lecture890708 - Workspace

Biosynthesis – Inspiration for Drug Discovery
Biosynthesis of Isoprenoids:
Terpenes (Including Steroids & Carotenoids)
Alan C. Spivey
a.c.spivey@imperial.ac.uk
Nov/Dec 2007

• What are isoprenoids?
Format & Scope of Lectures
– n × C 5 diversity: terpenes, steroids, carotenoids & natural rubber
– ‘the isoprene rule’
– mevalonate & 1-deoxyxylulose pathways to IPP & DMAPP
• Monoterpnes (C 10)
– regular (‘head-to-tail’) via geranyl pyrophosphate
– apparently irregular ‘iridoids’ (e.g. seco-loganin)
• Sesquiterpenes (C 15)
– farnesyl pyrophosphate derived metabolites
– sesquiterpene cyclases: pentalenene, aristolochene & 5-epi-aristolochene
• Diterpenes (C 20)
– gibberellins & taxol
• Triterpenes (C 30)
– steroids (2,3-oxidosqualene → lanosterol → cholesterol → estrone)
– ring-opened ‘steroids’: vitamin D 2 & azadirachtin
• Carotenoids (C 40)
– β-carotene, retinal & vitamin A

Isoprenoids
• isoprenoids are widely distributed in the natural world
– particularly prevalent in plants and least common in insects; >30,000 known
– composed of integral numbers of C 5 ‘isoprene’ units:
• monoterpenes (C 10); sesquiterpenes (C 15); diterpenes (C 20); sesterpenes (C 25, rare); triterpenes (C 30); carotenoids (C 40)
HO
H
H
OH
n
natural rubber (~10 5 x C 5)
β-carotene (C 40)
H
OH
cholesterol
(C 27 but C 30-derived)
O
O OH
HO
humulone (2x C5) H
O
BzHN
H H
thujone
(C 10)
OH
borneol
(C 10)
ISOPRENOIDS
OPP
dimethylallyl
pyrophosphate
(DMAPP)
Ph
O
AcO
HO O HO
BzOAcO
taxol (C20) isopentenyl
pyrophosphate
(IPP)
O
H
OH
O
HO
HO
lavandulol
(C 10) (Z)-α-bisabolene
OPP
H
O
H
O
CO 2H
OH
HO
(C 15)
O
H
O
OH
OH
O
H
O O H
O
OH
OH
OH
OH OH
gibberellic acid (C 20)
(gibberellin A 3)
artemisinin (C 15)
euonyminol (C 15)

Historical Perspective – ‘The Isoprenoid Rule’
• Early 1900s:
– common structural feature of terpenes – integral # of C 5 units
– pyrolysis of many monoterpenes produced two moles of isoprene:
• 1940s:
• 1953:
• 1964:
• 1993:
α-pinene (C 10)
200°C
– biogenesis of terpenes attributed to oligomers of isoprene – ‘the isoprene rule’
– Ruzicka proposes ‘the biogenetic isoprene rule’ to accomodate ‘irregular’ terpenoids:
• i.e. that terpenes were derived from a number of biological equivalents of isoprene initially joined in a ‘head-to-tail’
manner & sometimes subsequently modified enzymatically to provide greater diversity of structure
– Nobel prize awarded to Bloch, Cornforth & Popjak for elucidation of biosynthetic pathway to cholesterol
including the first steps:
• acetate → mevalonate (MVA) → isopentenylpyrophosphate (IPP) & dimethylallyl pyrophosphate (DMAPP)
– Rohmer, Sahm & Arigoni elucidate an additional pathway to IPP & DMAPP:
• pyruvate + glyceraldehyde-3-phosphate → 1-deoxyxylulose-5-phosphate → IPP & DMAPP
Δ
2x
isoprene (C 5)

