Anisotropic Synthetic Allomelanin Materials via Solid-State Polymerization of Self-Assembled 1,8-Dihydroxynaphthalene Dimers

Article abstract of DOI:10.1002/anie.202103447

Melanosomes in nature have diverse morphologies, including spheres, rods, and platelets. By contrast, shapes of synthetic melanins have been almost entirely limited to spherical nanoparticles with few exceptions produced by complex templated synthetic methods. Here, we report a non-templated method to access synthetic melanins with a variety of architectures including spheres, sheets, and platelets. Three 1,8-dihydroxynaphthalene dimers (4-4′, 2-4′ and 2-2′) were used as self-assembling synthons. These dimers pack to form well-defined structures of varying morphologies depending on the isomer. Specifically, distinctive ellipsoidal platelets can be obtained using 4-4′ dimers. Solid-state polymerization of the preorganized dimers generates polymeric synthetic melanins while maintaining the initial particle morphologies. This work provides a new route to anisotropic synthetic melanins, where the building blocks are preorganized into specific shapes, followed by solid-state polymerization.

Full text of DOI:10.1002/anie.202103447

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A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Anisotropic Synthetic Allomelanin Materials via Solid State
Polymerization of Self-Assembled 1,8-Dihydroxynaphthalene
Dimers
Authors: Xuhao Zhou, Xinyi Gong, Wei Cao, Christopher J. Forman,
Julia Oktawiec, Liliana D’ Alba, Hao Sun, Matthew P.
Thompson, Ziying Hu, Utkarsh Kapoor, Naneki C. McCallum,
Christos D. Malliakas, Omar K. Farha, Arthi Jayaraman,
Matthew D. Shawkey, and Nathan C. Gianneschi
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202103447
Link to VoR: https://doi.org/10.1002/anie.202103447

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RESEARCH ARTICLE
Anisotropic Synthetic Allomelanin Materials via Solid State
Polymerization of Self-Assembled 1,8-Dihydroxynaphthalene
Dimers
Xuhao Zhou, Xinyi Gong, Wei Cao, Christopher J. Forman, Julia Oktawiec, Liliana D’ Alba, Hao Sun,
Matthew P. Thompson, Ziying Hu, Utkarsh Kapoor, Naneki C. McCallum, Christos D. Malliakas, Omar
K. Farha, Arthi Jayaraman, Matthew D. Shawkey and Nathan C. Gianneschi*
[*]
X. Zhou, X. Gong, Dr. W. Cao, Dr. C. J. Forman, Dr. J. Oktawiec, Dr. H. Sun, Dr. M. P. Thompson, Dr. Z. Hu, Dr. N. C. McCallum, Dr. C. D. Malliakas, Prof.
O. K. Farha, Prof. N. C. Gianneschi
Department of Chemistry, International Institute of Nanotechnology, Simpson-Querrey Institute, Chemistry of Life Processes Institute, Lurie Cancer Center.
Northwestern University
Evanston, Illinois 60208, United States of America
E-mail: nathan.gianneschi@northwestern.edu
Prof. N. C. Gianneschi
Department of Materials Science and Engineering, and Department of Biomedical Engineering. Northwestern University
Evanston, Illinois 60208, United States of America
Dr. U. Kapoor, Prof. A. Jayaraman
Department of Chemical and Biomolecular Engineering, Colburn Laboratory and Department of Materials Science and Engineering, University of Delaware
Newark, Delaware 19716, United States of America
Dr. L. D’ Alba, Prof. M. D. Shawkey
Department of Biology, Evolution and Optics of Nanostructures Group, University of Ghent
Ghent 9000, Belgium
Supporting information for this article is given via a link at the end of the document.
Abstract: Melanosomes have diverse morphologies in nature,
including spheres, rods, and platelets. By contrast, shapes of
synthetic melanins have been almost entirely limited to spherical
nanoparticles with few exceptions produced by complex templated
synthetic methods. Here, we report a non-templated method to
access synthetic melanins with a variety of architectures including
spheres, sheets, and platelets. Three 1,8-dihydroxynaphthalene
dimers (4-4′, 2-4′ and 2-2′) were used as self-assembling synthons.
These dimers pack to form well-defined structures of varying
morphologies depending on the isomer. Specifically, distinctive
ellipsoidal platelets can be obtained using 4-4′ dimers. Solid-state
polymerization of the preorganized dimers generates polymeric
synthetic melanins while maintaining the initial particle morphologies.
