Dual-Responsive Ratiometric Fluorescent Probe for Hypochlorite and Peroxynitrite Detection and Imaging In Vitro and In Vivo.

Hypochlorite (ClO-) and peroxynitrite (ONOO-) are two crucial highly reactive oxygen/nitrogen species, which interplay with each other, and are implicated in numerous pathophysiological processes. The simultaneous detection of ClO- and ONOO- is immensely significant in evaluating the occurrence and progress of related diseases. Herein, a dual-responsive ratiometric fluorescent probe PTZ-H for the separate and simultaneous detection of ClO- and ONOO- was designed and synthesized. In this probe, the phenothiazine-based coumarin moiety was chosen as the ClO- responsive fluorescent fragment, and the precursor of 2-(benzo[d]thiazol-2-yl)aniline was employed as the sensor for ONOO-. The PTZ-H emitted red fluorescence (640 nm) can switch to green (520 nm) and turn on blue fluorescence (450 nm) in response to ClO- and ONOO-, respectively. This allowed the specific recognition and ratiometric quantification of ClO- and ONOO- with the detection limits of 17 and 21 nM, respectively. Notably, confocal laser scanning microscopy revealed that the PTZ-H probe could target-specifically image ClO- and ONOO- in living RAW 264.7 cells, zebrafish, and tissues with distinct fluorescence signals. With the aid of this single fluorescent probe, the endogenous accumulation of ClO- and ONOO- in inflammatory RAW 264.7 cells and zebrafish can be monitored through two distinct emission channels with fast responses. Moreover, the large fluorescence signal interval, high selectivity, and good biocompatibility may enable its application in deciphering the distribution and correlation of ClO- and ONOO- engaged in biological activity.

Bioluminescence imaging of fibroblast activation protein-alpha in vivo and human plasma with highly sensitive probe.

Fibroblast activation protein-alpha (FAPα) has emerged as a biomarker of tumor stromal fibroblasts. FAP was overexpressed in stromal fibroblasts of human malignancies and positively correlated with the depth of tumor invasion, lymphatic metastasis, distant metastases, high TNM stage and poor prognosis. However, the circulating FAP levels in the plasma of gastric cancer patients and the relationship between FAP levels and gastric cancer remain unknown. Moreover, probes with super selectivity, extremely high sensitivity, and excellent performance in quantitative detection are still lacking. Herein, we developed the bioluminescent probe BL-FAP for sensitive detection and imaging of endogenous FAP in gastric cancer cells and tissues and plasma from gastric cancer patients. The probe exhibited the high signal-to-noise ratio (15000∼fold), the excellent selectivity (FAP/DPP IV ratio and FAP/PREP ratio = 50000∼ fold), and the high sensitivity (18.1 pg/mL). BL-FAP facilitates monitoring of endogenous FAP in living cells and nude mice bearing MGC-803-luc tumors. More importantly, this probe was successfully applied to the measurement of FAP activity levels in plasma from gastric cancer patients for the first time. A significant enhancement in FAP levels was observed in patients with gastric cancer, suggesting that the FAP level may be a potential diagnostic parameter for gastric cancer.

A near-infrared fluorescent probe that can image endogenous hydrogen polysulfides in vivo in tumour-bearing mice.

Hydrogen polysulfides (H2Sn, n > 1), which are important reactive sulfur species, play crucial roles in H2S-related bioactivities, including antioxidation, cytoprotection, activation of ion channels, transcription factor functions and tumour suppression. Monitoring H2Snin vivo is of significant interest for exploring the physiological roles of H2Sn and the exact mechanisms of H2Sn-related diseases. Herein, we conceive a novel near-infrared fluorescent probe, NIR-CPS, that is used to detect H2Sn in living cells and in vivo. With the advantages of high sensitivity, good selectivity and a remarkably large Stokes shift (100 nm), NIR-CPS was successfully applied in visualizing H2Sn in living cells and mice. More importantly, NIR-CPS monitored H2Sn stimulated by lipopolysaccharide in tumour-bearing mice. These results demonstrate that the NIR-CPS probe is a potentially powerful tool for the detection of H2Snin vivo, thus providing a valuable approach in H2Sn-related medical research.

Design, synthesis, and cytotoxic activity of novel 2H-imidazo[1,2-c]pyrazolo[3,4-e]pyrimidine derivatives.