PO
glycolysis
CO 2
phosphoenol pyruvate
CO 2
O
pyruvate
SCoA
O
acetyl coenzyme A
CoAS
O
O
acetoacetyl coenzyme A
Primary Metabolism - Overview
PHOTOSYNTHESIS
Primary metabolism
CO 2 + H 2O
HO
HO
O
HO
HO
OH
glucose
& other 4,5,6 & 7 carbon sugars
+
PO
HO O
OH
erythrose-4-phosphate
Citric acid
cycle
(Krebs cycle)
1) 'light reactions': hv -> ATP and NADH
2) 'dark reactions': CO 2 -> sugars (Calvin cycle)
SCoA
O
malonyl coenzyme A
HO
CO 2
HO OH
OH
shikimate
aromatic amino acids
aliphatic amino acids
CO 2
HO
mevalonate
CO 2
Primary metabolites
oligosaccharides
polysaccharides
nucleic acids (RNA, DNA)
peptides
proteins
tetrapyrroles (porphyrins)
saturated fatty acids
unsaturated fatty acids
lipids
Secondary metabolites
SHIKIMATE METABOLITES
cinnamic acid derivatives
aromatic compounds
lignans, flavinoids
ALKALOIDS
penicillins
cephalosporins
cyclic peptides
FATTY ACIDS & POLYKETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids

Biosynthesis of Mevalonate
• Mevalonate (MVA) is the first committed step of isoprenoid biosynthesis
– this key 6-carbon metabolite is formed from three molecules of acetyl CoA via acetoacetyl CoA:
Claisen
condensation
aldol
reaction
then [R]
2x NADPH
O
O
SCoA
SCoA
CoAS
O
O
acetoacetyl CoA
HO
O
Primary metabolism
O
CoASH
NAD
CoASH
SCoA
CO 2
HO
mevalonate (MVA)
CO 2
2x CoASH
2x NADP
pyruvate GLYCOLYSIS
CO 2
NADH
acetyl CoA
O
oxidative decarboxylation
SCoA
CO 2
malonyl CoA
CITRIC ACID CYCLE
CO 2
acetyl CoA carboxylase
(biotin-dependent)
carboxylation
saturated fatty acids
unsaturated fatty acids
lipids
Secondary metabolism
FATTY ACIDS & POLYKETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids

Biosynthesis of IPP & DMAPP - via Mevalonate
• IPP & DMAPP are the key C 5 precursors to all isoprenoids
– the main pathway is via: acetyl CoA → acetoacetyl CoA → HMG CoA → mevalonate → IPP → DMAPP:
DMAPP
IPP
O
IPP isomerase
(overall anti
stereochemistry)
Glu 207
O
O
B-Enz
SCoA
SCoA
CoAS
H
acetyl CoA
OPP
OPP
H R
Cys 139
H s
T
H
D
T
D2O S
D
H
decarboxylative
elimination
P i + CO 2
O
Claisen condensation
CoAS
O
PPO
PO
O
Me
O
O
O SCoA
3x ATP
3x ADP
CoASH
sequential addition
H 2O
CoAS
O
O
CoASH
2x NADPH 2x NADP
HO
O
CoAS
O
CoAS HO
HO
CoASH
acetoacetyl CoA
aldol reaction
CO 2
CO 2
hydroxymethylglutaryl CoA
(HMG CoA)
HMG CoA reductase
RDS in cholesterol
biosynthesis
mevalonate (MVA)

HMG CoA reductase inhibitors - Statins
• HMG CoA → MVA is the rate determining step in the biosynthetic pathway to cholesterol
– 33 enzyme mediated steps are required to biosynthesise cholesterol from acetyl CoA & in principle the
inhibition of any one of these will serve to break the chain. In practice, control rests with HMG-CoA reductase
as the result of a variety of biochemical feedback mechanisms
• ‘Statins’ inhibit HMG CoA reductase and are used clinically to treat hypercholesteraemia -a
causative factor in heart disease
R
NADPH
– e.g. mevinolin (=lovastatin ® , Merck) from Aspergillus terreus is a competitive inhibitior of HMG-CoA reductase
N
H 2N
H
H
O
O
mevinolin (=lovastatin ® )
PRO-DRUG
NB. type I (iterative) PKS natural product
O
CoAS HO
O
OH
HMG CoA
O O
CO 2
OH 2
in vivo
NADP
O
HO
O
HO
CoAS O
H
OH
CO 2
ACTIVE DRUG
mimic of tetrahedral intermediate
in HMG reduction by NADPH
H
CO 2
Zn 2
NADPH
NADP
CoASH
HO
HO
mevalonate (MVA)
H
H
HO
H
cholesterol
CO 2
PhHN
O
Ph
i Pr
N
OH
OH
F
CO 2
LIPITOR

