Antimicrobial, Preservative, and Hazard Assessments from Eight Chemical Classes.

Preservatives, such as isothiazolinones and formaldehyde-releasing compounds, provide safety and stability in consumer products by preventing microbial contamination. Yet these ingredients present human and environmental hazards, including allergic contact dermatitis and aquatic toxicity. The development of safer alternatives has been stymied by trade-offs between safety and efficacy. To enable the identification of safer preservatives, substances from eight functional classes were assessed for antimicrobial efficacy and human and environmental hazards. First, 130 substances were evaluated for microbial inhibitory activity against two relevant model microorganisms, Aspergillus brasiliensis (filamentous fungi) and Pseudomonas aeruginosa (Gram-negative bacteria). High-performing compounds within each class were assessed for hazards across a broad suite of human and environmental health end points. Four promising compounds were selected for further testing based on microbial inhibition, hazard profiles, and commercial availability. These ingredients were tested for biocidal activity in model home care formulations using methods adapted from industrial preservative challenge guidelines (USP-51). Two substances were identified, caprylhydroxamic acid and caprylyl glycol, that provided adequate preservation and improved toxicity profiles compared to isothiazolinone and formaldehyde-releasing preservatives. This study highlights trade-offs between antimicrobial activity and hazards across a broad spectrum of chemical classes relevant to safer preservative development.

High-efficiency fungal pathogen intervention for seed protection: new utility of long-chain alkyl gallates as heat-sensitizing agents.

Control of food-contaminating fungi, especially pathogens that produce mycotoxins, is problematic since effective method for intervening fungal infection on food crops is often limited. Generally Regarded As Safe (GRAS) chemicals, such as natural compounds or their structural derivatives, can be developed as antimicrobial agents for sustainable food/crop production. This study identified that long-chain alkyl gallates, i.e., octyl-, nonyl-, and decyl gallates (OG (octyl 3,4,5-trihydroxybenzoic acid), NG, DG), can function as heat-sensitizing agents that effectively prevent fungal contamination. Out of twenty-eight candidate compounds and six conventional antifungal agents examined, the heat-sensitizing capacity was unique to the long-chain alkyl gallates, where OG exhibited the highest activity, followed by DG and NG. Since OG is a GRAS compound classified by the United States Food and Drug Administration (FDA), further in vitro antifungal studies were performed using OG. When OG and mild heat (57.5°C) were co-administered for 90 seconds, the treatment achieved > 99.999% fungal death (> 5 log reduction). Application of either treatment alone was significantly less effective at reducing fungal survival. Of note, co-application of OG (3 mM) and mild heat (50°C) for 20 minutes completely prevented the survival of aflatoxigenic Aspergillus flavus contaminating crop seeds (Brassica rapa Pekinensis), while seed germination rate was unaffected. Heat-sensitization was also determined in selected bacterial strains (Escherichia coli, Agrobacterium tumefaciens). Altogether, OG is an effective heat-sensitizing agent for control of microbial pathogens. OG-mediated heat sensitization will improve the efficacy of antimicrobial practices, achieving safe, rapid, and cost-effective pathogen control in agriculture/food industry settings.

Computational Studies of Rubber Ozonation Explain the Effectiveness of 6PPD as an Antidegradant and the Mechanism of Its Quinone Formation.

The discovery that the commercial rubber antidegradant 6PPD reacts with ozone (O3) to produce a highly toxic quinone (6PPDQ) spurred a significant research effort into nontoxic alternatives. This work has been hampered by lack of a detailed understanding of the mechanism of protection that 6PPD affords rubber compounds against ozone. Herein, we report high-level density functional theory studies into early steps of rubber and PPD (p-phenylenediamine) ozonation, identifying key steps that contribute to the antiozonant activity of PPDs. In this, we establish that our density functional theory approach can achieve chemical accuracy for many ozonation reactions, which are notoriously difficult to model. Using adiabatic energy decomposition analysis, we examine and dispel the notion that one-electron charge transfer initiates ozonation in these systems, as is sometimes argued. Instead, we find direct interaction between O3 and the PPD aromatic ring is kinetically accessible and that this motif is more significant than interactions with PPD nitrogens. The former pathway results in a hydroxylated PPD intermediate, which reacts further with O3 to afford 6PPD hydroquinone and, ultimately, 6PPDQ. This mechanism directly links the toxicity of 6PPDQ to the antiozonant function of 6PPD. These results have significant implications for development of alternative antiozonants, which are discussed.

