Intramolecular cyclization of isothiocyanyl amino acids/peptide: arrival at unnatural thioxoimidazolidinyl/thioxooxazolidinyl amino acids.

A novel intramolecular cyclization of isothiocyanyl amino acids/peptide is reported to arrive at unnatural thioxoimidazolidinyl (TOI)/thioxooxazolidinyl (TOO) amino acids for the first time. Interestingly, analogous isothiocyanyl amines under a similar reaction condition either follow 5-endo-dig cyclization to offer 5-membered thiourea or acyclic diethylaminyl thiourea derivative instead of 6-membered cyclic thiourea.

Design of "Click" Fluorescent Labeled 2'-deoxyuridines via C5-[4-(2-Propynyl(methyl)amino)]phenyl Acetylene as a Universal Linker: Synthesis, Photophysical Properties, and Interaction with BSA.

Microenvironment-sensitive fluorescent nucleosides present attractive advantages over single-emitting dyes for sensing inter-biomolecular interactions involving DNA. Herein, we report the rational design and synthesis of triazolyl push-pull fluorophore-labeled uridines via the intermediacy of C5-[4-(2-propynyl(methyl)amino)]phenyl acetylene as a universal linker. The synthesized nucleosides showed interesting solvatochromic characteristic and/or intramolecular charge transfer (ICT) features. A few of them also exhibited dual-emitting characteristics evidencing our designing concept. The HOMO-LUMO distribution showed that the emissive states of these nucleosides were characterized with more significant electron redistribution between the C5-[4-(2-propynyl(methyl)amino)]phenyl triazolyl donor moiety and the aromatic chromophores linked to it, leading to modulated emission property. The solvent polarity sensitivity of these nucleosides was also tested. The synthesized triazolyl benzonitrile (10C), naphthyl (10E), and pyrenyl (10G) nucleosides were found to exhibit interesting ICT and dual (LE/ICT) emission properties. The dual-emitting pyrenyl nucleoside maintained a good ratiometric response in the BSA protein microenvironment, enabling the switch-on ratiometric sensing of BSA as the only protein biomolecule. Thus, it is expected that the new fluorescent nucleoside analogues would be useful in designing DNA probes for nucleic acid analysis or studying DNA-protein interactions via a drastic change in fluorescence response due to a change in micropolarity.

Multipurpose isothiocyanyl alanine/lysine: Use as solvatochromic IR probes and in site specific labeling/ligation of short peptides.

The solvatochromic IR responsivity of small side chain -NCS in two unexplored unnatural amino acids, isothiocyanyl alanine (NCSAla = Ita) and lysine (NCSLys = Itl), without perturbing the conformation is demonstrated in two designed short tripeptide (BocAla-NCSAla-Ala-OMe) and hexapeptide (BocLeu-Val-Phe-Phe-NCSLys-Gly-OMe). Demonstration of site specific fluorescent labeling in both the peptides and ligation type reaction in NCSLys indicates the novelty of these two amino acids as alternative to the available canonical amino acids.

Isothiocyanyl Alanine as a Synthetic Intermediate for the Synthesis of Thioureayl Alanines and Subsequent Aminotetrazolyl Alanines.

The synthesis of unnatural amino acids with small side-chain functionalities usable for further transformations is highly demanding for the expansion of the genetic code and other possible biotechnological applications. To this end, we wanted to report the utility of an unexplored unnatural amino acid, isothiocyanyl alanine (NCSAla = Ita), for the synthesis of another class of unnatural amino acids, thioureayl alanines (TUAla = Tua). The synthesis of a third class of unnatural amino acids, amino tetrazolyl alanines (ATzAla = Ata), in a very good yield was subsequently achieved utilizing thioureayl alanines. Thus, a variety of aliphatic- and aromatic-substituted thioureayl alanines and aromatic-substituted amino tetrazolyl alanines were successfully synthesized in good to excellent yields. The photophysical properties of three of the fluorescent unnatural amino acids from two classes were also studied and presented herein.

Uracil-amino acid as a scaffold for ß-sheet peptidomimetics: Study of photophysics and interaction with BSA protein.

