Biomimetic pheomelanin to unravel the electronic, molecular and supramolecular structure of the natural product.

Herein, we investigate synthetic routes to a close mimic of natural pheomelanin. Three different oxidative polymerization routes were attempted to generate synthetic pheomelanin, each giving rise to structurally dissimilar materials. Among them, the route employing 5-cysteinyl-dihydroxyphenylalanine (5-CD) as a monomer was verified as a close analogue of extracted pheomelanin from humans and birds. The resulting biomimetic and natural pheomelanins were compared via various techniques, including solid-state Nuclear Magnetic Resonance (ssNMR) and Electron Paramagnetic Resonance (EPR). This synthetic pheomelanin closely mimics the structure of natural pheomelanin as determined by parallel characterization of pheomelanin extracted from multiple biological sources. With a good synthetic biomimetic material in hand, we describe cation-π interactions as an important driving force for pheomelanogenesis, further advancing our fundamental understanding of this important biological pigment.

Chemoenzymatic Isolation and Characterization of High Purity Mammalian Melanin.

Although melanin is one of the most ubiquitous polymers in living systems, our understanding of its molecular structure, biosynthesis and biophysical properties has been limited to only a small number of organisms other than humans. This is in part due to the difficulty associated with isolating pure melanin. While purification methods exist, they typically involve harsh treatments with strong acid/base conditions combined with elevated temperatures that can lead to the polymer backbone degradation. To be successful, a viable isolation method must deliver a selective, yet complete degradation of non-melanin biopolymers as well as remove small molecule metabolites that are not integrative to the melanin backbone. Here, we demonstrate the use of chemoenzymatic processing guided by fluorescent probes for the purification and isolation of native mammalian melanin without significant induction of chemical degradation. This multi-step purification-tracking methodology enables quantitative isolation of pure melanin from mammalian tissue for spectroscopic characterization.

Unraveling the Structure and Function of Melanin through Synthesis.

Melanin is ubiquitous in living organisms across different biological kingdoms of life, making it an important, natural biomaterial. Its presence in nature from microorganisms to higher animals and plants is attributed to the many functions of melanin, including pigmentation, radical scavenging, radiation protection, and thermal regulation. Generally, melanin is classified into five types-eumelanin, pheomelanin, neuromelanin, allomelanin, and pyomelanin-based on the various chemical precursors used in their biosynthesis. Despite its long history of study, the exact chemical makeup of melanin remains unclear, and it moreover has an inherent diversity and complexity of chemical structure, likely including many functions and properties that remain to be identified. Synthetic mimics have begun to play a broader role in unraveling structure and function relationships of natural melanins. In the past decade, polydopamine, which has served as the conventional form of synthetic eumelanin, has dominated the literature on melanin-based materials, while the synthetic analogues of other melanins have received far less attention. In this perspective, we will discuss the synthesis of melanin materials with a special focus beyond polydopamine. We will emphasize efforts to elucidate biosynthetic pathways and structural characterization approaches that can be harnessed to interrogate specific structure-function relationships, including electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. We believe that this timely Perspective will introduce this class of biopolymer to the broader chemistry community, where we hope to stimulate new opportunities in novel, melanin-based poly-functional synthetic materials.

Isolation and Characterization of Allomelanin from Pathogenic Black Knot Fungus-a Sustainable Source of Melanin.

Melanin, a widespread pigment found in many taxa, is widely recognized for its high refractive index, ultraviolet (UV) protection, radical quenching ability, metal binding, and many other unique properties. The aforementioned characteristic traits make melanin a potential candidate for biomedical, separation, structural coloration, and space applications. However, the commercially available natural (sepia) and synthetic melanin are very expensive, limiting their use in various applications. Additionally, eumelanin has been the primary focus in most of these studies. In the present study, we demonstrate that melanin can be extracted from the pathogenic black knot fungus Apiosporina morbosa with a yield of ∼10% using the acid-base extraction method. The extracted melanin shows irregular morphology. Chemical characterization using X-ray photoelectron spectroscopy, infrared spectroscopy, and solid-state nuclear magnetic resonance spectroscopy reveals that the melanin derived from black knots is the less explored nitrogen-free allomelanin. Additionally, the extracted melanin shows broadband UV absorption typical of other types of melanin. Because of the wide availability and low cost of black knots and the invasive nature of the fungus, black knots can serve as an alternative green source for obtaining allomelanin at a low cost, which could stimulate its use as an UV light absorber and antioxidant in cosmetics and packaging industries.

