Chemoenzymatic Synthesis of 9,11-Secosteroids Using an Enzyme Extract from a Marine Coral

Home , Hydroxysteroid, Pseudopterogorgia

Chemoenzymatic Synthesis of 9,11-Secosteroids Using an

Enzyme Extract from a Marine Coral

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

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

1 2

0 _:r-N 1:0s ~,

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. In exchange for providing organic matter to the gorgonians, the zooxanthellae obtain a place to live and a steady supply of

10 nutrients . At the same time, many marine organisms such as tunicates, byrozoans, hydroids and sponges attach themselves to gorgonians in order to feed, grow, or settle.

While the above organisms settle on gorgonians without producing any harm to them, others are harmful. Some of the gorgonian · s predators include the Cyphoma gibbosum,

8 snails from the genus Neosimnia, and some sea slugs .

The bluish gray sea plumes which are found on the reef belong to the genus

8 Pseudopterogorgia . There are about twelve species in the genus, with gorgonians

8 Pseudopterogorgia americana, P. acerosa, and P. bipinnata being the most common .

P. americana belongs to the family Gorgoniidae, the class Anthozoa, the suborder holaxonia, and its common name is slimy sea plume. They are found in shallow water, have a slimy surface, and their polyps always remain extended. The polyps are an important part of the gorgonian as they are key in the identification of the species.

Numerous natural products have been isolated from gorgonians. In 1943,

11 Bergmann et a!. isolated gorgosterol from the gorgonian Plaxaura jlexuosa •

5 Figure 2: Natural products from gorgonians

HO HO 4 5

0 6

7 8

OR 1 9 Ps A Rt = D-xylose, R2 = H 10 Ps B Rt = D-xylose-2-acetate, R2 = H 0~ II Ps C Rt = D-xylose-3-acetate, R2 = H 12 Ps D R 1 = D-xylose-4-acetate, R2 = H 13 Ps E Rt = H, R2 = D-arabinose

6 Gorgosterol (4). which is the most abundant steroid in gorgonians, has a cyclopropane ring in its side chain. Since Bergmann, other scientists have isolated gorgosterol from a number of gorgonians including Pseudopterogorgia americana, Gorgonia ventilina,

12 Plexaura sp., P. jlexuosa, Nephthea sp. , and Palythoa tuberculosa • Schmitz and

Pattabhiraman isolated 23-demethylgorgosterol (5) from the sea fan Gorgoniaflabellum.

This sterol also had an unusual side chain containing a cyclopropane ring. This sterol and others with unusual side chains have drawn attention to marine organisms as potential

11 sources of unique steroids . Cholestenone (6). a steroidal ketone, has been isolated from

11 the gorgonians Muricea atlantica and Plaxaurella mutans • The presence of steroidal ketones in these organisms shows that they have the enzyme or enzymes necessary for the

11 conversion of sterols to steroid hormones . These enzymes can be a very useful tool for the production of steroid hormones.

A large number of sesquiterpenes have been isolated from gorgonians. For example, (+ )-~-gorgonene (7) and (-)-1 ( 10)-aristolene (8) have been isolated from the

13 gorgonian Pseudopterogorgia americana . Another example are the pseudopterosins A

14 E (9-13), which have been characterized as diterpene-pentose-glycosides . The pseudopterosins have been isolated from the Caribbean sea whip Pseudopterogorgia elisabethae. Pseudopterosin A has potent anti-inflammatory and analgesic properties,

14 and it has been shown to inhibit pancreatic phospholipase A2 (lC50= 3.0 ).!M) in vitro .

Another pseudopterosin, pseudopterosin C, is the major component of the extract that is the key ingredient of the Estee Lauder skin care product Resilience. According to Estee

7 Lauder, this product maintains the skin firmness by preventing reactions that promote

15 aging • The pseudopterosins prevent aging by inhibiting the enzyme that triggers the

15 inflammatory reaction which produces premature aging . These compounds are believed

16 to be part ofthe defense mechanism of the gorgonian .

3. Secosteroids from marine sources

Monohydrox' · steroids are compounds present in the membranes of most

eukaryotic cells. They are found in both plants and animals, and they are essential to plant

and animal life. They contain the perhydroclopentanophenanthrene nucleus which is

17 planar and rigid . They are derived from six isopentenyl pyrophosphate units and thus

are classified as triterpenes.

Some steroids of significant importance include vitamin D, cholesterol, bile salts, ergosterol, lanosterol, and sex hormones. Cholesterol (14), one of the most common steroids, has been found in most animal tissue, and it is needed for membrane

18 19 construction · . It is widely found in the human body, where it also serves as an intermediate for the biosynthesis of all the other steroids in the body.

A large number of bioactive steroids have been isolated from marine sources.

Many of these steroids have unusual side chains and unique functionalization, which are their key features. For example, halistanol sulfate (15) has been isolated from the sponge

20 Halichondria moorei . This steroid is important because it has shown activity against

20 the HIV virus .

Secosteroids are a class of steroids characterized by a fission of the carbon

8 skeleton. They are named depending on the bond that is broken in the carbon skeleton.

The D vitamins are 9,10-secosteroids , this characterized by the lack of an intact Bring.

Vitamin D2 (16). D3 (17), D4 (18) are secosteroids while Vitamin D1 is a mixture of two

21 secosteroids . Vitamin D promotes calcium absorption from digestive tract, and it is

22 essential for the normal growth and maintenance of bone . Human skin contains 4.18 IU

2 vitamin D per cm . The specific case of vitamin D shows the biological significance of

one group of secosteroids.

A large number of secosteroids have been isolated from marine sources. A 5,6-

23 secosterol (19) has been isolated from the sponge Hippospongia communis . A 8,9-

secosteroid, jereisterol A (20), and a 8, 14-secosteroid, jereisterol B (21 ), were isolated

24 from the sponge Jereicopsis gradphidiophora . Swinhosterols A-B (22), (23) are very unusual 4-methylene 8, 14-secosteroids isolated from the marine sponge Theonella

25 swinhoei . Only a few methylene secosteroids have been isolated from marine sources.

Astrogorgiadiol (24), a 9,10-secosteroid isolated from the Japanese gorgonian

Astrogorgia sp. , showed inhibitory activity against cell division of fertilized starfish

26 eggs . Several 9,10-secosteroids , Calicoferols C-E (25-27), have been isolated from an undescribed gorgonian of the genus Muricella. Calicoferols C-E exhibit potent antiviral

27 activity and brine-shrimp lethality . Calicoferol D exhibits significant activity against

Herpes simplex viruses I and II (EC5o 1.2 f.lg/ml), polio virus (EC50 0.4 f.lg/ml), and it is

27 also toxic against brine-shrimp larvae . Several 5,6-secosterols (28-36) were isolated

8 from the marine sponge Hippospongia communii .