We report herein the uracil-di-aza-amino acid (UrAA) as a new family of molecular scaffold to induce ß-hairpin structure with H-bonded ß-sheet conformation in a short peptide. This has been demonstrated in two conceptual fluorescent pentapeptides wherein triazolylpyrenyl alanine and/or triazolylmethoxynapthyl alanine (TPyAlaDo and/or TMNapAlaDo) are embedded into two arms of the uracil-amino acid via an intervening leucine. Conformational analysis by CD, IR, variable temperature and 2D NMR spectroscopy reveals the ß-hairpin structures for both the peptides. Study of photophysical property reveals that the pentapeptide containing fluorescent triazolyl unnatural amino acids TMNapAlaDo and TPyAlaDo at the two termini exhibits dual path entry to exciplex emission-either via FRET from TMNapAlaDo to TPyAlaDo or via direct excitation of a FRET acceptor, TPyAlaDo. The other pentapeptide with TPyAlaDo/TPyAlaDo pair shows excimer emission. Furthermore, both the peptides maintaining their fundamental photophysics are found to interact with BSA as only a test biomolecule.

Tetrazolylpyrene unnatural nucleoside as a human telomeric multimeric G-quadruplex selective switch-on fluorescent sensor.

We report herein the specific sensing of dimeric H45 G-quadruplex DNA via a fluorescence light-up response using fluorescent tetrazolylpyrene nucleoside (TzPyBDo) as a probe. The strong binding of the probe via an intercalative stacking interaction inside the connecting loop of two G-quadruplex units of H45 and the discrimination to other monomeric and long DNA duplexes are accompanied by a drastic enhancement of the emission intensity without compromising the conformation and stability.

Mechanistic Studies on Garratt-Braverman Cyclization: The Diradical-Cycloaddition Puzzle.

In this work, we present the results of extensive multiprong studies involving the fate of deuterium-labeled substrates, EPR, trapping experiments, and LA-LDI mass spectrometry to sort out the controversies relating to the mechanism of Garratt-Braverman cyclization in two systems, namely bis-propargyl sulfones and ethers. The results are in conformity with a diradical mechamism for the sulfone, while for the ether, the anionic [4 + 2] appears to be the preferred pathway. This shows that the mechanistic pathway toward GB cyclization is dependent upon the nature of heteroatom (O or S in sulfone) bridging the propargyl arms.

Regioselective and stereoselective route to N2-ß-tetrazolyl unnatural nucleosides via SN2 reaction at the anomeric center of Hoffer's chlorosugar.

We are reporting a regioselective and stereoselective route to N2-ß-tetrazolyl aromatic donor/acceptor unnatural nucleosides as new class of possible DNA base analogs. The SN2 substitution reaction at the anomeric center of Hoffer's chlorosugar with various 5-substituted aromatic tetrazoles in THF in presence of K2CO3 proceeds with regioselectivity at N2-tetrazoles and stereoselectivity at α-chlorosugar with very good yield. The stereoelectronic and steric effects play a crucial role for the observed outcome which is also supported from a theoretical (DFT) study. The methodology is simple, eco-compatible and the tetrazolyl unnatural nucleosides might find applications in decorating DNA for various biotechnological and DNA based material science applications.

Donor/acceptor chromophores-decorated triazolyl unnatural nucleosides: synthesis, photophysical properties and study of interaction with BSA.

Much effort has been put forth to develop unnatural, stable, hydrophobic base pairs with orthogonal recognition properties and study their effect on DNA duplex stabilisation. Our continuous efforts on the design of fluorescent unnatural biomolecular building blocks lead us to the synthesis of some triazolyl donor/acceptor unnatural nucleosides via an azide-alkyne 1,3-dipolar cycloaddition reaction as a key step, which we want to report herein. We have studied their photophysical properties and found interesting solvatochromic fluorescence for two of the nucleosides. Photophysical interactions among two donor-acceptor ß-nucleosides as well as a pair of α/ß-nucleosides have also been evaluated. Furthermore, we have exploited one of the fluorescent nucleosides in studying its interaction with BSA with the help of UV-visible and steady state fluorescence techniques. Our design concept is based on the hypothesis that a pair of such donor/acceptor nucleosides might be involved in π-stacking as well as in photophysical interactions, leading to stabilization of the DNA duplex if such nucleosides can be incorporated into short oligonucleotide sequences. Therefore, the designed bases may find application in biophysical studies in the context of DNA.