Selenomelanin: An Abiotic Selenium Analogue of Pheomelanin.

Melanins are a family of heterogeneous biopolymers found ubiquitously across plant, animal, bacterial, and fungal kingdoms where they act variously as pigments and as radiation protection agents. There exist five multifunctional yet structurally and biosynthetically incompletely understood varieties of melanin: eumelanin, neuromelanin, pyomelanin, allomelanin, and pheomelanin. Although eumelanin and allomelanin have been the focus of most radiation protection studies to date, some research suggests that pheomelanin has a better absorption coefficient for X-rays than eumelanin. We reasoned that if a selenium enriched melanin existed, it would be a better X-ray protector than the sulfur-containing pheomelanin because the X-ray absorption coefficient is proportional to the fourth power of the atomic number (Z). Notably, selenium is an essential micronutrient, with the amino acid selenocysteine being genetically encoded in 25 natural human proteins. Therefore, we hypothesize that selenomelanin exists in nature, where it provides superior ionizing radiation protection to organisms compared to known melanins. Here we introduce this novel selenium analogue of pheomelanin through chemical and biosynthetic routes using selenocystine as a feedstock. The resulting selenomelanin is a structural mimic of pheomelanin. We found selenomelanin effectively prevented neonatal human epidermal keratinocytes (NHEK) from G2/M phase arrest under high-dose X-ray irradiation. Provocatively, this beneficial role of selenomelanin points to it as a sixth variety of yet to be discovered natural melanin.

Chemoenzymatic elaboration of the Raper-Mason pathway unravels the structural diversity within eumelanin pigments.

Melanin is a central polymer in living organisms, yet our understanding of its molecular structure remains unresolved. Here, we apply a biosynthetic approach to explore the composite structures accessible in one type of melanin, eumelanin. Using a combination of solid-state NMR, dynamic nuclear polarization, and electron microscopy, we reveal how a variety of monomers are enzymatically polymerized into their corresponding eumelanin pigments. We demonstrate how this approach can be used to unite structure with an understanding of enzymatic activity, substrate scope, and the regulation of nanostructural features. Overall, this data reveals how intermediate metabolites of the Raper-Mason metabolic pathway contribute to polymerization, allowing us to revisit the original proposal of how eumelanin is biosynthesized.

Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U-Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR.

Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.

In Situ Characterization of Pharmaceutical Formulations by Dynamic Nuclear Polarization Enhanced MAS NMR.

A principal advantage of magic angle spinning (MAS) NMR spectroscopy lies in its ability to determine molecular structure in a noninvasive and quantitative manner. Accordingly, MAS should be widely applicable to studies of the structure of active pharmaceutical ingredients (API) and formulations. However, the low sensitivity encountered in spectroscopy of natural abundance APIs present at low concentration has limited the success of MAS experiments. Dynamic nuclear polarization (DNP) enhances NMR sensitivity and can be used to circumvent this problem provided that suitable paramagnetic polarizing agent can be incorporated into the system without altering the integrity of solid dosages. Here, we demonstrate that DNP polarizing agents can be added in situ during the preparation of amorphous solid dispersions (ASDs) via spray drying and hot-melt extrusion so that ASDs can be examined during drug development. Specifically, the dependence of DNP enhancement on sample composition, radical concentration, relaxation properties of the API and excipients, types of polarizing agents and proton density, has been thoroughly investigated. Optimal enhancement values are obtained from ASDs containing 1% w/w radical concentration. Both polarizing agents TOTAPOL and AMUPol provided reasonable enhancements. Partial deuteration of the excipient produced 3× higher enhancement values. With these parameters, an ASD containing posaconazole and vinyl acetate yields a 32-fold enhancement which presumably results in a reduction of NMR measurement time by ∼1000. This boost in signal intensity enables the full assignment of the natural abundance pharmaceutical formulation through multidimensional correlation experiments.

Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR.

