microbial biosyntheses of contraceptive hormones

Microbial Biosyntheses of Contraceptive Hormones

Authored By:

Nicholas Gober; Edwards, L.; Sureka, H.

Report Submitted: 8 April 2014

CHEM 4765: Drug Design, Development, & Delivery

Having read the Georgia Institute of Technology Academic Honor Code, I understand and accept my

responsibility as a member of the Georgia Tech Community to uphold the Academic Honor Code at all times.

In addition, I understand my options for reporting honor violations as detailed in the code.

Inappropriate assistance was neither given nor received, at any time, during construction of this report.

Signature:____________________________________ Date:_____________________

Microbial Biosyntheses of Contraceptive Hormones Edwards, L.; Gober, N.; Sureka, H.

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Abstract

The vast and extensive medicinal applications of steroids and their derivatives represent a

well-established area of biopharmaceutical research. Because the overwhelming demand for

steroids by the pharmaceutical industry far exceeded the availability of these compounds from

natural sources many decades ago, potential synthetic pathways to efficiently mass-produce

steroids on an industrial scale are continually being investigated. One such approach involves the

biotransformation of steroids via microbial cells. The microbial syntheses of ethinyl estradiol and

norelgestromin, two steroid derivatives that are the active pharmaceutical ingredients in some

hormonal contraceptives, were analyzed. Ethinyl estradiol was found to be a good candidate for

biosynthesis because natural phytosterols could converted into the key intermediate, estrone, in

two biosynthetic steps. From here only one chemical reaction is required to give the desired

product, ethinyl estradiol. However, the functional groups on norelgestromin, especially the ethyl

group on C13 of the steroid backbone, are not amenable to biosynthesis. Thus, the proposed

synthesis only uses one biologically active step. The use of biosynthetic methods has the potential

to greatly simplify the synthesis of certain molecules; however, it has limitations, especially when

the target molecule varies from naturally occurring compounds too greatly.


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Introduction

History of hormonal contraception. The term ‘hormonal contraception’ refers to birth

control methods in which steroid hormones are the active pharmaceutical ingredients (API). There

are two types of hormonal contraception: progestogen-only, in which one steroid hormone

(specifically, one belonging to the progestogen class, hence the name) is used, and combined, in

which two hormones are used. In 1940, the chemist Russell Marker helped develop the first

progestogen-only drug by extracting the phytosterol diosgenin from barbasco, a wild Mexican

yam, and converting it into progesterone. However, because progesterone is destroyed by the

digestive system when taken orally, it was only available in injection form; thus, a chemical analog

that could be more easily and conveniently delivered was sought. In 1951, Carl Djerassi’s lab

chemically synthesized norethisterone, the first highly orally active progestin, which was eight

times more potent than the progesterone synthesized by the body. Subsequently, an isomer of

norethisterone was used as an API in Enovid, the first combined oral contraceptive pill (COCP),

which was approved by the FDA in 1960.[1]

Steroid pharmaceutical industry. Steroids represent one of the largest sectors of the

worldwide pharmaceutical industry. To date, there are over 300 approved steroid-based drugs to

treat a large variety of health complications, ranging from certain cancers to central nervous system

and metabolic disorders. In 2007, the global market of the steroid pharmaceutical industry was

around $10 billion, and it continues to consistently increase each year. Currently, only the

antibiotic sector is larger.[2]

Steroids and steroid derivatives. The primary characteristic structural feature of steroids is

an arrangement of four fused rings—three cyclohexane (rings A, B, and C) and one cyclopentane

ring (ring D)—that are comprised of 17 carbon atoms bonded together. This carbon skeleton is

called gonane (Figure 1-A), and it forms the core of all steroid molecules. However, because the

oxidation states of the rings can differ and various functional groups can be attached to the four-

ring core, steroids are a diverse group of compounds with many possible derivatives.[3]


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Figure 1: A (left) Structure of gonane, the simplest possible steroid, which is present in all substances called steroids.

B (right) Basic structure of sterol.

