New methods of analyzing pharmaceuticals by high performance thin-layer chromatography and microplate imaging were investigated using array imaging technology. Both techniques provide high sample throughput over more traditional analytical techniques for the analysis of pharmaceuticals. HPTLC provides high sample throughput by performing separations in parallel using a planar stationary phase. Imaging the entire plate with a single exposure performs quantitation of the analytes in the chromatographic medium. Fluorescence and fluorescence quenching detection modes are presented by employing tetracyclines, famotidine, and several over the counter drugs as model compounds. Studies conducted include sensitivity, separation efficiency and reproducibility of the system. Microplate imaging allows for the quantitation of numerous analytes in parallel. In this technique, solutions containing the analyte of interest are deposited into numerous self-contained wells on microplates, also known as 96-wellplates or ELISA plates. Light is passed through the wells of the plate allowing for absorption, fluorescence, or fluorescence quenching. Imaging with an array detector enables the researcher to simultaneously quantitate each well in parallel. The reaction between primary amines, ampicillin and amoxicillin, and fluorescamine was investigated. Microplate imaging was also tested for quantitating analytes in the low UV region (254 nm).

Based on the efficient substrate for p60ᶜ⁻ˢʳᶜ protein tyrosine kinase (PTK) YIYGSFK (1) (K(m) = 55 μM) obtained by combinatorial methods, we have designed and synthesized a series of conformationally and topographically constrained substrate-based peptide inhibitors for this enzyme. The inhibitors showed IC₅₀ values in low micromolar range (0.1-3 μM). A "rotamer scan" was performed by introducing four stereoisomers of β-Me(2')Nal in the postulated interaction site of peptide inhibitor (23) Y-c[D-Pen-(2')Nal-GSFC]KR-NH₂ (IC₅₀ = 1.6 μM). We found that the χ¹ space constraints imposed by the specialized amino acids, introduced at position 3 of peptide 23, were not as important as the configuration of the Cᵅ of that residue to recognize the active site of Src and Lck PTK, as reflected on the observed selectivity ratios. Cocrystallization studies between Lck and two of our inhibitors are in progress, in a collaboration with Dr. X. Zhu (Kinetix, Pharmaceuticals, Inc.). The results obtained may serve as the basis for the design of Lck and/or Src inhibitors, either peptide or nonpeptide. SL-3111 is a high affinity (IC₅₀ = 8.4 nM) and selective (μ/δ = 2020) δ-opioid receptor peptidomimetic ligand developed in Dr. Hruby's laboratory, as the result of extensive structure-activity relationship (SAR) studies based on peptide leads. However, bioassays (GPI and MVD) and in-vivo antinociception studies on the racemic mixture and both enantiomers of this compound, have shown particular problems such as low potency and toxicity. We have shown the importance of the piperazine ring in this molecule for binding toward the δ-opioid receptor. Thus, maintaining such scaffold we have studied a series of solution and solid-phase approaches toward the synthesis of SL-3111 analogues, which explore wider functional diversity at this heterocyclic ring. Compounds 64-67 were synthesized by solution methods. Analysis of the biological data and molecular modeling studies of these compounds, revealed an interesting trend in terms of the effects of the substituent at position two of the piperazine scaffold. Three different solid-phase protocols were explored toward the development of a combinatorial library of this type of compounds, which may facilitate future SAR studies.

Peptide ligands represent the most important hormones and neurotransmitters in physiological processes. Although native biologically active peptides have a great potential for medical applications, they often need to be modified to overcome certain inherent problems. A new research area called peptidomimetics has been developed in the last twenty years. The first generation of β-turn mimetics was focused on mimicking the β-turn backbone. In the last decade, many types of bicyclic β-turn dipeptides (BTD) have been design and synthesized. However, these methods do not have straightforward ways to introduce side chain groups on both rings. The introduction of functionalities on BTD, as the second generation of β-turn mimetics, is the major goal of my dissertation. By retrosynthetic analysis, convergent synthetic methodologies were initiated for [5,5]- and [6,5]-BTDs. Two kinds of nonproteinous amino acids are required in the strategies. One is the β-substituted cysteine derivatives and the other is β-substituted ω-unsaturated amino acids. The racemic β-vinylphenylalanine was synthesized by using Kazmaier-Claisen rearrangement, and the ω-unsaturated amino acids and β-substituted δ,ε-unsaturated amino acids were synthesized by using Ni(II)-complexes as chiral auxiliaries. Using these starting materials, [5,5]-BTD analogues were synthesized by a five-step strategy. The synthesis of [6,5]-BTDs has to proceed without formation of the 5-membered hemiaminal, which blocks further reaction. A Nᵅ-TFA protection group was used in this strategy and finally an efficient methodology was developed to generate the side chain groups into [6,5]-BTD analogues in nine steps. During the development of these methods, we solved the challenge to synthesize all 16 or 32 of the possible diastereomeric dipeptide mimetics. A novel idea to solve these problems was to synthesize the targeted peptide mimetics by solid phase methods in a combinatorial fashion, as the third generation of β-turn mimetics. We have succeeded in the synthesis of [3,3,0]-BTD²,³-Leu-enkephalins by unconventional solid phase synthesis, and four analogues have been synthesized and purified. This method is ready to expand to other sizes of BTD and to other target peptides with different functionalities.

