Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells.

Coevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host's endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood, and hosts are protected against experimental iNKT cell-mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids-exemplified by an isolated peak (MW = 717.6) called GSL-Bf717-reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive.

Oxazolone mediated peptide chain extension and homochirality in aqueous microdroplets.

Peptide formation from amino acids is thermodynamically unfavorable but a recent study provided evidence that the reaction occurs at the air/solution interfaces of aqueous microdroplets. Here, we show that i) the suggested amino acid complex in microdroplets undergoes dehydration to form oxazolone; ii) addition of water to oxazolone forms the dipeptide; and iii) reaction of oxazolone with other amino acids forms tripeptides. Furthermore, the chirality of the reacting amino acids is preserved in the oxazolone product, and strong chiral selectivity is observed when converting the oxazolone to tripeptide. This last fact ensures that optically impure amino acids will undergo chain extension to generate pure homochiral peptides. Peptide formation in bulk by wet-dry cycling shares a common pathway with the microdroplet reaction, both involving the oxazolone intermediate.

A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable Haemophilus influenzae virulence factor.

The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.

CD103 Regulates Dermal Regulatory T Cell Motility and Interactions with CD11c-Expressing Leukocytes to Control Skin Inflammation.

Dermal regulatory T cells (Tregs) are essential for maintenance of skin homeostasis and control of skin inflammatory responses. In mice, Tregs in the skin are characterized by high expression of CD103, the αE integrin. Evidence indicates that CD103 promotes Treg retention within the skin, although the mechanism underlying this effect is unknown. The main ligand of CD103, E-cadherin, is predominantly expressed by cells in the epidermis. However, because Tregs are predominantly located within the dermis, the nature of the interactions between E-cadherin and CD103-expressing Tregs is unclear. In this study, we used multiphoton intravital microscopy to examine the contribution of CD103 to Treg behavior in resting and inflamed skin of mice undergoing oxazolone-induced contact hypersensitivity. Inhibition of CD103 in uninflamed skin did not alter Treg behavior, whereas 48 h after inducing contact hypersensitivity by oxazolone challenge, CD103 inhibition increased Treg migration. This coincided with E-cadherin upregulation on infiltrating myeloid leukocytes in the dermis. Using CD11c-enhanced yellow fluorescent protein (EYFP) × Foxp3-GFP dual-reporter mice, inhibition of CD103 was found to reduce Treg interactions with dermal dendritic cells. CD103 inhibition also resulted in increased recruitment of effector CD4+ T cells and IFN-γ expression in challenged skin and resulted in reduced glucocorticoid-induced TNFR-related protein expression on Tregs. These results demonstrate that CD103 controls intradermal Treg migration, but only at later stages in the inflammatory response, when E-cadherin expression in the dermis is increased, and provide evidence that CD103-mediated interactions between Tregs and dermal dendritic cells support regulation of skin inflammation.

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis.

SignificanceMethanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.

Deuterium Labeling of Isoaspartic and Isoglutamic Acids for Mass Spectrometry Analysis.

Isoaspartic acid (isoAsp) is a common protein modification that spontaneously arises from asparagine or aspartic acid and has been linked to various diseases and health conditions. However, current methods for identifying isoAsp sites in proteins often suffer from ambiguity and have not gained widespread adoption. We developed a novel method that exclusively labels isoAsp with deuterium. This method capitalizes on the unique structural characteristics of isoAsp residues, which possess a free α-carboxyl group and can form an oxazolone ring. Once the oxazolone ring forms, it facilitates racemization at the Cα-position, incorporating a deuteron from a D2O solvent. The sites of deuterium-incorporated isoAsp in proteins can be unequivocally determined by comparing the precursor and product ion masses of the peptides from proteins reacted in H2O and D2O. The effectiveness of this method has been demonstrated through its application to model proteins lysozyme and rituximab. Furthermore, we have confirmed that the isoAsp deuterium-labeling reaction efficiently labels both l- and d-isoAsp without distinction, as well as isoglutamic acid (isoGlu), for which no effective detection methods currently exist.

The Influence of Clustered DNA Damage Containing Iz/Oz and OXOdG on the Charge Transfer through the Double Helix: A Theoretical Study.