In this study, a series of novel 2H-imidazo [1, 2-c] pyrazolo [3, 4-e] pyrimidine derivatives were designed, synthesized, and evaluated for their cytotoxic activities. The in vitro cell growth inhibition assay of the target compounds indicated their selectivity in inhibiting the proliferation of blood tumor cells (K562, U937) and solid tumor cells (HCT116, HT-29). Compound 9b exhibited the highest antiproliferative activities against K562 (IC50 = 5.597 µM) and U937 (IC50 = 3.512 µM). Based on the flow cytometry assays, compound 9b caused obvious induction of cell apoptosis and cell arrest at the S phase. Furthermore, western blot analysis revealed that compound 9b upregulated the expression of Bax, downregulated the levels of Bcl-2, and further activated caspase-3 in K562 cells. Therefore, compound 9b may be a potential anticancer agent that deserves further investigation.

Investigation of binding between fluoroquinolones and pepsin by fluorescence spectroscopy and molecular simulation.

In this paper, the interactions of pepsin with fluoroquinolones, including norfloxacin (NFX) or ofloxacin (OFX), were investigated using fluorescence spectroscopy. The effects of NFX or OFX on pepsin showed that the molecular conformation of pepsin and the microenvironment of tryptophan residues were changed under mimicked physiological conditions. Static quenching was suggested as a factor. Quenching constants and binding constants were determined and thermodynamic parameters were calculated at three temperatures (25°C, 31°C and 37°C). Molecular interaction distances (binding distance r) were obtained. Binding was enthalpy driven and the process was spontaneous. Synchronous fluorescence, three-dimensional fluorescence spectroscopy and molecular simulation were used for analysis. Interactions were further tested using molecular modelling. Quenching and binding constants of NFX with pepsin were the highest when testing NFX/OFX/fleroxacin/gatifloxacin with pepsin combinations. NFX was the strongest quencher, and affinity of NFX for pepsin was higher than that of OFX/fleroxacin/gatifloxacin.

A reaction-based near-infrared fluorescent probe that can visualize endogenous selenocysteine in vivo in tumor-bearing mice.

Monitoring the fluctuations of endogenous selenocysteine (Sec) in vivo is of significant interest to understand the physiological roles of Sec and the mechanisms of Sec-relevant diseases. Herein, a new near-infrared fluorescent probe, Fsec-1, has been developed for the determination of endogenous Sec in living cells and in vivo. Fsec-1 exhibits large fluorescence enhancement (136-fold) and a remarkably large Stokes shift (195 nm) when reacted with Sec. With the advantages of high sensitivity (a detection limit of 10 nM), good selectivity and low cytotoxicity, Fsec-1 was able to recognize both exogenous and endogenous Sec in living cells. The probe was also successfully applied in visualizing both exogenous and endogenous Sec in living mice. Notably, endogenously generated Sec in living tumors xenografted in nude mice was selectively detected by our reaction-based NIR probe for the first time. These results indicated that our new probe could serve as an efficient tool in monitoring endogenous Sec in vivo and exploring the anticancer mechanism of selenium.

Antiradical Activity and Mechanism of Coumarin-Chalcone Hybrids: Theoretical Insights.

In view of the multifunctional features of coumarins and chalcones, coumarin-chalcone hybrids have attracted much attention in recent years. Herein, the free radical scavenging activities of a series of coumarin-chalcone hybrids were investigated using the density functional theory (DFT) method. Three main reaction mechanisms were explored: hydrogen atom transfer (HAT), electron transfer followed by proton transfer (SET-PT), and sequential proton loss electron transfer (SPLET). Thermodynamic descriptors associated with these mechanisms were calculated in the gas phase and solvents. The results demonstrate that the predicted antioxidant efficiencies are generally in accordance with the experimental results. HAT is proposed as the thermodynamically favored mechanism in the gas phase and nonpolar solution, while SPLET is preferred in polar media. Our results indicate that compound MPHCC possesses potential for inactivating free radicals via double HAT and double SPLET mechanisms depending upon the polarity of environment. In addition, the SPLHAT mechanism provides an alternative pathway to HAT and SPLET for radical scavenging by MPHCC and OPHCC. The results confirmed the crucial role of hydroxyl groups on the chalcone moiety in trapping radicals. 4'-OH in the catechol group is proposed as the primary target for radical attack.

Density Functional and Kinetic Monte Carlo Study of Cu-Catalyzed Cross-Dehydrogenative Coupling Reaction of Thiazoles with THF.