Biosynthesis of IPP & DMAPP – via 1-Deoxyxylulose
• the mevalonate route to IPP & DMAPP has been proven in yeast & animals & in some plants & for
a long while was believed to be the only pathway to these key intermediates
– However, in some bacterial labelling studies:
• no incorporation of mevalonolactone was observed
• the pattern of label from glucose was inconsistent with derivation via catabolism to acetate
• in 1993 an additional pathway to IPP & DMAPP was discovered:
O
– Rohmer et al. Biochem. J. 1993, 295, 517 (DOI)
CO 2
pyruvate
DMAPP
IPP
+
OPP
OPP
H
O
OH
OP
glyceraldehyde
3-phosphate
NADP
NADPH
DXP
synthase
HO
CO 2
O
OH
OH
OP
1-deoxyxylulose
5-phosphate (DXP)
OPP
4-hydroxy-DMAPP
NADP
NADPH
• The pathway is prevalent in many pathogenic bacteria and so its inhibition represents an exciting
opportunity for antiinfective therapeutic development: Rohdich J. Org. Chem. 2006, 71, 8824 (DOI)
HO
H
OH
O
OH
OH
DXP
reductoisomerase
O
O
P
O
P O
O
O O
OP
NADPH
NADP
CMP
NB. CMP = cytidine monophosphate
CTP = cytidine triphosphate
HO
HO
ATP + CTP
OH
OH
OP
2-methyerythritol
4-phosphate
OP
OH
ADP + PP i
O
P
O
P
O O
O
O
O
OH
O
N
OH
cytosine
NH 2
N
O

Hemi-Terpenes – ‘Prenylated Alkaloids’
• DMAPP is an excellent alkylating agent
• C 5 units are frequently encountered as part of alkaloids (& shikimate metabolites) due to ‘latestage’
alkylation by DMAPP
– the transferred dimethyl allyl unit is often referred to as a ‘prenyl group’
– ‘normal prenylation’ – ‘S N2’-like alkylation; ‘reverse prenylation’ – ‘S N2’-like alkylation
4
OPP
• e.g. lysergic acid (recall the ergot alkaloids) – a ‘normal prenylated’ alkaloid (with significant subsequent processing)
• e.g. roquefortine (recall diketopiperazine alkaloids) – a ‘reverse prenylated’ alkaloid
N
H
'normal'
prenylation
cf. S N2
HO
tyrosine
O H
DMAPP
N
Me
H
N
H
lysergic acid
(halucinogen)
roquefortine
(blue cheese mould)
– review: R.M. Williams et al. ‘Biosynthesis of prenylated alkaloids derived from tryptophan’ Top. Curr. Chem.
2000, 209, 97-173 (DOI)
OPP
3
N
H
'reverse'
prenylation
cf. S N2'
DMAPP
tyrosine
N
H H
N
O
O
NH
histidine
H
N
N

Linear C 5n ‘head-to-tail’ Pyrophosphates
• head-to-tail C 5 oligomers are the key precursors to isoprenoids
– geranyl pyrophosphate (C 10) is formed by S N1 alkylation of DMAPP by IPP → monoterpenes
– farnesyl (C 15) & geranylgeranyl (C 20) pyrophosphates are formed by further S N1 alkylations with IPP:
evidence for S N1:
F 3C
OPP
H*
DMAPP
H
F 3C
OPP
S N1
OPP
ionisation is 1,000,000 times slower
OPP
OPP
intimate ion pair
H S H R
IPP
via
OPP
OPP inversion
H R
H S
OPP
*H
OPP
gerenyl pyrophosphate (C 10)
farnesyl pyrophosphate (C 15)
gerenylgeranyl pyrophosphate (C 20)
H
IPP
IPP
OPP
OPP
MONOTERPENES (C 10)
SESQUITERPENES (C 15)
TRITERPENES (C 30)
DITERPENES (C 20)
CAROTENOIDS (C 40)

Monoterpenes from Parsley & Sage
Parsley (Petroselinum sativum)
Sage (Salvia officinalis)
apiol
thujone
α-pinene
camphene

