Several methods have been proposed for the prediction of aqueous solubility. This study proposes the SCRATCH model for the aqueous solubility estimation of a compound directly from its structure. The algorithm utilizes predicted melting points and predicted aqueous activity coefficients for the solubility estimation, reflecting the truly predictive nature of the model. It uses two additive, constitutive molecular descriptors (enthalpy of melting and aqueous activity coefficient) and two non-additive molecular descriptors (symmetry and flexibility). The melting point prediction is trained on over 2200 compounds whereas the aqueous activity coefficient is trained on about 1640 compounds, making the model very rigorous and robust. The model is validated using a 10-fold cross- validation.A comparison with the General Solubility Equation suggests that the SCRATCH predicted aqueous solubilities have a slightly more average absolute error. This could result due to the fact that SCRATCH uses two predicted parameters whereas the GSE utilizes only one predicted property. Although the GSE is simpler to use, the drawback of requiring an experimental melting point is overcome in SCRATCH which can predict the aqueous solubility of a compound just based on its structure and no experimental values.

The United States Environmental Protection Agency was established in 1970 to control, limit, and regulate pollutant entry into the environment. The primary sources of pollutants are motor vehicle emissions, chemical plants, production factories, land fills, and natural or man-made catastrophes. Persistent organic pollutants have been known to cause such aliments as cancer, respiratory disease, and birth defects. These compounds can also cause irreversible environmental effects such as ozone depletion.The amounts of pollutants in air, water, soil, and organic matter can be correlated with the octanol solubility, vapor pressure, octanol-air partition coefficient, and air-water partition coefficient. The estimation of physical properties plays an important role in understanding the fate of organic pollutants. Although it is more desirable to measure such properties, their estimations can be of great importance in conserving resources and minimizing exposure.In this dissertation new equations for the estimation of these properties are generated. This is accomplished without the use of fitted parameters or regression analysis. The only experimental input parameters are the transition temperatures. The transition properties were estimated from molecular structure. The average absolute errors for the estimated properties are less than one log unit from the experimental values.

Motivated by the urgent need for new molecular targets and novel agents to treat pancreatic cancer, a target-based approach to drug discovery was implemented that led to the identification, validation and targeting of the Aurora family of kinases. The Aurora kinases (A, B and C) are mitotic serine/threonine kinases involved in various aspects of mitosis, including centrosome separation, bipolar spindle assembly, chromosome alignment and cytokinesis. In this dissertation, the potential use of the Aurora kinases as therapeutic targets to treat pancreatic cancer was investigated. It was found that both Aurora A and Aurora B are overexpressed in pancreatic adenocarcinomas, suggesting that some cancer cells are dependent upon their activity for continued proliferation and survival. To validate this hypothesis, antisense oligonucleotides were used in cell-based assays to evaluate the biological consequences of Aurora A and/or Aurora B inhibition. It was found that perturbations in Aurora kinase signaling result in cell cycle arrest and apoptosis. The biological fingerprints of Aurora A and Aurora B inhibition were compared and contrasted in an effort to identify the superior therapeutic target. It was concluded that an Aurora A-targeted therapy may have some beneficial consequences; however, a therapeutic approach discriminating between Aurora A and Aurora B is not straightforward. A fragment-based approach relying heavily on computer modeling was used to design and identify a nanomolar inhibitor of the Aurora kinases; however, it showed activity only at high micromolar concentrations in cell-based evaluations suggesting the compound possessed unfavorable characteristics that limited its biological activity. The preclinical development of analogues of the compound discovered by the work presented in this dissertation is ongoing. Finally, the pancreas-specific overexpression of Aurora A kinase was shown to be insufficient to induced pancreatic tumorigenesis in a mouse transgenic model.

