Thesis (M.B.A.)--Boston University

Zinc is an important micronutrient but the biological function of its labile form is poorly understood. Zinc selective fluorescence sensors, recognized as the major tool to gain information about the role of zinc in living systems, have been attracting more and more interest. The most promising solution currently being studied comes in the form of ratiometric sensors. Unlike sensors based on the switch-on mechanism, ratiometric sensors determine the free metal concentration directly from the ratio of the emission intensities at two wavelengths. The major restriction on the design of this type of sensor is from the necessity for a spectral-shift upon binding metal ions. To develop novel ratiometric sensors, we have developed designs based on excited-state intramolecular proton transfer (ESIPT). In the absence of ZnII at neutral pH, the 2-(2 -sulfonamidophenyl)benzimidazole family undergoes ESIPT to yield a highly Stokes-shifted emission from the proton-transfer tautomer. Coordination of ZnII inhibits the ESIPT process and yields a significant hypsochromic shift of the fluorescence emission maximum. By implementing structural modifications, we were able to gauge free ZnII concentrations in the millimolar to picomolar range. To tune the peak excitation towards lower energy, a property that is of particular importance in the light of biological applications, we modified the platform molecule with extended pi-conjugation and by substituent engineering. The position of the modification and the nature of the substituents strongly influenced the photophysical properties of the investigated derivatives. Several fluorophores revealed emission ratiometric properties with a large dynamic range combined with a peak absorption beyond 350 nm, rendering these probes promising candidates for applications. To further understand the origin of the substituent effect, we studied five derivatives for the solvatochromic shift analysis and quantum chemical studies. The results showed that the negative solvatochromic shift behavior was most pronounced in protic solvents presumably due to specific hydrogen-bonding interactions. The extrapolated gas-phase emission energies correlated qualitatively with the trends in Stokes shifts, suggesting that solute-solvent interactions do not play a significant role in explaining the divergent emission energy shifts. Detailed quantum chemical calculations not only confirmed the moderately polarized nature of the ESIPT tautomers but also provided a rationale for the observed emission shifts based on the differential change in the HOMO and LUMO energies. This study revealed the great potential of 2-(2 -arylsulfonamidophenyl)- benzimidazoles, such as tunable peak absorption and emission, a very wide dynamic range regarding to zinc binding, very little solvent polarity dependence, and especially, the emission ratiometric property. All these properties make this system a unique candidate to tackle the problems in the research of zinc biology.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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It is the purpose of this investigation to modify existing methods in an attempt to provide a rapid identification of members of the genus Streptomyces.

A new series of fluorescent prorin (promazine-coumarin hybrid) derivatives were synthesized via piperidine catalyzed cyclization with 3-formyl-2-hydroxypromazine and ethyl acetoacetate or Meldrum’s acid. The synthesis of these new compounds focused on the introduction of electron withdrawing carbonyl groups on the 3-position of the coumarin lactone ring in order to increase polarization of the molecule which results in a longer absorbance wavelength by stabilizing intramolecular charge transfer that occurs in the excited state. The 3-acetyl derivative was further subjected to an aldol condensation with either benzaldehyde or cinnamaldehyde in order to further extend the conjugation of the molecule. The four new prorin compounds exhibited excellent photophysical properties and had Stokes shifts ranging from 160-190 nm. These properties are ideal for use as fluorescent probes to detect RNA binding ligands. The optical properties of the new prorin compounds were compared with a previous series of prorins as well as theoretical calculations in order to gain a more comprehensive understanding of how the photophysical properties of the molecule can be tuned by introducing different substituents. Although increasing polarization of the molecule with electron withdrawing substituents does red-shift the absorbance wavelength, it has little effect on the emission wavelength and can actually cause a blue-shift in some cases. It was observed that introducing aromatic substituents has the greatest effect on red-shifting the emission wavelength and increasing Stokes shift.

