Arylgermanes As Linkers for Solid Phase Synthesis

Home , Polyketide synthase, Protein complex

Biosynthesis of Fatty Acids & Polyketides

Dec 2014 Format & Scope of Lectures

• What are fatty acids? – 1° metabolites: fatty acids; 2° metabolites: their derivatives – biosynthesis of the building blocks: acetyl CoA & malonyl CoA • Fatty acid synthesis by Fatty Acid Synthases (FASs) – the chemistry involved – the FAS protein complex & the dynamics of the iterative synthesis process • Fatty acid secondary metabolites – polyacetylenes – eiconasiods: prostaglandins, thromboxanes & leukotrienes – branched and cyclopropanated fatty acid derivatives • What are polyketides? – definitions & variety – 13C labelling techniques • Polyketide synthesis by PolyKetide Synthases (PKSs) – the chemistry involved – the PKS protein complexes & the dynamics of the iterative synthesis process • Polyketide secondary metabolites – Type I modular metabolites: macrolides – e.g. erythromycin & rapamycin – Type I iterative metabolites: e.g. mevinolin (=lovastatin®) – Type II iterative metabolites: aromatic compounds and polyphenols: e.g. actinorhodin etc. Fatty Acid Primary Metabolites OH OH • Primary metabolites: OH glycerol – fully saturated, linear carboxylic acids & derived (poly)unsaturated derivatives: 3x fatty acids • constituents of essential natural waxes, seed oils, glycerides (fats) & phospholipids • structural role – glycerides & phospholipids are essential constituents of cell membranes OCOR1 OCOR • energy storage – glycerides (fats) can also be catabolised into acetate → citric acid cycle 2 OCOR3 • biosynthetic precursors – for elaboration to secondary metabolites glycerides

SATURATED ACIDS [MeCH2(CH2CH2)nCH2CO2H (n = 2-8)] e.g.

CO2H caprylic acid (C8, n = 2) CO2H myristic acid (C14, n = 5) 8 1 14 1

CO2H CO2H palmitic acid (C16, n = 6) capric acid (C8, n = 3) 1 10 1 16

CO2H lauric acid (C12, n = 4) CO2H stearic acid (C18, n = 7) 12 1 18 1

MONO-UNSATURATED ACID DERIVATIVES (MUFAs) e.g. 9 9 CO2H CO2H 1 1 9 ∆9 palmitoleic acid (C16, Z -∆ ) 18 oleic acid (C18, Z- ) 16 (>80% of fat in olive oil )

POLY-UNSATURATED ACID DERIVATIVES (PUFAs) e.g. 8 5 8 5 CO H arachidonic acid (AA) CO2H eicosapentaenoic acid (EPA) 2 1 1 5 8 11 14, 17 (C20, Z -∆5, Z -∆8, Z -∆11, Z -∆14) (C20, Z-∆ , Z -∆ , Z-∆ , Z -∆ Z -∆ ) 20 11 14 17 20 (in cod liver oil) 11 14 Fatty Acids Derivatives – Secondary Metabolites

• Secondary metabolites – further elaborated derivatives of polyunsaturated fatty acids (PUFAs) • e.g. polyacetylenes & ‘eicosanoids’ (prostaglandins, thromboxanes & leukotrienes)

Br 9 O H H O H H MeO2C POLYACETYLENES O C e.g. CO2Me H 1 14 18 Br wyerone matricaria ester obtucallene II

HO PROSTAGLANDINS CO2H e.g. 1 prostaglandin F2α (PGF2α) 20 HO OH

THROMBOXANES CO2H EICOSANOIDS e.g. O 1 thromboxane A2 (TXA2) O 20 OH

O 1 CO2H LEUKOTRIENES leukotriene A4 (LTA4) e.g. 20

leukotriene A4(LTA4) Primary Metabolism - Overview

Primary metabolism Primary metabolites Secondary metabolites

CO2 + H2O

PHOTOSYNTHESIS 1) 'light reactions': hv -> ATP and NADH 2) 'dark reactions': CO2 -> sugars (Calvin cycle) HO O oligosaccharides HOHO polysaccharides HO nucleic acids (RNA, DNA) OH glycolysis glucose & other 4,5,6 & 7 carbon sugars

CO2 SHIKIMATE METABOLITES PO cinnamic acid derivatives CO2 + aromatic compounds HO O HO OH lignans PO OH OH phosphoenol pyruvate erythrose-4-phosphate shikimate

ALKALOIDS aromatic amino acids penicillins CO peptides 2 proteins cephalosporins aliphatic amino acids cyclic peptides O pyruvate tetrapyrroles (porphyrins) Citric acid cycle saturated fatty acids FATTY ACIDS & POLYKETIDES SCoA (Krebs cycle) SCoA unsaturated fatty acids polyacetylenes CO lipids O O 2 prostaglandins acetyl coenzyme A aromatic compounds, polyphenols malonyl coenzyme A macrolides

CoAS O HO ISOPRENOIDS CO2 terpenoids O HO steroids acetoacetyl coenzyme A mevalonate carotenoids Biosynthesis of Malonyl Coenzyme A

• Malonyl coenzyme A is the key ‘extender unit’ for the biosynthesis of fatty acids (& polyketides): – is formed by the carboxylation of acetyl coenzyme A mediated by a biotin-dependent enzyme – this is the first committed step of fatty acid/polyketide biosynthesis (& is a rate controlling step)

Primary metabolism Secondary metabolism

CO2 pyruvate GLYCOLYSIS O

CoASH CO2 oxidative decarboxylation NAD NADH

SCoA acetyl CoA CITRIC ACID CYCLE O

SCoA acetyl CoA carboxylase CO O CoASH 2 (biotin-dependent) CoAS O

saturated fatty acids O SCoA FATTY ACIDS & POLYKETIDES acetoacetyl CoA unsaturated fatty acids prostaglandins CO O 2 lipids polyacetylenes 2x CoASH 2x NADPH SCoA malonyl CoA aromatic compounds, polyphenols macrolides O 2x NADP

