Cationic styryl dyes for DNA labelling and selectivity toward cancer cells and Gram-negative bacteria.

Fluorescence-based methods are important tools for the analysis of nucleic acids in vitro and in cells. In this study, two cationic cyanine-styryl derivatives were produced using a two-step synthesis. Their optical properties were evaluated in different solvents, and frontier molecular orbital theory was utilized to interpret the findings. The DNA binding of these molecules was investigated to show fluorescence intensification. The molecular docking of both dyes in DNA illustrated the relevance of the electrostatic interaction between the quaternary ammonium of both dyes and the phosphate of the DNA backbone. Last but not least, applications of the synthesized styryl dyes were demonstrated to be selective towards cancer cells and particular kinds of bacteria.

Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade.

ß-Nicotinamide mononucleotide (NMN) has recently gained attention for a nutritional supplement because it is an intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+ ). In this study, we developed NMN synthesis by coupling two modules. The first module is to culture E. coli MG1655 ▵tktA ▵tktB ▵ptsG to metabolize xylose to generate D-ribose in the medium. The supernatant containing D-ribose was applied in the second module which is composed of EcRbsK-EcPRPS-CpNAMPT reaction to synthesize NMN, that requires additional enzymes of CHU0107 and EcPPase to remove feedback inhibitors ADP and pyrophosphate. The second module can be rapidly optimized by comparing NMN production determined by the cyanide assay. Finally, 10 mL optimal biocascade reaction generated NMN with a good yield of 84 % from 1 mM D-ribose supplied from the supernatant of E. coli MG1655 ▵tktA ▵tktB ▵ptsG. Our results can further guide researchers to metabolically engineer E. coli for NMN synthesis.

Design of a surrogate for high throughput screening of fatty aldehyde reductase engineering.

We designed and synthesized a fatty aldehyde surrogate containing a formyl thioester group, which can be reduced by fatty aldehyde reductase (FALR) with stoichiometric formaldehyde generation. It can be rapidly visualized and quantified using the Purpald assay. We demonstrated its successful application in the high throughput screening of FALR engineering.

Flavylium-Based Hypoxia-Responsive Probe for Cancer Cell Imaging.

A hypoxia-responsive probe based on a flavylium dye containing an azo group (AZO-Flav) was synthesized to detect hypoxic conditions via a reductase-catalyzed reaction in cancer cells. In in vitro enzymatic investigation, the azo group of AZO-Flav was reduced by a reductase in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) followed by fragmentation to generate a fluorescent molecule, Flav-NH2. The response of AZO-Flav to the reductase was as fast as 2 min with a limit of detection (LOD) of 0.4 µM. Moreover, AZO-Flav displayed high enzyme specificity even in the presence of high concentrations of biological interferences, such as reducing agents and biothiols. Therefore, AZO-Flav was tested to detect hypoxic and normoxic environments in cancer cells (HepG2). Compared to the normal condition, the fluorescence intensity in hypoxic conditions increased about 10-fold after 15 min. Prolonged incubation showed a 26-fold higher fluorescent intensity after 60 min. In addition, the fluorescence signal under hypoxia can be suppressed by an electron transport process inhibitor, diphenyliodonium chloride (DPIC), suggesting that reductases take part in the azo group reduction of AZO-Flav in a hypoxic environment. Therefore, this probe showed great potential application toward in vivo hypoxia detection.

Photophysical Study and Biological Applications of Synthetic Chalcone-Based Fluorescent Dyes.

A chalcone series (3a-f) with electron push-pull effect was synthesized via a one-pot Claisen-Schmidt reaction with a simple purification step. The compounds exhibited strong emission, peaking around 512-567 nm with mega-stokes shift (∆λ = 93-139 nm) in polar solvents (DMSO, MeOH, and PBS) and showed good photo-stability. Therefore, 3a-f were applied in cellular imaging. After 3 h of incubation, green fluorescence was clearly brighter in cancer cells (HepG2) compared to normal cells (HEK-293), suggesting preferential accumulation in cancer cells. Moreover, all compounds exhibited higher cytotoxicity within 24 h toward cancer cells (IC50 values ranging from 45 to 100 µM) than normal cells (IC50 value >100 µM). Furthermore, the antimicrobial properties of chalcones 3a-f were investigated. Interestingly, 3a-f exhibited antibacterial activities against Escherichia coli and Staphylococcus aureus, with minimum bactericidal concentrations (MBC) of 0.10-0.60 mg/mL (375-1000 µM), suggesting their potential antibacterial activity against both Gram-negative and Gram-positive bacteria. Thus, this series of chalcone-derived fluorescent dyes with facile synthesis shows great potential for the development of antibiotics and cancer cell staining agents.

Characterization of NucPNP and NucV involved in the early steps of nucleocidin biosynthesis in Streptomyces calvus.

Nucleocidin 1 produced by Streptomyces calvus is one of five characterized natural products containing fluorine. It was discovered in 1956, but its biosynthesis is not yet completely resolved. Recently, the biosynthetic gene cluster of 1 was identified. The nucPNP gene, which was initially annotated as orf206 and encodes a putative purine nucleoside phosphorylase, is essential for nucleocidin production. In this study, we performed in vitro assays and showed NucPNP produced adenine 3 from methylthioadenosine (MTA) 2 and adenosine 4. We also showed the downstream enzyme, NucV annotated as adenine phosphoribosyltransferase (APRT), catalyzes AMP formation from adenine 3 and 5-phospho-α-d-ribose-1-diphosphate (PRPP) 5. However, the catalytic efficiency of NucV was much slower than its homolog ScAPRT involved in the biosynthesis of canonical purine nucleoside in the same strain. These results provide new insights in nucleocidin biosynthesis and could guide future research on organofluorine formation.