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7. Triterpenes and Sterols7. Triterpenes and Sterols
RA Macahig
FM. Dayrit
HO H
CH3
CH3
H
H3C H
lanosterol HO
CH3
CH3
H
H3C H
H
cholesterol
7.0 Triterpenes & steroids (Dayrit) 2
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).
Introduction
7.0 Triterpenes & steroids (Dayrit) 3
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).
Squalene
Important features of squalene: • It does not have a -OPP leaving group.• It is a symmetric molecule.
7.0 Triterpenes & steroids (Dayrit) 4
Biosynthetic mechanism for the head-to-head dimerization of two farnesyl diphosphate chains to produce squalene.
OPP
PPOR
R
X_
RPPOR
X
HH
-H-X
+_
RPPO
R
H
-OPP
R
R
H
+
NADP(D)
D_
R
R
HD
H D
squalene
7.0 Triterpenes & steroids (Dayrit) 5
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.
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.
7.0 Triterpenes & steroids (Dayrit) 6
squalene, C30
+ PPO(farnesyl pyrophosphate, C15) x 2
OPP
OPP
OPP
DMAPP
IPP2 x
cyclization
HO
Cycloartenol
CholesterolHO
(29 Triterpene skeletal types)
O (3S)-2,3-oxidosqualene
epoxidation
23
1
5
3
6
10
1113
17
18
19
20
2122 26
27
1
3
10
1113
17
18
19
20
21 22 26
27
28
29 30
3029
28
2221
20
19
18
1713
11
10
1
LanosterolHO
3
Overview of squalene formation and the biogenetic relationships of triterpenes and sterols.
7.0 Triterpenes & steroids (Dayrit) 7
squalene, C30
+ PPO(farnesyl pyrophosphate, C15) x 2
OPP
OPP
OPP
DMAPP
IPP2 x
cyclization
HO
Cycloartenol
CholesterolHO
(29 Triterpene skeletal types)
O (3S)-2,3-oxidosqualene
epoxidation
23
1
5
3
6
10
1113
17
18
19
20
2122 26
27
1
3
10
1113
17
18
19
20
21 22 26
27
28
29 30
3029
28
2221
20
19
18
1713
11
10
1
LanosterolHO
3
7.0 Triterpenes & steroids (Dayrit) 8
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.
TEM of peroxisome showing crystalline and non-crystalline inclusions
7.0 Triterpenes & steroids (Dayrit) 9
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 kineticsand the apparent Km of (2,3)-oxidosqualene is 15 M and the apparent kcat/Km
is 200 M-1 min-1.
7.0 Triterpenes & steroids (Dayrit) 10
Terpene biosynthetic pathways differ in prokaryotes and eukaryotes. The two pathways shown here diverge after presqualene diphosphate.
(A) Staphyloxanthin biosynthesis in S. aureus. The NADPH reduction step is absent, resulting in formation of dehydrosqualene, not squalene.
(B) Steroid biosynthesis in humans and yeasts passes through squalene. (Liu et al., Science 319, 1391 -1394 (2008))
S. aureus: CrtM Humans and yeast: squalene synthase (SQS)
7.0 Triterpenes & steroids (Dayrit) 11
• 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: 1. Conformation of folding (regiochemistry)2. Conformation of folding (stereochemistry)3. Skeletal rearrangements4. Further chemical transformations, such as oxidation,
methylation, and glycosylation.
Overview of cyclization reactions in triterpenes
7.0 Triterpenes & steroids (Dayrit) 12
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.
Overview of cyclization reactions in triterpenes
7.0 Triterpenes & steroids (Dayrit) 13
Model for the oxidosqualene cyclase enzyme which controls the transition state conformation of triterpene formation.
