Structures of AT8 and PHF1 phosphomimetic tau: Insights into the posttranslational modification code of tau aggregation.

The microtubule-associated protein tau aggregates into amyloid fibrils in Alzheimer's disease and other neurodegenerative diseases. In these tauopathies, tau is hyperphosphorylated, suggesting that this posttranslational modification (PTM) may induce tau aggregation. Tau is also phosphorylated in normal developing brains. To investigate how tau phosphorylation induces amyloid fibrils, here we report the atomic structures of two phosphomimetic full-length tau fibrils assembled without anionic cofactors. We mutated key Ser and Thr residues to Glu in two regions of the protein. One construct contains three Glu mutations at the epitope of the anti-phospho-tau antibody AT8 (AT8-3E tau), whereas the other construct contains four Glu mutations at the epitope of the antibody PHF1 (PHF1-4E tau). Solid-state NMR data show that both phosphomimetic tau mutants form homogeneous fibrils with a single set of chemical shifts. The AT8-3E tau rigid core extends from the R3 repeat to the C terminus, whereas the PHF1-4E tau rigid core spans R2, R3, and R4 repeats. Cryoelectron microscopy data show that AT8-3E tau forms a triangular multi-layered core, whereas PHF1-4E tau forms a triple-stranded core. Interestingly, a construct combining all seven Glu mutations exhibits the same conformation as PHF1-4E tau. Scalar-coupled NMR data additionally reveal the dynamics and shape of the fuzzy coat surrounding the rigid cores. These results demonstrate that specific PTMs induce structurally specific tau aggregates, and the phosphorylation code of tau contains redundancy.

Structure of the nonhelical filament of the Alzheimer's disease tau core.

The microtubule-associated protein tau aggregates into neurofibrillary tangles in Alzheimer's disease (AD). The main type of aggregates, the paired helical filaments (PHF), incorporate about 20% of the full-length protein into the rigid core. Recently, cryo-electron microscopy data showed that a protease-resistant fragment of tau (residues 297-391) self-assembles in vitro in the presence of divalent cations to form twisted filaments whose molecular structure resembles that of AD PHF tau [S. Lövestam et al., Elife 11, e76494 (2022)]. To investigate whether this tau construct is uniquely predisposed to this morphology and structure, we fibrillized tau (297-391) under the reported conditions and determined its structure using solid-state NMR spectroscopy. Unexpectedly, the protein assembled predominantly into nontwisting ribbons whose rigid core spans residues 305-357. This rigid core forms a ß-arch that turns at residues 322CGS324. Two protofilaments stack together via a long interface that stretches from G323 to I354. Together, these two protofilaments form a four-layered ß-sheet core whose sidechains are stabilized by numerous polar and hydrophobic interactions. This structure gives insight into the fibril morphologies and molecular conformations that can be adopted by this protease-resistant core of AD tau under different pH and ionic conditions.

Milligram-scale assembly and NMR fingerprint of tau fibrils adopting the Alzheimer's disease fold.

In the Alzheimer's disease (AD) brain, the microtubule-associated protein tau aggregates into paired helical filaments in which each protofilament has a C-shaped conformation. In vitro assembly of tau fibrils adopting this fold is highly valuable for both fundamental and applied studies of AD without requiring patient-brain extracted fibrils. To date, reported methods for forming AD-fold tau fibrils have been irreproducible and sensitive to subtle variations in fibrillization conditions. Here, we describe a route to reproducibly assemble tau fibrils adopting the AD fold on the multi-milligram scale. We investigated the fibrillization conditions of two constructs and found that a tau (297-407) construct that contains four AD phospho-mimetic glutamate mutations robustly formed the C-shaped conformation. 2D and 3D correlation solid-state NMR spectra show a single predominant set of chemical shifts, indicating a single molecular conformation. Negative-stain electron microscopy and cryo-EM data confirm that the protofilament formed by 4E-tau (297-407) adopts the C-shaped conformation, which associates into paired, triple, and quadruple helical filaments. In comparison, NMR spectra indicate that a previously reported construct, tau (297-391), forms a mixture of a four-layered dimer structure and the C-shaped structure, whose populations are sensitive to the environmental conditions. The determination of the NMR chemical shifts of the AD-fold tau opens the possibility for future studies of tau fibril conformations and ligand binding by NMR. The quantitative assembly of tau fibrils adopting the AD fold should facilitate the development of diagnostic and therapeutic compounds that target AD tau.

