Topographical constraint is the most powerful approach for the design of bioactive peptides to explore the bioactive conformation of crucial side-chain pharmacophores of amino acid residues in peptide-receptor recognition and signal transduction. Novel topographically constrained amino acids β-isopropylphenylalanine and 2',6'-dimethyl-2,3-methanophenylalanine have been designed and synthesized. Incorporation of the four optically pure β-isopropylphenylalanine stereoisomers into deltorphin I produced four peptide analogues of [β-iPrPhe]Deltorphin I with differentiated bioactivities. The most potent and selective analogue, [(2S,3R)-β-iPrPhe]Deltorphin I showed an IC₅₀ nM binding affinity, and a 29000 fold selectivity for the δ-opioid receptor over the μ opioid receptor. Combined molecular modeling and NMR studies indicated that the (2S,3R)-β-iPrPhe³ residue in the analogue favors the trans rotamer, and can induce the linear peptide to form a low-energy folded conformation which was proposed as the bioactive conformation for the δ-opioid receptor. Coupling four optically pure, conformationally constrained β-methyl-2',6'-dimethyltyrosine (TMT) with L-Tic formed four dipeptide analogues of TMT-L-Tic. The most potent and selective analogue, (2S,3R)-TMT-L-Tic showed 9 nM binding affinity and 4000 fold selectivity to the δ vs μ opioid receptor. The lowest-energy conformation of (2S,3R)-TMT-L-Tic was suggested to be the bioactive one in which TMT side chain is trans and Tic side chain is in a gauche (+) conformation. Bicyclic oxytocin antagonist [dPen¹, cyclo(Glu⁴ Lys⁸)]OT (BC-OT) (pA₂ = 8.10) is an excellent template to examine further topographical ideas. Substitution of Tyr² with the topographically constrained para-methoxy-β-methyl-2',6'-dimethyltyrosine (p-MeOTMT) amino acids produced two very potent antagonists [(2S,3S)-p-MeOTMT²]BC-OT (pA₂ = 8.26) and [(2R,3R)-p-MeOTMT²]BC-OT(pA₂ = 7.80), and two inactive analogues [(2S,3R)-p-MeOTMT²]BC-OT and [(2R,3S)-p-MeOTMT²]BC-OT. These interesting results can be attributed to the biased side-chain conformation, gauche(+) and gauche(-) in (2S,3S)-p-MeOTMT and (2R,3R)-p-MeOTMT respectively, and trans in both (2S,3R)-p-MeOTMT and (2R,3S)-p-MeOTMT residues. Rational design of non-peptide mimetics from peptide leads is still elusive. Based on the δ-opioid selective lead [(2S,3R)-TMT¹]DPDPE and SAR of δ-opioid selective ligands, the first generation of non-peptide mimetics have been designed and synthesized. The new lead SL-3111 showed binding affinity IC₅₀ = 8 nM, and over 2000 fold selectivity for the δ-opioid receptor over the μ receptor.

The function of most proteins is regulated by post-translational modifications, of which phosphorylation in particular has been shown to be ubiquitous and of paramount importance to cell signaling. Two enzyme families, protein kinases and phosphatases, regulate phosphorylation, and aberrant activities of family members have been implicated in many diseases such as cancer and neurological disorders. Thus, understanding the function of these enzymes in living cells is important for understanding their biology and for designing new therapies, but a challenging task due to their highly conserved architecture. The major focus of the dissertation is on the development of a new approach to selectively turn-on multiple specific kinases and/or phosphatases using orthogonal ligands as chemical inducers of dimerization (CIDs). Specific kinases or phosphatases were dissected at particular sites into two inactive fragments or split-proteins. The split fragments are attached to interacting protein pairs of CID systems, such that upon addition of the specific ligand they heterodimerize with subsequent reassembly of the split-protein and concomitant activity. We demonstrated the in vitro and in cellulo feasibility of this approach using three orthogonal CIDs, rapamycin, abscisic acid, and gibberellic acid, to turn-on members of the tyrosine kinase group such as Lyn and Src, and of the tyrosine phosphatase group such as PTP1B and SHP1. We have also developed a new synthetic photocleavable di-trimethoprim CID that allows for ligand-gated turn-on of desired kinases in live cells. The new CID can be cleaved or turned-off by UV irradiation which results in a turn-off of kinase activity. Small molecule controlled split-proteins allow for developing logic gates and we demonstrate that the systems we have developed can be used to construct 7 out of the 10 basic, circuit-type Boolean phosphorylation-based logic gates in living cells. These post-translational logic gates may have interesting applications in synthetic biology. Finally, we present an initial approach to use redesigned kinases and redesigned ligands as potential scaffolds for developing new CIDs. Thus, we provide and extend new methodologies that potentially allow for posttranslational control over the activity of user defined split-kinases and split-phosphatases for interrogating and redesigning signaling pathways. The last section of this work focuses on understanding small-molecule selectivity toward protein kinases. We systematically analyzed different reported kinase screens to further understand the reliability of large scale data in the kinome field as the design of selective inhibitors is one the most useful approaches for understanding the function of enzymes or the development of drugs in a natural setting such as a primary cell or an organism.

