Preparation of Simple Bicyclic Carboxylate-Rich Alicyclic Molecules for the Investigation of Dissolved Organic Matter.

Dissolved organic matter (DOM) is a vast and complex chemical mixture that plays a key role in the mediation of the global carbon cycle. Fundamental understanding of the source and fate of oceanic organic matter is obscured due to poor definition of the key molecular contributors to DOM, which limits accurate sample analysis and prediction of the Earth's carbon cycle. Previous work has attempted to define the components of the DOM through a variety of chromatographic and spectral techniques. However, modern preparative and analytical methods have not isolated or unambiguously identified molecules from DOM. Therefore, previously proposed structures are based solely on the mixture's aggregate properties and do not accurately describe any true individual molecular component. In addition to this, there is a lack of appropriate analogues of the individual chemical classes within DOM, limiting the scope of experiments that probe the physical, chemical, and biological contributions from each class. To address these problems, we synthesized a series of analogues of carboxylate-rich alicyclic molecules (CRAM), a molecular class hypothesized to exist as a major contributor to DOM. Key analytical features of the synthetic CRAMs were consistent with marine DOM, supporting their suitability as chemical substitutes for CRAM. This new approach provides access to a molecular toolkit that will enable previously inaccessible experiments to test many unproven hypotheses surrounding the ever-enigmatic DOM.

Antimicrobial Peptides Incorporating Halogenated Marine-Derived Amino Acid Substituents.

Small synthetic mimics of cationic antimicrobial peptides represent a promising class of compounds with leads in clinical development for the treatment of persistent microbial infections. The activity and selectivity of these compounds rely on a balance between hydrophobic and cationic components, and here, we explore the activity of 19 linear cationic tripeptides against five different pathogenic bacteria and fungi, including clinical isolates. The compounds incorporated modified hydrophobic amino acids inspired by motifs often found in bioactive marine secondary metabolites in combination with different cationic residues to probe the possibility of generating active compounds with improved safety profiles. Several of the compounds displayed high activity (low µM concentrations), comparable with the positive controls AMC-109, amoxicillin, and amphotericin B. A higher activity was observed against the fungal strains, and a low in vitro off-target toxicity was observed against erythrocytes and HeLa cells, thereby illustrating effective means for tuning the activity and selectivity of short antimicrobial peptides.

Characterization of a Newly Available Coastal Marine Dissolved Organic Matter Reference Material (TRM-0522).

Recent methodological advances have greatly increased our ability to characterize aquatic dissolved organic matter (DOM) using high-resolution instrumentation, including nuclear magnetic resonance (NMR) and mass spectrometry (HRMS). Reliable DOM reference materials are required for further method development and data set alignment but do not currently exist for the marine environment. This presents a major limitation for marine biogeochemistry and related fields, including natural product discovery. To fill this resource gap, we have prepared a coastal marine DOM reference material (TRM-0522) from 45 m deep seawater obtained ∼1 km offshore of Sweden's west coast. Over 3000 molecular formulas were assigned by direct infusion HRMS, confirming sample diversity, and the distribution of formulas in van Krevelen space was typical for a marine sample, with the majority of formulas in the region H/C 1-1.5 and O/C 0.3-0.7. The extracted DOM pool was more nitrogen (N)- and sulfur (S)-rich than a typical terrestrial reference material (SRFA). MZmine3 processing of ultrahigh-performance liquid chromatography (UPLC)-HRMS/MS data revealed 494 resolvable features (233 in negative mode; 261 in positive mode) over a wide range of retention times and masses. NMR data indicated low contributions from aromatic protons and, generally speaking, low lignin, humic, and fulvic substances associated with terrestrial samples. Instead, carboxylic-rich aliphatic molecules were the most abundant components, followed by carbohydrates and aliphatic functionalities. This is consistent with a very low specific UV absorbance SUVA254 value of 1.52 L mg C-1 m-1. When combined with comparisons with existing terrestrial reference materials (Suwannee River fulvic acid and Pony Lake fulvic acid), these results suggest that TRM-0522 is a useful and otherwise unavailable reference material for use in marine DOM biogeochemistry.

Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling.

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

Does the Reaction of Cyclopropyl Acid Chlorides and Imines To Form 1,3-Oxazin-4-enone Heterocycles Proceed via a Ketene or an N-Acyl-iminium Mechanism?

The mechanism of the reaction between cyclopropyl acid chlorides and imines to form 1,3-oxazin-4-enones was probed through physical and computational experiments. The data gathered strongly support the reaction proceeding through an N-acyl iminium intermediate mechanism rather than a ketene intermediate mechanism.

Expedient metal-free synthesis of 1,3-oxazinen-4-ones.

1,3-Oxazinen-4-ones are medicinally important scaffolds which have traditionally been accessed using a hetero-Diels-Alder approach or more recently using a cobalt-catalyzed three-component cycloaddition. Herein we report a novel strategy to access this scaffold which allows for the rapid and high yielding synthesis of 1,3-oxazinen-4-ones under ambient temperature and pressures with improved substrate scope.