Snoring-generated fluid droplets as a potential mechanistic link between sleep-disordered breathing and pneumonia.

The soft palate and back of the throat represent vulnerable early infection sites for SARS-CoV-2, influenza, streptococci, and many other pathogens. We demonstrate that snoring causes aerosolization of pharyngeal fluid that covers these surfaces, which previously has escaped detection because the inspired airstream carries the micron-sized droplets into the lung, inaccessible to traditional aerosol detectors. While many of these droplets will settle in the lower respiratory tract, a fraction of the respirable smallest droplets remains airborne and can be detected in exhaled breath. We distinguished these exhaled droplets from those generated by the underlying breathing activity by using a chemical tracer, thereby proving their existence. The direct transfer of pharyngeal fluids and their pathogens into the deep lung by snoring represents a plausible mechanistic link between the previously recognized association between sleep-disordered breathing and pneumonia incidence.

Snoring may transmit infectious aerosols from the upper to the lower respiratory tract.

Migration to the lungs of an initial upper airway infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or other respiratory pathogens can lead to pneumonia, associated with progression from mild to severe symptoms. Chemical pneumonitis or bacterial pneumonia may be caused by the 'macroaspiration' of large volumes of oropharyngeal or gastroesophageal secretions into the lower respiratory tract. 'Microaspiration', i.e., a similar mechanism but involving much smaller amounts of oropharyngeal secretions, is considered the pathogenetic mechanism for most pneumonias, including that associated with COVID-19. Here, we hypothesize an alternative mechanism: Rather than by microaspiration, these fluids enter the lungs as microdroplets that are generated by snoring and then carried by the inspired airstream. Laboratory measurements indicate that snoring generates (a) comparable numbers and sizes of oral fluid droplets as loud speaking and (b) total fluid quantities that are similar to those reported for microaspiration. Snoring propensity is strongly correlated to known risk factors for severe COVID-19, including male gender, age, obesity, diabetes, obstructive sleep apnea, and pregnancy. Therefore, more research is urgently needed to determine if various methods that decrease snoring can prevent progression to pneumonia after initial infection of the upper airways.

Simultaneous Quantification of H2O2 and Organic Hydroperoxides by 1H NMR Spectroscopy.

Due to similar reactivity of organic hydroperoxides (OHPs), an HPLC separation step is typically required for their indirect (chemical) quantification in mixtures. The high sensitivity of chemical shifts to chemical structure makes NMR an ideal tool for the simultaneous quantification of OHPs in mixtures, but the concentration of these analytes in the samples of interest is usually well below the sensitivity of standard NMR experiments. This sensitivity problem can be mitigated by taking advantage of the fact that the z magnetization of the H2O2 resonance recovers at the rate of hydrogen exchange with water, which is significantly faster than longitudinal relaxation, thus enabling very fast scanning for signal-to-noise enhancement. An adaptation of the E-BURP2 pulse is described that suppresses the water signal by more than 4 orders of magnitude, yielding uniform excitation of peroxide signals without interference of the ca. 108-fold stronger H2O resonance. We demonstrate the method for a mixture of OHPs and report the chemical shifts for multiple OHPs that are of interest in atmospheric chemistry. As shown for hydroxymethyl hydroperoxide, the chemical decay of OHPs can be tracked directly by NMR spectroscopy.

Quantitative detection of hydrogen peroxide in rain, air, exhaled breath, and biological fluids by NMR spectroscopy.

Hydrogen peroxide (H2O2) plays a key role in environmental chemistry, biology, and medicine. H2O2 concentrations typically are 6 to 10 orders of magnitude lower than that of water, making its quantitative detection challenging. We demonstrate that optimized NMR spectroscopy allows direct, interference-free, quantitative measurements of H2O2 down to submicromolar levels in a wide range of fluids, ranging from exhaled breath and air condensate to rain, blood, urine, and saliva. NMR measurements confirm the previously reported spontaneous generation of H2O2 in microdroplets that form when condensing water vapor on a hydrophobic surface, which can interfere with atmospheric H2O2 measurements. Its antimicrobial activity and strong seasonal variation speculatively could be linked to the seasonality of respiratory viral diseases.

NMR characterization of H2O2 hydrogen exchange.

Quantification of H2O2 concentration in aqueous solutions is of interest in many fields. It usually is based on indirect methods that rely on oxidation reactions that turn on/off fluorescent probes. Such methods can suffer from reaction incompleteness and interfering chemical species. We describe optimization of NMR detection that enables direct quantification of H2O2 down to the nanomolar range. Taking advantage of fast hydrogen exchange (HX) between H2O2 and water permits the use of very short interscan delays, greatly increasing sensitivity. The specific acid-, base- and water-catalyzed HX rates at 2 °C were measured to be 2.1 × 107, 6.1 × 109, and 1.4 × 10-1 M-1s-1, respectively, which result in a minimum HX rate at pH 6.2. Furthermore, the exchange is accelerated by general acid/base catalysis. MES and phosphate buffers catalyze HX strongest in their unprotonated forms. For imidazole, only the unprotonated form catalyzes HX, which contrasts with acetic acid where only the protonated state catalyzes exchange. Inorganic salts such as sodium chloride and azide have negligible effect on HX. We present optimal conditions for accurate measurement of H2O2 concentrations as low as 40 nM in aqueous samples in a few hours.

