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Biosynthesis – Inspiration for Drug Discovery
Biosynthesis of Isoprenoids
Alan C. Spiveya.c.spivey@imperial.ac.uk
Dec 2008
Format & Scope of Lecture
• What are isoprenoids? – n × C5 diversity: terpenes, steroids, carotenoids & natural rubber– ‘the isoprene rule’– mevalonate pathway to IPP & DMAPP
• Monoterpnes (C10)– regular (‘head-to-tail’) via geranyl pyrophosphate– apparently irregular ‘iridoids’ (e.g. seco-loganin)
• Sesquiterpenes (C15)– farnesyl pyrophosphate derived metabolites
• Diterpenes (C20)– taxol
• Triterpenes (C30)– steroids (2,3-oxidosqualene → lanosterol → cholesterol → estrone)– ring-opened ‘steroids’: vitamin D2 & azadirachtin
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 C5 ‘isoprene’ units:
• monoterpenes (C10); sesquiterpenes (C15); diterpenes (C20); sesterpenes (C25, rare); triterpenes (C30); carotenoids (C40)
ISOPRENOIDS
thujone(C10)
OHborneol
(C10)
HO
lavandulol(C10) (Z)-α-bisabolene
(C15)
artemisinin (C15)
HH
H
HOHH
cholesterol(C27 but C30-derived)
OHn
natural rubber (~105x C5)
OPP
dimethylallylpyrophosphate
(DMAPP)
isopentenyl pyrophosphate
(IPP)
OPP
O
OH OHOH
OHOHHO
O
OH
OH
euonyminol (C15)
OH
CO2H
H
O
OH
gibberellic acid (C20)(gibberellin A3)HO
O
OH
AcO
HO
H
AcOBzO
O
OHO
PhBzHN
O
taxol (C20)
O
OHOHO
OH
humulone (2x C5)
β-carotene (C40)
OO
O
H
HO O H
H
Historical Perspective – ‘The Isoprenoid Rule’• Early 1900s:
– common structural feature of terpenes – integral # of C5 units– pyrolysis of many monoterpenes produced two moles of isoprene:
• 1940s:– biogenesis of terpenes attributed to oligomers of isoprene – ‘the isoprene rule’
• 1953:– 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
• 1964:– 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)
• 1993:– Rohmer, Sahm & Arigoni elucidate an additional pathway to IPP & DMAPP:
• pyruvate + glyceraldehyde-3-phosphate → 1-deoxyxylulose-5-phosphate → IPP & DMAPP
200°C
2x
isoprene (C5)
Δ
α-pinene (C10)
Primary Metabolism - Overview
CO2 + H2O
1) 'light reactions': hv -> ATP and NADPH2) 'dark reactions': CO2 -> sugars (Calvin cycle)
OHOHOHOOH
HO
glucose& other 4,5,6 & 7 carbon sugars
Primary metabolism Secondary metabolites
oligosaccharidespolysaccharidesnucleic acids (RNA, DNA)
phosphoenol pyruvate
glycolysis
CO2
HOOH
OHOOH
HO
POCO2
PO
erythrose-4-phosphate
SHIKIMATE METABOLITEScinnamic acid derivativesaromatic compoundslignans, f lavinoids+
shikimate
aromatic amino acids
aliphatic amino acidspeptidesproteinsCO2
Opyruvate
SCoA
Oacetyl coenzyme A
Citric acidcycle
(Krebs cycle)
ALKALOIDSpenicillinscephalosporinscyclic peptides
tetrapyrroles (porphyrins)
PHOTOSYNTHESIS
saturated fatty acidsunsaturated fatty acidslipids
FATTY ACIDS & POLYKETIDESprostaglandinspolyacetylenesaromatic compounds, polyphenolsmacrolides
ISOPRENOIDSterpenoidssteroidscarotenoids
SCoA
OCO2
malonyl coenzyme A
CoAS
O
O
HO
HOCO2
mevalonateacetoacetyl coenzyme A
Primary metabolites
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:
Primary metabolism Secondary metabolism
SCoA
Oacetyl CoA
CO2
SCoA
OCO2
malonyl CoA
SCoAO
CoAS
O
O
SCoAO
HO
HOCO2
mevalonate (MVA)
acetoacetyl CoAsaturated fatty acidsunsaturated fatty acidslipids