The genome-the source of life and platform of evolution-is continuously exposed to harmful factors, both extra- and intra-cellular. Their activity causes different types of DNA damage, with approximately 80 different types of lesions having been identified so far. In this paper, the influence of a clustered DNA damage site containing imidazolone (Iz) or oxazolone (Oz) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OXOdG) on the charge transfer through the double helix as well as their electronic properties were investigated. To this end, the structures of oligo-Iz, d[A1Iz2A3OXOG4A5]*d[T5C4T3C2T1], and oligo-Oz, d[A1Oz2A3OXOG4A5]*d[T5C4T3C2T1], were optimized at the M06-2X/6-D95**//M06-2X/sto-3G level of theory in the aqueous phase using the ONIOM methodology; all the discussed energies were obtained at the M06-2X/6-31++G** level of theory. The non-equilibrated and equilibrated solvent-solute interactions were taken into consideration. The following results were found: (A) In all the discussed cases, OXOdG showed a higher predisposition to radical cation formation, and B) the excess electron migration toward Iz and Oz was preferred. However, in the case of oligo-Oz, the electron transfer from Oz2 to complementary C4 was noted during vertical to adiabatic anion relaxation, while for oligo-Iz, it was settled exclusively on the Iz2 moiety. The above was reflected in the charge transfer rate constant, vertical/adiabatic ionization potential, and electron affinity energy values, as well as the charge and spin distribution. It can be postulated that imidazolone moiety formation within the CDL ds-oligo structure and its conversion to oxazolone can significantly influence the charge migration process, depending on the C2 carbon hybridization sp2 or sp3. The above can confuse the single DNA damage recognition and removal processes, cause an increase in mutagenesis, and harm the effectiveness of anticancer therapy.

Management of ulcerative colitis by dichloroacetate: Impact on NFATC1/NLRP3/IL1B signaling based on bioinformatics analysis combined with in vivo experimental verification.

The inflammatory response in ulcerative colitis (UC) could be relieved by the conventional immunomodulatory agents; 5-aminosalicylic acid, corticosteroids, or azathioprine. However, the low remission rates and the intolerance to these agents necessitate investigation of gene expression signature in UC that could influence the therapeutic efficacy of drugs, as well as the interference with persistence genes by novel therapeutic option. Three microarray datasets (GSE66407, GSE38713 and GSE14580) from the NCBI-GEO database were utilized. Differentially expressed genes between samples of patients with UC and healthy ones were analyzed using R software. In addition, in vivo study using oxazolone-induced UC in BALB/c mice was carried out to investigate the proposed therapeutic efficacy of dichloroacetate (DCA). The bioinformatics analysis revealed the persistence of NLRP3, NFATC1, and IL1B in UC despite treatment with common therapeutic agents. DCA administration to oxazolone-treated mice showed remarkable interference with those persistence genes. Western blotting analysis for NLRP3, NFATC1, nuclear/total NF-κB, and cleaved caspase-1 revealed the ability of DCA to reduce the expression levels of these proteins in oxazolone-treated mice. Additionally, the inflammatory cytokines IL-1ß and IL-13 were reduced in colonic tissue by DCA treatment. The therapeutic efficacy of DCA was further confirmed by the apparent reduction in histopathological scoring, disease activity index, and the normalization of colon length. Therefore, DCA could be suggested as a novel and promising therapeutic option in UC based on its ability to interfere with the persistence of NFATC1/NLRP3/IL1B signaling. That merits further safety/toxicological pre-clinical assessment and update of bioavailability/metabolism data prior to clinical investigation.

Age-Dependent Effects of Yolkin on Contact Sensitivity and Immune Phenotypes in Juvenile Mice.