Monoterpenes from Rosemary & Thyme
Rosemary (Rosmarinus officinalis)
Thyme (Thymus vulgaris)
camphor borneol cineol
thymol carvacrol

1. S-(-)-limonene (lemon)
2. R-(+)-limonene (orange)
3. RS-(±)-limonene (pleasant)
Limonene & Carvone
Chiroscience plc. (now Dow Inc.)
4. R-(-)-carvone (spearmint)
5. S-(+)-carvone (caraway)
6. RS-(±)-carvone (disgusting)

Monoterpenes – α-Terpinyl Cation Formation
• geranyl pyrophosphate isomerises readily via an allylic cation to linalyl & neryl pyrophosphates
– the leaving group abilty of pyrophosphate is enhanced by coordination to 3 × Mg 2+
– all three pyrophosphates are substrates for cyclases via an α-terpinyl cation:
OPP
(E)
OPP
(Z)
gerenyl
pyrophosphate
linalyl
pyrophosphate
OPP
neryl
pyrophosphate
allylic cation
intimate ion pair
O
O
P O O
Mg
P O
O
O
cyclase
Mg
initial
chiral centre
OPP
=
α-terpinyl cation
OPP
MONOTERPENES (C 10)

Monoterpenes – Fate of the α-Terpinyl Cation
• The α-terpinyl cation undergoes a rich variety of further chemistry to give a diverse array of
monoterpenes
• Some important enzyme catalysed pathways are shown below
– NB. intervention of Wagner-Meerwein 1,2-hydride- & 1,2-alkyl shifts
E 1 elimination
H 2O
O
b
c
H a
H e
thujone
a
limonene α-terpineol
trapping with
water
OPP OPP
d
=
c
d
α-terpinyl cation
1,2-hydride shift
H H
e
H
b
OH
d
c
=
trapping by alkene
at 'red' carbon
(anti-Markovnikov)
trapping by alkene
at 'blue' carbon
(Markovnikov)
H
=
H
trapping by PPO -
OPP
1,2-alkyl shift
E 1 elimination
E 1 elimination
H
OPP
bornyl
pyrophosphate
H
hydrolysis [O]
H
E 1 elimination
=
OH
O
borneol camphor
= =
=
=
camphene
β-pinene
α-pinene

Apparently Irregular Monoterpenes
• Apparently irregular monoterpenes can also occur by non-cationic cyclisation of geranyl PP
derivatives followed by extensive rearrangement
– e.g. iridoids – named after Iridomyrmex ants but generally of plant origin and invariably glucosidated
• e.g. seco-loganin (recall indole alkaloids) is a key component of strictosidine - precorsor to numerous complex
medicinally important alkaloids:
tryptophan
isoprene
geranyl PP
N
H H
H
MeO 2C
NH
OPP OPP
O
strictosidine
[O]
P 450
H
OGlu
10
NH 2
N
H
tryptamine
H 2O
enzymatic
Pictet-Spengler
reaction
OH
H
MeO2C H 2O
2x NADP
2x NADPH
O
PP i
secologanin
H
O
OGlu
H
O
O
10
10-oxo geranal
NADPH
NADP
O
OH
unusual reductive ring-closure -> cyclopentane
(NB. Non-cationic)
unusual fragmentation
[O] P 450
=
OH
O
HO 2C
H2O HO
MeO2C O
OP
OGlu
ATP
ADP
HO
H
MeO2C H
[O] P450 H
OGlu
methylation
O
SAM
HO2C loganin
O
[O]
P 450
O
OH
OH
glycoside
formation
O
OGlu

Strictosidine → Vinca, Strychnos, Quinine etc.
• The diversity of alkaloids derived from strictosidine is stunning and many pathways remain to be fully
elucidated:
MeO
N
N
camptothecin
MeO
N
H
MeO2C O
OH
O
O
H
HO
MeO
N
quinine
H
N
N
H
vinblastine
(vinca)
H
OH
N
H
Me
N
H
OH
CO2Me OAc
N
H H
N
H
H
MeO 2C
H
H
MeO2C NH
N
ajmalicine
(vinca)
3
H
O
strictosidine
(isovincoside)
Me
N
O
N O
H
gelsemine
(oxindole)
H
O
OGlu
N
H
H
H
MeO 2C
N
OH
yohimbine
O
N
H
H
N
H
H O
strychnine
(strychnos)
H
N
H
N H
H
aspidospermine
(vinca)