Cu-catalyzed cross-dehydrogenative coupling (CDC) reaction of thiazoles with THF has been studied with the density functional theory method and kinetic Monte Carlo (kMC) simulations. Our results show that the previously proposed concerted metalation-deprotonation mechanism is unfavorable. On the basis of the DFT calculation and kMC simulation results, a new mechanism is proposed. In the favorable mechanism, the Cu(II) catalyst first combines with the thiazoles, forming an organocopper species that then binds to the THF radical. The rate-limiting step, C-C bond formation, is realized through an intramolecular structural rearrangement. The Cu catalyst works as a matchmaker to render the C-C bond formation. Kinetic Monte Carlo simulations demonstrate that one should be careful with the conclusions drawn simply from the calculated barriers.

Computational insight into the cooperative role of non-covalent interactions in the aza-Henry reaction catalyzed by quinine derivatives: mechanism and enantioselectivity.

Density functional theory (DFT) calculations were performed to elucidate the mechanism and the origin of the high enantioselectivity of the aza-Henry reaction of isatin-derived N-Boc ketimine catalyzed by a quinine-derived catalyst (QN). The C-C bond formation step is found to be both the rate-determining and the stereo-controlled step. The results revealed the important role of the phenolic OH group in pre-organizing the complex of nitromethane and QN and stabilizing the in situ-generated nitronate and protonated QN. Three possible activation modes for C-C bond formation involving different coordination patterns of catalyst and substrates were studied, and it was found that both the ion pair-hydrogen bonding mode and the Brønsted acid-hydrogen bonding mode are viable, with the latter slightly preferred for the real catalytic system. The calculated enantiomeric excess (ee) favouring the S enantiomer is in good agreement with the experimental result. The high reactivity and enantioselectivity can be ascribed to the cooperative role of the multiple non-covalent interactions, including classical and non-classical H bonding as well as anionπ interactions. These results also highlight the importance of the inclusion of dispersion correction for achieving a reasonable agreement between theory and experiment for the current reaction.

Asymmetric aza-Morita-Baylis-Hillman reactions of chiral N-phosphonyl imines with acrylates via GAP chemistry/technology.

Chiral N-phosphonyl imines have been proven to be efficient electrophilic acceptors for asymmetric aza-Morita-Baylis-Hillman (aza-MBH) reactions with acrylates under convenient conditions. Thirty examples of ß-amino acrylates were generated in high yields (up to 99.4%) and diastereoselectivity (up to >99 : 1 dr) in an atom-economical fashion. The synthesis was proved to follow the GAP (group-assisted purification) chemistry, i.e., the pure products can be obtained simply by washing the crude products with hexane/ethyl acetate (v/v, 10/1) without the use of chromatography or recrystallization. DFT calculations were also conducted to support an asymmetric induction model accounting for high diastereoselectivity.

Design, synthesis, quantum chemical studies and biological activity evaluation of pyrazole-benzimidazole derivatives as potent Aurora A/B kinase inhibitors.

Novel pyrazole-benzimidazole derivatives have been designed and synthesized. The entire target compounds were determined against cancer cell lines U937, K562, A549, LoVo and HT29 and were screened for Aurora A/B kinase inhibitory activity in vitro. The compounds 7a, 7b, 7i, 7k and 7l demonstrated significant cancer cell lines and Aurora A/B kinase inhibitory activities. Molecular modeling studies suggested the derivatives have bound in the active site of Aurora A kinase through the formation of four hydrogen bonds. Quantum chemical studies were carried out on these compounds to understand the structural features essential for activity. The cellular activity of 7k was also tested by immunofluorescence.

An orthogonally activatable CRISPR-Cas13d nanoprodrug to reverse chemoresistance for enhanced chemo-photodynamic therapy.

Orthogonal therapy that combines CRISPR-based gene editing and prodrug-based chemotherapy is a promising approach to combat multidrug-resistant cancer. However, its potency to precisely regulate different therapeutic modalities in vivo is limited due to the lack of an integrated platform with high spatiotemporal resolution. Taking advantage of CRISPR technology, a Pt(iv)-based prodrug and orthogonal emissive upconversion nanoparticles (UCNPs), we herein rationally designed the first logic-gated CRISPR-Cas13d-based nanoprodrug for orthogonal photomodulation of gene editing and prodrug release for enhanced cancer therapy. The nanoprodrug (URL) was constructed by encapsulating a green light-activatable Pt(iv) prodrug and UV light-activatable Cas13d gene editing tool into UCNPs. We demonstrated that URL maintained excellent orthogonal emission behaviors under 808 and 980 nm excitations, allowing wavelength-selective photoactivation of Cas13d and the prodrug for downregulation of the resistance-related gene and induction of chemo-photodynamic therapy, respectively. Moreover, the photomodulation superiority of URL for overcoming drug resistance was highlighted by integrating it with a Boolean logic gate for programmable modulation of multiple cell behaviors. Importantly, in vivo studies demonstrated that URL can promote Pt(iv) prodrug activation and ROS generation and massively induce on-target drug accumulation by Cas13d-mediated drug resistance attenuation, delivering an ultimate chemo-photodynamic therapeutic performance in efficiently eradicating primary tumors and preventing further liver metastasis. Collectively, our results suggest that URL expands the Cas13d-based genome editing toolbox into prodrug nanomedicine and accelerates the discovery of new orthogonal therapeutic approaches.