HO ISOPRENOIDS CO2 terpenoids HO steroids mevalonate (MVA) carotenoids Oxidative Decarboxylation of Pyruvate

• Oxidative decarboxylation of pyruvate is catalysed by the Pyruvate Dehydrogenase Complex (PDC) – PDC is a huge complex comprising many copies of each of 3 enzymes:

• 24 × E1 Pyruvate dehydrogenase; 24 × E2 Dihydrolipoyl transferase; 12 × E3 Dihydrolipoyl dehydrogenase – Pyruvate dehydrogenase effects the key decarboxylation using thiamine pyrophosphate as a cofactor

NH2

N N R N OPP = N S R H S Thiamine pyrophosphate (TPP) a vitamin B derivative B 1 BH

TPP ylide TPP ylide R R N N R R S R CO2 R S CO O N R O 2 N CoSH R H N SCoA HO R H O O S S O R SH S S O pyruvate HO S S R'' acetyl CoA S S SH SH R'' R'' E lip R'' 1 pyruvate dehydrogenase oi c

NADH + H FADH ac 2 id E2 dihydrolipoyl transferase

NAD FAD

E3 Dihydrolipoyl dehydrogenase Oxidative Decarboxylation of Pyruvate

• The Pyruvate Dehydrogenase Complex (PDC) – http://www.bmsc.washington.edu/WimHol/figures/figs5/WimFigs5.html Biosynthesis of Malonyl Coenzyme A

• Bicarbonate is the source of the CO2: – the bicarbonate is first activated via phosphorylation by ATP – then the phosphorylated bicarbonate carboxylates biotin to give carboxybiotin – then the carboxybiotin carboxylates the enolate of acetyl CoA to give malonyl CoA:

O bicarbonate HO O Mg Nu O O Pi O N NH ATP H H O NHEnz CoAS S O enolate of acetyl CoA = Nu O Nu O O HN NH O O ADP P H H HO O OH O NHEnz CoAS O biotin S transcarboxylase carboxylase O malonyl CoA biotin = Nu

– the carboxylation of biotin & acetyl CoA are mediated by a single biotin-dependent enzyme (complex) having both biotin carboxylase and transcarboxylase active sites – NB. coupling to ATP ‘hydrolysis’ provides energy to drive carboxylation processes Acetyl CoA Carboxylase

• the biotin co-factor is swung between two active sites:

bicarbonate coupled to biotin transfer to acetyl CoA Biosynthesis of Fatty Acids – Iterative Oligomerisation

• fatty acids are biosynthesised from acetyl CoA as a starter unit by iterative ‘head-to-tail’ oligomerisation involving:

– condensation with malonyl CoA as an extender unit (with loss of CO2) – a decarboxylative Claisen condensation – 3-step reduction of the resulting ketone → methylene • after n = 3-8 iterations the C8-20 saturated fatty acid is released from the enzyme(s):

Malonyl CoA

O O

SEnz O O 2x NADPH O O EnzS O O O O CoAS EnzS EnzS EnzS EnzS CO 2x NADP 2 EnzS + H2O

decarboxylative #1 #1 dCc [R] #2 Claisen condensation reduction dCc

#3 #2 [R] dCc#3 [R] fatty O O O O O O acids EnzS EnzS EnzS EnzS The Decarboxylative Claisen Condensation (dCc)

• in vitro – the classical Claisen condensation:

EtO O O O O OEt O O H OEt OEt EtO EtO + O

EtO EtO

• in vivo - the decarboxylative Claisen condensation catalysed by a ketosynthase (KS)

O O O O SEnz O O O EnzS EnzS O + EnzS EnzS EnzS CO2

– the energy released upon loss of CO2 provides a driving force for the condensation – thioesters are also particularly reactive partners in this type of condensation... The Claisen Condensation - Why Thioesters?

• recall the chemistry of coenzyme A (1st lecture) – properties of alkyl thioesters (cf. alkyl esters) – good leaving group ability of RS- (cf. RO-)

• due to pKa (RSH) ~10 cf. pKa (ROH) ~16

Nu: Nu: O O Nu O O O Nu O cf. SR SR Nu OR OR Nu SR OR a good leaving group a poorer leaving group

– high acidity of protons α to the carbonyl of thioesters (cf. ester) & weak C-S bond (cf. C-O bond):

• due to poor orbital overlap between the lone pairs on sulfur (nS) [cf. nO] and the carbonyl anti bonding orbital π*C=O

O O O O H R H R cf. H R H R S O S less effective O 3p 2p

pKa = 20 pKa = 25 acyl-sulfur bond weak acyl-oxygen bond strong Ketone → Methylene - Reduction

• ketone → methylene reduction is achieved via a 3-step process:

1. NADPH-mediated ketone → alcohol reduction catalysed by a keto reductase (KR) 2. syn-eliminataion of water catalysed by a dehydratase (DH) 3. NADPH-mediated hydrogenation of the double bond catalysed by an enoyl reductase (ER)

ketone -> alcohol syn- alkene O O O OH reduction elimination O hydrogenation O O EnzS S Enz EnzS EnzS EnzS H S H O O H O O O HS H 2 H HR NH2 NH NH2 NH2 2

N N N N

N ADPH NADP NADPH NADP

• all steps are generally stereospecific but stereospecificity varies from organism to organism – indicated specificities are for human FAS Biosynthesis of Fatty Acids – Overview of FAS

• The in vivo process by which all this takes place involves a ‘molecular machine’ - Fatty Acid Synthase (FAS) – Type I FAS: single multifunctional protein complex (e.g. in mammals incl. humans) – Type II FAS: set of discrete, dissociable single-function proteins (e.g. in bacteria) – All FASs comprise 8 components (ACP & 7× catalytic activities): ACP, KS, AT, MT, KR, DH, ER & [TE] :

O O O O decarboxylative SH S O O S SH O S S O CoAS O Claisen O condensation AT MT KS ACP KS ACP CoAS KS ACP