O
AH+
hydrophobic pocket
Oxidosqualene cyclase enzyme
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.
chair chair
boatboat
7.0 Triterpenes & steroids (Dayrit) 14
2. The conformation of folding: stereochemistry.
(3S)-2,3-oxidosqualeneO
cyclization
"right-handed" coil "left-handed" coil
O
O
cyclization
HO
HO
(+) Tetrahymanol (-) Tetrahymanol
23
H+
H+
32
3
2
1
3
1
3
15
Triterpenes: an overview
B. c-b-c-b
steroid group
19
18
HO
CH3
RCH3
H
3
10
13 171713
14
810
3
lanosterol skeleton
HO
CH3
H
CH3
CH3
H
R1713
14
810
3
HO H
CH3
H
CH3
H
R
cycloartenol skeleton
A. c-c-c-b
euphane skeleton
CH3
HO
H3C CH3
CH3
CH3R
lupeol skeleton
H
CH3CH3
HO
H3C CH3
CH3
CH3
H
H
H
taraxasterol skeleton
H
CH3CH3
HO
H3C CH3
CH3
CH3
amyrin skeleton
CH3CH3
HO
H3C CH3
CH3
CH3
C. c-c-c-c-c
HO
CH3 CH3
H H
H
H CH3
CH3
CH3
CH3
tetrahymane skeletonhopane skeleton
HO
CH3 CH3
H H
H
H CH3
CH3
D. c-b-c-c-b
arborane skeleton
HO
CH3 H
CH3
CH3
H
CH3
H
H3C CH3
CH3
CH3H11
13
14
1718
21
16
Chair - chair - chair - boat conformation of
squalene folding to form tetracyclic triterpenes: the
Dammaranes.
squalene
[O]
OH+
HO
+
H
CH3CH3
HO
H3C CH3
CH3
+
dammarane skeleton
+
CH3
CH3CH3
CH3
H3C
HO
H
H
H
1
109
13
14
17
-H (9)+
17
1413
910
1CH3
CH3
CH3
H3C
HO
HCH3
CH3
HO
H3C CH3
CH3
CH3
euphol (euphane skeleton)
H
CH3CH3
HO
H3C CH3
CH3
OH
dammarenediol
OH_
to Figure 8.6
17
squalene
[O]
OH+
HO
+
H
CH3CH3
HO
H3C CH3
CH3
+
dammarane skeleton
+
CH3
CH3CH3
CH3
H3C
HO
H
H
H
1
109
13
14
17
-H (9)+
17
1413
910
1CH3
CH3
CH3
H3C
HO
HCH3
CH3
HO
H3C CH3
CH3
CH3
euphol (euphane skeleton)
H
CH3CH3
HO
H3C CH3
CH3
OH
dammarenediol
OH_
to Figure 8.6
Chair - chair - chair - boat
conformation: the
Dammaranes.
18
squalene
[O]
OH+
HO
+
H
CH3CH3
HO
H3C CH3
CH3
+
dammarane skeleton
+
CH3
CH3CH3
CH3
H3C
HO
H
H
H
1
109
13
14
17
-H (9)+
17
1413
910
1CH3
CH3
CH3
H3C
HO
HCH3
CH3
HO
H3C CH3
CH3
CH3
euphol (euphane skeleton)
H
CH3CH3
HO
H3C CH3
CH3
OH
dammarenediol
OH_
to Figure 8.6
19
Further modifications on
the dammarane skeleton.
dammarane skeleton (from Figure 8.6)
H
CH3C H3
HO
H3C C H3
C H3
+
HO
+
bond migration
+
HO
+
H
C H3C H3
HO
H3C CH3
CH3
CH3
ba
a b
a b
+CH3H
CH3
+
1,2-methyl migration;-H+
H
CH3CH3
HO
H3C CH3
CH3
CH3
taraxasterol
CH3CH3
HO
H3C CH3
CH3
CH3
1,2-methyl migration
-amyrin
O
CH3
CH3
CH3CH3
HH
CH3
H
CH3 friedelin
+-H
+
CH3H
CH3
+
H
CH3CH3
HO
H3C CH3
CH3
CH3
H
H
H
lupeol
20
dammarane skeleton (from Figure 8.6)
H
CH3C H3
HO
H3C C H3
C H3
+
HO
+
bond migration
+
HO
+
H
C H3C H3
HO
H3C CH3
CH3
CH3
ba
a b
a b
+CH3H
CH3
+
1,2-methyl migration;-H+
H
CH3CH3
HO
H3C CH3
CH3
CH3
taraxasterol
CH3CH3
HO
H3C CH3
CH3
CH3
1,2-methyl migration
-amyrin
O
CH3
CH3
CH3CH3
HH
CH3
H
CH3 friedelin
+-H
+
CH3H
CH3
+
H
CH3CH3
HO
H3C CH3
CH3
CH3
H
H
H
lupeol
Further modifications on
the dammarane skeleton.
