Terpenes and Steroids (2)

Terpenes and Steroids

Specific Natural Products

Methods of Separation

Method of Detection

Chemical Synthesis

Biosynthesis

Advances in Natural Products

Submitted in Partial Fulfillment for the Requirements in Chemistry 135 (Chemistry of Natural Products)

Submitted to:

Dr. Nelson Villarante

Submitted by:

Cua, Zabrina Tan

Pilar, Ron Benedict Ortega

Ronon, Mike Angel

August 5, 2011

I. Introduction

Terpenes

Terpenes are hydrocarbons occurring widely in several organisms, like plants and animals.

Turpentine, the so-called "resin of pine trees", is derived from the Latin word, balsamum terebinthinae. It

is the viscous pleasantly smelling balsam which flows upon cutting or carving the bark and the new wood

of several pine tree species. In 1880, Friedrich Kekulé discovered that turpentine is composed of

hydrocarbons, with the molecular formula of C10H16, and consequently he named these structures,

―terpenes‖. Terpenes with oxygen in the form of aldehydes, ketones, or alcohols, having the molecular

formula of C10H16O and C10H18O were assigned the name, ―camphor‖. Camphor has been known since

antiquity in the Orient, and at least the 11th century in Europe. It was said that the Arabs were responsible

of bringing it to Europe from the East. The term ―camphor‖ is then changed to ―terpenoids‖. (Stevens &

Verhé, 2004)

Otto Wallach, an assistant of Kekulé, after the analysis of several components of essential oils,

discovered that many contained 5n carbon atoms. Thus, in the year 1887, he proposed his isoprene rule,

stating that the structure of terpenes were composed of isoprene units, 2-methylbuta-1,3-diene. Thus

terpenes are also called isoprenoids. After thirty years, Sir Robert Robinson perfected Wallach‘s isoprene

rule, and stated that the joining of two isoprene units must be of head to tail fashion. But this rule can only

be used as guiding principle and not as a fixed rule. For example carotenoids are joined tail to tail at their

center.

Figure 1. Carbon skeleton of two Isoprene units with a bond between the tail of one and the head of another

In the case of cyclic compounds, the linkage of the head of one isoprene unit to the tail of another

is followed by an additional linkage to form the ring. The second linkage is not necessarily head-to-tail,

but whatever is necessary to form a stable five- or six-membered ring. A few years later, Leopold Ruzicka

proposed the rule of Ruzicka, classifying terpenes according to their number of isoprene units (Table 1)

and specifying that terpenes are derived from aliphatic precursors, namely geraniol, geranylgeraniol,

squalene, and farnesol, for monoterpenes, diterpenes, triterpenes, and sesquiterpenes, respectively.

(Berger, 2007)

Table 1. Isoprene Rule

Number of carbon atoms

Value of n Class

10 2 Monoterpenes(C10

H16

)

15 3 Sesquiterpenes(C15

H24

)

20 4 Diterpenes(C20

H32

)

25 5 Sesterpenes(C25

H40

)

30 6 Triterpenes(C30

H48

)

40 8 Tetraterpenes(C40

H64

)

>40 >8 Polyterpenes(C5H

8)n

After classification by the number of isoprene units, further classification can be done by the

number of rings present in the structure. Acyclic terpenes contain no ring but instead are open structures.

Monocyclic, bicyclic, tricyclic, and tetracyclic terpenes contain one, two, three, and four rings in the

structure, respectively.

Figure 2. Parent Hydrocarbon of Terpenes (Isoprenoids)

Terpenes are extracted or steam distilled, and these extracts or distillates are known as ethereal

or essential oils (―essence absolue‖). These essential oils are used widely as natural flavour additives,

fragrances, and traditional and alternative medicines, like aromatheraphy. Synthetic variations and

derivatives of these have expanded the diversity of aromas and flavours for fragrances and food additives,

respectively. The biological and ecochemical function of terpenes is not fully known, but many plants are

observed to produce volatile terpenes to either attract specific insects for pollination or to expel certain

animals or predators. Less volatile but strongly bitter-tasting or toxic terpenes also protect some plants

from being eaten by animals (antifeedants). (Breitmaier, 2006)

Many insects metabolize and synthesize these plant compounds to produce growth hormones

and pheromones. Pheromones are luring and signal compounds (sociohormones) that insects and other

organisms excrete in order to communicate with others like them, e.g. to warn (alarm pheromones), to

mark food resources and their location (trace pheromones), as well of assembly places (aggregation

pheromones) and to attract sexual partners for copulation (sexual pheromones). Harmless to the

environment, pheromones may replace conventional insecticides to trap harmful and damaging insects

such as bark beetles. (Breitmaier, 2006)

More than 30,000 terpenes have been isolated from plants, animals, and microorganisms.

(Berger, 2007) Some common examples of monoterpenes are citral, limonene, menthol, terpineol, α-

pinene, and, camphor. Citral is a constituent of lemon oil and is commercially obtained from lemon grass

oil. Limonene on the other hand is found in the skin of citrus fruits. Menthol is found in the essential oil of

the field mint, Mentha arvensis, and possesses useful physiological characteristics that include

anaesthetic and refreshing effects. Menthol is used to flavour sweets, tobacco, and toothpaste. Terpineol

and α-pinene were isolated from pine oil (turpentine), while camphor from camphor tree, Cinammomum

camphora. Camphor is now commercially made from α-pinene and is used to protect clothes from moths.

Figure 3. Structures of Citral, Limonene, Menthol, Terpineol, α-pinene, and Camphor. (Left to Right)

Sesquiterpenes are found in higher boiling portions of essential oils, when compared to

monoterpenes. Sesquiterpenoid lactones are commonly found in the Compositae family as biologically

active constituents. Santonin from wormwood, Artemisia maritime, was used as a medicine to eliminate

intestinal wounds, while a derivative of artemisinin, from Artemisia annua, is used to treat resistance

strains of malaria. Another example of a sesquiterpene is the plant hormone, abscisic acid, which

stimulates leaf fall and dormancy in plants.

Figure 4. Structures of Santonin, Artemisinin, and Abscisic acid. (Left to Right)

The most common examples for diterpenes, triterpenes, and tetraterpenes, are vitamin A,

squalene, and β-carotene and lycopene, respectively. Both vitamin A and β-carotene are involved in the

chemistry of vision, since β-carotene is converted to vitamin A. Vitamin A is also known as retinol.

Lycopene is the compound responsible for the red coloring of tomatoes and watermelon. Squalene on the

other hand is a precursor of steroid molecules, first isolated from fish liver oils, and then in plant oil and

mammalian fats. Lanosterol is a common tetracyclic triterpene and is a major constituent of wool fat.

(Erdman & Sandor, 1997)

Figure 5. Structure of Retinol.

Figure 6. Structure of Squalene.

Figure 7. Structure of Lycopene.

Figure 8. Structure of B-Carotene.