1) KR reduction DH SH SH 2) 3) ER n = 3-8 cycles KS ACP n

O S SH O S SH SH S O O translocation TE n OH KS ACP KS ACP KS ACP

= as = = = KS keto synthase (also known CE condensing enzyme); AT acetyl transferase; MT malonyl transferase; KR = keto reductase; DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein The Acyl Carrier Protein (ACP)

• the Acyl Carrier Protein (ACP) is the key protein that allows the growing oligomer to access the appropriate active sites • The ACP is first primed by the post-translational modification of one of its serine hydroxyl groups: – the introduction of a phosphopantetheine ‘swinging-arm’ by reaction with acetyl coenzyme A:

NH2

N N O O N N HS N N O P O P O H H O HO H O O O O coenzyme A (CoASH)

pantetheinate chain HO P O OH O O

ACP OH phpsphopantetheinyl transferase (ACP synthase)

O O Me Me HS O O ACP N N P primed acyl carrier protein H H - OH O O

phosphopantetheine chain ~ 20Å

– this swinging-arm provides flexibility for module-module acyl transfer & provides binding energy for catalysis – the ACP is inactive prior to priming Human Fatty Acid Synthase (FAS)

• Human FAS (EC 2-3-185) is a type I FAS – a homodimer of a multifunctional protein (272 kDa) – each monomer is ‘barrel’ shaped with diameter ~210 Å & length ~250 Å – each subunit protein contains seven catalytic activities plus the acyl carrier protein (ACP)

AT = acetyl transferase MT = malonyl transferase CE = condensing enzyme (=KS)

ACP = acyl carrier protein KR = keto reductase ER = enoyl reductase

DH = dehydratase

TE = thioesterase

– NB. keto synthases (KS) are also smetimes referred to as condensing enzymes (CE) Human Fatty Acid Synthase (FAS)

• the first three-dimensional structure of human fatty acid synthase at 4.5 Å resolution by X-ray crystallography: – Maier, Jenni & Ban Science 2006, 311, 1258 (DOI) ; also Fungal FAS @ 3.1 Å resolution see: Jenni et al. Science 2007, 316, 254 & 288

Structural overview. (A) Front view: FAS consists of a lower part comprising the KS (lower body) and MAT domains (legs) connected at the waist with an upper part formed by the DH, ER (upper body), and KR domains (arms). (B) Top view of FAS with the ER and KR domains resting on the DH domains. (C) Bottom view showing the arrangement of the KS and MAT domains and the continuous electron density between the KS and MAT domains FATTY ACID BIOSYNTHESIS (type II FAS)

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH Pantetheine

SH SH

NB. the following sequence of slides have been adapted from: http://www.courses.fas.harvard.edu/%7echem27/ FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH Pantetheine

O Me S Co SH SH Acetyl-CoA

• AT1 loads acetyl group onto KS1 FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys S Pantetheine O Me

SH SH FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys S Pantetheine O Me

SH SH O O - O S Co Malonyl-CoA

• AT1 loads malonyl group onto ACP1 FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys S Pantetheine O Me

S SH O O O - FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys S Pantetheine O Me

S CO2 SH O O O -

• KS1 catalyzes Claisen condensation FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH Pantetheine

S SH O O Me FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH Pantetheine S Me O O

• KR1 catalyzes reduction of ketone FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH Pantetheine S Me O OH

SH FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH S Me Pantetheine O OH

• DH1 catalyzes dehydration of alcohol FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH S Me Pantetheine O

SH FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH S Me Pantetheine O

• ER1 catalyzes reduction of alkene FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 Cys SH S Me Pantetheine O

SH FATTY ACID BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 MT2 KS2 KR Cys Cys SH SH S O Pantetheine

Me SH

• KS2 catalyzes translocation to module 2 FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH O Pantetheine

SH FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH O Pantetheine

SH O O - O S Co Malonyl-CoA

• MT2 loads malonyl group onto ACP2 FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH O Pantetheine

S O O O - FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH O Pantetheine

S CO2 O O O -

• KS2 catalyzes Claisen condensation FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH OH

Pantetheine

S O O

Me FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH S Me OH Pantetheine O O

• KR2 catalyzes reduction of ketone FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH S Me OH Pantetheine O OH FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Ser Cys S SH Me OH

Pantetheine O OH

• DH2 catalyzes dehydration of alcohol FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Ser Cys S SH Me OH

Pantetheine O FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Ser Cys S SH Me OH

Pantetheine O

• ER2 catalyzes reduction of alkene FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Ser Cys S SH Me OH

Pantetheine O FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH OH S Pantetheine O

• TE catalyzes transesterification FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH O

Pantetheine O SH

Me FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH O H OH Pantetheine O SH

• TE catalyzes hydrolysis FATTY ACID BIOSYNTHESIS

H1 ER1 ACP2 MT2 KS2 KR2 DH2 ER2 TE Cys Ser SH OH

Pantetheine SH OH O

Me Biosynthesis of Unsaturated Fatty Acids

• two mechanisms are known for the introduction of double bonds into fatty acids: – in BACTERIA: anaerobic [O] → monounsaturated FAs (MUFAs) – in MAMMALS, INSECTS & PLANTS: aerobic [O] → MUFAs & polyunsaturated FAs (PUFAs)

ac er a mamma s, nsec s an s ANAEROBIC ROUT E (b t i ) AEROBIC ROUTE ( l i t & pl t ) e ro ena on occurs ur n c a n e on a on e ro ena on occurs a er c a n e on a on (d hyd g t i d i g h i l g ti ) (d hyd g ti f t h i l g ti ) mainly MUFAs but some PUFAs MUFAs & PUFAs

H O - H H S Enz - S ACP OH O Enz S 1/2 O2 Enz S HOH Enz SH H2O Enz SH H - O S Enz - H S ACP O

thioesterase TE H2O ( ) (hydrolysis) H - nz O S E . - 9 NB in b oth cases cis 18 1 alkenes are produced x O OH 4 C2 oleic acid