Valorizing Tree-Nutshell Particles as Delivery Vehicles for a Natural Herbicide.

The United States is a principal producer of tree nuts (almonds, pistachios, and walnuts), resulting in the generation of excess of tree-nutshell by-products each year, with few market outlets. A nutshell is an essential, lignocellulosic layer that protects a kernel (seed) from the environment during cultivation. The objective of this study was to develop nutshell by-products as herbicide delivery systems, which would not only enable sustainable weed control in fields but also increases nutshell value and reduce the cost of waste disposal. We recently identified a natural salicylaldehyde (SA) that emits volatiles with both herbicidal and antifungal properties. In this study, walnut shell particles saturated with 0.8 to 1.6 M SA were developed as delivery vehicles for SA to soil, which allowed for the controlled release of an SA fumigant for weed control. The pre- and post-emergent herbicidal efficacy of SA was investigated using model monocot (Lolium arundinaceum (Schreb.) Darbysh; turfgrass) and dicot (Brassica rapa var. pekinensis; Chinese cabbage) plants. We compared (1) the effects of different types of solvents for dissolving SA (dimethyl sulfoxide (DMSO) and ethanol (60%, v/v)), and (2) the effect of covering soil with plastic layers (i.e., soil pasteurization) or not covering soil during SA fumigation using nutshells. Results: In the pre-emergent herbicidal testing with the soil covered, the dicot plants exhibited levels of higher susceptibility to SA in DMSO emitted from nutshells when compared to the monocot plants. The seed germination frequencies in the dicots were 15% and 1% with 0.8 and 1.6 M SA, respectively, while those in the monocots were 32% and 18%, respectively, under the same test conditions. In the post-emergent herbicidal testing with the soil covered, the growth of both the monocot and dicot plants was completely prevented after 5 to 7 days of SA fumigation, resulting in the deaths of entire plants. It was noteworthy that in the post-emergent herbicidal testing, SA dissolved in ethanol (60%, v/v) completely disrupted the growth of the monocot and dicot plants as early as 3 days after SA emission from the nutshells, even without the soil being covered. Tree-nutshell particles could serve as effective SA delivery vehicles with controlled release capabilities for SA. The SA exhibited pre- and post-emergent herbicidal activities against the monocot and dicot plants at most growth stages. SA (0.8 and 1.6 M) dissolved in ethanol (60%, v/v) might exert a synergism for higher herbicidal activity after emission from nutshells. Since tree nuts capture/store a substantial amount of carbon over their life-cycles, the new and sustainable utility of using nutshells not only reduces carbon emissions but also valorizes tree-nut by-products, thus benefitting the tree-nut industry.

Effect of an Eco-Friendly Cuminaldehyde Guanylhydrazone Disinfectant on Shiga Toxin Production and Global Transcription of Escherichia coli.

Antimicrobials have been important medicines used to treat various infections. However, some antibiotics increase the expression of Shiga toxin (Stx). Also, the pervasive use of persistent antibiotics has led to ecotoxicity and antibiotic resistance. In this study, a newly developed broad-spectrum and reversible antibiotic (guanylhydrazone disinfectant) was evaluated for its antibiotic activity and effects on Stx production and global transcription of bacteria. No Stx induction was observed in 25 Shiga toxin-producing E. coli (STEC) isolates treated with a sublethal concentration of the guanylhydrazone. A differential gene expression study comparing two guanylhydrazone-treated to non-treated E. coli strains indicated that the expression of a group of stress-responsive genes were enhanced. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that guanylhydrazone treatment significantly downregulated the pathways of ribosome and flagellar assembly in both pathogenic and non-pathogenic strains and differentially regulated some pathways essential for bacteria to maintain cell shape and gain survival advantage in two strains. In addition, upregulation of antibiotic resistant genes related to the multidrug efflux system and virulence genes coding for colibactin, colicin, and adhesin was observed in strains treated with the disinfectant. The knowledge obtained in this study contributes to our understanding of the mode of this disinfectant action and facilitates our effort to better use disinfectants for STEC treatments.

Production of polyhydroxyalkanoate copolymers containing 4-hydroxybutyrate in engineered Bacillus megaterium.

