Design and synthesis of fluorescent probes

The fundamental objective of this project is to design, synthesize, and characterize fluorescent dyes, which may be utilized in super resolution imaging techniques. In Chapters 1, 2 and 3, we concentrated on photoswitchable rhodamine dyes. We synthesized several rhodamine dyes and increased their water solubility, installed a bioconjugation unit and, more importantly, we optimized the absorption properties (close to 400 nm) of the rhodamine spirolactams in their closed state and studied their basic photophysical properties as well. In Chapter 4, we synthesized azido-DCDHF fluorogens that can be converted to the bright state after a 1,3-dipolar cycloaddition reaction between an azide-Ph-DCDHF and a strained alkene. We synthesized some strained alkenes, which may speed up the kinetics in 1,3-dipolar cycloaddition. This chemical method of turning the dyes from dark to bright state is a new dimension in the bioconjugation arena. In Chapter 5, we synthesized Nile red derivatives which can switch to a bright state from a dark state by collision on the cell surface utilizing PAINT methodology. We expected that the design of new Nile red derivatives may have better properties than the parent Nile red. Besides the PAINT technique, we worked on some active control of emission by enzymatic cleavage of fluorescent dyes in a dark state to the bright state, which can be utilized in super resolution imaging. Related to the 1,3-dipolar cycloaddition reaction between azido-DCDHF and norbornene, we have examined recently popularized tetrazine chemistry. We linked pyridyl tetrazines to DCDHF with short spacer. In Chapter 6, we describe the preparation of co-crystals between perfluorophenazine and several polynuclear aromatic compounds/polynuclear heteroaromatic compounds. In Chapter 7 we describe the preparation of some partially fluorinated heteropolynuclear aromatic compounds such phenzaine and acridine class of compounds for possible use in organic semiconductors

Genetic variability, heritability and genetic advance for growth, yield and yield related traits in maize genotypes

Evaluation of the genetic variability, heritability and genetic advance of traits is an essential task in any plant improvement program. Twenty maize genotypes were replicated twice in a randomized complete block design on a research plot of Prithu Technical College, Deukhuri Dang of Nepal from June 2017 to September 2017 to determine genetic variability, heritability, and genetic advance for different agronomic traits. Analysis of variance showed significant differences in the traits tassel length, ear height, days to fifty percent tasseling, days to fifty percent silking, kernels’ rows ear-1, kernels row-1 and grain yield. The highest GCV (31.53%) and PCV (39.20%) were recorded on grain yield. Grain yield and ear height recorded high heritability along with high genetic advance as a percent of mean (GAM). Tassel length and kernels row-1 showed high heritability integrated with moderate GAM and moderate heritability integrated with moderate GAM respectively. Further, grain yield showed a significant and positive correlation with plant height, tassel length, ear height, cob length, cob diameter, kernels’ rows ear-1, and kernels row-1. Thus the selection of ear height, tassel length and kernels row-1 is suggested as they performed better in terms of both heritability and GAM than other traits and they also recorded a significant and positive correlation with yield

Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photo­controllable small-molecule rhod­amine spiro­lactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhod­amine spiro­lactams photo­switch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photo­switching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spiro­lactam. Further, an <i>N</i>-hydroxy­succinimide-functionalized derivative enabled covalent labeling of amines on the surface of live <i>Caulobacter crescentus</i> cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in <i>x</i>, <i>y</i>, and <i>z</i>. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhod­amine spiro­lactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10–20 nm

Small-Molecule Labeling of Live Cell Surfaces for Three-Dimensional Super-Resolution Microscopy

Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photo­controllable small-molecule rhod­amine spiro­lactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhod­amine spiro­lactams photo­switch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photo­switching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spiro­lactam. Further, an <i>N</i>-hydroxy­succinimide-functionalized derivative enabled covalent labeling of amines on the surface of live <i>Caulobacter crescentus</i> cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in <i>x</i>, <i>y</i>, and <i>z</i>. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhod­amine spiro­lactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10–20 nm

Synthesis and properties of hydroxy tail-terminated cyanobiphenyl liquid crystals

<p>Two series of new hydroxy tail-terminated cyanobiphenyl compounds are described. The 4′-ω-hydroxyalkynyl-4-cyanobiphenyl compounds (<b>1a</b>–<b>1g</b>) were synthesised as the key intermediates to the 4′-?ω-hydroxyalkyl-4-cyanobiphenyl compounds (<b>2a</b>–<b>2g</b>) obtained upon reduction of the acetylenes. Many of these ω-hydroxyalkynyl and ω-hydroxyalkyl cyanobiphenyl compounds exhibit nematic mesophases and they also serve as precursors for the synthesis of other interesting materials. Using density functional theory, we calculate the dipole moment of all relevant ω-hydroxyalkynyl and ω-hydroxyalkyl cyanobiphenyl compounds and find a correlation between the calculated dipole moments and measured crystalline to nematic or isotropic liquid transition temperatures.</p

Design of Chemoresponsive Liquid Crystals through Integration of Computational Chemistry and Experimental Studies

We report the use of computational chemistry methods to design a chemically responsive liquid crystal (LC). Specifically, we used electronic structure calculations to model the binding of nitrile-containing mesogens (4′-<i>n</i>-pentyl-4-biphenylcarbonitrile) to metal perchlorate salts (with explicit description of the perchlorate anion), which we call the coordinately saturated anion model (CSAM). The model results were validated against experimental data. We then used the CSAM to predict that selective fluorination can reduce the strength of binding of nitrile-containing nematic LCs to metal-salt-decorated surfaces and thus generate a faster reordering of the LC in response to competitive binding of dimethylmethylphosphonate (DMMP). We tested this prediction via synthesis of fluorinated compounds 3-fluoro-4′-pentyl­[1,1′-biphenyl]-4-carbonitrile and 4-fluoro-4′-pentyl-1,1′-biphenyl, and subsequent experimental measurements of the orientational response of LCs containing these compounds to DMMP. These experimental measurements confirmed the theoretical predictions, thus providing the first demonstration of a chemoresponsive LC system designed from computational chemistry