Steroids

Hormones are chemical messengers- organic compounds synthesized in glands and delivered by

the bloodstream to certain tissues in order to stimulate or inhibit a desired process. Steroids are a type of

hormone, and derived from tetracyclic triterpenes and possess a cyclopentaperhydrophanthrene

backbone. They are recognized by their tetracyclic skeleton which consists of three fused six-membered

and one five-membered ring, as seen in Figure 9. Rings can either be trans fused or cis fused, and the

former is said to be more stable, and thus B,C,D rings of the carbon skeleton of steroids are trans fused.

While most naturally occurring steroids have the A,B rings trans fused as well.

Figure 9. Carbon Skeleton of Steroids

Many steroids have methyl groups at the 10- and 13- positions and are called angular methyl

groups, and these are shown to be above the plane of the steroid ring system when drawn. Substituents

on the same side of the steroid ring system as the angular methyl groups are designated as β-

substituents, which are indicated by a solid wedge, while those on the opposite side as α-substituents,

indicated by a hatched wedge.

The history of steroids can be traced back as far as antiquity. Adolf Butenandt isolated estrogen,

progesterone, and testosterone, in the year 1929, 1934, and 1935, respectively (Vasudevan, Sreekumari,

& Vaidyanathan, 2011). While in the year 1934, Ruzicka and his colleagues further developed

androsterone when he purified cholesterol. But the most significant event occurred during the year 1935,

when Karoly Gyula David et. al., purified the testicular hormone, which was later referred to as

testosterone, for the first time from bull testes. (2008 September « History of steroids)

Steroids are non-polar compounds thus are classified as lipids. The non-polarity of steroids

allows them to cross cell membranes, so they can leave the cells in which they are synthesized and enter

their target cells. Sterols are steroids with a hydroxyl group at C-3. Cholesterol is the most common and

most abundant steroid as well as sterol encountered by animals. It is also the precursor of all other

steroids. It is an important component of cell membranes. (Bruice, 1994)

Figure 10. Structure of Cholesterol.

Cholesterol is biosynthesized from squalene and its structure makes it more rigid than other

membrane lipids. Due to the eight asymmetric carbons, 256 stereoisomers are possible but only one

exists in nature (Figure 10). It is also a precursor of bile acids. In fact, the word cholesterol is derived from

the Greek words chole meaning ―bile‖ and stereos meaning ―solid.‖ The bile acids-cholic acid and

chenodeoxycholic acid—are synthesized in the liver, stored in the gallbladder, and secreted into the small

intestine, where they act as emulsifying agents so that fats and oils can be digested by water-soluble

digestive enzymes. Cholesterol is also the precursor of vitamin D.

Figure 11. Structure of Cholic acid (left) and Chenodeoxycholic acid (right).

Steroid hormones are divided into five classes: glucocorticoids, mineralocorticoids, androgens,

estrogens, and progestogens. Glucocorticoids and mineralocorticoids are known to be adrenal cortical

steroids due to it being synthesized in the adenal cortex. All adrenal cortical steroids contain an oxygen

on the carbon at position eleven. Glucocorticoids are involved in glucose metabolism as well as protein

and fatty acid metabolism, and an example is cortisone, which is used to treat arthritis and other

inflammatory conditions. Mineralcorticoids on the other hand cause an increased reabsorption of Na+, Cl

-,

and HCO3- by the kidneys, which leads to an increase in the blood pressure. Aldosterone is an example

of a mineralcorticoid.

Figure 12. Structure of Cortisone (left) and Aldosterone (right).

Androgens are male sex hormones, secreted by the testes, and are responsible for the

development of male secondary sex characteristics during puberty and promote muscle growth.

Testosterone and 5α-dihydrotestosterone are androgens. Estradiol and estrone, on the other hand, are

female sex hormones known as estrogens. They are secreted by the ovaries and are responsible for the

development of female secondary sex characteristics as well as regulation of the menstrual cycle.

Progesterone, a progestogen, is the hormone that prepares the lining of the uterus for implantation of an

ovum and is essential for the maintenance of pregnancy. It also prevents ovulation during pregnancy.

Modified estrogens and progesterons are currently used as oral contraceptives. (Hanson, 2003) One

example of which is Norethindrone, a synthetic steroid. (Ch26: Steroids)

Figure 13. Structure of Testosterone (left) and 5α-dihydrotestosterone (right).

Figure 14. Structure of Estradiol, Estrone, and Progesterone. (Left to Right)

Figure 15. Structure of Norethindrone.

Although the various steroid hormones have remarkably different physiological effects, their

structures are quite similar. For example, the only difference between testosterone and progesterone is

the substituent at C-17, and the only difference between 5α-dihydrotestosterone and estradiol is one

carbon and six hydrogens, but these compounds provides the barrier between being male and being

female. These examples show the extreme specificity of biochemical reactions. (Bruice, 1994)

Steroids can also be classified by their source. Phytosterols are sterols obtained from plants,

while zoosterols from animals. Ergosterols are from yeasts, fungi, algae, and protozoans. Ecdysteroids

are steroids obtained from insects.

II. Methodology

Extraction of Terpenes Tapping. Tapping is the deliberate damage and subsequent collection of the resin. This method

is used to collect latex for rubber production and for gum turpentine. Expression. Expression is the process of oil extraction through the use of physical pressure and

the product is called expressed oil. For example by squeezing a piece of orange peel, you will see the oil

bearing glands burst and eject a fine spray of orange oil. Many commercially available citrus oils, bergamot in particular, are prepared in this way.

Distillation. Distillation of terpenes from their natural sources can be done in three ways: dry or empyrumatic distillation, steam distillation or hydrodiffusion. Dry distillation involves high temperatures since heat, usually direct flame, is applied to the surface of the vessel containing the plant material. This technique is reserved for the highest boiling of the oils, typically those derived from wood, because the high temperatures are necessary to vaporize their chemical components. In steam distillation, water or steam is added to the still pot and the oils are co-distilled with the steam. The presence of water in the pot during steam distillation limits the temperature of the process to 100 "C. Thus much less degradation occurs in this process than in dry distillation. However, some degradation does occur, like in the case of tertiary alcohols, particularly the higher boiling sesquiterpenoid and diterpenoid alcohols. These are often dehydrated in the pot and distilled. The steam distilled oil is separated from the water by means of a Florentine flask which separates them based on their differing densities. The aqueous distillate is sometimes referred to as the waters of cohobation.

Hydrodiffusion. Hydrodiffusion is essentially a form of steam distillation. However, it is carried out upside down since the steam is introduced at the top of the pot and the water and oil are taken off as liquids at the bottom. The plant materials diffuse through the cell membranes into the steam and are carried to the bottom of the still by the descending flow of condensate. This technique therefore saves energy because it is not necessary to vaporize the oil. Terpenes can also be extracted using solvent extraction. There are many forms of solvent extraction; the following are examples of procedures used in extracting terpenes:

1. Ethanolic extraction is seldom used for plant materials because of the high proportion of water compared to oil in the plant..

