Primary Metabolism, Enzyme Cofactor Chemistry & Shikimate Metabolites

Home , Camphor, DAHP synthase, Reagent, Shikimate dehydrogenase, Shikimic acid

Nov 2014 Lessons in synthesis - Azadirachtin

• Azadirachtin is a potent insect anti-feedant from the Indian neem tree: – exact biogenesis unknown but certainly via steroid modification:

O MeO C OAc O 2 H O OH O H O OH 12 O O C 11 O 14 OH oxidative 8 O H 7 cleavage highly hindered C-C bond HO OH AcO OH AcO OH for synthesis! H H of C ring H MeO2C O AcO H

tirucallol azadirachtanin A azadirachtin (cf. lanosterol) (a limanoid = tetra-nor-triterpenoid)

– Intense synhtetic efforts by the groups of Nicolaou, Watanabe, Ley and others since structural elucidation in 1987. – 1st total synthesis achieved in 2007 by Ley following 22 yrs of effort – ~40 researchers and over 100 man-years of research! – 64-step synthesis

– Veitch Angew. Chem. Int. Ed. 2007, 46, 7629 (DOI) & Veitch Angew. Chem. Int. Ed. 2007, 46, 7633 (DOI)

Inspiration for med chem - statins

• HMG CoA → MVA is the rate determining step in the biosynthetic pathway to cholesterol

• ‘Statins’ inhibit HMG CoA reductase and are used clinically to treat hypercholesteraemia - a causative factor in heart disease – e.g. lipitor (Atorvastatin calcium, Pfizer) is a competitive inhibitior of HMG-CoA reductase and the worlds biggest selling drug [first drug to reach $10 billion sales (2004: $10.8 bn]

HO NADPH CoAS HO CO2 Zn2 HO H CO2 O CO2 R N CoAS O HO H H O NADPH HMG CoA NADP H2N NADP mevalonate (MVA) CoASH Lipitor inhibits this step

CO2 H

i O Pr OH H H HO PhHN N cholesterol

F Format & Scope of Lectures

• What is biosynthesis? – some definitions – phototrophs, chemotrophs; metabolism (catabolism/anabolism), 1° & 2° metabolites • Overview of primary metabolism → secondary metabolites – photosynthesis & glycolysis → shikimate formation → shikimate metabolites – acetylCoA & the citric acid cycle → a-amino acids → penicillins, cephalosporins, alkaloids – acetylCoA → malonylCoA → fatty acids, prostaglandins, polyketides, macrolide antibiotics – acetylCoA → mevalonate → isoprenoids, terpenoids, steroids, carotenoids • Biological/biosynthetic reactions – enzyme & cofactor chemistry – free energy source – ATP – C-C & C-O bond formation – CoASH, SAM, DMAPP, biotin – oxidation – NAD+, FAD/FMN, haem iron oxo monooxygenases – reduction – NADPH – C-N bond formation – pyridoxal • The shikimate biosynthetic pathway – the core shikimate pathway - mechanisms of the key enzymes – aromatic amino acids: Phe, Tyr & Trp

– ArC3 metabolites – coumarins, lignans & lignins • mixed shikimate/malonylCoA (polyketide): flavonoids

– ArC2, ArC1 & ArC0 metabolites • mixed shikimate/mevalonate (isoprenoid): ubiquinones, menaquinones & tocopherols Metabolism & Natural Product Diversity

O Me Me HO C 2 Me N N NMe O H H H O N N H camphor caffeine HO N H MeO NH

lysergic acid CO2 H2O Pi N2 N hv quinine

H Me O O OH O N Me O O H CO2H N clavulanic acid O Me androstenedione N O OH nicotine patulin Phototrophs & Chemotrophs

• Living organisms are not at equilibrium. They require a continuous influx of free energy to perform mechanical work & for cellular growth/repair:

– Phototrophs (e.g. green plants, algae & photosynthetic bacteria): derive free energy from the sun via

photosynthesis (‘CO2 fixation’): • 1015 kg/year by green plants, which constitute 99% of Earths biomass (i.e. 1012 tons of dry matter) • 1g of carbon processed = >6250 litres of air

hv CO2 + H2O (CHO) + O 2 PHOTOSYNTHESIS

– Chemotrophs (e.g. animals, fungi, most bacteria): derive free energy by oxidising nutrients (carbohydrates, lipids, proteins) obtained from other organisms, ultimately phototrophs • some bacteria & fungi require just D-glucose • mammals require sugars, essential amino acids (~half total used) & certain vitamins (enzyme co-factors or precursors)

• Degradation of the nutrients is coupled to the stoichiometric production of ‘high energy’ phosphate compounds, particularly adenosine triphosphate (ATP, see later). All metabolic function is underpinned by ATP energetic coupling. • By analogy with a money-based economy, the metabolic cost of production of a given metabolite from another can be quantified in terms of ‘ATP equivalents’ defined as the # of moles of ATP consumed/produced per mole of substrate converted in the reaction or sequence Metabolism

• Metabolism is the term used for in vivo processes by which compounds are degraded, interconverted and synthesised: – Catabolic or degradative: primarily to release energy and provide building blocks • generally oxidative processes/sequences (glycolysis, Krebs cycle) – Anabolic or biosynthetic: primarily to create new cellular materials (1° & 2° metabolites) • generally reductive processes/sequences • These two types of process are coupled – one provides the driving force for the other:

Natural product CO2 + H2O secondary metabolites

'CO2 fixation' hv complex metabolites (photosynthesis)

Nutrients ATP Cell components energy energy & Growth storage release Catabolism ADP + Pi O2 Anabolism oxidative NAD(P)H reductive Energy & degredation biosynthesis Building blocks NAD(P) + H Building Blocks respiration 'nitrogen fixation' simple products (by diazatrophs, lightening, the Haber process)

CO2 + H2O N2 Types of Metabolite & Biosynthesis

• Biosynthesis is the term for the in vivo synthesis of metabolites/natural products: – These are divided into two camps: • Primary metabolites: These are the universal and essential components for the survival of living organisms. e.g. sugars, amino acids, nucleotides, ‘common’ fats and polymers such as proteins, DNA, RNA, lipids and polysaccharides • Secondary metabolites: Compounds produced by organisms which are not required for survival, many of which have no apparent utility to the host organism. Frequently a given metabolite will only be produced in a single organism or in a set of closely related organisms. Provide a rich source of pharmacologically active compounds. e.g. shikimate derivatives, alkaloids, fatty acids, polyketides, isoprenoids – Although the boundary is imprecise the term biosynthesis is most commonly applied, by organic chemists, to the in vivo synthesis of secondary metabolites:

“Now ever since Perkin, failing to make quinine, founded the dyestuffs industry, organic chemists have found the study of ‘natural products’ an inexhaustable source of exercises, which can be performed out of pure curiosity even when paid for in the hope of a more commercial reward. As a result the organic chemist’s view of nature is unbalanced, even lunatic but still in some ways more exciting than that of the biochemist. While the enzymologist’s garden is a dream of uniformity, a green meadow where the cycles of Calvin and Krebs tick round in disciplined order, the organic chemist walks in an untidy jungle of uncouthly named extractives, rainbow displays of pigments, where in every bush there lurks the mangled shapes of some alkaloid, the exotic perfume of some new terpene, or some shocking and explosive polyacetylene...’’ ... Since these intriguing derivatives AND e.g. lysine or ATP are ALL in a sense ‘natural products’ we may prefer the term ‘secondary metabolite’ for the former Bu’Lock Adv. Appl. Microbiol. 1961, 3, 293 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 HO OH lignans, flavinoids PO O OH OH phosphoenol pyruvate erythrose-4-phosphate shikimate

ALKALOIDS aromatic amino acids peptides penicillins CO 2 proteins cephalosporins aliphatic amino acids cyclic peptides O pyruvate tetrapyrroles (porphyrins) Citric acid cycle (Krebs cycle) saturated fatty acids FATTY ACIDS & POLYKETIDES SCoA SCoA unsaturated fatty acids prostaglandins CO2 lipids polyacetylenes O O acetyl coenzyme A aromatic compounds, polyphenols malonyl coenzyme A macrolides

CoAS O HO ISOPRENOIDS CO2 terpenoids O HO steroids acetoacetyl coenzyme A mevalonate carotenoids Biological/Biosynthetic Reactions – Enzyme Catalysis & Cofactors

• Most biosynthetic steps are catalysed by specific, individual enzymes. They generally perform familiar processes such as oxidation, reduction, alkylation, hydrolysis, acylation, hydroxylation, elimination etc. • Different enzymes carrying out related reactions often employ common co-factors: small organic functional fragments and/or metal ions. e.g.

