Progress 01/01/06 to 12/31/06
Outputs
New intermediates have been identified on the anaerobic pathway to vitamin B12. Oxidized forms of taxadiene have been prepared using SeO2 and characterized by NMR spectroscopy. Intermediates on the pathway to taxadiene have been intercepted by use of altered starting materials, giving insight into the cyclization mechanism. Ribozymes have been used to prepare Claisen condensation products. A series of insect neuropeptide analogs containing -amino acid residues have been prepared and bioassayed.
Impacts
Our research aim is to use enzymes to perform reactions and produce chemical products which would be prohibitively expensive to synthesize using normal chemical synthetic methods. In several cases we have shown that mixtures of enzymes can carry out several synthetic steps in a procedure leading to optically active, highly functionalized products from simple, cheap precursors. Studies of insect neuropeptide analogs may eventually lead to highly specific, environmentally friendly insect control agents.
Publications
- Q.Huang, J. D. Pennington, H. J. Williams, and A. Ian Scott. 2006. Models for Taxol Biosynthesis: SeO2 Oxidation of Taxadiene. Synthetic Commun. 36, 2577.
- P. J. Santander, Y. Kajiwara, H. J. Williams, and A. I. Scott. 2006 Structural Characterization of Novel Cobalt Corrinoids Synthesized by Enzymes of the Vitamin B12 Anaerobic Pathway. Bioorg. Med. Chem. 14, 724.
- Y. Ryu, K-J. Kim, C. A. Roessner and A. I. Scott. 2006. Decarboxylative Claisen Condensation Catalyzed by in vitro Selected Ribozymes. Chem. Commun., 1439.
- C. Pichon-Santander, P. J. Santander and A. I. Scott. 2006. Synthesis of Substrate Analogs of Methyltransferases in the Vitamin B12 Biosynthetic Pathway and Characterization of Their Enzymatic Products. Bioorganic and Medicinal Chemistry, 3904.
- Y. Dajiwara, P. J. Santander, C. A. Roessner, L. M. Perez, and A. Ian Scott. 2006. Genetically Engineered Synthesis and Structural Characterization of Cobalt-Precorin 5A and 5B, Two New Intermediates on the Anaerobic Pathway to Vitamin B12: Definition of the Roles of the CbiF and CbiG Enzymes. J. Amer. Chem. Soc. 128, 9971.
- C. A. Roessner and A. I. Scott. J. Bacteriol. 2006. Fine-Tuning Our Knowledge of the Anaerobic Route to Cobalamin (Vitamin B12).7331 (226).
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Progress 08/30/00 to 08/29/06
Outputs
New intermediates have been identified on the anaerobic pathway to vitamin B12. Oxidized forms of taxadiene have been prepared using SeO2 and characterized by NMR spectroscopy. Intermediates on the pathway to taxadiene have been intercepted by use of altered starting materials, giving insight into the cyclization mechanism. Ribozymes have been used to prepare Claisen condensation products. A series of insect neuropeptide analogs containing -amino acid residues have been prepared and bioassayed.
Impacts
Our research aim is to use enzymes to perform reactions and produce chemical products which would be prohibitively expensive to synthesize using normal chemical synthetic methods. In several cases we have shown that mixtures of enzymes can carry out several synthetic steps in a procedure leading to optically active, highly functionalized products from simple, cheap precursors. Studies of insect neuropeptide analogs may eventually lead to highly specific, environmentally friendly insect control agents.
Publications
- Q.Huang, J. D. Pennington, H. J. Williams, and A. Ian Scott. 2006. Models for Taxol Biosynthesis: SeO2 Oxidation of Taxadiene. Synthetic Commun. 36, 2577.
- P. J. Santander, Y. Kajiwara, H. J. Williams, and A. I. Scott. 2006. Structural Characterization of Novel Cobalt Corrinoids Synthesized by Enzymes of the Vitamin B12 Anaerobic Pathway. Bioorg. Med. Chem. 14, 724.
- Y. Ryu, K-J. Kim, C. A. Roessner and A. I. Scott. 2006. Decarboxylative Claisen Condensation Catalyzed by in vitro Selected Ribozymes. Chem. Commun., 1439.
- C. Pichon-Santander, P. J. Santander and A. I. Scott. 2006. Synthesis of Substrate Analogs of Methyltransferases in the Vitamin B12 Biosynthetic Pathway and Characterization of Their Enzymatic Products. Bioorganic and Medicinal Chemistry, 3904.