FATTY ACIDS & POLYKETIDESprostaglandinspolyacetylenesaromatic compounds, polyphenolsmacrolides
ISOPRENOIDSterpenoidssteroidscarotenoids
CO2
NADH
CoASH
NAD
CO2
Opyruvate GLYCOLYSIS
oxidative decarboxylation
CITRIC ACID CYCLE
acetyl CoA carboxylase(biotin-dependent)
carboxylation
2x NADPH 2x NADP
CoASH
2x CoASH
Claisencondensation
aldolreactionthen [R]
Biosynthesis of IPP & DMAPP - via Mevalonate• IPP & DMAPP are the key C5 precursors to all isoprenoids
– the main pathway is via: acetyl CoA → acetoacetyl CoA → HMG CoA → mevalonate → IPP → DMAPP:
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
– e.g. mevinolin (=lovastatin®, Merck) from Aspergillus terreus is a competitive inhibitior of HMG-CoA reductase
HOH
H
H
cholesterol
mevinolin (=lovastatin®)
PRO-DRUGNB. type I (iterative) PKS natural product
O
O
O O
OH
O
O
OH
HO
in vivo
OH2
mevalonate (MVA)
CO2
CoASH
HO
HOCO2
O
HOCO2
CoAS
HMG CoANADPH NADP
H
CO2
CoAS OH
HO
ACTIVE DRUGmimic of tetrahedral intermediate
in HMG reduction by NADPH
NH
H
H2NO
RZn2 NADPH
NADP
CO2
OH
NPhHN
O iPr
F
Ph
OH
LIPITOR
Hemi-Terpenes – ‘Prenylated Alkaloids’• DMAPP is an excellent alkylating agent• C5 units are frequently encountered as part of alkaloids (& shikimate metabolites) due to ‘late-
stage’ alkylation by DMAPP – the transferred dimethyl allyl unit is often referred to as a ‘prenyl group’– ‘normal prenylation’ – ‘SN2’-like alkylation; ‘reverse prenylation’ – ‘SN2’-like alkylation
• e.g. lysergic acid (recall the ergot alkaloids) – a ‘normal prenylated’ alkaloid (with significant subsequent processing)• e.g. roquefortine – a ‘reverse prenylated’ alkaloid
– review: R.M. Williams et al. ‘Biosynthesis of prenylated alkaloids derived from tryptophan’ Top. Curr. Chem.2000, 209, 97-173 (DOI)
lysergic acid(halucinogen)
NH
N
OH
H
Me
DMAPP
HO
roquefortine(blue cheese mould)
NNH
O
O
N
HN
NH
Htyrosine
DMAPP
histidine
tyrosine
3
OPP
NH
4
'normal'prenylation
OPP
NH
cf. SN2cf. SN2'
'reverse'prenylation
Linear C5n ‘head-to-tail’ Pyrophosphates• head-to-tail C5 oligomers are the key precursors to isoprenoids
– geranyl pyrophosphate (C10) is formed by SN1 alkylation of DMAPP by IPP → monoterpenes– farnesyl (C15) & geranylgeranyl (C20) pyrophosphates are formed by further SN1 alkylations with IPP:
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 Mg2+ ions– all three pyrophosphates are substrates for cyclases via an α-terpinyl cation:
gerenylpyrophosphate
linalylpyrophosphate
OPP
nerylpyrophosphate
allylic cationintimate ion pair
OPP
(E)
(Z)cyclase
=
α-terpinyl cation
MONOTERPENES (C10)
OPP OPP
OPPO
P O POMg
O
O
O
O
Mg
initialchiral centre
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
E1 elimination
OPP
limonene α-terpineolH
bornyl pyrophosphate
OH
H2O
=
α-terpinyl cation
OPP OPP
OH
H
borneol camphorO
H
c d
e
c
H
α-pinene
H
camphene
β-pinene
c d
OPP
= =
= =
=
H a
a
d
e
b
H
O
thujone
btrapping withwater
=
=
trapping by alkeneat 'red' carbon
(anti-Markovnikov)
trapping by alkeneat 'blue' carbon(Markovnikov)
1,2-hydride shift
trapping by PPO-
1,2-alkyl shift
H
E1 elimination
E1 elimination
hydrolysis [O]
E1 elimination
HH
Limonene & Carvone
Chiroscience plc. (now Dow Inc.)