– FREE ENERGY RELEASING COUPLE: Adenosine triphosphate (ATP)

– C-C & C-O BOND FORMATION: Coenzyme A (CoASH); S-adenosyl methionine (SAM); dimethylallylpyrophosphate (DMAPP); biotin

+ – OXIDATION: NAD(P) ; FAD/FMN; Haem iron oxo species (e.g. P450)

– REDUCTION: NAD(P)H; (FADH2/FMNH2)

– C-N BOND FORMATION: Pyridoxal

Free Energy Releasing Couple - ATP

• Adenosine triphosphate (ATP) – phosphorylation of an alcohol by adenosine diphosphate (ADP) is highly exothermic (i.e. liberates energy):

NH NH2 2 N N N N

N N N HO O O O N HO O O P P P enzyme P P O O RO P OH O O O O O O + ROH O O O O + -1 O O G = -31 kJmol Mg2+ or Mn2+ PPPO- OH OH PPO- OH OH -OP adenosine adenosine t riphosphate (ATP) ADP

– The phosphorylated alcohol (ROP) is then activated towards e.g. nucleophilic displacement:

O OH Nu + ROP R-Nu + OP OP = Pi = orthophosphate = P O O

– So, overall the endothermic process ROH + Y- → RY + OH- has been achieved by ‘coupling’ the process to the ‘hydrolysis of ATP’ – The situation is analogous to the use of tosylate activation to achieve nucleophilic displacement of an alcohol – In general, the exothermicity associated with phosphorylation shifts the equilibria of ‘coupled’ process by a factor of ~108 Acylation & C-C Bond Formation a to C=O – CoASH

• Coenzyme A (CoASH) – Coenzyme A acts as an acyl transfer/a-carbon activation reagent by forming reactive acyl thioesters:

Y O R ACYL TRANSFER Y NH2 enzyme O N N R'-LG R a-CARBON ALKYLATION O O O SCoA N N R R' HS N N O P O P O X O H H O O HO H O O O O R SCoA O R SCoA ALDOL REACTIONS pantetheinate HO P O OH OH coenzyme A (CoASH) O O adenosine O O SCoA R CLAISEN-type C-ACYLATION SCoA

– Acyl CoA derivatives can act as nucleophiles or electrophiles depending on the circumstances – These modes of reactivity are inherent properties of alkyl thioesters: - - • The good leaving group ability of RS (cf. RO ) reflects: pKa (RSH) ~10 cf. pKa (ROH) ~16 • The enhanced acidity of protons a to the carbonyl of thioesters cf. normal esters reflects the poor orbital overlap

between the lone pairs on sulfur (nS) [cf. nO] and the carbonyl anti bonding molecular orbital p*C=O

H B: R H R nX-p*C=O resonance makes carbonyl less susceptible to enolisation p*C=O R H H Sulfur is in the 2nd period C O H O OH + BH so its lone pair has poor size/energy match with the p* orbital nOp X C=O X : pK ~20 cf. RCH COOR’ ~25 X R' R' Hence a(RCH2COSR’) 2 R' i.e. a to a thioester is similar to a to a ketone Methylation/Dimethylallylation – SAM & DMAPP

• S-Adenosyl methionine (SAM) – SAM acts as a versatile O-, C-, N- & S- methylating reagent in vivo

e.g. Nu NH2 Me SR2 N N enzyme Me O OMe N O C S N 2 Nu-Me Me Me O Me SR2 NH3 O O OH OH OH Me SR methionine 2 S-adenosyl methionine (SAM) R2N R2NMe