Synthesis, photophysical properties of triazolyl-donor/acceptor chromophores decorated unnatural amino acids: Incorporation of a pair into Leu-enkephalin peptide and application of triazolylperylene amino acid in sensing BSA.

The research in the field of design and synthesis of unnatural amino acids is growing at a fast space for the increasing demand of proteins of potential therapeutics and many other diversified novel functional applications. Thus, we report herein the design and synthesis of microenvironment sensitive fluorescent triazolyl unnatural amino acids (UNAA) decorated with donor and/or acceptor aromatic chromophores via click chemistry. The synthesized fluorescent amino acids show interesting solvatochromic characteristic and/or intramolecular charge transfer (ICT) feature as is revealed from the UV-visible, fluorescence photophysical properties and DFT/TDDFT calculation. HOMO-LUMO distribution shows that the emissive states of some of the amino acids are characterized with more significant electron redistribution between the triazolyl moiety and the aromatic chromophores linked to it leading to modulated emission property. A pair of donor-acceptor amino acid shows interesting photophysical interaction property indicating a FRET quenching event. Furthermore, one of the amino acid, triazolyl-perylene amino acid, has been exploited for studying interaction with BSA and found that it is able to sense BSA with an enhancement of fluorescence intensity. Finally, we incorporated a pair of donor/acceptor amino acids into a Leu-enkephalin analogue pentapeptide which was found to adopt predominantly type II ß-turn conformation. We envisage that our investigation is of importance for the development of new fluorescent donor-acceptor unnatural amino acids a pair of which can be exploited for generating fluorescent peptidomimetic probe of interesting photophysical property for applications in studying peptide-protein interaction.

Wavelength shifting oligonucleotide probe for the detection of adenosine of a target DNA with enhanced fluorescence signal.

The modulated photophysical property of strong electronically coupled naphthyl uridine linked via a single C-C bond was explored in DNA detection via wavelength shifting and enhanced fluorescence emission by a simple 'Just-Mix & Read' strategy of homogeneous DNA detection.

Precise and rapid point-of-care quantification of albumin levels in unspiked blood using organic field-effect transistors.

Nanowire-based field-effect transistors (FETs) are widely used to detect biomolecules precisely. However, the fabrication of such devices involves complex integration procedures of nanowires into the device and most are not easily scalable. In this work, we report a straightforward fabrication approach that utilizes the grain boundaries of the semiconducting film of organic FETs to fabricate biosensors for the detection of human serum albumin (HSA) with an enhanced sensitivity and detection range. We used trichromophoric pentapeptide (TPyAlaDo-Leu-ArTAA-Leu-TPyAlaDo, TPP) as a receptor molecule to precisely estimate the concentration of HSA protein in human blood. Bi-layer semiconductors (pentacene and TPP) were used to fabricate the OFET, where the pentacene molecule acted as a conducting channel and TPP acted as a receptor molecule. This approach of engineering the diffusion of receptor molecules into the grain boundaries is crucial in developing OFET-based HSA protein sensors, which cover a considerable detection range from 1 pM to 1 mM in a single device. The point-of-care detection in unspiked blood samples was confirmed at 4.2 g dL-1, which is similar to 4.1 g dL-1 measured using a pathological procedure.

Triazolyl donor/acceptor chromophore decorated unnatural nucleosides and oligonucleotides with duplex stability comparable to that of a natural adenine/thymine pair.

We report the design and synthesis of triazolyl donor/acceptor unnatural nucleosides via click chemistry and studies on the duplex stabilization of DNA containing two such new nucleosides. The observed duplex stabilization among the self-pair/heteropair has been found to be comparable to that of a natural A/T pair. Our observations on the comparable duplex stabilization has been explained on the basis of possible π-π stacking and/or charge transfer interactions between the pairing partners. The evidence of ground-state charge transfer complexation came from the UV-vis spectra and the static quenching of fluorescence in a heteropair. We have also exploited one of our unnatural DNAs in stabilizing abasic DNA.