Yolkin, an egg yolk immunoregulatory protein, stimulates the humoral but inhibits the cellular immune response in adult mice. The aim of this investigation was to evaluate the effects of yolkin administration on the immune response using a model of juvenile, i.e., 28-day- and 37-day-old, mice. We examined the yolkin influence on the magnitude of the cellular immune response, which was determined as contact sensitivity (CS) to oxazolone (OXA), and the humoral immune response, which was determined as the antibody response to ovalbumin (OVA). Yolkin was administered in drinking water, followed by immunization with OXA or OVA. In parallel, the phenotypic changes in the lymphoid organs were determined following yolkin treatment and prior immunization. The results showed that yolkin had a stimulatory effect on CS in the mice treated with yolkin from the 37th day of life but not from the 28th day of life. In contrast, no regulatory effect of yolkin on antibody production was found in 28-day- and 37-day-old mice. Phenotypic studies revealed significant changes in the content of B cells and T cell subpopulations, including CD4+CD25+Foxp3 regulatory T cells. The association between the effects of yolkin on the magnitude of CS and phenotypic changes in main T- and B-cell compartments, as well the importance of changes in T-regulatory and CD8+ cells in the age categories, are discussed. We conclude that the immunoregulatory effects of yolkin on the generation of CS in mice are age dependent and change from stimulation in juvenile to suppression in adult mice.

Co-occurrence of enzyme domains guides the discovery of an oxazolone synthetase.

Multidomain enzymes orchestrate two or more catalytic activities to carry out metabolic transformations with increased control and speed. Here, we report the design and development of a genome-mining approach for targeted discovery of biochemical transformations through the analysis of co-occurring enzyme domains (CO-ED) in a single protein. CO-ED was designed to identify unannotated multifunctional enzymes for functional characterization and discovery based on the premise that linked enzyme domains have evolved to function collaboratively. Guided by CO-ED, we targeted an unannotated predicted ThiF-nitroreductase di-domain enzyme found in more than 50 proteobacteria. Through heterologous expression and biochemical reconstitution, we discovered a series of natural products containing the rare oxazolone heterocycle and characterized their biosynthesis. Notably, we identified the di-domain enzyme as an oxazolone synthetase, validating CO-ED-guided genome mining as a methodology with potential broad utility for both the discovery of unusual enzymatic transformations and the functional annotation of multidomain enzymes.

Early Stress-Response Gene REDD1 Controls Oxazolone-Induced Allergic Contact Dermatitis.

REDD1 is an energy sensor and stress-induced mTOR inhibitor. Recently, its novel role in linking metabolism and inflammation/immune responses has emerged. In this study, we assessed the role of REDD1 in murine oxazolone-induced allergic contact dermatitis (ACD), a T cell-dependent model with features of human ACD. A variety of immune indices, including edema, cellular infiltration, inflammatory gene expression, and glucocorticoid response, were compared in Redd1 knockout (KO) and isogenic (C57BL/6 × 129)F1 wild-type mice after sensitization and subsequent ear challenge with oxazolone. Despite relatively normal thymic profiles and similar T cell populations in the lymph nodes of naive Redd1 KO mice, early T cell expansion and cytokine production were profoundly impaired after sensitization. Surprisingly, higher steady-state populations of CD4+ and CD8+ T cells, as well as macrophages (CD45+/Ly-6G-/CD11b+), dendritic cells (CD45+/Ly-6G-/CD11c+), neutrophils (CD45+/Ly-6G+/CD11b+), and innate lymphoid cells (CD45+/Lineage-/IL-7Ra+/ST2+/c-Kit+), were observed in the ears of naive Redd1 KO mice. Upon challenge, ear edema, T cell, macrophage, neutrophil, and dendritic cell infiltration into the ear was significantly reduced in Redd1 KO animals. Accordingly, we observed significantly lower induction of IFN-γ, IL-4, and other cytokines as well as proinflammatory factors, including TSLP, IL-33, IL-1ß, IL-6, and TNF-α, in challenged ears of Redd1 KO mice. The response to glucocorticoid treatment was also diminished. Taken together, these data establish REDD1 as an essential immune modulator that influences both the initiation of ACD disease, by driving naive T cell activation, and the effector phase, by promoting immune cell trafficking in T cell-mediated skin inflammation.

Synthesis of Bis(amino acids) Containing the Styryl-cyclobutane Core by Photosensitized [2+2]-Cross-cycloaddition of Allylidene-5(4H)-oxazolones.