9 Figure 3: Examples of bioactive steroids

14 15

HO HO

16 17

110

10 Figure 4: Examples of secosteroids from marine sources

A cO 20

22R= /

23 R= ' HO

24 Rt = H, RZ = OH, X= H 2 25 Rt = H, RZ= OH, X= CH 2 26 Rt. RZ=O, X=H 2

11 Figure 5: Further examples of secosteroids from marine sources

28 R= ''ql.Y.,,,,, 33 R= ········~ = 29R= ······~ 34R= -~.,,,, 30 R= .,,,,,, ""' -~ 35 R= 31 R= M

I~ I 0 36 R= ''''l.Y 32 R= yJY

12 Other steroids that have been isolated from marine sources are the 9, 11 -

secosteroids. In these steroids, the C9-C 11 bond is absent. Different groups can be attached to carbo:.' nine and eleven in place of the carbon bond. For example, acetyl groups, aldehydes !o roups. carbonyl groups, and hydroxy groups can be at carbons nine and eleven. All the steroids studied in this project have a carbonyl group at carbon nine and a hydroxy group al carbon eleven.

In the last few years, many 9, 11-secosteroids with biological activity have been isolated from sponges anl: soft corals. 9, 11-Secogorgosterol (37), which was isolated from the gorgonian Pseudcpterogorgia americana, has anti-inflammatory and anti

28 29 proliferative activity, and exhibits activity against protein kinase C · . It is an unusual steroid because ofthe cyclopropane ring in its side chain. Another 9,11-secosteroid isolated from the gorgonian Pseudoptervgurgia americana is 9, 11-seco-24-

31 hydroxydinosterol (38) . Two new 9.11-secosteroids (39), (40) have been isolated from

30 the gorgonian Pseudopterogorgia sp . These secosteroids inhibited the human PKC

25 enzymes a, ~I, ~II, y, £, 11, and s with IC 50 values in the range 12-50 !-!M . These

30 compounds also exhibit activity against MCF-7 human breast carcinoma proliferation .

9, 11-Seco-24-hydroxydinosterol (38) was also isolated with the above 9, 11-secosteroids,

30 and it showed similar biological activities . A 9, 11-secosteroid (41), which was isolated from the soft coral Gersemiafruticosa. has shown anti-proliferative activity against

32 Ehrlich carcinoma and human erythroleukemia K-562 cells . This secosteroid has shown a growth inhibitory activity (IC 5u) in two different tumor lines in vitro at 1 and

13 2 3)lg!ml for Ehrlich carcinoma and human erythroleukemia cells, respectiveli .

Blancasterol (42), a 9,11 secosteroid, has been isolated from the marine sponge

33 Pleraplysilla, sp . Blancasterol exhibits in vitro cytotoxicity against Ll21 0 murine leukemia (ED5o 8)lg/ml), drug sensitive MCF-7 human breast cancer (ED50 10)lg/ml), and

33 drug resistant MCF-7 Adr human breast cancer (ED5o 1O)lg /ml) . A further example of a biologically active 9, 11-secosteroid is herbasterol ( 43). which was isolated from the sponge Dysidea herbacea34 Herbasterol exhibits ichthyotoxic and anti-microbial

34 activity . Herbasterol also inhibits growth of the bacteria Bacillus subtilis and

34 Staphylococcus aureus . Glaciasterols A and B (44), (45) are cytotoxic 9,11-

35 secosteroids, which were isolated from the marine sponge Aplysilla glacialis .

Glacisterol A showed in vitro cytotoxicity in murine leukemia Ll21 0 (ED50 7)lg/ml) and

35 human breast cancer (ED50 7)lg/ml) cell line assays . 9, 11-Secocholesterol (46) is among other polyhydroxylated and oxygenated 9, 11-secosteroids isolated from a soft

36 coral (Sinularia sp. ) . Luffasterol A-C ( 47), ( 48), (49), which are 9, 11-secosteroids,

37 were isolated from the Palauan sponge Luffariella sp. . These 9, 11-secosteroids include an acetoxy ··,.oup · · C 3, an epoxide group at C 5 and C6. and an aldehyde group at C 11 .

38 New 9, 11- ~ .osteiv1 ds (50-51) were isolated from a soft coral of Sinularia sp . Finally, a 9,11-secosteroid (52), 3~ , 6a-dihydroxy-9-oxo-9,11-seco-5a-cholest-7-en-11-al, has been isolated from the sponge Spongia ojjicianalis and its structure confirmed by

39 synthesis . As shown above, a large number of secosteroids with wide ranging biological activity have been isolated from marine sources. Besides their biological activity, these

14 Figure 6: 9, 11-secosteroids from marine sources

HO 1-10 37 38

HO HO 39 40

01-1 41

IS Figure 7: Examples of 9, 11-secosteroids from marine sources

=ffi-1 O"Ac 42 43

44 45

HO AcO

46 47

16 Figure 8: Further examples of 9,11-secosteroids from marine sources

ArO

49 48

50 R =Cit

51 R= H, Me

H OH 52

17 compounds are also interesting because of their unusual side chains, and in some cases,

highly functionalized carbon skeletons.

4. Research Goals

Three 9, 11-secosteroids have been isolated from the gorgonian

Pseudopterogorgia americana, 9, 11-secodinosterol (53), 9.11-secogorgosterol (54) and

40 7-hydroxy-9,11-secogorgosterol (55) . These steroids appear to be biosynthetically

related, but have marked differences in their side chains and nuclei. According toW.

Fenical at Scripps Institute of Oceanography, 9.11-secosteroids are the most potent fish

41 deterrent of any marine natural product . 9, 11-Secogorgosterol exhibits inhibitory

29 activity against protein kinase C, and anti-inflammatory and anti-proliferative activities .

9, 11-Secogorgosterol has intriguing biological activity; unfortunately, it must be isolated from the marine organism every time it is needed, which presents a serious environmental problem. By collecting the source organism, the population of the gorgonians is diminished and the reefs destroyed. Thus, it is necessary to find a solution for the commercial production ofthe 9,11-secosteroids. One ofthe solutions involves the synthesis of the 9, 11-secosteroids, which has been completed for a number of 9,11- secosteroids; however, it usually involves a large number of steps, and it is an expensive process. This is not a possible solution for the commercial production of 9,11- secosteroids. Other solutions involve aquaculture and cell culture; however, the development ofthese processes is still in its early stages, and no conclusion can be drawn regarding their success. A final solution involves the enzyme based synthesis of

18 Figure 9: Examples of 9,11-secosteroids from the gorgonian P. americana

54 55

19 9, 11-secosteroids. This has been completed for terrestrial natural products such as

42 vinblastine .