Two types of steroid derivatives are sterols and steroid hormones. Sterols, or “steroid

alcohols,” are a steroid subgroup characterized by the presence of a hydroxyl group at position

three of ring A (Figure 1-B). They are found naturally in plants (phytosterols), animals

(zoosterols), and fungi. One well known zoosterol that is vital to animal cell membrane structure

is cholesterol, a precursor to numerous vitamins and steroid hormones. Steroid hormones,

according to the particular receptor to which they bind, can be grouped into five distinct classes:

estrogens, progestogens, androgens, glucocorticoids, and mineralocorticoids. Steroid hormones

are signaling molecules that are found naturally in humans, assisting with metabolism, immune

functions, and the development of sexual characteristics.[3]

Estrogens and progestogens. Estrogens are a class of steroid hormones that are

characterized by an estrane skeleton made up of 18 carbons. Although they are found naturally in

both sexes, estrogens are significantly more prevalent in females than males; they are the major

female sex hormones and are produced primarily in the ovaries. The three major estrogens that

naturally occur in women are estrone, estradiol, and estriol (Figure 2-A). Estriol is released during

pregnancy and is the least potent of the three. Estrone, which is released only during menopause,

is the least prevalent of the three. Because it is released throughout the reproductive years of

women, estradiol is the most prevalent of the three; fittingly, it is also the most potent—around 80

times more potent than estriol.[4]

Progestogens, which are named after their pro-gestational functions, are a class of steroid

hormones characterized by a pregnane skeleton made up of 21 carbon atoms. Because they precede

other steroids in the first step of the steroidogenic pathway, certain progestogens are the precursors

to all other steroids—thus, all steroid-producing tissues must be capable of producing

1-A 1-B


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progestogens. The major naturally occurring progestogen is progesterone (Figure 2-B). Synthetic

progestogens are called progestins.[5]

Figure 2: A (left) Structures of estrone (note ketone group attached to ring D), estradiol (note single hydroxyl group

attached to ring D), and estriol (note two hydroxyl groups attached to ring D). B (right) Structure of progesterone.

“The Patch.” Ethinyl estradiol (EE) and norelgestromin, an estrogen and progestogen,

respectively, are the APIs in the transdermal combined contraceptive patch (matrix-type) Ortho

Evra®, known simply as “the Patch”. Its primary mechanism of action is ovulation prevention, but

the Patch also inhibits sperm penetration through the cervix by increasing the amount of and

viscosity of cervical mucus. Covering an area of 20 cm2, the Patch is applied once-weekly for three

consecutive weeks (21 days), followed by a patch-free week. Each Ortho Evra® patch contains 6

mg norelgestromin and 0.75 mg EE and delivers 150 µg norelgestromin and 20 µg EE daily to the

systemic circulation for 7 full days.[6]

Microbial Steroid Biotransformation

Because of the numerous widespread medicinal applications of steroids, pharmaceutical

companies and scientists alike are continually looking for better, more efficient methods (chemical

and biosynthetic) to mass-produce steroids. Microbial conversion (or transformation) is one

biosynthetic method that has been used to industrially produce steroids for many decades. Steroid

biotransformation is achieved through hemisynthesis that mainly starts with β-sitosterol (or

Estrone Estradiol

Estriol

2-A 2-B


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diosgenin and other phytosterols) and involves a varying number of sophisticated chemical and

microbial bioconversion steps.[2, 7]

Advantages. One major advantage of microbial steroid conversion is that functionalization

(namely hydroxylation) can be performed both regio- and stereo-specifically—thus, conversions

can be made at certain sites on the sterol that would otherwise be unavailable using chemical

reactions. Additionally, even under relatively mild conditions, several reactions can be completed

in one step which is not feasible in chemical-based approaches. Furthermore, metabolic pathways

can be constructed in specific sequences in the newly generated strain. Lastly, biosynthetic

pathways are, in general, more ecologically friendly than chemical syntheses.[8]

Needs and issues. The overarching need in this area of pharmaceutical research and

development is cost-efficient, economical processes to produce steroids. Because many chemical

reactions are economical, the number of steroid biotransformations that can compete with chemical

reactions on a cost basis on an industrial scale is limited. The primary issue with microbial steroid

conversion is the low aqueous solubility of steroids, resulting in poor availability of substrate to

whole-cell biocatalysts. Biotransformation in organic media has been developed to help

circumvent this issue; however, the high toxicity of organic solvents to cells is a major limiting

factor of this approach. Other limiting factors that are of concern include: the formation of side

products; yield variations due to biological variations; undesirable degradation of the steroid

product by whole cells; and low selectivity of whole-cell biocatalysts due to the inhibition effect.