- Y. Dajiwara, P. J. Santander, C. A. Roessner, L. M. Perez, and A. Ian Scott. 2006. Genetically Engineered Synthesis and Structural Characterization of Cobalt-Precorin 5A and 5B, Two New Intermediates on the Anaerobic Pathway to Vitamin B12: Definition of the Roles of the CbiF and CbiG Enzymes. J. Amer. Chem. Soc. 128, 9971 (2006).
- C. A.Roessner and A. I. Scott. J. Bacteriol. 2006. Fine-Tuning Our Knowledge of the Anaerobic Route to Cobalamin (Vitamin B12). 7331 (226).
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Progress 01/01/05 to 12/31/05
Outputs
We have made further progress determining the role of individual enzymes and identifying intermediate chemicals synthesized on the anaerobic pathway to vitamin B12. We have also identified products produced when unusual substrates are metabolized to determine steps involved in transformation of geranylgeranyl diphosphate to taxadiene by the enzyme taxadiene synthase. New insect neuropeptide analogs have been prepared, their structures have been determined by NMR spectroscopy and molecular modeling, and their mode of action has been studied.
Impacts
Our research aim is to use enzymes to perform reactions and produce chemical products which would be prohibitively expensive to synthesize using normal chemical synthetic methods. In several cases we have shown that mixtures of enzymes can carry out several synthetic steps in a procedure leading to optically active, highly functionalized products from simple, cheap precursors. Studies of insect neuropeptide analogs may eventually lead to highly specific, environmentally friendly insect control agents.
Publications
- R. J. Nachman, T. Vercammen, H. Williams, K Kaczmarek, J. Zabrocki, and L. Schoofs, Peptides 26, 115-120 (2005). Aliphatic Amino Diacid Asu Functions as an Effective Mimic of Tyr(SO3H) in Sulfakinins for Myotropic and Food Intake-inhibition Activity in Insects.
- C. A. Roessner, H. J. Williams , and A. I. Scott. J. of Biological Chemistry. 280, 16748-16753 (2005). Genetically Engineered Production of 1-Desmethylcobyrinic Acid, 1-Desmethylcobyrinic Acid a,c-Diamide, and Cobyrinic Acid a,cDiamide in Escherichia coli implies a Role for CbiD in C-1 Methylation in the Anaerobic Pathway to Cobalamin.
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Progress 01/01/04 to 12/31/04
Outputs
New compounds have been synthesized using improved methods to study polyketide biosynthesis by ribonucleic acid enzymatic action. New peptide analogs have been synthesized and their conformations analyzed by NMR spectroscopy and molecular modeling in a program to develop insect neuropeptide based pesticides. Further studies of enzymes involved in vitamin B-12 have led to identification of new intermediates and new understanding of enzyme mechanisms.
Impacts
Use of enzymes to prepare complicated and expensive chemicals should be cost effective and produce minimum environmental damage. Development of insecticidal agents based on insect neuropeptide analogs should lead to selective control without adversely affecting the environment and other organisms. Studies of ribonucleic acid enzymatic activity may lead to new insights on early life processes.
Publications
- S. Nanda and A. I. Scott. 2004. Asymmetric Synthesis of (E)- and (Z)-3,7-Dimethyl-2-octene-1,8-diol and Callosobruchusic Acid. Tetrahedron: Asymmetry, 15, 963-970.
- S. Nanda and A. I. Scott. 2004. A Highly Efficient Chemoselective Synthesis of 3,5-Diketoesters by Lipase-catalyzed Transesterification: Application to the Resolution of Secondary Alcohols. Journal of Molecular Catalysis B: Enzymatic, 30, (1), 1-12.
- J. Vevodova, R. M. Graham, E. Raux, H. L. Schubert, D. I.. Roper, A. A. Brindley, A. I. Scott, C. A. Roessner, N. Patrick, J. Stamford, M. E. Stroupe, E. D. Getzoff, M. J. Warren and K. S. Wilson. 2004. Structure/Function Studies on a S-Adenosyl-L-methionine-dependent Uroporphyrinogen III C Methyltransferase (SUMT), a Key Regulatory Enzyme of Tetrapyrrole Biosynthesis. J. Mol. Biol. 344, 419-433.