The overall goal of this thesis research was to develop an efficient method for the chemoenzymatic synthesis of 9 _i 1-secogorgosterol. The second goal was to determine the specificity of the enzymes responsible for the process, and if possible, use it to synthesize known and novel 9, 11-secosteroids.

Gorgosterol was believed to be the precursor of 9, 11-secogorgosterol since the only difference between them is the fission of the C9-C 11 bond. In order to confirm this, a cell-free extract of the gorgonian, which is a simple aqueous cellular extract, was made.

A former student at R. G. Kerr's lab, J. Kellman, incubated radioactive gorgosterol with a cell-free extract from the gorgonian P. americana and radioactive 9, 11-secogorgosterol

43 was obtained . This proved that gorgosterol was indeed the precursor of 9, 11- secogorgosterol.

20 CHAPTER 2 DEVELOPMENT AND OPTIMIZATION OF THE

CHEMOENZYMATIC SYNTHESIS OF 9,11-SECOGORGOSTEROL

A major goal ofthis research project was the development and optimization ofthe chemoenzymatic synthesis of 9.11-secogorgosterol. Previously, trace quantities of radioactive 9.11-secogorgosterol were synthesized using a cell-free extract (CFE) derived of the gorgonian P. americana. Even thought a CFE was used in many incubations successfully producing radioactive 9, 11-secogorgosterol, a CFE is not a very efficient way to produce mg quantities of 9, 11-secogorgosterol. A CFE contains not only the enzymes responsible for the synthesis of 9, 11-secogorgosterol, but other biological macromolecules that could interfere with its biosynthesis resulting in lower yields of

9,11-secogorgosterol. Thus, the development of a method for the concentration ofthe biosynthetic enzymes was essential. A classical method used to concentrate enzymes is the production of an acetone powder, which contains all the proteins contained in the

CFE, but none of the smaller biomolecules.

The method of protein precipitation by water-miscible solvents has been

44 employed since the early days ofprotein precipitation . Addition of a solvent such as acetone or ethanol to an aqueous extract containing proteins has a series of effects which lead to protein precipitation. The solvating power of water for a charged, hydrophilic protein molecule is decreased as the concentration of organic solvent increases, which

4 results in precipitation of the proteins in the organic solvent"" . The solvent must be

44 completely water-miscible and have a good precipitating effect . This procedure must

21 take place at low temperatures since at above I 0 °C the enzymes can be denatured. One

advantage to the use of acetone instead of ethanol to precipitate the proteins is that

acetone is more volatile which enables the powder to be dried faster. Acetone was used to

concentrate the proteins present in the cell-free extract of the gorgonian P. americana

successfully producing an acetone powder. The acetone powder was made by diluting 25

ml of the CFE with phosphate buffer followed by centrifugation at 18,000 x g for 4 hours

at 0 C. The supernatant underwent vacuum filtration through a 0.45 jlm filter followed by

filtration through a 0.2 jlm filter. The supernatant was slowly poured into 800 ml of

acetone, which was previously cooled to -78 °C in a bath of xylenes, while vigorously

stirred. Then, the precipitate was filtered and washed with cold acetone. In order to

eliminate all the solvent, the acetone powder was lyophilized overnight. The acetone

powders can be maintained at -80 °C until further use.

As an initial test for the activity of this protein precipitate, 50 mg of acetone

powder was incubated with phosphate buffer at pH 7.7, 2 mg ofNAD+, and 5 mg of

cholesterol for 24 hours at 30 °C resulting in 16 % of 9 ,11-secocholesterol. Cholesterol

was used in all these reactions due its availability and low cost. Since cholesterol and

gorgosterol have the same nucleus, it was believed that cholesterol and gorgosterol would

be similarly modified.

1. Optimization of co-factors

The above result was very encouraging and we therefore undertook a study to optimize the conditions for this process. There were several factors that could be

22 optimized: 1) whether to use NAD+. NADP+ or both, and whether a constant flow of air

could increase the production of 9.11-secogorgosteroL 2) the optimum incubation time,

and 3) the concentration of acetone powder.

The biosynthesis of 9, 11-secosteroids involves oxidoreductase enzymes. It is well

known that enzymes carrying out redox reactions often use coenzymes as the electron

45 transfer agents . Two common oxidative coenzymes are nicotinamide adenine

dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). These

coenzymes are water-soluble co-factors that undergo reversible oxidation and reduction in

45 many processes . NAD+ and NADP+ are composed oftwo nucleotidesjoined through their phosphate groups by a phosphoester bond. Their reactive site is the nicotinamide ring. As a substrate molecule undergoes oxidation giving up two hydrogen atoms, the oxidized form of the nucleotide accepts a hydride ion and is transformed into its reduced

18 form, NADH or NADPH . These coenzymes can be readily reduced by those dehydrogenases for which they are specific such as L-glutamate dehydrogenase. Riva et

46 a!. used L-glutamate dehydrogenase to recycle NAD+ in the oxidation of cholic acid .

L-Glutamate dehydrogenase consists of six identical subunits, and it is involved in the transdeamination process. In this process, glutamate undergoes oxidative deamination catalyzed by L-glutamate dehydrogenase. This enzyme requires either NAD+ or NADP+ as the acceptor of the reducing equivalents. In this process, NAD+ and NADP+ are

19 reduced .

23 Figure 10: Reduction ofNicotinamide Adenine Dinucleotide (NAD+) and Nicotinamide Adenine Dinucleotide Phosphate (NADP+)

o--c~ N

OH OX

NAD+ (oxidized)

X= H Nicotinamide adenine dinucleotide (NAD+) X= po43-Nicotinamide adenine dinucleotide phosphate (NADP+)

24 NAD+ and the enzyme L-glutamate dehydrogenase had been used in the biosynthesis of radioactive 9, 11-secogorgosterol by a former graduate student in Kerr's

43 research lab . Since NADP+ has the same general function as NAD+, experiments including both co-factors were performed. Incubations including L-glutamate dehydrogenase were performed to determine if it could make the oxidation process more efficient. In order to determine which coenzymes could give the optimum yield of 9,11- secosteroids, several experiments were carried out with radioactive gorgosterol. Table 1 shows the effects of the co-factors in the synthesis of 9, 11-secogorgosterol. In the first trial, no co-factors were added resulting in a 6% yield. In the second trial, 2 mg ofNAD+ were added to the incubation and the yield increased to 18% while in the third trial, 2 mg ofNADP+ were added to the incubation resulting in a 50% yield of 9, 11-secogorgosterol.