Highly solvatochromic fluorescent naphthalimides: design, synthesis, photophysical properties and fluorescence switch-on sensing of ct-DNA.

We report the design, synthesis and photophysical properties of highly solvatochromic donor/acceptor substituted naphthalimide based fluorophores. The synthesized naphthalimides containing propargyl ends showed highly solvatochromic intramolecular charge transfer (ICT) feature as was revealed from the UV-visible, fluorescence photophysical properties of these fluorophores and DFT/TDDFT calculation. Fluorescence life times for the imide fluorophores were also measured in different solvents. The solid state photophysical property of donor substituted naphthalimide 1 showed promising for future application in material sciences. Furthermore, both the donor/acceptor substituted naphthalimide fluorophores 1-2 were exploited in sensing calf-thymus DNA via switch-on fluorescence response. The propargyl linker containing naphthalimides can further be exploited for the synthesis of labeled biomolecular building blocks.

Sensing of micellar microenvironment with dual fluorescent probe, triazolylpyrene (TNDMBPy).

We report a dual fluorescent triazolylpyrene ((TNDMB) Py) as an efficient fluorescent light-up probe of various micellar microenvironments. The absorption spectra of (TNDMB) Py in an aqueous solution of varying surfactant concentration, CTAB, SDS and TX-100 showed that as the surfactant concentration was increased the absorbance increased with no shift in wavelength maxima. The increase of absorbance in each surfactant solution with increase in surfactant concentration was due to the enhanced solubilization of (TNDMB) Py in surfactant solutions. Our investigations based on steady state and time resolved fluorescence techniques showed that the probe reports the microenvironment of ionic surfactant solutions (CTAB and SDS) via dual emission (LE and ICT) at low surfactant concentration. The ICT band showed a blue shifting pattern with enhanced intensity that disappeared as the concentration of surfactant increases (> 1 mM for CTAB and > 3 mM for SDS). In non-ionic surfactant (Triton X-100) solution, the fluorophore showed dual emission with dominant ICT behaviour over LE emission at low concentration (up to 0.35 mM). In reverse micelle we observed a blue shifted ICT band with no LE band with increasing molar concentration of water. We found 100 nm blue shifting when we moved from R = 0 to R = 7, where R is the molar ratio of water to TX-100 (R = [H2O]/[TX-100]). The blue shifting of ICT band is because of the movement of the probe from hydrophilic core to hydrophobic core (surface) of the reverse micelle. Thus from the steady-state fluorescence study it was observed that the ICT band of the probe, (TNDMB) Py was more influenced by the micellar environment in comparison to the LE band. This difference in behaviour of the fluorophore is probably because of varying extent of hydrophobic/hydrogen bonding interactions experienced by the probe and its relative disposition inside the various micellar nanocores.

Targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) with synthetic/designer unnatural nucleoside analogs: an in silico study.

CONTEXT: Since the outbreak of COVID-19 in December 2019, it developed into a pandemic affecting all the countries and millions of people around the globe. Until now, there is no medicine available to contain the spread of the virus. As an aid to drug discovery, the molecular docking and molecular dynamic tools were applied extensively. In silico studies made it possible for rapid screening of potential molecules as possible inhibitors/drugs against the targeted proteins. As a continuation of our drug discovery research, we have carried out molecular docking studies of our 12 reported unnatural nucleosides and 14 designer Avigan analogs with SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), which we want to report herein. The same calculation was also carried out, taking 11 known/under trail/commercial nucleoside drug molecules for a comparison of the binding interactions in the catalytic site of RdRp. The docking results and binding efficiencies of our reported nucleosides and designer nucleosidic were compared with the binding energy of commercially available drugs such as remdesevir and favipiravir. Furthermore, we evaluated the protein-drug binding efficiency and stability of the best docked molecules by molecular dynamic studies (MD). From our study, we have found that few of our proposed drugs show promising binding efficiency at the catalytic pocket of SARS-CoV-2 RdRp and can be a promising RdRp inhibitor drug candidate. Hence, this study will be of importance to make progress toward developing successful nucleoside-based drugs and conduct the antiviral test in the wet lab to understand their efficacy against COVID-19. METHOD: All the docking studies were carried out with AutoDock 4.2, AutoDock Vina and Molegro Virtual Docker. Following the docking studies, the MD simulations were carried out following the standard protocol with the GROMACS ver. 2019.6. by applying the CHARMM36 all-atom biomolecular force field. The drug-protein interaction was studied using the Biovia Discovery Studio suite, Ligplot software, and Protein-Ligand Interaction Profiler (PLIP).