2. Enfleurage was used by ancient Egyptians to extract perfume ingredients from plant material and

exudates. In enfleurage, the purified fat absorbs the oil through intimate physical contact. For flowers, for example, the petals were pressed into a thin bed of fat. The perfume oils diffuse into the fat over time and then the fat can be melted and the whole mixture filtered to remove solid matter. On cooling, the fat forms a pomade. Nowadays odorous oil is extracted using ethanol, but during ancient times the pomade was used directly. The odorous oils are soluble in alcohol because of their degree of oxygenation. The fat used in the extraction and any fats and waxes extracted from the plant along with the oil, are insoluble in ethanol and so are separated from the oil. Removal of the ethanol by distillation produces what is known as an absolute.

3. The most important extraction technique for terpenes would be simple solvent extraction.

Traditionally benzene was used as the solvent but it has been replaced with the use of other solvents because of possible toxic effects. Petroleum ether, acetone, hexane and ethyl acetate, together with various combinations of these, are typical solvents for extraction. Recently, there has been a great deal of interest in the use of liquid carbon dioxide as an extraction solvent. The pressure required to liquefy carbon dioxide at ambient temperature is considerable and thus the necessary equipment is expensive. This is reflected in the cost of the oils produced; however, carbon dioxide has the advantage that it is easily removed and there are no concerns about residual solvent levels. The product of such extractions is called a concrete or resinoid. It can be extracted with ethanol to yield an absolute or distilled to give an essential oil

Figure 16. Simple Schematic for Solvent Extraction

Extraction of Steroids

Solvent Extraction. When deciding upon the best solvent for such extractions, consideration must be given to the polarity of the steroid of interest and the interaction of the steroid to binding proteins. The extraction solvent must ideally do two things; it must totally disrupt the binding of the steroid to protein and must extract the steroid of interest quantitatively and leave behind in the aqueous medium other steroids and non-specific interfering substances. In practice, of course, this is never possible and by its very nature such solvent extraction will also extract a number of other steroids of similar polarity and thus similar structure to the analyte which may well interfere in the final quantization. Depending upon the relative concentration of the steroid analyte in comparison to that of the potentially interfering steroids, it may be necessary to interpolate further purification steps prior to quantization. For steroids which are incorporated into the lipoproteins, such as cholesterol and vitamin D, it may be necessary to add chemicals to disrupt the lipoprotein structure prior to extraction since without this disruption the recovery of steroid analyte may be very low. When discussing the extraction of steroids, it must be remembered that many steroids often bind very tightly to glass and it is therefore advisable to silanise all glassware prior to use by treatment with dimethyldichlorosilane (1% v/v in toluene) or similar reagent washing afterwards with methanol. It is probably obvious, but still needs emphasising that solvent extraction generally precludes the use of plastic, silicone grease, etc. Considerable care must be taken to exclude all plastic since the occurrence of plasticisers (phthalates) in extracts may interfere in the final analysis.

Figure 17. Suitable Solvent for Steroids

Solid Phase Extraction. There are a wide variety of solid-phase materials available for use in the extraction of steroids. SPE material appears to fall into two distinct groups. Firstly, those systems based on Keiselguhr (Celite – a diatomaceous earth) treated in various and sometimes unspecified ways to inactivate the material and then sieved into different size ranges. The material is then packed into syringes, cartridges, etc. of various sizes and shapes made from a variety of different plastics. The aqueous medium is poured onto the material, which takes up the water, and the steroids are then eluted with organic solvents. This process would appear to be a simple liquid– liquid partition chromatography process similar to the celite partition column chromatography of the past. Since these columns have a finite capacity to absorb water, it is possible inadvertently to overload and if aqueous material passes through the column, steroids of interest will also pass through still dissolved in the aqueous matrix. Tox Elut (Varian), a similar type of system but using a more granular material designed for the analysis of drugs of abuse has a dye incorporated into it, which indicates how far down the column the added aqueous medium has reached. Such systems are still occasionally used for preliminary purification before quantization.

The second type of SPE material is based upon microparticulate silica either used directly or modified in an ever increasing variety of different ways. Some of the non-polar, polar and ion-exchange sorbents, which are in use today and can be obtained from a variety of sources are examples. These sorbents can be packed into syringe-like reservoirs or pre-packed cartridges of different sizes, which can cope with differing loads. These SPE systems are based on a variety of absorption and partition.

Immunoaffinity Extraction. In an ideal world, the initial extraction procedure would not be necessary as the quantization method would be so specific that neither the biological matrix nor steroids with similar structures to that of the analyte would interfere. Unfortunately, although some direct immunoassays are described, there are very few of these analytical methods for steroids which cannot be improved by some form of extraction and/or pre-purification before quantization. Extraction procedures, which removed only the steroid of interest from the matrix, would be of considerable value in some circumstances and one extraction method has shown considerable promise in this area. Use of columns packed with immobilised antibodies (immunoaffinity columns - IAC) for extraction can provide a means of selective extraction and purification. The selectivity depends of course upon the specificity of the antibody for the analyte. In these methods, the antibody is chemically attached to a support and kits are available to carry out this procedure. The immobilised antibody is then packed into a column and the matrix percolated through it. After washing, the analyte can be released from the antibody and thus eluted from

the column by altering the salt concentration. Highly specific antibodies obviously provide a selective extraction but there may be situations when a less selective, broad spectrum extraction is required. This can be achieved by using an antibody with broader specificity.

Molecularly Imprinted Polymers and Restricted Access Material.New forms of selective

extraction have been introduced but not yet widely applied to steroid analysis. Molecularly imprinted polymers, which could be regarded as synthetic ‗antibodies‘ are one. While another development uses the so-called restricted access material (RAM) coupled on-line to LC-MS or by the use of column switching – after absorption onto a C4-alkyl-diol silica RAM. The absorbed steroid was backflushed onto a conventional C18 column followed by ES-MS. Isolation and Detection of Sample

TLC Chromatography. The adsorptive material is coated onto a plate for thin-layer chromatography (TLC). Thin-layer plates can be either prepared in the laboratory or alternatively bought from a number of suppliers. While original TLC was carried out using glass plates, today the support is usually coated onto aluminum foil, which has the advantage that it is light, unaffected by solvents used for elution and areas of interest can be removed by cutting with scissors rather than the alternative procedure of scraping off the adsorbent material. TLC of steroids is still widely carried out and the adsorptive material used is usually silica gel although aluminum oxide has been used for the separation of C19 androgens. TLC has the advantage that a large number of samples can be processed in a single chromatographic run. The difficulty with TLC is the need to identify the areas on the plate which correspond to the steroid of interest. Steroids which are ultraviolet (UV) absorbing (e.g., delta-4-3-ones absorb at 240 nm) can be visualized with the use of UV light and adsorptive material is available which contains a fluorescent compound which enhances the UV absorbance of the steroid of interest. Steroids which do not absorb in the UV may have to be visualized by spraying part of the plate to identify the position of standards which have been run together with the samples of interest. There are a wide variety of methods of visualizing steroids on TLC plates usually involving spraying and/or heating with a variety of reagents which may or may not be specific for particular types of steroids. If standards have not been run, a narrow side strip of the TLC plate can be removed and the steroids located.