21
dammarane skeleton (from Figure 8.6)
H
CH3C H3
HO
H3C C H3
C H3
+
HO
+
bond migration
+
HO
+
H
C H3C H3
HO
H3C CH3
CH3
CH3
ba
a b
a b
+CH3H
CH3
+
1,2-methyl migration;-H+
H
CH3CH3
HO
H3C CH3
CH3
CH3
taraxasterol
CH3CH3
HO
H3C CH3
CH3
CH3
1,2-methyl migration
-amyrin
O
CH3
CH3
CH3CH3
HH
CH3
H
CH3 friedelin
+-H
+
CH3H
CH3
+
H
CH3CH3
HO
H3C CH3
CH3
CH3
H
H
H
lupeol
22
dammarane skeleton (from Figure 8.6)
H
CH3C H3
HO
H3C C H3
C H3
+
HO
+
bond migration
+
HO
+
H
C H3C H3
HO
H3C CH3
CH3
CH3
ba
a b
a b
+CH3H
CH3
+
1,2-methyl migration;-H+
H
CH3CH3
HO
H3C CH3
CH3
CH3
taraxasterol
CH3CH3
HO
H3C CH3
CH3
CH3
1,2-methyl migration
-amyrin
O
CH3
CH3
CH3CH3
HH
CH3
H
CH3 friedelin
+-H
+
CH3H
CH3
+
H
CH3CH3
HO
H3C CH3
CH3
CH3
H
H
H
lupeol
23
Chair-boat-chair-boat
conformation: Cycloartenol
and Lanosterol.
OH+
CH3
H3C
CH3
CH3CH3
HO
CH3
H
H
H
H
+
HO
H
H
H
+
1
3
10 8
17
18
19
20
21 22
26
2728
29 30
protostane skeleton
HO
H
H
H
protosterol
3
8
1013
14 17 20
-H+
H: 9 to 8H: 17 to 20
H: 13 to 17
Me: 14 to 13
Me: 8 to 14
-H (9)+
20171413
10
8
3 +
CH3
H3C
CH3
CH3CH3
HO
CH3
H
H
H
H
+
3
8
1013
14
17
20
CH3
H3C
CH2-H
HO
HCH3
CH3
H
H3CH
H9
9
-H (19)+
19
19
19
19
9CH3
H3C
HO
HCH3
CH3
H
H3CH
H20
17
14
1310
8
3
in plants
HO H
CH3
H
CH3
H
H3C H
cycloartenol
3
109
8
14
13 17
20
-H (8)+
in animals
3
8
1013
14
17
20
CH3
H3C
CH3
HO
HCH3
CH3
H
H3CH
9
19
HO H
CH3
CH3
H
H3C H
lanosterol
3
109
8
14
13 17
20
20
1713
10
3
HO
CH3
CH3
H
H3C H
H
18
19
cholesterol
24
OH+
CH3
H3C
CH3
CH3CH3
HO
CH3
H
H
H
H
+
HO
H
H
H
+
1
3
10 8
17
18
19
20
21 22
26
2728
29 30
protostane skeleton
HO
H
H
H
protosterol
3
8
1013
14 17 20
-H+
H: 9 to 8H: 17 to 20
H: 13 to 17
Me: 14 to 13
Me: 8 to 14
-H (9)+
20171413
10
8
3 +
CH3
H3C
CH3
CH3CH3
HO
CH3
H
H
H
H
+
3
8
1013
14
17
20
CH3
H3C
CH2-H
HO
HCH3
CH3
H
H3CH
H9
9
-H (19)+
19
19
19
19
9CH3
H3C
HO
HCH3
CH3
H
H3CH
H20
17
14
1310
8
3
in plants
HO H
CH3
H
CH3
H
H3C H
cycloartenol
3
109
8
14
13 17
20
-H (8)+
in animals
3
8
1013
14
17
20
CH3
H3C
CH3
HO
HCH3
CH3
H
H3CH
9
19
HO H
CH3
CH3
H
H3C H
lanosterol
3
109
8
14
13 17
20
20
1713
10
3
HO
CH3
CH3
H
H3C H
H
18
19
cholesterol
Chair-boat-chair-boat: Cycloartenol and
Lanosterol.
