a more likely structural moiety). Notably, moreover, even though mass data compatible with cyclic structures have been reported, alternative N-confused isomeric porphyrin scaffolds with the same molecular formula but predictably different properties should be considered as well. Even more fundamentally, pseudomolecular ion mass data are intrinsically unable to distinguish between a cyclic oligomer and the corresponding noncyclic structure at a higher oxidation level. Likewise, UV-visible and FT-IR spectroscopy can be decisive only after diagnostic bands that can be unequivocally attributed to those kinds of structures are identified. Based on the above survey, specific aims of this paper are: a) to compare the structure and film-forming properties of PDA and DHI eumelanin in order to assess to what extent film


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formation depends on intact DHI-based components present in PDA; b) to assess whether PDA films and bulk precipitate are made up of the same components or show different structural features, and to provide an insight into main film components; c) to verify whether porphyrin tetramers do form under favorable conditions, i.e. by autoxidation of DHI in the presence of FA as templating agent, or by oxidation of the 2,7’-dimer of DHI, the putative precursor of porphyrin tetramers. Although porphyrins have not been directly implicated in adhesion, addressing this issue seems a relevant goal given the recurrent recourse to this structural model in the literature. To this aim, a computational survey of realistic porphyrin structures and their simulated NMR, UV-visible absorption and Raman properties will be reported as a function of the oxidation state, to look for diagnostic signatures guiding identification in PDA films. To avoid possible interference of the amine-containing Tris buffer with polymer buildup and film deposition for the purpose of structural investigation, PDA synthesis was carried out in carbonate buffer unless otherwise stated.12,16 Experimental section Synthesis of PDA and DHI melanin and coating experiments PDA and DHI melanin were prepared respectively by autoxidation of dopamine hydrochloride (30 mg, Sigma Aldrich) or DHI (30 mg, prepared according to Edge et al.26) dissolved in 0.1 M bicarbonate buffer (pH 9.0)3 or 0.05 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.5)3 (final concentration 10 mM). The reaction mixtures were left under vigorous stirring and after 24 h were acidified to pH 2 with 4 M HCl. The dark pigments were collected by centrifugation at 7000 rpm at 4 °C, washed 3 times with water and lyophilized. The yields of PDA and DHI melanin were 28% and 79% respectively.


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Quartz substrates were cleaned by soaking in piranha solution (96% H2SO4 /30% H2O2 5:1) overnight, rinsed with distilled water, dried under vacuum and dipped in autoxidation mixtures. After 24 h, substrates were sonicated in a 1:1 v/v methanol/water solution and dried. UV-Vis analysis of the coated substrates obtained were performed on a Jasco V-730 Spectrophotometer. In separate experiments, dopamine or DHI (0.3 mg/mL) in the absence or in the presence of FA (0.15 mg/mL) were first dissolved in deionized water and stirred for 1 day at 60 °C in the dark, then NaOH aqueous solution (0.1 M) was used to adjust the pH value (pH ca. 8.5) and the mixture was stirred for 3 h at 60 °C in the dark22. In the case of DHI, the phase of pre-incubation with FA was performed sealing the reaction mixture under argon to prevent the indole oxidation. Then, the dark suspension was centrifuged at 10000 rpm for 30 min to collect the precipitate. The sediment was washed several times with water and then dried by lyophilization. Sample preparation for MALDI-MS analysis22 α-Cyano-4-hydroxycinnamic acid (CHCA, 98% purity) was purchased from Sigma Aldrich. HPLC-grade acetonitrile, trifluoroacetic acid (TFA) and bidistilled water were used. The solution of matrix CHCA (10 mg/mL) was prepared in acetonitrile/water (1:1, v/v) containing 0.1% TFA. 1 mL of the analyte was premixed with 1 mL of the matrix in a centrifuge tube, and then 2 µL of the resulting mixture was pipetted on the MALDI target plate and air-dried for MALDI-ToF MS analysis. MALDI spectra were recorded on a Sciex 4800 MALDI ToF/ToF instrument. The laser was operated at 3700 Hz in the positive reflectron mode. The mass spectrometer parameters were set as recommended by the manufacturer and adjusted for optimal acquisition performance. The laser spot size was set at medium focus (B50 mm laser spot diameter). The mass spectra data were acquired over a mass range of m/z 100–4000 Da and each mass spectrum was collected


