Tuning the Reactivity of a Substrate for SNAP-Tag Expands Its Application for Recognition-Driven DNA-Protein Conjugation

Recognition-driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site-specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA as they consist of both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence-specific reaction. However, combining an MA consisting of a reactive protein-tag and its substrate, for example, SNAP-tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP-tag have been designed to realize sequence-selective rapid crosslinking reactions of MAs with SNAP-tag. The reactions of substrates with SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP-tag against CLIP-tag to achieve orthogonal DNA-protein crosslinking by six unique MAs.     

Towards Photochromic Azobenzene-Based Inhibitors for Tryptophan Synthase

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene-based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto-unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light.     

Stereochemical preference of Candida parapsilosis ATCC 7330 mediated deracemization: E- versus Z-aryl secondary alcohols

The stereochemical preference of the biocatalyst, Candida parapsilosis ATCC 7330, was investigated with respect to the E/Z configuration in the deracemization and the asymmetric reduction of aryl secondary alcohols and prochiral ketones, respectively. The biocatalyst preferred the E-isomers over Z-isomers as substrates as evidenced from the experimental results of >99% ee and up to 86% isolated yield for E-secondary alcohols. The synthesis of enantiomerically pure E-4-phenylbut-3-ene-1,2-diol (ee >99%, isolated yield 86%) by whole cell mediated deracemization is reported here for the first time. The geometric preference of the enzymes was confirmed by using the cell free extract of this biocatalyst. Mechanistic insights using in silico studies showed that the E-isomers when located in the active site are favourably placed with respect to the catalytic triad (Ser-Tyr-Lys) for hydride transfer from NADPH.     

New mesogen with thermotropic cubic phase: 3,4,5‐tris‐(11,11,12,12,13,13,14,14,15,15,16,16,16‐tridecafluorohexadecyloxy)benzoic acid

A new mesogen, 3,4,5‐tris(11,11,12,12,13,13,14,14,15,15,16,16,16‐tridecafluoro‐hexadecyloxy)benzoic acid, with a thermotropic cubic phase was synthesized. The phase behaviour of the fluorinated compound and its corresponding non‐fluorinated analogue was investigated by differential scanning calorimetry, polarizing optical microscopy, and synchrotron small‐angle X‐ray diffraction. The fluorinated compound, which contains three partially perfluorinated alkoxy moieties and one carboxylic acid group capable of forming hydrogen bonding, exhibits a very interesting sequence of isotropic, cubic and hexagonal columnar phases, whereas its non‐fluorinated compound does not have a liquid crystalline phase. The cubic phase exists over a wide temperature range (from ～200°C to ～30°C on cooling) and is hence amenable to various physical measurements and potential applications. The ability to self‐assemble molecules into ordered superstructure via both partially perfluorinated chains and hydrogen bonding provides new insight for the development of novel liquid crystalline materials.     

Bioanalytical method development,validation and quantification of dorsomorphin in rat plasma by LC‐MS/MS

The present investigation describes the development and validation of a sensitive liquid chromatography–mass spectrometry/mass spectrometry (LC‐MS/MS) method for the estimation of dorsomorphin in rat plasma. A sensitive LC‐MS/MS method was developed using multiple reaction monitoring mode, with the transition of m/z (Q1/Q3) 400.2/289.3 for dorsomorphin and m/z (Q1/Q3) 306.2/236.3 for zaleplon. Chromatographic separation was achieved on a reverse phase Agilent XDB C₁₈ column (100 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile and 5 mm ammonium acetate buffer (pH 6.0) 90:10 v/v, at a flow rate of 0.8 mL/min. The effluence was ionized in positive ion mode by electrospray ionization (ESI) and quantitated by mass spectrometry. The retention times of dorsomorphin and internal standard were found to be 2.13 and 1.13 min, respectively. Mean extraction recovery of dorsomorphin and internal standard in rat plasma was above 80%. Dorsomorphin calibration curve in rat plasma was linear (r² ≥ 0.99) ranging from 0.005 to 10 µg/mL. Inter‐day and intra‐day precision and accuracy were found to be within 85–115% (coefficient of variation). This method was successfully applied for evaluation of the oral pharmacokinetic profile of dorsomorphin in male Wistar rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Nonspecific Effect of Double-Stranded (ds) RNA on Prophenoloxidase (proPO) Expression in Penaeus monodo n