Finally, in the fourth trial, both co-factors were added resulting in a 97% yield. These experiments were repeated using 5 mg of cholesterol with a yield of 78 % of 9,11- secocholesterol resulting when both co-factors were added to the incubations.

As previously stated, the biosynthesis of 9, 11-secogorgosterol is an oxidation reaction. This is also supported by the need to include the coenzymes NAD+ and NADP+ to obtain the maximum yield of9,11-secogorgosterol. To determine if vigorous aeration would increase the rate of the process, an experiment was performed in which the incubation mixture was aerated by means of an air pump. However, no increase in the

9, 11-secocholesterol yield was observed.

25 Figure II : Enzymatic oxidation of cholic acid

+ NAD(P)+

or 7a-HSDH or 12a-HSDH

+ NA D(P)H

a -Ketoglutarate + NAD( P)H + NH3 + H+ GIDIL_ L-Giutamatc + NA D(P)+ + H20

NAD was used with 3a-hydroxysteroid dehydrogenase (3a-HSDI-l) and 7a-hydroxysteroid dehydrogenase (7a-HSDH) and NADP with 12a -hydroxysteroid dehydrogenase ( 12a-HSDH ). Coenzyme regeneration was carried out with glutamate dehydrogenase (GIDH).

26 Table 1: Optimization of Co factors

Trial NAD (mg) NADP (mg) %Yield

1 0 0 6

2 2 0 18

.., _) 0 2 50

4 2 2 97

2. Optimization of incubation time

A kinetic study was necessary in order to determine the optimum incubation

period. In this experiment, the yield of 9, 11-secocholestero1 was measured with time.

Cholesterol was incubated with the coenzymes NAD+, NADP+, the enzyme GLDH, and phosphate buffer at pH 7.7. Every 4 hours, an aliquot was removed and the yields of cholesterol and 9,11-secocholesterol determined. As described in Table 2 and Figure 12,

48 hours was the optimum incubation time. The optimization experiments were performed using cholesterol as the precursor for reasons explained earlier; however, an experiment using gorgosterol as the precursor was performed and the yield was 97%,

27 Table 2 : Optimization of incubation time

Aliquot Time (hr) Cholesterol Secocholesterol % Conversion

(mg) (mg)

1 8 1.1 0.4 21.3

2 12 0.7 0.5 26.7

3 20 1.2 0.5 26.7

4 24 0.8 0.7 37.3

5 36 0.7 0.8 42.7

6 48 2.0 1.2 64.0

7 52 2.2 0.7 37.3

8 56 0.5 0.3 16.0

%Conversion: % conversion of cholesterol to 9, 11-secocholesterol

28 Figure 12 : Optimization of incubation time

0.------~------~ 0 8 12 24 36 48 52

Tirre(IT)

29 confirming that indeed 48 hours was the optimum incubation time. After 48 hours, the amount of 9, 11-secocholesterol in the incubation greatly decreased. It is not clear what happens to the 9, 11-secosterol after this time.

3. Acetone powder concentration

It is important to determine the concentration of acetone powder necessary for the optimization of the chemoenzymatic synthesis of 9, 11-secogorgosterol. Before an acetone powder is made, thin layer chromatography (TLC) of the gorgonian' s organic extract is performed. The concentration of 9, 11-secogorgosterol in each gorgonian determines the effectiveness of the acetone powder. If the TLC shows a small concentration of the 9, 11 -secosterol in that particular gorgonian, it is concluded that the acetone powder resulting from that gorgonian would produce low yields of 9,11- secogorgosterol. Thus, when performing steroid incubations, the yields cannot be compared unless the acetone powders used in the incubations are made from the same gorgoman.

Another factor affecting the concentration of the acetone powder is the nucleic acids and phosphate salts which it contains. For example, when 50 mg of acetone powder is incubated with a steroid, it is not clear how much of that 50 mg are enzymes responsible for the biosynthesis of 9, 11-secogorgosterol. Taking those factors in consideration, several experiments were performed to determine the optimum concentration of acetone powder. Experiments with incubation times of 24 hours and 48

30 Table 3: Optimization of acetone powder

Incubation Acetone NAD+ NADP+ 9,11- 0/o Conversion Time (hr) Powder (mg) (mg) Secocholesterol (mg)

24 50 2 2 0.8 16

24 200 2 2 1.3 26

24 400 2 2 2.2 44

48 50 2 2 2.7 54

48 200 2 2 3.2 64

48 400 2 2 3.8 72

hours with varying amounts of acetone powders were performed and the results are summarized in Table 3.

For reasons explained above, the acetone powders used in the incubations were made from the same gorgonian. From those experiments, it was concluded that the yield increases with increasing amounts of acetone powder. However, for economic reasons,

100 mg of acetone powder was selected for future incubations. Until an incubation is performed, it is difficult to know what yields of 9,11-secogorgosterol will be obtained using a particular acetone powder.

31 After all these experiments, it was concluded that the optimum conditions for

9, 11-secogorgosterol production on a 5 mg scale are: I 00 mg of acetone powder, 2 mg of

NAD+, 2 mg ofNADP+, 45 ml of phosphate buffer at pH 7.7, and 0.5 mg ofL-glutamate dehydrogenase with an incubation time of 48 hours at 30 °C.

This is the first enzyme based production method of a marine natural product, and represents an efficient and inexpensive method for the production of a potentially useful group of natural products.

32 CHAPTER 3 PRODUCTION OF KNOWN

AND NOVEL 9,11-SECOSTEROIDS

A major goal of this project was to determine the specificity of the enzymes involved in the chemoenzymatic synthesis of 9, 11-secosteroids. This was important because the developed method discussed in the above sections could allow for the synthesis of a wide range of secosteroids.

As mentioned previously, the gorgonian P. americana produces three secosteroids: 9, 11-secodinosterol (53), 9, 11-secogorgosterol (54), and 7-hydroxy-9, 11- secogorgosterol (55). As these 9, 11-secosteroids have marked differences in their side chains and nuclei, the enzymes responsible for their production are likely relatively nonspecific. Thus, our acetone powder could provide a general synthetic route to a variety of 9, 11-secosteroids. The fact that 5 mg of cholesterol were transformed to 9,11- cholesterol in high yields when incubated with the acetone powder of the gorgonian also led us to believe that other 9, 11-secosteroids could be synthesized. 9, 11-Secocholesterol, is not found in the gorgonian P. americana, but it is a known metabolite of the soft coral

6 (Sinularia sp.i .