Despite being used as a common platform for the commercial production of many biochemicals, Bacilli are often overlooked as a source of industrial polyhydroxyalkanoates (PHAs), biodegradable plastic replacements. In addition to having a robust expression system, the lack of lipopolysaccharides and ease of lysis make Bacilli an attractive host for the production of PHAs. In this work, a Bacillus megaterium strain was engineered to generate poly(3-hydroxybutyrate-co-4-hydroxybutryate) (P[3HB-co-4HB]) copolymers, which are among the most useful and industrially-relevant copolymers. These copolymers had lower modulus and increased toughness, thus making the copolymer suitable for a broader range of applications. Due to high metabolic flux through succinate, the engineered B. megaterium strain produced P(3HB-co-4HB) with >10% mol fraction 4HB from glucose, without the use of highly regulated and expensive precursors or potentially damaging truncation of central biochemical pathways.

Penicillium camemberti galacturonate reductase: C-1 oxidation/reduction of uronic acids and substrate inhibition mitigation by aldonic acids.

The enzyme galacturonate oxidoreductase PcGOR from Penicillium camemberti reduces the C-1 carbon of D-glucuronate and C-4 epimer D-galacturonate to their corresponding aldonic acids, important reactions in both pectin catabolism and ascorbate biosynthesis. PcGOR was active on both glucuronic acid and galacturonic acid, with similar substrate specificities (kcat/Km) using the preferred co-substrate NADPH. Substrate acceptance extended to lactone congeners, and D-glucurono-3,6-lactone was converted to L-gulono-1,4-lactone, an immediate precursor of ascorbate. Reaction with glucuronate showed only minor substrate inhibition, and the product L-gulonate and L-gulono-1,4-lactone were both found to be competitive inhibitors with Ki in the low mM range. In contrast, reaction with C-4 epimer galacturonate displayed marked substrate inhibition. Moreover, the product L-galactonate and L-galactono-1,4-lactone were observed to mitigate substrate inhibition by galacturonate, with the lactone having a greater effect than the acid.

Protein-like proton exchange in a synthetic host cavity.

The mechanism of proton exchange in a metal-ligand enzyme active site mimic (compound 1) is described through amide hydrogen-deuterium exchange kinetics. The type and ratio of cationic guest to host in solution affect the rate of isotope exchange, suggesting that the rate of exchange is driven by a host whose cavity is occupied by water. Rate constants for acid-, base-, and water-mediated proton exchange vary by orders of magnitude depending on the guest, and differ by up to 200 million-fold relative to an alanine polypeptide. These results suggest that the unusual microenvironment of the cavity of 1 can dramatically alter the reactivity of associated water by magnitudes comparable to that of enzymes.

The effect of host structure on the selectivity and mechanism of supramolecular catalysis of Prins cyclizations.

The effect of host structure on the selectivity and mechanism of intramolecular Prins reactions is evaluated using K12Ga4L6 tetrahedral catalysts. The host structure was varied by modifying the structure of the chelating moieties and the size of the aromatic spacers. While variation in chelator substituents was generally observed to affect changes in rate but not selectivity, changing the host spacer afforded differences in efficiency and product diastereoselectivity. An extremely high number of turnovers (up to 840) was observed. Maximum rate accelerations were measured to be on the order of 105, which numbers among the largest magnitudes of transition state stabilization measured with a synthetic host-catalyst. Host/guest size effects were observed to play an important role in host-mediated enantioselectivity.

Chiral amide directed assembly of a diastereo- and enantiopure supramolecular host and its application to enantioselective catalysis of neutral substrates.

The synthesis of a novel supramolecular tetrahedral assembly of K12Ga4L6 stoichiometry is reported. The newly designed chiral ligand exhibits high diastereoselective control during cluster formation, leading exclusively to a single diastereomer of the desired host. This new assembly also exhibits high stability toward oxidation or a low pH environment and is a more robust and efficient catalyst for asymmetric organic transformations of neutral substrates.

Selective monoterpene-like cyclization reactions achieved by water exclusion from reactive intermediates in a supramolecular catalyst.

A polyanionic supramolecular assembly (1) is shown to catalytically cyclize the monoterpene citronellal and two homologues. In contrast to cyclization in acidic aqueous solution, the hydrophobic interior of 1 prevents the capture of reactive intermediates by water. This effect was also observed in the gold-catalyzed cycloisomerization of an enyne. Due to the steric confinement of the catalyst's interior, Prins cyclizations in 1 proceed cleanly both for substrates containing and lacking gem-dimethyl substitution. Encapsulation in 1 consequently imposes a degree of mechanistic control that, similar to enzyme catalysis, is not observed in bulk aqueous solution.