RNA interference-mediated silencing is an effective way of controlling white spot syndrome virus (WSSV). However, the effect of RNAi on the innate immune mechanism is not well understood. Prophenoloxidase (proPO) is an important component of the shrimp innate immunity. In the present study, nonspecific effect of two double-stranded (ds)RNA-expressing constructs, one targeting vp28 gene of WSSV (pCMV-VP28-LH) and another targeting green fluorescent protein (GFP) (pCMV-GFP-LH) on proPO2 gene expression, is investigated. mRNA expression levels of proPO2 in hemocytes of DNA construct-injected shrimp were estimated using real-time PCR with elongation factor 1-α as internal control. Empty vector (pcDNA)-injected shrimp were used as experimental control. In pCMV-VP28-LH-injected shrimp, proPO2 showed significant upregulation until 48 h post-injection (p.i.). Similarly, pCMV-GFP-LH-injected animals showed high levels of expression until 72 h p.i. WSSV-challenged animals, compared to pcDNA-injected control group, showed no significant change in expression of the gene until 24 h. However, an increased expression was noticed at 48 h p.i. Our results suggest that neither the plasmids nor the long hairpin RNA expressed by the constructs has any nonspecific silencing effect on the proPO2 expression. On the contrary, the consistent upregulation of proPO2 observed in shrimp injected with dsRNA at early time-points indicates the possibility of nonspecific protection against WSSV infection.     

Pyrene-Hydrazone-π···Hole Coupled Turn-on Fluorescent and Naked- Eye Colorimetric Sensor for Cyanide: Role of Homogeneous π-Hole Dispersion in Anion Selectivity

A pyrene based probe associated with π···hole – hydrazone as one of the recognizing elements is synthesized and its turn in to a selective colorimetric and turn-on fluorescent sensor, (L³) for cyanide anion. This chemo sensor show high selectivity towards cyanide anion through photo electron transfer (PET) mechanism. The binding strength and sensitivity of the chemo sensor L³ towards cyanide are found to be 2.0 X 10⁴, and 4.44 x 10^-4 respectively. We have compared this high selectivity of the receptor towards cyanide, with our previously reported receptors L¹ and L². The detailed UV-Vis, Emission, ¹H-NMR, IR spectroscopic and Molecular Electrostatic Potential (MEP) studies reveals that the homogeneous π···hole dispersion in the aromatic ring governing the selectivity of the receptor towards cyanide anion. Such a positive π···hole homogeneous dispersion is missing in the case of sensor L², instead we have polarized π···hole dispersion towards 2^nd and 4^th position of di-nitrophenyl chromophoric unit in L².

Graphical Abstract

     

Vanadium dodecylamino phosphate: A novel efficient catalyst for synthesis of polyhydroquinolines

A novel vanadium dodecylamino phosphate was synthesised by an instant reaction between phosphoric acid and vanadyl acetylacetonoate using dodecylamine as the structure-directing agent at ambient temperature. The physicochemical characteristics of the material were investigated by a variety of analytical techniques. XRD studies revealed the presence of vanadium phosphate and hydrated vanadium phosphate phases in the framework of the material. The catalytic application of this material toward in the synthesis of polyhydroquinolines via Hantzsch condensation was investigated at ambient temperature. This method affords high yields within short reaction times. The influence of various reaction parameters such as different solvents, catalyst dosage, effect of aldehydes, and reusability was studied and a plausible mechanism proposed.     

Novel preparation method of nanosilver doped sol gel TiO2 photocatalysts for dye pollutant degradation

In the present study, a novel method for the silver nanoparticle incorporation into TiO₂ sol is reported which provides efficient dispersion of inserted metal in the calcined gel. Glucose mediated metal nanoparticle synthesis eliminates the additional steps for the introduction of dopant over the support. Ag nanoparticle acts as competent trapping centers preventing the recombination of electron?Chole pairs. Thus, in spite of the low UV intensity present in the sunlight, considerable enhancement in the catalytic efficiency was observed for the degradation of dye pollutants over nanosilver loaded TiO₂. Transmission electron microscopic images display the well isolated nanoparticles of silver having uniform dimensions of less than 5?nm over the best system, 2?wt% nanosilver loaded TiO₂, which is accountable for its effectiveness as a photocatalyst. Anatase is found to be the photocatalytic active phase, as evident from the X-ray diffraction studies.