Installation/modulation of the emission response via click reaction.

We have demonstrated the installation of a fluorescence property into a nonfluorescent precursor and modulation of an emission response of a pyrene fluorophore via click reaction. The synthesized fluorophores show different solvatochromicity and/or intramolecular charge transfer (ICT) feature as is revealed from the UV-visible, fluorescence photophysical properties of these fluorophores, and DFT/TDDFT calculation. We observed that some of the synthesized fluorophores showed purely ICT character while emission from some of them arose from the LE state. A structureless and solvent polarity-sensitive dual emission behavior was observed for one of the triazolylpyrene fluorophores that contains an electron-donating -NMe(2) substituent (fluorophore, 7a). Conversely, triazolylpyrene with an electron-withdrawing -CN group (fluorophore, 7b) showed a solvent polarity-independent vibronic emission. The effect of ICT on the photophysical properties of these fluorophores was studied by fluorescence emission spectra and DFT/TDDFT calculations. Fluorescence lifetimes were also measured in different solvents. All of our findings revealed the delicate interplay of structure and emission properties and thus having broader general utility. As the CT to LE intensity ratio can be employed as a sensing index, the dual emissive fluorophore can be utilized in designing the molecular recognition system too. We envisage that our investigation is of importance for the development of new fluorophores with predetermined photophysical properties that may find a wide range of applications in chemistry, biology, and material sciences.

Click-reagent version of Sonogashira coupling protocol to conjugated fluorescent alkynes with no or reduced homocoupling.

A click-reagent version of the Sonogashira-coupling protocol has been developed. Diarylalkynes with donor and/or acceptor substituents have been synthesized via this protocol at moderate to excellent yields and with no or drastically reduced quantities of undesired homocoupled side products. This protocol is green-solvent compatible, air-insensitive, and effective under microwave conditions.

Bio-inspired PtNPs/Graphene nanocomposite based electrocatalytic sensing of metabolites of dipyrone.

Noble metal nanoparticles are known to electrocatalyze various redox reactions by improving the electron transfer kinetics. In the present study, we have introduced a facile bioinspired synthesis of PtNPs and their integration for the formation of PtNPs/graphene nanocomposite using Psidium guajava (guava) leaves extract. Graphene used in nanocomposite formulation was synthesized by exfoliation of graphite in water/acetone (25:75 v/v) mixture followed by mechanical shearing using ultrasonication and microwave irradiation. PtNPs/graphene nanocomposite was drop-cast onto a glassy carbon electrode (GCE, 3 mm dia). The electrocatalytic activity of PtNPs/graphene nanocomposite was tested in a three-electrode system for sensing of metabolic products of dipyrone (DIP) formed through 1 e- and 2 e- transfer reactions. The modified electrode exhibited almost 50% reduction in electrode resistance. The limit of detection was found to be 0.142 µM with sensitivities of 0.820 and 0.445 µA․µM-1cm-2 for DIP concentration below and above 100 µM, respectively, using square wave voltammetry. The signal of sensing of metabolites of DIP was almost invariant in the presence of glucose, dopamine, uric acid, and ciprofloxacin; however, the response current was decayed by 20% within the 10th cycle. The sensing of DIP spiked in treated sewage-water and running tap-water samples was ∼100% recoverable and comparable with HPLC.

Singly and doubly labeled base-discriminating fluorescent oligonucleotide probes containing oxo-pyrene chromophore.

We have developed new oxo-pyrene labeled fluorescent nucleoside, (Oxo-Py)U which showed a strong fluorescence dependency on solvent polarity at long wavelength. The designed singly and doubly (Oxo-Py)U labeled fluorescent oligonucleotide probes were found highly efficient for the discrimination of A and consecutive AA bases of target DNA opposite to the labeled base via generation of enhanced fluorescence signal.