The advantages and disadvantages of the use of biosynthesis for the production of two APIs, EE

and norelgestromin, have been considered and are discussed below.[8]

Ethinyl Estradiol

Use as an API. Ethinyl estradiol belongs to the estrogen class of steroid hormones, and it

is a commonly used API in various hormonal contraceptives. Coupled with a progestin, EE is an

API in both COCPs (i.e., Ortho-Cyclen®) and transdermal contraceptive patches (i.e., Ortho-

Evra®), and its primary mechanism of action is to prevent ovulation—this is achieved by inhibiting

follicle-stimulating hormone from being released.[7] The main difference between COCPs and

contraceptives patches is the varying pharmacokinetic properties of EE.

The range of daily EE dosage delivered by Ortho Evra® (~20 µg) is comparable to that of

the COCP Ortho-Cyclen® (~35 µg),[7] but drug delivery via the Patch is much more consistent over


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a given time period. The average steady state concentration of EE and the area under its time-vs.-

concentration curve are approximately 60% higher in women using the Patch than in those using

Ortho-Cyclen®; however, the peak concentration of EE is 25% lower in women who use the Patch

(Figure 3).[6] The adverse effects of these differences are not yet known, but increased estrogen

exposure has been shown to increase the risk of certain health complications such as venous

thromboembolisms. Additionally, when delivered via the Patch, EE can stay in the blood for up to

ten full days, suggesting that a patient could wait up to two days to apply a new patch after

removing one and still be protected.[7]

Figure 3: Mean serum concentration-versus-time profiles of norelgestromin and EE after a single seven-day patch is

applied (Graph A) and after taking one Ortho-Cyclen® pill (Graph B). Image taken from Abrams et al.[7]

Synthesis of ethinyl estradiol. EE can be synthesized via both chemical and biosynthetic

pathways. The chemical process requires numerous reaction steps and toxic chemicals, and it is

quite burdensome and inefficient. Conversely, a biosynthesis of EE can be performed in three

relatively straightforward steps, which are discussed below. The first two steps of this synthesis

are carried out using biological catalysts, and the final step is a straightforward chemical reaction.

Bioconversion of phytosterols to androstenedione. The first step of the synthesis of EE is

converting phytosterols to androstenedione (AD), an important precursor to many steroid-based

drugs. Peréz et al.[9] tested three different soybean oil samples containing various proportions of

three different phytosterols: stigmasterol, β-sitosterol, and campesterol (Figure 4). Increased

concentration of β-sitosterol appears to correlate with the higher yield of AD (Figure 5). The final


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optimized reaction is shown in Scheme 1.[9] After the discovery of Mycobacterium, this method

became widely used in industry because of its low cost and ease of transformation into AD.[10]

Figure 4: Percent (w/w) of phytosterol in each oil sample and the concentration of each different type of phytosterol

in each oil sample. Table taken from Peréz et al.[9]

Figure 5: Results of the trial for each type of oil used. The MB3683 was used because it had the highest conversion

yield of AD. Table taken from Peréz et al.[9]

Scheme 1: Optimum reaction found by Perez et al.[9] for the biotransformation of phytosterols to AD.


9

Mycobacterium MB3683 was chosen because it gave the highest yield of AD and the

lowest yield of 1,4-androstadiene-3,17-dione (ADD). These cells were grown in NB medium for

48 hours, at 30°C and with shaking at 200 rpm. The cultures were grown to 10 % (v/v), and placed

in either 50 mL NB or MS media containing the phytosterols. The media were prepared with a

phytosterol concentration of 1 mg mL-1. After 5 days of incubation at the same temperature and

shaking speed, the cultures were autoclaved and the product concentrations were tested.[9]

As shown in Scheme 1, the yield of AD was 65%, while the yield of the side product was

only 2%. Based on the data presented in Figure 5 for yield of AD from the various soybean oils

tested, it appears that the β-sitosterol concentration is weakly correlated to higher AD yield. VN-

3 had a lower concentration of β-sitosterol and resulted in a lower percent yield of AD as compared

to VN-1. The paper suggests that this could be due to a better bioaccessibility to substrates of

mycobacterial cells or that a stigmasterol regulating mechanism in the steroid 1,2-dehydrogenase

could be at work.[9]

Malaviya and Gomes[10] present a mechanism for the biotransformation of β-sitosterol to

AD by Mycobacterium. The side chain cleavage, which is the main step, requires the regeneration

of cofactors such as NAD+ and FAD. This process starts by hydroxylating the C27, which is

subsequently oxidized to a carbonyl group, followed by the carboxylation of C28.[10] The carbon-

numbering convention can be seen in Figure 6.