OH 1 - 18 11 s P FAs O PUFA C11 C12 U vaccenic acid plants animals Biosynthesis of Polyacetylenes

• A family of over 1000 natural products! – review: Tykwinski Angew. Chem. Int. Ed. 2006, 45, 1034 (DOI) • Few detailed pathways have been established but generally involve sequential dehydrogenations: – e.g. biosynthesis of matricaria ester (Matricaria chamomilla): • component of chamomile tea

9 9 1 1 1 6 COSEnz 6 COS 16COSEnz 18 Enz stearate linoleate 18 oleate 12

9 9 9 1 1 1 6 COSEnz 6 COSEnz 6 COSEnz 12 crepenynate 12 12

14 18 14 18 18

O O 1 9 1 9 MeO 6 COSEnz 2C 6 COSEnz

18 12 12 18

14 14 O2 18 matricaria ester Matricaria chamomilla Biosynthesis of Prostaglandins & Thromboxanes

• prostaglandins & thromboxanes are derived from further oxidative processing of arachiodonic acid • both are important hormones which control e.g. smooth muscle contractility (blood pressure), gastric secretion, platelet aggregation & inflammation (

inhibited by asprin, celebrex, vioxx etc.

cyclooxygenase CO2H CO2H O 'COX' CO2H CO H H 2 O O O O O OH O PGG2 O arachidonic acid OH rearrangement release from cellular stores peroxidase O inhibited by corticosteroids O CO2H CO2H O 1 H O 20 O H OH OH HO OH

thromboxane A2 (TXA2) prostacyclin (induces platelet aggregation) (PGI2) (inhibits platelet aggregation)

HO H OH 1 H CO2H CO2H 1 20 H HO O 20 HO OH H OH prostaglandin F2α (PGF2α)

thromboxane B2 (TXB2) (used to induce labour) Biomimetic Synthesis of Prostaglandins

• In 1984 Corey published a classsic biomimetic total synthesis of prostaglandins – Corey, Shimoji & Shih J. Am. Chem. Soc. 1984, 106, 6425 (DOI)

(Z) OMe H H 1) MsCl, Et N H H OMe ClHg OMe 3 1) Hg(OCOCH2Cl)2 OMe 2) H2O2 2) NaCl O OMe OMe C H O OMe 5 11 C5H11 C H HO HOO 5 11 1) Bu3SnH 2) O O 2 H OMe OMe OMe H (E) OMe O O O 1) MsCl, Et3N H 1) Hg(OCOCH2Cl)2 ClHg OMe 2) H2O2 OMe 2) NaCl O OMe H H C5H11 O C5H11 C H C H disrotatory HO HOO 5 11 5 11 (only syn products) 5-exo-trig OMe CO2H radical cyclisation O OMe C H O 5 11 OOH 1 OMe + .. 1) PPh OMe O O 3 2 2) PPTS O OMe C5H11 O HO OH HO OH OOH prostacyclin (PGI2)

– review: E.J. Corey & X.-M. Cheng ‘The logic of chemical synthesis’ Wiley, New York, 1989, pp297 Biosynthesis of Leukotrienes

• leukotrienes are the other main class of 2° metabolites derived from arachidonic acid – they are potent (

O O O OH 5 O CO2H CO2H CO2H 5-lipoxygenase H H H H

arachidonic acid leukotriene A4 (LTA4)

LTC4 synthase LTA4 hydrolase

OH OH OH

fatty acid CO2H CO2H

S C5H11 O H H N N CO H leukotriene B (LTB ) NRPS 2 N 2 4 4 (glutathione) H CO2H O

leukotriene C4 (LTC4) Branched & Cyclopropanated Fatty Acids

• fatty acid metabolites occaisionally contain 'extra' methyl groups: – there are two methods by which these are added: • by use of a different extender unit – methyl malonyl CoA:

CoAS O biotin-dependent O O carboxylase CO2H Met, Val or Ile CoAS CoAS O FAS propionyl CoA methyl malonyl CoA

• by SAM-mediated methylation/cyclopropanation process:

NADPH Me O

NADP C8H16 SCoA Me S SAM tuberculostearic acid O Me O

C8H16 SCoA C8H16 SCoA oeyl-CoA CH O H 2 H

C8H16 SCoA H dihydrosterculic acid The Polyketide Pathway

• Polyketides are also sometimes known as acetogenins • acetyl CoA is also the starting point for the biosynthesis of polyketide secondary metabolites • these metabolites are topologically very different to the fatty acid metabolites but are in fact synthesised in a very similar fashion. The significant difference is that during the iterative cycle of chain extension the β-keto group is generally not completely reduced out. This gives rise to huge structural diversity based around a 1,3-oxygenation pattern & cyclisation to give aromatic compounds

O O O O × (n+1)

CoAS O n SEnz n OH FATTY ACIDS (FAs) O

SCoA

O O O HO O O SEnz CoAS O × (n+1) n CO2H OH

POLYKETIDES (PKs)

the polyketide pathway • NB. unlike fatty acids. polyketides are NOT biosynthesised by humans – only microorganisms (bacteria) & fungi Polyketides • the structural variety of polyketide secondary metabolites is very wide: – NB. starter units marked in red; extender units in bold black; post oligomerisation appended groups in blue

Me Me OMe O OH O HO O O O O 2 CO H O H CO2H 2 CO2H MeO CO2H OH OH OH Cl OH O 6-methylsalicylic acid orsellinic acid citrinin Griseofulvin actinorhodin (antibiotic) (kidney toxin (treatment for ring (antibiotic) 'yellow rice disease') worm infections) O O OMe HO O O H OH O O MeO POLYKETIDES O H O O MeO aflatoxin B1 (mycotoxic carcingen) N O O O MeO OH H OH O HO O O OH NMe OH OH 2 Me HO O Me O O OH O O O O

rapamycin O OH O O OMe (immunosuppressant) NB. a mixed polypropionate/acetate O OH 6-deoxyerythronolide B erythromycin A mevinolin NB. a polypropionate (antibiotic) (=lovastatin®) (anti-cholesterol) Historical Perspective – ‘The Acetate Hypothesis’