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Progress 01/01/03 to 12/31/03
Outputs
New compounds have been identified on the anaerobic pathway leading to vitamin B-12 through incubation experiments using labeled substrates and cloned enzymes which have a role in the biochemical transformations. New insect neuropeptide analogs have been prepared and tested as pesticide agents based on our earlier conformational analyses and structure-activity relationship studies.
Impacts
Use of enzymes to prepare complicated and expensive chemicals should be cost effective and produce minimum environmental damage. Development of insecticidal agents based on insect neuropeptide analogs should lead to selective control without adversely affecting the environment and other organisms.
Publications
- An Improved Transient Method for the Synthesis of N-Benzoylated Nucleosides. X-F Zhu, H. J. Williams and A. I. Scott. Synthetic Communications 33, 1233-1243 (2003).
- Genetic and Mechanistic Exploration of the Two Pathways of Vitamin B12 Biosynthesis. A. I. Scott, C. A. Roessner and P. J. Santander, Chapter 76 in 'The Porphyrin Handbook II, Vol. 12' K. M. Kadish, K. M. Smith, R. Guilard, Eds. Academic Press, New York, 2003, p. 211.
- Discovering Nature's Diverse Pathways to Vitamin B12: A 35-year Odyssey. A. I. Scott. Journal of Organic Chemistry 68 (7), 2529-2539 (2003).
- Aqueous Trichloroacetic Acid: Another Useful Reagent for Highly Selective 5'-Desilylation of Multisilylated Nucleosides. X-F Zhu, H. J. Williams and A. I. Scott. Synthetic Communications, 33, 2011-2016 (2003).
- Self-condensation of activated malonic acid half esters: a model for the decarboxylative Claisen condensatin in polyketide biosynthesis. Y. Ryu and A. I. Scott. Tetrahedron Letters, 44, 7499-7502 (2003).
- Efficient One-Step Syntheses of Isoprenoid Conjugates of Nucleoside 5'-Diphosphates. Y. Ryu and A. I. Scott. Organic Letters, 5, 4713-4715 (2003).
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Progress 01/01/02 to 12/31/02
Outputs
Genes responsible for production of enzymes from the anaerobic pathway leading to vitamin B-12 have been expressed in E. coli and new products of these genes are now being identified using NMR and mass spectroscopy. New insect neuropeptide analogs have been prepared for structure activity correlations. Syntheses of important substrates for enzymatic activity studies were developed.
Impacts
Use of enzymes to prepare complicated and expensive chemicals should be cost effective and produce minimum environmental damage. Development of insecticidal agents based on insect neuropeptide analogs should lead to selective control without adversely affecting other organisms.
Publications
- Cis-peptide bond mimetic tetrazole analogs of the insect kinins identify the active conformation. Nachman, . R. J.; Zabrocki, J.; Olczak, J.; Williams, H.J.; Moyna, G.; Scott, A. I.; and . Coast,G. M.; Peptides, 23, 709-716 (2002).
- Biosynthesis of cobalamin (vitamin B12). Scott, A. I.; Roessner, C. A. Biochemical Society Transactions (2002), 30(4), 613-620.
- Studies on the tetramerization of substituted monopyrroles to type I porphyrins. Pichon-Santander, C.; Scott, A. I.. Tetrahedron Letters (2002), 43(39), 6967-6969.
- Mutagenesis identifies a conserved tyrosine residue important for the activity of uroporphyrinogen III synthase from Anacystis nidulans. Roessner, Charles A.; Ponnamperuma, Krishan; Scott, A. I.. FEBS Letters (2002), 525(1-3), 25-28.
- Isolation and characterization of 14 additional genes specifying the anaerobic biosynthesis of cobalamin (vitamin B12) in Propionibacterium freudenreichii (P. Shermanii). Roessner, Charles A.; Huang, Ke-Xue; Warren, Martin J.; Raux, Evelyne; Scott, A. Ian. Microbiology (Reading, United Kingdom) (2002), 148(6), 1845-1853.
- Structure of the Methanococcus jannaschii mevalonate kinase, a member of the GHMP kinase superfamily. Yang, Dong; Shipman, Lance W.; Roessner, Charles A.; Scott, A. Ian; Sacchettini, James C. Journal of Biological Chemistry (2002), 277(11), 9462-9467.
- Sterol carrier protein-2: structure reveals function. Stolowich, N. J.; Petrescu, A. D.; Huang, H.; Martin, G. G.; Scott, A. I.; Schroeder, F. Cellular and Molecular Life Sciences (2002), 59(2), 193-212.