Binding Sites of a Positron Emission Tomography Imaging Agent in Alzheimer's ß-Amyloid Fibrils Studied Using 19F Solid-State NMR.

Amyloid imaging by positron emission tomography (PET) is an important method for diagnosing neurodegenerative disorders such as Alzheimer's disease. Many 11C- and 18F-labeled PET tracers show varying binding capacities, specificities, and affinities for their target proteins. The structural basis of these variations is poorly understood. Here we employ 19F and 13C solid-state NMR to investigate the binding sites of a PET ligand, flutemetamol, to the 40-residue Alzheimer's ß-amyloid peptide (Aß40). Analytical high-performance liquid chromatography and 19F NMR spectra show that flutemetamol binds the current Aß40 fibril polymorph with a stoichiometry of one ligand per four to five peptides. Half of the ligands are tightly bound while the other half are loosely bound. 13C and 15N chemical shifts indicate that this Aß40 polymorph has an immobilized N-terminus, a non-ß-sheet His14, and a non-ß-sheet C-terminus. We measured the proximity of the ligand fluorine to peptide residues using 19F-13C and 19F-1H rotational-echo double-resonance (REDOR) experiments. The spectra show that three segments in the peptide, 12VHH14, 18VFF20, and 39VV40, lie the closest to the ligand. REDOR-constrained docking simulations indicate that these three segments form multiple binding sites, and the ligand orientations and positions at these sites are similar across different Aß polymorphs. Comparison of the flutemetamol-interacting residues in Aß40 with the small-molecule binding sites in other amyloid proteins suggest that conjugated aromatic compounds preferentially bind ß-sheet surface grooves lined by aromatic, polar, and charged residues. These motifs may explain the specificity of different PET tracers to different amyloid proteins.

Solid-State NMR 19F-1H-15N Correlation Experiments for Resonance Assignment and Distance Measurements of Multifluorinated Proteins.

Several solid-state NMR techniques have been introduced recently to measure nanometer distances involving 19F, whose high gyromagnetic ratio makes it a potent nuclear spin for structural investigation. These solid-state NMR techniques either use 19F correlation with 1H or 13C to obtain qualitative interatomic contacts or use the rotational-echo double-resonance (REDOR) pulse sequence to measure quantitative distances. However, no NMR technique is yet available for disambiguating 1H-19F distances in multiply fluorinated proteins and protein-ligand complexes. Here, we introduce a three-dimensional (3D) 19F-15N-1H correlation experiment that resolves the distances of multiple fluorines to their adjacent amide protons. We show that optimal polarization transfer between 1H and 19F spins is achieved using an out-and-back 1H-19F REDOR sequence. We demonstrate this 3D correlation experiment on the model protein GB1 and apply it to the multidrug-resistance transporter, EmrE, complexed to a tetrafluorinated substrate. This technique should be useful for resolving and assigning distance constraints in multiply fluorinated proteins, leading to significant savings of time and precious samples compared to producing several singly fluorinated samples. Moreover, the method enables structural determination of protein-ligand complexes for ligands that contain multiple fluorines.

Structurally Based Design of Glucagon Mutants That Inhibit Fibril Formation.

The peptide hormone glucagon is prescribed as a pharmaceutical compound to treat diabetic hypoglycemia. However, at the acidic pH where it is highly soluble, glucagon rapidly aggregates into inactive and cytotoxic amyloid fibrils. The recently determined high-resolution structure of these fibrils revealed various stabilizing molecular interactions. On the basis of this structure, we have now designed four arginine mutants of glucagon that resist fibrillization at pharmaceutical concentrations for weeks. An S2R, T29R double mutant and a T29R single mutant remove a hydrogen-bonding interaction in the wild-type fibril, whereas a Y13R, A19R double mutant and a Y13R mutant remove a cation-π interaction. 1H solution nuclear magnetic resonance spectra and ultraviolet absorbance data indicate that these mutants remain soluble in pH 2 buffer under quiescent conditions at concentrations of ≤4 mg/mL for weeks. Under stressed conditions with high salt concentrations and agitation, these mutants fibrillize significantly more slowly than the wild type. The S2R, T29R mutant and the T29R mutant exhibit a mixture of random coil and α-helical conformations, while the Y13R mutant is completely random coil. The mutation sites are chosen to be uninvolved in strong interactions with the glucagon receptor in the active structure of the peptide. Therefore, these arginine mutants of glucagon are promising alternative compounds for treating hypoglycemia.

