Paul Allan Bartlett - US grants

We propose to synthesize five representative members of the biologically important class of naturally-occurring polyethers, including the antibiotics septamycin, nigericin, lasalocid A, and ionomycin, and the algal metabolite thyrsiferol. Versatile stereoselective methods for producing substituted tetrahydrofurans and tetrahydropyrans by cyclization of olefinic alcohol derivatives with relative asymmetric induction will be developed. These methods involve electrophilic cyclization of ether derivatives and hydroperioxides (for the selective formation of cis-2,5-disubstituted tetrahydrofurans) and cyclization of the alcohols themselves or ring contraction of tetrahydropyrans (for the selective formation of the trans-isomers).

We propose to synthesize members of four classes of biologically important natural products: the macrocyclic rifamycins, streptovaricins, and polyene macrolides, as well as related compounds such as tirandamycin. These materials exhibit significant antitumor, antibiotic, and antifungal properties, and provide a continuing challenge to the synthetic chemist. In the preceding grant period, we developed a number of versatile methods for acyclic stereocontrol with relative asymmetric induction, based on the functionalization of olefins via cyclic intermediates. We intend to employ these methods in the syntheses proposed, as well as to develop a number of new ones.

We propose a number of synthetic and mechanistic studies related to chorismic acid, a key intermediate in the biosynthesis of aromatic metabolites. 1. We plan to elucidate the stereochemistry of the Claisen rearrangement of chorismate to prephenate by developing a synthesis of stereospecifically labeled deuterochorismate. 2. We will synthesize a transition state analog of the rearrangement and evaluate it as an inhibitor of chorismate mutase enzymes and as a potential antibiotic. 3. We will develop total syntheses of the still-virgin intermediates in the shikimic acid pathway: shikimate-3-phosphate, 5-enolpyruvylshikimate-3-phosphate, and chorismate itself.

Three enzymes which are involved in the metabolism of the nucleoside bases will be the targets of inhibitor studies: cytidine deaminase, adenosine deaminase, and carbamyl phosphate synthetase II. With cytidine deaminase, we will investigate the kinetics and mode of inhibition of our phosphorus-containing pyrimidine transition state analog, which we have already demonstrated to be the most potent inhibitor known for this enzyme. For application to adenosine deaminase, the analogous phosphorus-containing purine derivatives will be evaluated. A series of multi-substrate analogs for carbamyl phosphate synthetase will be studied. The deaminase inhibitors may shed light on the mechanism of action of these enzymes, as well as act in a synergistic manner with a number of clinically important anti-neoplastic agents. Selective inhibitors of carbamyl phosphate synthetase could prove to have useful anti-cancer activity in their own right.

The proposed research involves synthesis of a number of enzyme inhibitors, evaluation of their binding behavior, and correlation of this binding behavior with turnover of related substrates. Our intention is to develop effective strategies for the design of biologically active molecules and to provide insight into the relationship between inhibitor binding and enzyme mechanism. The inhibitors that are developed during the course of this work should be of medicinal importance in the areas of antitumor therapy, hypertension, and analgesia. The key elements to the program are the following: A. Cytidine Deaminase: The stereoselectivity of phosphapyrimidine inhibitors will be evaluated, whether they are transition state analogs will be determined, and the relationship between their slow-binding behavior and protein conformational changes will be examined. B. Zinc Peptidases: The correlation between phosphonamidate and transition state binding will be extended, the mechanism of slow-binding for these inhibitors will be elucidated, thermolysin and carboxypeptidase A will be compared in this respect, and a new class of phosphonamide inhibitors will be explored. C. Aspartic Peptidases: The nature of inhibition by a phosphorous-containing pepstatin analog will be elucidated, an explanation for its slow, two-step binding behavior will be sought, and phosphinamides and phosphonic acid inhibitors will be developed as more accurate transition state analogs for this class of peptidases. D. Glutamine-Dependent Amidotransferases: A general strategy for the inhibition of these enzymes will be developed and applied to the synthesis of multisubstrate analogs for carbamyl phosphate synthetase and phosphoribosyl pyrophosphate amidotransferase. E. Adenosine Deaminase: The phosphinate and phosphinamide analogs of coformycin will be synthesized as better mimics of the tetrahedral intermediate, their inhibition and slow-binding will be analyzed in relation to enzyme conformational changes. F. Sulfoxides as Inhibitors of Dehydrases: A program on suicide inhibitors based on enzyme-induced Pummerer-type reactions will be initiated (enzymes addressed: carnitine acetyltransferase, Beta-hydroxydecanoyl thiolester dehydrase, and mevalonate pyrophosphate decarboxylase).

