Microwave irradiation: a green approach for the synthesis of functionalized N-methyl-1,4-dihydropyridines

An eco-friendly and cost-effective, microwave-assisted green approach has been developed for the synthesis of diverse functionalized N-methyl-1,4-dihydropyridines (1,4-DHPs). This pseudo three-component reaction was carried out between two equivalents of (E)-N-methyl-1-(methylthio)-2-nitroethenamine (NMSM) and one equivalent of aromatic aldehydes under microwave irradiation at 100 °C without catalyst and solvent. Short reaction times, avoidance of toxic solvents or expensive, metallic and corrosive catalysts and no need for column chromatographic purification are among the valuable features of the presented method. Moreover, the “greenness” of the method was evaluated within the ambits of the defined green metrics such as atom economy, carbon efficiency, E-factor, reaction mass efficiency, overall efficiency, process mass intensity and solvent intensity and the method exhibited a good to excellent score.

Microwave-assisted green synthesis of N-methyl-1,4-dihydropyridines under eco-friendly conditions.     

2′-Deoxy-5-(hydroxymethyl)cytidine: estimation in human cancer cells with a simple chemosensor

A pyrrole-based rhodamine conjugate (CS-1) has been developed and characterized for the selective detection and quantification of 2′-deoxy-5-(hydroxymethyl)cytidine (5hmC) in human cancer cells with a simple chemosensing method.

A new chemosensor, CS-1, has been developed and characterized for the selective detection and quantification of 2′-deoxy-5-(hydroxymethyl)cytidine (5hmC) in human cancer cells.