Novel biotin-linked amphiphilic calix[4]arene-based supramolecular micelles as doxorubicin carriers for boosted anticancer activity.

Supramolecular carrier-mediated chemotherapy is a highly attractive strategy for targeted drug delivery. In this study, four novel biotin-linked calix[4]arenes BPCA1-BPCA4 have been rationally designed to construct nano-complex with doxorubicin. The in vitro and in vivo assessments reveal that BPCA4-DOX with excellent stability are capable of affording significantly superior anti-tumor activity and lower side effects.

Antioxidant Activity and Mechanism of Avenanthramides: Double H+/e- Processes and Role of the Catechol, Guaiacyl, and Carboxyl Groups.

Avenanthramides (AVAs), unique phenolic compounds in oats, have attracted increasing interest due to their health benefits. Eight representative AVAs were studied using the density functional theory (DFT) method to elucidate their antioxidant activity and mechanism. Preference of different mechanisms was evaluated based on thermodynamic descriptors involved in double (2H+/2e-) free radical scavenging reactions. It was found that the hydrogen atom transfer (HAT) mechanism is more favorable in the gas and benzene phases, while sequential proton loss electron transfer (SPLET) is preferred in polar media. The results suggest the feasibility of the double HAT and double SPLET mechanisms for 2s and c-series AVAs. The sequential triple proton loss double electron transfer (StPLdET) mechanism represents the dominant pathway in aqueous solution at physiological pH. In addition, the sequential proton loss hydrogen atom transfer (SPLHAT) mechanism provides an alternative pathway to trap free radicals. Results also revealed the important role of the catechol, guaiacyl, and carboxyl moieties.

TDDFT study on the photophysical properties of coumarinyl chalcones and sensing mechanism of a derived fluorescent probe for hydrogen sulfide.

Coumarin-chalcone hybrids have attracted much attention in recent years due to their important optical properties. Herein, the photophysical properties of a series of coumarinyl chalcones and the sensing mechanism for H2S of a related fluorescent probe CC-DNP were investigated using density functional theory (DFT) and time-dependent density functional theory (TDDFT) methods. The predicted spectral properties agree well with the experimental results, which allowed an assignment of the spectra. Our calculations successfully clarified the experimental observed fluorescence "off-on" effect and the fluorescent quenching mechanism of the probe. The results revealed that the first excited state (S1) of the probe CC-DNP is a dark state with obvious charge transfer from coumarin unit to 2,4-dinitrophenyl (DNP) moiety, which results in the fluorescence quenching via the nonradiative photoinduced electron transfer (PET) process. On the other hand, the excited state S1 in the thiolysis product CC-OH decayed directly to S0, and thus the fluorescence is recovered.

Computational study on the antioxidant property of coumarin-fused coumarins.

Fused coumarins recently attracted strong scientific interest due to their potent pharmacological activities. In this study, density functional theory (DFT) calculations were performed to evaluate the antiradical activities of a series of coumarin-fused coumarins. By calculating the thermodynamic parameters, three primary mechanisms including hydrogen atom transfer (HAT), electron transfer followed by proton transfer (SET-PT) and sequential proton loss electron transfer (SPLET) were examined. It was found that in the gas and benzene phases, the studied compounds prefer to undergo HAT mechanism, while SPLET is more favored in polar media. The results also reveal the possibility of double HAT and double SPLET mechanisms for compound CC-6. Interestingly, a new polycyclic compound was generated by forming a new C5-O5' bond during the second HAT process at the 5'-OH in CC-6-R6 radical. In addition, the SPLHAT mechanism is proposed as a competitive pathway for radical scavenging by CC-4, CC-5 and CC-6.

Antioxidant activity and mechanism of dihydrochalcone C-glycosides: Effects of C-glycosylation and hydroxyl groups.