25
OH+
CH3
H3C
CH3
CH3CH3
HO
CH3
H
H
H
H
+
HO
H
H
H
+
1
3
10 8
17
18
19
20
21 22
26
2728
29 30
protostane skeleton
HO
H
H
H
protosterol
3
8
1013
14 17 20
-H+
H: 9 to 8H: 17 to 20
H: 13 to 17
Me: 14 to 13
Me: 8 to 14
-H (9)+
20171413
10
8
3 +
CH3
H3C
CH3
CH3CH3
HO
CH3
H
H
H
H
+
3
8
1013
14
17
20
CH3
H3C
CH2-H
HO
HCH3
CH3
H
H3CH
H9
9
-H (19)+
19
19
19
19
9CH3
H3C
HO
HCH3
CH3
H
H3CH
H20
17
14
1310
8
3
in plants
HO H
CH3
H
CH3
H
H3C H
cycloartenol
3
109
8
14
13 17
20
-H (8)+
in animals
3
8
1013
14
17
20
CH3
H3C
CH3
HO
HCH3
CH3
H
H3CH
9
19
HO H
CH3
CH3
H
H3C H
lanosterol
3
109
8
14
13 17
20
20
1713
10
3
HO
CH3
CH3
H
H3C H
H
18
19
cholesterol
cycloartenol and lanosterol.
26
OH+
CH3
H3C
CH3
CH3CH3
HO
CH3
H
H
H
H
+
HO
H
H
H
+
1
3
10 8
17
18
19
20
21 22
26
2728
29 30
protostane skeleton
HO
H
H
H
protosterol
3
8
1013
14 17 20
-H+
H: 9 to 8H: 17 to 20
H: 13 to 17
Me: 14 to 13
Me: 8 to 14
-H (9)+
20171413
10
8
3 +
CH3
H3C
CH3
CH3CH3
HO
CH3
H
H
H
H
+
3
8
1013
14
17
20
CH3
H3C
CH2-H
HO
HCH3
CH3
H
H3CH
H9
9
-H (19)+
19
19
19
19
9CH3
H3C
HO
HCH3
CH3
H
H3CH
H20
17
14
1310
8
3
in plants
HO H
CH3
H
CH3
H
H3C H
cycloartenol
3
109
8
14
13 17
20
-H (8)+
in animals
3
8
1013
14
17
20
CH3
H3C
CH3
HO
HCH3
CH3
H
H3CH
9
19
HO H
CH3
CH3
H
H3C H
lanosterol
3
109
8
14
13 17
20
20
1713
10
3
HO
CH3
CH3
H
H3C H
H
18
19
cholesterol
7.0 Triterpenes & steroids (Dayrit) 27
Unusual triterpenes.
Ambrein and malabaricol
are examples of
interrupted cyclization.
H+ O
OH+
OH-
A.
OH
H
O
H+
OH
H
OH
Ambrein
B.
OH-
OH
OH+
O OHH
H
H
(+) Malabaricol
1. cyclization2. reduction at C33. epoxidation at terminal olefin
1. cyclization2. reduction of terminal alcohol
7.0 Triterpenes & steroids (Dayrit) 28
Some important triterpenesCorosolic 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.
HO
CO2HHO
Corosolic acid(2-hydroxyursolic acid)
2
3
12
14
17
1920
HO
CO2HHO
Maslinic acid(2-hydroxyoleanolic acid)
2
3
12
14
17
20
7.0 Triterpenes & steroids (Dayrit) 29
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.
7.0 Triterpenes & steroids (Dayrit) 30
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.
O
HO
HOHO
O
O
O
OHO
AcO
HOHO
OH
OO
OOHOH
HO
OHOH
HO
ginsenoside Rs2
glucose
glucose-6-acetyl
glucosearabinose
31
Triterpenes: summary of cyclization modes: c-c-c-b
OH+
HO
+
H
CH3CH3
HO
H3C CH3
CH3
+
dammarane skeleton
+
CH3
CH3CH3CH3
H3C
HO
H
H
H
1
109
13
14
17
-H (9)+
17
1413
910
1CH3
CH3CH3
H3C
HO
HCH3
CH3
HO
H3C CH3
CH3
CH3
euphol (euphane skeleton)
7.0 Triterpenes & steroids (Dayrit) 32
Introduction to sterolsThe 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-ring3. Other modifications4. 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).