1. S-(-)-limonene (lemon)
2. R-(+)-limonene (orange)
3. RS-(±)-limonene (pleasant)
4. R-(-)-carvone (spearmint)
5. S-(+)-carvone (caraway)
6. RS-(±)-carvone (disgusting)
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:
OPP OPP
10
[O]
OHOH
O
O
OH
HO2CO
OH
[O]P450
[O] P450
methylation SAM
NH
NH2
tryptamine
enzymaticPictet-Spengler
reaction
H2OO
MeO2C
H
HOGlu
strictosidine
NHNH Htryptophan
isoprene
geranyl PP
P450
=
2x NADP
2x NADPH
H2O
PPi
O
O
HO
OH
10-oxo geranal10
NADPH
NADP
unusual reductive ring-closure -> cyclopentane(NB. Non-cationic)
glycosideformation
HO2CO
OGlu
unusual fragmentation
O
OMeO2C
H
HOGlu
MeO2CO
OGlu
HOOP
secologaninH2O MeO2C
O
OGlu
HO
loganin
H
H
H
[O] P450
ATP
ADP
Strictosidine → Vinca, Strychnos, Quinine etc.• The diversity of alkaloids derived from strictosidine is stunning and many pathways remain to be fully
elucidated:
OMeO2C
H
HOGlu
strictosidine(isovincoside)
NHNH H
yohimbine
NH
N
OHMeO2C
H
H
H
N
MeON
H
H
HHO
quininestrychnine(strychnos)
N
O
N
O
H
H
H
H
vinblastine(vinca)
NH
N
N
N
OH
MeO2C
HCO2Me
OHOAc
H
MeO
H
Me
NH
N
O
H
H
H
MeO2Cajmalicine
(vinca)
N
MeON
O
O
O
OH
camptothecin
NH
H
NH
aspidospermine(vinca)
NH
gelsemine(oxindole)
O
O
MeN
3
Sesquiterpenes – Farnesyl Pyrophosphate (FPP)• ‘SN2’-like alkylation of geranyl PP by IPP gives farnesyl PP:
• just as geranyl PP readily isomerises to neryl & linaly PPs so farnesyl PP readily isomerises to equivalent compounds – allowing many modes of cyclisation & bicyclisation
OPPHRHS
E,E-farnesyl PP (FPP)
pro-R hydrogen is lost
(E)OPP
(E)
geranyl PP
OPPOPP
IPP
OPP
OPP
nerolidyl PP
E,Z-FPP
(Z)
E,E-FPP
(E) (E)OPP
OP O P
OMg
O
O
O
O
allylic cationintimate ion pair
Mg
cyclases vast array ofmono- & bicyclic
SESQUITERPENES
6-memb10-memb 11-memb
ring cyclised'CATIONS'
- further cyclisation- 1,2-hydride & alkyl shifts
- trapping with H2O- elimination to alkenes
(E)
NB. control by:1) enzyme to enforce conformation & sequestration of reactive intermediates2) intrinsic stereoelectronics of participating orbitals
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:
– 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/
• 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
geranylgeranyl PPOPP H
H
O
OH
AcO
HO
H
AcOBzO
O
OHO
PhBzNH
O
cembrene
cyclisation
b
taxol
bsesquiterpene(isoprenoid)
β-amino acid(shikimate)
OPP
a
a14-exo
Triterpenes – FPP → Squalene• triterpenes (C30) 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)
H
H
squalene
OPP
OPP
OPP
H H
EnzB:
OPP
presqualene PP
FPP (donor)
FPP (acceptor)
OPP
OPP
NADPHNADP
H
+ PPi
blocked by squalestatinssqualene synthase
H H
O
OO
OH
HO2CHO2C
OH CO2H
OAcO
zaragozic acid A(squalestatin S1)
Triterpenes – Squalene → 2,3-Oxidosqualene • squalene is oxidised to 2,3-oxidosqualene by squalene oxidase – which is an O2/FADH2-
dependent enzyme:
• the key oxidant is a peroxyflavin:
FADH2
NRHN
NH
HN OO
NRHN
NH
HN OO
OO
peroxyflavin
OO
NRN
N
HN OO
O
HOH
H2ONR
NN
HN OO
FADhydroxyflavin
reductive recycling
squalene
squaleneoxidase
O2 + FADH2
H2O + FAD 2,3-oxidosqualene
O
Oxidosqualene-Lanosterol Cyclase – Mechanism• oxidosqualene-lanosterol cyclase catalyses the formation of lanosterol from 2,3-oxidosqualene:
– this cascade establishes the characteristic ring system of ALL steroids– ring-expansion sequence to establish the C ring– the process is NOT concerted, discrete cationic intermediates are involved & stereoelectronics dictate
the regio- & stereoselectivity although the enzyme undoubtedly lays a role in pre-organising the ~chair-boat-chair conformation
– “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)
Lanosterol → Cholesterol – Oxidative Demethylation• Several steps are required for conversion of lanosterol to cholesterol:
H
HOHH
cholesterolHO
lanosterolH
H H
1
4
8
5
14
24
1) Δ24 hydrogenation2) 14α DEMETHYLATION
3) 4α & 4β DEMETHYLATION4) Δ8 to Δ5 rearrangement
H
8
5isomerase
H
H
NADH
NAD
HH
[-> vitamin D]
4) Δ8 to Δ5 rearrangement
24
NADPH
NADP
H
14
1) Δ24 hydrogenation 2) 14α DEMETHYLATION
H
H
NADPH
NADP
NADPH
NADP
HOH
4 OH H
HO
P450
4x H2O
4x O2
OH
O O
3) 4α & 4β DEMETHYLATION
CO2
P450
2x H2O
2x O2 H
O
HP450O2
HO
H
O
OFeIII
H :BEnz
HCO2H
OH
O O
OH
OH
H
P450
4x H2O
4x O2
CO2
NADH
NAD
HO=
flat, rigid structure
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 P450 enzymes– estrone is produced from androstendione by oxidative demethylation with concomitant aromatisation:
H
HOHH
cholesterol
H P450
2x H2O
2x O2 P450O2
H
HOHH
H
OHOH
H
HOHH
H
O
O
H
OHH
H
O
progesterone
H
OHH
androstendione (X = O)testosterone (X = H, β−OH)
X
H
HH
estrone(œstrone)
O
HO
NADH
NAD
[O]
HCO2H
H
OHH
O
O
OH
P450
2x H2O
2x O2P450O2O OHO
FeIII
HEnzB:
DEMETHYLATIVE aromatisation by 'aromatase' enzyme
Steroid Ring Cleavage - Vitamin D & Azadirachtin• vitamin D2 is biosynthesised by the photolytic cleavage of Δ7-dehydrocholesterol by UV light:
– a classic example of photo-allowed, conrotatory electrocyclic ring-opening:
– 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:
H
HOHH
cholesterol
H
NADPH
NADP
H
H
HO
vitamin D2
[O]
H
H
HO
1,5-hydrideshift
OH
OHOH
calcium ion chelator
HOHH
H UV light (hv)
HOH
H
electrocyclicring-opening7
ψ4SOMO
Me HMe
H
[6π]
conrotatory
OMeO2CAcO OH
OO
OO
OO OH
MeO2C
H
OH
azadirachtin
HO
tirucallol(cf. lanosterol)
H
H
OH7
AcO
azadirachtanin A(a limanoid =
tetra-nor-triterpenoid)
H
H
OH
O
OOAc
OH
O
AcO H
H oxidativecleavage of C ring
highly hindered C-C bondfor synthesis!
C 1112
814
Primary Metabolism - Overview
CO2 + H2O
1) 'light reactions': hv -> ATP and NADPH2) 'dark reactions': CO2 -> sugars (Calvin cycle)
OHOHOHOOH
HO
glucose& other 4,5,6 & 7 carbon sugars
Primary metabolism Secondary metabolites
oligosaccharidespolysaccharidesnucleic acids (RNA, DNA)
phosphoenol pyruvate
glycolysis
CO2
HOOH
OHOOH
HO
POCO2
PO
erythrose-4-phosphate
SHIKIMATE METABOLITEScinnamic acid derivativesaromatic compoundslignans, f lavinoids+
shikimate
aromatic amino acids
aliphatic amino acidspeptidesproteinsCO2
Opyruvate
SCoA
Oacetyl coenzyme A
Citric acidcycle
(Krebs cycle)
ALKALOIDSpenicillinscephalosporinscyclic peptides
tetrapyrroles (porphyrins)
PHOTOSYNTHESIS
saturated fatty acidsunsaturated f atty acidslipids
FATTY ACIDS & POLYKETIDESprostaglandinspolyacetylenesaromatic compounds, polyphenolsmacrolides
ISOPRENOIDSterpenoidssteroidscarotenoids
SCoA
OCO2
malonyl coenzyme A
CoAS
O
O
HO
HOCO2
mevalonateacetoacetyl coenzyme A
Primary metabolites
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