• 1907: James Collie (University of London) converts dehydroacetic acid to orcinol by boiling with Ba(OH)2 (while trying to deduce the structure of the former):

H2O H OH O O HO O HO O O HO OH O O OH

H2O O OH O O O O O OH 2,4,6-triketone dehydroacetic acid (triketide) orcinol

– Collie perceptively postulated the triketone as an intermediate & suggetsed that this might also be an intermediate in the biosynthesis of orcinol (the ‘polyketide hypothesis’)

• 1955: Arthur Birch used 14C labelled acetate to show that 6-methylsalicylic acid (ex. Penicillium patulum) was biosynthesised by head-to-tail oligomerisation of 4 × acetate units and proposed the following biogenesis – proceeding via a tetraketide intermediate (cf. Collie!):

O HO OH O HO 4x O O OH OH OH OH OH

O O 2x H O OH O 3x H2O O O O O 2 14C labelled tetraketide acetate 6-methylsalicilic acid Isotopic Labelling Studies – Use of 13C

• 1970s: Commercial availability of 13C & 2H labelled precursors & NMR instruments allowed rapid determination of labelling patterns of polyketides (cf. radiolabelling/degradation)

• single labelled acetate {[1-13C]- or [2-13C]-acetate, cf. 14C label used by Birch} 1. feed ~99.9% 13C enriched acetate 2. verify uniform incorporation along backbone (ideally obtain incorporation to give ~ doubling of signal size) 3. assign positions of labelled carbons by reference to standard 13C spectrum

• double labelled acetate {[1,2-di-13C]-acetate} 1. feed >90% 2× 13C enriched acetate 2. observe pairs of 13C-13C coupled doublets (NB. again incorporation to a level representing ~ doubling of signal sizes is standard; since natural abundance is ~1% this amounts to ~1% incorporation...this ensures that statistically very few (1 in 104) labelled acetates will be sequentially incorporated & result in inter-unit coupling patterns) 3. assign positions of labelled carbons by reference to standard 13C spectrum – e.g.

O2 OH O O O SAM O2 PKS HO O O J = 44Hz 4x HO O OH O OH CO2H CO2H O O O 13 O OH OMe Me [1,2- C]-acetate 3x H2O CO2 OMe tetraketide penicillic acid orsellinic acid J = 77Hz Isotopic Labelling Studies – Use of 13C/2H

• the ‘α-shift’ technique for following the fate of hydrogens: – allows # of hydrogens (deuteriums) retained at the labelled carbon following biosynthesis → evidence of redox processing etc.

C CD CD2 CD3

upfield shift and multiplicity t quin sept of 13C signal gives 2H connectivity

δ X X + 0.3 X + 0.6 X + 0.9

– useful for identifying starter units e.g.

13 2 13 2 • C & 3× H labelled acetate {[2- C, H3]-acetate} 1. feed multiply labelled acetate 2. observe shifts of labelled carbons 3. assign positions of labelled carbons & determine fate of attached hydrogens

D D D D D D D O O D PKS HO D 5x D D O D OH O O D O D OH D HO OH 4x H O OH O 13 2 2 O O [2- C, H3]-acetate pentaketide terrein Biosynthesis of Polyketides – Oligomerisation Steps • polyketides are biosynthesised by a process very similar to that for fatty acids – the key differences are: • greater variety of starter units, extender units & termination processes • absent or incomplete reduction of the iteratively introduced β-carbonyl groups: ie. each cycle may differ in terms of KR, DH & ER modules & stereochemistry

. . e g O O ' = #1 R H f unct ' = e [ ] o R M C AS O R' = Et R' O O O O no KR EnzS R SEnz O O R' ' ' O O R R O OH O OH EnzS O O O no DH (R) (S) o EnzS R nz or nz C AS R EnzS R E S R E S R EnzS R ' ' R' R CO R R = Me 2 EnzS O O R = Z E R Et no ( ) ( ) = r ER R P decarboxylative EnzS R or EnzS dCc#1 ' Claisen condensation R' R O EnzS R ' R #2 dCc

#3 #2 H H [f unct] O O OH O OH [f unct] O O near O O O #3 li & no dCc no c c se DH DH y li d z nz EnzS R EnzS R o e es En S R E S R p lyk tid ' ' ' ' ' ' ' R' R' R' R R R R R R R

– this leads to enormous diversity... Polyketide Diversity

O R = Me O O O O • starter units: R = Et CoAS R CoAS CoAS CoAS CoAS R = nPr & iPr acetyl CoA propionyl CoA butyryl CoA isobutyryl CoA

O O O O O O O O • extender units: R' = H CoAS O CoAS O R' = Me CoAS O CoAS O R' = Et R' Me Et malonyl CoA (C2) Me-malonyl CoA (C3) Et-malonyl CoA (C4) • non-functional or missing KR, DH, ER:

OH OH none H H O (S) (Z) (E) no KR no ER (R) no DH missing or or

• stereochemistry:

1) side chain stereochemistry (determined by KSn) 2) OH stereochemistry (determined by KRn+1) 3) alkene stereochemistry (determined by DHn+1)

• termination step: – depends on nucleophile that releases product at TE stage:

Nu = OH O O Nu = H O O O 2 intramolecular Nu = CoA NADH O EnzS O HO CoAS H Biosynthesis of Polyketides – Overview of PKS • the in vivo process of polyketide synthesis involves PolyKetide Synthases (PKSs): – PKSs (except Type II, see later) comprise the same 8 components as FASs. i.e. (ACP & 7× catalytic activities): ACP, KS, AT, MT, [KR, DH, ER & TE] – Type I PKSs: single (or small set of) multifunctional protein complex(es) • modular (microbial) - each ‘iteration’ has a dedicated set of catalytic site s (→ macrolides) • iterative (fungal) – single set of catalytic sites, each of which may operate in each iteration (cf. FASs) (→ aromatics/polyphenols - generally) – Type II PKSs: single set of discrete, dissociable single-function proteins (see later) • iterative (microbial) - each catalytic module may operate in each iteration (cf. FASs) (→ aromatics/polyphenols)