Influenza is an enveloped virus, consisting of two surface glycoproteins, neuraminidase and hemagglutinin. The viral receptor is a glycoconjugate on which sialic acid is the terminal sugar. Neuraminidase catalyses the cleavage of the terminal sialic acid from the adjoining carbohydrate moiety, thereby assisting the virus to spread, and infect new cells. Thus development of neuraminidase inhibitors has been of great interest. Our studies are based on synthesis of new potential neuraminidase inhibitors. The synthetic strategy that was adopted for the preparation of the potential inhibitors, required the introduction of glycine ethyl ester at C1 of 1,4-lactone derivatives of N-acetylneuraminic acid. Furthermore, the rate of the ring opening of the 1,4-lactones was studied via proton NMR. Structural determination of the lactones are reported using specialized NMR techniques (Inverse Detected Single Quantum Filtered Long Range Spectroscopy). Conformational studies of the lactones were also determined with computational models.

The use of immobilized enzymes has steadily increased in recent years. Based upon the advantages that immobilized enzymes possess over soluble enzymes, numerous applications have emerged in medical and analytical fields. This work demonstrates the applicability of a liquid chromatographic system based upon coupled on-line immobilized enzyme reactors (IMERs) to organic synthesis, biochemistry and pharmacology. It is envisioned that the model system will grow into a modular process where synthetic chemists can add or subtract the enzymes necessary for their particular synthetic goal. The system allows for on-line chromatographic purification and structural identification of products and could greatly reduce time required to discover new synthetic pathways. In addition, the construction of a coupled enzyme system provides a number of approaches to basic research into synthetic and metabolic pathways as well as a rapid method for the discovery of new pharmaceutical substances. / A coupled system using extremely different enzymes with incompatible cofactors and reaction conditions has been constructed. The significance of the proposed project not only lies in the development of the liquid chromatographic on-line enzyme cascade but also in the biosynthetic pathway chosen for this study. The biosynthetic pathway involving dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase encompass the synthesis of the key transmitters, norepinephrine and epinephrine. The results demonstrate for the first time the immobilization of dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase. The IMERs are active and can be used in a liquid chromatographic format for qualitative and quantitative determinations. Studies with the IMER-HPLC systems have also shown that the activity of the immobilized enzymes reflects the non-immobilized enzymes. Thus, the IMER-HPLC system can be used to carry out standard Michaelis-Menten enzyme kinetic studies and to quantitatively determine enzyme kinetic constants, identify specific enzyme inhibitors, provide information regarding the mode of inhibition and the inhibitor constants (Ki). The immobilized enzyme reactors used independently or as a combination will provide a unique opportunity to explore the interrelationships between these enzymes, to investigate the source of catecholamine-related disorders and to design new drug entities for identified clinical syndromes.

Through the serendipitous discovery of a palladium catalyzed conversion of ethylthiol esters to their corresponding aldehydes, the pyrrolo (1,4) benzodiazepine systems found in (+)-anthramycin 1a* and (+)-neothramycins A 2a and B 2b have been constructed from the diethylthiol esters 3 and 4 respectively. A key intermediate 5 for the total synthesis of (+)-anthramycin and a facile route to (+)-neothramycins A and B are thus provided. ftn*Please refer to dissertation for diagrams.

