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Flavins and their alloxazine isomers are key chemical scaffolds for bioinspired electron transfer strategies. Their properties can be fine‐tuned by functional groups, which must be introduced at an early stage of the synthesis as their aromatic ring is inert towards post‐functionalization. We show that the introduction of a remote metal‐binding redox site on alloxazine and flavin activates their aromatic ring towards direct C−H functionalization. Mechanistic studies are consistent with a synthetic sequence involving ground‐state single electron transfer (SET) with an electrophilic source followed by radical‐radical coupling. This unprecedented reactivity opens new opportunities in molecular editing of flavins by direct aromatic post‐functionalization and the utility of the method is demonstrated with the site‐selective C6 functionalization of alloxazine and flavin with a CF3 group, Br or Cl, that can be further elaborated into OH and aryl for chemical diversification. [ABSTRACT FROM AUTHOR]

Flavins and their alloxazine isomers are key chemical scaffolds for bioinspired electron transfer strategies. Their properties can be fine‐tuned by functional groups, which must be introduced at an early stage of the synthesis as their aromatic ring is inert towards post‐functionalization. We show that the introduction of a remote metal‐binding redox site on alloxazine and flavin activates their aromatic ring towards direct C−H functionalization. Mechanistic studies are consistent with a synthetic sequence involving ground‐state single electron transfer (SET) with an electrophilic source followed by radical‐radical coupling. This unprecedented reactivity opens new opportunities in molecular editing of flavins by direct aromatic post‐functionalization and the utility of the method is demonstrated with the site‐selective C6 functionalization of alloxazine and flavin with a CF3 group, Br or Cl, that can be further elaborated into OH and aryl for chemical diversification. [ABSTRACT FROM AUTHOR]

TDP-43 forms aggregates in the neurons of patients with several neurodegenerative diseases. Human TDP-43 also aggregates and is toxic in yeast. Here, we used a yeast model to investigate (1) the nature of TDP-43 aggregates and (2) the mechanism of TDP-43 toxicity. Thioflavin T, which stains amyloid but not wild-type TDP-43 aggregates, also did not stain mutant TDP-43 aggregates made from TDP-43 with intragenic mutations that increase or decrease its toxicity. However, 1,6-hexanediol, which dissolves liquid droplets, dissolved wild-type or mutant TDP-43 aggregates. To investigate the mechanism of TDP-43 toxicity, the effects of TDP-43 mutations on the autophagy of the GFP-ATG8 reporter were examined. Mutations in TDP-43 that enhance its toxicity, but not mutations that reduce its toxicity, caused a larger reduction in autophagy. TOROID formation, which enhances autophagy, was scored as GFP-TOR1 aggregation. TDP-43 inhibited TOROID formation. TORC1 bound to both toxic and non-toxic TDP-43, and to TDP-43, with reduced toxicity due to pbp1Δ. However, extragenic modifiers and TDP-43 mutants that reduced TDP-43 toxicity, but not TDP-43 mutants that enhanced toxicity, restored TOROID formation. This is consistent with the hypothesis that TDP-43 is toxic in yeast because it reduces TOROID formation, causing the inhibition of autophagy. Whether TDP-43 exerts a similar effect in higher cells remains to be determined. [ABSTRACT FROM AUTHOR]

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be −0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: A label‐free deoxyribozyme (DNAzyme) biosensor for m6A demethylase activity detection is developed. When demethylase FTO (Fat mass and obesity‐associated protein), an important m6A demethylase of ALKBH demethylase family is present, the m6A group is specifically demethylated, and the cleavage activity of DNAzyme is restored. This allows the cleavage product that contains G‐quadruplex sequence to bind to thioflavin T (ThT) and generate fluorescence signals. The biosensor shows high specificity and sensitivity, and fast reaction speed. Our study demonstrates a new design of allosteric DNAzyme for sensing. This method represents the first label‐free nucleic acid biosensor for FTO assay, providing a feasible route towards inhibitors screening. [ABSTRACT FROM AUTHOR]

The isoalloxazine heterocycle of flavin cofactors reacts with various nucleophiles to form covalent adducts with important functions in enzymes. Molecular flavin models allow for the characterization of such adducts and the study of their properties. A fascinating set of reactions occurs when flavins react with hydroxide base, which leads to imidazolonequinoxalines, ring‐contracted flavins, with so far unexplored activity. We report a systematic study of the photophysical properties of this new chromophore by absorption and emission spectroscopy as well as cyclic voltammetry. Excited, ring‐contracted flavins are significantly stronger hydrogen atom abstractors when compared to the parent flavins, which allowed the direct trifluoromethylthiolation of aliphatic methine positions (bond dissociation energy (BDE) of 400.8 kJ mol−1). In an orthogonal activity, their increased triplet energy (E(S0←T1)=244 kJ mol−1) made sensitized reactions possible which exceeded the power of standard flavins. Combining both properties, ring‐contracted flavin catalysts enabled the one‐pot, five‐step transformation of α‐tropolone into trans‐3,4‐disubstituted cyclopentanones. We envision this new class of flavin‐derived chromophores to open up new modes of reactivity that are currently impossible with unmodified flavins. [ABSTRACT FROM AUTHOR]

The isoalloxazine heterocycle of flavin cofactors reacts with various nucleophiles to form covalent adducts with important functions in enzymes. Molecular flavin models allow for the characterization of such adducts and the study of their properties. A fascinating set of reactions occurs when flavins react with hydroxide base, which leads to imidazolonequinoxalines, ring‐contracted flavins, with so far unexplored activity. We report a systematic study of the photophysical properties of this new chromophore by absorption and emission spectroscopy as well as cyclic voltammetry. Excited, ring‐contracted flavins are significantly stronger hydrogen atom abstractors when compared to the parent flavins, which allowed the direct trifluoromethylthiolation of aliphatic methine positions (bond dissociation energy (BDE) of 400.8 kJ mol−1). In an orthogonal activity, their increased triplet energy (E(S0←T1)=244 kJ mol−1) made sensitized reactions possible which exceeded the power of standard flavins. Combining both properties, ring‐contracted flavin catalysts enabled the one‐pot, five‐step transformation of α‐tropolone into trans‐3,4‐disubstituted cyclopentanones. We envision this new class of flavin‐derived chromophores to open up new modes of reactivity that are currently impossible with unmodified flavins. [ABSTRACT FROM AUTHOR]

