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Plasmodium falciparum is the most lethal species of malaria. In infected human red blood cells, P. falciparum digests hemoglobin as a nutrient source, liberating cytotoxic free heme in the process. Sequestration and subsequent conversion of this byproduct into hemozoin, an inert biocrystalline heme aggregate, plays a key role in parasite survival. Hemozoin has been a longstanding target of antimalarials such as chloroquine (CQ), which inhibit the biocrystallization of free heme. In this study, we explore heme-binding interactions with histidine-rich-protein 2 (HRP2), a known malarial biomarker and purported player in free heme sequestration. HRP2 is notoriously challenging to target due to its highly repetitious sequence and irregular secondary structure. We started with three protein-catalyzed capture agents (PCCs) developed against epitopes of HRP2, inclusive of heme-binding motifs, and explored their ability to inhibit heme:HRP2 complex formation. Cocktails of the individual PCCs exhibit an inhibitory potency similar to CQ, while a covalently linked structure built from two separate PCCs provided considerably increased inhibition relative to CQ. Epitope-targeted disruption of heme:HRP2 binding is a novel approach towards disrupting P. falciparum-related hemozoin formation., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

Pulsed dipolar electron spin resonance spectroscopy (PDS) is a powerful tool for measuring distances in solution-state macromolecules. Paramagnetic metal ions, such as Cu 2+ , are used as spin probes because they can report on metalloprotein features and can be spectroscopically distinguished from traditional nitroxide (NO)-based labels. Here, we demonstrate site-specific incorporation of Cu 2+ into non-metalloproteins through the use of a genetically encodable non-natural amino acid, 3-pyrazolyltyrosine (PyTyr). We first incorporate PyTyr in cyan fluorescent protein to measure Cu 2+ -to-NO distances and examine the effects of solvent conditions on Cu 2+ binding and protein aggregation. We then apply the method to characterize the complex formed by the histidine kinase CheA and its target response regulator CheY. The X-ray structure of CheY-PyTyr confirms Cu labeling at PyTyr but also reveals a secondary Cu site. Cu 2+ -to-NO and Cu 2+ -to-Cu 2+ PDS measurements of CheY-PyTyr with nitroxide-labeled CheA provide new insights into the conformational landscape of the phosphotransfer complex and have implications for kinase regulation.

Amyotrophic lateral sclerosis, or Lou Gehrig's disease, is characterized by motor neuron death, with average survival times of two to five years. One cause of this disease is the misfolding of superoxide dismutase 1 (SOD1), a phenomenon influenced by point mutations spanning the protein. Herein, we used an epitope-specific high-throughput screen to identify a peptide ligand that stabilizes the SOD1 native conformation and accelerates its folding by a factor of 2.5. This strategy may be useful for fundamental studies of protein energy landscapes as well as designing new classes of therapeutics., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Acid-catalyzed, thermally activated ester cleavage reactions are critical for lithographic patterning processes used in the semiconductor industry. The rates of these high-temperature reactions within polymer thin films are difficult to characterize because of the thermal instability of many polymers and a lack of temperature-resolved measurement techniques. Here we introduce the use of transient laser irradiation to heat a methyladamantane-protected acrylate copolymer to 600 °C in less than a millisecond. These conditions mediate the removal of the protecting groups and enable accurate kinetic measurements. At sub-millisecond exposure to high temperatures (∼600 °C), the rate of the ester cleavage reaction exhibits the expected first-order dependence on acid concentration. In contrast, the reaction exhibits more complex kinetics when the polymer film is heated to lower temperatures (115 °C) on a conventional hot-plate. We identify distinct methyladamantane-derived deprotection products under the high- and low-temperature conditions that are consistent with the observed rate differences. The acid-catalyzed dimerization of 1-methyleneadamantane occurs at low temperature, which reduces the acid concentration available for the ester cleavage. This dimerization reaction is minimized during transient laser-induced heating because bimolecular reactions are disfavored under these conditions. We constructed a mathematical model based on these observations that accounts for the competition for the catalyst between the dimerization and ester cleavage processes. This laser-induced, sub-millisecond heating technique provides a means to probe and model temperature and time regimes of thermally activated reactions in polymer films, and these regimes exhibit distinct and advantageous reaction pathways that will inform future advances in high-performance photolithography.

Two-dimensional (2D) polymers assemble organic subunits into covalently linked, high-aspect-ratio networks with long-range order. Despite recent advances in 2D polymerization, scalable and general methods to access few- and single-layer materials are limited. Here we exfoliate a hydrazone-linked covalent organic framework (COF) to yield bulk quantities of few-layer two-dimensional (2D) polymers. Immersing the COF powder in several laboratory solvents exfoliates and disperses thin COF-43 samples, which maintain their characteristic periodic hexagonal structure. This phenomenon was characterized using infrared spectroscopy, dynamic light scattering, atomic force microscopy, transmission electron microscopy, and selected area electron diffraction. 2D COFs with reduced interlayer interaction energies offer a new means to access high-aspect-ratio 2D polymers whose structure may be designed using established principles of COF synthesis.

We demonstrate a tandem truncation-functionalization strategy using the 3D covalent organic framework, COF-102. Allyl groups incorporated within the pore walls were subjected to thiol-ene coupling conditions, achieving high conversions and maintaining the crystallinity and permanent porosity of the parent framework.

Framework materials have attracted intense interest for gas storage, separations, catalysis, and other applications as a consequence of their periodicity, high specific surface area, and rational synthesis. Cocrystallizing multiple monomers with identical linking chemistry represents an emerging route to access materials with increased complexity and advanced functions. This Concept Article highlights three strategies for framework synthesis that employ mixtures of monomers with 1) identical linking geometries, 2) different linking geometries, or 3) in which one monomer is truncated with respect to the other. These approaches offer a diverse toolbox to modify framework topology, incorporate active functionality, and rationally control crystallite size and morphology., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)