– Equivalent to performing an SN2 methylation using MeI in the laboratory

• Dimethylallyl pyrophosphate (DMAPP) – DMAPP acts a dimethylallylating reagent – the pyrophosphate (+ Mg2+/Mn2+) is an excellent leaving group

O P O P O enzyme Nu O O O O Mg2+ or Mn2+ dimethylallyl pyrophosphate (DMAPP)

– Equivalent to performing an SN2 allylation using allyl bromide in the laboratory Carboxylation – Biotin

• Biotin – Biotin in the presence of bicarbonate, ATP and Mg2+ enables nucleophile carboxylation in vivo:

O Mg Nu O O HN NH enzyme O H H O N NH NHR H H HCO Nu O S 3 NHR O ATP S 2+ biotin (R = carrier protein) Mg O

– a very similar reaction can be carried out in the laboratory • Sakurai et al. Tetrahedron Lett. 1980, 21,1967 (DOI)

BrMg MgBr MgBr O O 1) MeMgBr (2 eq) O O O O O O CH N O O 2) CO2 BrMg 2 2 HN NH N N O N N O MgBr DMF O OMe 110 °C [74%] white powder Oxidation – NAD+

• Nicotinamide-adenine dinucleotide (NAD+) [and its phosphorylated analogue (NADP+)] are mediators of biological oxidation (e.g. alcohol to ketone oxidation) – In general, the couple NAD+/NADH is used by enzymes in catabolic oxidation (degradation) – The reagent is a stereospecific hydride acceptor:

stereospecific: O NH enzyme 2 O HS HR NH N N + H H H H H 2 NH 2 H + H N N N R OH R O R O O P O P O N - H O O O O O O O H H O OH O H NADH OH OR NH2 NH2 N N nicotinam ide-adenine dinucleotide (NAD+) R = H NB. H = H + 2e + - + NB. NADP : R = PO3H NAD NADH

– Different enzymes show different absolute specificities but are generally specific for the pro-R or pro-S hydrogens both for removal and delivery – The Oppenauer oxidation is a similar (non-stereoselective) laboratory reaction: • for asymmetric variants see: Nishide et al. Chirality 2002, 14, 759 (DOI)

HO H

O H H H H + Al(OtBu) H OtBu + Al(OtBu) R OH 3 Al 3 R O t R O O O Bu Oxidation Reactions Mediated by NAD(P)+

NAD H OH O maleate dehydrogenase R R' R R' lactate dehydrogenase NADH alcohol dehydrogenase

NAD O H NH3 + NH3 glutamate dehydrogenase R R' R R' NADH

Me OH O NAD + CO CO H 2 isocitrate dehydrogenase R 2 R aldehyde dehydrogenase H H NADH Me R R H NAD R R dihydrosteroid dehydrogenase R R (steroid reductase) R H NADH R

R H R NAD R R dihydrosteroid dehydrogenase R N R N (steroid reductase) R H R NADH

H OH NAD O glucose oxidase R OR' R OR' NADH

NAD O O O O O O glyceraldehyde-3-phosphate + P P dehydrogenase R H HO O R O O NADH

• Adapted from C.T. Walshe, 'Enzymatic Reaction Mechanisms', Freeman, 3rd ed. Oxidation – Flavins (FAD & FMN) • Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are also mediators of biological oxidations (e.g. dehydrogenations – alkane to alkene) – Unlike NAD+, which readily diffuses from enzyme to enzyme, FAD/FMN is usually tightly bound to a given enzyme, sometimes covalently

flavin mononucleotide (FMN) R H R H H O N O NH enzyme O N O R R 2 R R N R H N N + H2 NH R N OH OH HN H H N N N N O N O O O O P O P O N O - H2 N OH O O O O N HN NH N N ribose OH OH isoa lloxazine FADH2/ flavin-adenine dinucleotide (FAD) = 'flavin' FMNH2 FAD/ FADH2/ FMN FMNH 2 + – Re-oxidation of the FADH2 back to FAD is generally by molecular oxygen (although NAD is also sometimes used). The intermediate peroxyflavin can also mediate hydroxylation, epoxidation & other oxygen transfer reactions (see next slide):