Gas-Liquid Chromatography. Gas–liquid chromatography (GLC or GC) is a partition system where the steroid solute is in the vapor phase. Because of the relatively high molecular weight of steroids and their derivatives, GLC has to be carried out at high temperatures, usually in excess of 200°C. The vaporized steroid, once introduced into the GLC column, is carried through the system by a gas, usually helium, because it is less dense than nitrogen, gives improved separation but is of course much more expensive to buy. Better separation can be achieved with hydrogen but there are safety issues to be considered. For many years, the separation procedure with the greatest resolving power was gas chromatography, originally carried out using packed columns but today capillary columns of glass or fused silica are more popular.

High Pressure Liquid Chromatography. I. High temperatures are not required. II. Choice of stationary and mobile phases for optimal separation. III. Material can be recovered from the column eluates for further analytical procedures. IV. The resolution achieved by HPLC is superior to TLC and paper chromatography, V. HPLC offers the potential and versatility for separation of intact conjugates. VI. Although most steroid metabolites are virtually without ultraviolet absorption, which is the

most useful of current detectors, some further metabolites of steroids can be detected with a refractive index or an electrochemical detector or by the use of pre- or post-column reaction with compounds which cause enhanced UV absorbance, fluorescence etc.

VII. Methods of linking HPLC to mass spectrometers have greatly improved, allowing the routine use of LC-MS and LC-MS-MS

Mass Spectroscopy. For steroid analysis a number of different types of ionization methods are used to generate gas-phase ions and include; electron ionization (EI), chemical ionization (CI), electrospray (ES), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and the recently introduced, desorption electrospray ionization (DESI) technique. The selection of the appropriate ionization mode is one of the key decisions for the analyst to make, and thus we discuss the most important ionization modes in some detail below. Enormous technological progress, in both instruments and computers, now enables spectroscopists to interpret mass spectra directly without the need to speculate. Exact mass measurements made at high resolution on all fragments provide information about their elemental composition, and tandem mass spectrometry (MS/MS) techniques make it possible to follow the fate of each fragment. Thus, the structure of unknown compounds can now often be found merely by using mass spectrometry. It will become evident that vacuum ionization techniques i.e. EI, are most useful for volatile compounds; while atmospheric ionization methods such as ES and APCI are more suitable for less volatile compounds. Mass spectroscopy can be done in concert with either gas chromatography or liquid chromatography or mass spectroscopy also.

Colorimetric Tests Bromine Water Test. Bromine will add to the carbon-carbon double bond of alkenes to produce

dibromoalkanes and with alkynes to produce tetrabromoalkanes. When this reaction occurs, molecular

bromine is consumed, and its characteristic dark red‑brown color disappears if bromine is not added in

excess. The rapid disappearance of the bromine color is a positive test for unsaturation. The test is not

unequivocal, however, because some alkenes do not react with bromine, and some react very slowly. In

the case of a negative test, therefore, the potassium permanganate test should be performed.

Lieberman-Burchard Test. The Lieberman–Burchard or acetic anhydride test is used for the

detection of cholesterol. The formation of a green or green-blue color after a few minutes is a positive

result. Lieberman–Burchard test is a colorimetric test to detect cholesterol, which gives a deep green

color. This color begins as a purplish, pink color and progresses through to a light green then very dark

green color. The color is due to the hydroxyl group (-OH) of cholesterol reacting with the reagents and

increasing the conjugation of the un-saturation in the adjacent fused ring. Since this test uses acetic

anhydride and sulfuric acid as reagents, caution must be exercised so as not to receive severe burns.

Lifschutz Test. The sterol is combined with perbenzoic acid and heated in glacial acetic acid;

sulfuric acid is then added. A bluish green color means a positive result. Alternative method uses

FeCl3∙6H2O with glacial acetic acid and concentrated sulfuric acid. Same bluish green color is observed.

Phosphomolybdic Acid Test. Phosphomolybdic acid, also known as dodeca

molybdophosphoric acid or PMA is a component of Masson's trichrome stain. It is a yellow-green

compound, freely soluble in water and polar organic solvents such as ethanol. It is used as a reagent in

thin layer chromatography for staining phenolics, hydrocarbon waxes, alkaloids and steroids. Conjugated,

unsaturated compounds reduce PMA to molybdenum blue. The color intensifies with increasing number

of double bonds in the molecule being stained.

Potassium Permanganate (Baeyer Test). The Baeyer test, depends on the ability of potassium

permanganate to oxidize the carbon‑carbon double bond to give alkanediols or the carbon-carbon triple

bond to give carboxylic acids. The permanganate is destroyed in the reaction, and a brown precipitate of

MnO2 is produced. The disappearance of the characteristic color of the permanganate ion is a positive

test for unsaturation. However, care must be taken, since compounds containing certain other types of

functional groups (for example, aldehydes, containing the --CH=O group) also decolorize permanganate

ion.

Rosenheim Test. A chloroform solution of sterol with 90% tricholoracetic acid which gives a blue

or pink color.

Salkowski Reaction. Cholesterol is oxidized in the presence of an excess amount of phosphoric

acid and ferric ions (FeCl3) to give a reddish purple color whose absorbance is measured at 560 nm.

Other Salkowski reaction uses cholesterol dissolved in chloroform and mixed with sulfuric acid. A two-

phase system forms where the upper layer is red and the lower layer is green.

Zlatkis Test. A solution of cholesterol is added to a solution of ferric chloride in concentrated

sulfuric acid and glacial acetic acid to give a purple color. This test is used to detect cholesterol in plasma.

III. Elucidation of Structure

Terpene Structure Elucidation

In exceptional cases, terpenes crystallize after chromatographic purification, thus enabling

determination of their three-dimensional structure in the solid state by X-ray crystallography. For the most

part, an amorphous or oily material rather than a single crystal is obtained after chromatographic

separation, and high-resolution NMR has been identified as the most efficient method to elucidate the

three-dimensional structure of molecules in solution, requiring sample quantities of less than one mg.

Other spectroscopic methods such as UV- and visible light absorption- and IR spectroscopy

predominantly permit the identification of known terpenes, e.g., by computer-assisted spectral

comparison on the basis of digitized spectroscopic data files or spectra catalogues. In the case of

unknown terpenes, chromophores such as carbonyl groups and their structural environment can be

detected by UV spectroscopy. Nearly all functional groups of a terpene are identified in the IR spectrum

by means of characteristic vibration frequencies; OH single bonds, for example, vibrate with 3600 cm-1

,

carbonyl double bonds with 1700 cm-1, and carbon-oxygen single bonds with 1200 cm

-1 detected as

absorption bands in the IR spectra. Mass spectrometry (MS) detects the molecular mass of a compound

with a precision of 10-4 mass units. Owing to the isotope mass defect of elements the molecular formula

of a terpene can be determined by high-resolution mass spectrometry of the molecular ion. For example,

the molecular formula C17H22O4 of acanthifolin from Senecio acanthifolius (Asteraceae) is calculated from

the molecular mass of 290.1525 determined by high-resolution mass spectrometry of the molecular ion.

