Chemoenzymatic Synthesis of 9,11-Secosteroids Using an Enzyme Extract from a Marine Coral by Lesbeth C. Rodriguez A Thesis Submitted to the Faculty of the College of Science in Partial Fulfillment of the Requirements for the Degree of Master of Science Florida Atlantic University Boca Raton. Florida August 1997 Chemoenzymatic Synthesis of 9,11-Secosteroids Using an Enzyme Extract from a Marine Coral by Lesbeth C. Rodriguez This thesis was prepared under the direction of the candidate's thesis advisor, Dr. Russell G. Kerr, Department of Chemistry and Biochemistry, and has been approved by the members of her supervisory committee. It was submitted to the faculty of The College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science. TTEE Thesis Advisor Chairperson, Department of Chemistry 7-/.7--f? ch Date ii Acknowledgments I would like to extend my gratitude to my thesis advisor, Dr. Russell G. Kerr, for his time, patience, and advice. I would also like to thank my parents and my husband Cristobal for their constant support and encouragement. Finally, I would like to acknowledge the Florida Sea Grant College Program for their financial support. iii Abstract Author: Lesbeth C. Rodriguez Title: Chemoenzymatic Synthesis of 9, 11-Secosteroids Using an Enzyme Extract from a Marine Coral Institution: Florida Atlantic University Thesis Advisor: Dr. Russell G. Kerr Degree: Master of Science Year: 1997 9, 11-Secogorgosterol, a secondary metabolite from the gorgonian Pseudopterogorgia americana, exhibits inhibitory activity against protein kinase C, and potent anti-proliferative and anti-inflammatory activity. An efficient method for the production of 9, 11-secogorgosterol has been deve loped and optimized using an enzyme extract from the gorgonian P. americana. The gorgonian also produces two other 9 ,11-secosteroids which have marked differences in their side chains and nuclei, which suggested that the enzymes responsible fo r their production were likely relatively nonspecific. Novel 9, 11-secosteroids have been synthesized using the enzyme extract from the gorgonian. iv Table of Contents Page List of Tables VII List of Figures Vlll Chapter 1 Introduction 1. Biomedical significance of marine natural products 2. Natural products from gorgonians . 4 3. Secosteroids from marine sources . 8 4. Research goals 18 Chapter 2 Development and optimization of the chemoenzymatic synthesis of 9,11- secogorgosterol 21 1. Optimization of co-factors . 22 2. Optimization of incubation time 27 -'" · Acetone powder concentration 30 v Chapter 3 Production of known and novel 9, 11 -secosteroids 1. Structural variation ""-'-' 2. Characterization of novel secosteroids 34 Chapter 4 Preliminary studies directed at enzyme isolation and elucidation of biosynthetic pathway 41 l . Importance of enzyme purification 41 2. Attempts to isolate intermediates . 45 3. Mechanism of 9, 11-secosteroid production 48 Chapter 5 Experimental section 52 1. Instrumentation 2. Materials 52 3. Analysis of 9, 11-secogorgosterol in specimens of P. americana 53 4. Isolation of gorgosterol 54 5. Cell-free extract 54 6. Acetone powder preparation 55 7. Synthesis of cholestanol 55 8. Optimum incubation conditions for secosteroid biosynthesis 56 9. Preparation of 9, 11-secosteroids 56 References 57 VI List of Tables Table Page Optimization of co-factors 27 2 Optimization of incubation time 28 3 Optimization of acetone powder 31 4 HPLC retention times of 9, 11-secosteroids 38 5 TLC analysis of novel 9, 11-secosteroids 39 vii List of Figures Figure Page Examples of biologically active marine natural products. 2 2 Natural products from gorgonians 6 Examples of bioactive steroids 10 4 Examples of secosteroids from marine sources 11 5 Further examples secosteroids from marine sources 12 6 9, 11-Secosteroids from marine sources 15 7 Examples of 9, 11-secosteroids from marine sources . 16 8 Further examples of 9, 11-secosteroids from marine sources . 17 9 Examples of 9, I 1-secosteroids from the gorgon ian P. americana 19 I 0 Reduction of Nicotinamide Adenine Dinucleotide (NAD+) and Nicotinamide Adenine Dinucleotide Phosphate (NADP+) 24 11 Enzymatic oxidation of cholic acid 26 12 Optimization of incubation time 29 13 Yields of '·natural" and ''unnatural" 9, 11-secosteroids 35 14 Yields of '·unnatural" 9, 11-secosteroids 36 15 Structural diversity of9,11-secosteroids 37 16 1H-NMR of 9, 11-secogorgosterol 40 17 18 %T SDS gel 46 viii 18 Proposed mechanism of secosteroid production 49 19 Synthesis of 3~ , 6a-dihydroxy-9-oxo-9.11-seco-5a-cholest-7-en-11-al 51 ix CHAPTER I INTRODUCTION 1. Biomedical significance of marine natural products Natural products are secondary metabolites which are a significant source of biologically active compounds. By definition. these compounds are not necessary for the basic sustenance of life, but in some manner provide an adaptive advantage to the producing organism. Recently. the marine environment has shown to be an important source of compounds with potent