H H H O N O O N O O N O O O O NH HO N N HN HN N + HO OH N N N hydrogen peroxide

FADH 2 FAD peroxyflavin Oxidation Reactions Mediated by Flavins • Dehydrogenation by flavins – e.g. dehydrogenation of succinate → fumarate:

succinate H H H H dehydrogenase CO2 H CO CO 2 O2C H (Krebs cycle) H 2 = H O2C H CO2 H CO H H 2 H H CO2 succinate gauche anti H H fumarate O N O O N O (i.e. 100% trans) anti conformation N NH imposed by enzyme via N HN argenine salt bridges N N FAD FADH2

• Baeyer-Villiger-type oxidation by peroxyflavins – e.g. ketone monooxygenase:

H H O O O O N O O O O N HN N H H H O N O O N O O N O O HO HO N N N HN HN N N N N

H2O peroxyflavin NADPH + H + O 2 FAD

NADP Biomimetic Oxidation using FAD Models

• A stoichiometric flavin model oxidising system (alcohol → aldehyde): – Shinkai Chem. Lett. 1982, 812 & Bull. Soc. Chim. Fr. 1983, 56, 1694

intermolecular oxidation O N O O N O NB. simple N-alkylated nicotinamide 4+ + M N NH salts (cf. NAD ) perform poorly N HN N N N N e.g.

CONMe (1 eq) (1 eq) 2 H H N X OH O Ph ZrCl4 ~100%

• A catalytic peroxyflavin model oxidising system (amine → amine N-oxide): – Bäckvall Chem. Eur. J. 2001, 7, 297 (DOI)

O N O Et N via N H O N O O O NH Et N N (2.8 mol%) NH F O F N N >90% F H2O2 (2.6 eq), MeOH, 50 min F amine amine N-oxide

Oxidation – Haem Iron oxo Species (P450)

• Haem iron oxo species e.g. in cytochrome P450 (a ubiquitous heam monooxygenase) are also mediators of biological oxidation (e.g. phenolic coupling, epoxidation, hydroxylation):

H H H N O N OH N N N N HO H V IV Fe Fe FeIII + N N N N N N

SEnz HO2C CO H SEnz SEnz 2 HO2C CO2H HO2C CO2H haem Fe(V) oxo species haem Fe(III) species

O 2 – The porphyrin ring acts as a tetradentate ligand for the octahedral iron. The two axial positions are occupied by an enzyme amino acid ligand (typically a histidine nitrogen) and hydroxy/hydroperoxy residue respectively – Ferricyanide effects similar oxidative processes in the lab (e.g. phenolic coupling, see ‘alkaloids’, later) • Barton & Kirby J. Chem. Soc. 1962, 806 (DOI)

OH O O

K Fe(CN) NMe 3 6 NMe NMe O HO HO 0.92% (!)

MeO MeO MeO OMe-belladine narwedine Reduction - NADPH

• Dihydro-nicotinamide-adenine dinucleotide phosphate (NADPH) [and its de-phosphorylated analogue (NADH)] are mediators of biological reduction (e.g. ketone to alcohol reduction) – In general, the couple NAPH/NADP+ is used by enzymes in anabolic reduction (biosynthesis) – The reagent is a stereospecific hydride donor:

stereospecific: O NH HS HR 2 enzyme O

NH N N 2 - H NH H H H H H H 2 N N N O P O P O N R O R O R OH + H O O O O O O H O O OH OH NADP + H OH OR NH2 NH2 - NB. H = H + 2e N N Hydro-nicotinamide-adenine dinucleotide (NADPH) R = PO3H + NB. NADH: R = H NADPH NADP

– As for the reverse process, different enzymes show different absolute specificities but are generally specific for the pro-R or pro-S hydrogens both for removal and delivery

– NADPH acts like a biochemical equivalent of ‘laboratory’ metal hydride reductants (e.g. LiAlH4, NaBH4) or their chiral equivalents (e.g. CBS-borane):

HPh Ph O B-Me CBS·BH3(10 mol%) HO H 0.6 eq BH3·DMS O N B CH2Cl2, rt, 45 min BH3Me