Iron is the most abundant transition metal found in living systems and plays a crucial role in DNA biosynthesis. To accommodate higher replication rates, cancer cells require higher amounts of iron compared to non-neoplastic counterparts. This higher demand for iron renders cancer cells susceptible to iron deprivation, and exposure to iron chelators leads to growth arrest and cell death. Iron chelation strategies employing a wide variety of iron-binding scaffolds are currently under investigation for use in cancer treatment. Although these chelation approaches are effective against several cancer cell types, their use is limited due to toxicity ascribed to indiscriminate metal sequestration and induction of oxidative stress. Prochelation strategies in which the chelating unit remains inactive until triggered by a disease-specific event are expected to increase the specificity of chelation-based therapeutics. Chapter 1 provides an overview of chelation and prochelation based therapies as well as disulfide-based approaches in the design of prodrugs. In Chapter 2, the reduction activation mechanism of disulfide-masked thiosemicarbazone prochelators is described. Whereas disulfide-masked prochelators do not bind iron, reduction of the disulfide bond upon cellular uptake produces active chelators that readily bind intracellular iron. These systems are not active extracellularly; rather, they target the intracellular labile iron pool. We found that the antiproliferative activity of these disulfide-masked prochelators is dependent on the intracellular redox environment, with enhanced toxicity in more reducing conditions. The iron complexes resulting from exposure of cultured cells to the chelation systems were detected intracellularly by electron paramagnetic resonance in intact frozen cells. The compounds in our first series do not engage in intracellular redox chemistry and do not cause oxidative stress. In Chapter 3, the synthesis and characterization of a larger series of disulfide-masked prochelators featuring several classes of tridentate ligands is described. We investigated the iron-binding efficacy of the corresponding chelators, their ability to induce oxidative stress and their cell-cycle effects. We found that these prochelator systems, regardless of the identity of the donor set of atoms, do not result in the intracellular generation of oxidative stress. We also found that treatment of cultured cancer cells with prochelators results in cell-cycle arrest at G1/0 in non-synchronized cells and G2/M in G2-synchronized cells. In addition, we found that all classes of prochelators exhibit antiproliferative effects likely through induction of apoptosis. In Chapter 4, the syntheses and biological evaluations of disulfide-masked prochelators that feature carbohydrate targeting units are described. The sugar conjugates present increased aqueous solubility, compete as effectively as D-glucose for transporter-mediated cellular uptake, and are 6 to 11-fold more selective towards colorectal cancer compared to an aglycone that does not contain a targeting unit. The design of more potent prochelator systems, as well as the design of systems with improved selectivity and aqueous solubility are discussed in Chapter 5.

Applications of planar integrated optical waveguide (IOW) technology to problems in surface spectroscopy and optical chemical sensing have been partly limited by the difficulty of producing high quality glass IOWs is. The fabrication of IOWs by the sol-gel method from methyltriethoxysilane and titanium tetrabutoxide precursors has therefore been developed. The physical, chemical, and optical properties of the films were studied using a variety of analytical techniques. The results show that the catalyst used to accelerate the sol-gel reaction strongly influenced the optical quality of the IOW. A novel optical sensing platform was subsequently developed using a sol-gel derived, laminate planar IOW structure. The sensing element is fabricated by coating a sol-gel IOW with a second, porous sol-gel layer in which optical indicator molecules are physically entrapped, yet remain sterically accessible to analytes that diffuse into the pore network. Formation of a complex between the analyte and entrapped indicator is detected via attenuated total reflection (ATR) of light guided in the IOW. Feasibility was evaluated by constructing IOW-ATR sensors for Pb2+ and pH, based on entrapped xylenol orange and bromocresol purple respectively. The response of both sensors was sensitive and rapid. This work was further extended to the development of a new class of gaseous iodine sensors. The sensing principle is based on the detection of a charge transfer complex formed between iodine and phenyl groups that have been incorporated into a porous, methylated glass film. The sol-gel iodine sensor exhibits a linear response to gaseous I2 in the range of 100 ppb to 15 ppm with response and recovery times less than 15 sec. Langmuir-Blodgett (LB) films have also been deposited on a sol-gel IOW from zinc 1,4,8,11,15,18,22,25-octabutoxy-phthalocyanine (ZnPc). Planar waveguide linear dichroism was used to determine molecular orientation in a ZnPc LB monolayer. The IOW-supported ZnPc monolayer was found to exhibit a sensitive spectral response to gaseous I2. The overall optical sensing approach described in this dissertation is technically simple, inexpensive, and applicable to a wide variety of chemical sensing problems.