Type I (modular & iterative): O O O O [Type II see later] decarboxylative SH S O O S SH O S S O CoAS O Claisen O condensation AT MT KS ACP KS ACP CoAS KS ACP

1) ± KR reduction ? 2) ± DH SH SH 3) ± ER n O cycles KS ACP O O n O O S SH O S SH SH S O O O O translocation TE n OH CE ACP KS ACP KS ACP

KS = keto synthase; AT = acetyl transferase; MT = malonyl transferase; KR = keto reductase; DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein POLYKETIDE BIOSYNTHESIS [Type I – (modular)]

ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A Cys SH Pantetheine Pantetheine Pantetheine

SH SH

NB. the following sequence of slides has also been adapted from: http://www.courses.fas.harvard.edu/%7echem27/ POLYKETIDE BIOSYNTHESIS

ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A Cys SH Pantetheine Pantetheine Pantetheine

SH SH O Me S Co Propionyl-CoA

• AT0 loads starting group (propionyl) onto ACP0 POLYKETIDE BIOSYNTHESIS

ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A Cys SH Pantetheine Pantetheine Pantetheine

S SH O

Me POLYKETIDE BIOSYNTHESIS

ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A Cys SH Pantetheine Pantetheine S Pantetheine O

• KS1 catalyzes translocation to module 1 POLYKETIDE BIOSYNTHESIS

ACP0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 A Cys S Pantetheine Pantetheine O Pantetheine

SH SH POLYKETIDE BIOSYNTHESIS

P0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 Cys S Pantetheine O

SH SH O O SH - O S Co Me Methylmalonyl-CoA

• AT1 loads methylmalonyl group onto ACP1 POLYKETIDE BIOSYNTHESIS

P0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 Cys S Pantetheine O

S SH O Me SH O O - POLYKETIDE BIOSYNTHESIS

P0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 Cys S Pantetheine O

CO2 S SH O Me SH O O -

• KS1 catalyzes Claisen condensation POLYKETIDE BIOSYNTHESIS

P0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 Cys SH Pantetheine

S SH O * Me SH O

Me Stereocenter POLYKETIDE BIOSYNTHESIS

P0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 Cys SH Me S * Pantetheine Me O O

• KR1 catalyzes reduction of ketone POLYKETIDE BIOSYNTHESIS

P0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 Cys SH Me Pantetheine S * * Me O OH

Stereocenter SH

SH POLYKETIDE BIOSYNTHESIS

P0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 Cys Me SH S * Pantetheine * Me O OH

• no DH1 activity POLYKETIDE BIOSYNTHESIS

P0 AT0 ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 Cys Me SH S * Pantetheine * Me O OH

• no ER1 activity POLYKETIDE BIOSYNTHESIS

ACP1 AT1 KS1 KR1 DH1 ER1 ACP2 AT2 KS2 KR Cys Cys SH SH S O Pantetheine * Me HO * Me

• KS2 catalyzes translocation to module 2 POLYKETIDE BIOSYNTHESIS

H1 ER1 ACP2 AT2 KS2 KR2 DH2 ER2 TE Cys Ser S OH

Pantetheine O * Me HO *

Me SH

• Electron cryo-microscopy has recently thrown additional light on how this process works for the Type 1 PKS that synthesises pikromycin in Streptomyces venezuelae. – Dutta et al. Nature 2014, 510, 512-517 (DOI) and Whicher et al. Nature 2014, 510, 560-564 (DOI) – For a video of the process see: http://cen.acs.org/articles/92/i25/Polyketide-Synthase-Secrets-Revealed.html

‘Deconvolution’ of Type I(modular) PKSs

O O OH OH • deduce the module structure for the type I modular PKS hexaketide responsible for the synthesis of this hexaketide: HO

direction O O OH OH 1. identify the last building block: of synthesis i.e. O O HO or #5 HO O

O O OH OH 2. identify each extender unit (working back from the last one): – 2C in the backbone HO #3 #1 – + 0, 1, 2 (or more) C in the sidechain #2 #4 3. identify the starter unit: O O OH OH – the module that appended this unit is designated module 0 HO #0

module# #5 #4 #3 #2 #1 #0 starting material Mal-CoA Et-Mal-CoA Mal-CoA Me-Mal-CoA Mal-CoA Ac-CoA 4. deduce what happens to each ketone: ACP Y Y Y Y Y Y NB. module n modifies the ketone of the AT Y Y Y Y Y Y KS Y Y Y Y Y - building block added by module n-1 KR Y - Y Y Y - DH Y - - - Y - ER Y - - - - - TE Y(H2O) - - - - - Biosynthesis of Erythromycin – Type I(modular) PKS

• 6-deoxyerthronolide is a precursor to erythromycin A – bacterial antibiotic (Streptomyces erythreus): – propionate based heptaketide; 3 multifunctional polypeptides (DEBS1, DEBS2 & DEBS3, all ~350 kDa) – Katz et al. Science 1991, 252, 675 (DOI); Staunton, Leadley et al. Science 1995, 268, 1487 (DOI); Khosla et al. J. Am. Chem. Soc. 1995, 9105 (DOI); review: Staunton & Weissman Nat. Prod. Rep. 2001, 18, 380 (DOI)

DEBS1 DEBS2 DEBS3

module 1 module 2 module 3 module 4 module 5 module 6 O

KR KR DH ER KR KR KR AT KS AT KS AT KS AT KS AT KS AT KS AT TE OH release S S S S S S S O OH O O O O O O O O OH HO HO O HO HO

HO HO O HO loading O HO HO O

HO HO O OH OH HO HO O OH

HO O OH = Acyl Carrier Protein erythronolide B TE = Thioesterase Biosynthesis of Rapamycin – Type I(modular) PKS