100% yield, 95% ee B-Me CBS·BH3 Biomimetic Reduction using NAD(P)H Models

• A catalytic, enantioselective NADPH model reducing system (a,b-unsaturated aldehyde → aldehyde): – highlight: Adolfsson Angew. Chem. Int. Ed. 2005, 44, 3340 (DOI)

BnO O N via N 20 mol% H2 Cl BnO O N dioxane, rt, 12 h N O O Cl

O2N O2N H H [81% yield, 81% ee] EtO2C CO2Et EtO2C CO2Et H O2N N N H iminium salt (1 eq) 'LUMO-lowering catalysis' Transamination - PLP

• Pyridoxine (vitamin B6) → pyridoxal-5’-phosphate (PLP) – PLP forms imines (Schiffs bases) with primary amines. This forms the basis of in vivo transamination of a-ketoacids to give a-amino acids (& also racemisation/decarboxylation processes, see ‘alkaloids’)

Enz-NH3 Enz-NH2 H Enz-NH2 OH R CO2 R CO2 R CO2 Enz OH NH3 R CO2 H N N N N HO OP OP H OP H OP H OP H O imine O O O O exchange O N Pyridoxine (vitamin B ) 6 N N N N N H H2O H H H H H R CO2 pyridoxamine imine pyridoxal phosphate phosphate formation NH bound as imine transaminase 3

– The a-carbon protonation is stereospecific and gives the (S) configured chiral centre – Jørgensen has developed a catalytic, enantioselective lab equivalent of this process: • Jørgensen et al. Chem. Comm. 2003, 2602 (DOI)

NH O 2 O O O 1) Zn-cat (20 mol%) H NHBoc OMe OMe MeNO2, 40h, rt N N + Zn O + O TfO OTf 2) Boc2O, Et3N, MeOH N N N 50°C N H H Zn-cat 15% yield, 37%ee Primary Metabolism - Overview

Primary metabolism Primary metabolites Secondary metabolites

CO2 + H2O

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

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

H NH3 H NH3 O2C

CO2 CO2 NH3 NH H n O Me O (S)-tryptophan (S)-phenylalanine (ArC0) OH (ArC ) O O 3 (S)-tyrosine (ArC3) MeO scopoletin SHIKIMATE METABOLITES OH menaquinone (vitamin K2) (ArC3) (ArC ) 1 OH H H HO OH O O 3 H O O H H O O OH Me Me OH MeO OMe a-tocopherol (vitamin E) OMe HO OH (ArC1) podophyllotoxin OH (ArC3) epigallocatechin (EGC) (ArC3) The Shikimate Biosynthetic Pathway - Overview

• Phosphoenol pyruvate & erythrose-4-phosphate → shikimate → chorismate → prephenate:

CO2 PEP PO CO2 CO2 CO2 PO NAD NADH HO CO2 non-concerted CO2 syn-elimination PO O PO O O H HO O NADH OH Pi HO OH HO OH NAD O OH O OH O OH H O OH O OH OH 2 OH Pi erythrose-4-phosphate DAHP 3-dehydroquinate 3-dehydroshikimate (E-4-P) (3-DHQ) (3-DHS) NADPH NADP

FMNH -mediated CO2 CO 2 CO2 CO2 O2C CO Claisen 2 non-concerted PEP CO2 2 H PO rearangement 1,4-anti-elimination ATP O PO O CO2 PO OH O CO2 HO OH OH P OH ADP OH OH Pi i OH pr ephenate chorismate 5-EPS-3-P 3-phosphoshikimate shikimate (3-PS)

phenylalanine tryptophan tyro sine

– The detailed mechanisms of these steps have been studied intensively. Most are chemically complex and interesting. For additional details see: • Mann Chemical Aspects of Biosynthesis Oxford Chemistry Primer No. 20, 1994 (key details) • Haslam Shikimic Acid – Metabolism and Metabolites Wiley, 1993 (full details and primary Lit. citations) • http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/misc/shikim.html (interesting web-site with many biosynethtic pathways) PEP + E-4-P → DAHP