In DNP MAS NMR experiments at ∼80-110 K, the structurally important -13CH3 and -15NH3+ signals in MAS spectra of biological samples disappear due to the interference of the molecular motions with the 1H decoupling. Here we investigate the effect of these dynamic processes on the NMR line shapes and signal intensities in several typical systems: (1) microcrystalline APG, (2) membrane protein bR, (3) amyloid fibrils PI3-SH3, (4) monomeric alanine-CD3, and (5) the protonated and deuterated dipeptide N-Ac-VL over 78-300 K. In APG, the three-site hopping of the Ala-Cß peak disappears completely at 112 K, concomitant with the attenuation of CP signals from other 13C's and 15N's. Similarly, the 15N signal from Ala-NH3+ disappears at ∼173 K, concurrent with the attenuation in CP experiments of other 15N's as well as 13C's. In bR and PI3-SH3, the methyl groups are attenuated at ∼95 K, while all other 13C's remain unaffected. However, both systems exhibit substantial losses of intensity at ∼243 K. Finally, with spectra of Ala and N-Ac-VL, we show that it is possible to extract site specific dynamic data from the temperature dependence of the intensity losses. Furthermore, 2H labeling can assist with recovering the spectral intensity. Thus, our study provides insight into the dynamic behavior of biological systems over a wide range of temperatures, and serves as a guide to optimizing the sensitivity and resolution of structural data in low temperature DNP MAS NMR spectra.

Atomic Resolution Structure of Monomorphic Aß42 Amyloid Fibrils.

Amyloid-ß (Aß) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-ß amyloid fibrils that are a hallmark of Alzheimer's disease (AD). The most prominent forms of Aß are Aß1-40 and Aß1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aß42 is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of AßM01-42 amyloid fibrils derived from over 500 (13)C-(13)C, (13)C-(15)N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3 ) shows that the fibril core consists of a dimer of Aß42 molecules, each containing four ß-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular (13)C-(15)N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aß42 aggregation.

Structure and Mechanism of the Influenza A M218-60 Dimer of Dimers.

We report a magic angle spinning (MAS) NMR structure of the drug-resistant S31N mutation of M218-60 from Influenza A. The protein was dispersed in diphytanoyl-sn-glycero-3-phosphocholine lipid bilayers, and the spectra and an extensive set of constraints indicate that M218-60 consists of a dimer of dimers. In particular, ∼280 structural constraints were obtained using dipole recoupling experiments that yielded well-resolved (13)C-(15)N, (13)C-(13)C, and (1)H-(15)N 2D, 3D, and 4D MAS spectra, all of which show cross-peak doubling. Interhelical distances were measured using mixed (15)N/(13)C labeling and with deuterated protein, MAS at ωr/2π = 60 kHz, ω0H/2π = 1000 MHz, and (1)H detection of methyl-methyl contacts. The experiments reveal a compact structure consisting of a tetramer composed of four transmembrane helices, in which two opposing helices are displaced and rotated in the direction of the membrane normal relative to a four-fold symmetric arrangement, yielding a two-fold symmetric structure. Side chain conformations of the important gating and pH-sensing residues W41 and H37 are found to differ markedly from four-fold symmetry. The rmsd of the structure is 0.7 Å for backbone heavy atoms and 1.1 Å for all heavy atoms. This two-fold symmetric structure is different from all of the previous structures of M2, many of which were determined in detergent and/or with shorter constructs that are not fully active. The structure has implications for the mechanism of H(+) transport since the distance between His and Trp residues on different helices is found to be short. The structure also exhibits two-fold symmetry in the vicinity of the binding site of adamantyl inhibitors, and steric constraints may explain the mechanism of the drug-resistant S31N mutation.

Low-temperature polymorphic phase transition in a crystalline tripeptide L-Ala-L-Pro-Gly·H2O revealed by adiabatic calorimetry.

We demonstrate application of precise adiabatic vacuum calorimetry to observation of phase transition in the tripeptide L-alanyl-L-prolyl-glycine monohydrate (APG) from 6 to 320 K and report the standard thermodynamic properties of the tripeptide in the entire range. Thus, the heat capacity of APG was measured by adiabatic vacuum calorimetry in the above temperature range. The tripeptide exhibits a reversible first-order solid-to-solid phase transition characterized by strong thermal hysteresis. We report the standard thermodynamic characteristics of this transition and show that differential scanning calorimetry can reliably characterize the observed phase transition with <5 mg of the sample. Additionally, the standard entropy of formation from the elemental substances and the standard entropy of hypothetical reaction of synthesis from the amino acids at 298.15 K were calculated for the studied tripeptide.

Paramagnet induced signal quenching in MAS-DNP experiments in frozen homogeneous solutions.