Additionally, partial structures of molecules can be derived from the masses of the ions arising from

fragmentation of the molecular ion and detected in the mass spectrum.

Steroid Structure Elucidation

Infrared. The several tables show the different important wavenumbers and their corresponding

responsible groups.

NMR. NMR spectra of steroids are usually measured in CDCl3. Dispersion of signals can be to a

certain extent influenced by a change of solvent (mainly benzene or pyridine). Even small induced shifts

can in some cases significantly simplify the analysis of spectra and enable the extraction of J(H,H) values

in the region of strongly overlapping signals. The shifts induced with aromatic solvent (ASIS) are largest

in steroids containing polar substituents and for protons in their neighbourhood. Much larger shifts can be

induced by lanthanide shift reagents (LSR). Paramagnetic lanthanide ion brings about dramatic changes

in chemical shifts of nuclei in the substrate molecule. The size of lanthanide induced shifts (LIS) depends

on the ratio of LSR and substrate. The relative LIS values for individual protons depend on the distance

and orientation of a given proton and the lanthanide in the dynamic complex of LSR with a steroid. The

disadvantage of LSR application is the line-broadening of the proton signal that increases with the

LSR/substrate ratio and also with the increasing magnetic field of the spectrometer.

In 1D proton NMR spectra of steroids overlapping signals of CH2 and CH groups appear as a

characteristic hump in the region 0.5–2.5 ppm that is difficult to analyze. Most of the structure information

from low-frequency (<200 MHz) 1H NMR data was therefore traditionally obtained from easily visible

strong signals of methyl groups and from rare signals of protons shifted from the steroid hump either by

hybridization (olefinic protons) or by substituent effects (mainly CH–O protons). The much larger range of

carbon-13 chemical shifts eliminates this problem with resolution of signals in 13

C NMR spectra of steroids.

X-Ray Crystallography. This determines the arrangement of atoms within a crystal and provides

information about: 1) 3D picture of the density of electrons within a crystal, 2) mean positions and sizes of

the atoms in the crystal, 3) lengths, angles, and types of chemical bonds and 4) chemical functions,

interactions, and processes.

Some Spectra of Terpenes

Figure 18. UV-Vis spectrum of menthol. (Kumar, Choudhary, Joshi, Ramya, Sahithi, & Veena, 2011)

Figure 19. NMR spectrum of menthol. (File:Menthol Proton Spectrum.jpg)

Figure 20. IR spectrum of menthol. (AIST:RIO-DB Spectral Database for Organic Compounds,SDBS)

Figure 21. MS spectrum of menthol. (AIST:RIO-DB Spectral Database for Organic Compounds,SDBS)

Some Spectra of Steroids

Figure 22. UV-Vis spectrum of cholesterol.

Figure 23. NMR spectrum for cholesterol.

Figure 24. IR spectrum for cholesterol.

Figure 25. MS spectrum for cholesterol.

IV. Chemical Synthesis

Terpenes Chemical Synthesis

A. Chemical Synthesis of Menthol

Figure 26. Synthesis of Menthol from Myrcene.

The process begins by forming an allylic amine from myrcene, which undergoes asymmetric

isomerisation in the presence of a BINAP rhodium complex to give (after hydrolysis) enantiomerically pure

R-citronellal. This is cyclized by a carbonyl-ene-reaction initiated by zinc bromide to isopulegol which is

then hydrogenated to give pure (1R,2S,5R)-menthol.

Racemic menthol can be prepared simply by hydrogenation of thymol, and menthol is also

formed by hydrogenation of pulegone.

B. Chemical Synthesis of Camphor

Camphor can be produced from alpha-pinene, which is abundant in the oils of coniferous trees

and can be distilled from turpentine produced as a side product of chemical pulping. With acetic acid as

the solvent and with catalysis by a strong acid, alpha-pinene readily rearranges into camphene, which in

turn undergoes Wagner-Meerwein rearrangement into the isobornyl cation, which is captured by acetate

to give isobornyl acetate. Hydrolysis into isoborneol followed by oxidation gives camphor.

Figure 27. Synthesis of Camphor from α-pinene.

Steroid Chemical Synthesis

A. Diosgenin to Progesterone Synthesis: The Marker Degradation

Figure 28. The Marker semisynthesis of progesterone from diosgenin.

An economical semisynthesis of progesterone from the plant steroid diosgenin isolated from

yams was developed by Russell Marker in 1940 for the Parke-Davis pharmaceutical company. This

synthesis is known as the Marker degradation. First, the spiroketal is opened, then acetylation of 26-

hydroxyl occurs, followed by loss of proton. Then esterification of 3-hydroxyl follows. After which, a

selective oxidation of the 20,22 double bond occurs. Upon ester hydrolysis, the resultant alcohol

dehydrates to form a conjugated system, called dehydropregnenolone acetate. Selective catalytic

hydrogenation of 16,17 double bond occurs at the less hindered α face, and the resulting pregnenolone

undergoes Oppenauer oxidation of 3-hydroxyl to ketone and tautomerism to give progesterone.

Additional semisynthesis of progesterone have also been reported starting from a variety of

steroids. For the example, cortisone can be simultaneously deoxygenated at the C-17 and C-21 position

by treatment with iodotrimethylsilane in chloroform to produce 11-keto-progesterone (ketogestin), which in

turn can be reduced at position-11 to yield progesterone.

B. The Johnson Total Progesterone Synthesis

A total synthesis of progesterone was reported in 1971 by W.S. Johnson. The synthesis begins

with reacting the phosphonium salt 7 with phenyl lithium to produce the phosphonium ylide 8. The ylide 8

is reacted with an aldehyde to produce the alkene 9. The ketal protecting groups of 9 are hydrolyzed to

produce the diketone 10, which in turn is cyclized to form the cyclopentenone 11. The ketone of 11 is

reacted with methyl lithium to yield the tertiary alcohol 12, which in turn is treated with acid to produce the

tertiary cation 13. The key step of the synthesis is the π-cation cyclization of 13 in which the B-, C-, and

D-rings of the steroid are simultaneously formed to produce 14. This step resembles the cationic

cyclization reaction used in the biosynthesis of steroids and hence is referred to as biomimetic. In the next

step the enol orthoester is hydrolyzed to produce the ketone 15. The cyclopentene A-ring is then opened

by oxidizing with ozone to produce 16. Finally, the diketone 17 undergoes an intramolecular aldol

condensation by treating with aqueous potassium hydroxide to produce progesterone.

Figure 29. The Johnson total synthesis of progesterone.