• Phosphoenol pyruvate (PEP) + erythrose-4-phosphate (E-4-P) → 3-deoxy-D-arabino- heptulosonate-7-phosphate (DHAP) • Enzyme: 3-deoxy-7-phosphoheptulosonate synthase = DAHP synthase [EC 2.5.1.54] – chemistry catalysed: an aldol reaction

O CO2 Enz O new C-C bond Pi Enz O PO O CO2 CO CO2 O 2 PO O PO HO OH PEP H-B-Enz H Enz O PO OH :B-Enz DAHP H2O: HO O H-B-Enz OH erythrose-4-phosphate (E-4-P)

– Floss et al. J. Biol. Chem. 1972, 247, 736 (DOI) DAHP → 3-DHQ

• 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DHAP) → 3-dehydroquinate (3-DHQ) • Enzyme: 3-dehydroquinate synthase [EC 4.2.3.4] – chemistry catalysed: alcohol → ketone → alcohol redox cycle & cyclisation via aldol reaction

CO2 CO 2 CO PO O 2 PO O O HO OH HO OH OH HO OH O DAHP O NAD H O OH O OH O OH OH HO O CO PO O CO2 O CO PO 2 NADH PO O CO2 2 OH P OH H OH i OH :B-Enz NADH H OH NAD HO CO2 H H H O OH OH OH OH HO HO HO = O CO new C-C bond CO2 2 O CO2 CO O HO 2 O= O O H Enz-B: CO H-B-Enz 2 O OH O OH 3-dehydroquinate O OH (3-DHQ) OH

– Knowles et al. Biochemistry 1989, 28, 7555 (DOI) 3-DHQ → 3-DHS

• 3-Dehydroquinate (3-DHQ) → 3-dehydroshikimate (3-DHS) • Enzyme: 3-dehydroquinate dehydratase [EC 4.2.1.10] – chemistry catalysed: stereoselective syn-elimination

double bond introduced Enz-B-H Enz-B: CO H O CO2 HO CO2 HO CO2 HO CO2 2 2 H H

Enz Enz O OH N OH N OH N OH O OH Enz-NH2 H O OH H2O H OH H OH 2 Enz OH OH 3-dehydroquinate 3-dehydroshikimate (3-DHQ) (3-DHS)

– Abell et al. Biochem. J. 1996, 319, 333 (DOI) – Coggins et al. J. Biol. Chem. 1995, 270, 25827 (DOI) – Coggins et al. Nature Struct. Biol. 1999, 6, 521 (DOI) 3-DHS → 3-PS

• 3-Dehydroshikimate (3-DHS) → shikimate → 3-phosphoshikimate (3-PS) • Enzymes: shikimate dehydrogenase [EC 1.1.1.25] then shikimate kinase [EC 2.7.1.71] – chemistry catalysed: stereoselective ketone → alcohol reduction then alcohol phosphorylation

CO2 NADPH CO CO2 2 ATP

O OH HO OH PO OH OH ADP OH NADP OH 3-dehydroshikimate shikimate 3-phosphoshikimate (3-DHS) (3-PS)

– Ye et al. J. Bacteriol. 2003, 185, 4144 (DOI) – Morell et al. J. Biol. Chem. 1968, 243, 676 (DOI)

3-PS → 5-EPS-3-P

• 3-Phosphoshikimate (3-PS) → 5-enolpyruvylshikimate-3-phosphate (5-EPS-3P) • Enzyme: 3-phosphoshikimate 1-carboxyvinyltransferase [EC 2.5.1.19] – chemistry catalysed: vinyl ether formation

CO 2 CO2 CO2

CO2 CO2 :B-Enz PO OH PO H OH PO O PO O CO2 PEP H-B-Enz PO 3-phosphoshikimate OH Pi OH (3-PS) 5-EPS-3-P glyphosate (Roundup®)

PO N CO2 H2

– Glyphosate (‘Roundup’) – a Monsanto agrochemical is a potent inhibitor of this biosynthetic step • a non-selective herbicide

– Lewis et al. Biochemistry 1999, 38, 7372 (DOI) – Jakeman et al. Biochemistry 1998, 37, 12012 (DOI) 5-EPS-3-P → Chorismate