The folates are a family of structurally similar, water-soluble, B vitamins, documented to play prominently in human health and disease. The potential impact of folate nutrition has been demonstrated by large-scale epidemiological and nutritional studies indicating that decreased folate intake increases the risk of cancer development. Human skin is particularly prone to the development of carcinomas and it is established that skin cancer risk correlates with exposure to the complete carcinogen ultraviolet radiation (UVR) in the form of sunlight. Recently a link between skin, sunlight, and folate has emerged from studies demonstrating that folate species are degraded by exposure to wavelengths of UVR contained within the solar spectrum. It is hypothesized that the unique physiology, function, and environment of skin combine to make skin tissue prone to folate deficiencies and that folate supplementation is a promising strategy for the prevention of skin cancer. However, many questions regarding folate nutrition within human skin must be answered before strategies to modulate folate nutrition may be rationally designed and safely implemented. This work presents novel means to examine skin-specific folate nutrition, including an analytical method to quantify individual folate species in human keratinocytes adaptable for the analysis of intact skin tissue and innovative cultured keratinocyte models of both acute and chronic folate deficiencies. It is demonstrated that folate deficiencies in skin tissue are possible and even likely as proliferating human keratinocytes are unable to maintain intracellular folate concentrations when nutrient conditions are limited and exposure to UVR results in biologically relevant folate degradation. Folate deficiency in human keratinocytes is observed to have potential pro-carcinogenic consequences including S-phase proliferation arrest, increased inherent DNA strand breaks, increased uracil misincorporation into DNA, and deficiencies in DNA damage repair, which are reversed when folate nutrient levels are optimized. The presented work characterizes the relationship between intracellular folate species and environmental carcinogens known to induce skin cancer and addresses challenges facing supplementation strategies for specifically improving folate nutriture in human skin. In total, this report broadens our understanding of folate nutrition in human skin and demonstrates that optimization of folate nutrition holds promise as a cancer preventive strategy.

A topochemical model has been derived to account for the diverse selectivity of cyclic opioid peptides related to enkephalins and for narcotic alkaloids towards the $ mu$ and $ delta$ opiate subclasses. Efforts toward demonstrating its validity have later been accomplished. The model is based on a computer molecular modeling study using the opiate pharmacophore as a minimum prerequisite to align the molecules and benefit also of informations obtained from spectroscopic study in solution concerning the macrocycles conformation. The conformational properties of N$ sp alpha$Cbz-c((D)A$ sb2$Bu-Gly-Phe-Leu) were studied by nuclear Overhauser effect which provided semi-quantitative internuclear distances. One of the important elements that the model brings out is that the eventual presence of an aromatic ring on the beta carbon of Cys of the opioid peptide HTyr-(D)Pen-Gly-Phe-Cys-NH$ sb2$ should induce an increase in selectivity toward the 6 opiate subclass. Consequently, we engaged in preparing the special amino acid, P-phenylcysteine, using a method based on the addition of mercaptan to unsaturated azlactone. The diastereoisomers obtained were separated, and their relative configurations assigned by X-ray crystallography. The pure enantiomers were obtained by resolution with the enzyme carboxypeptidase A. Several difficulties were encountered during this preparation, the main concern being the S protecting group. This protecting group should be chosen with the property that it can be quantitatively removed when desired and compatible with the conditions of peptides synthesis. Moreover, the size of the protecting group should be minimized in order to be able to realize the resolution with the enzyme. We explored, by the same token, the size of the hydrophobic pocket of the exopeptidase. Using the structural information obtained from the X-Ray diffraction study of several of the $ beta$-phenycysteine, we have determined with the help of computer molecular modeling, the

For the first time, carbon nanostructures have been designed and synthesized to form immunoconjugates with monoclonal antibodies (mAb) for use in cell-targeted cancer diagnosis and therapy. The immunoconjugates are derived from various nanoscale carbon-based building blocks, specifically fullerenes (C60 ), gadofullerenes (M C60) and ultra-short carbon nanotubes (US-tubes). The exterior of each nanostructure has been derivatized with water-solubilizing addends using Bingel-type (nucleophilic cyclopropanation) addition chemistry to facilitate biocompatibility. Initially, conjugation to the murine anti-gp240 melanoma antibody (ZME-018 mAb) was completed with two different water-soluble C60 derivatives, only one of which had the potential to covalently attach to the ZME-018 mAb. After conjugation, this covalently linked C 60-SPDP conjugate incorporated 15 C60 moieties per antibody, while retaining 80% of the antibody's target specificity. In a second experiment a non-covalently linked C60-Ser conjugate incorporated 38 fullerenes per antibody but retained only 4% of the antibody's target specificity. These findings suggest that covalent attachment of C60 derivatives to antibodies may not be essential for the development of fullerene immunotherapy (FIT), although the ratio of C60: antibody may need to be minimized so as not to inhibit antibody targeting. To study the cell internalization characteristics of the fullerene immunoconjugates, two water-soluble Gd C60 derivatives, which allowed for Gd 3+ monitoring by inductively-coupled plasma mass spectrometry (ICP-MS) at concentrations <10 ppb, have been utilized. These studies have provided evidence that the fullerene-based immunoconjugates retain the ability to effectively internalize into target cells, with approximately 20% of the available Gd 3+ internalizing into the A375m melanoma cells. These results suggest that immunoconjugates derived from C60-based chemotherapeutics may become new-targeted therapies against cancer. Of the carbon-based nanomaterials studied in this work, US-tubes are perhaps the most attractive candidates for nanomedicine platforms, due to the possibility of internally loading medically interesting materials, such as Gd3+ ions for magnetic imaging resonance (MRI) or iodine (I2) for computed tomography (CT). Toward this end, single-molecule US-tubes have been isolated by chemical reduction of the US-tubes, followed by immediate functionalization using Bingel chemistry to produce debundled and derivatized US-tube materials. Three different malonate addends have been attached to the US-tubes, including serinol, polyethylene glycol (PEG) and amide malonates. Each of the US-tube derivatives exhibited varying degrees of solubilities in water ranging from 0.25 mg/mL to 1.00 mg/mL. An n-octanol/water partition coefficient has also been determined for each derivative, with values ranging from 0.25 to 1.20, which suggest that these derivatized nanocapsules might readily internalize into cells. Loaded internally with medically-useful materials such as Gd3+ ions, I2 or radionuclides for imaging and therapeutic applications, these biocompatible carbon nanocapsules may be engineered into a universal platform for the containment and delivery of an array of medical agents in vivo.