Inclusion of the C-Terminal Domain in the ß-Sheet Core of Heparin-Fibrillized Three-Repeat Tau Protein Revealed by Solid-State Nuclear Magnetic Resonance Spectroscopy.

Many neurodegenerative diseases such as Alzheimer's disease are characterized by pathological ß-sheet filaments of the tau protein, which spread in a prion-like manner in patient brains. To date, high-resolution structures of tau filaments obtained from patient brains show that the ß-sheet core only includes portions of the microtubule-binding repeat domains and excludes the C-terminal residues, indicating that the C-terminus is dynamically disordered. Here, we use solid-state NMR spectroscopy to identify the ß-sheet core of full-length 0N3R tau fibrillized using heparin. Assignment of 13C and 15N chemical shifts of the rigid core of the protein revealed a single predominant ß-sheet conformation, which spans not only the R3, R4, R' repeats but also the entire C-terminal domain (CT) of the protein. This massive ß-sheet core qualitatively differs from all other tau fibril structures known to date. Using long-range correlation NMR experiments, we found that the R3 and R4 repeats form a ß-arch, similar to that seen in some of the brain-derived tau fibrils, but the R1 and R3 domains additionally stack against the CT, reminiscent of previously reported transient interactions of the CT with the microtubule-binding repeats. This expanded ß-sheet core structure suggests that the CT may have a protective effect against the formation of pathological tau fibrils by shielding the amyloidogenic R3 and R4 domains, preventing side-on nucleation. Truncation and post-translational modification of the CT in vivo may thus play an important role in the progression of tauopathies.

Physicochemical Changes in Biomass Chars by Thermal Oxidation or Ambient Weathering and Their Impacts on Sorption of a Hydrophobic and a Cationic Compound.

This study examined conditions that mimic oxidative processes of biomass chars during formation and weathering in the environment. A maple char prepared at the single heat treatment temperature of 500 °C for 2 h was exposed to different thermal oxidation conditions or accelerated oxidative aging conditions prior to sorption of naphthalene or the dication paraquat. Strong chemical oxidation (SCO) was included for comparison. Thermal oxidation caused micropore reaming, with ambient oxidation and SCO much less so. All oxidative treatments incorporated O, acidity, and cation exchange capacity (CEC). Thermal incorporation of O was a function of headspace O2 concentration and reached a maximum at 350 °C due to the opposing process of burn-off. The CEC was linearly correlated with O/C, but the positive intercept together with nuclear magnetic resonance data signifies that, compared to O groups derived by anoxic pyrolysis, O acquired through oxidation by thermal or ambient routes contributes more to the CEC. Thermal oxidation increased the naphthalene sorption coefficient, the characteristic energy of sorption, and the uptake rate due to pore reaming. By contrast, ambient oxidation (and SCO) suppressed naphthalene sorption by creating a more hydrophilic surface. Paraquat sorption capacity was predicted by an equation that includes a CEC2 term due to bidentate interaction with pairs of charges, predominating over monodentate interaction, plus a term for the capacity of naphthalene as a reference representing nonspecific driving forces.

Hydration and Dynamics of Full-Length Tau Amyloid Fibrils Investigated by Solid-State Nuclear Magnetic Resonance.

The microtubule-associated protein tau aggregates into distinct neurofibrillary tangles in brains afflicted with multiple neurodegenerative diseases such as Alzheimer's disease and corticobasal degeneration (CBD). The mechanism of tau misfolding and aggregation is poorly understood. Determining the structure, dynamics, and water accessibility of tau filaments may provide insight into the pathway of tau misfolding. Here, we investigate the hydration and dynamics of the ß-sheet core of heparin-fibrillized 0N4R tau using solid-state nuclear magnetic resonance spectroscopy. This ß-sheet core consists of the second and third microtubule-binding repeats, R2 and R3, respectively, which form a hairpin. Water-edited two-dimensional (2D) 13C-13C and 15N-13C correlation spectra show that most residues in R2 and R3 domains have low water accessibility, indicating that this hairpin is surrounded by other proteinaceous segments. However, a small number of residues, especially S285 and S316, are well hydrated compared to other Ser and Thr residues, suggesting that there is a small water channel in the middle of the hairpin. To probe whether water accessibility correlates with protein dynamics, we measured the backbone N-H dipolar couplings of the ß-sheet core. Interestingly, residues in the fourth microtubule-binding repeat, R4, show rigid-limit N-H dipolar couplings, even though this domain exhibits weaker intensities in the 2D 15N-13C correlation spectra. These results suggest that the R4 domain participates in cross-ß hydrogen bonding in some of the subunits but exhibits dynamic disorder in other subunits. Taken together, these hydration and dynamics data indicate that the R2-R3 hairpin of 0N4R tau is shielded from water by other proteinaceous segments on the exterior but contains a small water pore in the interior. This structural topology has various similarities with the CBD tau fibril structure but also shows specific differences. The disorder of the R4 domain and the presence of a small water channel in the heparin-fibrillized 4R tau have implications for the structure of tau fibrils in diseased brains.