Chemoenzymatic Synthesis of 9NHAc-GD2 Antigen to Overcome the Hydrolytic Instability of O-Acetylated-GD2 for Anticancer Conjugate Vaccine Development.

Ganglioside GD2 is an attractive tumor-associated carbohydrate antigen for anti-cancer vaccine development. However, its low immunogenicity and the significant side effects observed with anti-GD2 antibodies present significant obstacles for vaccines. To overcome these, a new GD2 derivative bearing an N-acetamide (NHAc) at its non-reducing end neuraminic acid (9NHAc-GD2) has been designed to mimic the 9-O-acetylated-GD2 (9OAc-GD2), a GD2 based antigen with a restricted expression on tumor cells. 9NHAc-GD2 was synthesized efficiently via a chemoenzymatic method and subsequently conjugated with a powerful carrier bacteriophage Qß. Mouse immunization with the Qß-9NHAc-GD2 conjugate elicited strong and long-lasting IgG antibodies, which were highly selective toward 9NHAc-GD2 with little cross-recognition of GD2. Immunization of canines with Qß-9NHAc-GD2 showed the construct was immunogenic in canines with little adverse effects, paving the way for future clinical translation to humans.

A lowly populated, transient ß-sheet structure in monomeric Aß1-42 identified by multinuclear NMR of chemical denaturation.

Chemical denaturation is a well-established approach for probing the equilibrium between folded and unfolded states of proteins. We demonstrate applicability of this method to the detection of a small population of a transiently folded structural element in a system that is often considered to be intrinsically fully disordered. The 1HN, 15N, 13Cα, and 13C' chemical shifts of Aß1-40 and Aß1-42 peptides and their M35-oxidized variants were monitored as a function of urea concentration and compared to analogous urea titrations of synthetic pentapeptides of homologous sequence. Fitting of the chemical shift titrations yields a 10 ± 1% population for a structured element at the C-terminus of Aß1-42 that folds with a cooperativity of m = 0.06 kcal/mol·M. The fit also yields the chemical shifts of the folded state and, using a database search, for Aß1-42 these shifts identified an antiparallel intramolecular ß-sheet for residues I32-A42, linked by a type I' ß-turn at G37 and G38. The structure is destabilized by oxidation of M35. Paramagnetic relaxation rates and two previously reported weak, medium-range NOE interactions are consistent with this transient ß-sheet. Introduction of the requisite A42C mutation and tagging with MTSL resulted in a small stabilization of this ß-sheet. Chemical shift analysis suggests a C-terminal ß-sheet may be present in Aß1-40 too, but the turn type at G37 is not type I'. The approach to derive Transient Structure from chemical Denaturation by NMR (TSD-NMR), demonstrated here for Aß peptides, provides a sensitive tool for identifying the presence of lowly populated, transiently ordered elements in proteins that are considered to be intrinsically disordered, and permits extraction of structural data for such elements.

Mechanistic Insights into the Origin of Stereoselectivity in an Asymmetric Chlorolactonization Catalyzed by (DHQD)2PHAL.

Electrophilic halofunctionalization reactions have undergone a resurgence sparked by recent discoveries in the field of catalytic asymmetric halocyclizations. To build mechanistic understanding of these asymmetric transformations, a toolbox of analytical methods has been deployed, addressing the roles of catalyst, electrophile (halenium donor), and nucleophile in determining rates and stereopreferences. The test reaction, (DHQD)2PHAL-catalyzed chlorocyclization of 4-arylpent-4-enoic acid with 1,3-dichloro-5,5-dimethylhydantoin (DCDMH), is revealed to be first order in catalyst and chlorenium ion donor and zero order in alkenoic acid substrate under synthetically relevant conditions. The simplest interpretation is that rapid substrate-catalyst binding precedes rate-limiting chlorenium attack, controlling the face selectivity of both chlorine attack and lactone closure. ROESY and DFT studies, aided by crystal structures of carboxylic acids bound by the catalyst, point to a plausible resting state of the catalyst-substrate complex predisposed for asymmetric chlorolactonization. As revealed by our earlier labeling studies, these findings suggest modes of binding in the (DHQD)2PHAL chiral pocket that explain the system's remarkable control over rate- and enantioselection-determining events. Though a comprehensive modeling analysis is beyond the scope of the present work, quantum chemical analysis of the fragments' interactions and candidate reaction paths point to a one-step concerted process, with the nucleophile playing a critical role in activating the olefin for concomitant electrophilic attack.

Cycloaddition/Electrocyclic Ring Opening Sequence between Alkynyl Sulfides and Azodicarboxylates To Provide N,N-Dicarbamoyl 2-Iminothioimidates.