V. Biosynthesis

Terpene Biosynthesis

Terpenes appear structurally unrelated but all contain a multiple of 5 carbons. This is so because

terpenes biosynthetically arise from the 5-carbon precursor isopentyl diphosphate, formerly called

isopentyl pyrophosphate, abbreviated as IPP.

IPP is biosynthesized by two different pathways. In animals and higher plants, the mevalonate

pathway is used to biosynthesize 15C sesquiterpenes and 30C triterpenes, while the deoxyxylulose

phosphate pathway is utilized to biosynthesize 10C monoterpenes, 20C diterpenes and 40C

tetraterpenes. In bacteria, both pathways are used.

Figure 30. Basic pathway of synthesis of terpenes.

A. Mevalonate Pathway to IPP

Step 1: Claisen Condensation

An acetyl group is first bound to acetoacetylCoA acetyltransferase by nucleophilic acyl

substitution reaction with a cysteine –SH group. Formation of an enolate ion from a second molecule of

acetyl CoA, followed by Claisen condensation, give acetoacetyl CoA.

Figure 31. Claisen condensation to form acetoacetyl coA

Step 2: Aldol Condensation

Acetoacetyl-CoA reacts with another equivalent of acetyl-CoA as a carbon nucleophile. It first

binds to the cysteine –SH group in the 3-hydroxy-3-methylglutaryl-CoA synthase. Following the pattern of

an aldol reaction, the acetyl-CoA enolate ion adds on the re face of the ketone carbonyl group and

hydrolysis follows to give 3-hydroxy-3-methylglutaryl-CoA, or HMG-CoA.

Figure 32. Aldol condensation to form HMG-coA

Step 3: Reduction

Enzymatic reduction with dihydronicotinamide adenine dinucleotide (NADPH + H+) follows to

produce (R)- mevalonic acid. NADPH transfers a hydride to the thioester carbonyl group of HMG-CoA.

This requires an acid catalyst, possibly the protonated side chain of a lysine residue, from the enzyme 3-

hydroxy-3-methylglutaryl-CoA reductase, and gives a hemithioacetal intermediate. After coenzyme A

expulsion, the aldehyde intermediate undergoes another hydride-transfer addition to give mevalonate.

Figure 33. Reduction to form Mevalonate.

Step 4: Phosphorylation and Decarboxylation

Mevalonate is phosphorylated by reaction with ATP catalyzed by mevalonate kinase to produce

mevalonate 5-phosphate (phosphomevalonate), and then reacts with another molecule of ATP to produce

mevalonate 5-diphosphate (diphosphomevalonate). Another phosphorylation occurs, this time on the free

–OH group of diphosphomevalonate, catalyzed by mevalonate-5-diphosphate decarboxylase. Inorganic

phosphate is expelled to give a tertiary carbocation. The positive charge acts as a electron acceptor to

facilitate decarboxylation to produce isopentyl diphosphate.

Figure 34. Formation of IPP.

Figure 35. More specific mechanism on the formation of IPP.

B. Deoxyxylulose Phosphate Pathway to IPP

Step 1: Nucleophilic Addition

TPP ylid adds to the ketone carbonyl group of pyruvate to give an α–hydroxy iminium ion,

containing the C=N+ functional group.

Figure 36. Nucleophilic addition to dorm an iminium ion from pyruvate.

Step 2: Decarboxylation

The C=N+ group can accept electrons, allowing the thiamin addition product to undergo loss of

CO2, producing an enamine, a compound with an amino substituent on a C=C bond.

Figure 37. Formation of an enamine.

Step 3: Nucleophilic Addition

The enamine double bond does a nucleophilic addition to the si face of the glyceraldehyde 3-

phosphate (G3P) carbonyl group, producing a β–hydroxy iminium ion.

Figure 38. Formation of iminium ion from G3P.

Step 4: Cleavage

This is the reverse of the first step and is similar to a retro-aldol reaction. The β–hydroxy iminium

ion decomposes, regenerating TPP ylid and producing 1-Deoxy-D-xylulose 5-phosphate.

Figure 39. Formation of 1-deoxy-d-xylulose-5-phosphate.

Step 5: Rearrangement and Reduction

A base from the enzyme deoxyxylulose-5-phosphate reductoisomerase removes a proton from

the C3 hydroxyl group, and the alkoxide ion undergoes an acyloin rearrangement to yield an isomeric α–

hydroxy aldehyde. Reduction of the aldehyde carbonyl group by NADPH gives 2C-Methyl-D-erythritol 4-

phosphate.

Figure 40. Formation of 2C-methyl-D-erythritol-4-phosphate.

Step 6. Phosphorylations

2C-Methyl-D-erythritol 4-phosphate reacts with cytidine triphosphate (CTP), and the

diphosphocytidil-2C-methyl-D-erythritol is further phosphorylated by ATP to form 2-Phospho-4-

diphosphocytidyl-2C-methyl-D-erythritol. Cyclization then occurs, expelling CMP, and forming 2C-methyl-

D-erythritol 2,4-cyclodiphosphate.

Figure 41. Formation of 2C-methyl-D-erythritol-2,4-cyclodiphosphate.

Step 7. Reduction

Reduction by steps and mechanisms not yet known in detail give IPP, although it appears that

radical reactions, perhaps mediated by iron-sulfur clusters, are involved. One possibility is that the

cyclodiphosphate is converted to an epoxide, which then undergoes a deoxygenation and subsequent

reduction of the allylic alcohol.

Figure 42. Formation of IPP from 2C-methyl-D-erythritol-2,4-cyclodiphosphate.

C. IPP to GPP and FPP

IPP isomerizes to dimethylallyl diphosphate, formerly called dimethylallyl pyrophosphate and

abbreviated as DMAPP. The isomerization is catalyzed by IPP isomerase. The protonation of the IPP

double bond by a hydrogen-bonded cysteine residue gives a tertiary carbocation intermediate, and the

deprotonation by glutamate removes the pro-R hydrogen from C2, yielding DMAPP.

Figure 43. Formation of DMAPP.

The diphosphate group of DMAPP contains an Mg2+

acting as an electron sink, and upon leaving,

a carbocation is generated, which is then susceptible to the nucleophilic substitution attack by the double

bond of IPP. The intermediate tertiary carbocation is then deprotonated by a basic residue in the farnesyl

diphosphate synthase, forming geranyl diphosphate. Subsequent coupling of GPP with a second

molecule of IPP gives farnesyl diphosphate, also catalyzed by farnesyl diphosphate synthase.

Figure 44. Formation of FPP.

D. GPP to Limonene

Monoterpene cyclase catalyzes the isomerization of geranyl diphosphate to its allylic isomer

linalyl diphosphate (LPP). The diphosphate dissociates in an SN1 like mechanism, forming an allylic

carbocation, and resonance to form a tertiary carbocation, which then recombines with the diphosphate.

The diphosphate once again dissociates, and cyclization occurs by electrophilic addition of the cationic

carbon to the terminal double bond then gives a cyclic cation, which might either rearrange, undergo a

hrdride shift, be captures by a nucleophile or be deprotonated to give any of the several hundred known

monoterpenes. Limonene arises by deprotonation of the methyl hydrogen.