- An efficient synthesis of 5-amino[3-13C] levulinic acid. Kajiwara, Y.; Scott, A. I.; Tetrahedron Letters, 43, 8795-8796 (2002).
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Progress 01/01/01 to 12/31/01
Outputs
During the past year, our studies of insect neuropeptide structure activity relationships and biosynthetic pathways leading to terpenes and vitamin B12 have continued to make good progress. Preparation of amino acids in protected form to be used in production of a new set of insect neuropeptide analogs less subject to peptidase degradation is well underway, with gram quantities of several examples now on hand. Several new enzymes in terpenoid, polyactetate, and vitamin B12 biosynthetic pathways have been cloned and overexpressed and biosynthetic products produced by these enzymes using natural and unnatural substrates have been identified using NMR and mass spectroscopy. One enzyme structure has been determined by xray crystallography. New synthetic methods for protection of nucleic acid precursers useful in these studies have been developed.
Impacts
Use of enzymes to prepare complicated and expensive chemicals should be cost effective and produce minimum environmental damage. Development of insect control agents based on insect neuropeptide analogs should lead to selective control without affecting other organisms.
Publications
- Roessner, C.A., Santander, P.J. and Scott, A.I. (2001) 'Multiple Biosynthetic Pathways for Vitamin B12:Variations on a Central Theme,' Vitamins and Hormones, Vol 61, T. P. Begley, Ed., Academic Press, New York,267.
- Scott, A.I. (2001)'Reflections on the Discovery of Nature's Pathways to Vitamin B12,' The Chemical Record, 1, 212-227.
- Zhu, X. F. and Scott, A.I. (2001) 'An Improved Synthesis of the Dinucleotides pdCpA and pdCpdA,' Nucleosides, Nucleotides & Nucleic Acids, 20, 197-211.
- Huang, Q., Roessner, C.A., Croteau, R., Scott, A.I.,(2001) 'Engineering Escherichia coli for the Synthesis of Taxadiene, a Key Intermediate in the Biosynthesis of Taxol.',Bioorganic & Medicinal Chemistry, 9, 2237-2242.
- Shipman, L.W., Li, D., Roessner, C.A., Scott, A.I. and Sacchettini, J.C., (2001) 'Crystal Structure of Precorrin-8x Methyl Mutase', Structure, 9, 587-596.
- Benedict, C.R., Lu, J.L., Pettigrew, D.W., Liu, J., Stipanovic, R.D., Williams, H.J. (2001), 'The Cyclization of Farnesyl Diphosphate and Nerolidyl Diphosphate by a Purified Recombinant Cadinene Synthase.', Plant Physiology, 125, 1754-1765.
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Progress 01/01/00 to 12/31/00
Outputs
Studies of substrate specificity and unusual products produced by mutated taxadiene synthase enzymes gave insights concerning active site configuration and also led to increased production of the desired diterpene product. Biosynthetic pathways producing fungal metabolites were studied. Further work concerning insect neuropeptide conformation by NMR spectroscopy and molecular modeling were performed with the object of designing a new class of pesticides. New methods of preparing nucleosides were developed.
Impacts
Preparation of commercially valuable natural products using recombinant DNA techniques and use of neuropeptides as insecticides are now more feasible.
Publications
- X. Zhu, H. J. Williams, and A. I. Scott. 2000. Facile and Highly Selective 5'-Desilylation of Multisilylated Nucleosides, J. Chem. Soc., Perkin Trans. 1, 2305.
- G. Moyna, H. J. Williams, R. J. Nachman, and A. I. Scott. 1999. Detection of Nascent Polyproline II Helices in Solution by NMR in Synthetic Insect Kinin Neuropeptide Mimics Containing the X-Pro-Pro-X Motif, J. Peptide Res. 53, 294.
- P. Spencer, F. Agnelli, H. J. Williams, N. P. Keller, and G. A. Sulikovski. 2000. Biosynthetic Studies on the Fungal Secondary Metabolites CP-225,917 and CP-263,114, J. Amer. Chem. Soc. 122, 420.
- R. J. Nachman, G. Moyna, H. J. Williams, J. Zabrocki, J. E. Zadina, G. M. Coast, and J. Vanden Brock. 1999. Comparison of Active Conformations of the Insecttachykinin/tachykinin and Insect Kinin/Tyr-W-MIF-1 Neuropeptide Family Pairs, Ann. N. Y. Acad. Sci. 897, 388.