Structure of the Polymer Backbones in polyMOF Materials.

The molecular connectivity of polymer-metal-organic framework (polyMOF) hybrid materials was investigated using density functional theory calculations and solid-state NMR spectroscopy. The architectural constraints that dictate the formation of polyMOFs were assessed by examining poly(1,4-benzenedicarboxylic acid) (pbdc) polymers in two archetypical MOF lattices (UiO-66 and IRMOF-1). Modeling of the polyMOFs showed that in the IRMOF-1-type lattice, six, seven, and eight methylene (-CH2-) groups between 1,4-benzenedicarboxylate (terephthalate, bdc2-) units can be accommodated without significant distortions, while in the UiO-66-type lattice, an optimal spacing of seven methylene groups between bdc2- units is needed to minimize strain. Solid-state NMR supports these predictions and reveals pronounced spectral differences for the same polymer in the two polyMOF lattices. With seven methylene groups, polyUiO-66-7a shows 7 ± 3% of uncoordinated terephthalate linkers, while these are undetectable (<4%) in the corresponding polyIRMOF-1-7a. In addition, NMR-detected backbone mobility is significantly higher in the polyIRMOF-1-7a than in the corresponding polyUiO-66-7a, again indicative of taut chains in the latter.

Quantifying Molecular Mixing and Heterogeneity in Pharmaceutical Dispersions at Sub-100 nm Resolution by Spin Diffusion NMR.

Molecular miscibility and homogeneity of amorphous solid dispersions (ASDs) are critical attributes that impact physicochemical stability, bioavailability, and processability. Observation of a single glass transition is utilized as a criterion for good mixing of drug substance and polymeric components but can be misleading and cannot quantitatively analyze the domain size at high resolution. While imaging techniques, on the other hand, can characterize phase separation on the particle surface at the nanometer scale, they often require customized sample preparation and handling. Moreover, a mixed system is not necessarily homogeneous. Compared to the numerous studies that have evaluated the mixing of drug substance and polymer in ASDs, inhomogeneity in the phase compositions has remained significantly underexplored. To overcome the analytical challenge, we have developed a 1H spin diffusion NMR technique to quantify molecular mixing of bulk ASDs at sub-100 nm resolution. It combines relaxation filtering (T2H and T1ρ) that leaves the active pharmaceutical ingredient (API) as the main source of 1H magnetization at the start of spin diffusion to the polymer matrix. A spray-dried nifedipine-poly(vinylpyrrolidone) (Nif-PVP) ASD at a 5 wt % drug loading was a homogeneous reference system that exhibited equilibration of magnetization transfer from API to polymer within a short spin diffusion time of ∼3 ms. While fast initial magnetization transfer proving mixing on the 1 nm scale was also observed in Nif-PVP ASDs prepared by hot-melt extrusion (HME) at 186 °C at a 40 wt % drug loading, incomplete equilibration of peak intensities documented inhomogeneity on the ≥30 nm scale. The nonuniformity was confirmed by the partial inversion of the Nif magnetization in the filter that resulted in an even more pronounced deviation from equilibration and by 1H-13C heteronuclear correlation (HETCOR) NMR. It is consistent with the observed differential 1H spin-lattice relaxation of Nif and PVP as well as a domain structure on the 20 nm scale observed in atomic force microscopy (AFM) images. The incomplete equilibration and differential relaxation were consistently reproduced in a model of two mixed phases of different compositions, e.g., 40 wt % of the ASD with a 15 wt % drug loading and the remaining 60 wt % with a 56 wt % drug loading. Hot-melt extrusion produced more inhomogeneous samples than spray drying for the samples examined in our study. To the best of our knowledge, this spin diffusion NMR method provides currently the highest-resolution quantification of inhomogeneous molecular mixing and phase composition in bulk samples of pharmaceutical dispersions produced with equipment, procedures, and drug loadings that are relevant to industrial drug development.