• rapamycin – bacterial immunosuppressant used in organ transplant surgery: – mixed polyketide (acetate & propionate)/peptide with novel cyclohexyl carboxamide starter unit – 3 multifunctional polypeptides with 70 catalytic functions! – RAPS1 (~900 kDa, 4 modules), RAPS2 (1.07 MDa, 6 modules), RAPS3 (660 kDa, 4 modules) – Staunton, Leadley et al. Proc. Natl. Acad. Sci. USA 1995, 92, 7839 (DOI); ibid. Gene 1996, 169, 9 (DOI)

Biomimetic Decarboxylative Thioester Aldol

• recall the key C-C bond forming process in both FAS and PKS chain extension is a decarboxylative Claisen condensation of enzyme thioester-bound acetyl and malonyl residues:

O Me

Me O O decarboxylative SH S O O S S O KS = keto synthase Claisen ACP = acyl carrier protein condensation CO 2 KS KS ACP ACP

• Shair has developed an exceptionally mild aldol reaction of malonic acid half thioesters (MAHTs) inspired by this process: – Shair et al. J. Am. Chem. Soc. 2003, 125, 2852 (DOI)

aldehyde outcome

O H Ph O 3.5h, 82% O Cu (20 mol%) 2O R Et 3.5h, R O 85% decarboxylative + O HO + CO2 thioester aldol O N OMe O 2.5h, 81% BnS O (22 mol%) BnS O N H O2N wet THF, air, 23°C O 8h, 82% Biomimetic Iterative Claisen-Like Condensations • Harrison has developed a glycoluril ‘template’ to mimic the proximal ketosynthase (KS) & acyl carrier protein (ACP) units in FAS and PKS and achieved iterative chain extension of up to eight carbons: – Harrison et al. J. Chem. Soc., Perkin Trans. 1 1998, 437 (DOI)

nBuLi, THF, 1h t ° Ac2O, ∆ BuOLi, THF, 0 C N N 20h (=A) N N then AcCl (=B) N N 20min (=C) N N N N O O O O O O O O O O [89%] N N N N [78%] N N N N Li [93%] N N H H H H O O O O O O glycoluril O NaBH4, MeOH, 10min then AcOH (=D) [93%] TFAA Et3N, CH2Cl2 N N D N N C N N B N N 20min (=E) N N O O O O O O O O O O N N [44%] N N [60%] N N [72%] N N [83%] N N H H H H O O O O O O

OH O OH E [86%]

BnOLi THF

N N B N N C N N O O O O O O BnO N N [38%] N N [60%] N N O H H O O O O [55%]

O + [86%] N N O O N N cleavage from 'tetraketide' H H template Biosynthesis of Mevinolin – Type I(iterative) PKS

• mevinolin (=lovastatin®) – cholesterol lowering metabolite of filamentous fungus Aspergillus terreus – inhibits HMG-CoA → mevalonate (see next lecture) – rate-limiting step in biosynthesis of cholesterol – acetate based polyketide composed of a diketide and nonaketide linked by an ester – 2 × Type I (iterative) PKSs: LNKS and LDKS...both contain MeT (methyl transferase) activities – Hutchinson et al. Science 1999, 284, 1368 (DOI)

O O O O O O 3x 3x 2x OH O CoAS O CoAS O O O CoAS O SEnz Me Me (MeT) SEnz SEnz Me O LNKS SAM OH CoAS Diels- Alder nonaketide 3x CO2 3x CO2 2x CO2

P450 OH O OH O 1x O O CoAS O OH OH Me OH Me (MeT) OH OH P450 O LDKS SAM O OH Me CoAS O SEnz Me 1x CO 2 Me O O O diketide

mevinolin (=lovastatin®) Type II PKSs – Enzyme Clusters (Microbial) • Type II PKSs: single set of discrete, dissociable single-function proteins (ACP & 6× catalytic functions): ACP, KSα, KSβ, [KR, DH, ER, & TE] [NB. NO acetyl or malonyl transferases (AT, MT)] – iterative - each catalytic module may operate in each iteration (cf. FASs) (→ aromatics/polyphenols) • these clusters (generally) use malonate as BOTH starter & extender unit • their ACP proteins are able to load malonate direct from malonyl CoA (no MT required)

– the starter malonate is decarboxylated by ‘ketosynthase’ β (KSβ) to give S-acetyl-ACP

– the extender malonates undergo decarboxylative Claisen condensations by ketosynthase α (KSα) • these clusters rarely utilise KR, DH or ER activities and produce ‘true’ polyketides:

O O Type II (iterative): CONH2 O O S O SH S O O S SH O S S O decarboxylative SH S O CoAS O Claisen O O condensation α α α CoAS O KSβ ACP KSα ACP KS ACP KS ACP KS ACP

1) ± KR CONH2 reduction 2) ± DH (rarely) 3) ± ER SH n O cycles

KSβ ACP O n O O O S SH O S SH SH S O O O O translocation TE n OH KSα ACP KSα ACP KSα ACP

KSβ = 'keto synthase β' (=decarboxylase!); KSα = 'keto synthase α' (=ketosynthase!); KR = keto reductase; DH = dehydratase; ER = enoyl reductase; TE = thioesterase; ACP = acyl carrier protein Biosynthesis of Actinorhodin – Type II PKS • actinorhodin – octaketide bacterial antibiotic (Streptomyces coelicolor) – Hopwood Chem. Rev. 1997, 97, 2465 (DOI)

7x O O CoAS O O O O O O O O O O PKS 16 16 16 O O [ACP, KSβ, KSα] O O O O cyclase O O O KR O O O CoAS O O 1 SEnz O SEnz HO SEnz OH 1 OH 1 8x CO2 octaketide aromatase

OH O OH O O OH O O 16 16 O cyclase 2 H O O O O O CO2H 1 SEnz 1 SEnz OH O OH actinorhodin