Theaflavins (TFs) are a kind of bioactive compound that formed during the fermentation stage of black tea. TFs play a vital role both in the sensory quality and the biological activity of black tea. However, the complex composition of black tea and the low content of TFs in black tea (0.3%–0.7%) make the technology for the preparation of high‐purity TFs a great challenge. This review provides an updated overview for extracting, synthesizing and purifying of TFs. Molecular imprinting technology, an efficient method for extracting bioactive compounds, will be a promising method for the preparation of TFs with high purity. Overall, this review can provide a scientific basis and technical support for the preparation of TFs with high purity and will be helpful for the in‐depth development and further research of TFs. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein (BMP) signaling plays a vital role in differentiation, organogenesis, and various cell processes. As a member of TGF‐β superfamily, the BMP initiation usually accompanies crosstalk with other signaling pathways and simultaneously activates some of them. It is quite challenging to solely initiate an individual pathway. In this study, an opsin‐free optical method to specifically activate BMP receptors (BMPR) and subsequent pSmad1/5/8 cascades by a single‐time scan of a tightly‐focused femtosecond laser in the near infrared range is reported. Via transient two‐photon excitation to intrinsic local flavins near the cell membrane, the photoactivation drives conformational changes of preformed BMPR complexes to enable their bonding and phosphorylation of the GS domain in BMPR‐I by BMPR‐II. The pSmad1/5/8 signaling is initiated by this method, while p38 and pSmad2 are rarely perturbed. Based on a microscopic system, primary adipose‐derived stem cells in an area of 420 × 420 µm2 are photoactivated by a single‐time laser scanning for 1.5 s and exhibit pSmad1/5/8 upregulation and osteoblastic differentiation after 21 days. Hence, an opsin‐free, specific, and noninvasive optical method to initiate BMP signaling, easily accomplished by a two‐photon microscope system is reported. [ABSTRACT FROM AUTHOR]

Many studies have been conducted on the microbial reduction of Pd (II) to palladium nanoparticles (Pd-NPs) due to the environmental friendliness, low cost, and the decreased toxicity of Pd (II) ions. In this study, we investigate the reduction mechanism of Pd (II) by Bacillus megaterium Y-4 through proteomics. The data are available via ProteomeXchange with identifier PXD049711. Our results revealed that B. megaterium Y-4 may use the endogenous electron donor (NAD(P)H) generated by nirB, tdh, and fabG and reductase to reduce Pd (II) to Pd-NPs. The expression levels of fabG, tdh, gudB, and rocG that generate NAD(P)H were further increased, and the number of reduced Pd-NPs was further increased with the exogenous electron donor sodium formate. Endogenous electron mediators such as quinones and flavins in B. megaterium Y-4 can further enhance Pd (II) reduction. The findings provided invaluable information regarding the reduction mechanism of Pd (II) by B. megaterium Y-4 at the proteome level. [ABSTRACT FROM AUTHOR]

We report a simple, inexpensive design of a fluorescence microscope with light-emitting diode (LED) excitation for detection of labeled and unlabeled microorganisms in mineral substrates. The use of deep UV (DUV) excitation with visible emission requires no specialized optics or slides and can be implemented easily and inexpensively using an oblique illumination geometry. DUV excitation (<280 nm) is preferable to near UV (365 nm) for avoidance of mineral autofluorescence. When excited with DUV, unpigmented bacteria show two emission peaks: one in the near UV ∼320 nm, corresponding to proteins, and another peak in the blue to green range, corresponding to flavins and/or reduced nicotinamide adenine dinucleotide (NADH). Many commonly used dyes also show secondary excitation peaks in the DUV, with identical emission spectra and quantum yields as their primary peak. However, DUV fails to excite key biosignature molecules, especially chlorophyll in cyanobacteria. Visible excitation (violet to blue) also results in less mineral autofluorescence than near UV, and most autofluorescence in the minerals seen here is green, so that red dyes and red autofluorescence of chlorophyll and porphyrins are readily distinguished. The pairing of DUV and near UV or visible excitation, with emission across the visible, represents the most thorough approach to detection of labeled and unlabeled bacteria in soil and rock. [ABSTRACT FROM AUTHOR]

Theaflavins are the characteristic polyphenols in black tea which can be enzymatically synthesized. In this review, the effects and molecular mechanisms of theaflavins on obesity and its comorbidities, including dyslipidemia, insulin resistance, hepatic steatosis, and atherosclerosis, were summarized. Theaflavins ameliorate obesity potentially via reducing food intake, inhibiting pancreatic lipase to reduce lipid absorption, activating the adenosine monophosphate-activated protein kinase (AMPK), and regulating the gut microbiota. As to the comorbidities, theaflavins ameliorate hypercholesterolemia by inhibiting micelle formation to reduce cholesterol absorption. Theaflavins improve insulin sensitivity by increasing the signaling of protein kinase B, eliminating glucose toxicity, and inhibiting inflammation. Theaflavins ameliorate hepatic steatosis via activating AMPK. Theaflavins reduce atherosclerosis by upregulating nuclear factor erythropoietin-2-related factor 2 signaling and inhibiting plasminogen activator inhibitor 1. In randomized controlled trails, black tea extracts containing theaflavins reduced body weight in overweight people and improved glucose tolerance in healthy adults. The amelioration on the hyperlipidemia and the prevention of coronary artery disease by black tea extracts were supported by meta-analysis. [ABSTRACT FROM AUTHOR]

Drying is a crucial and energy-consuming process in black tea manufacturing that is aimed at reducing moisture content and terminating enzymatic reactions in tea leaves. In Sri Lanka, an endless chain pressure (ECP) dryer is commonly used for drying, but it consumes a significant amount of energy, necessitating the optimization of drying conditions. The current drying temperature at the Houpe tea factory in Ratnapura, Sri Lanka is 121 °C (250 °F), and it has not been optimized for a considerable period. As a result, energy consumption and wastage are high, leading to an inferior quality of black tea. To optimize factory conditions, tea leaves were dried under different temperatures: 115 (T1), 118 (T2), 121 (T3), 124 (T4), and 127 (T5) °C. Energy consumption, energy wastage, and specific energy consumption (SEC) for tea drying were calculated. Additionally, chemical and sensory analyses of samples of made tea were performed. SEC and energy wastage were significantly (p < 0.05) lower for treatments T1 and T2 than for other treatments. The theaflavin and thearubigin contents were significantly (p < 0.05) higher while total phenolic content was moderate for treatment T2. The sensory parameters of T2 outperformed other treatments. Based on these results, the optimum drying temperature for the ECP dryer was determined to be 118 °C and this temperature has been recommended for this factory. [ABSTRACT FROM AUTHOR]