2′-Deoxy-5-(hydroxymethyl)cytidine (5hmC) is found in both neuronal cells and embryonic stem cells. It is a modified pyrimidine and used to quantify DNA hydroxymethylation levels in biological samples^1–3 as it is capable of producing interstrand cross-links in double-stranded DNA. It is produced through an enzymatic pathway carried out by the Ten-Eleven Translocation (TET1, TET2, TET3) enzymes, iron and 2-oxoglutarate dependent dioxygenase.^4–7 In the DNA demethylation process, methylcytosine is converted to cytosine and generates 5hmC as an intermediate in the first step of this process which is then further oxidized to 5-formylcytosine (fC) and 5-carboxycytosine (caC) of very low levels compared to the cytosine level.⁸ Though the biological function of 5hmC in the mammalian genome is still not revealed, the presence of a hydroxymethyl group can regulate gene expression (switch ON & OFF). Reports say that in artificial DNA 5hmC is converted to unmodified cytosine when introduced into mammalian cells.^9,10Levels of 5hmC substantially vary in different tissues and cells. It is found to be highest in the brain, particularly in nervous system and in moderate percentage in liver, colon, rectum and kidney tissues, whereas it is relatively low in lung and very low in breast and placenta.^11,12 The percentage of 5hmC content is much less in cancer and tumor tissues compared to the healthy ones. The reason behind this loss is the absence of TET1, TET2, TET3, IDH1, or IDH2 mutations in most of the human cancer cells which means decrease of methylcytosine oxidation.^13–15 This loss of 5hmC in cancer cells is being used as a diagnostic tool for the detection of early-stage of malignant disease. Few analytical methods^16–19 such as glucosyltransferase assays, tungsten-based oxidation systems, and TET-assisted bisulfite sequencing (TAB-Seq) or oxidative bisulfite sequencing (oxBS-Seq) protocols are now developed to differentiate 5hmC from other nucleotide which are naturally occurred. There are also few methods such as liquid chromatography/tandem mass spectroscopy (LC/MS-MS), which determine the level of 5hmC in mammalian cancer cell.^20–22 However, these procedures are highly toxic and expensive due to requirement of catalyzation through enzymes or heavy metal ion and these techniques require expertise, facilities, much time and costs even beyond standard DNA sequencing. As a result, these detection techniques are currently inappropriate for the high-throughput screening of genome-wide 5hmC levels (performance comparison is shown in Table S1, ESI^†).Among all reputed methods fluorescence detection method using chemosensors is significantly important due to its indispensable role in medicinal and biological applications.^23–27 Chemosensors have been effectively explored to monitor biochemical processes and assays through in situ analysis in living systems and abiotic samples with much less time and cost.In this contribution we prepared and characterize (Scheme S1 and Fig. S1–S3, ESI^†) a pyrrole–rhodamine based chemosensor (CS-1) which shows efficient and selective fluorescence signal for 5hmC in aqueous medium (Scheme 1). A transparent single crystal of CS-1 (Fig. 1) was obtained by slow evaporation of the solvent from a solution of CS-1 in CH₃CN. It crystallizes as monoclinic with space group P2₁/n (Fig. S4 and Table S2, ESI^†).Open in a separate windowScheme 15hmC-induced FRET OFF–ON mechanism of the chemosensor CS-1.Open in a separate windowFig. 1ORTEP diagram of CS-1 (ellipsoids are drawn at 40% probability level).Spectrophotometric and spectrofluorimetric titrations were carried out to understand the CS-1–5hmC interaction with 1 : 1 binding stoichiometry (Fig. S5, ESI^†) upon adding varying concentrations of 5hmC to a fixed concentration of CS-1 (1 μM) in aqueous medium at neutral pH. Upon the addition of increasing concentrations of the 5hmC, a clear absorption band (K_a = 4.47 × 10⁵ M⁻¹, Fig. S6, ESI^†) appeared to be centered at 556 nm with increasing intensity (Fig. 2a). On the other hand, for the fluorescence emission spectra of CS-1 (Fig. 2b), upon irradiation at 325 nm, an emission maxima at 390 nm was observed, which was attributed to the fluorescence emission from the donor unit i.e. the pyrrole moiety of CS-1. When 5hmC were added, due to rhodamine moiety CS-1 showed a 95-fold increase in fluorescence at 565 nm (K_a = 4.61 × 10⁵ M⁻¹, Fig. S7, ESI^†) with the detection limit of 8 nM (Fig. S8, ESI^†). The binding of 5hmC induces opening of the spirolactam ring in CS-1, inducing a shift of the emission spectrum. Subsequently, increased overlap between the emission of the energy-donor (pyrrole) and the absorption of the energy-acceptor (rhodamine) greatly enhances the intramolecular FRET process,^28,29 producing an emission from the energy acceptor unit in CS-1.Open in a separate windowFig. 2(a) UV-vis absorption spectra of CS-1 (1 μM) upon gradual addition of 5hmC up to 1.2 equiv. in H₂O–CH₃CN (15 : 1, v/v) at neutral pH. (b) Fluorescence emission spectra of CS-1 (1 μM) upon addition of 1.2 equiv. of 5hmC in H₂O–CH₃CN (15 : 1, v/v) at neutral pH (λ_ex = 325 nm).In order to establish the sensing selectivity of the chemosensor CS-1, parallel experimentations were carried out with other pyrimidine/purine derivatives such as 5-methylcytosine, cytosine, cytidine, thymine, uracil, 5-hydroxymethyluracil, adenine and guanine. Comparing with other pyrimidine/purine derivatives the abrupt fluorescence enhancement was found upon addition of 5hmC to CS-1 while others do not make any fluorescence changes under UV lamp (Fig. 3, lower panel). Furthermore, the prominent color change from colorless to deep pink allows 5hmC to be detected by naked eye (Fig. 3, upper panel). The above observation shows consistency with the fluorescence titration experiments where no such binding of CS-1 with other pyrimidine/purine derivatives was found (Fig. S9, ESI^†).Open in a separate windowFig. 3Visible color (top) and fluorescence changes (bottom) of CS-1 (1 μM) in aqueous medium upon addition of 1.2 equiv. of various pyrimidine/purine derivatives (λ_ex = 325 nm) in H₂O–CH₃CN (15 : 1, v/v) at neutral pH.pH titration reveals that CS-1 becomes fluorescent below pH 5 due to the spirolactam ring opening of rhodamine. However, it is non-fluorescent at pH range of 5–13. Upon addition of 5hmC to CS-1 shows deep red fluorescence in the pH range of 5–8 (Fig. S10, ESI^†). Considering the biological application and the practical applicability of the chemosensor pH 7.4 has been preferred to accomplish all experiments successfully.In ¹H NMR titration (Fig. S11, ESI^†), the most interesting feature is the continuous downfield shift of aromatic protons on the pyrrole moiety of CS-1 upon gradual addition of 5hmC. This may be explained as the decrease in electron density of the pyrrole moiety upon binding with 5hmC through hydrogen bonding. Xanthene protons to be shifted downfield upon spirolactam ring opening indicates the probe to coordinate with 5hmC and electrons are accumulated around 5hmC. In ¹³C NMR titration the spiro cycle carbon peak at 65 ppm was shifted to 138 ppm along with a little downfield shift of the aromatic region of CS-1 (Fig. S12, ESI^†). This coordination led to the spiro cycle opening and changes to the absorption and emission spectra, further evident by mass spectrometry (Fig. S13, ESI^†), which corroborates the stronger interaction of CS-1 with 5hmC.The experimental findings were validated by density functional theory (DFT) calculations using the 6-31G+(d,p) method basis set implemented at Gaussian 09 program. Energy optimization calculations presented the conformational changes at the spirolactam position of CS-1 while 5hmC takes part to accommodate a probe molecule. After CS-1–5hmC complexation the energy is minimized by 19.45 kcal from the chemosensor CS-1, indicating a stable complex structure (Fig. 4 and Table S3, ESI^†). This theoretical study strongly correlates the experimental findings.Open in a separate windowFig. 4Energy diagram showing the energy differences between CS-1 and CS-1–5hmC complex.The desirable features of CS-1 such as high sensitivity and high selectivity at physiological pH encouraged us to further evaluate the potential of the chemosensor for imaging 5hmC in live cells (Fig. 5). A549 cells (Human cancer cell A549, ATCC no. CCL-185) treated with CS-1 (1 μM) exhibited weak fluorescence, whereas a deep red fluorescence signal was observed in the cells stained with CS-1 (1 μM) and 5hmC (10 μM), which is in good agreement with the FRET OFF–ON profile of the chemosensor CS-1 in presence of 5hmC, thus corroborating the in-solution observation (Fig. S14, ESI^†). Cytotoxicity assay measurement shows that the chemosensor CS-1 does not have any toxicity on the tested cells and CS-1–5hmC complex does not exert any significant adverse effect on cell viability at tested concentrations (Fig. S15, ESI^†). As far as we are aware, this is the first report where we are executing the possible use of the pyrrole–rhodamine based chemosensor for selective recognition of 5hmC in living cells. These findings open an avenue for future biomedical applications of the chemosensor to recognize 5hmC.Open in a separate windowFig. 5Confocal microscopic images of A549 cells treated with CS-1 and 5hmC. (a) Cells treated with only CS-1 at 1 μM concentration. (b) Bright field image of (a). (c) Cells treated with CS-1 and 5hmC at concentration 10 μM. (d) Bright field image of (c). All images were acquired with a 60× objective lens with the applied wavelengths: For (a) and (b), E_ex = 341 nm, E_em = 414 nm, filter used: DIDS; for (c) and (d) E_ex = 550 nm, E_em = 571 nm, filter used: Rhod-2.The concentration of 5hmC was also quantified from A549 human cancer cells. Lysate of 10⁷ A549 cells was added to 1 μM of CS-1 and the fluorescence signal was recorded. Presence of 5hmC in these cancer cells was detected with the help of CS-1–5hmC standard fluorescence curve (Fig. 6) using the selective detection ability of the chemosensor CS-1.Open in a separate windowFig. 6(a) Calibration curve obtained for the estimation of 5hmC. (b) Estimation of the concentration of 5hmC (red point) from the calibration curve.From the standard curve it was found that the concentration of 5hmC in the tested sample was 0.034 μM present in 16.7 mm³ A549 cell volume (†). Assay of 5hmC was further validated from multiple samples of A549 human cancer cells using CS-1. Increasing fold of fluorescence signals was also statistically validated after calculating the Z′ value (Table S5, ESI^†). All tested samples shows the Z′ score value more than 0.9, indicating an optimized and validated assay of 5hmC.Quantification of 5hmC in human cancer cell A549