Multinuclear solid-state NMR of complex nitrogen-rich polymeric microcapsules: Weight fractions, spectral editing, component mixing, and persistent radicals.

The molecular structure of a crosslinked nitrogen-rich resin made from melamine, urea, and aldehydes, and of microcapsules made from the reactive resin with multiple polymeric components in aqueous dispersion, has been analyzed by 13C, 13C{1H}, 1H-13C, 1H, 13C{14N}, and 15N solid-state NMR without isotopic enrichment. Quantitative 13C NMR spectra of the microcapsules and three precursor materials enable determination of the fractions of different components. Spectral editing of non-protonated carbons by recoupled dipolar dephasing, of CH by dipolar DEPT, and of C-N by 13C{14N} SPIDER resolves peak overlap and helps with peak assignment. It reveals that the N- and O-rich resin "imitates" the spectrum of polysaccharides such as chitin, cellulose, or Ambergum to an astonishing degree. 15N NMR can distinguish melamine from urea and guanazole, NC=O from COO, and primary from secondary amines. Such a comprehensive and quantitative analysis enables prediction of the elemental composition of the resin, to be compared with combustion analysis for validation. It also provides a reliable reference for iterative simulations of 13C NMR spectra from structural models. The conversion from quantitative NMR peak areas of structural components to the weight fractions of interest in industrial practice is derived and demonstrated. Upon microcapsule formation, 15N and 13C NMR consistently show loss of urea and aldehyde and an increase in primary amines while melamine is retained. NMR also made unexpected findings, such as imbedded crystallites in one of the resins, as well as persistent radicals in the microcapsules. The crystallites produce distinct sharp lines and are distinguished from liquid-like components by their strong dipolar couplings, resulting in fast dipolar dephasing. Fast 1H spin-lattice relaxation on the 35-ms time scale and characteristically non-exponential 13C spin-lattice relaxation indicate persistent radicals, confirmed by EPR. Through 1H spin diffusion, the mixing of components on the 5-nm scale was documented.

A molecular fluorophore in citric acid/ethylenediamine carbon dots identified and quantified by multinuclear solid-state nuclear magnetic resonance.

The composition of fluorescent polymer nanoparticles, commonly referred to as carbon dots, synthesized by microwave-assisted reaction of citric acid and ethylenediamine was investigated by 13 C, 13 C{1 H}, 1 H─13 C, 13 C{14 N}, and 15 N solid-state nuclear magnetic resonance (NMR) experiments. 13 C NMR with spectral editing provided no evidence for significant condensed aromatic or diamondoid carbon phases. 15 N NMR showed that the nanoparticle matrix has been polymerized by amide and some imide formation. Five small, resolved 13 C NMR peaks, including an unusual ═CH signal at 84 ppm (1 H chemical shift of 5.8 ppm) and ═CN2 at 155 ppm, and two distinctive 15 N NMR resonances near 80 and 160 ppm proved the presence of 5-oxo-1,2,3,5-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid (IPCA) or its derivatives. This molecular fluorophore with conjugated double bonds, formed by a double cyclization reaction of citric acid and ethylenediamine as first shown by Y. Song, B. Yang, and coworkers in 2015, accounts for the fluorescence of the carbon dots. Cross-peaks in a 1 H─13 C HETCOR spectrum with brief 1 H spin diffusion proved that IPCA is finely dispersed in the polyamide matrix. From quantitative 13 C and 15 N NMR spectra, a high concentration (18 ± 2 wt%) of IPCA in the carbon dots was determined. A pronounced gradient in 13 C chemical-shift perturbations and peak widths, with the broadest lines near the COO group of IPCA, indicated at least partial transformation of the carboxylic acid of IPCA by amide or ester formation.

Polymer Infiltration into Metal-Organic Frameworks in Mixed-Matrix Membranes Detected in Situ by NMR.