Amyloid fibril formation by the extracellular protein β2-microglobulin (β2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human β2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N β2m), which readily forms amyloid fibrils in vitro under physiological conditions. In this study, we examined the role of the extracellular molecular chaperone clusterin in modulating D76N β2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N β2m fibril formation, implying that electrostatic interactions are involved in the protein's aggregation. Thioflavin T (ThT) and 1-anilinonaphthalene-8-sulfonate fluorescence assays indicated that clusterin interacts via hydrophobic and electrostatic forces with the monomeric, prefibrillar and fibrillar species of D76N β2m. As a result, clusterin was incorporated into D76N β2m aggregates during the latter's fibril formation, as indicated by SDS-PAGE of depolymerised fibrils. SYPRO Orange and ThT fluorescence assays suggested that, compared to pure D76N β2m fibrils, those formed in the presence of clusterin are chemically more stable with a reduced ability to act as nucleation seeds. Detailed 15N NMR relaxation studies of mixtures of 15N-labelled β2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric β2m. Clusterin inhibits both primary and secondary nucleation of D76N β2m fibril formation. In doing so, clusterin binds to D76N β2m fibrils and stabilises them to prevent possible fragmentation. In vivo , the multifaceted chaperone action of clusterin may delay, if not prevent, β2m amyloid proliferation and deposition in tissues. Accumulation of β2-microglobin in tissues is responsible for the disease dialysis-related amyloidosis. The D76N mutant is highly amyloidogenic. An in vitro biophysical and spectroscopic investigation of D76N β2-microglobin amyloid fibril formation and its inhibition by the common extracellular chaperone, clusterin, is described. In vivo , clusterin may play an important role in preventing β2-microglobin deposition. CH23082_TOC.jpg [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) play a significant role in the pathogenesis of various diseases throughout biological processes, and the accurate detection of miRNA biomarkers holds great potential for early stage disease diagnosis and treatment. In this study, a novel method is developed to detect miRNA-21, a biomarker for drug-induced liver injury, by combining target sequence recycling with G-quadruplex-based signal production. This approach is highly sensitive and does not require the use of labels. The target sequence facilitates the cyclic exposure of G-rich regions in the detection probe by toehold-mediated strand displacement processes, with the aid of the catalytic chain. The G-quadruplex sequences that have been produced subsequently interact with thioflavin T (ThT), resulting in a significant increase in its fluorescence intensity. This enhanced fluorescence is utilized for the purpose of detecting miRNA-21, with a remarkably low detection limit of 4.4 fM. The suggested technique also allows for the very specific identification of the target miRNA-21. Due to its non-label format, excellent selectivity, and sensitivity, this technology presents a straightforward and versatile approach for detecting a wide range of biomarkers in the early phases of illness detection. [ABSTRACT FROM AUTHOR]

Objective To study the impact of theaflavin on the proliferation, apoptosis and epithelial mesenchymal transformation of oral squamous cell carcinoma (OSCC) cells by regulating Snail/Slug signaling pathway. Methods MTT method was used to detect the survival rate of human OSCC cells SCC-25 treated with 25, 50, 100, 150 and 175 mg/L theaflavin, and the appropriate concentration of theaflavin was selected. SCC-25 cells were cultured in vitro and OSCC transplanted tumor model was constructed. Cells were randomly divide into the control group, the low dose theaflavin group, the high dose theaflavin group, the high dose theaxanthin+empty group and the high dose theaflavin+Snail overexpression group. The expression levels of Snail and Slug in cells of each group were detected by real-time fluorescent quantitative PCR and Western blot assay. MTT assay and flow cytometry were applied to detect cell proliferation and apoptosis. The migration and invasion of SCC-25 cells in each group were detected by cell scratch and Transwell invasion tests. The apoptosis of SCC-25 cells and the expression of epithelial mesenchymal transformation related proteins were detected by Western blot assay. The tumor volume and tumor weight of nude mice in each group were detected. Results Compared with the control group, the cell survival rate, migration rate, invasion number, Snail mRNA and protein expression, Slug mRNA and protein expression, Bcl-2, N-cadherin protein expression, tumor weight and volume of nude mice were decreased in the low dose theaflavin group and the high dose theaxanthin group (P＜0.05), and the apoptosis rate, Bax, ZO-1 and E-cadherin protein expression increased (P＜0.05). There was no significant difference in each index between the high-dose theaxanthin group and the high-dose theaflavin+no-load group (P＞0.05). Overexpression of Snail can reverse the effect of theaflavin on cells and nude mice. Conclusion Theaflavin can down regulate the expression of Snail/Slug pathway, thus inhibit the epithelial mesenchymal transformation, proliferation, migration and invasion of OSCC cells, promote their apoptosis, and delay the growth of transplanted tumors in nude mice. [ABSTRACT FROM AUTHOR]

We carried out transient absorption spectroscopy of thioflavin T (ThT) molecules in various solvents employing an asynchronous optical sampling (ASOPS) scheme with dual synchronized and frequency up-converted mode-lock lasers in the near UV (NUV) spectral region. We developed a pair of synchronized femtosecond lasers with tunable center wavelengths ranging from 380 to 430 nm and spectral bandwidths of 30 nm. As a proof-of-principle experiment, we measured interferometrically detected time and frequency-resolved pump–probe signals of ThT in various solvents to study the twisted intramolecular charge transfer process of photo-excited ThT molecules. Both single-color NUV-NUV and two-color NUV-near IR (NIR) pump–probe measurements reveal that the vibronic coupling strengths of two vibrational modes with frequencies of 214 and 526 cm−1 in the excited state of ThT are reduced when ThT is dissolved in a chlorine-containing solvent, e.g., chloroform. We confirm theoretically that these vibrational modes have relatively high electric dipole moments in the excited state. As a result, the intramolecular charge transfer process of ThT in chloroform, which is driven by the solvation process of surrounding polar solvent molecules, could occur less efficiently, which results in an increase in the fluorescence quantum yield. Here, we demonstrate that the NUV-NUV and NUV-NIR ASOPS-transient absorption could be useful techniques for studying ultrafast photochemical reactions in condensed phases. [ABSTRACT FROM AUTHOR]

The N-terminal fragment of apolipoprotein A-I (apoA-I), comprising residues 1–83, contains three segments prone to aggregation: residues 14–22, 53–58, and 67–72. We previously demonstrated that residues 14–22 are critical in apoA-I fibril formation while residues 53–58 entropically drove the nucleation process. Here, we investigated the impact of amyloidogenic mutations (Δ60–71/VT, Δ70–72, and F71Y) located around residues 67–72 on fibril formation by the apoA-I 1–83 fragment. Thioflavin T fluorescence assay demonstrated that the Δ60–71/VT mutation significantly enhances both nucleation and fibril elongation rates, whereas the Δ70–72 and F71Y mutations had minimal effects. Circular dichroism measurements and microscopic observations revealed that all variant fragments formed straight fibrils, transitioning from random coils to β-sheet structures. Kinetic analysis demonstrated that primary nucleation is the dominant step in fibril formation, with fibril elongation reaching saturation at high protein concentrations. Thermodynamically, both nucleation and fibril elongation were enthalpically and entropically unfavorable in all apoA-I 1–83 variants, in which the entropic barrier of nucleation was almost eliminated for the Δ60–71/VT variant. Taken together, our results suggest the presence of new aggregation-prone segment in the Δ60–71/VT variant that promotes nucleation through entropic effects. [ABSTRACT FROM AUTHOR]