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from the accumulation of 1000 laser shots. Raw data were analyzed using the computer software provided by the manufacturers and reported as monoisotopic masses. AFM and micro-Raman analysis The combined AFM and micro-Raman analysis was conducted with the integrated apparatus Alpha300 RS (WITec, Germany). The system can be switched at will between AFM and confocal micro-Raman configurations, allowing a combined topographical and spectral characterization of a specified micro-region of the sample. The film topography was investigated by AFM operating in AC mode. For the micro-Raman analysis, a laser beam at λ = 488 nm was used as excitation light source. The beam was focused onto the sample surface by means of a microscope objective (NA=0.75) working in epi-illumination mode. The diffraction-limited focused spot in the objective focal plane had a FWHM of approximately 320 nm. The light backscattered from the sample was collected by the same objective and sent to the spectrograph through a confocal optical collection path. Computational methods All DFT calculations were performed with the Gaussian package of programs27, with the PBE0 hybrid functional28. The 6-31+G(d,p) basis set was employed for geometry optimizations. For each species, an extensive exploration of the different tautomers/conformers, as well as different protonation states was carried out. In those cases where conformational enantiomers exist, a single enantiomeric series has been examined. Computations were performed either in vacuo, or by adoption of a polarizable continuum medium (PCM)29–32 to account for the influence of the solution environment. In view of the faster convergence, a scaled van der Waals cavity based on universal force field (UFF) radii33


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was used, and polarization charges were modeled by spherical Gaussian functions34,35; nonelectrostatic contributions to the solvation free energy were disregarded at this stage: these terms were accounted for in single-point PCM calculations (at the PCM geometries) employing radii and non-electrostatic terms of the SMD solvation model.36. Vibrational-rotational contributions to the free energy were also computed. For the preparation of Raman scattering activity plots, harmonic frequencies were scaled by a factor of 0.954737 and Gaussian line width of 20 cm-1 was used. UV-Vis spectra of the main species were computed in vacuo or in solution using the timedependent density functional theory (TD-PBE0) approach38–42 with 6-311++G(2d,2p) basis set. To produce graphs, transitions below 5.6 eV were selected, and an arbitrary Gaussian line width of 0.25 eV was imposed; the spectra were finally converted to a wavelength scale. NMR shielding tensors were computed within the Gauge-Including Atomic Orbitals (GIAO) ansatz43,44 with the 6-311+G(d,p) basis set. Computed

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N isotropic shieldings were converted

into chemical shifts using as reference the values obtained at the same level for methyl 5,6dimethoxyindole-2-carboxylate (at 140.0 ppm from ammonia, cfr.).45 Results and Discussion Comparative structural analysis of PDA and DHI eumelanin and their film-forming properties In an initial set of experiments, the formation on a quartz surface of thin films from DHI melanin was assessed in comparison with PDA based on the typical dip-coating methodology, to assess whether DHI-related units play any specific role in PDA film formation. Figure 1 shows the


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absorption spectra of the quartz substrates after 24 h incubation in solutions of dopamine or DHI at alkaline pH. UV-Vis analysis clearly indicated that film deposition from DHI was null or below detection. For comparison the UV-Vis absorption spectra of the corresponding PDA and DHI melanin solutions are reported in the Supporting Information (Figure S1-S2).

(a) (b)

Figure 1. a) Quartz substrates immersed in the autoxidation mixture for dip-coating experiments comparing PDA (left) and DHI (right) melanin. b) UV-visible absorption spectra of quartz substrates subjected to dip-coating with dopamine (blue curve) and DHI (red curve). To gain additional insight into PDA films and to definitely rule out film deposition from DHI polymerization, the quartz substrates immersed into PDA and DHI oxidation mixtures were investigated by a combined atomic force microscopy (AFM) and micro-Raman analysis. Data for PDA film (Figure 2a) indicated a mean thickness of 70 nm and dispersed sub-micron sized grains (Figure 2b). In the same sample region, the micro-Raman analysis was also performed. The Raman spectrum resulting from the average of 625 spectra collected in different positions of