The presence of sitosterol helps to induce the two enzymatic reactions. The first of these is

catalyzed by three enzymes, while the second is dependent on the dissolved CO2 concentration. In

Mycobacterium, side chain cleavage of β-sitosterol can be induced by propionate or by propinyl-

SCoA. Dissolved CO2 (1%) affects the yield of AD positively, possibly because excess aeration

changes the way the cell metabolizes the substrate. Through the cleavage of one sitosterol

molecule, three molecules each of propionyl-SCoA, FADH2 , and NADH, and one molecule of

acetic acid were generated; these products can then be used for the production of ATP. If the

sitosterol is broken down completely, 18 molecules of NADH and 7 molecules of FADH2 are

created, which means 80 molecules of ATP can be produced from one molecule of β-sitosterol.

This presents a challenge for the biological catalysis of this cleavage because it is energetically

favorable for the cell to entirely break down the molecule.[10]


10

Figure 6: Carbon-numbering convention of β-sitosterol. Image taken from Wikimedia Commons.

The chemical synthesis goes through multiple reaction steps making it unfavorable when

compared to the biosynthetic step. The main challenges are cleaving the side chain of the C17 and

dealing with the very sensitive steroid ring structure. This, combined with required use of some

harmful and toxic reagents, such as pyridine, leads to a very lengthy, costly, and low-yield process,

making the biosynthetic route the more adequate method.[10] However, regarding the conversion

of sitosterols to AD, there are still problems and areas for improvement with the biosynthetic

pathway, namely the degradation of the steroid nucleus and inhibition of the side-chain

degradation by the reaction products.

Some potential solutions proposed by Malaviya and Gomes[10] include inhibiting the 9-α-

hydroxylase and screening for the improvement of the microorganism to give a higher yield of

AD. In order to maintain the steroid nucleus of AD, the action of 9-α-hydroxylase and 3-

ketosteroid-1(2)-dehydrogenase must be blocked. The activation of 3-ketosteroid-1(2)-

dehydrogenase triggers the formation of a double bond between C1 and C2, leading to the

formation of ADD. The activation of 9-α-hydroxylase leads to the addition of a hydroxyl group on

C9 forming 9-α-hydroxy-4-androstene-3,17-dione. Inhibition of these enzymes would help to

boost yields, but can also be lethal to the cells. 9-α-hydroxylase is a monooxygenase that is

important to the electron transport chain and contains proteins that require Fe2+; therefore, a good

way to inhibit this enzyme is to chelate Fe2+ using a chemical such as 8-hydroxyquinolone. Also,

in the commercial arena, scientists are trying to screen and improve the bacteria used. This


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improvement can be achieved by developing strains which are less sensitive to phytosterol toxicity

or by mutagenic treatment that increases the efficiency with which the bacteria cleaves the side

chain.[10]

There is also the major problem of low solubility of the phytosterols in aqueous media,

which is the factor that makes this the bottleneck of EE production. Malaviya and Gomes[10]

propose five major ways to get around this problem. These include: 1) biotransformation in two-

phase systems; 2) biotransformation in cloud-point systems; 3) biotransformation via immobilized

biocatalysts; and employing 4) microemulsions and/or 5) liposomes as alternative

biotransformation systems. However, as of 2008,[10] these methods have proven inadequate for

application industry. While research is being conducted to find solutions to the issues with

biosynthesis, this process is used in industry because it is a better way to make AD than the

chemical method.

Androstenedione (AD) to estrone. The next step of the synthesis is the conversion of the

AD formed in the previous section to estrone (Scheme 2). To accomplish this, the A-ring has to

be aromatized, the C19 has to be removed, and the carbonyl group at C3 has to be oxidized. This

step currently is not done biosynthetically in industry, but we propose a biosynthetic method to

form estrone biosynthetically using P450arom, a human aromatase.