A broadly based bioorganic program for the study of some of the key enzymes in the shikimate-chorismate pathway is outlined. The key elements are the following: 1. Chorismate mutase: New transition state analog inhibitors will be synthesized and evaluated; a synthesis of chorismic acid itself will be completed. 2. 3-Dehydroquinate synthase: The stereochemistry of the individual steps in the enzymic transformation will be determined by stereoselective synthesis of a key intermediate; blocked-substrate as well as transition state analog inhibitors will be synthesized and evaluated. 3. Chorismate synthetase: An allylic isomer of the natural substrate will be synthesized and evaluated as an alternative substrate or inhibitor. 4. 5-Enolpyruvylshikimate-3-phosphate synthase: The postulated tetrahedral adduct will be synthesized and its enzymatic conversion to product evaluated; transition state and multisubstrate analog inhibitors will also be synthesized. 5. Prephenate dehydratase: A series of sulfoxide and sulfilimine analogs of prephenate will be synthesized and evaluated as transition state analog or suicide inhibitors. 6. A number of other analogs of intermediates in the shikimate-chorismate pathway are proposed as substrates and inhibitors.

The College of Chemistry at the University of California (Berkeley) is requesting funds for the purchase of a high mass (10,000 amu) high resolution mass spectrometer with Fast Atom Bombardment and Field Desorption capabilities. An instrument of this description is required in our facility because we cannot fulfill our users needs with our present equipment. This instrument will be used for the analysis of samples submitted by nine faculty members whose biomedical research encompasses the following: Synthesis of Peptides and Studies of Enzyme Inhibitors Characterization of Complex Carbohydrates Structural Analysis of Phycobiliproteins Structure and Function of Oligonucleotides Structural Determination and Studies of Protein-Bound Pigments Characterization of Siderophore Complexes Design and Synthesis of Enzyme Catalysts Oligonucleotide Directed Mutagenesis Novel Synthetic Routes of Potentially Physiologically Active Compounds Stereoselective Synthesis of Transition Metal Enolates and Related Organometallic Compounds

The goal of the proposed research is to developed methods for the design of biologically active compounds based on a knowledge of the structure and conformation of oligopeptide ligands and protein binding sites. Five distinct systems will be explored through the synthesis, evaluation, and refinement of a series of peptide mimics. The compounds to be synthesized include: Inhibitors of alpha-Amylase: Tricyclic molecules incorporating tyrosine, arginine, and tryptophan sidechains of the key triad of amino acids of the amylase inhibitor tendamistat. Serine Protease Inhibitors: Macrocyclic mimics of the active loop of bovine pancreatic trypsin inhibitor, incorporating elements of residues 13-17, the lysine-15 side chain, and a reactive ketone in place of the scissile amide. This series of inhibitor mimics can be readily extended to other serine proteases. Alpha-Helix Mimics: A pair of bicyclic molecules containing three hydrogen bonding groups fixed in the orientation found within a peptide alpha-helix. These molecules will be appended to oligopeptides as their N- or C-terminal residues and evaluated for their ability to induce or stabilize the alpha-helical conformation. Thermolysin Inhibitors: Macrocyclic derivatives of high affinity phosphonate inhibitors which adopt a specific loop conformation in the active site of thermolysin. These compounds will be used to explore in a structurally verifiable manner the effect of conformational restriction on protein-ligand binding. Haloalkene Isosteres of the Peptide Bond: Dipeptide analogs in which the amide linkage is replaced with a fluorine- or chlorine- substituted double bond. Such analogs will be prepared for enkephalin, thyrotropin releasing hormone, aspartame, and as ground-state substrate analogs for peptidases. The synthetic methodology will also be adapted for the stereospecific synthesis of ketomethylene dipeptide isoteres. In addition to direct evaluation of the ability of the above compounds to exert their intended biochemcial or biophysical effect, collaborative studies will be undertaken to determine their solution structure through 2-D NMR or their bound conformation through X-ray crystallography, where appropriate.