1. Structure variation

Initially, steroids such as stigmasterol (58), ergosterol (59), and lanosterol (60) were incubated with the acetone powder under the optimized conditions since they were the most likely to be transformed to their secosteroid derivatives. Later, other steroids with different nuclei and side chains including pregnenolone (63) progesterone (64), and

33 cortisone (65) were also transformed into their secosteroid derivatives.

Figure 13 and 14 show the 9, 11-secosteroids synthesized using our chemoenzymatic method. As seen, these compounds have great structural diversity, which is summarized in Figure 15. For example, 9, 11-secholestanol has a hydroxy group at c3and no double bonds while 9, 11-secoprogesterone has a carbonyl group at c3and a double bond at C4 . Also, these compounds' side chains differ greatly. For example, 9,11- secocholestanol has eight carbon atoms in its side chain while 9, 11-secoprogesterone has a side chain with a carbonyl group and only two carbons. The relevance of this efficient method is clearly demonstrated when these compounds were synthesized at a low cost, in one step, and in a short period of time.

2. Characterization of novel secosteroids

Eight novel 9, 11-secosteroids have been synthesized in a 5 mg scale using 100 mg of an acetone powder of the gorgonian P. americana, 2 mg ofNAD+, 2 mg ofNADP+,

0.5 mg ofL-Glutamate dehydrogenase, and 45 ml of phosphate buffer at pH 7.7. These are 9, 11-secostigmasterol (58), 9, 11-secoergosterol (59), 9, 11-secolanosterol (60), 9,11- secocholestanol (61), 9, 11-seco-4-cholesten-3-one (62), 9, 11-secopregnenolone (63),

9,11-secoprogesterone (64), and 9, 11-secocortisone (65). These compounds were first isolated by thin layer chromatography (TLC). An NMR spectrum was obtained for each of the 9, 11-secosteroids synthesized. Some of the 9,1 1-secosteroid peaks can be seen in

34 Figure 13: Yields of "natural" and "unnatural" 9, ll-secosteroids

HO HO

(56) 97% (57) 78%

HO HO

(58) 18% (59) 15%

(60) 12%

35 Figure 14 : Yields of "unnatural" 9,11-secosteroids

(61) 26% (62) 16%

(63) 22% (64) 24%

OH OH

(65) 84%

36 Figure 15: Structural diversity of9.1 1-secosteroids

Side Chain : C2-C 10 sa tu rated ~22 C20 ketone C20 alcohol

X=-OH(a&~j,=O

Yields: 16%-97% on Smg sca le

the NMR spectra; however, they show an unknown impurity. The compounds were synthesized again; however, the impurity again appeared in the samples, which resulted in further purification steps with the use of HPLC.

Table (4) shows the retention times each of these compounds and the solvents used to purify them by HPLC. Retention time (tR) is the time that takes after the sample

37 47 injection for the analyte peak to reach the detector . Retention times are very useful in

the identification of9,11-secosteroids isolated through HPLC.

Table (5) shows the retardation factor (R1) of each 9, 11-secosteroid and the solvent used. The retardation factor is the ratio between the linear distance from the

47 origin solvent line and the sample spot, and the origin solvent line and the solvent front .

Table 4: HPLC retention times of 9, 11-secosteroids

9,11-Secosteroid Conditions Retention Times (min.)

9, 11-secostigmasterol NP, I: I EtOAc/Hex. 26

9, 11-secoergosterol NP, 3:2 Hex./EtOAc. 42

9,11-secolanosterol NP, 3:2 Hex./EtOAc. 28

9 .11-secocholestanol NP, 3:2 Hex./EtOAc 27

9, 11-seco-4-cholesten-3-one NP, 1: I EtOAc/Hex. 27

9, 11-secopregnenolone NP, 4: I EtOAc/Hex. 26

9, 11-secoprogesterone NP, 4: I EtOAc/Hex. 26

9.1 1-secocortisone RP, 95% CH30H/S% H20 10

38 Table 5: TLC analysis of novel 9, Il-secosteroids

9,11-Secosteroid Conditions Retardation factor

9, 11-secostigmasterol NP, 1: I EtOAc/Hex. 0.28

9, II-secoergosterol NP, 1:1 EtOAc/Hex. 0.26

9, 11-secolanosterol NP, 1:1 EtOAc/Hex. 0.3 0

9.11-secocholestanol NP, 1: I EtOAc/Hex. 0.30

9,11-seco-4-cholesten-3-one NP. I: I EtOAc/Hex. 0.29

9, 11-secopregnenolone NP, EtOAc 0.60

9, 11-secoprogesterone NP. EtOAc 0.62

9,I1-secocortisone NP, Methanol 0.10

Analysis ofthe 1H-NMR of9J 1-secogorgosterol shows two multiplets at 3.60 and 3.80 ppm representing the hydrogen atoms at C 11 , and a multiplets at 3.05 ppm representing the hydrogen at C8. These peaks are characteristic of 9, 11-secosteroids and correspond to the spectra of the 9.1I-secogorgosterol isolated from a soft coral (Sinularia

36 sp.) . Other 9, I1-secosteroids showed an unknown impurity in their NMR spectra, superimposed on the resonances of the secosteroid, which has not been identified.

39 Lurrent Data Paraseters rKerr96 EXPNO 10 ·PllOLNO I

f 2 - lt cc tl.~!S.tJofl P4rll•eters Odte %0/23 n.. t4 .~3 Pll.PAOG tg.lO SOLVEIIT COCJ3 1.0 1. 16 JC 8 . ~ q :t e l. F I OAF S (I -4 29tk5 Hz o• 7i .0 ... ~eL II(, <56 ~EU S Ill ILl taB 01 1. ~ooocco , •• Pt 14 . 0 uset DE 101.4 useL SFO I 500 . t 3b !HOO lttz SIIH ) 042 . <5 Hz TO 1D!b4 NS 58 OS

F2 - PrOL8 5!S .flQ para.eter~ Sl HHk SF 500 13~ 4 205 lttz 110. EM sse I) LB 0 10 ltz GB 0 ... PC 0 • . 00

10 ~ PiOt par-aeecera LX ~b ('0 l l. F1P 6 r iJ PP• Fl 3009. 45 HZ F2P -0 . 48.::: PP• F< -240 bq Hl. PPI4CM 0 l BO'B op•lc• HlCW 90 d1/22 Hl.h:•

l .AA _A__ __LA_ll tL.ll .....