Fungal unspecific peroxygenases (UPOs) have gained substantial attention for their versatile oxyfunctionalization chemistry paired with impressive catalytic capabilities. A major drawback, however, remains their sensitivity towards their co‐substrate hydrogen peroxide, necessitating the use of smart in situ hydrogen peroxide generation methods to enable efficient catalysis setups. Herein, we introduce flavin‐containing protein photosensitizers as a new general tool for light‐controlled in situ hydrogen peroxide production. By genetically fusing flavin binding fluorescent proteins and UPOs, we have created two virtually self‐sufficient photo‐enzymes (PhotUPO). Subsequent testing of a versatile substrate panel with the two divergent PhotUPOs revealed two stereoselective conversions. The catalytic performance of the fusion protein was optimized through enzyme and substrate loading variation, enabling up to 24300 turnover numbers (TONs) for the sulfoxidation of methyl phenyl sulfide. The PhotUPO concept was upscaled to a 100 mg substrate preparative scale, enabling the extraction of enantiomerically pure alcohol products. [ABSTRACT FROM AUTHOR]

Fungal unspecific peroxygenases (UPOs) have gained substantial attention for their versatile oxyfunctionalization chemistry paired with impressive catalytic capabilities. A major drawback, however, remains their sensitivity towards their co‐substrate hydrogen peroxide, necessitating the use of smart in situ hydrogen peroxide generation methods to enable efficient catalysis setups. Herein, we introduce flavin‐containing protein photosensitizers as a new general tool for light‐controlled in situ hydrogen peroxide production. By genetically fusing flavin binding fluorescent proteins and UPOs, we have created two virtually self‐sufficient photo‐enzymes (PhotUPO). Subsequent testing of a versatile substrate panel with the two divergent PhotUPOs revealed two stereoselective conversions. The catalytic performance of the fusion protein was optimized through enzyme and substrate loading variation, enabling up to 24300 turnover numbers (TONs) for the sulfoxidation of methyl phenyl sulfide. The PhotUPO concept was upscaled to a 100 mg substrate preparative scale, enabling the extraction of enantiomerically pure alcohol products. [ABSTRACT FROM AUTHOR]

Many redox reactions are being catalyzed by oxidoreductase enzymes. Although they have high structural similarity, these enzymes undergo different reaction mechanisms and rates depending on the types of substrate, structure, and protonation state. Many pharmaceutical and food industry applications of these enzymes depend on the structural characterization and elucidation of the reaction mechanisms. Combined experimental studies with computational tools provided evidence on the isoalloxazine ring of the oxidoreductase's flavin cofactor reacting through a step-wise process of consecutive single-electron transfers. Moreover, a reaction mechanism is enabled due to the protonation state of the proximity of the cofactor and substrate binding pocket. This paper aims to review the recent advances concerning the structural aspects and factors affecting the mechanism of reactions involving several representatives of the flavin-dependent oxidase family. [ABSTRACT FROM AUTHOR]

The use of the cationic, dye thioflavin T (ThT), to estimate the electric plasma membrane potential difference (PMP) via the fluorescence changes and to obtain its actual values from the accumulation of the dye, considering important correction factors by its binding to the internal components of the cell, was described previously for baker's yeast. However, it was considered important to explore whether the method developed could be applied to other yeast strains. Alternative ways to estimate the PMP by using flow cytometry and a multi-well plate reader are also presented here. The methods were tested with other strains of Saccharomyces cerevisiae (W303-1A and FY833), as well as with non-conventional yeasts: Debaryomyces hansenii, Candida albicans, Meyerozyma guilliermondii, and Rhodotorula mucilaginosa. Results of the estimation of the PMP via the fluorescence changes under different conditions were adequate with all strains. Consistent results were also obtained with several mutants of the main monovalent transporters, validating ThT as a monitor for PMP estimation. [ABSTRACT FROM AUTHOR]

Sinorhizobium meliloti 1021 bacteria secretes a considerable amount of flavins (FLs) and can form a nitrogen-fixing symbiosis with legumes. This strain is also associated with non-legume plants. However, its role in plant growth promotion (PGP) of non-legumes is not well understood. The present study evaluated the growth and development of lettuce (Lactuca sativa) and kale (Brassica oleracea var. acephala) plants inoculated with S. meliloti 1021 (FL+) and its mutant 1021ΔribBA, with a limited ability to secrete FLs (FL−). The results from this study indicated that inoculation with 1021 significantly (p < 0.05) increased the lengths and surface areas of the roots and hypocotyls of the seedlings compared to 1021ΔribBA. The kale and lettuce seedlings recorded 19% and 14% increases in total root length, respectively, following inoculation with 1021 compared to 1021ΔribBA. A greenhouse study showed that plant growth, photosynthetic rate, and yield were improved by 1021 inoculation. Moreover, chlorophylls a and b, and total carotenoids were more significantly (p < 0.05) increased in kale plants associated with 1021 than non-inoculated plants. In kale, total phenolics and flavonoids were significantly (p < 0.05) increased by 6% and 23%, respectively, and in lettuce, the increments were 102% and 57%, respectively, following 1021 inoculation. Overall, bacterial-derived FLs enhanced kale and lettuce plant growth, physiological indices, and yield. Future investigation will use proteomic approaches combined with plant physiological responses to better understand host-plant responses to bacteria-derived FLs. [ABSTRACT FROM AUTHOR]

In order to investigate the antibacterial mechanism of tea polyphenols and theaflavins against oral cariogenic bacteria, the pH value of the culture medium, the number of bacteria adhering to the smooth glass tube wall, and the electrical conductivity value within 10 h were measured, respectively. The effects of four concentrations of tea polyphenols and theaflavins below the MIC value were studied on acid production, adhesion, and electrical conductivity of oral cariogenic bacteria. The live/dead staining method was used to observe the effects of four concentrations of tea polyphenols and theaflavins below the MIC value on the biofilm formation of oral cariogenic bacteria under a laser scanning confocal microscope. With the increase in concentrations of tea polyphenols and theaflavins, the acid production and adhesion of the cariogenic bacteria gradually decreased, and the conductivity gradually increased. However, the conductivity increase was not significant (p < 0.05). Compared with the control group, the 1/2MIC and 1/4MIC tea polyphenols and theaflavins treatments significantly reduced the biomass of the cariogenic biofilm (p < 0.05). The confocal laser scanning microscope showed that the integrated optical density of green fluorescence of the cariogenic biofilm gradually decreased with the increase in agent concentration after the action of tea polyphenols and theaflavins. [ABSTRACT FROM AUTHOR]