Sesquiterpenes – Farnesyl Pyrophosphate (FPP)
• ‘S N2’-like alkylation of geranyl PP by IPP gives farnesyl PP:
geranyl PP
OPP
pro-R hydrogen is lost
• just as geranyl PP readily isomerises to neryl & linaly PPs so farnesyl PP readily isomerises to
equivalent compounds – allowing many modes of cyclisation & bicyclisation
E,Z-FPP
nerolidyl PP
(E) (E) OPP
E,E-FPP
(E)
(Z)
OPP
OPP
OPP
allylic cation
intimate ion pair
H S
H R
IPP
OPP
O
O
P O O
Mg
P
O
O
cyclases
O
Mg
6-memb
10-memb
11-memb
ring cyclised
'CATIONS'
(E)
E,E-farnesyl PP (FPP)
OPP
(E)
- further cyclisation
- 1,2-hydride & alkyl shifts
- trapping with H 2O
- elimination to alkenes
vast array of
mono- & bicyclic
SESQUITERPENES
NB. control by:
1) enzyme to enforce conformation & sequestration of reactive intermediates
2) intrinsic stereoelectronics of participating orbitals

Sesquiterpene Cyclases
Christianson et al. Curr. Opin. Struct. Biol. 1998, 695 (DOI)

Terpene Cyclases – Control of Cyclisation
• Functional aspects of terpenoid cyclases:
– Templating: Active site provides a template for a specific conformation of the flexible linear isoprenoid starting
material.
– Triggering: Cyclase initiates carbocation formation.
• Metal-assisted leaving group departure (e.g. pyrophosphate ionization aided by Mg 2+ )
• C=C bond protonation (e.g. squalene-hopene cyclase, see later).
• Epoxide protonation (e.g. oxidosqualene cyclase, see later).
– Chaperoning: Chaperones conformations of carbocationic intermediates through the reaction sequence,
ordinarily leading to one specific product.
– Sequestering: Sequesters the carbocation intermediates by burying the substrate in a hydrophobic cavity that is
generally solvent-inaccessible. Carbocations are concomitantly stabilized by the presence of aromatic residues in
the active site that exert their effects via cation-π interactions
RECALL:
δ
= δ
δ
δ on edges
δ on faces
– Adapted from: Christianson et al. Curr. Opin. Struct. Biol. 1998, 695 (DOI)
• BUT:
– individual terpene cyclases can give multiple products, see: Matsuda J. Am. Chem. Soc. 2007, 129, 11213 (DOI)
R
R
R
Enz
cation - π
stabilisation
(~40-80 kJmol -1 )

Sesquiterpene Cyclase Crystal structures
pentalenene synthase 5-epi-aristolochene synthase aristolochene synthase
→ pentalenolactone (antibiotic) → fungal (myco)toxins (e.g. bipolaroxin, PR-toxin)

Aristolochene & 5-Epi-Aristolochene Synthases
• molecular modelling studies indicate that the shape of the active sites determines the conformation
of FPP and thus the stereochemistry of the final product
– Penicillium roqueforti aristolochene synthase – Felicetti & Cane J. Am. Chem. Soc. 2004, 126, 7212 (DOI)
PPO
10-exo
Phe-112
E,E-FPP
PPO H
Phe-112
germacrene A
(+)-aristolochene
H
H
– tobacco 5-epi-aristolochene synthase – Starks, Back, Chappell & Noel Science 1997, 277, 1815 (DOI)
PPO
cyclisation
elimination
H
E,E-FPP
Tyr–O
cyclisation
10-exo
elimination
1,2-alkyl
shift
TyrOH
held by enzyme active site in different conformations from above
(R)
H
H
(S)
H
cation
formation
1,2-hydride
shift
H
H
transannular
cyclisation
eudesmane cation
germacrene 5-epi-aristolochene

effects of gibberellin A 1 and
brassinolide on rice seedlings
Diterpenes - Gibberellins
Dwarf rice
seedlings (on the
left) have a defect
in the gibberellindependent
signalling
mechanism
gibberellin A 20