Scheme 2: Bioconversion of AD to estrone using a human aromatase (P450arom) in E. coli.

Currently, the P450arom-expression plasmids have to be constructed. Kagawa et al.[11]

describe a method to accomplish this. In brief, the GroES/GroEL expression plasmid pGro12 was

obtained from an outside source and the gene of interest was spliced in using site directed

mutagenesis. Kagawa et al.[11] also describe a process for the preparation of P450arom, which is

summarized below. In order to prepare the P450arom, E. Coli DH5α cells with the expression


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plasmids were incubated overnight in 5 mL TB with 100 µg mL-1 ampicillin at 37°C. These

cultures (2 mL) were diluted into 250 mL TB media in a 3 L culture flask and incubated for 4

hours at 37°C.[11] IPTG, gamma-aminolevulinic acid, and arabinose (for induction of molecular

chaperones GroES/GroEL) were added to the culture, and it was subsequently incubated for 28

hours at 28°C.[11] Cells were harvested by centrifugation and lysed, then centrifuged in order to

separate supernatant. This solution was purified to extract the P450arom. The yield for this reaction

was 13.4 nmol P450 (mg protein)-1.[11]

This P450arom was then added directly to the AD solution. First, the P450arom would

oxidize C19, and then activate the concerted elimination of C19. The C19 is eliminated as formic

acid, and the 1-β and 2-β hydrogen atoms are eliminated from the A-ring.[11] The third step that

oxidizes the carboxyl group is unclear, but it results in an estrone. We propose doing this step of

the synthesis in a fermentation setup. While this change would introduce new issues, such as

diffusion and solubility limitations, it could potentially decrease both capital and operating costs

by eliminating the protein purification steps; however, both methods would still need to be

analyzed to ensure that the most economical process is used.

Estrone to ethinyl estradiol. The ethinylation process is a very old one that started in the

1930s.[12] This process includes adding ethyne, sodium, and sodium amide to the estrone (Scheme

3). The ethyne will attack the carbonyl carbon causing the oxidation of the carboxyl group forming

a hydroxyl group—this leaves us with the final product, EE. This process is necessary to change

the bioavailability of the estradiol.[13]

Scheme 3: Chemical ethinylation process of estrone to yield ethinyl estradiol.[6] Figure adapted from Wikimedia

Commons.


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Norelgestromin

The progestin norelgestromin (Figure 7) is the second API of Ortho Evra®. It is the

progestational component of the patch and acts by preventing the release of luteinizing hormone,

which is associated with the initiation of ovulation. The molecule is derived from norgestrel,

another progestin, and is the active metabolite of norgestimate.[14] In a study by Abrams et al.[7],

administration of norelgestromin through a patch was shown to maintain more consistent levels of

drug in the body, which helps to ensure the drug is at a therapeutically effective concentration

throughout the administration period. The patch was designed to deliver 150 µg day-1, compared

to the pill’s 250 µg day-1. The average concentration achieved via oral delivery was found to be

0.75 ± 0.23 ng mL-1, while the steady state concentration delivered by the patch was 0.83 ± 0.21

ng mL-1. The area under the curve was comparable for the two delivery methods as well.[7]

Norelgestromin presents significant challenges for biosynthesis because of the variations

between the API and naturally occurring progestogens. The presence of an ethyl group at C13 of

the molecule makes this synthesis particularly difficult because no naturally occurring progestogen

has this group,[5] and selective methylation of C18 (methyl group on C13) cannot be carried out

biologically. Similarly, the addition of the oxime group at C3 and the ethinyl group at C17 must

be done chemically. As such, norelgestromin is not a good candidate for a microbial based

synthesis; nonetheless, a synthesis involving a small biological component can be done.

Figure 7: Structure of norelgestromin.

Synthesis of norgestrel. The synthesis of norelgestromin begins with the synthesis of

norgestrel, the key intermediate in the synthesis. The proposed synthesis was adapted from Gibian

et al.[14] and Kleemann et al.[13], and is shown in Scheme 4. Beginning with a two-ring structure,

6-methoxy-1-tetralone, the first step is the addition of a vinyl group via a Grignard reaction, which


14

must be carried out in an organic solvent such as THF or diethyl ether. Following this, a Michael

addition is carried out with Product I and 2-ethyl-1,3-cyclopentadione, in the presence of a base.