Theaflavins (TFs), the primary bioactive components in black tea, are poorly absorbed in the small intestine. However, the biological activity of TFs does not match their low bioavailability, which suggests that the gut microbiota plays a crucial role in their biotransformation and activities. In this study, we aimed to investigate the biotransferred metabolites of TFs produced by the human gut microbiota and these metabolites' function. We profiled the microbial metabolites of TFs by in vitro anaerobic human gut microbiota fermentation using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. A total of 17 microbial metabolites were identified, and their corresponding metabolic pathways were proposed. Moreover, full-length 16S rRNA gene sequence analysis revealed that the TFs altered the gut microbiota diversity and increased the relative abundance of specific members of the microbiota involved in the catabolism of the TFs, including Flavonifractor_plautii, Bacteroides_uniformis, Eubacterium_ramulus, etc. Notably, the antioxidant capacity of the TF sample increased after fermentation compared to the initial sample. In conclusion, the results contribute to a more comprehensive understanding of the microbial metabolites and antioxidant capacity of TFs. [ABSTRACT FROM AUTHOR]

The spectroscopy and photo-induced dissociation of flavin mononucleotide anions in vacuo are investigated over the 300–500 nm wavelength range. Comparison of the dependence of fragment ion yields as a function of deposited photon energy with calculated dissociation energies and collision-induced dissociation measurements performed under single-collision conditions suggests that a substantial fraction of photo-activated ions decompose through non-statistical fragmentation pathways. Among these pathways is the dominant photo-induced fragmentation channel, the loss of a fragment identified as formylmethylflavin. The fragment ion specific action spectra reveal electronic transition energies close to those for flavins in solution and previously published gas-phase measurements, although the photo-fragment yield upon excitation of the S2 ← S0 transition appears to be suppressed. [ABSTRACT FROM AUTHOR]

Enzyme driven oxidations catalyzed by alcohol dehydrogenases rely on the in situ NAD(P)+ regeneration. A wide variety of chemoenzymatic and fully enzymatic methods have been reported over the last 30 years to integrate the cofactor regeneration in biocatalytic oxidations. However, the majority of examples are limited to homogeneous systems where the reuse of both enzymes and chemical catalysts are challenging. In this work, we co‐immobilize an alcohol dehydrogenase from Bacillus stearothermophilus with a flavin derivative (FMN), which performs as an organocatalyst that oxidizes NADH back to NAD+. This latter oxidized cofactor is sequentially utilized by the dehydrogenase to oxidize 1,ω‐diols. Remarkably, the immobilization chemistry of FMN determines its efficiency to oxidize NADH and, unlike in its free state, the immobilized FMN can recycle NAD+ in dark. This is possible because the support where both enzyme and FMN are immobilized also captures NADH, making the electron transfer from the substrates to the cofactors more efficient. This work illustrates how the co‐immobilization and confinement of bio and chemical catalysts on solid materials (heterogeneous phase) enable chemoenzymatic cascades that are precluded in solution (homogeneous phase). [ABSTRACT FROM AUTHOR]

Possible use of Thioflavin T based fluorescent molecular rotors as temperature sensors was assessed in this work. Fluorescent properties of Thioflavin T and its derivative 6-Me-BTA-2C in 99% glycerol were studied using steady-state and time-resolved fluorescence spectroscopy methods. For Thioflavin T in glycerol it has been found that temperature growth from 261 to 353 K results in ~3 orders of magnitude increase of non-radiative decay rate constant knr for the excited state of the molecule. Fluorescence decay studies by time-correlated single photon counting method showed that fluorescence decay kinetics of Thioflavin T in glycerol can be used for temperature measurements in the range 260–290 K. For 6-Me-BTA-2C molecule the range of the maximal sensitivity was shifted to higher temperatures (280–320 K). The obtained results about fluorescent properties and decay kinetics of Thioflavin T based dyes in highly viscous media can be used to develop nanoscale temperature sensors. [ABSTRACT FROM AUTHOR]

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested. [ABSTRACT FROM AUTHOR]

This short review reports the surprising phenomenon of nuclear hyperpolarization occurring in chemical reactions, which is called CIDNP (chemically induced dynamic nuclear polarization) or photo-CIDNP if the chemical reaction is light-driven. The phenomenon occurs in both liquid and solid-state, and electron transfer systems, often carrying flavins as electron acceptors, are involved. Here, we explain the physical and chemical properties of flavins, their occurrence in spin-correlated radical pairs (SCRP) and the possible involvement of flavin-carrying SCRPs in animal magneto-reception at earth's magnetic field. [ABSTRACT FROM AUTHOR]

The UbiX/UbiD system is widespread in microbes and responsible for the reversible decarboxylation of unsaturated carboxylic acids. The UbiD enzyme catalyzes this unusual reaction using a prenylated flavin (prFMN) as cofactor, the latter formed by the flavin prenyltransferase UbiX. A detailed picture of the biochemistry of flavin prenylation, oxidative maturation, and covalent catalysis underpinning reversible decarboxylation is emerging. This reveals the prFMN cofactor can undergo a wide range of transformations, complemented by considerable UbiD‐variability. These provide a blueprint for biotechnological applications aimed at producing hydrocarbons or aromatic C–H activation through carboxylation. [ABSTRACT FROM AUTHOR]

Flavins are a class of organic compounds with the basic structure of 7,8-dimethy-10-alkyl isoalloxazine. They are ubiquitous in nature and participate in many biochemical reactions. Due to various existing forms, there is a lack of systematic research on the absorption and fluorescence spectra of flavins. In this study, employing the density functional theory (DFT) and time-dependent (TD) DFT, we calculated the pH-dependent absorption and fluorescence spectra of flavin of three redox states (quinone, semiquinone, and hydroquinone) in solvents. The chemical equilibrium of three redox states of flavins and the pH effect on the absorption spectra and fluorescence spectra of flavins were carefully discussed. The conclusion helps with identifying the existing forms of flavins in solvent with different pH values. [ABSTRACT FROM AUTHOR]

Theaflavins (TFs), as the major polyphenolic components of fermented tea, possess beneficial effects on human health. In this study, the effervescent tablets based on theaflavins were developed. The optimal formulation of TF effervescent tablets was obtained by response surface methodology with the Box-Behnken design. Then, the physiochemical properties were evaluated, including hardness, friability, effervescent time and pH of the solution. At last, the antioxidant ability of TF effervescent tablets was studied through DPPH and ABTS radical scavenging assay. According to the results, the optimal formulation of the tablets contained TF powder 9.09%, disintegrating agent 43.80% (the weight ratio of citric acid and sodium bicarbonate was 1:1), aspartame 1.86%, PEG-600 3%, and mannitol in balance. With the wet granulation method, the TF effervescent tablets displayed suitable hardness, fast disintegration time, good color, pleasant taste and high antioxidant activity. This study demonstrated that the TF effervescent tablets could be a valuable product for the supplement market and contribute to promoting practical application of TFs. [ABSTRACT FROM AUTHOR]