The α-oxidized thioimidates are useful bidentate ligands and are important motifs in pharmaceuticals, pesticides, and fungicides. Despite their broad utility, a direct route for their synthesis has been elusive. Herein, we describe a one-step synthesis of N,N-dicarbamoyl 2-iminothioimidates from easily accessible thioacetylenes and commercially available azodicarboxylates (20 examples, ≤99% yield). Additionally, the mechanism of the transformation was extensively explored by variable-temperature NMR, in situ IR, and quantum mechanical simulations. These experiments suggest that the reaction commences with a highly asynchronous [2 + 2] cycloaddition, which leads to a four-membered diazacyclobutene intermediate with a barrier consistent with the observed reaction rate. This intermediate was then isolated for subsequent kinetic measurements, which yielded an experimental barrier within 1 kcal/mol of the calculated barrier for a subsequent 4π electrocyclic ring opening leading to the observed iminothioimidate products. This method represents the first direct route to α-oxidized thioimidates from readily accessible starting materials.

Chemoenzymatic synthesis of glycopeptides bearing rare N-glycan sequences with or without bisecting GlcNAc.

N-Linked glycopeptides have highly diverse structures in nature. Herein, we describe the first synthesis of rare multi-antennary N-glycan bearing glycan chains on 6-OH of both α1,6- and α1,3-linked mannose arms. To expedite divergent generation of N-glycan structures, four orthogonal protective groups were installed at the branching points on the core tetrasaccharide, which could be removed individually without affecting one another. In addition, the synthetic route is flexible, allowing a bisecting glucosamine moiety to be introduced at a late stage of the synthesis, further expanding the diversity of sequences that could be achieved. The bisecting glucosamine unit significantly reduced the glycosylation yields of adjacent mannoses, which was attributed to steric hindrance imposed by the glucosamine based on molecular modelling analysis. The N-glycans were then transformed to oxazoline donors and ligated with a glycopeptide acceptor from haptoglobin promoted by the wild type Arthrobacter endo-ß-N-acetylglucosaminidase (Endo-A). Endo-A exhibited interesting substrate preferences depending on donor sizes, which was rationalized through molecular dynamics studies. This is the first time that a glycopeptide bearing a bisecting N-acetyl glucosamine (GlcNAc), the rare N-glycan branch, and two LewisX trisaccharide antennae was synthesized, enabling access to this class of complex glycopeptide structures.

Substrate Controlled Regioselective Bromination of Acylated Pyrroles Using Tetrabutylammonium Tribromide (TBABr3).

Electrophilic bromination of pyrroles bearing carbonyl substituents at C-2 typically results in a mixture of the 4- and 5-brominated species, generally favoring the 4-position. Herein, we describe a substrate-controlled regioselective bromination in which tetra-butyl ammonium tribromide (TBABr3) reacts with pyrrole-2-carboxamide substrates to yield the 5-brominated species as the predominant (up to >10:1) product.

High-Field NMR Spectroscopy Reveals Aromaticity-Modulated Hydrogen Bonding in Heterocycles.

From DNA base pairs to drug-receptor binding, hydrogen (H-)bonding and aromaticity are common features of heterocycles. Herein, the interplay of these bonding aspects is explored. H-bond strength modulation due to enhancement or disruption of aromaticity of heterocycles is experimentally revealed by comparing homodimer H-bond energies of aromatic heterocycles with analogs that have the same H-bonding moieties but lack cyclic π-conjugation. NMR studies of dimerization in C6 D6 find aromaticity-modulated H-bonding (AMHB) energy effects of approximately ±30 %, depending on whether they enhance or weaken aromatic delocalization. The attendant ring current perturbations expected from such modulation are confirmed by chemical shift changes in both observed ring C-H and calculated nucleus-independent sites. In silico modeling confirms that AMHB effects outweigh those of hybridization or dipole-dipole interaction.

AMHB: (Anti)aromaticity-Modulated Hydrogen Bonding.

This in silico survey shows that changes in the (anti)aromatic character of π-conjugated heterocycles can be used to fine-tune their hydrogen (H-)bond strengths. Upon H-bonding dimerization, the π-electrons of these rings can be polarized to reinforce or disrupt their (anti)aromatic π-conjugated circuits (πCCs) and stabilize or destabilize the resulting H-bonded complexes. H-bonding interactions that enhance aromaticity or relieve antiaromaticity are fortified, whereas those that intensify antiaromaticity or disrupt aromaticity are weakened, relative to analogues lacking full π-circuits. Computed dissected nucleus-independent chemical shifts, NICS(1)(zz), reveal a uniform pattern and document changes in the magnetic (anti)aromatic character of the heterocycles considered. Recognition of this (anti)aromaticity-modulated H-bonding (AMHB) phenomenon offers insights into a range of fields from organocatalysis and self-assembly to pharmaceutical chemistry and molecular biology.