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Synthesis and in vitro applications of fluorescent imaging agentsBrunet, Aurelie Claude Laure January 2014 (has links)
Fluorescent imaging technologies that offer new ways to visualise and quantify fluorescently labelled molecules are increasing, necessitating the development of fluorescent molecules that can efficiently and specifically label targets in vitro and in vivo. The first aim of this thesis was the study of human neutrophil elastase. Human neutrophil elastase is an important enzyme in the regulation of inflammation but if over expressed can become part of the cause of inflammation itself. To elucidate this dual function and have a greater understanding of this enzyme, an imaging probe for neutrophil elastase was designed. Firstly, the syntheses of fluorescently labelled three branched dendron core structures were optimised, and studied in neutrophils. The selected core structure was functionalised with an elastase specific peptide sequence and fluorescently labelled. The probe was specifically cleaved by neutrophil elastase in an enzymatic assay and in the presence of activated neutrophils (Chapter 1). Fluorescein and rhodamine are dyes that are readily available, are affordable and have convenient wavelengths for microscopy and flow cytometry. Carboxyfluorescein diacetate N-succinimidyl ester (CFDA-SE) is a commonly used fluorescein derivative, widely used in cell proliferation assay. It is mainly used as a mixture of isomers and its synthesis is not reported. Herein a short and simple synthesis of the two individual isomers of carboxyfluorescein diacetate N-succinimidyl ester as well as the equivalent rhodamine variation (carboxytetraethylrhodamine N-succinimidyl ester) is reported (Chapter 2). The labelling properties of these probes were studied in proliferation assays on mouse and human T lymphocytes. Finally, the nuclear penetration of the dendron structure combined with nuclear localisation sequences (NLS) was investigated. Attachment of nuclear localisation sequences to the probe in the presence of fluorescein demonstrated successful entry into the nucleus in human alveolar adenocarcinoma cell line (A549) (Chapter 3).
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Synthesis, characterization and capillary electrophoretic use of new, single-isomer hexasulfated alpha-cyclodextrinsLi, Shulan 29 August 2005 (has links)
The first three, pure, single-isomer, 6-O-sulfo a-cyclodextrins, the sodium salts of hexakis(6-O-sulfo)-a-CD (HxS), hexakis(2,3-di-O-methyl-6-O-sulfo)-a-cyclodextrin (HxDMS) and hexakis(2,3-di-O-acetyl-6-O-sulfo)-a-cyclodextrin (HxDAS) have been synthesized, analytically characterized and utilized as chiral resolving agents in capillary electrophoresis. The purity of each synthetic intermediate and of the final product was determined by HPLC-ELSD and indirect UV-detection capillary electrophoresis. The structural identity of each intermediate and final product was verified by 1D and 2D NMR, and mass spectrometry.HxS, HxDMS and HxDAS have been used to separate a series of neutral, basic, ampholytic and acidic enantiomers in pH 2.5 and pH 9.5 aqueous and acidic methanol background electrolytes using capillary electrophoresis. Rapid separations with satisfactory peak resolution values were obtained for most of the analytes, indicating that HxS, HxDAS and HxDMS can serve as chiral resolving agent for a wide range of analytes. The observed separation patterns follow the predictions of the CHArged Resolving agent Migration (CHARM) model. The separation patterns observed with HxS, HxDAS and HxDMS as chiral resolving agent were compared with those of (1) b-cyclodextrin analogues, such as, heptakis(6-O-sulfo)-b-cyclodextrin (HS), heptakis(2,3-di-O-acetyl-6-O-sulfo)-b-cyclodextrin (HDAS) and heptakis(2,3-di-O-methyl-6-O-sulfo)-b-cyclodextrin (HDMS); (2) g-cyclodextrin analogues, such as, octakis(6-O-sulfo)-g-cyclodextrin (OS), octakis(2,3-di-O-acetyl-6-Osulfo)- g-cyclodextrin (ODAS) and octakis(2,3-di-O-methyl-6-O-sulfo)-g-cyclodextrin (ODMS). The effects of the structure of the analytes, and those of the pH and the solvent of the background electrolyte were also studied.
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Synthesis of Heptakis-2-O-Sulfo-Cyclomaltoheptaose, a Single-Isomer Chiral Resolving Agent for Enantiomer Separations in Capillary ElectrophoresisTutu, Edward 2010 December 1900 (has links)
Single-isomer sulfated cyclodextrins (SISCDs) have proven to be reliable,
effective, robust means for separation of enantiomers by capillary electrophoresis (CE).
SISCD derivatives used as chiral resolving agents in CE can carry the sulfo groups either
at the C2, C3 or C6 positions of the glucopyranose subunits which provides varied
intermolecular interactions to bring about favorable enantioselectivities.