Dihydrochalcones (DHCs), an important subgroup of flavonoids, have recently received much attention due to their diverse biological activities. In contrast to their O-glycosides, understanding of the antioxidant property and mechanism of DHC C-glycosides remains limited. Herein, the free radical scavenging activity and mechanism of two representative C-glycosyl DHCs, aspalathin (ASP) and nothofagin (NOT) as well as their aglycones, 3-hydroxyphloretin (HPHL) and phloretin (PHL) were evaluated using the density functional theory (DFT) calculations. The results revealed the crucial role of sugar moiety on the conformation and the activity. The o-dihydroxyl in the B-ring and the 2',6'-dihydroxyacetophenone moiety were found significant in determining the activity. Our results showed that hydrogen atom transfer (HAT) is the dominant mechanism for radical-trapping in the gas and benzene phases, while the sequential proton loss electron transfer (SPLET) is more preferable in the polar environments. Also, the results revealed the feasibility of the double HAT and double SPLET as well as the SPLHAT mechanisms, which provide alternative pathways to trap radical for the studied DHCs. These results could deepen the understanding of the antiradical activity and mechanism of DHCs, which will facilitate the design of novel efficient antioxidants.

Design, synthesis, and biological evaluation of novel 4-anilinoquinazoline derivatives bearing amino acid moiety as potential EGFR kinase inhibitors.

In this study, a series of 4-anilinoquinazoline derivatives bearing amino acid moiety were designed, synthesized and evaluated for biological activities. The synthesized compounds were screened for anticancer activity against human hepatocellular carcinoma cell HepG2 using SRB assay. In vitro cell growth inhibition assays indicated that compound 6m exhibited moderate inhibitory activities only against human hepatocellular carcinoma cells HepG2 with IC50 of 8.3 µM. Synthetic derivatives showed excellent selectivity, such as compound 6m demonstrated a strong inhibition of EGFR (IC50 = 0.0032 µM), with selectivity of over 2000-fold over other kinases. Apoptosis analysis revealed that compound 6m caused obvious induction of cell apoptosis. 6m significantly down-regulated the expression of Bcl-2 and up-regulated the expression of Bax, decreased mitochondrial membrane potential (ΔΨm), promoted the mitochondrial cytochrome c release into the cytoplasm, activated caspase-3, and finally induced apoptosis of HepG2 cells. Molecular docking indicated that compound 6m could bind well with EGFR. Therefore, compound 6m may be a potential agent for cancer therapy deserving further research.

Synthesis, X-ray crystal structure and anti-tumor activity of calix[n]arene polyhydroxyamine derivatives.

Calixarene-based compounds are highly effective therapeutic agents against cancer. This study aims to prepare a series of calix [n]arene (n = 4, 6, 8) polyhydroxyamine derivatives (3a-3m) and to study their potential antitumor activities. The single crystal structure of calixs[4]arene derivative 3a was determined through X-ray diffraction. We assessed the ability of the prepared calix [n]arene polyhydroxyamine derivatives to induce cytotoxicity in six cancer cell lines by performing cancer cell growth inhibition assays. Results demonstrated that compounds 3a-3d achieved IC50 values ranging from 1.6 µM to 11.3 µM. Among the different compounds, 3a and 3b exerted the strongest cytotoxic effect in inhibiting the growth of SKOV3 cells. In relation to the underlying mechanisms of cytotoxic effects, cell cycle analysis revealed that the exposure of SKOV3 cells to 3a induced cell cycle arrest in the G0/G1 phase, suggesting a reduction in DNA synthesis. Immunofluorescent staining indicated that the protein expression levels of caspase-3 and p53 in cells significantly increased, whereas that of Bcl-2 was effectively suppressed. Meanwhile, no significant changes in Bax were observed in SKOV3 cells. These results highlight that calixarene 3a can be further studied as a potential anticancer agent.

Theoretical study on the structural and antioxidant properties of some recently synthesised 2,4,5-trimethoxy chalcones.

The free radical scavenging activity of a series of 2,4,5-trimethoxy chalcones has been computationally explored using the density functional theory (DFT) method. Three potential working mechanisms, hydrogen atom transfer (HAT), stepwise electron transfer proton transfer (SET-PT) and sequential proton loss electron transfer (SPLET) have been investigated. The physiochemical parameters including O-H bond dissociation enthalpy (BDE), ionisation potential (IP), proton dissociation enthalpy (PDE), proton affinity (PA) and electron transfer enthalpy (ETE) have been calculated in gas phase and solvents. The order of antioxidant efficiencies predicted theoretically in this work is in good agreement with that reported by experimental results. The results obtained demonstrate that HAT would be the most favourable mechanism in the gas and benzene phases, whereas the SPLET mechanism is the thermodynamically preferred pathway in polar media. In addition, the importance of the A-ring on the radical scavenging capabilities of chalcones was also confirmed.