- HU, H., Dun, D-1, and Scott, A. I. 2000. An efficient synthesis of 4(5), 11(12)-taxadiene derivatives and microbial mediated 20-hydroxylation of taxoids. Tetrahedron Letters, 41, 1703-1705.
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Progress 01/01/99 to 12/31/99
Outputs
Further NMR and molecular modeling studies of the relationship between conformation and biological activity in taxol analogs were completed. New force field parameters for use in conformational analysis of insect neuropeptides containing unnatural amino acids were developed and tested. Efficient new methods were developed for synthesizing S-adenosyl-L-methionine, taxol side chain precursors, and deuterium labeled cadinene. Novel saccharides, caryophyllene derivitives, and diterpenoids were isolated from cotton and other plant sources and their structures were determined by NMR spectroscopy and X-ray crystallography. Biotransformations of terpenes with cell suspension cultures produced new fragant hydroxy compounds.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/98 to 12/31/98
Outputs
New natural products were identified from cotton or Gossypium and Iva frutescens. Work progressed well in the development of insect neuropeptide analogs for use as environmentally friendly pesticide reagents. New methods were developed which allowed determination of neuropeptide conformation based on chemical shift perturbation by aromatic groups in the molecule. Use of this method and coupling constant constraint methods developed earlier allowed determination of conformation of new analogs with turn promoting moieties which cause the molecule to take on an active conformation but prevent degradation by peptidases present in insect hemolymph or blood. Knowledge gained from these studies has led to preparation of new constrained neuropeptide analogs which are both highly active and stable to peptidase inactivation.
Impacts
(N/A)
Publications
- H. J. Williams, G. Moyna, S. B. Vinson, A. Ian. Scott, A. A. Bell, and R. D. Stipanovic, 1998, _-Caryophyllene Derivatives from the Wild Cottons Gossypium armourianum Kearn., Gossypium harknessii Brandg., and Gossypium turneri Fryx., Nat. Prod. Lett., 11, 25.
- G. Moyna, R. Zauhar, H. J. Williams, R. J. Nachman, and A. I. Scott, 1998, Comparison of Ring Current Methods for use in Molecular Modeling Refinement of NMR Derived Three Dimensional Structures, J. Chem. Inf. Comput. Sci., 38, 702.
- A. A. Ahmed, B. A. A. Balboul, A. I. Scott, H. J. Williams, K. B. Miao, and T. J. Mabry, 1998, Eudesmane derivatives from Iva frutescens, Phytochem., 47, 411.
- R. J. Nachman, G. Moyna, H. J. Williams, S. S. Tobe, and A. I. Scott, 1998, Synthesis, Biological Activity, and Conformational Studies of Insect Allatostatin Neuropeptide Analogues Incorporating Turn-Promoting Moieties, Bioorg. & Med. Chem., 1379.
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Progress 01/01/97 to 12/31/97
Outputs
Efficient new methods were developed for synthesizing S-adenosyl-L-methionine, taxol side chain precursors, and deuterium labeled cadinene. Novel saccharides, caryophyllene derivitives, and diterpenoids were isolated from cotton and other plant sources and their structures were determined by NMR spectroscopy and X-ray crystallography. Biotransformations of terpenes with cell suspension cultures produced new fragrant hydroxy compounds. Further NMR and molecular modeling studies of the relationship between conformation and biological activity in taxol analogs were completed. New force field parameters for use in conformational analysis of insect neuropeptides containing unnatural amino acids were developed and tested.
Impacts
(N/A)
Publications
- SCOTT, A.I., STIPANOVIC, R.D., WILLIAMS, H.J., SATTLER, I., VINSON, S.B., AND LIU, J. 1997. Preparation of Two Stereochemically Defined Isomers of Deuterium Labeled d- Cadiene. J. Of Labeled Compounds and Radiopharmaceuticals, 39:223-230.
- SCOTT, A.I., MOYNA, G., AND WILLIAMS, H.J. 1997. Preparation of Aminated Taxol Side Chain Precursors, A Simple Approach to 2,3-Diamino Acids Using the Beta-Lactam Synthon Method. Synthetic Communications, 27:1561-1567.