– timing of 1st cyclisation and mechanism of control of chain length uncertain • octaketide synthesis then cyclisation? (as shown above) • hexaketide synthesis then cyclisation then two further rounds of extension? – indications can sometimes be gleaned from biomimetic syntheses... Biomimetic Synthesis of Quinone Antibiotics • Pioneered by Harris. e.g. classic biomimetic synthesis of chrysophanol: – position of ‘reduced’ ketone dictates cyclisation site – Harris et al. J. Am. Chem. Soc. 1976, 98, 6065 (DOI); see also Barrett et al. J. Chem. Soc., Perkin Trans. 1. 1980, 2272 (DOI)

O Li Li O O O O OH O OH OH O O O OH O OH OEt O O O O O O N OEt N N [24%] O NOT Et, typo in chrysophanol original paper! 'heptaketide' mimic

• Abell & Staunton’s biomimetic syntheses of rubrofusarin & alternariol: – timing of pyrone ring formation dictates subsequent cyclisation-aromatisation pathway – Abell, Bush, Staunton J. Chem. Soc., Chem. Commun. 1986, 15 (DOI)

O THF OH H2O-MeOH MeO O 2+ MeO O LDA, 0C pH 6-9, Mg 30 min MeO 14h O O O MeO SEt O [75%] [>75%] OH OH O OH O SEt OH O rubrofusarin 'heptaketide' mimic OH O alternariol Me ether 'heptaketide' mimic – review of biomimetic quinone antibiotic synthesis: Krohn Eur. J. Org. Chem. 2002, 1351 (DOI) Biosynthesis of Citrinin - Type II PKS

• Citrinin is a liver toxic metabolite of the mould Penicillium citrinum

4x O O Me Me CoAS O Me Me aldol O O cyclisation/ OH O O PKS 3x SAM aromatisation O O O O O EnzS EnzS Me CoAS O EnzS Me O OH H2O 5x CO2 O O O O pentaketide NADPH SAM mediated methylation NADP

Me Me Me Me 3x NADP O2 Me Me NADPH Me Me O OH OH OH P450 O O OH O CO2H Me CO2H Me OH H O OH OH O OH O OH 2 3x NADPH NADP citrinin + P450/NADP mediated oxidation

• Note: – role of SAM for introduction of methyl groups + – P450 then NADP for Me → CO2H oxidation... Methylation by SAM

• methylation at carbon by SAM takes place at the 2-position of 1,3-dicarbonys (or at the 2-position of phenols):

Me Me Me O O O SAM SAM O O O O O EnzS O O O O EnzS S EnzS Enz Me O O NH2 O O O O pentaketide O O N N Me N N O2C S O2C S O NH3 NH3 OH OH S-adenosyl methionine (SAM) Oxidation by P450 Enzymes

• Hydroxylation at unactivated CH positions is achieved by the haem co-factor in P450 enzymes:

OH O2 NADPH O O O N N N N N III III III N N N N N Fe Fe Fe FeV FeIV N N N N N N N N N N SEnz SEnz SEnz SEnz SEnz NADP OH Me Me N N N N III OH FeIII = Fe O H N N N N H OH OH H SEnz

Me Me Me Me SEnz Me Me 2x NADP OH HO2C CO2H OH N N OH N N OH III IV + Fe + Fe haem Fe(III) species N N N N O O H O H S CO2H OH Enz SEnz OH OH 2x NADPH OH OH H OH OH H Griseofulvin Biosynthesis - Type II PKS

• Griseofulvin is a mould metabolite (Penicillium griseofulvum, Penicillium janczewskii) used to treat worm infections in animals and humans – Birch delineated the basic biogenesis in the 1950s & in 1959 griseofulvin was marketed as an oral fungicide by ICI & Glaxo (as Fulcin® & Grisovin®, respectively)

6x O O in vivo in vitro CoAS O O O O OH O OH OMe O OMe PKS O O O O o O C AS O HO OH OH MeO OH OH x 7 CO2 O SEnz - Cl O/p phenolic - heptaketide Fe CN 3 coupling ( )6

OMe e e OH O OH e e O OM NADPH ? OM O OMe OM O OM 3x SAM O O O MeO MeO O HO O O MeO O O Cl NADP ? Cl

OMe e O OM e OM O OMe O O MeO O MeO O Cl Cl r seo u v n g i f l i - (±) griseof ulvin Scope of Structures - Type II PKS

• microbial polyphenolic metabolites:

OH O OH pentaketides (5x C2) O O O Me octaketides (8x C2) emodin eugenone HO MeO OMe O

hexaketides (6x C2) nonaketides (9x C2) OH O OH O O O NH2 plumbagin tetracycline OH Me H H OH NMe2 OH O

O O heptaketides (7x C2) MeO O OH decaketides (10x C2) rubrofusarin rabelomycine

OH OH O OH O OH

• many display interesting biological activities... Primary Metabolism - Overview

Primary metabolism Primary metabolites Secondary metabolites

CO2 + H2O

PHOTOSYNTHESIS 1) 'light reactions': hv -> ATP and NADH 2) 'dark reactions': CO2 -> sugars (Calvin cycle)

HO O oligosaccharides HOHO polysaccharides HO nucleic acids (RNA, DNA) OH glycolysis glucose & other 4,5,6 & 7 carbon sugars

CO2 SHIKIMATE METABOLITES PO cinnamic acid derivatives CO2 + aromatic compounds HO O HO OH lignans, flavinoids PO OH OH phosphoenol pyruvate erythrose-4-phosphate shikimate

ALKALOIDS aromatic amino acids penicillins CO peptides 2 proteins cephalosporins aliphatic amino acids cyclic peptides O pyruvate tetrapyrroles (porphyrins) Citric acid cycle saturated fatty acids FATTY ACIDS & POLYKETIDES SCoA (Krebs cycle) SCoA unsaturated fatty acids prostaglandins CO lipids O O 2 polyacetylenes acetyl coenzyme A aromatic compounds, polyphenols malonyl coenzyme A macrolides

CoAS O HO ISOPRENOIDS CO2 terpenoids O HO steroids acetoacetyl coenzyme A mevalonate carotenoids