The flavin derivatives 10-methyl-isoalloxazine (MIA) and 6-fluoro-10-methyl-isoalloxazine (6F-MIA) were incorporated in two alternative metal-organic frameworks, (MOFs) MIL-53(Al) and MOF-5. We used a post-synthetic, diffusion-based incorporation into microcrystalline MIL-53 powders with one-dimensional (1D) pores and an in-situ approach during the synthesis of MOF-5 with its 3D channel network. The maximum amount of flavin dye incorporation is 3.9 wt% for MIA@MIL-53(Al) and 1.5 wt% for 6F-MIA@MIL-53(Al), 0.85 wt% for MIA@MOF-5 and 5.2 wt% for 6F-MIA@MOF-5. For the high incorporation yields the probability to have more than one dye molecule in a pore volume is significant. As compared to the flavins in solution, the fluorescence spectrum of these flavin@MOF composites is broadened at the bathocromic side especially for MIA. Time-resolved spectroscopy showed that multi-exponential fluorescence lifetimes were needed to describe the decays. The fluorescence-weighted lifetime of flavin@MOF of 4 ± 1 ns also corresponds to those in solution but is significantly prolonged compared to the solid flavin dyes with less than 1 ns, thereby confirming the concept of "solid solutions" for dye@MOF composites. The fluorescence quantum yield (ΦF) of the flavin@MOF composites is about half of the solution but is significantly higher compared to the solid flavin dyes. Both the fluorescence lifetime and quantum yield of flavin@MOF decrease with the flavin loading in MIL-53 due to the formation of various J-aggregates. Theoretical calculations using plane-wave and QM/MM methods are in good correspondence with the experimental results and explain the electronic structures as well as the photophysical properties of crystalline MIA and the flavin@MOF composites. In the solid flavins, π-stacking interactions of the molecules lead to a charge transfer state with low oscillator strength resulting in aggregation-caused quenching (ACQ) with low lifetimes and quantum yields. In the MOF pores, single flavin molecules represent a major population and the computed MIA@MOF structures do not find π-stacking interactions with the pore walls but only weak van-der-Waals contacts which reasons the enhanced fluorescence lifetime and quantum yield of the flavins in the composites compared to their neat solid state. To analyze the orientation of flavins in MOFs, we measured fluorescence anisotropy images of single flavin@MOF-5 crystals and a static ensemble flavin@MIL53 microcrystals, respectively. Based on image information, anisotropy distributions and overall curve of the time-resolved anisotropy curves combined with theoretical calculations, we can prove that all fluorescent flavins species have a defined and rather homogeneous orientation in the MOF framework. In MIL-53, the transition dipole moments of flavins are orientated along the 1D channel axis, whereas in MOF-5 we resolved an average orientation that is tilted with respect to the cubic crystal lattice. Notably, the more hydrophobic 6F-MIA exhibits a higher degree order than MIA. The flexible MOF MIL-53(Al) was optimized essentially to the experimental large-pore form in the guest-free state with QuantumEspresso (QE) and with MIA molecules in the pores the structure contracted to close to the experimental narrow-pore form which was also confirmed by PXRD. In summary, the incorporation of flavins in MOFs yields solid-state materials with enhanced rigidity, stabilized conformation, defined orientation and reduced aggregations of the flavins, leading to increased fluorescence lifetime and quantum yield as controllable photo-luminescent and photo-physical properties. [ABSTRACT FROM AUTHOR]

Flavin is involved in a wide range of life processes. Flavin can exist in different forms due to its ability to transfer electron(s) in different physiological conditions. It is difficult to experimentally obtain the absorption and fluorescence spectra at the vibrational level. A systematically theoretical simulation is a necessary way to explore the fine structure of these spectra of all forms of flavin molecules. In this study, the vibrationally resolved absorption spectra and fluorescence spectra of five forms of flavins (fully oxidized form FLox, neutral radical form HFLsq∙, anionic radical form FLsq−∙, neutral reduced form H2FLred, and anionic reduced form HFLred−) are simulated by adiabatic or vertical methods combined with time‐dependent or time‐independent (TD/TI) formalisms, with displace, Duschinsky, and Herzberg−Teller (HT) effects under the framework of the Franck‐Condon approximation. The statistical vibronic transition analysis reveals the unity of the spectrum transition property, the relevant normal modes, and the primary geometrical variations. The correlation between geometry and spectral calculation is explained. [ABSTRACT FROM AUTHOR]

Thearubigins (TRs) is a type of heterogeneous mixture from black tea, usually accounts for 60–70% of dry substance of black tea infusion. It is an important tea pigment for food industry and also contributes much to the sensory properties and biological activities of black tea. TRs therefore have a practical application value for the development of tea processing, natural food colorants and healthy products. The review paper summarizes the current knowledge from the perspectives of formation mechanism, chemical structure, sensory characteristics and health benefits of TRs. Through the oxidation mechanism and products of flavan-3-ols, the primary characteristics and reaction pathways of catechin, gallocatechin and galloylated catechin are profiled. Furthermore, the sensory properties and biological activities of TRs are comprehensively discussed. Gallocatechins containing vic -trihydroxy are more liable to lose protons and produce o -quinone, and subsequently condense through B–B ring to form dehydrotheasinensin, theasinensin, dehydrotheasinensin AQ in a pathway, or form bicyclo[3.2.1]octane to produce theacitrins, theacitrinins and proepitheaflagallin in another pathway. Furthermore, the oxidation of a couple of catechol- and pyrogallol-type catechins produce theaflavins (TFs), theanaphthoquinone and theanaphththeaflavins. Catechin can undergo nucleophilic reaction to form polymers, while catechin gallate also forms polymers through condensing consecutive benzotropolone moieties. The ratio of TRs to TFs is a critical index for color and brightness of black tea infusion, thus regulating enzymatic oxidation of tea polyphenols is not only an effective strategy in high-quality black tea processing, but also provides more options for tea pigment producing in beverage industry. • Formation mechanism of thearubigins is profiled. • Basic units of thearubigins are clarified. • Thearubigins health benefits are discussed. • Sensory properties of thearubigins are proposed. [ABSTRACT FROM AUTHOR]