The irradiation of 2-aryl-4-(E-3'-aryl-allylidene)-5(4H)-oxazolones 1 with blue light (456 nm) in the presence of [Ru(bpy)3](BF4)2 (bpy = 2,2'-bipyridine, 5% mol) gives the unstable cyclobutane-bis(oxazolones) 2 by [2+2]-photocycloaddition of two oxazolones 1. Each oxazolone contributes to the formation of 2 with a different C=C bond, one of them reacting through the exocyclic C=C bond, while the other does so through the styryl group. Treatment of unstable cyclobutanes 2 with NaOMe/MeOH produces the oxazolone ring opening reaction, affording stable styryl-cyclobutane bis(amino acids) 3. The reaction starts with formation of the T1 excited state of the photosensitizer 3[Ru*(bpy)3]2+, which reacts with S0 of oxazolones 1 through energy transfer to give the oxazolone T1 state 3(oxa*)-1, which is the reactive species and was characterized by transient absorption spectroscopy. Measurement of the half-life of 3(oxa*)-1 for 1a, 1b and 1d shows large values for 1a and 1b (10-12 µs), while that of 1d is shorter (726 ns). Density functional theory (DFT) modeling displays strong structural differences in the T1 states of the three oxazolones. Moreover, study of the spin density of T1 state 3(oxa*)-1 provides clues to understanding the different reactivity of 4-allylidene-oxazolones described here with respect to the previously reported 4-arylidene-oxazolones.

Human Umbilical Cord Mesenchymal-Stem-Cell-Derived Extracellular Vesicles Reduce Skin Inflammation In Vitro.

The protective roles of extracellular vesicles derived from human umbilical cord mesenchymal stem cells against oxazolone-induced damage in the immortalized human keratinocyte cell line HaCaT were investigated. The cells were pretreated with or without UCMSC-derived extracellular vesicles 24 h before oxazolone exposure. The pretreated UVMSC-EVs showed protective activity, elevating cell viability, reducing intracellular ROS, and reducing the changes in the mitochondrial membrane potential compared to the cells with a direct oxazolone treatment alone. The UCMSC-EVs exhibited anti-inflammatory activity via reducing the inflammatory cytokines IL-1ß and TNF-α. A mechanism study showed that the UCMSC-EVs increased the protein expression levels of SIRT1 and P53 and reduced P65 protein expression. It was concluded that UVMSC-EVs can induce the antioxidant defense systems of HaCaT cells and that they may have potential as functional ingredients in anti-aging cosmetics for skin care.

Moderating Gut Microbiome/Mitochondrial Axis in Oxazolone Induced Ulcerative Colitis: The Evolving Role of ß-Glucan and/or, Aldose Reductase Inhibitor, Fidarestat.

A mechanistic understanding of the dynamic interactions between the mitochondria and the gut microbiome is thought to offer innovative explanations for many diseases and thus provide innovative management approaches, especially in GIT-related autoimmune diseases, such as ulcerative colitis (UC). ß-Glucans, important components of many nutritious diets, including oats and mushrooms, have been shown to exhibit a variety of biological anti-inflammatory and immune-modulating actions. Our research study sought to provide insight into the function of ß-glucan and/or fidarestat in modifying the microbiome/mitochondrial gut axis in the treatment of UC. A total of 50 Wistar albino male rats were grouped into five groups: control, UC, ß-Glucan, Fidarestat, and combined treatment groups. All the groups were tested for the presence of free fatty acid receptors 2 and 3 (FFAR-2 and -3) and mitochondrial transcription factor A (TFAM) mRNA gene expressions. The reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and ATP content were found. The trimethylamine N-oxide (TMAO) and short-chain fatty acid (SCFA) levels were also examined. Nuclear factor kappa ß (NF-kß), nuclear factor (erythroid-2)-related factor 2 (Nrf2) DNA binding activity, and peroxisome proliferator-activated receptor gamma co-activator-1 (PGC-1) were identified using the ELISA method. We observed a substantial increase FFAR-2, -3, and TFAM mRNA expression after the therapy. Similar increases were seen in the ATP levels, MMP, SCFA, PGC-1, and Nrf2 DNA binding activity. The levels of ROS, TMAO, and NF-kß, on the other hand, significantly decreased. Using ß-glucan and fidarestat together had unique therapeutic benefits in treating UC by focusing on the microbiota/mitochondrial axis, opening up a new avenue for a potential treatment for such a complex, multidimensional illness.

New Heterocyclic Compounds from Oxazol-5(4H)-one and 1,2,4-Triazin-6(5H)-one Classes: Synthesis, Characterization and Toxicity Evaluation.