A catalyst- and solvent-free protocol for the sustainable synthesis of fused 4H-pyran derivatives

An efficient and cost-effective method was developed for the synthesis of two kinds of fused 4H-pyran derivatives, namely, dihydropyrano[2,3-c]pyrazole 4 and pyrano[3,2-c]chromenone 6. The reactions of 3-methyl-1-phenyl-5-pyrazolone/4-hydroxycoumarin with aromatic aldehydes and (E)-N-methyl-1-(methylthio)-2-nitroethenamine (NMSM), involving the Knoevenagel, Michael-addition, O-cyclization and elimination reactions under thermal heating, afforded the desired products. The synthesized compounds were characterized by standard spectroscopic techniques. Further, the structures of pyrazole-fused 4H-pyran 4a and coumarin-fused 4H-pyran 6b were confirmed by single-crystal XRD analysis. The short reaction time, good-to-excellent yields, elimination of the use of expensive, metallic and toxic catalysts or hazardous organic solvents and high atom-economy are some noteworthy features of this protocol.

A practical and greener method for the synthesis of highly functionalized pyrazole and coumarin fused 4H-pyran derivatives by exploring NMSM under neat conditions at 110 °C is described.     

Retracted Article: Physics of excitons and their transport in two dimensional transition metal dichalcogenide semiconductors

Two-dimensional (2D) group-VI transition metal dichalcogenide (TMD) semiconductors, such as MoS₂, MoSe₂, WS₂ and others manifest strong light matter coupling and exhibit direct band gaps which lie in the visible and infrared spectral regimes. These properties make them potentially interesting candidates for applications in optics and optoelectronics. The excitons found in these materials are tightly bound and dominate the optical response, even at room temperatures. Large binding energies and unique exciton fine structure make these materials an ideal platform to study exciton behaviors in two-dimensional systems. This review article mainly focuses on studies of mechanisms that control dynamics of excitons in 2D systems – an area where there remains a lack of consensus in spite of extensive research. Firstly, we focus on the kinetics of dark and bright excitons based on a rate equation model and discuss on the role of previous ‘unsuspected’ dark excitons in controlling valley polarization. Intrinsically, dark and bright exciton energy splitting plays a key role in modulating the dynamics. In the second part, we review the excitation energy-dependent possible characteristic relaxation pathways of photoexcited carriers in monolayer and bilayer systems. In the third part, we review the extrinsic factors, in particular the defects that are so prevalent in single layer TMDs, affecting exciton dynamics, transport and non-radiative recombination such as exciton–exciton annihilation. Lastly, the optical response due to pump-induced changes in TMD monolayers have been reviewed using femtosecond pump–probe spectroscopy which facilitates the analysis of underlying physical process just after the excitation.