This work provides a new route to anisotropic synthetic melanins,
where the building blocks are preorganized into specific shapes,
followed by solid-state polymerization.
In contrast to this diversity found in nature, synthetic melanin is
mostly produced as spherical nanoparticles. Although some
anisotropic structures of polydopamine have been achieved
a more convenient,
scalable, and direct route towards non-spherical synthetic
melanin materials is desirable.
using DNA-origami-based templates,[9]
Allomelanin, the class of melanin targeted in this work, is a
heterogeneous group of polymers found mostly in fungi, bacteria,
10]
and plants.[7,
These organisms synthesize allomelanins
through oxidative polymerization of nitrogen-free precursors,
such as 1,8-dihydroxynaphthalene (1,8-DHN) and catechols.[10b,
11]
Typically, allomelanin generated by fungi utilizes 1,8-DHN
and is referred to as DHN melanin. The existence of DHN
melanin may be critical in the survival of fungi in hostile
environments, protecting them against ionizing radiation,
extreme temperatures, and oxidizing agents.[12] Previously, we
reported
a route for the chemical synthesis of artificial
allomelanin nanoparticles via oxidative oligomerization of 1,8-
DHN.[13] The resulting nanoparticles showed well-defined
spherical nanostructures and free radical scavenging activity.
Experimentally, we determined that spherical nanostructures
formed upon dimerization of 1,8-DHN, inspiring Kapoor and
Jayaraman to probe the self-assembly behavior of the three
DHN dimers (4-4′, 2-4′ and 2-2′) (Figure 1) via atomistic
molecular dynamics simulations.[14] These simulations revealed
that 4-4′ dimers adopt an anisotropic shape as they assemble,
as opposed to 2-2′ dimers that self-assemble into spherical
morphologies.[14] Inspired by the simulations, we hypothesized
that different isomers of DHN dimers would exhibit differences in
hierarchical self-assembly behavior that would be reflected in
their nanoscale or microscale morphologies. Specifically, 4-4′
dimers would have the propensity to generate synthetic
allomelanins with anisotropic morphologies. Furthermore, we
Introduction
Melanin is the term for a class of natural pigments ubiquitously
found in most organisms.[1] Melanin serves a variety of functions,
including as a pigment in human skin and hair,[2] in thermal
regulation,[3] in radiation protection,[4] in metal ion
sequestration,[5] and in neuroprotection.[6] In nature,
melanosomes, or melanin-containing organelles, possess
various morphologies and sizes.[7] Unique melanosome shapes,
including rods, disks, and platelets, are ubiquitous across
different biological kingdoms, particularly in bird feathers.[7-8]
A
wide array of spectacular bird colors emerges as a direct
consequence of the diversity of melanosomes morphologies. For
example, the green hermit hummingbird (Phaethornis guy)
applies densely packed ellipsoidal platelet-like melanosomes in
the gorget feathers to generate bright structural color (Figure S1).
1
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RESEARCH ARTICLE
Figure 1. Synthesis of DHN dimers. (A) Separation from oligomer mixtures. (B) Synthetic route to 2-2′ DHN dimer. (C) Synthetic route to 4-4′ DHN dimer.
reasoned that once assembled, the DHN dimers would yield to
further polymerization and crosslinking.
Acidic workup gave the desired boronic acid 2 ((1,8-
dimethoxynaphthalen-2-yl)boronic acid) in moderate yield.
