7. Triterpenes and Sterols

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


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.


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


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.


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.


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


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


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.


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)


• 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


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


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


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


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


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


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

+


H

CH3CH3

HO

H3C CH3

CH3

CH3

taraxasterol

CH3CH3

HO

H3C CH3

CH3

CH3


-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


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

+


H

CH3CH3

HO

H3C CH3

CH3

CH3

taraxasterol

CH3CH3

HO

H3C CH3

CH3

CH3


-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


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

+


H

CH3CH3

HO

H3C CH3

CH3

CH3

taraxasterol

CH3CH3

HO

H3C CH3

CH3

CH3


-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


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


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


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.


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



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


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).



• 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).



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


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


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.


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


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


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


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


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


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


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


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


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


cholesterol (C27)HO

CH3

CH3

HH3C

CH3

CH3


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


cholesterol (C27)HO

CH3

CH3

HH3C

CH3

CH3


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



HO

CH3

CH3

H

H3C CH3

H

CH3

H3CCH3

CH3


cholesterol (C27)HO

CH3

CH3

HH3C

CH3

CH3


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


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


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.


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)


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.


Some carotenoids.

XanthophyllHO

OH

Carotene

Carotene

HO

O

O

OH

Violoxanthin


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


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.)


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.


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.


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.


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.

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7. Triterpenes and Sterols

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