Figure 45. Formation of limonene.

E. FPP to epi-Aristolochene

Loss of diphosphate ion from FPP gives an allylic cation, which undergoes cationic cyclization to

yield a macrocyclic, tertiary carbocation. Loss of a proton then gives a neutral triene, which is then

reprotonated on C6 to give an isomeric cation. Further cyclization yields a bicyclic cation, which

undergoes a 1,2-hydride migration, a methyl shift and a deprotonation, resulting to the sesquiterpene epi-

Aristolochene.

Figure 46. Formation of epi-aristoloochene.

Steroid Biosynthesis

A. Lanosterol to Cholesterol

From lanosterol, the pathway for steroid biosynthesis continues on to yield cholesterol. Three

methyl groups are removed, one double bond is reduced, and another double bond is relocated.

Cholesterol then becomes a branch point, serving as the common precursor from which other steroids

are derived.

Figure 47. Formation of cholesterol.

Figure 48. Synthesis of several steroids

B. Cholesterol to Progesterone

Cholesterol undergoes side-chain cleavage by hydroxylation at C22 and C20 and cleavage of

C20-C22 bond, catalyzed by 20 α -hydroxylase, 22 hydroxylase & 20,22 desmolase, to produce

pregnenolone. Pregnenolone undergoes reduction catalyzed by 3β -hydroxysteroid dehydrogenase & D5

- D4 isomerase, producing progesterone.

C. Progesterone to Testosterone

Progesterone undergoes steroid α -hydroxylation at C17 and is catalyzed by 17 α - hydroxylyase

to produce 17 α - hydroxyprogesterone. Then, steroid 17,20-lyase catalyzes the cleavage of steroid C17-

C20 bond to form androstenedione. Androstenedione undergoes a redox reaction involving 17β -

hydroxysteroid dehydrogenase as the enzyme and zinc as its cofactor to produce testosterone. However,

this enzyme has known inhibitors, such as ethanol, licorice, phytoestrogens (flavonoids, coumarins,

coumestans), zearalenone, coumestrol, genistein, quercetin and biochanin A, and excessive copper.

VI. Recent Discoveries

Terpenes

Many of the world‘s most dangerous diseases like that of malaria and dengue fever are

transmitted by blood-feeding arthropods. Just recently, an outbreak of infection cases caused by tick

biting has been reported resulting in a fatality rate as high as 30% in some area of central China, and was

said to be caused by a new type of Bunyavirus. This virus is currently considered as a new emerging

deadly virus, thus there is a need for the development of antiplasmodium/antivirus agents as well as new

safe and effective repellents against blood-feeding arthropods.

In the year 1946, the US Army developed a potent and easily synthesized insect repellent known

as DEET (N,N-diethyl-3-methylbenzamide, 4), initially thought to be safe, but was later known to a

carcinogen. Another synthetic repellent known is picaridin (5), said to be as potent as DEET but not toxic

to humans. Aside from synthetic repellents, natural-product repellents were also used. PMD (p-

menthane-3,8-diol, 6) is one, the main component in citrus/eucalyptus solutions. Due to the growing

demand for new arthropod repellents, the genus Beautyberry (Callicarpa) was studied, since it was used

as a traditional biting-insect repellent in certain southern states of the US.

Cantrell et al. studied the chemical origins of the insect-repellent property of beautyberry extract,

and he found out that three potent mosquito-repellent compounds were responsible: callicarpenal (1) and

intermedeol (2), from the American plant, and spathulenol (3), from the Japanese plant. The study lead to

low isolation yields of callicarpenal, and bulk isolation and purification techniques were tedious. To add to

that, the compound was not that abundant in natural resources, thus leading to the recent study made by

Ling and his colleagues. They performed a stereoselective synthesis of (-)-callicarpenal, a potent

arthropod repellent. (Ling, Xu, Smith, Ali, Cantrell, & Theodorakis, 2011)

Figure 49. Structures of different mosquito repellent compounds.

Figure 50. Synthesis of (-)-callicarpenal from 5,8a-Dimethyl-3,4,8,8a-tetrahydronaphthalene-1,6 (2H,7H)-dione ((-)-7).

The synthesis of (-)-callicarpenal started from a readily available enantiomeric pure diketone, (R)-

5,8a-Dimethyl-3,4,8,8a-tetrahydronaphthalene-1,6 (2H,7H)-dione ((-)-7). Addition of ethylene glycol to the

(-)-7 solution and PTSA (p-toluenesulfonic acid) dissolved in benzene solution mixed together, was done

to selectively protect the C-4 carbonyl group to form the corresponding C-4 ketal. This process was

performed at 80°C for 24 hours in an argon atmosphere. After protection at C-4, the C-8 enone was

reduced under lithium/ammonia conditions, done by placing ketal in THF (tetrahydrofuran) and dropwise

addition of a solution of lithium in liquid ammonia at -78°C. The resulting enolate was alklated in situ with

allyl bromide to form ketone 8 as a single isomer. Ketone 8 is (4a‘R,5‘R,8a‘R)-50-Allyl-5‘,8a‘-

dimethylhexahydro-2‘H-spiro[[1,3]dioxolane-2,10-naphthalen]-6‘(7‘H)-one.

In order to convert 8 to 10, three steps is necessary. First the ozonolysis of the terminal alkene

followed by reduction of both carbonyl groups, performed by exposure to ozone at -78°C of the ketone 8

solution, with the resulting mixture flushed with argon, treated with PPh3 (triphenylphosphate) and

warmed up to 25°C. NaBH4 was added to the mixture at 0°C and in turn obtaining the corresponding diol.

The diol obtained was then selectively silylated by placing the diol in CH2Cl2 and adding 2,6-lutidine and

TISOP (triisopropylsilyl trifluoreomethanesulfonate). The last step is the oxidation of the remaining C-8

hydroxyl groups, being in the same solvent , making use of NMO (4-methylmorpholine-N-oxide) as the co-

oxidant and catalytic amount of TPAP (tetrapropylammonium perruthenate). Thus forming ketone 10,

(4a‘R,5‘R,8a‘R)-5‘,8a‘-Dimethyl-5‘-(2-((triisopropylsilyl) oxy) ethyl)hexahydro-2‘H spiro[[1,3]dioxolane-

2,10-naphthalen]-6‘(7‗H)-one. The authors also tried to remove the ozonolysis reaction and exchange it

with a sequence of alkene dihydroxylation which is followed by oxidative cleavage of the resulting 1,2-diol.

But on the contrary, treatment of 8 using OsO4 (osmium tetroxide) /NMO can lead to the formation of

hemiacetal 9, thus, complicating subsequent functionalization strategies. This part was done by addition

of OsO4, NMO, and dissolving 8 in acetone-water solution.