Solid-state NMR has been used to study mixed-matrix membranes (MMMs) prepared with a metal-organic framework (MOF, UiO-66) and two different high molecular weight polymers (PEO and PVDF). 13C and 1H NMR data provide overwhelming evidence that most UiO-66 organic linkers are within 1 nm of PEO, which indicates that PEO is homogeneously distributed throughout the MOF. Systematic changes in MOF 13C NMR peak positions and 1H NMR line widths, as well as dramatic reductions in the MOF 1H T1ρ relaxation times, are observed as the PEO content increases, and when the pores have been filled, a further increase in PEO results in the formation of semicrystalline PEO outside the UiO-66 particles. In contrast, similar studies on PVDF MMMs show that the polymer contacts only a small fraction (<20%) of the MOF linkers. Simulations confirm that PEO penetrates into UiO-66 more easily than does PVDF. These studies are among the first to provide experimental insights into MOF-polymer interactions in an MMM.

Postsynthetic Metal Exchange in a Metal-Organic Framework Assembled from Co(III) Diphosphine Pincer Complexes.

A Zr metal-organic framework (MOF) 1-CoCl3 has been synthesized by solvothermal reaction of ZrCl4 with a carboxylic acid-functionalized CoIII-PNNNP pincer complex H4(L-CoCl3) ([L-CoCl3]4- = [(2,6-(NHPAr2)2C6H3)CoCl3]4-, Ar = p-C6H4CO2-). The structure of 1-CoCl3 has been determined by X-ray powder diffraction and exhibits a csq topology that differs from previously reported ftw-net Zr MOFs assembled from related PdII- and PtII-PNNNP pincer complexes. The Co-PNNNP pincer species readily demetallate upon reduction of CoIII to CoII, allowing for transmetalation with late second and third row transition metals in both the homogeneous complex and 1-CoCl3. Reaction of 1-CoCl3 with [Rh(nbd)Cl]2 (nbd = 2,5-nobornadiene) results in complete Rh/Co metal exchange at the supported diphosphine pincer complexes to generate 1-RhCl, which has been inaccessible by direct solvothermal synthesis. Treating 1-CoCl3 with PtCl2(SMe2)2 in the presence of the mild reductant NEt3 resulted in nearly complete Co substitution by Pt. In addition, a mixed metal pincer MOF, 1-PtRh, was generated by sequential substitution of Co with Pt followed by Rh.

Constraining Carbon Nanothread Structures by Experimental and Calculated Nuclear Magnetic Resonance Spectra.

A one-dimensional (1D) sp3 carbon nanomaterial with high lateral packing order, known as carbon nanothreads, has recently been synthesized by slowly compressing and decompressing crystalline solid benzene at high pressure. The atomic structure of an individual nanothread has not yet been determined experimentally. We have calculated the 13C nuclear magnetic resonance (NMR) chemical shifts, chemical shielding tensors, and anisotropies of several axially ordered and disordered partially saturated and fully saturated nanothreads within density functional theory and systematically compared the results with experimental solid-state NMR data to assist in identifying the structures of the synthesized nanothreads. In the fully saturated threads, every carbon atom in each progenitor benzene molecule has bonded to a neighboring molecule (i.e., 6 bonds per molecule, a so-called "degree-6" nanothread), while the partially saturated threads examined retain a single double bond per benzene ring ("degree-4"). The most-parsimonious theoretical fit to the experimental 1D solid-state NMR spectrum, constrained by the measured chemical shift anisotropies and key features of two-dimensional NMR spectra, suggests a certain combination of degree-4 and degree-6 nanothreads as plausible components of this 1D sp3 carbon nanomaterial, with intriguing hints of a [4 + 2] cycloaddition pathway toward nanothread formation from benzene columns in the progenitor molecular crystal, based on the presence of nanothreads IV-7, IV-8, and square polymer in the minimal fit.

The Chemical Structure of Carbon Nanothreads Analyzed by Advanced Solid-State NMR.