This research presents the synthesis and spectroscopic characterization of I2-filled single-walled carbon nanotubes (I2 SWNTs) and I2-filled ultra-short carbon nanotubes (I2 US-tubes). These I2-internally-loaded SWNTs and US-tubes have applications both as a model system for the filling of SWNTs with other medically-interesting materials, such as alpha-radiotherapeutic radionuclides like At-211, and as synthons for a new class of computed-tomography (CT) X-ray contrast agents (CAs). Existing CT technology lacks the ability to diagnose critical diseases such as coronary artery vulnerable plaque, recently discovered as the cause of 70% of heart attacks, because current CAs circulate in the bloodstream rather than being targeted to specific cell-types. SWNT-based CAs offer several advantages over commercially-available CT CAs, such as the ability to sequester toxic ion and molecule imaging agents within the SWNT, to be targeted to specific cell types, and to translocate into targeted cells for intracellular molecular imaging. The synthesis of I2-SWNTs and I2-US-tubes is achieved via sublimation of molecular iodine (I2), a method proven to produce high yields for other filling materials. X-ray photoelectron spectroscopy (XPS) along with inductively-coupled plasma analysis (ICP-AE) and Raman spectroscopy have been used to define the location and quantify the amount of I2 in I2-SWNT and I2-US-tube samples. The exterior-adsorbed I2 can be removed (as I-) from I2-SWNTs by reduction with Na0/THF or by heating the I2-SWNTs to 300°C (without reduction), leaving I2 contained only within the interior of the SWNTs (I2 SWNTs). These I2 SWNTs contain ∼25 weight % of I2 and are stable without the loss of I2 even after exposure to additional reduction with Na 0/THF or upon heating to ca. 500°C. Micro CT experiments confirm that I2 SWNTs, with a radiodensity of 28,400 HU, are functional CT contrast agents. In contrast to I2-SWNTs, the internal I 2 in I2-US-tubes is unstable and is removed by either Na 0/THF reduction or temperatures of 300°C. To date, this instability has resulted in the inability to synthesize PEG-I2 US-tubes, a biocompatible water-soluble derivative of I2-US-tubes. However, initial experiments toward the synthesis of serinol-aide-I2 US-tubes have shown promising results, indicating a water-soluble derivative of I 2 US-tubes will be synthesized in the very near future. The I2 SWNTs and I2 US-tubes prepared in this research represent the first medical applications of filled SWNTs for CT imaging. I 2 SWNTs can now be used as a model system for the filling of SWNTs with other materials such as 211AtX (x = any halogen) for targeted alpha-radioimmunotherapy. They also signify the first step toward revolutionary intracellular CT imaging for the diagnosis of many diseases including coronary artery vulnerable plaque and cancer.

The biosynthesis of three natural products, L-2-amino-4-pentynoic acid (propargylglycine) (1), 3-(3-isocyanocyclopent-2-enylidene) propionic acid (2), and (+)-(alpha)-(S)-amino-(2,5-dihydro-5(S)-methyl)-furan-2(R)-acetic acid (3) (furanomycin), produced by an unidentified Streptomyces, Trichoderma hamatum, and Streptomyces threomyceticus respectively, have been investigated. Although two prospective labeled precursors of the amino acid (1), {1-('14)C}-norvaline and {1-('14)C}-allylglycine were synthesized, and procedures for the isolation, detection and degradation of (1) devised, the elucidation of the biogenesis of (1) was hampered due to lack of production of (1) by the microorganism. The isonitrile acid (2) has been shown by using ('14)C- and ('3)H-labeled precursor incorporation experiments to be derived from the amino acid tyrosine which undergoes an oxidative cleavage of the aromatic ring followed by recyclization. In addition to specific incorporation studies with {('14)C}- and {('3)H}-labeled precursors, experiments using {('2)H}, {('18)O}, and {('13)C}-labeled precursors in conjunction with ('2)H-NMR and ('13)C-NMR have been undertaken to investigate the biosynthetic origin of the amino acid (3). The evidence indicates that (3) arises from one unit of propionic acid and two units of acetic acid.

