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
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Microbial Biosyntheses of Contraceptive Hormones Edwards, L.; Gober, N.; Sureka, H.
2
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.

3
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]

4
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

5
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

6
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

7
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

8
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

11
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

12
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.

13
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.

15
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.

18
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
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