H
Diterpenes – Geranylgeranyl Pyrophosphate
• S N2 alkylation of farnesyl PP by IPP gives geranylgeranyl PP:
FPP
OPP bicyclisation OPP cyclisation cyclisation
H
6-endo
H
6-exo
H
geranylgeranyl PP
6-endo H
labdadienyl PP
H
HO
OPP
• geranylgeranyl PP readily cyclises to give numerous multicyclic diterpenes
– e.g. gibberellins – plant growth hormones
H
H
O
CO
O 2H
gibberellic acid
(gibberellin A3) OH
OPP
• NB. cyclisation initiated by alkene protonation NOT loss of PPO -
H
HO 2C H CHO
pinacol
rearrangement (?)
HO 2C
H
H
H
H
OP
• review: L.N. Mander ‘Twenty years of gibberellin research’ Nat. Prod. Rep. 2003, 20, 49-69 (DOI)
H
H
IPP
OPP
gerenylgeranyl PP (C 20)
OH
1,2-alkyl
shift
4x [O]
H
H
OPP
H
H
kaurene
elimination
H

Diterpenes - Taxol

Diterpenes – Geranylgeranyl PP → Taxol
• Taxol is a potent anti-cancer agent used in the treatment of breast & ovarian cancers
– comes from the bark of the pacific yew (Taxus brevifolia)
– binds to tubulin and intereferes with the assembly of microtubules
• biosynthesis is from geranylgeranyl PP:
OPP
geranylgeranyl PP
cyclisation
14-exo
– for details see: http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/terp/taxadiene.html
– home page is: http://www.chem.qmul.ac.uk/iubmb/enzyme/
OPP
b
H
a
H
a
b
• recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the
Nomenclature and Classification of Enzyme-Catalysed Reactions
• based at Department of Chemistry, Queen Mary University of London
BzNH
HO
Ph
β-amino acid
(shikimate)
O
O
AcO
HO
BzO AcO
cembrene
O
H
OH
O
taxol
sesquiterpene
(isoprenoid)

Triterpenes – FPP → Squalene
• triterpenes (C 30) arise from the ‘head to head’
coupling of two fanesyl PP units to give
squalene catalysed by squalene synthase:
– squalene was first identified as a steroid precursor
from shark liver oil
– the dimerisation proceeds via an unusual mechanism
involving electrophilic cyclopropane formation -
rearrangement to a tertiary cyclopropylmethyl cation
and reductive cyclopropane ring-opening by NADPH
(NB. exact mechanism disputed)
– Zaragozic acids (squalestatins) mimic a
rearrangement intermediate and inhibit squalene
synthase. They constitute interesting leads for
development of new treatments for
hypercholesteraemia & heart disease (cf. statins)
O
O
HO2C zaragozic acid A
(squalestatin S1) HO2C OH
O
O
OH CO2H OAc
FPP (acceptor)
EnzB:
squalene synthase
H
NADPH
H
H H
H H
H
OPP
OPP
OPP
OPP
OPP
OPP
NADP
FPP (donor)
blocked by squalestatins
+ PP i
presqualene PP
squalene

Triterpenes – Squalene → 2,3-Oxidosqualene
• squalene is oxidised to 2,3-oxidosqualene by squalene oxidase – which is an O 2/FADH 2dependent
enzyme:
• the key oxidant is a peroxyflavin:
O
O O
HN
FADH 2
squalene
H
N O
NR
NH
O
O
O
HN
H
N O
NR
peroxyflavin
NH
squalene
oxidase
O 2 + FADH 2
H 2O + FAD
reductive recycling
O
O
HO
H N
O
H
N O
NR
N
hydroxyflavin
H 2O
2,3-oxidosqualene
O
N
FAD
H
N O
NR
N

Modes of Cyclisation of Squalene
• all triterpenes (steroids, hopanoids etc.) are formed by the action of cyclase enzymes on either
squalene or 2,3-oxidosqualene
squalene oxidase
O
– i.e. different methods of ‘triggering’ cyclisation
squalene
2,3-oxidosqualene
squalene
cyclases
triggering by
ALKENE protonation
oxidosqualene
cyclases
triggering by
EPOXIDE protonation
OH
diplopterol H tetrahymanol
HO HO HO
H β-amyrin H cycloartenol
STEROID/TRITERPENE NOMENCLATURE: β-face = top face (as drawn here)
α-face = bottom face (as drawn here)
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
hopene
H H
= hydrogen up
(on β-face)
H
H
lanosterol
= hydrogen down
(on α-face)
HOPANOIDS
STEROIDS

