7. Triterpenes and Sterols

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7. Triterpenes and Sterols

• RA Macahig
• FM. Dayrit

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Introduction

• Triterpenes and sterols comprise a large group of
  distinctive polycyclic terpenes.
• Triterpenes are C30 compounds which have four or
  five fused cyclohexyl rings.
• Sterols are C21 to C29 compounds with a
  characteristic fused ring system of three
  cyclohexyl rings and one cyclopentyl ring.
• Triterpenes and sterols can be isolated in free
  form, glycosylated, or bound to fatty acids.
• Sterols are well known to have important
  hormonal activity in insects and mammals
  (including humans).

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Squalene

• Triterpenes arise from the reductive dimerization
  of two farnesyl diphosphate chains (2 x C15)
  which condense in a head-to-head manner to form
  squalene.
• Squalene is the key intermediate in the
  biosynthetic pathway to the two key triterpene
  intermediates cycloartenol (in plants) and
  lanosterol (in animals).

• Important features of squalene
• It does not have a -OPP leaving group.
• It is a symmetric molecule.

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Biosynthetic mechanism for the head-to-head
dimerization of two farnesyl diphosphate chains
to produce squalene.
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Squalene
Squalene itself is found in large quantities in
shark liver oil, thus its name (squalus shark).
It is also obtained from vegetable oils, such as
rice bran, wheat germ, and olives. It is marketed
as a cosmetic and health supplement for
protection against oxidative processes which lead
to aging, atherosclerosis and other immune
diseases.
Squalene is found only in the all-trans
double-bond configuration. All variations of
structures among triterpenes arise from the
conformation of folding there are no geometric
isomers of double bonds.
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Overview of squalene formation and the biogenetic
relationships of triterpenes and sterols.
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Early studies on the enzymes involved in
cholesterol biosynthesis proposed that all the
enzymes involved in the conversion of acetyl-CoA
to farnesyl diphosphate (FPP), with the exception
of 3-hydroxy-3-methylglutaryl-coenzyme A
reductase (HMG-CoA reductase), are cytosolic or
are located on the endoplasmic reticulum. This
study shows that FPP is found in peroxisomes.
Peroxisomes are ubiquitous organelles in
eukaryotic cells that participate in the
metabolism of fatty acids and other metabolites.
Peroxisomes have a single lipid bilayer membrane
that separates their contents from the cytosol
(the internal fluid of the cell) and contain
membrane proteins critical for various functions.
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2,3-Oxidosqualene cyclase is an integral membrane
enzyme that catalyzes the cyclization of squalene
epoxide to lanosterol. The solubilized enzyme was
purified to homogeneity by fast protein liquid
chromatography. The purified enzyme consists of a
single subunit that has an apparent molecular
weight of 65,000 as determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The enzyme obeys saturation
kinetics and the apparent Km of
(2,3)-oxidosqualene is 15 mM and the apparent
kcat/Km is 200 M-1 min-1.
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• Terpene biosynthetic pathways differ in
  prokaryotes and eukaryotes. The two pathways
  shown here diverge after presqualene diphosphate.
  
• Staphyloxanthin biosynthesis in S. aureus. The
  NADPH reduction step is absent, resulting in
  formation of dehydrosqualene, not squalene.
• Steroid biosynthesis in humans and yeasts passes
  through squalene.
• (Liu et al., Science 319, 1391 -1394 (2008))

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Overview of cyclization reactions in triterpenes

• The triterpene structures are formed from the
  enzyme-catalyzed cyclization of squalene. The
  first step involves the activation of the
  asymmetric squalene by epoxidation of a terminal
  olefin group producing the chiral intermediate
  (3S)-2,3-oxidosqualene.
• Various modes of cyclization produce 29 major
  triterpene skeletal types plus about 5 irregular
  ones. The main sources of variation among the
  triterpenes are due to
• Conformation of folding (regiochemistry)
• Conformation of folding (stereochemistry)
• Skeletal rearrangements
• Further chemical transformations, such as
  oxidation, methylation, and glycosylation.