Carbon nanothreads are a new type of one-dimensional sp3-carbon nanomaterial formed by slow compression and decompression of benzene. We report characterization of the chemical structure of 13C-enriched nanothreads by advanced quantitative, selective, and two-dimensional solid-state nuclear magnetic resonance (NMR) experiments complemented by infrared (IR) spectroscopy. The width of the NMR spectral peaks suggests that the nanothread reaction products are much more organized than amorphous carbon. In addition, there is no evidence from NMR of a second phase such as amorphous mixed sp2/sp3-carbon. Spectral editing reveals that almost all carbon atoms are bonded to one hydrogen atom, unlike in amorphous carbon but as is expected for enumerated nanothread structures. Characterization of the local bonding structure confirms the presence of pure fully saturated "degree-6" carbon nanothreads previously deduced on the basis of crystal packing considerations from diffraction and transmission electron microscopy. These fully saturated threads comprise between 20% and 45% of the sample. Furthermore, 13C-13C spin exchange experiments indicate that the length of the fully saturated regions of the threads exceeds 2.5 nm. Two-dimensional 13C-13C NMR spectra showing bonding between chemically nonequivalent sites rule out enumerated single-site thread structures such as polytwistane or tube (3,0) but are consistent with multisite degree-6 nanothreads. Approximately a third of the carbon is in "degree-4" nanothreads with isolated double bonds. The presence of doubly unsaturated degree-2 benzene polymers can be ruled out on the basis of 13C-13C NMR with spin exchange rate constants tuned by rotational resonance and 1H decoupling. A small fraction of the sample consists of aromatic rings within the threads that link sections with mostly saturated bonding. NMR provides the detailed bonding information necessary to refine solid-state organic synthesis techniques to produce pure degree-6 or degree-4 carbon nanothreads.

Carbon Nitride Nanothread Crystals Derived from Pyridine.

Carbon nanothreads are a new one-dimensional sp3 carbon nanomaterial. They assemble into hexagonal crystals in a room temperature, nontopochemical solid-state reaction induced by slow compression of benzene to 23 GPa. Here we show that pyridine also reacts under compression to form a well-ordered sp3 product: C5NH5 carbon nitride nanothreads. Solid pyridine has a different crystal structure from solid benzene, so the nontopochemical formation of low-dimensional crystalline solids by slow compression of small aromatics may be a general phenomenon that enables chemical design of properties. The nitrogen in the carbon nitride nanothreads may improve processability, alters photoluminescence, and is predicted to reduce the bandgap.

Zirconium Metal-Organic Frameworks Assembled from Pd and Pt PNNNP Pincer Complexes: Synthesis, Postsynthetic Modification, and Lewis Acid Catalysis.

Carboxylic acid-functionalized Pd and Pt PNNNP pincer complexes were used for the assembly of two porous Zr metal-organic frameworks (MOFs), 2-PdX and 2-PtX. Powder X-ray diffraction analysis shows that the new MOFs adopt cubic framework structures similar to the previously reported Zr6O4(OH)4[(POCOP)PdX]3, [POCOP = 2,6-(OPAr2)2C6H3); Ar = p-C6H4CO2-, X = Cl-, I-] (1-PdX). Elemental analysis and spectroscopic characterization indicate the presence of missing linker defects, and 2-PdX and 2-PtX were formulated as Zr6O4(OH)4(OAc)2.4[M(PNNNP)X]2.4 [M = Pd, Pt; PNNNP = 2,6-(HNPAr2)2C5H3N; Ar = p-C6H4CO2-; X = Cl-, I-]. Postsynthetic halide ligand exchange reactions were carried out by treating 2-PdX with Ag(O3SCF3) or NaI followed by PhI(O2CCF3)2. The latter strategy proved to be more effective at activating the MOF for the catalytic intramolecular hydroamination of an o-substituted alkynyl aniline, underscoring the advantage of using halide exchange reagents that produce soluble byproducts.

A Major Step in Opening the Black Box of High-Molecular-Weight Dissolved Organic Nitrogen by Isotopic Labeling of Synechococcus and Multibond Two-Dimensional NMR.

Dissolved organic nitrogen (DON) comprises the largest pool of fixed N in the surface ocean, yet its composition has remained poorly constrained. Knowledge of the chemical composition of this nitrogen pool is crucial for understanding its biogeochemical function and reactivity in the environment. Previous work has suggested that high-molecular-weight (high-MW) DON exists only in two closely related forms, the secondary amides of peptides and of N-acetylated hexose sugars. Here, we demonstrate that the chemical structures of high-MW DON may be much more diverse than previously thought. We couple isotopic labeling of cyanobacterially derived dissolved organic matter with advanced two-dimensional NMR spectroscopy to open the "black box" of uncharacterized high-MW DON. Using multibond NMR correlations, we have identified novel N-methyl-containing amines and amides, primary amides, and novel N-acetylated sugars, which together account for nearly 50% of cyanobacterially derived high-MW DON. This study reveals unprecedented compositional details of the previously uncharacterized DON pool and outlines the means to further advance our understanding of this biogeochemically and globally important reservoir of organic nitrogen.