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the sample is shown in Figure 2c, together with the positions of the observable peaks. Main bands at 1583, 1416, 1347 and 1242 cm-1 were detected, which were compatible with the presence of aromatic rings, while the broadband around 2900 cm-1 was attributable to strongly hydrogen-bonded OH and NH stretching vibrations. Notably, no intense carbonyl band in the 1650-1700 cm-1 range was detected. These results are in line with previous Raman spectroscopy studies, indicating two main bands at 1360 and 1588 cm-1 in one case,11 and at 1340 and 1600 cm-1 in the other case, due to catechol stretching vibrations.46 The combined micro-Raman imaging of the film presented in Figure S3 of the Supporting Information revealed furthermore a homogeneous chemical composition of the film presenting the typical topography shown in Figure 2a. A similar AFM-Raman analysis was conducted onto the films of PDA-Tris as a reference sample (see Figure S4), confirming a complete overlap of the topographical and chemical features of the PDA-carbonate with the PDA-Tris films. This conclusion was in accord with a previous study based on the ESI(+) spectra of PDA samples obtained in Tris or phosphate buffer.47 The same AFM-Raman analysis revealed the absence of any material attached to the substrate in the DHI melanin reaction (see Figure S5).


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Figure 2. (a) AFM image of a representative region of the PDA film sample. (b) AFM height profile measured along the red line in the panel (a). (c) Micro-Raman spectrum resulting from the average of 625 spectra collected with a spatial resolution of 2 µm in the sample scanning over an area of 50 × 50 µm2. Overall, these data suggested that preformed DHI-based cyclized structures are unlikely to be the major determinants of PDA film-formation properties. To support this conclusion, in separate experiments different PDA films that adhered to the walls of reaction beakers and to immersed glass substrates were carefully washed with water, solubilized and analyzed by MALDI-MS in comparison with the bulk precipitate from the same mixtures and with DHI melanin. Salient regions of representative spectra are reported in Figure 3, whereas the main peaks in common to at least two different samples, which are considered for structural analysis, are reported in Table 1.


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Intensity (A.U.)

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Mass (m/z)

Figure 3. Segmental spectrum of MALDI-ToF (m/z: 300-600 Da) characterizations of (a) PDA film in carbonate buffer at pH=9, (b) PDA precipitate in carbonate buffer at pH=9, (c) PDA


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synthesized in the presence of FA (dopamine dissolved in water for over 24 h in the dark at 60°C + NaOH 0.1M to adjust the pH value to ~ 8), and (d) DHI melanin in carbonate buffer at pH=9 , respectively. Asterisks indicate signals due to matrix or impurities.

Table 1. Main MALDI-MS peaks considered for structural investigation of PDA and melanins from DHI and its 2,7’-dimer. All spectra were obtained using α-cyano-4-hydroxycinnamic acid unless otherwise indicated. Selected peaks are identified on the basis of their presence in all spectra examined (at least duplicate samples).

n-mer Dopamine oligomer calcd.1 (m/z) 456 (478, 494) 3

PDA + FA4 (m/z)

402

402

549

402

DHI oligomer calcd.1 (m/z) 444 (466, 482) 591 (613, 629)

DHI melanin2 (m/z)

Melanin from 2,7’dimer5 (m/z)

416, 430

549, 533, 577, 551, 591 563, 591, 621 581 1 M+H (M+Na, M+K). 2Precipitate from the reaction in carbonate. 3Solubilized in MeOH/DMSO. 4 Precipitate obtained in the presence of FA according to Fan et al22. 52,5-dihydroxybenzoic acid as the matrix. 4

607 (629, 645)

PDA2 PDA (m/z) film3 (m/z)

Comparative inspection of traces a and b in Figure 3 confirmed for both the film and the precipitate the intense peak at m/z 402 reported previously.16 Additional minor peaks were noted in the precipitate which were not detectable in the film. Neither the peak at m/z 402, nor the other peaks detected in PDA precipitate could be detected in the spectra of DHI melanin samples (see trace d). No significant peak attributable to cyclic porphyrin-type tetramers could be detected in any of spectra examined.