Aerobic homocoupling of the boronic acid 2, catalyzed by
copper(I) chloride (CuCl), generated the methylated 2-2′ dimer 3
While DHN dimers have been identified in previous studies as
key intermediates and natural products, the dimers have not
been individually isolated or used in the preparation of artificial
melanin-like materials.[15] By studying each DHN dimer
individually, we aim to gain critical information on the process of
nanoparticle formation and to better understand the structure of
both natural and synthetic DHN melanins. Here, we describe the
synthesis and self-assembly of 4-4′, 2-4′ and 2-2′ DHN dimers in
(1,1′,8,8′-tetramethoxy-2,2′-binaphthalene),[16]
which
was
subsequently treated with BBr3 to remove the methyl protecting
groups to give the desired 2-2′ DHN dimer with an overall yield
of 14%. A similar synthetic approach was applied to selectively
synthesize 4-4′ DHN dimer (Figure 1C). Regioselective
bromination of 1 was accomplished by treatment with N-
bromosuccinamide (NBS), giving rise to the para-substituted
aqueous
solution,
followed
by
solid-state
oxidative
polymerization to generate corresponding synthetic allomelanins. compound
4
(1,8-dimethoxy-4-bromo-naphthalene). After
borylation, homocoupling and deprotection, the desired 4-4′
DHN dimer was obtained with an overall yield of 13%. The
structures of all three dimers were identified by nuclear magnetic
resonance (NMR) spectroscopy and mass spectrometry (MS)
(Supporting Information, section 5). Analytical HPLC traces were
used to confirm the purity and elution time for each dimer
(Figure S2).
The self-assembly of the three DHN dimers was performed via
dialysis to leverage this relatively slow process to produce
thermodynamically favorable structures (Figure 2). Briefly, self-
assembly was performed via dialysis using a slide-A-Lyzer MINI
dialysis device. DHN dimers were dissolved in CH3CN at a
concentration of 1.0 mg/mL, and then added to the dialysis
device (Figure 2A), which was then placed into a conical tube
containing ultrapure water and a stir bar. After stirring for two
days at room temperature, the samples were collected by
centrifugation and washed with ultrapure water three times to
remove unassembled dimers. The resulting self-assembled
materials are referred to as 4-4′-DHN-SA, 2-4′-DHN-SA and 2-2′-
DHN-SA (SA = self-assembly) (Figure S3).
Results and Discussion
The three dimers were first synthesized as a mixture and
isolated by separation using high-performance liquid
chromatography (HPLC) (Figure 1A). Specifically, the oxidizing
agent KMnO4 was added to a solution containing 1,8-DHN to
generate a mixture of DHN oligomers. By controlling the feed
ratio of the oxidizing agent and quenching the reaction early,
dimers of DHN were isolated as the main product. Preparatory
high-performance liquid chromatography (prep-HPLC) was used
to separate and purify each dimer. However, since the oxidative
oligomerization process is not well-controlled and regioselective,
only 4-4′ and 2-4′ dimers were obtained in appreciable yields
(6% yield for 4-4′ dimer, 27% yield for 2-4′ dimer). Only trace
amounts of the 2-2′ dimer was detected. Therefore, a selective
multistep synthetic strategy was adopted to synthesize the 2-2′
dimer, with the key step being the copper-mediated
homocoupling of the boronic acid derivative of 1,8-DHN (Figure
1B). The hydroxy groups of 1,8-DHN were first protected as
methyl ethers using methyl iodide, to avoid unwanted side
The DHN-SA samples were first characterized by transmission
electron microscopy (TEM) and scanning electron microscopy
(SEM) (Figure 2). Interestingly, the 4-4′ dimer self-assembled
into rectangular structures composed of oriented sheets with
well-defined edges. The length of 4-4′-DHN-SA structures
reactions.
Regioselective
borylation
of
1
(1,8-
dimethoxynaphthalene) was accomplished by treatment at
−78 °C with n-BuLi, followed by addition of trimethyl borate.
2
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Figure 2. (A) Dialysis-mediated self-assembly of three DHN dimers. Supramolecular structure of (B) 4-4′-DHN-SA. (ⅰ) TEM image, (ⅱ) SEM image. (C) 2-4′-DHN-
SA. (ⅰ) TEM image, (ⅱ) SEM image. (D) 2-2′-DHN-SA. (ⅰ) TEM image, (ⅱ) SEM image, scale bars 600 nm.
ranges from 5 to 10 µm, with a width of 1 to 2 µm (Figure 2B).