13 17
18
19
10
3
R
steroid skeleton
A B
C D
5
8
11
14
7.0 Triterpenes & steroids (Dayrit) 33
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.
• 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.
• 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).
7.0 Triterpenes & steroids (Dayrit) 34
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).
7.0 Triterpenes & steroids (Dayrit) 35
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
36
From triterpenes to sterols: A. lanosterol to cholesterol in mammals; B. cycloartenol to stigmasterol in plants and fungi.
HO
CH3
CH3
H
H3C CH3
H
CH3
H3CCH3
CH3
lanosterol
A. Zoosterols B. Plant sterols
cycloartenolHO
CH3
H
H3C CH3
H
CH3
H3CCH3
CH3
H
2,3-Oxidosqualene
in mammalsin plants and fungi
cholesterol (C27)HO
CH3
CH3
HH3C
CH3
CH3
other mammalian steroids other plant and fungal steroids
HO
CH3
CH3
HH3C
CH3
CH3
stigmasterol (C29)
2228
29
3 5
10
13 17
18
19
20
21
3 5
10
19
18
13 17
20
3
8
3
9810
19
7.0 Triterpenes & steroids (Dayrit) 37
Major mammalian
sterol skeletal structures.
(Estrogens and some androgens
do not have the C3 alcohol
group.)
sterols (C27)HO
3
10
19
18
17
20
A. Mammalian steroids
20
17
18
19
10
3
HO
CO2H
bile acids
24
corticosteroidsand gestogensHO
10
19
18
17
20
3 3
18
19
10
(O) androgens
estrogens
18
13
13
13
1313
7.0 Triterpenes & steroids (Dayrit) 38
HO
R
phytosterols (C28, C29)
3
10
17
18
19
20
13
B. Plant and fungal steroids
13
20
19
18
17
10
3spirostane (C27), steroidal saponins
RO
O
O
RO
N
steroid alkaloids (C27)
3
10
17
18
19
20
13 13
20
19
18
17
10
3
RO
N
Major sterol skeletal
structures in plants and
fungi.
7.0 Triterpenes & steroids (Dayrit) 39
Survey of some important sterolsCholesterol is the principal zoosterol. It is found in all body tissues of animals, especially in cell membranes. Animal tissues contain between 0.05% to 5% of cholesterol; the brain contains 17% cholesterol by weight. It is also found in some higher plants.
14
5
13
20
17
18
19
10
3
HO
HH
H
cholesterol
-Sitosterol and -stigmasterol are the most common phytosterols. These compounds are often isolated as 3-O-glycosides. The ethyl substituent at C24 has the S-configuration. -Sitosterol has a saturated side-chain while -stigmasterol is 22,23. -sitosterol
-stigmasterol if 22,23 HO
HH
H
H
3
10
19
18
17
20
13
5
14
22
21
24S
Ergosterol is a typical mycosterol (fungi). It differs from the phytosterols in two aspects: there is a methyl group at C24 with the R configuration. Ergosterol has been shown to arise from lanosterol in fungi.
24R
21
22
14
5
13
20
17
18
19
10
3
HO
HH
H
7 ergosterol
7.0 Triterpenes & steroids (Dayrit) 40
Insect moulting hormones are steroidal compounds called ecdysones. Insects generally ingest steroids (e.g., cholesterol) in their diet and metabolize these into insect hormones.
ecdysone
76
14
25
2
HO
OH
H
HO
OH
O
20
3 5
17
22
Tomatidine is a steroidal alkaloid found in the roots of the tomato plant. Tomatine, the 3-O-glycosylated derivative is found in the leaves of the tomato plant.
O
N
HO
CH3
CH3CH3
H
H
H3C
3
17
20
22
27
5
14 16
Tomatidine
25S
Soft corals (Coelenterata) produce marine sterols which are derived from the 24-methylene-cholesterane and pregnane skeletons. Many of these compounds feature extra hydroxyl and glycosidic groups.