Oxidosqualene-Lanosterol Cyclase (OSC)
• oxidosqualene-lanosterol cyclase catalyses the formation of lanosterol from 2,3-oxidosqualene:
– this cascade establishes the characteristic ring system of ALL steroids
– until recently, as for SHC, the enzyme was believed to enforce a chair-boat-chair conformation to allow a
concerted cationic ring-closure cascade followed by a series of suprafacial 1,2-shifts (shown below)
– however, this requires anti-Markovnikov regioselectivity for the C ring-closure...
HO
O
2,3-oxidosqualene
H
C
lanosterol
H
OSC
4x ring-closures
2x 1,2-hydride shifts
2x 1,2-Me shifts
elimination
O
EnzB-H
HO
C ring-closure by
anti-Markovnikov addition?
H
H
H
6-endo, 6-endo, 6-endo, 5-endo ?
chair-boat-chair conformation ?
prosterol cation
NB all bolded bonds are anti-peri planar

Oxidosqualene-Lanosterol Cyclase – Mechanism
• In fact, this process has also been shown to involve a Markovnikov ring-closure/1,2-alkyl-shift
ring-expansion sequence to establish the C ring
EnzB-H
– again, the conformation enforced by the enzyme is NOT strictly a chair-boat-chair one (although probably
very close), the process is NOT concerted, discrete cationic intermediates are involved &
stereoelectronics dictate the regio- & stereoselectivity
O
2,3-oxidosqualene
1) epoxide opening
2) 2x Markovnikov
ring-closures
(6-memb rings)
6-endo, 6-endo
HO
HO
H
H
lanosterol
H
E 1 elimination
Markovnikov
ring-closure
(5-memb ring)
HO HO
– “The enzyme’s role is most likely to shield intermediate carbocations… thereby allowing the hydride and
methyl group migrations to proceed down a thermodynamically favorable and kinetically facile cascade”
• Wendt et al. Angew. Chem. Int. Ed. 2000, 39, 2812 (DOI) & Wendt ibid 2005, 44, 3966 (DOI)
5-endo
HO
H
H
H
:BEnz
H
ring
expansion
(5 -> 6)
1,2-alkyl shift
1) 1,2-hydride shift
2) 1,2-hydride shift
3) 1,2-Me shift
4) 1,2-Me shift
(ALL suprafacial)
5-endo
H
Markovnikov
ring-closure
(5-memb ring)
H
H
HO
H
protosterol cation
H

Lanosterol → Cholesterol – Oxidative Demethylation
• Several steps are required for conversion of lanosterol to cholesterol:
HO
1
4
H
5
24
8
H
5
14
H
lanosterol
8
1) Δ 24 hydrogenation
2) 14α DEMETHYLATION
3) 4α & 4β DEMETHYLATION
4) Δ 8 to Δ 5 rearrangement
1) Δ 24 hydrogenation 2) 14α DEMETHYLATION
NADPH
24
NADP
3) 4α & 4β DEMETHYLATION
HO
4) Δ 8 to Δ 5 rearrangement
4
H
4x O 2
4x H 2O
P 450
O
H
O O
isomerase
CO 2
H
14
H
O
H
H
NAD
NADH
HO
H
H
H
H
[-> vitamin D]
H
4x O 2
4x H 2O
cholesterol
2x O2 P450 H O2 P450 H
H
2x H 2O
O
O
H
HO
=
P 450
NADH
NAD
O
HO
H :BEnz
O
O III
Fe
H
O O
HCO 2H
H
CO 2
O
flat, rigid structure
H
NADPH
NADP
NADPH
H
HO
H
NADP
H
H