The effects of nuclear signal quenching induced by the presence of a paramagnetic polarizing agent are documented for conditions used in magic angle spinning (MAS)-dynamic nuclear polarization (DNP) experiments on homogeneous solutions. In particular, we present a detailed analysis of three time constants: (1) the longitudinal build-up time constant TB for (1)H; (2) the rotating frame relaxation time constant T1ρ for (1)H and (13)C and (3) T2 of (13)C, the transverse relaxation time constant in the laboratory frame. These relaxation times were measured during microwave irradiation at a magnetic field of 5 T (140 GHz) as a function of the concentration of four polarizing agents: TOTAPOL, 4-amino-TEMPO, trityl (OX063), and Gd-DOTA and are compared to those obtained for a sample lacking paramagnetic doping. We also report the EPR relaxation time constants T1S and T2S, the DNP enhancements, ε, and the parameter E, defined below, which measures the sensitivity enhancement for the four polarizing agents as a function of the electron concentration. We observe substantial intensity losses (paramagnetic quenching) with all of the polarizing agents due to broadening mechanisms and cross relaxation during MAS. In particular, the monoradical trityl and biradical TOTAPOL induce ∼40% and 50% loss of signal intensity. In contrast there is little suppression of signal intensity in static samples containing these paramagnetic species. Despite the losses due to quenching, we find that all of the polarizing agents provide substantial gains in signal intensity with DNP, and in particular that the net enhancement is optimal for biradicals that operate with the cross effect. We discuss the possibility that much of this polarization loss can be regained with the development of instrumentation and methods to perform electron decoupling.

High frequency dynamic nuclear polarization.

During the three decades 1980-2010, magic angle spinning (MAS) NMR developed into the method of choice to examine many chemical, physical, and biological problems. In particular, a variety of dipolar recoupling methods to measure distances and torsion angles can now constrain molecular structures to high resolution. However, applications are often limited by the low sensitivity of the experiments, due in large part to the necessity of observing spectra of low-γ nuclei such as the I = 1/2 species (13)C or (15)N. The difficulty is still greater when quadrupolar nuclei, such as (17)O or (27)Al, are involved. This problem has stimulated efforts to increase the sensitivity of MAS experiments. A particularly powerful approach is dynamic nuclear polarization (DNP) which takes advantage of the higher equilibrium polarization of electrons (which conventionally manifests in the great sensitivity advantage of EPR over NMR). In DNP, the sample is doped with a stable paramagnetic polarizing agent and irradiated with microwaves to transfer the high polarization in the electron spin reservoir to the nuclei of interest. The idea was first explored by Overhauser and Slichter in 1953. However, these experiments were carried out on static samples, at magnetic fields that are low by current standards. To be implemented in contemporary MAS NMR experiments, DNP requires microwave sources operating in the subterahertz regime, roughly 150-660 GHz, and cryogenic MAS probes. In addition, improvements were required in the polarizing agents, because the high concentrations of conventional radicals that are required to produce significant enhancements compromise spectral resolution. In the last two decades, scientific and technical advances have addressed these problems and brought DNP to the point where it is achieving wide applicability. These advances include the development of high frequency gyrotron microwave sources operating in the subterahertz frequency range. In addition, low temperature MAS probes were developed that permit in situ microwave irradiation of the samples. And, finally, biradical polarizing agents were developed that increased the efficiency of DNP experiments by factors of ∼4 at considerably lower paramagnet concentrations. Collectively, these developments have made it possible to apply DNP on a routine basis to a number of different scientific endeavors, most prominently in the biological and material sciences. This Account reviews these developments, including the primary mechanisms used to transfer polarization in high frequency DNP, and the current choice of microwave sources and biradical polarizing agents. In addition, we illustrate the utility of the technique with a description of applications to membrane and amyloid proteins that emphasizes the unique structural information that is available in these two cases.

Continuously Tunable 250 GHz Gyrotron with a Double Disk Window for DNP-NMR Spectroscopy.

In this paper, we describe the design and experimental results from the rebuild of a 250 GHz gyrotron used for Dynamic Nuclear Polarization enhanced Nuclear Magnetic Resonance spectroscopy on a 380 MHz spectrometer. Tuning bandwidth of approximately 2 GHz is easily achieved at a fixed magnetic field of 9.24 T and a beam current of 95 mA producing an average output power of >10 W over the entire tuning band. This tube incorporates a double disk output sapphire window in order to maximize the transmission at 250.58 GHz. DNP Signal enhancement of >125 is achieved on a 13C-Urea sample using this gyrotron.