This paper describes the synthesis of new heterocycles from oxazol-5(4H)-one and 1,2,4-triazin-6(5H)-one classes containing a phenyl-/4-bromophenylsulfonylphenyl moiety. The oxazol-5(4H)-ones were obtained via condensation of 2-(4-(4-X-phenylsulfonyl)benzamido)acetic acids with benzaldehyde/4-fluorobenzaldehyde in acetic anhydride and in the presence of sodium acetate. The reaction of oxazolones with phenylhydrazine, in acetic acid and sodium acetate, yielded the corresponding 1,2,4-triazin-6(5H)-ones. The structures of the compounds were confirmed using spectral (FT-IR, 1H-NMR, 13C-NMR, MS) and elemental analysis. The toxicity of the compounds was evaluated on Daphnia magna Straus crustaceans and on the budding yeast Saccharomyces cerevisiae. The results indicate that both the heterocyclic nucleus and halogen atoms significantly influenced the toxicity against D. magna, with the oxazolones being less toxic than triazinones. The halogen-free oxazolone had the lowest toxicity, and the fluorine-containing triazinone exhibited the highest toxicity. The compounds showed low toxicity against yeast cells, apparently due to the activity of plasma membrane multidrug transporters Pdr5 and Snq2. The predictive analyses indicated an antiproliferative effect as the most probable biological action. The PASS prediction and CHEMBL similarity studies show evidence that the compounds could inhibit certain relevant oncological protein kinases. These results correlated with toxicity assays suggest that halogen-free oxazolone could be a good candidate for future anticancer investigations.

Inhibitory Activity of 4-Benzylidene Oxazolones Derivatives of Cinnamic Acid on Human Acetylcholinesterase and Cognitive Improvements in a Mouse Model.

We synthesized seven (Z)-benzylidene-2-(E)-styryloxazol-5(4H)-ones derivatives of cinnamic acid and evaluated the ability of these compounds to inhibit human acetylcholinesterase (hAChE). The most potent compound was evaluated for cognitive improvement in short-term memory. The seven compounds reversibly inhibited the hAChE between 51 and 75% at 300 µM, showed an affinity (Ki) from 2 to 198 µM, and an IC50 from 9 to 246 µM. Molecular docking studies revealed that all binding moieties are involved in the non-covalent interactions with hAChE for all compounds. In addition, in silico pharmacokinetic analysis was carried out to predict the compounds' blood-brain barrier (BBB) permeability. The most potent inhibitor of hAChE significantly improved cognitive impairment in a modified Y-maze test (5 µmol/kg) and an Object Recognition Test (10 µmol/kg). Our results can help the rational design of hAChE inhibitors to work as potential candidates for treating cognitive disorders.

IL-25 contributes to development of chronic contact dermatitis in C57BL/6 mice, but not BALB/c mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by type 2 immune responses. Interleukin-25 (IL-25) is produced predominantly by epithelial cells. It can activate Th2 cells to produce type 2 cytokines such as IL-4, IL-5 and IL-13, contributing to host defense against nematodes. However, excessive/inappropriate production of IL-25 is considered to be involved in development of type 2 cytokine-associated allergic disorders such as asthma. On the other hand, the contribution of IL-25 to the pathogenesis of AD remains poorly understood. In the present study, we found that expression of Il25 mRNA was significantly increased in the skin of mice during oxazolone-induced chronic contact hypersensitivity (CHS), which is a mouse model of human AD. In addition, development of oxazolone-induced chronic CHS was significantly reduced in IL-25-deficient (Il25-/-) mice compared with wild-type mice on the C57BL/6, but not BALB/c, background, although IL-25 was not essential for IL-4 production by hapten-specific T cells. Therefore, IL-25 is crucial for development of chronic CHS, although that is partly dependent on the genetic background of the mice.

MbnC Is Not Required for the Formation of the N-Terminal Oxazolone in the Methanobactin from Methylosinus trichosporium OB3b.