This step simultaneously performs a base-catalyzed hydrolysis resulting in Product II, which has

three of the four rings of the steroid backbone formed.

The next step is the only biologically active step of the synthesis of norelgestromin. Product

II is added to a culture of yeast, Saccharomyces uvarum, to stereo-specifically reduce one of the

ketone functional groups on what will become ring D (see Product III). The enzyme responsible

is a keto reductase, and as such the media may need to be doped with NADP+.[15] The reaction has

an overall yield of 44-52%.[14] One method of boosting this yield would be isolating the enzyme

and performing the reaction independent of a fermentation process. This would help to rid the

system of some of the mass transport limitations that exist in whole-cell catalyzed processes.[13, 14]

Following the biosynthetic step, acetic anhydride and a strong acid, in this case

toluenesulfonic acid, are used to protect the remaining hydroxyl group on ring D and to hydrolyze

the ketone group, allowing ring C to form (see Product IV). Unnecessary double bonds are then

saturated in the presence of hydrogen and a palladium-carbon catalyst, followed by potassium

hydroxide, methanol, lithium, ammonia, and aniline. This step also unprotects the hydroxyl group

on ring D and leaves two unsaturated bonds on ring A (see Product V). The addition of the ethinyl

group and the oxidation of the ester to a ketone are then carried out to give norgestrel. This is done

by addition of a hindered base (Al(O-iPr)3) in MEK, followed by reaction with lithium acetylide,

and, finally, by addition of a strong base.

Overall, this synthesis consists of fairly simple organic reactions. Again, this is because of

the ethyl group at C13. This group is not naturally occurring, so the use of a natural precursor is

not feasible for this synthesis. Therefore, a total synthesis beginning with commercially available

reagents is necessary. The use of S. uvarum helps in providing in enantiomerically pure product,

but does not shorten the overall synthesis as in the synthesis of EE. With this in mind, a biological

synthesis of norgestrel does not provide large advantages over a traditional chemical route.


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Scheme 4: Synthesis of norgestrel with S. uvarum, based on synthetic approaches reported by Kleemann et al.[13] and

Gibian et al.[14].


16

Scheme 5: Synthesis of norelgestromin from norgestrel, which was developed by Tuba et al.[16] (yield = 70%).

Synthesis of norelgestromin. The norgestrel from the previous synthesis can be

transformed into norelgestromin via a three-step synthesis. The synthesis described was developed

in the patent by Tuba et al.[16], and is shown in Scheme 5. The only modification that needs to be

completed is the addition of the oxime group at C3. In order to ensure that the group is only added

to the appropriate location on the molecule, the hydroxyl group must be protected.

This protection is again accomplished with acetic anhydride and a strong acid, in this case

hydrochloric acid. Under nitrogen, a suspension of norgestrel with acetic acid (100 mL), acetic

anhydride (6 mL), zinc chloride (2 g), and 6.7% hydrochloric acid in acetic acid (1.6 mL) is stirred

for 20 min, then water (5 mL) is added to the mixture. After stirring for an additional 15 min, 18%

aqueous hydrochloric acid (3 mL) is added, and the mixture is stirred for another 45 min. At this

point, the reaction is complete, and a crude product is isolated by adding ice water (600 mL),

filtering, washing with water, and drying. The total reaction time was about 80 minutes.[16]

Following this step, the intermediate, norgestrel acetate, must be purified. This is

accomplished by dissolving the product in dichloromethane (100 mL) and stirring with silica gel

(10 g). The mixture is stirred for 30 min, and the gel is then filtered off. The solvent is then boiled

off. The remaining product is refluxed in isopropyl ether:acetonitrile (9:1 v/v ratio) mixture for 15


17

min. The mixture is then cooled in ice water to 0°C, precipitating the product, which is then filtered

and dried. The mother liquor can be reprocessed using the same purification method to obtain more

product. This step has a yield of 15.4 g (90.5%).[16]

