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