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Overview of cyclization reactions in triterpenes
1. The conformation of folding regiochemistry.
(3S)-2,3-oxidosqualene is folded into specific
geometries under enzyme control during the
cyclization process. These conformations are
either chair or boat (c chair b boat).
There are four conformational folding patterns
a. To form tetracyclic products, two folding
conformations are observed (c - c - c - b) and
(c - b - c - b) b. To form pentacyclic products,
the folding conformations are (c - c - c - c -
c) and (c - b - c - c - b) and c. Irregular
cyclization leads to other types of triterpenes.
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Model for the oxidosqualene cyclase enzyme which
controls the transition state conformation of
triterpene formation.
chair
chair
boat
boat
Each folding conformation and product is presumed
to require a unique enzyme. A number of cyclases
have been isolated and sequences from a number of
plants and microorganisms, as well as from pig
liver, has been determined.
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2. The conformation of folding stereochemistry.
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Triterpenes an overview
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Chair - chair - chair - boat conformation of
squalene folding to form tetracyclic triterpenes
the Dammaranes.
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Chair - chair - chair - boat conformation the
Dammaranes.
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Further modifications on the dammarane skeleton.
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Further modifications on the dammarane skeleton.
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Chair-boat-chair-boat conformation Cycloartenol
and Lanosterol.
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Chair-boat-chair-boat Cycloartenol and
Lanosterol.
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cycloartenol and lanosterol.
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Unusual triterpenes. Ambrein and malabaricol are
examples of interrupted cyclization.
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Some important triterpenes
Corosolic acid (2?-hydroxyursolic acid) has been
identified as one of the active constituents in
the leaves of Banaba (Lagerstroemia speciosa). It
has been shown to lower blood sugar in animals.
The leaves are very popular as a remedy for
diabetes. Maslinic acid (2?-hydroxyoleanolic
acid), a structurally-related triterpene, has
also been identified in the leaves.
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Triterpene glycosides

• In plants, triterpenes have been isolated in
  either of two forms in its free form as an
  aglycone (that is, without any sugar attached) or
  as the glycoside (aglycone sugar).
• It is likely that in many cases, the aglycone
  may have been an artifact of the isolation
  process for example, enzymes present in the
  leaves may hydrolyze the triterpene glycosides,
  or the extraction process itself may cause
  hydrolysis.
• At any rate, it is reasonable to assume that
  triterpene glycosides are common constituents of
  plants and that many triterpenes may exist in the
  glycoside form in the intact plant.

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Ginseng is a very complex mixture of at least 31
ginsenoside triterpenes. Ginseng is used in both
traditional and modern forms, especially for
maintenance of health in old-age. It is widely
used in both Europe and East Asia. In 1994 retail
sales in Europe reached about 50M.
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Triterpenes summary of cyclization modes c-c-c-b
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Introduction to sterols
The sterols make up a large group of compounds
which are derived from triterpenes and have the
characteristic tetracyclic ring ranging from C27
to C29. The various sterols are differentiated by
the following main structural features 1. Side
chain on C17 position 2. Modifications on the
A-ring 3. Other modifications
4. Alcohol in the C3 position with the exception
of some human hormones.

• Notes on the structure
• The C3-hydroxy is always ?.
• The A/B ring fusion can be cis or trans (5-H is
  ? or ?, respectively).

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Introduction to sterols

• Sterols are found in all kingdoms. In many
  organisms, these are synthesized de novo from
  mevalonic acid ? squalene in some, they are
  obtained from the diet in others, they are both
  synthesized and ingested.

• The sterols in the various kingdoms have
  characteristic structural features, and so are
  classified according to their respective
  kingdoms e.g., mycosterols (fungi), phytosterols
  (plants), marine sterols (sponges and other
  invertebrate marine organisms), and zoosterols
  (animals).

• However, it is important to note that the
  occurrence of specific sterol compounds is not
  always unique to an organism. For example,
  although cholesterol is the prototypical
  zoosterol, it is also produced by some algae and
  plants. On the other hand, stigmasterol, a
  typical plant sterol, is not produced by animals.

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Introduction to sterols

• Sterols perform diverse functions in various
  organisms. For example, sterols are essential
  constituents of biological membranes of different
  organisms. They function as hormones and
  pheromones in a wide range of organisms ranging
  from mammals to insects. Synthetic steroids have
  also been developed to control or mediate in
  various aspects of human physiology, disease and
  reproduction.

• Sterols are derived from the group of
  tetracyclic triterpenes using the c-b-c-b folding
  conformation via a series of transformations
  starting with lanosterol (in mammals) and
  cycloartenol (in plants). The primary mammalian
  sterol is cholesterol (C27) while the primary
  plant sterol is stigmasterol (C29).