The proposed research involves synthesis of a number of enzyme inhibitors, evaluation of their binding behavior, and correlation of this binding behavior with turnover of related substrates. Our intention is to develop effective strategies for the design of biologically active molecules and to provide insight into the relationship between inhibitor binding and enzyme mechanism. The inhibitors that are developed during the course of this work should be of medicinal importance in the areas of antitumor therapy, hypertension, and analgesia. The key elements to the program are the following: A. Cytidine Deaminase: The stereoselectivity of phosphapyrimidine inhibitors will be evaluated, whether they are transition state analogs will be determined, and the relationship between their slow-binding behavior and protein conformational changes will be examined. B. Zinc Peptidases: The correlation between phosphonamidate and transition state binding will be extended, the mechanism of slow-binding for these inhibitors will be elucidated, thermolysin and carboxypeptidase A will be compared in this respect, and a new class of phosphonamide inhibitors will be explored. C. Aspartic Peptidases: The nature of inhibition by a phosphorous-containing pepstatin analog will be elucidated, an explanation for its slow, two-step binding behavior will be sought, and phosphinamides and phosphonic acid inhibitors will be developed as more accurate transition state analogs for this class of peptidases. D. Glutamine-Dependent Amidotransferases: A general strategy for the inhibition of these enzymes will be developed and applied to the synthesis of multisubstrate analogs for carbamyl phosphate synthetase and phosphoribosyl pyrophosphate amidotransferase. E. Adenosine Deaminase: The phosphinate and phosphinamide analogs of coformycin will be synthesized as better mimics of the tetrahedral intermediate, their inhibition and slow-binding will be analyzed in relation to enzyme conformational changes. F. Sulfoxides as Inhibitors of Dehydrases: A program on suicide inhibitors based on enzyme-induced Pummerer-type reactions will be initiated (enzymes addressed: carnitine acetyltransferase, Beta-hydroxydecanoyl thiolester dehydrase, and mevalonate pyrophosphate decarboxylase).

Continuation of a broadly based bioorganic program to study some of the key enzymes in the shikimate-chorismate pathway is proposed. The major elements are the following: 1.5-Enolpyruvylshikimate-3-phosphate Synthase. We propose to continue our work on the synthesis of stabilized analogs of the tetrahedral intermediate and on the synthesis of this species itself; we will also study the mechanism and stereochemistry of the elimination step. 2.Chorismate Synthase. Through the use of isotopically labeled substrate analogs and of 5-deazaflavin, we will study the role of the flavin cofactor and the mechanism of the 1,4-elimination reaction catalyzed by this enzyme. 3.Chorismate Mutase. We expect to complete our study of this enzyme with the synthesis of ammonium and aziridinium inhibitor analogs and an investigation of an electronically perturbed substrate analog. 4.Dehydroquinate Synthase. We propose to resolve the remaining mechanistic issue, namely the extent of enzymatic involvement in the final step of the transformation, and to synthesize some potential suicide inhibitors of the enzyme. 5.Dehydroquinase. We will initiate a program to develop suicide inhibitors for this early enzyme in the pathway. 6.Isochorismate Synthase and Isochorismatase. We will initiate programs to develop inhibitors and reaction intermediate analogs for these enzymes of a branch pathway.