The first single-isomer, sulfated β-CD that carries the sulfo group at the C2
position, the sodium salt of heptakis(2-O-sulfo-3-O-methyl-6-Oacetyl)
cyclomaltoheptaose (HAMS) has been synthesized. The purity of each synthetic
intermediate and of the final product was determined by HILIC and reversed phase
HPLC. The structural identity of each intermediate and the final product was verified by
1D, and 2D NMR, and MALDI-TOF mass spectrometry.
HAMS has been used as chiral resolving agent for the CE separation of a set of
nonionic, weak base and strong acid enantiomers in pH 2.5 background electrolytes.
Rapid separations with satisfactory peak resolution values were obtained for the enantiomers of most of the nonionic and weak base analytes. Typically, low
concentrations of HAMS were required to effect good enantiomer resolution.
The trends in the effective mobilities and separation selectivities as a function of
HAMS concentrations followed the predictions of the ionic strength-corrected charged
resolving agent migration model (CHARM model). HAMS showed poor complexation
with the anionic strong electrolyte enantiomers for which no peak resolution was
observed. The separation patterns observed with HAMS as chiral resolving agent were
compared with those of other β-cyclodextrin analogues, including heptakis(2-O-methyl-
3-O-acetyl-6-O-sulfo)-b-cyclodextrin (HMAS), heptakis(2-O-methyl-3,6-di-O-sulfo)-b-
cyclodextrin (HMdiSu), heptakis(2,3-di-O-acetyl-6-O-sulfo)-b-cyclodextrin (HDAS)
and heptakis(2,3-di-O-methyl-6-O-sulfo)-b-cyclodextrin (HDMS).
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Synthesis, characterization and capillary electrophoretic use of new, single-isomer hexasulfated alpha-cyclodextrinsLi, Shulan 29 August 2005 (has links)
The first three, pure, single-isomer, 6-O-sulfo a-cyclodextrins, the sodium salts of hexakis(6-O-sulfo)-a-CD (HxS), hexakis(2,3-di-O-methyl-6-O-sulfo)-a-cyclodextrin (HxDMS) and hexakis(2,3-di-O-acetyl-6-O-sulfo)-a-cyclodextrin (HxDAS) have been synthesized, analytically characterized and utilized as chiral resolving agents in capillary electrophoresis. The purity of each synthetic intermediate and of the final product was determined by HPLC-ELSD and indirect UV-detection capillary electrophoresis. The structural identity of each intermediate and final product was verified by 1D and 2D NMR, and mass spectrometry.HxS, HxDMS and HxDAS have been used to separate a series of neutral, basic, ampholytic and acidic enantiomers in pH 2.5 and pH 9.5 aqueous and acidic methanol background electrolytes using capillary electrophoresis. Rapid separations with satisfactory peak resolution values were obtained for most of the analytes, indicating that HxS, HxDAS and HxDMS can serve as chiral resolving agent for a wide range of analytes. The observed separation patterns follow the predictions of the CHArged Resolving agent Migration (CHARM) model. The separation patterns observed with HxS, HxDAS and HxDMS as chiral resolving agent were compared with those of (1) b-cyclodextrin analogues, such as, heptakis(6-O-sulfo)-b-cyclodextrin (HS), heptakis(2,3-di-O-acetyl-6-O-sulfo)-b-cyclodextrin (HDAS) and heptakis(2,3-di-O-methyl-6-O-sulfo)-b-cyclodextrin (HDMS); (2) g-cyclodextrin analogues, such as, octakis(6-O-sulfo)-g-cyclodextrin (OS), octakis(2,3-di-O-acetyl-6-Osulfo)- g-cyclodextrin (ODAS) and octakis(2,3-di-O-methyl-6-O-sulfo)-g-cyclodextrin (ODMS). The effects of the structure of the analytes, and those of the pH and the solvent of the background electrolyte were also studied.
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Two new, single-isomer, sulfated β-cyclodextrins for use as chiral resolving agents for enantiomer separations in capillary electrophoresisBusby, Michael Brent 16 August 2006 (has links)
Two novel, single-isomer, sulfated cyclodextrins, the sodium salts of heptakis(2-
O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose (HMAS) and heptakis(2-O-methyl-
6-O-sulfo)cyclomaltoheptaose (HMS) were used as chiral resolving agents in both
aqueous and non-aqueous electrophoretic separation of a set of pharmaceutically active
weak base enantiomers. Enantiomers of twenty one of the twenty four weak bases were
baseline resolved in one or more of the background electrolytes (BGE’s) used.
An eight-step synthetic method was used to produce, on a large scale, the title
compounds in greater than 97% purity. The purity of the synthetic intermediates and the
final products were characterized by HPLC-ELSD and indirect UV-detection capillary
electrophoresis (CE), respectively. X-ray crystallography, MALDI-TOF mass
spectrometry and 1H as well as 13C NMR spectroscopy allowed for unambiguous
characterization of the structure of each intermediate and the final product.
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