- SCOTT, A.I., AHMED, A., MOHAMED, T.K., WILLIAMS, H.J., AND REIBENSPIES, J.H. 1997. Structure Revision of Cleoamblynol A From Cleome Amblyocarpa. National Product Letters, 10:239-244.
- SCOTT, A.I., WILLIAMS, H.J., HAMADA, H., YASUMUNE, H., FUCHIKAMI, Y., HIRATA, T. AND SATTLER, I. 1997. Biotransformation of Geraniol, Nerol and (+)- and (-)-Carvone by Suspension Cultured Cells of Catharanthus Roseus. Phytochemistry, 44:615-621.
- SCOTT, A.I., MOYNA, G. AND WILLIAMS, H.J. 1997. Conformational Studies of Paclitaxel Analogs Modified at the C-12' Position in Hydrophobic and Hydrophilic Solvent Systems. J. Med. Chem., 40:3305-3311.
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Progress 01/01/96 to 12/30/96
Outputs
A novel inhibitor for the enzyme urogen III synthase was synthesized. A new procedure was developed for the synthesis of taxol side chain and the conformation and reactions of taxol studied. Several substituted pyrroles for use in enzyme inhibition were prepared and our work on genetically engineered synthesis was reviewed. The conformation of cephalomannine, an anti-tumor substance was deduced by NMR.
Impacts
(N/A)
Publications
- MOYNA, G., WILLIAMS, H. J., and SCOTT, A.I. 1996. An Improved Procedure for the Epoxidation of Methyl Cinnamate Derivatives and Production of Acid Sensitive Expoxides. Synthetic Communications, 26: No. 11.
- WILLIAMS, H.J., MOYNA, G., SCOTT, A.I., SWINDELL, L.E., CHIRLIAN, J. M., HEERDING, and WILLIAMS, D.K. 1996. NMR and Molecular Modeling Study of the Conformations of Taxol 2'-Acetate in Chloroform and Aqueous Dimethyl Sulfoxide Solutions. Med HAMADA, H., SANADA, K., IKEDA, S., ODA, T., WILLIAMS, H.
- J., MOYNA, G., and SCOTT, A.I. 1996. Epimerization of Taxol in Human Cancer Cells. Natural Product Letters, 8: 1130117.
- WANG, J. and SCOTT, A.I. 1996. A General Synthesis of Beta-Aryl and Heteroarylpyrroles by Palladium-catalyzed Coupling Reaction of Beta-Tributylstannylpyrrole with Aryl and Heteroaryl Halides. Tetrahedron Letters, 37: 3247-3250. ROESSNER C.A. and SCOTT, A.I. 1996. Genetically Engineered Synthesis of Natural Products: From Alkaloids to Corrins. Ann Rev. Microbiol, 50: 467-490.
- MOYNA, G., MEDIWALA, S., WILLIAMS, H. J., and SCOTT, A.I. 1996. A Simple Algorit.
- PICHON, C. and SCOTT, A.I. 1996. Synthesis of a novel tetrapyrrolic macrolactam and its activity towards Uro'gen III synthase. Recl. Trav. Chim. Pays-Bas, 115: 1-8.
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Progress 01/01/95 to 12/30/95
Outputs
A wide array of slow substrates and inhibitors were synthesized and used to investigate the binding specificity of porphoblinogen (PBG) deaminase and uro'gen III synthase, two enzymes essential for the synthesis of hemes, chlorophylls, and Vitamin B12. Of particular interest, an a-fluoro analog of PBG was synthesized and shown to be an irreversible inhibitor of PBG deaminase, while the a-bromo- and a-methyl analogs of HMB, the substrate for uro'gen III synthase, were shown to be competitive inhibitors of that enzyme. Further NMR studies of the enzyme-inhibitor complexes are now in progress Model studies performed using NMR spectroscopy of the mechanism by which Vitamin B12 facilitates methyl migration in methyl malonyl-CoA mutase indicated that initial cobalt-carbon bond formation is involved. An extensive study of the conformation of active and inactive analogs of taxol in aqueous and organic solvents showed that two conformations were predominant and activity
correlated best with the organic conformation, indicating a possible hydrophobic active site for this anticancer agent.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