The goal of the proposed research is to developed methods for the design of biologically active compounds based on a knowledge of the structure and conformation of oligopeptide ligands and protein binding sites. Five distinct systems will be explored through the synthesis, evaluation, and refinement of a series of peptide mimics. The compounds to be synthesized include: Inhibitors of alpha-Amylase: Tricyclic molecules incorporating tyrosine, arginine, and tryptophan sidechains of the key triad of amino acids of the amylase inhibitor tendamistat. Serine Protease Inhibitors: Macrocyclic mimics of the active loop of bovine pancreatic trypsin inhibitor, incorporating elements of residues 13-17, the lysine-15 side chain, and a reactive ketone in place of the scissile amide. This series of inhibitor mimics can be readily extended to other serine proteases. Alpha-Helix Mimics: A pair of bicyclic molecules containing three hydrogen bonding groups fixed in the orientation found within a peptide alpha-helix. These molecules will be appended to oligopeptides as their N- or C-terminal residues and evaluated for their ability to induce or stabilize the alpha-helical conformation. Thermolysin Inhibitors: Macrocyclic derivatives of high affinity phosphonate inhibitors which adopt a specific loop conformation in the active site of thermolysin. These compounds will be used to explore in a structurally verifiable manner the effect of conformational restriction on protein-ligand binding. Haloalkene Isosteres of the Peptide Bond: Dipeptide analogs in which the amide linkage is replaced with a fluorine- or chlorine- substituted double bond. Such analogs will be prepared for enkephalin, thyrotropin releasing hormone, aspartame, and as ground-state substrate analogs for peptidases. The synthetic methodology will also be adapted for the stereospecific synthesis of ketomethylene dipeptide isoteres. In addition to direct evaluation of the ability of the above compounds to exert their intended biochemcial or biophysical effect, collaborative studies will be undertaken to determine their solution structure through 2-D NMR or their bound conformation through X-ray crystallography, where appropriate.

The University of California Berkeley will receive ARI Facilities support to create modern facilities for researchers working in a unique, integrated Program in Biological Chemistry and Engineering within the College of Chemistry. The renovated facilities will promote a strong interaction among bioinorganic chemists, biochemical engineers, and bioorganic chemists working in the areas of biotechnology and environmental research. The renovations activity funded by this award will be directed at the improvement of 1,305 square meters in 30-year old Latimer Hall and will consolidate researchers. Program activity will be enhanced as researchers are currently located in geographically distributed campus laboratories that are between 32 and 77 years old. A 7 month- long space assessment precedes the project which will involve gutting much of the interior space, installation of modular laboratory units and new fume hoods and upgrades to the mechanical, electrical and plumbing services for increased safety and improved efficiency. Provision of adequate facilities will enable the basic discovery processes to be linked more closely with the development process. Biochemical engineers will study separation techniques for the recovery of biological products, biomimetic adsorbents for metal removal and recovery and new approaches to bioremediation. Bioinorganic chemists will study metal ion transport that is essential to life processes, the use of chelating agents in the sequestration of heavy metals, and lanthanide complexes for possible use in enhancing MRI. A total of 8 professors and 117 graduate students and postdoctoral fellows will benefit from these improvements.

This award from the Chemistry Research Instrumentation and Facilities (CRIF) Program will assist the Department of Chemistry at the University of California Berkeley acquire a NMR. This equipment will enhance research in a number of areas including the following: (1) application of organic synthesis to biologial problems including helix nucleators and macrocyclic peptidase inhibitors (2) synthesis and exploratory chemistry of organometallic compounds and the investigations of their reaction mechanisms (3) organic synthesis methodology and total synthesis of natural products including zaragozic acid and dictyoxetane (4) molecular recognition and catalysis in biological systems and applications of these studies to the design and synthesis of new molecules with novel biological properties, and (5) exploratory synthetic, structural and reactivity studies on novel inorganic systems including transition metal-silicon chemistry and metal-mediated routes to charge-transporting polymers. Nuclear Magnetic Resonance (NMR) spectroscopy is the most powerful tool available to chemists for the elucidation of the structure of molecules. It is used to identify unknown substances, characterize specific arrangements of atoms within molecules, and to study the dynamics of interactions between molecules in solution. Access to state-of-the-art NMR spectrometry is essential to chemists who are carrying out frontier research. The results from these NMR studies are useful in the areas such as polymers, catalysis, and in biology