A new method for threo-selective synthesis of β-amino alcohols is described. This method employs N-diphenylmethylene-protected α-amino esters as starting materials. The α-amino ester is reduced to the oxidation state of an aldehyde with DIBAL or DIBAL:TRIBAL (1:1) followed by sequential addition of various Grignards and alkenyllithiums. The method is highly threo-selective (stereoselectivities ranged from 8:1 to > 20:1) and provides norpseudoephedrine and threo-sphingosine analogs in enantiomerically enriched form (> 97% ee). The mechanism of C-C bond formation was examined. A stable aluminoxy-acetal intermediate (generated by DIBAL reduction of the ester) was trapped with TMS-imidazole and isolated as the corresponding siloxy-acetal. The stereochemical outcome of the C-C bond forming step was shown to correlate with the steric bulk of the ester moiety. Bulky ester groups showed the greatest degree of threo-selectivity. These results suggest that the aluminoxy-acetal intermediate may be involved in determining stereoselectivity via either a tight-ion S(N)1-like or S(N)2-like reaction mechanism. A series of glycosyl acceptors was synthesized from N-diphenylmethylene-protected threo-sphingosine derivatives. These glycosyl acceptors undergo β-specific glycosylation using the method developed in this laboratory. This method capitalizes on a favorable hydrogen-bonding pattern imparted by the N-diphenylmethylene-protection. The favorable hydrogen-bonding enhances the nucleophilicity of the glycosyl acceptor relative to glycosyl acceptors with more conventional N-protection (i.e. Cbz, Boc, acyl etc.). This enhanced nucleophilicity allows the glycosylation to be carried out under mild conditions (AgOTfl, CH₂CI₂, RT overnight) and provides the corresponding β-glycosphingolipids in approximately 70% chemical yield.

Surface enhanced Raman scattering (SERS) is a powerful means for obtaining vibrational data from the metal/electrolyte or metal/gas interfacial environment. However, SERS is only observed for a limited number of metal surfaces under certain experimental conditions. Before this method can become a universal tool, the enhancement mechanism(s) must be understood. The results reported in this dissertation assess both electronic and chemical contributions to the SERS mechanism. The electronic properties of the metal are altered by systematic deposition of Pb or Cu onto a substrate that supports intense SERS, Ag. The chemical nature of the interface is altered with different probe molecules. The effect of Pb deposition on the SERS enhancing ability of Ag electrodes has previously been investigated with strongly adsorbed probe molecules. The behavior of cyanide species in the presence of Pb⁺² is complicated by the necessity of maintaining low solution pH to prevent Pb(OH)₂ precipitation; thus, the predominant solution species is HCN. Although previous reports state that no SERS can be detected from cyanide-containing solutions below pH 6, intense SERS signals can be obtained at pH 2 if sufficiently positive electrode potentials are maintained. The two unresolved SERS bands observed in acidic solutions are attributed to HCN which interacts with the Ag surface in end-on and side-on configurations. The predominant effect of Pb deposition on HCN SERS is HCN displacement. Enhancement due to charge transfer processes is not significant, while electromagnetic effects dictate the residual SERS intensity remaining after the initial HCN displacement. The supporting electrolyte anion affects the rate of change of the potential dependent C≡N stretch in basic CN⁻ media. A correlation between the rate of frequency change and anion charge/radius ratio was observed at potentials near and slightly negative of the Ag potential of zero charge in basic CN⁻ media. These results demonstrate the extraordinary sensitivity of SERS to interfacial conditions. The contributions from chemical and electromagnetic enhancement are further assessed by following excitation wavelength dependence of the SERS intensity of pyridine and Cl⁻ as a function of Cu coverage. Contributions from both are observed, but chemical enhancement is less evident for Cu than for Pb deposition. This is related to the smaller change in work function that occurs as a consequence of Cu versus Pb deposition on Ag surfaces.