The pure product can then undergo oximation to form norgestimate, a reaction that is again

carried out under nitrogen. Hydroxylammonium chloride (76 g) is added to a stirred solution of

norgestrel acetate (120 g), acetic acid (1200 mL), and anhydrous sodium acetate (90.2 g). The

reaction takes 1 hour and must be maintained under 30°C. The resulting mixture is added to water

(10 L) and stirred for 30 min, after which the crude precipitated product is filtered off and dried at

40°C.[16]

The crude product is then dissolved in boiling ethanol (2500 mL), clarified with charcoal

(12 g), and filtered. The filtrate is concentrated under partial vacuum (below 40°C) to 400 mL,

then cooled to 0°C for 3 hours to precipitate the product. The product is then filtered off, washed

with two portions of ethanol (125 mL each), and dried under 40°C. The yield of norgestimate was

102 g (81.6%), with a purity of over 99.5%.[16]

The final step of the synthesis is un-protection of the hydroxyl group on ring D. Again

under nitrogen, norgestimate (10 g), methanol (100 mL), and sodium hydroxide (3.25 g) are stirred

together at 22°C. After approximately 10 min, the temperature rises by about 10°C and a

homogeneous mixture is obtained. The mixture is then stirred for 3 hours at 25°C and subsequently

added to chilled water (1000 mL). The pH is adjusted with acetic acid (3 mL) to 7-7.5, and the

suspension is stirred for 20 min more. The product is then filtered, washed with water, and dried

over phosphorous pentoxide at 40°C under partial vacuum. The yield of norgestimate was 8.4 g

(94.8%), with a purity of 99.9%.[16]

The overall yield for this synthesis was 70.0%, which seems acceptable. However, the

amount of solvent used for each step is quite large, especially when considering scale up of the

process. Before a scale up is proposed, a pilot scale operation meant to optimize the solvent use

would be prudent to minimize operating and capital cost for a commercial process. Some

optimization can also be done when incorporating the two processes. For example, the hydroxyl

group on ring D is protected and un-protected twice, so protecting the group early on and leaving

it protected until the final step of the synthesis.


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Figure 8: Structure of norelgestromin, with problematic functionalities circled.

Challenges of norelgestromin biosynthesis. Norelgestromin is not a good candidate for

microbial synthesis, unlike EE, because of the functional groups circled in Figure 8. The ethyl

group is again the most challenging of these groups because it is not naturally occurring, and both

selective methylation and ethylation are difficult to accomplish. Thus, using a common natural

precursor such as AD for both of the syntheses is not practical. The other two problematic reactions

are the oximation and the ethinylation, both of which must be done chemically; hence, the majority

of the functional groups on norelgestromin would have to be added chemically, and the backbone

itself has to be made with a total synthesis to ensure the presence of the ethyl group.

Conclusions

Microbial synthesis provides a myriad of benefits in the manufacture of steroid molecules.

While it has limitations caused by mass transport, solubility, etc., microbes are able to perform

complicated chemistry, which would often require multiple chemical steps, in one process. A very

good example of this is the microbial synthesis of AD from phytosterols, where the microbe

removes all of the unnecessary sidechains and leaves the target intermediate product in a single

fermentation process. Because the biosynthesis of estrone can be done in two steps and forming

EE only requires one further reaction, it is, indeed, an ideal candidate for biosynthesis.

Microbial synthesis does however have severe limitations when the target molecule does

not resemble natural compounds well enough as shown with the synthesis of norelgestromin. The

presence of the ethyl group at C13 severely limits the potential for biosynthesis of norelgestromin.


19

Because of this group, the backbone needs to be totally synthesized from raw materials, as we have

shown.

Future work in microbial synthesis includes strain improvement,[10] continued work with

two-phase reactor systems, development of green solvent systems, and development of novel

hydrophobic delivery methods.[8] These goals are driven by the need to continue improving the

productivity of various microbial strains in order to make them more competitive compared to

traditional chemical syntheses.

References

[1] C., Anna. The History of the Birth Control Pill, Parts 1-6.

http://advocatesaz.org/tag/hormonal-contraceptives/ (accessed 5 April 2014), Planned

Parenthood Advocates of Arizona.

[2] Microbial Steroid Biotransformations Using Cytochrome P450 Enzymes. Modern

Biooxidation: Enzymes, Reactions and Applications; Schmid, R. D.; Urlacher, V. B.,

Ed.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2007; pp 155-170.

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