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Introduction to sterols

• In the literature, the sterols are also
  classified the following groups, partly based on
  structure, and partly based on activity
• sterols steroidal skeleton with a C3 alcohol
  group
• cardiotonic sterols and steroidal saponins
  physiologic activity
• ecdysones insect hormones
• bile acids modified steroids in humans
• steroidal hormones steroids which affect
  physiology

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From triterpenes to sterols A. lanosterol to
cholesterol in mammals B. cycloartenol to
stigmasterol in plants and fungi.
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Major mammalian sterol skeletal structures.
(Estrogens and some androgens do not have the
C3 alcohol group.)
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Major sterol skeletal structures in plants and
fungi.
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Survey of some important sterols
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Phytosterols

• The path from squalene to sterols In plants and
  fungi
• squalene ? cycloartenol ? phytosterols and fungal
  sterols

1. Artificially introduced cycloartenol in plants
is incorporated into phytosterols. 2. In plants,
cycloartenol is found in large amounts while
lanosterol is rare. 3. The conversion of
cycloartenol to the phytosterols has also been
shown to follow a metabolic grid. Cycloartenol
can be funneled into two main pathways
methylation of the intermediate to C28 and C29
phytosterols, or formation of C27 sterols (which
includes cholesterol). The most common
phytosterol is stigmasterol which is a C29
compound.
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Proposed pathway for the conversion of
cycloartenol in plants to sitosterol.
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Proposed mechanism for the conversion of
cycloartenol to phytosterols.
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Phytosterols mixed metabolites
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Cardiotonic sterols
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Steroidal saponins
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Zoosterols
The conversion of lanosterol to cholesterol in
mammals has been extensively studied using rat
liver tissues and in vitro enzyme preparations.
It has been found that there is no unique,
sequential biosynthetic pathway. That is, the
enzymes which catalyze the various
transformations are not absolutely specific for a
particular substrate. It has also been observed
that different enzymes are able to catalyze the
same reaction on different substrates. The
biosynthesis of cholesterol is best described as
a metabolic grid. Lanosterol is converted into
cholesterol, the entry compound for all of the
steroids in mammals, and are called zoosterols.
In the animal liver, lanosterol is converted into
cholesterol, while cycloartenol is not
metabolized. This sequence of transformations in
mammals is as follows squalene ? lanosterol ?
cholesterol ? steroid hormones
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The pathway for the conversion of lanosterol to
cholesterol in mammals is believed to occur
through a metabolic grid where there is no single
pathway and transformations occur in random
order.
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Lanosterol to cholesterol.
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Lanosterol to cholesterol.
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Lanosterol to cholesterol.
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Cholesterol is the most decorated small molecule.
The discovery of LDL by Goldstein and Brown is
the most recent Nobel Prize (1985) in the field
of cholesterol metabolism.
Lipoprotein metabolism. Abbreviations apoB48,
apolipoprotein B48 apo A1, apolipoprotein A1
apoB100, apolipoprotein B100 TG, Triglyceride
LRP, LDL receptor-like protein ABCA1,
ATP-binding cassette A1 SRA1, SRB1, CD36, 3
members of the scavenger receptor family CETP,
cholesteryl ester transfer protein VLDL, very
low density lipoprotein IDL, intermediate
density lipoprotein LDL, low density
lipoprotein and HDL, high density lipoprotein.
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• Cholesterol is an important component of cell
  membranes, reducing its fluidity.
• Cholesterol is the precursor molecule for the
  synthesis of steroid hormones, vitamin D and bile
  salts.
• Cholesterol is derived from the diet or
  synthesized within the body. The typical human
  diet contains 200500 mg of cholesterol. 3060
  of intestinal cholesterol is absorbed.
• The principal sites of cholesterol biosynthesis
  are the liver and the CNS, using fatty acids.
• Cholesterol can be lost from the body as bile
  salts and intestinal cholesterol which are not
  absorbed, and in sebum. The daily faecal loss of
  cholesterol from bile and desquamated cells is
  550 mg and as unabsorbed bile salts 250 mg. Daily
  losses in sebum are 100 mg.
• A total of some 900 mg must therefore be derived
  from the diet or synthesized each day.

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• Cholesterol circulates as a component of
  lipoproteins. Its concentration in humans is
  typically in the range 100300 mg dl-1. In many
  Asian countries, adult levels are often less than
  200 mg dl-1 (5 mmol l-1), whereas in Europe and
  the USA they are generally greater than 200 mg
  dl-1.
• The principal plasma lipoproteins are the
  chylomicrons, VLDL, LDL and HDL. Chylomicron
  remnants, VLDL and LDL, cause atherosclerosis and
  HDL opposes this.
• Chylomicrons are secreted by enterocytes into the
  intestine and enter blood circulation from lymph
  via the thoracic duct. They are rich in
  triglycerides.

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• Triglycerides, the principal fat in the diet, are
  absorbed from mixed micelles formed in the
  intestinal lumen as fatty acids and
  monoglycerides after its hydrolysis by intestinal
  and pancreatic lipases.
• In the enterocyte, triglyceride is resynthesized
  and complexed with Apo B48, a process involving
  microsomal triglyceride transfer protein (MTP),
  to form chylomicrons.
• Short chain fatty acids (C810) escape this
  process and enter the portal blood directly.
• Free cholesterol is absorbed from mixed micelles
  by the enterocytes from the gut lumen, and is
  re-esterified and is packaged with triglyceride
  to form the core of chylomicrons.