and novel biological activity. Compounds isolated from marine sources are believed to have potent activity due to the intense competition for space in reef communities and the age (in evolutionary terms) of marine invertebrates. Biologically active compounds have been isolated from the major marine phyla with the majority of novel corrnounds being isolated from sponges, micro algae, coelenterates, and tunicates. Three examples of marine natural products with promising therapeutic activity are the bryostatins, the ecteinascidins, and the dolastatins. Bryostatins are a group of macrocyclic lactones isolated from the bryozoan 1 Bugula neritina • One of the bryostatins, bryostatin 1 (1), has been found to have antileukemic properties. It has shown inhibitory activity against P 388 murine lymphocyte leukemia cell line and other tumors'. It has been isolated fro m the source organism trace amounts, which presents a supply problem. It has been estimated that about 10 kg of bryostatin 1 could be required on an annual basis, which requires that a large number of Figure 1: Examples of biologically active natural products OCI-!1 Cl) OAc Meqc OH 1 2 0 _:r-N 1:0s ~, 3 2 the organisms be collected. Eventually, the population of Bugufa neritina could be greatly diminished. It is believed that bryostatin I will be an important anticancer drug; however, there is not enough bryostatin I to satisfy its demand. Because of its promising results as a potent anti-cancer drug. bryostatin I is being extensively studied by scientists all over the world who are working on possible solutions for the supply and demand problem ofbryostatin 1. It is undergoing several clinical trials in United States as well as in the U.K . A second example of a class of potent biologically active compounds are the ecteinascidins. These alkaloids have been isolated from the tunicate Ecteinascidia turbinata. Ecteinas -..,~ din 74j (2) is the most abundant of the ecteinascidins and has been isolated in trace amounts. These compounds have shown very promising activity against solid tumors. They exhibit activity against P388 lymphoma. B 16 melanoma, M5076 ovarian sarcoma, lewis carcinoma. and the LX- I human lung and MC-1 human mammary 2 carcinoma xenografts . As in the case of the bryostatins, ecteinascidin 743 is obtained by isolation from the producing organism. Ecteinascidin 743 has been synthesized, and its 3 synthesis involves several steps . This process is expensive, time consuming and labor intensive. Currently, there are many research groups trying to find better methods of producing ecteinascidin 743, which is undergoing clinical trials in the United States and Europe. The third example of a class of biologically active compounds are the dolastatins. 4 small peptides isolated from the marine sea hare Do/abe/fa auricufaria . They exhibit 3 4 very potent activity with dolastatin 10 (3) being the most active one . Dolastatin 10 has shown a 17-67% curative response at 3.25-26 f..lg/kg against the NCI human melanoma xenograph, 42-138% life extension at 1.44-11.1 f..lg/kg using the Bl6 melanoma, and 69- 5 102% life extension at 1-4 f..lg/kg against the PS leukemia . Dolastatins have been shown to inhibit cell growth, and have antimitotic and anti-proliferative activitl. The effects of dolastatins 10 and 15 given after treatment with bryostatin 1 on human diffuse large cell 7 lymphoma cell line are also being investigated . The dolastatins, especially dolastatin 10, are undergoing clinical trials in the US and other countries. 2. Natural products from gorgonians More than half of the marine natural products have been isolated from corals and sponges. Many of these corals are found in the warm shallow waters of the Caribbean, 8 Bahamas, Florida and Bermuda . Gorgonians are plant-like seafans, sea rods, flat sea 8 whips, sea whips, and sea feather plumes, which are found in the above-mentioned areas . They have flexible skeletons made of a substance called gorgonin, and thousands of individual polyps cover the surface of each colonl. Gorgonians belong to the order Gorgonacea and the animal phylum Coelenterata. Their outer crust consists of calcium 9 carbonate spicules surrounding flexible skeletons . They are permanently or semi­ permanently attached to the sea bottom. In the sea. there are a large number of animals and plants called plankton, which are made up of small crustaceans, worms, snails, fish, 8 and other small organisms . To feed, the gorgonians wait for the current to bring these organisms to them. They feed on small animals by discharging their nematocysts, which 4 10 are cells that have stinging properties . Besides feeding on the plankton, gorgonians are known to feed by extracting organic matter from seawater. They are also believed to feed on the symbiotic algae which live in their tissue. The algae are single-celled 8 dinoflagellates called zooxanthellae . The zooxanthellae provide nourishment to the coral by passing some of the organic matter to the coral.