~.-rr~rr~~r-rrrT~ r-rro-rr~-r~.-rr~rr~-rrro-rr~-rro~rr~-rrT-rTT.-rr~-r~_-rr,-•rrrro-roo-rrrT-rrrorrr~-rrr.-rr~rrrT-rrr-rr 5 . 5 s.o 4 . 5 4 .0 3 . ~ 3 o 2.~ 2.r 1.5 1. 0 o . ~ o o

Figure 16: 1H-NMR of9,11-secogorgosterol CHAPTER 4 PRELIMINARY STUDIES DIRECTED AT ENZYME ISOLATION

AND ELUCIDATION OF BIOSYNTHETIC PATHWAY

The chemoenzymatic synthesis of 9 ,11-secogorgosterol has been shown to be an effective method for the production of known and novel 9, 11-secosteroids with a wide range of structural diversity. The significance of this effective and inexpensive process is demonstrated when the biological activity ofthe 9,11-secosteroids are analyzed. The activity of these compounds as shown before ranges from anti-inflammatory to anti proliferative30. As mentioned before, most 9, 11-secosteroids can only be obtained by isolation from the source organism, which can create a serious environmental problem.

Our method is efficient and could potentially be used for the commercial production of9,11-secosteroids, but it can be further optimized. Thus, there is still a great deal to learn about the biosynthesis of 9, 11-secosteroids, which could make this process even more efficient.

Our ultimate goal is to purity the individual enzymes responsible for this process, and elucidate the biosynthetic origin ofthe 9.11-secosteroids. This would allow for the isolation and identification of the biosynthetic intermediates. Very preliminary studies have been conducted dealing with the isolation of the enzymes as well as with the elucidation of the mechanism of 9,11-secosteroid production.

41 1. Importance of enzyme purification

We have made preliminary attempts to isolate the enzymes responsible for the biosynthesis of 9, 11-secosteroids as enzymes could be made use of in the production of secosteroid intermediates. Further, once the enzymes responsible for the process are purified, there will be no need to collect the source organism from nature. Thus, the reefs would not be affected in the process. The enzymes could be obtained in a laboratory by means of molecular techniques.

The enzymes responsible for 9, 11-secosteroid production are contained in the acetone powder of the gorgonian's CFE. Through TLC, it was possible to relatively determine the amount of the enzymes in each animal indirectly. An organic extract of each gorgonian would be applied to a TLC plate, which would show the relative amount of 9, 11-secogorgosterol in the gorgonian. We assumed that the amount of 9,11- secogorgosterol in the gorgonian was directly related to the concentration of the enzymes responsible for their production in the gorgonian.

Standard techniques for enzyme purification include affinity chromatography and gel filtration. However, in this case, because we have gorgonians which produce 9,11- secogorgosterol while others of the same species do not, we may be able to take some

"short cuts''. A potential method to identify these particular enzymes was to find several gorgonians which did not produce 9, 11-secogorgosterol and make an acetone powder of each. Six acetone powders were made: four from gorgonians which did not produce

9, 11-secogorgosterol and 2 gorgonians which were rich in 9, 11-secogorgosterol.

42 Electrophoresis gel of each of the acetone powders was run in an attempt to identify proteins present only in the secosteroid-containing individuals.

48 Electrophoresis is the motion of charged particles in an electric field . During electrophoresis, externally applied electric fields drive charged molecules through various media. A main goal of electrophoresis is to maximize differences in mobility and

49 maximize the separations of molecules in the region of interest . Electrophoresis on a particular sample must be run in conjunction with a standard of known mobility since it is difficult to reproduce gels. Once the gel is run, separated molecules are fixed in their positions, and then stained with a developing solution in order to see the bands.

Polyacrylamide gels are gels formed by copolymerization of acrylamide and a cross linking monomer, usually N, N'-methylenebisacrylamide (bisacrylamide). The polymer chains are crosslinked by bis, resulting in a gel with a porosity which depends on the polymerization conditions. The reaction is initiated by a free radical generating system by

50 means ofTEMED (tetramethylethylenediamine) and ammonium persulfate . TEMED accelerates the rate of formation of free radicals from persulfate and these in tum catalyze polymerization. This occurs when persulfate free radicals convert acrylamide monomers to free radicals which react with unactivated monomers to begin the polymerization chain

50 reaction . There are various developing methods depending on the sample, resolution needed and other parameters. There are several factors that affect gel reproducibility: 1) the purity of the reagents, 2) the concentration of the initiators, and 3) whether the buffer and stock solutions has been degassed properly. Polyacrylamide gels are characterized by

43 % T and %C. % T is the weight percentage of total monomer including the cross-linker (in g/ 100 ml) and %C is the proportion of cross-linker as a percentage of total monomer.

The pore size is inversely proportional to % T. When % T is increased at a constant cross linker concentration, the number of chains increase and the pore size decrease.

An important factor to consider when running gels is which buffer system to use.

All the gels cast in the course of this research were Laemmli gels, which use discontinuous and denaturing buffer systems. Discontinuous buffer systems employ

49 different buffer ions in the gel and in the electrode solutions . These systems are designed to increase resolution and consist of a stacking and resolving gel. All ionic species form moving fronts containing buffering ions that move ahead of the sample molecules while the electrode buffer ions move behind the sample molecules. The stacking gel has a large pore size, where the sample is stacked. The sample molecules then enter the resolving gel of smaller pores where they separate on the basis of size and charge. The Laemmli method allows for molecular weight determination by incorporating 0.1% sodium dodecyl sulfate (SDS) in the buffers which denatures the sample proteins to their polypeptide chains prior electrophoresis. The samples are heated with a buffer containing SDS and the reducing agent 2-mercaptoethanol, which breaks the disulfide bonds between the proteins. SDS coats most of the polypeptides, which are then extended with dimensions proportional to their molecular weight. In SDS gels, the

51 mobility of the protein is generally proportional to its sub-unit molecular weight •

SDS gels were performed in order to determine the differences, if any, between

44 the acetone powders from P. americana individuals both rich in, and devoid of 9,11- secosteroids. A molecular weight standard was also included every time a gel was run.

Figure 17 shows a 18% T SDS gel. The gel contains six samples and a low range molecular weight standard. The first column at the left is the molecular standard, the next four samples numbered l, 5, 11, and 54 are the proteins from the individuals that do not produce 9, 11-secogorgosterol, and the last two numbered samples 25 and 45 are the samples containing the proteins from individuals that produce 9,11-secogorgosterol.