3-Mannosyl-7-hydroxy-24-methylenecholesterol
O
HH
H
OOH
HOHO
HO
OH
3 7
24
7.0 Triterpenes & steroids (Dayrit) 41
Phytosterols
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.
• The path from squalene to sterols In plants and fungi:
squalene cycloartenol phytosterols and fungal sterols
7.0 Triterpenes & steroids (Dayrit) 42
Proposed pathway for the conversion of cycloartenol in plants to sitosterol.
HO
H3C CH3
H
CH3
cycloartenol
24
1. [O] (C28,C29,C30)2. -3CO (C28,C29,C30)28
29 30
2
24
HO H
HO
H
24
22
ergosterol
24
HO
H
sitosterol
53
28
29 28
3 5 7
*
**
(*) : from methyl methionine
7.0 Triterpenes & steroids (Dayrit) 43
Proposed mechanism for the conversion of cycloartenol to phytosterols.
3
8
910
19
HB2
B3 HO
B1
H
Enzyme
Enzyme pocket
Cycloeucalenol
B2
B3 HO
B1
CH3 H
8
910
109
8
HB2
B3 HO
B1
CH3
Obtusifoliol
_
19
7.0 Triterpenes & steroids (Dayrit) 44
Phytosterols: mixed metabolites
B. Fatty acylglucosyl sterol from ampalaya (Momordica charantia ). (Guevara, Sylianco, Dayrit,and Finch, Phytochem., 28 , 1721-1724 (1989).
OO
OHHO
HO
O
O
3-O-[6'-O-Palmitoyl- -glucosyl]-stigmasta-5,25(27)-diene
6'3 5
25
27
7.0 Triterpenes & steroids (Dayrit) 45
Cardiotonic sterols
5
20
17
3
HO
R
zoostero l or phytosterol HO
O
3
17
20
5
pregnenolone progesterone
5
20
17
3
O
O
in toadsin plants
HOH
OH
OAc
O
O
3
17
5
bufotalin
141614
digitoxigenin
5
17
3
HOH
OH
OO
A. Cardiotonic glycosides
7.0 Triterpenes & steroids (Dayrit) 46
OH
HHO
OO
47
Steroidal saponins
cholestero l HO
20
A. Steroidal saponins
3 5
1617
2226
2[H]
[O]
[O]
[O] 2622
1716
53
20
HOH
OH
O
OH
H+
HOH
O
OH
OH
20
3 5
1617
22
26
26
22
1716
53
20
RO
O
O
H+
25R
diosgenin, R=Hdioscin, R= rhamnose(1->4)glucose- (1->2) rhamnose
digitogenin, R=Hdigitonin, R= xyl(1->3)glu(1->4)gal-- (1->2) glu(1->3)gal
RO
O
O
HO
H
OH
20
3 5
1617
22
26
7.0 Triterpenes & steroids (Dayrit) 48
ZoosterolsThe 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
49
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.
HO
CH3
CH3
H
H3C CH3
H
CH3
H3CCH3
CH3
24,25-dihydrolanosterol
cholesterol (C27)HO
CH3
CH3
HH3C
CH3
CH3
other mammalian steroids3 5
10
19
18
13 17
20
2425
[O]
HO
CH3
CH3
H
H3C CH3
H
CHO
H3CCH3
CH3
HO
CH3
CH3
H
H3C CH3
H
H3CCH3
CH3
-CH O2
1. [O]2. +2[H] (-14,15)
HO
CH3
CH3
H
H3C CO2H
H
H3CCH3
CH3
-CO2
14
8 8
88
8
HO
CH3
CH3
H
H3C
H
H3CCH3
CH3
H
[O]
HO
CH3
CH3
H
HO2C
H
H3CCH3
CH3
H
8
1. -CO 2. +2[H] (-8,9)
3. -2[H] (+5,6)
2
50
HO
CH3
CH3
H
H3C CH3
H
CH3
H3CCH3
CH3
24,25-dihydrolanosterol
cholesterol (C27)HO
CH3
CH3
HH3C
CH3
CH3
other mammalian steroids3 5
10
19
18
13 17
20
2425
[O]
HO
CH3
CH3
H
H3C CH3
H
CHO
H3CCH3
CH3
HO
CH3
CH3
H
H3C CH3
H
H3CCH3
CH3
-CH O2
1. [O]2. +2[H] (-14,15)
HO
CH3
CH3
H
H3C CO2H
H
H3CCH3
CH3
-CO2
14
8 8
88
8
HO
CH3
CH3
H
H3C
H
H3CCH3
CH3
H
[O]
HO
CH3
CH3
H
HO2C
H
H3CCH3
CH3
H
8
1. -CO 2. +2[H] (-8,9)
3. -2[H] (+5,6)
2
Lanosterol to cholesterol.