A wide array of slow substrates and inhibitors were synthesized(superscript 1-4)and used to investigate the binding specificity of porphoblinogen (PBG) deaminase and urogen III synthase, two enzymes essential for the synthesis of hemes, chlorophylls, and Vitamin B(subscript 12). Of particular interest, an (alpha)-fluoro analog of PBG was synthesized and shown to be an irreversible inhibitor of PBG deaminase(superscript 2), while the (alpha)-bromo- and (alpha)-methyl analogs of HMB, the substrate for Uro'gen III synthase, were shown to be competitive inhibitors of the enzyme(superscript 3,4). Further NMR studies of the enzyme-inhibitor complexes are now in progress. Model studies performed using NMR spectroscopy of the mechanism by which Vitamin B(subscript 12) facilitates methyl migration in methyl malonyl-CoA mutase indicated that initial cobalt-carbon bond formation is involved(superscript 5). An extensive study of the conformation of active and inactive analogs of
taxol in aqueous and organic solvents showed that two conformations were predominant and activity correlated best with the organic conformation, indicating a possible hydrophobic active site for this anticancer agent.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
(superscript 1)H and (superscript 13)C NMR studies of sirohydrochlorin (Factor II) and its 20-methyl derivative (Factor III).(superscript 1) (superscript 13)C NMR studies of plant cell culture metabolism using stirred aerated media: metabolism of 1-(superscript 13)C-glucose in Catharanthus roseus cell suspensions.(superscript 2) Sequence of the Candida albicans erg7 gene.(superscript 3) NMR and molecular modeling study of the conformations of taxol and of its side chain methylester in aqueous and non-aqueous solution.(superscript 4) Interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 17(beta)estradiol in MCF-7 human breast cancer cells.(superscript 5) New sesquiterpene (alpha)-methylene lactones from the Egyptian plant Jasonia candicans.(superscript 6).
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Conformation of vinblastine in aqueous solution determined by 2D (superscript 1)H- and (superscript 13)C-NMR spectroscopy.(superscript 1) Observation of enzyme bound intermediates in the biosynthesis of preuroporphyrinogen by PBG deaminase.(superscript 2) Enzymatic synthesis and structure of precorrin-3, a trimethyldipyrrocorphin intermediate in vitamin B(subscript 12) biosynthesis.(superscript 3) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on 17(beta)-estradiol-induced glucose metabolism in MCF-7 human breast cancer cells: (superscript 13)C-nuclear magnetic resonance spectroscopy studies.(superscript 4) Crystal and molecular structure of echinocystic acid diacetate, a secondary metabolite isolated from Cucurbita faetidissima H.B.K.(superscript 5) Studies on the mechanism of porphobilinogen deaminase: design, synthesis and enzymatic reactions of novel porphobilinogen analogs.(superscript 6) Isolation and identification of xochitloldione and isoxochitlolone from
Cnidosculus urens.(superscript 7).
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
Bacteriochlorophyll c is formed from glutamate.(superscript 1) The biologically active porphyrin, corallistin A has been synthesized.(superscript 2) NMR devices have been constructed to follow plant cell metabolism.(superscript 3,4). The points at which cobalt and oxygen (from water) are inserted in the B(subscript 12) biosynthetic pathway have been established.(superscript 5,6) Several complex natural products have been synthesized by multi-enzyme technology.(superscript 7,8) New enzyme bound intermediates have been observed.(superscript 9,10).