Samples 25 and 45 show strong bands at about 35 ,000 daltons that are barely visible in samples 1, 5, 11, and 54. Also, a strong band is seen in samples 25 and 45 at 30,000 daltons which is barely visible in the other samples. This is very preliminary work, and further studies are required, however, these results indicate proteins which may well be involved in secosteroid production.

2. Attempts to isolate intermediates

Preliminary experiments have been performed to try to isolate the intermediates involved in the biosynthesis of 9,11-secosteroids. In the first set of experiments, the objectives were to determine if, by using only NAD+, NADP+, or neither, biosynthetic intermediates could be produced. In each experiment, cholesterol was incubated with phophate buffer at pH 7.7, 100 mg of acetone powder, 0.5 mg ofL-glutamate

45 Figure 17: 18 %T SDS gel

- ~ .. --:..,-..;. ... ~......

46 dehydrogenase, and 2 mg of either of the co-factors at 30 °C for 48 hours. However, in incubations with only NAD+. NADP+, or neither, the only products visible by TLC were the steroid starting material or the secosteroid product. Further studies by gas chromatography (GC) and high performance liquid chromatography (HPLC) also failed to reveal any byproducts.

A second set of experiments designed to produce secosteroid intermediates involved eliminating oxygen during the incubation. There are two possible sources of oxygen in the biosynthesis of 9, 11-secosteroids: water and oxygen gas. In order to determine which one is involved in the process, an experiment in which the incubation was kept under an argon atmosphere was performed. Performing the experiment under an argon atmosphere would remove air from the experiment revealing whether water or air was the source of oxygen in the process. The incubation was set up with the use of a argon filled balloon. The system was purged with argon to eliminate any oxygen gas present in the incubation, and then a argon filled balloon was attached to the incubation.

This experiment produced the 9, 11-secocholesterol in a yield comparable to the reaction performed in the presence of air, thus indicating that water is likely the source of oxygen.

Unfortunately, no intermediates were observed.

The third set of experiments involved quenching the incubations at regular intervals. These incubations contained both co-factors and all the components ofthe optimized incubations. When analyzing the kinetic study of the biosynthesis of 9,11- secosteroids, the yields of 9,11-secosteroids increased with time up to 48 hours and

47 decreased after this period. Thus, experiments were set up with incubation times of24

and 48 hours. The objective was to detect any byproducts chromatographically. The

samples did not show any other compounds on TLC other than the steroid used as the

starting material, in these experiments cholesterol, and the corresponding 9,11-

secosteroid. However, since it was possible that the concentration of any intermediates

would be so small to be invisible in TLC, GC was employed as a more sensitive form of

analysis. Several GC runs were performed of 9, 11-secogorgosterol following different

methods; however, 9, 11-secogorgosterol decomposed under all conditions. Further,

HPLC analysis revealed that 9, 11-secocholesterol was the only compound present in the

9, 11-secosteroid samples. Thus, no intermediates in secosteroid biosynthesis could be

isolated.

3. Mechanism of 9,11-secosteroid production

Very preliminary studies have been performed trying to identify the mechanism of

9,11-secosteroid production. Figure 18 represents the likely biosynthetic origins of 9,11-

secosteroids. First, the steroid undergoes a dehydrogenation which resulted in a 5, 9,11-

triene. At this point, the triene can undergo oxidation to the 3, 9, 11-triol or to a C9-C 11 epoxide. The epoxide will eventually be transformed into the 3, 9,11-triol. Further, oxidative cleavage of the vicinal diol will result in the 9, 11-secosteroid. The other

mechanism involves starting from the steroid in which C9 or C 11 is oxidized making the diol, which in turn will be further oxidized to the 3,9, 11-triol. The trio] compound which

48 Figure 18: Proposed Mechanism of Secosteroid Production

H / / R R R

H H I

R = Sterol side chain

49 seems to be key in all three proposed pathways has been isolated with 9,11-

13 secogorgosterol which suggests that it may be a precursor of9,11-secosteroid .

Even though a synthesis should not be compared to a biosynthesis, there are several similarities between the synthesis of a 9, 11-secosterol, 3~, 6a-dihydroxy-9-oxo-

9, 11-seco-Sa-cholest-7-en-11-al (figure 19), from the sponge Spongia and the proposed biosynthetic origins of 9, 11-secogorgosterol. The structure of 3 ~, 6a- dihydroxy-9-oxo-

9, 11-seco-Sa-cholest-7-en-11-al was confirmed by synthesis starting from

38 7-dehydrocholesterol . Several ways to synthesize this compound were attempted including the cleavage of the C9-C 11 bond of the steroidal nucleus via the C9-C 11 epoxide intermediate. Even though the C9-C 11 epoxide was synthesized, it was not possible to

39 obtain the 9, 11-secosteroid since a complex mixture of products was obtained . One of the proposed biosynthetic pathways for the production of 9, 11-secosteroids (figure 18) also suggests the C9-C 11 epoxide as a possible intermediate. Also, the fact that the trio!, which is a possible precursor of 9, 11-secosteroids, is an intermediate in the synthesis of

3 ~' 6a-dihydroxy-9-pxo-9, 11-seco-Sa-cholest-7 -en-11-al also seems to support the idea that the 9 .11-secosteroids could follow one of the proposed pathways. However, further research is needed to confirm this. The chemoenzymatic synthesis of 9, 11-secosteroids is an ongoing investigation in R. G. Kerr's laboratory. Once finished, this investigation will allow for the commercial production 9, 11-secosteroids which is a key factor due to their significant biological properties.

50 Figure 19: Synthesis of3~, 6a-dihydroxy-9-oxo-9,11-seco-5a-cholest-7-en-11-al

a-c ,..

HO - - i1 : H : 01-1 OH

Reagents: (a) BH3.THF; (b) H202, NaOH; (c) Ac20, Py; (d) Hg (Oac)2, CHCb/AcOH; (e) Os04, dioxane; (f) LiAlH4; (g) Pb(OAc)4, AcOH

51 CHAPTER 5 EXPERI MENTAL SECTION

1. Instrumentation

The radioactivity of the samples was checked on a 1219 Rackbeta liquid

scintillation counter from LKB Wallac. HPLC was performed using Waters instruments.

A M45 solvent delivery system and a Perkin-Elmer LC-30 refractive index detector were

used for the normal and reversed-phase HPLC. The normal phase HPLC was carried out

using an Altex Ultrasil-Si column (I Omm i.d. x 25cm) to purify various secosteroids. It

was also used to separate various sterols with the mobile phase 8% ethyl acetate/hexane.