Methanobactins (MBs) are ribosomally synthesized and posttranslationally modified peptides (RiPPs) produced by methanotrophs for copper uptake. The posttranslational modification that defines MBs is the formation of two heterocyclic groups with associated thioamines from X-Cys dipeptide sequences. Both heterocyclic groups in the MB from Methylosinus trichosporium OB3b (MB-OB3b) are oxazolone groups. The precursor gene for MB-OB3b is mbnA, which is part of a gene cluster that contains both annotated and unannotated genes. One of those unannotated genes, mbnC, is found in all MB operons and, in conjunction with mbnB, is reported to be involved in the formation of both heterocyclic groups in all MBs. To determine the function of mbnC, a deletion mutation was constructed in M. trichosporium OB3b, and the MB produced from the ΔmbnC mutant was purified and structurally characterized by UV-visible absorption spectroscopy, mass spectrometry, and solution nuclear magnetic resonance (NMR) spectroscopy. MB-OB3b from the ΔmbnC mutant was missing the C-terminal Met and was also found to contain a Pro and a Cys in place of the pyrrolidinyl-oxazolone-thioamide group. These results demonstrate MbnC is required for the formation of the C-terminal pyrrolidinyl-oxazolone-thioamide group from the Pro-Cys dipeptide, but not for the formation of the N-terminal 3-methylbutanol-oxazolone-thioamide group from the N-terminal dipeptide Leu-Cys. IMPORTANCE A number of environmental and medical applications have been proposed for MBs, including bioremediation of toxic metals and nanoparticle formation, as well as the treatment of copper- and iron-related diseases. However, before MBs can be modified and optimized for any specific application, the biosynthetic pathway for MB production must be defined. The discovery that mbnC is involved in the formation of the C-terminal oxazolone group with associated thioamide but not for the formation of the N-terminal oxazolone group with associated thioamide in M. trichosporium OB3b suggests the enzymes responsible for posttranslational modification(s) of the two oxazolone groups are not identical.

A Modified Oxazolone Dye Dedicated to Spectroscopy and Optoelectronics.

Here we present a newly synthesized bifunctional organic chromophore with appealing spectroscopic and nonlinear optical features. The positions of absorption and emission maxima of the dye vary with increasing solvent polarity and exhibit positive solvatochromism. The determined change in the dipole moment upon excitation based on the Bilot and Kawski theory is 5.94 D, which corresponds to the intermolecular displacement of a charge equal to 1.24 Å. An investigated organic-based system represents a significant, repeatable, and stable over time optical signal modulation in the manner of the refractive index value. Its magnitude is varied both by optical pumping intensity as well as by external frequency modulation, which indicates that such system is an alluring and alternative core unit for optoelectronic devices and complex networks. Then, the same active system, due to the nonresonant mechanism of higher harmonics of light inducement, can provide second and third harmonic signals. According to the introduced laser line spatial modifications (parallel or perpendicular polarization directions), it is resulted in output SHG signal with magnitude varied about 100%. Its magnitude is noticeably small; however, to construct sensitive optical sensors or infrared indicators, such feature may guarantee satisfying circumstances.

Syntheses and Aromaticity Parameters of Hexahydroxypyrrocorphin, Porphotrilactones, and Their Oxidation State Intermediates.

Triple OsO4-mediated dihydroxylation of meso-tetrakis(pentafluorophenyl)porphyrin formed a non-aromatic hexahydroxypyrrocorphin as a single stereo-isomer. A one-step oxidative conversion of all three diol functionalities to lactone moieties generated three out of the four possible porphotrilactone regioisomers that were spectroscopically and structurally characterized. This conversion recovered most of the porphyrinic macrocycle aromatic ring current, as seen in their 1H NMR spectra and modeled using DFT computations. Stepwise OsO4-mediated dihydroxylations of porpho-mono- and -di-lactones generated intermediate oxidation state compounds between the pyrrole-three pyrroline macrocycle of the pyrrocorphin and the pyrrole-three oxazolone chromophore of the trilactones. The aromaticity of these chromophores was reduced with increasing number of oxazolone to pyrroline replacements, showing the importance for the presence of three lactone moieties for the retention of the macrocycle aromaticity in the tris-ß,ß'-modified macrocycles. This work first describes hexahydoxypyrrocorphins, porphotrislactones, and the oxidation state intermediates between them; furthers the understanding of the roles of ß-lactone moieties in the expression of porphyrinic macrocycle aromaticity; and generally broadens access to chemically stable pyrrocorphins and pyrrocorphin analogues.