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Taking these results with due caution because of the limitations inherent to the MALDI-MS methodology, it could concluded that both PDA precipitate and film consist of the species at m/z 402 as the main detectable component, though the film does not show the peaks for the minor species seen in the precipitated polymer. Interestingly, PDA synthesized in the presence of FA leads to a peak pattern similar to that observed in the carbonate reaction but with the presence of additional minor species (m/z 563, 581) the last of which could in principle be compatible with a cyclic tetramer at the 8-electron oxidation level. However, formation of this species was independent of the presence of FA (Figure S6), was not observed under the usual reaction conditions in carbonate buffer, and was not detected in DHI melanin. Moreover, FA did not affect the structure of DHI melanin (Figure S7-S8). Though not entirely conclusive, these data suggest that the species at m/z 581 cannot be unambiguously identified as a cyclic tetramer and can be accounted for by other structures (see below). It is noticed that the general spectral features in the control sample of Figure S4 are different from those of PDA film (Figure 3a) and PDA precipitate (Figure 3b). This may arguably be due to the different reaction conditions but it is difficult to provide a more detailed explanation. To explore further conditions under which the postulated porphyrin tetramer could be obtained in subsequent experiments the 2,7’-dimer of DHI was oxidized and the resulting black eumelanin-type precipitate was subjected to MALDI-MS analysis. The spectrum showed two small peaks at m/z 551 and 591 (Figure S9), suggesting a tetramer and a degradation product thereof, but no peak compatible with the postulated porphyrin structures.


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Toward rational models for PDA and DHI melanin building blocks To rationalize the origin of the species responsible for the main MALDI-MS peaks of PDA film and bulk polymer the main oxidation pathways accessible to dopamine were considered (see overall view in Supporting Information, Scheme S1). These include: a) intramolecular cyclization of dopamine quinone; b) isomerization of dopamine quinone to quinonemethide, followed by autoxidative deamination and chain breakdown; and c) muconic acid-type oxidative fission of o-quinones. The latter pathway was carefully tracked in the oxidative conversion of DHI to pyrrolecarboxylic acids.48 Taking Scheme S1 as a reference, the origin of the main peak of PDA at m/z 402, previously attributed to a physical trimer of two DHI units and a pyrrolecarboxylic acid, was addressed. Its odd mass (giving an even pseudomolecular ion peak) was compatible with a set of alternative structures containing either 1 or 3 nitrogen atoms, some of which are illustrated in Scheme 3 together with plausible key formation steps.


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NH2 NH 2

A

HO

NH 2

C

HO

HO

B

HO

HO

+N O

N H

HO HO

OH HO

OH

HO HO

OH OHC

CHO H 2N

HO

N H

HO

N

O

NH HO

N

H N

OH

OH

OH HO

H 2N

HO

O

HO

OH

OH

OH

O

OH

O

O

O

N H

N

N

O

N

OH

NH HO O

N H

HO OH

NH2

m/z 402

O O OH

m/z 402

m/z 402

Scheme 3. Mechanism-based identification of possible structures accounting for the main peak at m/z 402 in PDA films and bulk polymer. In this Scheme, path A involves a sequence of oxidative condensation and two chain breakdown and deamination processes, leading to 7-(3,4-dihydroxyphenethyl)pyrano[3,4,5kl]acridine-6,9,10(7H)-trione, which is representative of the one-nitrogen option. Notably, the proposed coupling of the primary amine with the o-quinone is akin to the reaction of caffeic acid with amine-containing substrates leading to film-forming benzacridine derivatives.49,50 A critical step of this pathway would be the formation of the pyran ring by ring closure of aldehyde groups derived from a combination of carbonyl-forming chain fission induced by hydrogen peroxide51 (for a detailed mechanism see Scheme S2 Supporting Information). Path B involves sequential