Two-dimensional (2D) group-VI transition metal dichalcogenide (TMD) semiconductors, such as MoS₂, MoSe₂, WS₂ and others manifest strong light matter coupling and exhibit direct band gaps which lie in the visible and infrared spectral regimes.     

Estimation of Quizalofop Ethyl Residues in Black Gram (Vigna mungo L.) by Gas Liquid Chromatography

Quizalofop ethyl, a phenoxy propionate herbicide is used for post emergence control of annual and perennial grass weeds in broad-leaved crops in India. The experiments were designed to study the harvest time residues of quizalofop ethyl in black gram for two seasons. At harvest time, the residues of quizalofop ethyl on black gram seed, foliage and soil were found to be below the determination limit of 0.01 mg kg^?1 following a single application of the herbicide at 50 and 100 g a.i. ha^?1 for both the periods. Application of the herbicide is quite safe from a consumer and environmental point of view.     

Development and in vitro/in vivo evaluation of controlled release provesicles of a nateglinide–maltodextrin complex

The aim of this study was to characterize the provesicle formulation of nateglinide (NTG) to facilitate the development of a novel controlled release system of NTG with improved efficacy and oral bioavailability compared to the currently marketed NTG formulation (Glinate™ 60). NTG provesicles were prepared by a slurry method using the non-ionic surfactant, Span 60 (SP), and cholesterol (CH) as vesicle forming agents and maltodextrin as a coated carrier. Multilamellar niosomes with narrow size distribution were shown to be successfully prepared by means of dynamic laser scattering (DLS) and field emission scanning electron microscopy (FESEM). The absence of drug-excipient interactions was confirmed by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) studies. In vitro release of NTG in different dissolution media was improved compared to pure drug. A goat intestinal permeation study revealed that the provesicular formulation (F4) with an SP:CH ratio of 5:5 gave higher cumulative amount of drug permeated at 48 h compared to Glinate™ 60 and control. A pharmacodynamic study in streptozotocin-induced diabetic rats confirmed that formulation F4 significantly (P<0.05) reduced blood glucose levels in comparison to Glinate 60. Overall the results show that controlled release NTG provesicles offer a useful and promising oral delivery system for the treatment of type II diabetes.KEY WORDS: Provesicles, Niosomes, Maltodextrin, Nateglinide, In vitro release, Goat intestinal permeation, Hypoglycemic     

Mid Versus Late Secondary Alveolar Cleft Grafting Using Iliac Crest Corticocancellous Bone Graft

Background

Mid-secondary alveolar cleft repair performed at ages 9–12, in the mixed dentition stage, prior to eruption of the permanent canine, is generally accepted as the ideal time for residual alveolar cleft closure in cleft lip and palate cases with a cleft alveolus.

Methods

In our study, four cases of mid-secondary and five cases of late–secondary alveolar cleft grafting were carried out using iliac crest corticocancellous bone graft. Clinical defect closure and radiographic bone fill were compared.

Results

All the nine cases performed in the two different age groups showed excellent results, clinically, with complete closure of the cleft defect and achievement of continuity of the dental arches. One case was planned for a two-stage procedure owing to the large bilateral maxillary defects. Good bone fill was visualized radiographically in all nine cases.

Conclusion

Precise timing for undertaking alveolar cleft repair may not be all that crucial for a successful alveolar cleft grafting procedure.     

A Review on Basic Biology of Bacterial Biofilm Infections and Their Treatments by Nanotechnology-Based Approaches

Biofilms are responsible for causing 80% of human infections including chronic infections like-cystic fibrosis, endocarditis and osteomyelitis. The growing ability of the biofilm to resist most of the available antibiotics has caused a serious threat to different life forms. Plenty of research work has already been reported, and some are ongoing to combat this serious health issue worldwide. Recent developments in nanotechnology have given a great boost in dealing biofilm infections. The unique size-dependent properties for antibacterial and antibiofilm activities provide the nanoparticles better options to eradicate biofilms. Here, the authors have discussed the basic biology of bacterial biofilm and their impact on human health. In addition, different nanotechnology-based strategies to overcome serious health issues caused by biofilm infections have been highlighted.