A reversed-phase HPLC with two Altex Ultrasphere ODS columns (I Omm i.d. x 25cm)

connected in series using I 00% methanol as the mobile phase was used to isolated

gorgosterol. The same columns were used to purify the 9,11 -secosteroids. A Perkin

Elmer 8500 GC was used for the steroid and secosteroid analysis. A refrigerated

centrifuge, Marathon 21 K/BR, was used in the preparation of CFEs and acetone powders.

A Fisher Biotech Photo-Documentation Hood model FB-PDH-81 0 was used to photograph the gels. /

2. Materials

The HPLC solvents were Optima grade obtained from Fisher Scientific and they were all degassed and filtered using Gelman Sciences, 0.45 fJ.m membrane filters and vacuum filtration. Extraction solvents were Optima grade.

Preparative TLC plates were Anal tech silica gel G (20x20) with a thickness of 250

52 J...lm or 1000 J...lm. Analytical TLC plates were Whatman 250 J...lm layer, AI Sil G/UV.

After centrifugation, CFEs were first filtered through Gelman Sciences, 0.45 J...lm membrane filters followed by filtration through Sterile Acrodisc, 0.2 J...lm , low protein binding filters.

Gels were run on 0.75 mm thick gels in the Mini-PROTEAN II cell. Silver stain gels used 10 well combs. Bio-Rad low molecular weight standards were employed in gel electrophoresis. The silver stain plus kits were used to stain the SDS gels. A Polaroid type camera of 35 mm with a 667 film was used to photograph the gels. All regeants used during electrophoresis were electrophoresis purity reagents.

Gorgonians for the development and optimization of the biosynthesis of 9,11- secogorgosterol were collected off Ft. Lauderdale. Gorgonians for enzyme work were collected from two sites: Marker 44 (15ft deep) and Craig Key (6ft deep) off Long Key,

Florida.

3. Analysis of 9,11-secogorgosterol in specimens of the gorgonian P. americana

Gorgonians were tagged, a small piece of each was cut, and brought to the laboratory. The pieces were left to dry for I 1/2 hours. In a numbered vial, they were extracted with 10 ml of a chloroform/methanol (I: I) mixture. Each sample was applied to analytical TLC plates into a ethyl acetate/hexane ( 1: 1) mixture, and 9,11- secogorgosterol was used as the standard. The samples were compared for their 9,11- secogorgosterol concentration shown by the TLC plates.

53 4. Isolation of gorgosterol

P. americana was collected off Ft. Lauderdale, Florida at a depth of 30 ft. The gorgonian was cut in pieces and blended with chloroform, allowed to stand for 2 hours, and then vacuum filtered. The supernatant was concentrated by rotovap. The residue was re-extracted with chloroform and let stand overnight at 4 °C. The extract was filtered and rotovaped again and an oily brown residue was obtained. The crude extract was run through through a silica column with the solvent system 100% hexane, 5% ethyl acetate/hexane, and 15% ethyl acetate/hexane. The steroids were found in the 5% ethyl acetate/hexane fractions, which was verified by TLC and by comparing it with a cholesterol standard. The sterol mixture was purified by NP HPLC (8% ethyl acetate/hexane). A cholesterol standard was run to determine the retention time of the sterols. Finally, gorgosterol was purified by RP HPLC with 100 % methanol.

5. Cell-free extract

The gorgonian (200g) was homogenized in a Waring blender with 300 ml of phosphate buffer at pH 7.7 and ca. 500 mlliquid nitrogen. The homogenate was then centrifuged at 5000 x g at °C for 10 minutes to remove insoluble debris. The supernatant was kept at -80 °C until further use.

Phosphate buffer, pH 7.7

The 0.2 M phosphate buffer A was prepared with 13.9 g ofmonobasic sodium phosphate dissolved in 500 ml of water. The 0.2 M phosphate buffer B was prepared

54 with 53.65 g of dibasic sodium phosphate dissolved in 1 L of water. 10.5 ml of 0.2 M

phosphate buffer A is added to 90.5 ml of 0.2 M phosphate buffer B and then diluted to a total volume of200 ml. 0.157 g ofDTT, 0.2192 g ofEDTA were then added to the solution.

6. Acetone powder preparation

25 Ml ofthe CFE was diluted once with 25 ml of phosphate buffer followed by centrifugation at 18,000 x g for 4 hours at 0 C. The supernatant underwent vacuum filtration through a 0.45 filter followed by filtration through a 0.2 f.!ID filter. 800 Ml of acetone was cooled to -78 °C in a bath of xylenes and dry ice. The supernatant was slowly poured into the cold acetone while vigorously stirred. The precipitate was left stirring for about 5 minutes. Then, it was filtered and washed with 100 ml of cold acetone. In order to eliminate all the solvent, the acetone powder was lyophilized for 12 hours. The acetone powder (ca. 1.0 g) was kept at -80 °C until further use.

7. Synthesis of cholestanol

25 Mg of cholesterol was added to 30 ml of ethyl acetate in a round bottom flask.

A spatula tip of the palladium in charcoal catalyst was added to the reaction. Hydrogen gas was added to the reaction using a balloon, which was kept filled during the course of the reaction. After hydrogen was purged through the reaction, the vacuum was set on for about 2 minutes to eliminate any other gases present in the reaction flask. Then, the vacuum was closed and only hydrogen gas remained. After 16 hours, the reaction was

55 stopped, and cholestanol was isolated from the charcoal mixture by means of a silica

column made with a pasteur pipette. The ethyl acetate was removed leaving the cholestanol.

8. Optimum incubation conditions for secosteroid biosynthesis

Acetone powder (100 mg) was suspended in 45 ml of phosphate buffer at pH 7.7 for 48 hours, at 30 °C in a shaker bath. 2 mg ofNAD+ and 2 mg ofNADP+, 0.5 mg ofL glutamate dehydrogenase as well as 5 mg of the steroid starting material were added.

9. Preparation of 9,11-secosteroids

Each incubation was stopped by flash freezing using liquid nitrogen and then left to lyophilized overnight to eliminate the water from the phosphate buffer. The resulting powder was extracted and passed through a silica column made by the use of a pasteur pipette. The solvent was evaporated with nitrogen gas, resulting in a mixture of the steroid starting material and the secosteroid. This was applied to a preparative TLC plate.

Two bands were obtained. The steroid was found in the top band while the secosteroid was found in the bottom band.

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