Since formation of 9 made subsequent fucntionalization strategies more complicated, structure 10

was the focus. A solution of CH3PPh3Br (methyltriphenylphosphonium bromide) in THF was treated

dropwise with NaHMDS (sodium bis(trimethylsilyl)amide) in THF at 0°C and the resulting orange solution

was treated with a solution of ketone 10 in THF at the same temperature. This process involves the

conversion of 10 to 11, (2-((4a‘R,5‘R,8a‘R)-5‘,8a‘-Dimethyl-6‘-methyleneoctahydro-2‘H-spiro

[[1,3]dioxolane-2,1‘-naphthalen]-5‘-yl) ethoxy) triisopropylsilane, under standard Wittig conditions. 11

dissolved in THF was treated with TBAF (tetrabutylammonium fluoride) for desilylation and lead to the

formation of an alcohol which was then hydrogenated in a Parr apparatus along with Crabtree‘s catalyst

in CH2Cl2, thus forming a reduced product, 12, 2-((4a‘R,5‘S,6‘R,8a‘R)-5‘,6‘,8a‘-trimethyloctahydro-2‘H-

spiro [[1,3] dioxolane-2,1‘-naphthalen]-5‘-yl)ethanol. Crabtree catalyst is the given namen to the complex

of iridium with 1,5-cyclooctadiene, tris-cyclohexylphosphine, and pyridine ([Ir(COD)(PCy3)(Py)]. The

produced 12 is a single diastereomer at C8 center. Then the produced 12 in THF was mixed HCl for 8

hours at room temperature, and then neutralized with saturated NaHCO3. This process of removing the

acetonide in acidic conditions led to the formation of a hydroxyl ketone 13, (4aR,5S,6R,8aR)-5-(2-

Hydroxyethyl)-5,6, 8a-trimethylocta hydronaphthalen-1(2H)-one. Same procedure for the formation of 11

was performed on 13 to form 14, 2-((1S,2R,4aR,8aR)-1,2,4a-trimethyl-5-methylenedeca hydro-

naphthalen-1-yl)ethanol. Then RuCl3.H2O (rhodium (III) chloride hydrate) was added to a solution of 14 in

ethanol for the isomerisation of the exocyclic double bond to form endo-alkene 15, 2-((1S,2R,4aR,8aR)-

1,2,4a,5-tetramethyl-1,2,3,4,4a,7,8,8a-octahydro naphthalen-1-yl) ethanol. Then the last step, oxidation of

15, to form (-)-1 was performed. This involved addition of NMO and TPAP to the solution of alcohol 15.

The synthesized (-)-1 and its analogues, 13, 14, and15, were tested for its mosquito-biting deterrent

effects against Aedes aegypti. The results obtained revealed that all four compounds were significantly

more effective than ethanol, but DEET was still more effective (Table 3).

Table 3. Mosquito biting-deterrent effects of (-)-callicarpenal (1) and its analogues 13, 14, and 15 Ae. Aegypti.

Steroids

Malika Ibrahim-Ouali and Hamze Khalil in the year 2010 have recently synthesized a pentacyclic

steroid through the use of stereoselective epoxide ring opening. Classical approach for drug development

when using a known lead structure is varying the substitution pattern and alteration of the stereochemical

relationships. Given a biological active compound, modifications can lead to a very useful homologue

showing a subtle biological difference than the patent substance. The change in the biological activity is

unpredictable, meaning it could result to a more potent agonist, a more selective agonist, or an antagonist.

For steroids, changes in the C-11 position leads to a major effect on the biological properties, like

corticoid activities. A recent study conducted by Salunke et.al. shown that 11-amino-12-hydroxy/keto

steroids are potential HIV-1 protease. HIV-1 protease is essential to the life-cycle of HIV. Also many

examples of pentacyclic steroidal derivatives are of pharmacological and biological importance. A popular

strategy to produce biologically active steroid hormone analogues is to incorporate short carbon bridges

spanning characteristic portions of the steroid backbone.

These recent advances has lead the authors to combine the two known process of synthesizing

biologically active steroid compound, introduction of a new ring and a new function at the C-11 position,

forming a new class of steroids. The authors stereoselectively synthesized pentacyclic steroids with an

amino function at the C-11 position using cholic acid as the lead structure.

In order to synthesize the aminolactone from six steps were performed. The first step was the

esterification of the acid group in the substituent at C-12 of cholate (1). The esterification process was

done by addition of methanol and PTSA (p-toluene sulfonic acid) in the presence of heat. After which, the

acetylation of the hydroxyl groups at the C-3 and C-7 carbon were performed by the addition of Ac2O

(acetic anhydride), pyridine, and DMAP (4-dimethylaminopyridine). This step led to the formation of

methyl-3,7-diacetoxycholate (3), which was then treated with a slight excess of mesyl chloride in

presence of pyridine at 0°C to room temperature for 24 hours, which gave a quantitative yield of methyl

3,7-diacetyl-12-mesyl cholate (4). Dehydromesylation of 4 was done by applying the conditions described

by Chen. This made use of AcOK (potassium acetate) and HMPT (hexamethylphosphoric triamide) at

100°C at 48 hours. The isolated methyl 3,7-diacetylchol-11-enate (5) was obtained in a good yield (80%).

The next step involved the epoxidation of the steroidal double bond (C11-C12) by the addition of m-

CPBA (meta-chloroperoxybenzoic acid) in CH2Cl2 (dichloromethane) at room temperature for 24 hours,

and thus forming the 11α,12 α-epoxide (6) in excellent yield. It must be noted the epoxide ring was

formed with high selectivity in the least hindered face of steroid. The α-configuration of the 11,12-epoxide

was assigned due to the presence of the C-18 angular methyl group blocking the β-face of the C ring

when the Δ11

double bond of 5 was epoxidized. The observed 1H-NMR spectrum showed characteristic

doublets (J = 4 Hz) for the CH-11 and CH-12 protons at δH 2.98 ppm and 3.16 ppm, respectively.

The last step involved the selective epoxide ring opening with nucleophilic amines. This was done

by the addition of different amines in ethanol/toluene (1:1) solution, as seen in Figure 52, at reflux for 24

hours. This step made use of two reactions- stereoselective epoxide ring opening and intramolecular

lactonization and in turn forming the steroidal lactone (7). The assignment of structure 7 was supported

by the NMR spectra that showed a disappearance of the characteristic singlet of the lateral chain at δH

3.66ppm (s, C-23-OMe) and the coupling constant between 11-H and 9-H is 3.8 Hz, which shows the 11-

H should be in the α-position. Furthermore, the positions of the angular methyl groups at δH ~ 0.76 ppm (s,

C-13-Me) and δH ~0.98ppm (s, C-10-Me) are consistent with the 11-amino-12-oxa-structure. The stereo

and regioselectivity of the epoxide ring opening can be understood in Figure 53. (Ibrahim-Ouali & Khalil,

2010)

Figure 51. Synthesis of aminolactone from cholic acid.

Figure 52. Nucleophilic amine used with their respective yields.

Figure 53. Proposed mechanism of the epoxide ring opening.

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