Thioflavin T, a cationic benzothiazole dye, is typically used to detect amyloid fibrils. In this study, we analyzed the staining properties of Bacillus subtilis cells using several fluorescent dyes, including thioflavin T analogs, 2- (4 '-methylaminophenyl) benzothiazole (BTA-1), and 2- (4-aminophenyl) benzothiazole (APBT). Thioflavin T stained vegetative cells in the early log phase and outer layer structures of forespores and mature spores. The inner parts of forespores and heat-killed mature spores were also stained with thioflavin T. Congo red, auramine O, and rhodamine B stained forespores and mature spores similar to thioflavin T. In contrast, APBT and BTA-1 fluorescence was detected in the outer layers of vegetative cells, mother cells, forespores, and mature spores, indicating that they bind to the cell membrane and/or cell wall. The combination of the fluorescent dyes used in this study will help analyze morphogenetic processes during the sporulation and the damage mechanisms of vegetative cells and spores. [ABSTRACT FROM AUTHOR]

Tea polyphenol (TPs) oxidation caused by polyphenol oxidase (PPO) in manufacturing is responsible for the sensory characteristics and health function of fermented tea, therefore, this subject is rich in scientific and commercial interests. In this work, an in vitro catalysis of TPs in liquid nitrogen grinding of sun-dried green tea leaves by PPO was developed, and the changes in metabolites were analyzed by metabolomics. A total of 441 metabolites were identified in the catalyzed tea powder and control check samples, which were classified into 11 classes, including flavonoids (125 metabolites), phenolic acids (67 metabolites), and lipids (55 metabolites). The relative levels of 28 metabolites after catalysis were decreased significantly (variable importance in projection (VIP) > 1.0, p < 0.05, and fold change (FC) < 0.5)), while the relative levels of 45 metabolites, including theaflavin, theaflavin-3′-gallate, theaflavin-3-gallate, and theaflavin 3,3′-digallate were increased significantly (VIP > 1.0, p < 0.05, and FC > 2). The increase in theaflavins was associated with the polymerization of catechins catalyzed by PPO. This work provided an in vitro method for the study of the catalysis of enzymes in tea leaves. [ABSTRACT FROM AUTHOR]

With the emergence of host-guest systems, a novel branch of complexation chemistry has found wide application in industries such as food, pharmacy, medicine, environmental protection and cosmetics. Along with the extensively studied cyclodextrins and calixarenes, the innovative cucurbiturils (CB) have enjoyed increased popularity among the scientific community as they possess even better qualities as cavitands as compared to the former molecules. Moreover, their complexation abilities could further be enhanced with the assistance of metal cations, which can interestingly exert a dual effect on the complexation process: either by competitively binding to the host entity or cooperatively associating with the CB@guest structures. In our previous work, two metal species (Mg2+ and Ga3+) have been found to bind to CB molecules in the strongest fashion upon the formation of host–guest complexes. The current study focuses on their role in the complex formation with three dye molecules: thiazole orange, neutral red, and thioflavin T. Various key factors influencing the process have been recognized, such as pH and the dielectric constant of the medium, the cavity size of the host, Mn+ charge, and the presence/absence of hydration shell around the metal cation. A well-calibrated DFT methodology, solidly based and validated and presented in the literature experimental data, is applied. The obtained results shed new light on several aspects of the cucurbituril complexation chemistry. [ABSTRACT FROM AUTHOR]

Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm−1, which covers only part of the spectrum of biologically relevant time scales and Raman shifts. Here we report on a broadband (~200–2200 cm−1) dual transient visible absorption (visTA)/FSRS set-up that can accommodate time delays from a few femtoseconds to several hundreds of microseconds after illumination with an actinic pump. The extended time scale and wavenumber range allowed us to monitor the complete excited-state dynamics of the biological chromophore flavin mononucleotide (FMN), both free in solution and embedded in two variants of the bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The observed lifetimes and intermediate states (singlet, triplet, and adduct) are in agreement with previous time-resolved infrared spectroscopy experiments. Importantly, we found evidence for additional dynamical events, particularly upon analysis of the low-frequency Raman region below 1000 cm−1. We show that fs-to-sub-ms visTA/FSRS with a broad wavenumber range is a useful tool to characterize short-lived conformationally excited states in flavoproteins and potentially other light-responsive proteins. [ABSTRACT FROM AUTHOR]

A label- and enzyme-free fluorescence assay based on thioflavin T–induced G-quadruplexes is developed to sensitively and specifically detect telomerase activity. Thioflavin T has a dual role as an efficient inducer and fluorescent probe, and the incorporation of thioflavin T into the thioflavin T–induced G-quadruplexes results in an intense fluorescence enhancement. In the presence of thioflavin T and K+, G-quadruplexes are formed by elongation of the telomerase substrate primer that is catalyzed by telomerase extracted from cancer cells. Thus, the telomerase activity in cancer cell extracts can be evaluated by measuring the thioflavin T fluorescence. More importantly, thioflavin T can specifically recognize and bind to G-quadruplexes, whereas it cannot recognize single- and double-stranded DNAs, which leads to the thioflavin T–based fluorescence assay exhibiting a reduced background and improved signal-to-noise ratio. As a result, the proposed assay has the linear range from 5 to 200 HeLa cells and the detection limit is 34 HeLa cells, which holds great potential for use in the detection of telomerase activity and the diagnosis of cancer. [ABSTRACT FROM AUTHOR]

Lactiplantibacillus plantarum is a lactic acid bacterium that is commonly found in the human gut and fermented food products. Despite its overwhelmingly fermentative metabolism, this microbe can perform extracellular electron transfer (EET) when provided with an exogenous quinone, 1,4-dihydroxy-2-naphthoic acid (DHNA), and riboflavin. However, the separate roles of DHNA and riboflavin in EET in L. plantarum have remained unclear. Here, we seek to understand the role of quinones and flavins in EET by monitoring iron and anode reduction in the presence and absence of these small molecules. We found that addition of either DHNA or riboflavin can support robust iron reduction, indicating electron transfer to extracellular iron occurs through both flavin-dependent and DHNA-dependent routes. Using genetic mutants of L. plantarum, we found that flavin-dependent iron reduction requires Ndh2 and EetA, while DHNA-dependent iron reduction largely relies on Ndh2 and PplA. In contrast to iron reduction, DHNA-containing medium supported more robust anode reduction than riboflavin-containing medium, suggesting electron transfer to an anode proceeds most efficiently through the DHNA-dependent pathway. Furthermore, we found that flavin-dependent anode reduction requires EetA, Ndh2, and PplA, while DHNA-dependent anode reduction requires Ndh2 and PplA. Taken together, we identify multiple EET routes utilized by L. plantarum and show that the EET route depends on access to environmental biomolecules and on the electron acceptor. This work expands our molecular-level understanding of EET in Gram-positive microbes and provides additional opportunities to manipulate EET for biotechnology. [ABSTRACT FROM AUTHOR]