The first total synthesis of ($-$)-hapalindole G, a member of novel chlorine- and isonitrile-containing hapalindoles from the cultured cyanophyte Hapalosiphon fontinalis, is accomplished. Our 21-step synthesis of ($-$)-hapalindole G from ($-$)-carveol features a stereospecific introduction of chlorine next to a quaternary center via cleavage of the cyclopropane intermediate and facile elaboration of the indole moiety through a conjugate addition of lithium methyl methylthiomethyl sulfoxide to an enone followed by hydrolysis of the resultant adduct. The absolute configuration of ($-$)-hapalindole G has therefore been confirmed on the basis of the specific rotation of our synthetic sample. Also described herein is a novel tin-mediated radical indole synthesis by using o-isocyanostyrene derivatives as starting materials via 2-tri-n-butylstannyl-3-substituted indoles as intermediates. The 2-tri-n-butylstannyl-indoles were also subjected to the one-pot Stille coupling reaction and iodination. The iodoindoles were capable of further manipulation. Our efficient synthesis paves the way for a facile construction of a variety of 3- or 2,3-substituted indoles from readily accessible isonitriles.

An advanced intermediate 2 for the synthesis of antitumor antibiotic ($\pm$)-Fredericamycin $A$, 1, has been prepared. The synthetic route features a novel palladium(0)-mediated intramolecular spiroarylation of 3. This methodology allows access to the unusual spirocyclic ring skeleton characteristic of Fredericamycin $A$ from more readily accessible precursors, isoquinoline 4 and naphthalide 5. The syntheses of 4 and 5 are discussed. Preliminary model studies established an efficient pathway to structures related to 3. These initial studies also disclosed a means by which to generate spirocyclic ring systems of the type found in Fredericamycin $A$. (DIAGRAM, TABLE OR GRAPHIC OMITTED...PLEASE SEE DAI)

A new method for the enantioselective synthesis of unusual amino acids has been developed. The chemoenzymatic aza-Achmatowicz rearrangement of appropriate furylglycine derivatives provided nitrogenous synthons that were readily converted to amino acid building blocks. An application of the new chemistry to the synthesis of the unique pyridazine carboxylic acid component of luzopeptins is presented. Luzopeptin C has potent inhibitory activity towards the reverse transcriptase of HIV as the causative agent of AIDS.

Five new metalloporphyrins, $\rm \lbrack Al\sp{III} (TPPCM)Cl\rbrack,$ $\rm \lbrack Al\sp{III} (TPPCN)Cl\rbrack,$ $\rm \lbrack Al\sp{III} (TPPC)Cl\rbrack,$ $\rm \lbrack Cr\sp{III} (TPPCM)Cl\rbrack,$ and $\rm \lbrack Cr\sp{III} (TPPCN)Cl\rbrack,$ have been synthesized and characterized as a first step toward the bulk synthesis of water-soluble C$\sb{60}\sp{.-}$ salts. According to a literature report, Cr$\rm \sp{II}$(TPP) and (Al$\rm \sp{III}$(TPP)$\sp{.-}\rbrack$ (TPP$\sp{2{-}}$ = tetraphenylporphyrinato) reduce C$\sb{60}$ to C$\sb{60}\sp{.-}$ under proper solvent conditions to form an insoluble $\rm \lbrack Cr\sp{III}(TPP)\rbrack\sp+(C\sb{60}\sp{.-})$ or (Al$\rm \sp{III}$(TPP)$\rbrack\sp+$(C$\sb{60}\sp{.-})$ salt. Here it is proposed to derivatize these Cr$\rm \sp{II}$ and Al$\rm \sp{III}$ tetraphenylporphyrins with substituents on the phenyl rings to produce water-soluble $\rm \lbrack Cr\sp{III} (TPPR)\rbrack\sp+(C\sb{60}\sp{.-})$ and $\rm \lbrack Al\sp{III} (TPPR)\rbrack\sp+(C\sb{60}\sp{.-})$ salts. Initial electrochemical data for precursor $\rm \lbrack Al\sp{III} (TPPCM)Cl\rbrack,$ $\rm \lbrack Al\sp{III} (TPPCN)Cl\rbrack,$ $\rm \lbrack Al\sp{III} (TPPC)Cl\rbrack,$ $\rm \lbrack Cr\sp{III} (TPPCM)Cl\rbrack,$ and $\rm \lbrack Cr\sp{III} (TPPCN)Cl\rbrack$ compounds suggest that adding water-solubilizing substituents does not interfere with the electron transfer between the reduced Al$\rm \sp{III}$ and C$\rm \sp{II}$ metalloporphyrins and C$\sb{60}.$ Hence, there is potential to produce bulk amounts of water-soluble C$\sb{60}\sp{.-}$ by this method. The radical anion has 150 A$\sp2$ of paramagnetic surface area (S = 1/2), and possible utility as a new magnetic resonance imaging contrast agent.