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
The methyl transferase enzymes M-1 and M-2 have been over expressed from the appropriate genes from E. coli and Salmonella. These enzymes are responsible for the commitment of porphyrin substrates to the vitamin B(12) pathway. In a breakthrough experiment the gene for alkaloid synthesis in Catharanthus roseus has been sequenced and expressed in E. coli thus making the biotechnology of plant alkaloid synthesis viable for the first time. Finally the yeast steroid cyclase enzyme O.S.C. has been purified for the first time.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
Progress in 1988-89 has been excellent in the elucidation of the pathway of vitamin B(12) biosynthesis in vitro and in vivo, and the exploration of the single steps of selected enzyme-catalyzed reactions. We have established the complete methylation sequence in Vitamin B(12) biosynthesis from Uro'gen III. The genes encoding for each of the methyl transferases are clustered in one region of the Salmonella genetic map so that the remaining intermediates can now be discovered. Site directed mutagenesis of the enzyme PBG deaminase shows that the dipyrromethane cofactor is attached to Cys-242 of the enzyme. Crystals of this enzyme have now been obtained for X-ray studies. Studies of glucose metabolism in cancer cell cultures and the effect of experimental anti-cancer drugs on metabolic rates and product profiles have been carried out using our newly designed mixing system. Similar studies utilizing Catharanthus roesus plant cell suspensions with the mixing apparatus have
demonstrated the utility of the device and will allow terpene biosynthesios studies to be performed. In vivo insect cuticle formation studies utilizing new isotopomers of labeled tyrosine are in progress. H NMR imaging continues to be useful in in vivo studies of physiology and parasitoid development in insects.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
Considerable progress has been achieved in the area of Vitamin B(12)/porphyrin biochemistry. C Pulse labeling experiments have led to the resolution of the methylation sequence in the synthesis of Vitamin B(12). The combination of site-specific mutagenesis, H and C NMR spectroscopy has defined the active site of porphobilinogen deaminase to contain a novel dipyrromethane cofactor covalently bound to cysteine-242 of the enzyme. The exclusive operation of the C(5) pathway of ALA biosynthesis has been demonstrated in Clostridia, a anaerobic bacterium. Solution C and N NMR techniques have been employed to investigate the mechanisms of action or mode of substrate/inhibitor binding of several important enzymes in vitro. Examples of ongoing research in this area include the enzymes asparate aminotransferase, carboxypeptidase, elastase, and tryspin. Solid state NMR spectroscopy has been used to correlate solution and crystallographic data in several systems. To this end,
construction of a (15)N CPMAS probe has been successfully completed. (1)H NMR imaging has been proven to be useful in in vivo studies of physiology and parasitoid development in insects.
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Progress 01/01/87 to 12/30/87
Outputs
Studies of the biosynthesis of vitamin B(12) have led to identification of a newmetabolite of related structure. The origin of hydrogen atoms of vitamin B(12) has been studied by growing organisms producing the compound in D(2)0 and then determining the C NMR signal shifts in the vitamin which are caused by H incorporation. In vivo and solid state C NMR spectroscopy have been used to follow the metabolism of tyrosine during insect cuticle formation. Further studies of mechanisms of action of digestive enzymes have been performed using C and H NMR spectroscopy. Solid state NMR spectroscopy has been used to correlate solution studies of enzyme mechanisms with data obtained from X-ray crystallography of enzyme-inhibitor complexes.
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Progress 01/01/86 to 12/30/86
Outputs
Further inhibitors for thiol and serine proteases have been synthesized. These have been used with trypsin and with the thiol protease enzyme, papain, from Papaya for NMR experiments at 500 MHz. The results have now been completely confirmed by X-ray diffraction. The use of NMR has led to the discovery of the structures of intermediates with both of the above enzymes. The same techniques are being applied to other enzymes from higher plants. In vivo metabolism of parasitic organisms has been further developed using 300 MHz NMR spectroscopy. Three-dimensional computer graphics have been developed to map the active site of the above enzymes. The biosynthesis of vitamin B(12) has been further refined by NMR experiments. Several new metabolites of B(12)-producing organisms have been identified. The total synthesis of the antibiotic, tetracycline, has been completed, based on a biogenetic model. Covalent complexes of porphyrin synthesizing enzymes have been
characterized by NMR.
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Progress 01/01/85 to 12/30/85
Outputs
Inhibitors for thiol and serine proteases have been synthesized. These have been used with the thiol protease enzyme, papain, from Papaya for NMR experiments at 500MHz and compared with results obtained with the serine protease trypsin. The use of NMR has led to the discovery of the structures of intermediates with both of the above enzymes. The same techniques are being applied to other enzymes from higher plants. In vivo metabolism of parasitic organisms has been further developed using 300 MHz NMR spectroscopy. 3 dimensional computer graphics have been developed to map the active site of the above enzymes. The biosynthesis of Vitamin B(12) has been further refined by NMR experiments.
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Progress 01/01/84 to 12/30/84
Outputs
The thiol protease enzyme, papain, from Papaya has been studied by NMR experiments at 500 MHz and compared with results obtained with the serine protease trypsin. The use of NMR has led to the discovery of the structures of intermediates with both of the above enzymes. The same techniques are being applied to other enzymes from higher plants. In vivo metabolism of parasitic organisms has been developed usin 300 MHz NMR spectroscopy.
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Progress 01/01/83 to 12/30/83
Outputs
The PGDTC depleted chamber has been constructed and the requisite tissue cultures grown for the experiments described in the original proposal. Enzyme work has resulted in the publications listed below. Progress is thus in phase with our proposed timetable.
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