Access to new non‐canonical amino acid residues is crucial for medicinal chemistry and chemical biology. Analogues of the amino acid methionine have been far less explored—despite their use in biochemistry, pharmacology and peptide bioconjugation. This is largely due to limited synthetic access. Herein, we exploit a new disconnection to access non‐natural methionines through the development of a photochemical method for the radical α‐C−H functionalization of sulfides with alkenes, in water, using inexpensive and commercially‐available riboflavin (vitamin B2) as a photocatalyst. Our photochemical conditions allow the two‐step synthesis of novel methionine analogues—by radical addition to unsaturated amino acid derivatives—and the chemoselective modification of peptide side‐chains to yield non‐natural methionine residues within small peptides. The mechanism of the bio‐inspired flavin photocatalysis has been probed by experimental, DFT and TDDFT studies. [ABSTRACT FROM AUTHOR]

Access to new non‐canonical amino acid residues is crucial for medicinal chemistry and chemical biology. Analogues of the amino acid methionine have been far less explored—despite their use in biochemistry, pharmacology and peptide bioconjugation. This is largely due to limited synthetic access. Herein, we exploit a new disconnection to access non‐natural methionines through the development of a photochemical method for the radical α‐C−H functionalization of sulfides with alkenes, in water, using inexpensive and commercially‐available riboflavin (vitamin B2) as a photocatalyst. Our photochemical conditions allow the two‐step synthesis of novel methionine analogues—by radical addition to unsaturated amino acid derivatives—and the chemoselective modification of peptide side‐chains to yield non‐natural methionine residues within small peptides. The mechanism of the bio‐inspired flavin photocatalysis has been probed by experimental, DFT and TDDFT studies. [ABSTRACT FROM AUTHOR]

From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726 nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins. [ABSTRACT FROM AUTHOR]

The riboflavin derivatives FMN and flavin adenine dinucleotide (FAD) are critical cofactors for wide-ranging biological processes across all kingdoms of life. Although it is well established that these flavins can be readily interconverted, in plants, the responsible catalysts and regulatory mechanisms remain poorly understood. Here, we report the cloning and biochemical characterization of an FAD synthetase encoded by the gene At5g03430, which we have designated AtFADS1 (A. thaliana FADS1). The catalytic properties of the FAD synthetase activity are similar to those reported for other FAD synthetases, except that we observed maximum activity with Zn2+ as the associated divalent metal cation. Like human FAD synthetase, AtFADS1 exists as an apparent fusion with an ancestral FAD pyrophosphatase, a feature that is conserved across plants. However, we detected no pyrophosphatase activity with AtFADS1, consistent with an observed loss of a key catalytic residue in higher plant evolutionary history. In contrast, we determined that algal FADS1 retains both FAD synthetase and pyrophosphatase activity. We discuss the implications, including the potential for yet-unstudied biologically relevant noncatalytic functions, and possible evolutionary pressures that have led to the loss of FAD pyrophosphatase activity, yet universal retention of an apparently nonfunctional domain in FADS of land plants. [ABSTRACT FROM AUTHOR]

A structural study on the solid, microcrystalline chromophore roseolumiflavin was carried out. Based on rational design considerations three binary co-crystals were prepared with a primary focus set on utilizing the imide functional group. Therefore, robust supramolecular synthons, including a carboxylic acid–amide dimer, halogen bonding, as well as a hydrogen-bonded three-point binding motif, were successfully incorporated into the multicomponent systems. All the co-crystals showed a blue-shifted fluorescence in the red range compared to the near-infrared-emitting solid roseolumiflavin. Solvatochromic studies finally elucidated that the degree of close packing between the flavin molecules within the crystal structures was decisive for the emission colour. These results render new possibilities to investigate flavins in the aspect of crystal engineering to tune optical properties of organic chromophores in the solid state. [ABSTRACT FROM AUTHOR]

Lumichrome (LC) is the major photodegradation product of biologically important flavin cofactors. Since LC serves as a structural comparison with the flavins; understanding excited states of LC is fundamentally important to establish a connection with photophysics of different flavins, such as lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Herein, we deduce the initial excited state structural dynamics of LC using UV resonance Raman (UVRR) intensity analysis. The UVRR spectra at wavelengths across the 260 nm absorption band of LC were measured and resulting Raman excitation profiles and absorption spectrum were self‐consistently simulated using a time‐dependent wave packet formalism to extract the initial excited state structural and solvent broadening parameters. These results are compared with those obtained for other flavins following UV excitations. We find that LC undergoes a very distinct instantaneous charge redistribution than flavins, which is attributed to the extended π‐conjugation present in flavins but missing in LC. The homogeneous broadening linewidth of LC appears to be lower than that of LF, while the inhomogeneous broadening values are comparable, indicating greater solvent interaction with excited flavin on ultrafast timescale compared with LC, whereas on longer timescale these interactions are almost similar. [ABSTRACT FROM AUTHOR]

Flavin with defined helical self-assembly helps to understand chemical designs for obtaining high-purity semiconducting (s)-single-walled carbon nanotubes (SWNT) in a diameter (dt)-selective manner for high-end applications. In this study, flavins containing 8, 12, 16, and 20 n-alkyl chains were synthesized, and their single/tandem effects on dt-selective s-SWNT dispersibility were investigated at isomolarity. Flavins with n-dodecyl and longer chain lengths (FC12, FC16, and FC20) act as good surfactants for stable SWNT dispersions whereas n-octyl flavin (FC8) exhibits poor dispersibility owing to the lack of SWNT buoyancy. When used with small-dt SWNT, FC8 displays chirality-selective SWNT dispersion. This behavior, along with various flavin helical motifs, prompts the development of criteria for 'side chain length (lS)' required for stable and dt-selective SWNT dispersion, which also explains lS-dependent dt-enrichment behavior. Moreover, SWNT dispersions with flavins with dodecyl and longer lS exhibit increased metallic (m)-SWNT, background absorption-contributing carbonaceous impurities (CIs) and preferential selectivity of s-SWNT with slightly larger dt. The increased CIs that affect the SWNT quantum yield were attributed to a solubility parameter. Furthermore, the effects of flavin lS, sonication bath temperature, centrifugal speed, and surfactant concentration on SWNT purity and s-/m-SWNT ratio were investigated. A tandem FC8/FC12 provides fine-tuning of dt-selective SWNT dispersion, wherein the FC8 ratio governs the tendency towards smaller dt. Kinetic and thermodynamic assemblies of tandem flavins result in different sorting behaviors in which wide dt-tunability was demonstrated using kinetic assembly. This study highlights the importance of appropriate side chain length and other extrinsic parameters to obtain dt-selective or high-purity s-SWNT. [ABSTRACT FROM AUTHOR]

Background: CYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae. Results: Investigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses. Conclusions: This study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains. [ABSTRACT FROM AUTHOR]