The rectangular sheet structure can also be visualized via
optical microscopy (Figure S4). In contrast to the 4-4′ dimer, the
2-4′ and 2-2′ dimers formed well-defined spherical nanoparticles,
with diameters ranging from 100 nm to 500 nm for 2-4′-DHN-SA,
and 150 nm to 500 nm for 2-2′-DHN-SA based on the TEM
images (Figure 2C and Figure 2D). Given the spherical
morphology of 2-4′-DHN-SA and 2-2′-DHN-SA structures,
dynamic light scattering (DLS) was used to determine the
hydrodynamic diameter of the particles and was found to be 244
nm and 342 nm respectively (Figure S5). Considering that
phenol and naphthol materials can be slowly oxidized by O2 from
air in solution, we wished to confirm whether the resulting self-
assembled structures were composed solely of DHN dimers and
not higher molecular weight oligomers generated by aerobic
oxidation during the dialysis process. To verify this, DHN-SAs
were re-dissolved in CH3CN and subjected to HPLC analysis
(Figure S6). The HPLC traces showed that DHN-SAs had
identical elution times compared to the corresponding DHN
dimers. Additionally, ESI-MS data of DHN-SAs revealed the
DHN dimer peak at m/z 317 [M-H]- (Figure S7). These data
confirm that the DHN dimers do not oxidize during the self-
assembly process.
underscores the lack of order within these self-assembled
structures. Electron diffraction data of DHN-SAs are similar to
the powder X-ray diffraction patterns, with electron diffraction of
4-4′-DHN-SA showing specific diffraction patterns (Figure 3B),
while for the two spherical structures, no diffraction patterns
were observed (Figure S9A and Figure S9B).
To understand the molecular packing and interactions of 4-4′-
DHN-SA, the compound was studied using single crystal X-ray
diffraction (SCXRD) analysis. Needle-like crystals of 4-4′-DHN-
SA suitable for SCXRD were prepared using the solvent switch
method described above, without stirring at 4 °C for 2 days
(Figure 3C). Analysis of the diffraction data reveals that 4-4′-
DHN-SA crystallizes in the P21/c space group, with one 4-4′
dimer molecule and two H2O molecules in the asymmetric unit
(Table S2, Figure S10). The other enantiomer of the 4-4′ dimer
is generated by the symmetry elements of the space group. The
dihedral angle of the two naphthalene rings is 108.0(1)°. This
value is in reasonable agreement with that calculated in
atomistic molecular dynamics simulations,[14] which found a
narrow distribution of 90°–100° for the 4-4′ dimer, suggesting
that its reduced flexibility that could lead to anisotropic packing.
Further examination of the crystal structure reveals that aromatic
stacking of the naphthalene rings and hydrogen bonding
between the hydroxyl groups and co-crystalized water molecules
are key driving forces that contribute to this particular packing of
the molecules (Figure 3D). The aromatic rings of the DHN
dimers form edge-to-face interactions and off-center parallel
stacking between neighboring molecules.[17] The closest carbon-
carbon distances associated with these two types of interactions
are 3.596(2) and 3.533(2) Å, respectively. The oxygen atoms of
the hydroxyl groups are located 2.555(1) Å from each other, and
2.590(2) and 2.722(2) Å from the oxygen atoms of the two water
molecules in the asymmetric unit. As a result, the DHN dimer
molecules orient to create layers of aromatic moieties bridged by
regions of hydrogen-bonded water along the [100] direction.
These results are also supported by the observation of aromatic
stacking and hydrogen bonding with water in simulations.[14]
The experimental PXRD pattern of 4-4′-DHN-SA displays distinct
diffraction peaks that match well in position and relative
intensities to the predicted pattern (Figure S11). Similarly, the
To further investigate the atomic structure of DHN-SAs, powder
X-ray diffraction (PXRD) and electron diffraction data were
collected. The PXRD pattern of 4-4′-DHN-SA displayed many
sharp reflections, characteristic of a highly crystalline material
(Figure 3A). In contrast, the diffraction patterns of 2-4′-DHN-SA
and 2-2′-DHN-SA exhibit broad peaks attributed to a loss of
long-range order (Figure S8A and Figure S8B). Specifically, only
three features can be located at approximately 14.9, 21.7, and
23.4° in the 2-4′-DHN-SA sample and 15.6, 21.0, and 22.5° in
the 2-2′-DHN-SA sample. These values correspond to
d
spacings of approximately 6.0, 4.2, and 3.9 Å and 5.7, 4.3, and
4.0 Å, respectively. The broadness of these peaks is consistent
with the nanoparticle nature of these samples observed in TEM
and SEM. Scherrer analysis yields an average crystallite size of
2.56±0.06-3.15±0.08 nm for 2-4′-DHN-SA and 5.1±0.1 nm for 2-
2′-DHN-SA (Table S1). The difference of these values from the
sizes of the particles determined by electron microscopy
3
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