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Page 22 of 36

condensation of intact dopamine units, while path C is entirely derived from DHI coupling and oxidative quinone breakdown. Although these pathways are supported by extensive literature, some of them do not appear to be compatible with experimental evidence. In particular, path C should be involved also in DHI melanin, but no peak at m/z 402 could be detected in the relevant spectrum. As to Path B, a strength is the integrity of dopamine units, but its weakness lies in the dibenzofuran forming pathway, usually favored in acids but not under basic conditions, and in the o-quinone diimine, usually not observed in the conjugation of amines with o-quinones. Conversely, Path A is the sole pathway among those considered here that leads to a flat visible light-absorbing species, purportedly ensuring adhesion. The presence of the 9,10-quinone system incorporated into the acridine ring can be justified by the expected oxidizability of the aminocatechol form. Of course, definitive experimental evidence for the proposed structure is lacking and requires further work currently in progress. Attention is also called to the possibility that the main peak at m/z 402 arises from a mixture of species sharing the same molecular weight. Assuming path A as a working hypothesis, the peak at m/z 549 in PDA polymer could arise by coupling of the species at m/z 402 with a cyclized DHI-type unit (+ 147 mass units), while the peak at m/z 581 observed in the presence of FA could arise by muconic-type oxidative ring cleavage of an o-quinone from the species at m/z 549. The possible origin of the main peaks in DHI melanin is summarized in Scheme S3 (Supporting Information). DFT investigation of putative PDA building blocks The results reported so far indicated that porphyrin components are unlikely to be present to a significant proportion in PDA precipitate and film, and that a MALDI MS-detectable species at


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m/z 402 is a component of PDA films. With a view to settling these issues, a detailed DFT investigation of the postulated porphyrin compounds at the most stable oxidation level and of the postulated pyranoacridinetrione structure was carried out (Figure 4), to identify diagnostic signals in their UV-visible, Raman and 15N NMR spectra that may be revealing of their presence in PDA films. A systematic and detailed inventory of the possible porphyrin structures at various oxidation levels was out of the scope of this study and will be reported elsewhere. Herein, we report computed UV-visible absorption and Raman spectra as well as the

15

NMR resonances for the

most stable oxidation form of the porphyrin system (the 6-electron oxidation level) which was determined by assessing the relative stability of various oxidation states to disproportionation. The underlying assumption was that the porphyrin species at the oxidation level with the lowest tendency to disproportionate can be taken as a plausible reference for theoretical analysis.

O

OH

HO

O N

O

O

HN NH N

O

O

N O

OH HO

O

Figure 4. Porphyrin-type DHI tetramer at the 6-electron oxidation level and truncated pyranoacridinetrione structures considered for DFT calculations. Figure 5 shows the simulated UV-visible and Raman spectra for the 6-electron oxidation state of the porphyrin tetramer.


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Page 24 of 36

(b)

(a)

Figure 5. Computed UV-visible (a: in vacuo, black line, and in water, red line) and Raman (b: in vacuo) spectrum of the most significant tautomer/conformer of the porphyrin tetramer at the 6electron oxidation level. Interestingly, the species concentrated most of the absorption properties in the high energy region of the visible spectrum, whereas the region between 600-880 nm displayed very poor absorption, in contrast with the PDA spectrum (Figure 1). The computed Raman spectrum for the most stable tautomer of the 6-electron oxidation indicated an intense band in the range 16001700 cm-1, i.e. in a region where the Raman spectrum of PDA film is devoid of features (Figure 5 and Table S1). 15

N chemical shifts (ppm) computed in water for the most significant tautomers /conformers in

the 6-electron oxidation state are provided in Supporting Information (Table S2). Compared to pyrrole-type nitrogens, pyridine-type nitrogens in the porphyrin system resonate relatively downfield (ca. 275 ppm) giving an average values around 217 ppm. Again, this result is in contrast with the reported

15

N NMR spectrum of PDA12 in which no resonance above ca. 180

ppm was detected.


24

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Based on these results, the UV-visible, Raman and

15

N NMR spectra of the

pyranoacridinetrione derivatives proposed for the main species at m/z 402 were next investigated. To simplify calculations, a model structure truncated on the phenylethyl chain was considered. The computed absorption spectrum displayed a series of bands spanning the entire UV-visible range, thus being compatible with PDA broadband chromophore (Figure 6). Likewise, intense bands were predicted for the Raman spectrum, which matched fairly well those of PDA film in Figure 1.