51
HO
CH3
CH3
H
H3C CH3
H
CH3
H3CCH3
CH3
24,25-dihydrolanosterol
cholesterol (C27)HO
CH3
CH3
HH3C
CH3
CH3
other mammalian steroids3 5
10
19
18
13 17
20
2425
[O]
HO
CH3
CH3
H
H3C CH3
H
CHO
H3CCH3
CH3
HO
CH3
CH3
H
H3C CH3
H
H3CCH3
CH3
-CH O2
1. [O]2. +2[H] (-14,15)
HO
CH3
CH3
H
H3C CO2H
H
H3CCH3
CH3
-CO2
14
8 8
88
8
HO
CH3
CH3
H
H3C
H
H3CCH3
CH3
H
[O]
HO
CH3
CH3
H
HO2C
H
H3CCH3
CH3
H
8
1. -CO 2. +2[H] (-8,9)
3. -2[H] (+5,6)
2
Lanosterol to cholesterol.
7.0 Triterpenes & steroids (Dayrit) 52
HO
CH3
CH3
H
H3C CH3
H
CH3
H3CCH3
CH3
24,25-dihydrolanosterol
cholesterol (C27)HO
CH3
CH3
HH3C
CH3
CH3
other mammalian steroids3 5
10
19
18
13 17
20
2425
[O]
HO
CH3
CH3
H
H3C CH3
H
CHO
H3CCH3
CH3
HO
CH3
CH3
H
H3C CH3
H
H3CCH3
CH3
-CH O2
1. [O]2. +2[H] (-14,15)
HO
CH3
CH3
H
H3C CO2H
H
H3CCH3
CH3
-CO2
14
8 8
88
8
HO
CH3
CH3
H
H3C
H
H3CCH3
CH3
H
[O]
HO
CH3
CH3
H
HO2C
H
H3CCH3
CH3
H
8
1. -CO 2. +2[H] (-8,9)
3. -2[H] (+5,6)
2
Lanosterol to cholesterol.
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.
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.
• 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 200–500 mg of cholesterol. 30–60% 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.
54
• Cholesterol circulates as a component of lipoproteins. Its concentration in humans is typically in the range 100–300 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.
55
• 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 (C8–10) 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.
56
7.0 Triterpenes & steroids (Dayrit) 57
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.
7.0 Triterpenes & steroids (Dayrit) 58
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.
carotene
lycopene
lycopersene
2 x geranylgeranyl pyrophosphate (C20)
7.0 Triterpenes & steroids (Dayrit) 59
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.
7.0 Triterpenes & steroids (Dayrit) 60
Some carotenoids.
XanthophyllHO
OH
Carotene
Carotene
HO
O
O
OH
Violoxanthin
7.0 Triterpenes & steroids (Dayrit) 61
Some well-known commercial carotenoids: retinol (Vitamin A) and all-trans retinoic acid (tretinoin), a topic cream for skin care and acne.
CH2OH
H3C CH3
CH3
CH3CH3
Vitamin A (Retinol) All-trans retinoic acid (Tretinoin)
CO2H
H3C CH3
CH3
CH3CH3
7.0 Triterpenes & steroids (Dayrit) 62
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).)
lycopene
bixin aldehyde
OHCCHO
HO2CCO2CH3
bixin
Dioxygenase
1. Aldehyde dehydrogenase2. Methyl transferase
(Camara et al., Science, 300, 2089 (2003.)
7.0 Triterpenes & steroids (Dayrit) 63
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.
7.0 Triterpenes & steroids (Dayrit) 64
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.
7.0 Triterpenes & steroids (Dayrit) 65
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.
7.0 Triterpenes & steroids (Dayrit) 66
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.