C. roseus is the source of the important anticancer indole alkaloids, vincristine and vinblastine. Hairy root cultures are an attractive alternative for the production of plant derived secondary metabolites. C. roseus hairy root cultures were established in our laboratory by A. rhizogenes mediated transformation of C. roseus seedlings prior to the beginning of this project. The objectives of this project were twofold: (i) Alkaloid analysis of C. roseus hairy root cultures, (ii) Scaleup of hairy root cultures from shake flasks to bioreactors. An extraction method was adapted for the rapid quantification of C. roseus hairy root extracts, and was used for quantifying alkaloid yields from five different root clones. Optimum analytical wavelengths were chosen for quantification after analyzing chromatograms at five different wavelengths using PDA detection. Extracts were analyzed using different analytical techniques--GC/MS, MS, HPLC-PDA and TLC--and several alkaloids were identified. The presence of ajmalicine, akuammine, apparicine, catharanthine, hoerhammericine, lochnericine, serpentine and tabersonine was confirmed. The presence of vindoline--an important precursor of vinblastine and vincristine--could not be confirmed although GC/MS indicated the presence of vindoline fragment ions. A novel method based on non-linear regression analysis was developed for online measurement of oxygen uptake rates. Thus, mass transfer rates could be adjusted in bioreactor cultures to provide sufficient oxygen. Three different bioreactor configurations--two recirculation reactors (60 mL, 1L) and a 500 mL spinner flask--were designed for scaleup studies. Growth, nutrient utilization and alkaloid productivities were measured. This is the first study where several biochemical parameters have been measured in multiple bioreactor configurations and found to be comparable--without exception--to shake flask cultures. Cultures were cultivated in different gas phase environments--'air' and '2.5% CO$\sb2$'--in each bioreactor configuration. This is the first study of the effect of CO$\sb2$ on hairy roots cultivated in liquid culture. A beneficial effect ($>$98% confidence) of CO$\sb2$ was observed on biomass accumulation. Possible metabolic importance of CO$\sb2$ on heterotrophic plant cultures is discussed.

Enantiomerically-pure compounds are becoming increasingly important particularly to the pharmaceutical industry. Enzymes are useful tools in the synthesis of such compounds. To better understand how lipases discriminate between enantiomers of chiral secondary alcohols we obtained x-ray crystal structures of covalent complexes of Candida rugosa lipase with the transition-state analogs (1R)-menthyl hexylphosphonate and (1S)-menthyl hexylphosphonate. These compounds are transition-state analogs for the hydrolysis of menthyl esters. We observed that the transition-state analog of the unfavored (1S)-enantiomer of menthol disrupted the hydrogen bond between N$ varepsilon$2 of histidine 449 and the menthol oxygen. His 449 is part of the catalytic triad in CRL. This may account for the slower reaction of the (1S)-enantiomer of menthol. The crystal structures also identified binding site regions for secondary alcohols in CRL. These regions are common among many lipases and esterases and account for their common enantiopreference toward secondary alcohols. / Lipases and esterases can resolve compounds with phosphorus and sulfur stereocenters by hydrolysis of a pendant acetoxy group. Both CRL and cholesterol esterase have high selectivity for (2-acetoxy-1-naphthyl)methylphenylphosphine oxide. They resolved this substrate with and E of 81 and 32 respectively. A synthetic scale resolution of this substrate with subsequent recrystallization and chemical transformation followed by stereospecific reduction gave both enantiomers of (2-methoxy-1-naphthyl)methylphenyl-phosphine with 96-97% ee. This chiral phosphine is potentially useful in asymmetric syntheses. / CE is the most selective enzyme for the sulfur substrates tested but these enantioselectivities were moderate, E's ranged from 5 to 25. From the CE resolution of the phosphorus and sulfur compounds and others we propose an empirical model that predicts which enatiomer reacts faster. The model is based on the size of the substituents and their conformational preferences. / Crude Aspergillus niger resolves esters of pipecolic acid with an E of 20 $ pm$ 4. A simple partial purification of ANL by fractional precipitation with ammonium sulfate increased the enantioselectivity to $>$100. The partially purified ANL can be used in a synthetic scale resolution of ($ pm$)-n-octyl pipecolate to give (S)-($-$)-pipecolic acid (93% ee) and (R)-(+)-pipecolic acid (97% ee).