Cholesterol → Human Sex Hormones
• cholesterol is the precursor to the human sex hormones – progesterone, testosterone & estrone
– the pathway is characterised by extensive oxidative processing by P 450 enzymes
– estrone is produced from androstendione by oxidative demethylation with concomitant aromatisation:
HO
HO
H
H
H
cholesterol
H
H
estrone
(œstrone)
H
O
H
2x O 2
2x H 2O
HCO 2H
P 450
HO
EnzB:
Fe III
O
H
H
OH
OH
H
H
O
O OH
H
H
O 2
O 2
P 450
O
P 450
HO
DEMETHYLATIVE aromatisation by 'aromatase' enzyme
O
O
H
H
H
H
H
H
O
O
H
2x O 2
NAD
2x H 2O
NADH
P 450
O
O
H
H
progesterone
H
H
[O]
H
H
X
O
H
androstendione (X = O)
testosterone (X = H, β−OH)

Steroid Ring Cleavage - Vitamin D & Azadirachtin
• vitamin D 2 is biosynthesised by the photolytic cleavage of Δ 7 -dehydrocholesterol by UV light:
HO
– a classic example of photo-allowed, conrotatory electrocyclic ring-opening:
H
H
H
cholesterol
H
NADP
NADPH
HO
H
– D vitamins are involved in calcium absorption; defficiency leads to rickets (brittle/deformed bones)
• Azadirachtin is a potent insect anti-feedant from the Indian neem tree:
– exact biogenesis unknown but certainly via steroid modification:
HO
H
7
C
OH
tirucallol
(cf. lanosterol)
H
Me
7
H
H
H
O
OH
O
AcO
H
AcO H
H
UV light (hv)
electrocyclic
ring-opening
[6π]
conrotatory
OAc
11 12
OH
azadirachtanin A
(a limanoid =
tetra-nor-triterpenoid)
O
H
HO
oxidative
cleavage
of C ring
Me H
H
H
ψ 4
SOMO
1,5-hydride
shift
O
MeO2C O
O
OH
O
AcO OH
MeO H
2C O
8
azadirachtin
14
HO
H
vitamin D 2
H
O
O
OH
highly hindered C-C bond
for synthesis!
[O]
HO
OH OH
H
OH
H
calcium ion chelator

Carotenoids – β-Carotene & vitamin A 1
• Carotenoids (C 40) are coloured pigments made by photosynthetic plants & certain algae, bacteria &
fungi. Dietary ingestion by birds and further processing gives rise to bright feather pigments etc.
– biosynthesised by head-to-head coupling of two geranylgeranyl PP units to give lycopersene:
GGPP (acceptor)
prephytoene synthase
PPO
NADPH
H
OPP
OPP
[O]
NADP + PP i
GGPP (donor)
prephytoene PP
(cf. presqualene PP)
lycopersene
β-carotene
– subsequent oxidative degradation (cf. ozonolysis!) gives retinal (mediator of vision) & vitamin A 1:
11
(E)
retinal
O
vitamin A 1
OH

PO
glycolysis
CO 2
phosphoenol pyruvate
CO 2
O
pyruvate
SCoA
O
acetyl coenzyme A
CoAS
O
O
acetoacetyl coenzyme A
PHOTOSYNTHESIS
Primary Metabolism - Overview
Primary metabolism
CO 2 + H 2O
HO
HO
O
HO
HO
OH
glucose
& other 4,5,6 & 7 carbon sugars
+
PO
HO O
OH
erythrose-4-phosphate
Citric acid
cycle
(Krebs cycle)
1) 'light reactions': hv -> ATP and NADH
2) 'dark reactions': CO 2 -> sugars (Calvin cycle)
SCoA
O
malonyl coenzyme A
HO
CO 2
HO OH
OH
shikimate
aromatic amino acids
aliphatic amino acids
CO 2
HO
mevalonate
CO 2
Primary metabolites
oligosaccharides
polysaccharides
nucleic acids (RNA, DNA)
peptides
proteins
tetrapyrroles (porphyrins)
saturated fatty acids
unsaturated fatty acids
lipids
Secondary metabolites
SHIKIMATE METABOLITES
cinnamic acid derivatives
aromatic compounds
lignans, flavinoids
ALKALOIDS
penicillins
cephalosporins
cyclic peptides
FATTY ACIDS & POLYKETIDES
prostaglandins
polyacetylenes
aromatic compounds, polyphenols
macrolides
ISOPRENOIDS
terpenoids
steroids
carotenoids