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Carotenoids, C40

• The carotenoids make up an important and
  ubiquitous group of C40 terpenes. Many of the
  yellow, orange, red and purple colors of
  organisms are due to carotenoids. Approximately
  600 naturally-occurring carotenoids have been
  isolated.

• The carotenes arise from the head-to-head
  condensation of geranylgeranyl diphosphate (2 x
  C20) to form lycopersene, the biosynthetic
  equivalent of squalene. This process takes place
  in the chloroplasts of plants or the
  chromatophores of bacteria and fungi. It is
  believed to be a secondary metabolite of ancient
  origin.

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The carotenoids are C40 all-trans polyolefinic
constituents of green plants, as well as in a
number of algae, bacteria and fungi. Many of them
are accessory pigments in photosynthesis.
?-Carotene is the most important member of this
group.
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Carotenoids

• ?-Carotene is widely distributed in the plant
  kingdom. It is almost always found with
  chlorophyll. ?-Carotene is the most important
  pro-vitamin A compound in humans. It is believed
  to be one of the natural anti-oxidants.
• Isomers of ?-carotene are known which are
  double-bond isomers (e.g., ?-carotene) or
  open-ring analogues (?- and ?-carotene). The
  xanthines are oxidized derivatives which are also
  widely found in nature.
• Industrially, it is obtained by chemical
  synthesis or fermentation.

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Some carotenoids.
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Some well-known commercial carotenoids retinol
(Vitamin A) and all-trans retinoic acid
(tretinoin), a topic cream for skin care and
acne.
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Bixin is a commerically-important food colorant
which is obtained from the seeds of Bixa orellana
(annato, achuete). Bixin imparts the
yellow-orange color of Cheez Whiz. It was
recently discovered that bixin is derived from
lycopene. Molecular biologists are now trying to
engineer tomatoes with dioxygenase genes to
convert lycopene to bixin aldehyde, then an
aldehyde dehydrogenase and methyoltransferase to
yield bixin. (Camara et al., Science, 300, 2089
(2003).)
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Bixin biosynthesis (from http//www.plantcyc.org
)
Bixin, also known as 'annatto', is a pigment
synthesized naturally by a single terrestrial
plant, Bixa orellana, native from the tropical
Americas. The pigments are found on the surface
of the seeds where they accumulate in a resinous,
oily substance. Norbixin is a strong colorant
one liter of a 1 norbixin solution is sufficient
for the coloring of 16 tons of cheese. It is also
used in the cosmetic industry and as a dye for
leather.
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Annato

• Annatto is used in traditional medicine to cure
  diabetes, as an antimicrobial, against snake
  bites or sunburns. Today, annatto is used as a
  food colorant and dye for traditional silk. It is
  also still used in the cosmetic industry for body
  care products.

• A recent paper in Food Chemistry claimed that
  annatto can be used to replace nitrites in cured
  meats without affecting the microbial or sensory
  profiles of the finished product. Nitrite salts
  (sodium nitrite) have traditionally been added to
  meat to retard rancidity, stabilize flavor, and
  establish the characteristic pink color of cured
  meat. Many studies warned against the use of
  nitrites for curing meats. The results showed
  that the sample formulated with 60 annatto was
  the best for its color. In addition, this sausage
  formulation also did not differ significantly
  from the control (100 nitrite) sausage in terms
  of flavor and aroma, and microbial contamination.

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Summary

• Head-to-head reductive dimerization of two
  farnesyl diphosphate units yields squalene.
  Squalene, a symmetric C30 all-trans hexaene, is
  the starting point of the triterpenes.
• The various triterpenes are formed from
  conformational folding of squalene, in particular
  chair and boat. There are four main folding
  conformations (c - c - c - b) and
  (c - b - c - b) form tetracyclic triterpenes, and
  (c - c - c - c - c) and
  (c - b - c - c - b) form pentacyclic
  triterpenes.
• The chair - boat - chair - boat conformation
  yields the important intermediates cycloartenol
  (in plants) and lanosterol (in animals). The
  various sterols arise from these compounds.

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Summary

• Sterols are modified by various organisms to
  yield compounds which are characteristic of the
  organism. These include the steroidal hormones,
  sterol glycosides, phytoecdysones, and bile
  acids.
• Head-to-head reductive dimerization of two
  geranylgeranyl diphosphate units yields
  lycopersene, the squalene equivalent of the C40
  terpenes. Lycopersene is starting point of the
  carotenoids.