(a)

(b)

Figure 6. UV-visible (a: in vacuo, black line, and in water, red line) and Raman (b: in vacuo) spectra computed of the pyranoacridinetrione model. Finally, a

15

N NMR resonance at 137 ppm (in water) was predicted for the N-substituted

acridine nitrogen, which falls within the range of

15

N resonances observed in the PDA

spectrum.12


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Conclusions The combined experimental and theoretical approach reported herein has addressed critical issues concerning the structural basis of PDA film forming properties. The results can be summarized as follows: a)

PDA and DHI melanin show different peak patterns in the MALDI-MS spectra,

suggesting that PDA does not contain main structural components related to indole-based oligomers. b)

PDA films and bulk precipitate samples show a single main peak in common at m/z 402

on MALDI-MS analysis. c)

Mechanism-based analysis of plausible structures for the main peak in PDA film at m/z

402, based also on the lack of this peak in DHI melanin, would support a covalent species: at the present stage, a pyranoacridinetrione structure seems the most likely candidate based on simulated UV-visible, Raman and 15N NMR spectra. d)

Under the same dip-coating conditions used for PDA, DHI polymerization does not

produce AFM detectable films on a quartz surface. e)

No MALDI MS-detectable species matching the m/z values expected for cyclic

porphyrin-type tetramers was found in DHI melanin even in the presence of FA as templating agent under reported conditions; likewise, no evidence for a significant cyclodimerization was obtained by oxidation of the 2,7’-dimer of DHI, a putative porphyrin precursor.


26

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f)

Spectral features predicted by extensive DFT calculations for the most stable porphyrin-

like structures at the 6-electron oxidation state did not match experimental data for PDA or DHI melanin. Before drawing conclusions about the nature of the species accounting for PDA adhesion properties, a note of caution is in order concerning the results discussed herein. In addition to those reported in previous studies, the main information on PDA films obtained in the present study that can be confidently used for structural characterization derives from the Raman and the MALDI-MS spectra. In particular, interpretation of mass spectrometric data is subject to caveats relating to the different ionization properties of the various structural components. Notwithstanding this limitation, it can be confidently stated that PDA contains specific molecular constituents that are involved in film formation and that do not appear to relate to DHI-based units derived from cyclization of dopamine or fragmentation products thereof. Thus, it is concluded that PDA is different from DHI melanin and that it is the difference, rather than the analogy that determines adhesion and film-forming properties. Several arguments illustrated in this study, moreover, would not support the recent claim that the primary building block of PDA at m/z 402 is a trimer complex consisting of two 5,6-dihydroxyindole (DHI) units and one pyrrolecarboxylic acid (PCA) moiety. Considering accumulating evidence that amine containing aliphatic chains are a crucial structural element favoring formation of adhesive films from catecholic substrates,49,52 it can be speculated that PDA film formation requires amine-containing structural components, for which various hypotheses have been proposed. Based on these results and a critical analysis of relevant literature data it is also concluded that any interpretation of PDA or eumelanin properties based on porphyrin-type structures is at present not supported by solid experimental evidence and needs to be revisited.


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Page 28 of 36

ASSOCIATED CONTENT Supporting Information AFM, Raman and MALDI-MS analysis of PDA and DHI melanin, computed Raman bands and 15N chemical shifts and reaction mechanism. The following files are available free of charge (PDF).

AUTHOR INFORMATION Corresponding Author *Email: [email protected]; phone: +39 081-674132. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was carried out in part with financial support from Kao Corporation.

ACKNOWLEDGMENT Computational resources were provided by the SCoPE Data Center of the University Federico II of Naples. ABBREVIATIONS DHI, 5,6-dihydroxyindole; PDA, polydopamine; MALDI-MS matrixassisted laser desorption/ionization – mass spectrometry; AFM, atomic force microscopy; Mefp-


28

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5, Mytilus edulis foot protein-5; HPLC, high performance liquid chromatography; Tris, tris(hydroxymethyl)aminomethane; CPPI-MAS NMR, cross polarization polarization inversion magic angle spinning nuclear magnetic resonance; ES-MS electrospray mass spectrometry; HRMS, high resolution mass spectroscopy; XPS, X-ray photoelectron spectroscopy; ToF-SIMS, time-of-flight secondary ion mass spectrometry; PCA, pyrrolecarboxylic acid; DFT, density functional theory; TEM, transmission electron microscopy; FA, folic acid; EPR, electron paramagnetic resonance; UV, ultraviolet; FT-IR, Fourier transform infrared spectroscopy; CHCA, α-cyano-4-hydroxy cinnamic acid.

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