Cellular secretion regulates cell communication and function. The ability to detect and quantify the release of hormones and neurotransmitters provides deeper understanding of cell signaling pathways. Ion channel sensors demonstrate a high potential for detecting cellular secretion with high sensitivity and selectivity, as well as adequate spatial and temporal resolution for real-time subcellular detection. Ion channel sensors utilize ligand-gated ion channels (LGICs) as recognition elements, enabling detection of ligand-receptor binding with high specificity. LGICs serve as signal transducers that transduce ligand binding events into highly sensitive current measurements, allowing label-free detection of hormone and neurotransmitters that are neither optically, nor electrochemically active. The work within this dissertation describes three new approaches to further advance ion channel sensor development. First, in vitro expression of eGFP-Kir6.2 was explored and verified using fluorescence microscopy, SDS-PAGE and dot blot. Electrophysiological measurement confirmed the successful expression of functional ion channels with expected pore conductance and antagonist sensitivity. The new expression method allowed fast and purification-free protein production, greatly reducing the time and technical barrier for ion channel sensor fabrication. Second, a dual-barrel ion channel probe was described to provide precise positioning of sniffer sensor using access resistance as feedback signal. Selective formation of polymer scaffold stabilized black lipid membrane across one barrel was confirmed and enabled membrane protein insertions. Precise positioning of the sensor will increase sensor reproducibility, thus providing accurate measurements of cellular release. Finally, a surface modified microfluidic valve was fabricated with > 70 fold enhancement in electrical resistance, enabling the isolation of ion channel signals in pA regime. The microfluidic valve provides a simple but cost-effective alternative for high throughput parallel electrophysiology. The efforts to advance the development of ion channel sensors will greatly improve our understanding of the biological system, benefiting disease diagnosis and treatment.

Ultrathin organized films of organic electronic materials, such as phthalocyanines (Pc), are promising for both fundamental and applied studies due to their special optical, electronic and photoconductive properties. The studies presented in this dissertation include fabrication of ultrathin molecular assemblies by molecular beam epitaxy and Langmuir-Blodgett techniques. The degree of molecular order, extent of charge transfer and the morphology within these films, assessed by methodologies, such as photoelectrochemistry, electrochemistry, surface analysis and optical spectroscopy were discussed and characterized. Under high vacuum condition, a wide range of ordered structures of some trivalent metal phthalocyanines, such as GaPc-Cl, InPc-Cl and AlPc-F, can be fabricated. These materials exhibit "layer-by-layer" growth on the single crystal SnS₂ surface when deposited by molecular beam epitaxy (MBE). The MBE technique allows for closer packing of these highly ordered phthalocyanines than in self-assembled (SA) or Langmuir-Blodgett (LB) thin films, due to the lack of hydrocarbon side chains which are necessary for control of molecular architecture during SA or LB depositions. Several new solution processable substituted phthalocyanines are introduced, which due to their strong self-assembled tendency, may be suitable for the formation of well organized thin films by SA and LB techniques. It is found that the types of the substituents attached to the Pc rings play a significant role in determining both the aggregation tendency and the electrochemical properties of Pcs. Surface pressure-area isotherms of these substituted phthalocyanines show that there can be one or two stable phase transition regimes for monomolecular film at the air/water interface. On-trough spectroscopic studies of benzylalkoxy substituted phthalocyanines show that in the pressure-area region prior to the formation of the first stable phase extensive aggregation has occurred. Electrochemical studies of fully compressed films of substituted phthalocyanines on certain substrates show the presence of multiple electroactive domains, controlling the oxidation or reduction process of the Pc rings. Spectroelectrochemical studies of LB films of CuPcOC₂OBz suggest that the presence of both monomer and aggregates leads to the two separate oxidation processes.

Antibodies specific for insulin and human immunoglobulin G (HlgG) were attached to controlled pore glass (CPG) particles which had been silanized with a diol-bearing silane. Up to 20 mg of antibody protein could be attached covalently to 1 gram of CPG. Such immobilized antibodies, or immunosorbents, would bind specific antigens, but not unrelated proteins, when used in a high pressure liquid chromatographic configuration. This technique was given the name "high performance immunoaffinity chromatography" (HPIC). The HPIC properties of these immunosorbents were evaluated by an equilibrium theory and were found to be comparable to batch values. An immunosorbent for HIgG antigen showed an HPIC association constant of 10⁷·⁶; the batch equilibrium constant for the same immunosorbent was 10⁷·⁸. Two different anti-insulin immunosorbents retained the intrinsic affinity (10⁶ and 10⁹) of the antibody used to make them. The total active antibody concentrations of these immunosorbents were evaluated by HPIC and batch methods with good agreement between the two. The immobilization reaction was seen to result typically in the loss of 90% of the original antibody activity. HPIC was shown to be applicable to the rapid analysis of antigens at levels as low as ng/mL. This was found to be possible in part because of the rapid forward kinetics which were assessed by HPIC. A forward rate constant of 3 X 10⁷ L·mol⁻¹·sec⁻¹ for the binding of insulin by a specific HPIC column could be determined. The possibility of HPIC fluorescence immunoassays was investigated using a highly sensitive fluorescence detector. An Eimac collimated xenon arc lamp provided sufficient power to detect picomolar levels of fluorescamine labeled insulin and other compounds. The limitations of HPIC in performing picomolar immunoassays were thus shown to be immunochemical rather than instrumental. The ability of immunoaffinity purifications to overcome these limitations was demonstrated.