Chan, Man Hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 141-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

The structural complexity of glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) and their numerous biological roles, brings forth the need to develop new methods, capable of studying GAGs and their interactions with peptides and proteins under native settings. This thesis explores the development of chemical tools to study heparin/HS binding interactions under physiologically relevant conditions using fluorescence. In chapter 2, we designed peptide-based quinolinium probes to study the structural requirements of cationic peptides required for high affinity peptide-heparin interactions. These fluorescent probes enabled the study of peptide-heparin interactions at nM concentrations allowing the calculation of peptide-heparin binding constants. It was observed that peptides with positive charge displayed on one face of an α-helix in a continuous arrangement bound to heparin with the highest affinity and that heparin likely prefers to bind to these peptides while remaining in an extended conformation. In chapter 3, we set out to study an important biological role of HS which involves the binding and sequestering of proteins at the cell surface, facilitating endocytosis. HS has been implicated in the mechanism of cell penetrating peptide (CPP) cell uptake, with different CPPs showing different degrees of HS dependence on uptake as well as different mechanisms of entry. The role of HS in the mechanism of CPP uptake was investigated in chapter 3 using fluorescent peptide-based probes incorporating fluorophore/quencher pairs. These were used to identify and characterize the ability of heparin/HS to bind and cluster with CPPs to form colloidally stable aggregates. It was shown that the CPP Antp formed much more stable clusters with heparin than the TAT peptide despite both peptides having similar binding affinity for a single heparin chain. These findings were used to explain the cell surface HS dependence of Antp on cell uptake via endocytosis in contrast to the low dependance of TAT on HS and its uptake via translocation. A general model relating the ability of a CPP to cluster surface HS to its preferred mechanism of cell entry was proposed. In chapter 4, a strategy to selectively, and site specifically acylate carbohydrate binding proteins was developed using thioester-based affinity conjugates. It was possible to label maltose binding protein, a periplasmic protein, with high yield and selectivity at a single lysine residue proximal to the maltose binding site. Selective protein labeling could be carried out in bacterial cell extracts and in live bacterial cells. This strategy can potentially be applied to develop protein-based carbohydrate biosensors as well as profile carbohydrate binding proteins in biological samples.

The structural complexity of glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) and their numerous biological roles, brings forth the need to develop new methods, capable of studying GAGs and their interactions with peptides and proteins under native settings. This thesis explores the development of chemical tools to study heparin/HS binding interactions under physiologically relevant conditions using fluorescence. In chapter 2, we designed peptide-based quinolinium probes to study the structural requirements of cationic peptides required for high affinity peptide-heparin interactions. These fluorescent probes enabled the study of peptide-heparin interactions at nM concentrations allowing the calculation of peptide-heparin binding constants. It was observed that peptides with positive charge displayed on one face of an α-helix in a continuous arrangement bound to heparin with the highest affinity and that heparin likely prefers to bind to these peptides while remaining in an extended conformation. In chapter 3, we set out to study an important biological role of HS which involves the binding and sequestering of proteins at the cell surface, facilitating endocytosis. HS has been implicated in the mechanism of cell penetrating peptide (CPP) cell uptake, with different CPPs showing different degrees of HS dependence on uptake as well as different mechanisms of entry. The role of HS in the mechanism of CPP uptake was investigated in chapter 3 using fluorescent peptide-based probes incorporating fluorophore/quencher pairs. These were used to identify and characterize the ability of heparin/HS to bind and cluster with CPPs to form colloidally stable aggregates. It was shown that the CPP Antp formed much more stable clusters with heparin than the TAT peptide despite both peptides having similar binding affinity for a single heparin chain. These findings were used to explain the cell surface HS dependence of Antp on cell uptake via endocytosis in contrast to the low dependance of TAT on HS and its uptake via translocation. A general model relating the ability of a CPP to cluster surface HS to its preferred mechanism of cell entry was proposed. In chapter 4, a strategy to selectively, and site specifically acylate carbohydrate binding proteins was developed using thioester-based affinity conjugates. It was possible to label maltose binding protein, a periplasmic protein, with high yield and selectivity at a single lysine residue proximal to the maltose binding site. Selective protein labeling could be carried out in bacterial cell extracts and in live bacterial cells. This strategy can potentially be applied to develop protein-based carbohydrate biosensors as well as profile carbohydrate binding proteins in biological samples.