• 5-Enolpyruvylshikimate-3-phosphate (5-EPS-3P) → chorismate • Enzyme: chorismate synthase [EC 4.2.3.5] – chemistry catalysed: non-concerted anti-1,4-elimination

CO2 FMNH 2 CO2 FMNH CO2 H

PO O CO2 O CO OH 2 O CO2 FMNH OH FMNH 5-EPS-3-PS 2 OH chorismate Pi



O O

allyl vinyl ether Claisen [3,3] sigmatropic rearrangement – Abell et al. Bioorg. Chem. 2000, 282, 191 (DOI) – Abell et al. J. Biol. Chem. 2000, 275, 35825 (DOI) – Bornemann et al. Biochemistry 1996, 35, 9907 (DOI)

Chorismate → Tryptophan, Tyrosine & Phenylalanine

• Chorismate → anthranilate → tryptophan

• Chorismate → prephenate → tyrosine & phenylalanine – NB. The enzyme chorismate mutase [EC 5.4.99.5] which mediates the conversion of chorismate to prephenate is the only known ‘Claisen rearrangementase’ Tyrosine/Phenylalanine → ArC3 Metabolites

• Tyrosine & phenylalanine → cinnamate derivatives → ArC3 metabolites – coumarins, lignans (stereoselective enzymatic dimerisation) & lignins (stereorandom radical polymerisation) Biomimetic Lignan Synthesis

• Oxidative dimerisation of cinnamyl alcohols gives symmetric furanofuran lignans – review: Brown & Swain Synthesis 2004, 811 (DOI) – IN VIVO: Lewis et al. Science 1997, 275, 362 (DOI) (oxidase → single enantiomer of product)

– IN VITRO: Vermes et al. Phytochem. 1991, 30, 3087 (DOI) [CuSO4 (cat.), O2, acetone-H2O (90%)]

OH OMe O MeO OH OMe HO 2 x coniferyl alcohol OMe MeO HO

O OMe OMe O

OH OMe OMe OH H MeO O HO OMe H H O MeO OMe O syringaresinol H HO OMe NATURAL = single enantiomer SYNTHETIC = racemic - BUT a single diastereoisomer Tyrosine/Phenylalanine → Flavonoids

• 4-Hydroxycinnamic acid → flavonoids: flavanones, flavanonols, flavones & anthocyanins

– Glycosides of these ArC3 metabolites (esp. anthocyanins) constitute coloured pigments in flowers and insects. They also confer bitter and astringent flavours (e.g. tannins & catechins in tea are polymerised flavonoids) – NB. ‘Mixed’ biosynthetic origin: shikimate/malonylCoA (polyketide)

HO OH

O O HO OH HO OH

O CoA CoA O O O RO OR CO2 OH H O OH O flavones ArC3

HO OH HO OH chalcone SCoA 3 x synthase O CO RO OR O 2 OH OH OH OH OH O O malonyl coenzyme A OH OH flavanones ArC3

RO OR RO OR OH OH

flavanonols ArC3 anthocyanins ArC3 Chorismate → Coenzymes Q & Vitamins E & K

• Chorismate → p- & o-hydroxybenzoic acids → coenzymes Q & vitamins E & K – NB. ‘Mixed’ biosynthetic origin: shikimate/mevalonate (isoprenoid)

H O C CO2 CO2 2 CO2 H H O O CO2 OH OH O O 3 O 3

H OH 1 2 CO2 CO2 R , R = H or Me CO2 isoprenoid 1 2 prephenate R R OH OH OH OH OH homogentisate tocopherols (vitamins E) electron transport, antioxidant ArC1

CO2 OH2 CO2 CO2 O isoprenoid MeO Me

O CO2 ubiquinones (coenzymes Q) H OH R MeO R MeO H lipid-soluble electron transport CO n chorismate 2 OH OH OH O O R ArC0 pyruvate

a-ketoglutarate (aKG) aKG H R isoprenoid O SCoA n CO2 R CO O C CO 2 2 2 OH O Me O OH O menaquinones (vitamins K) CO2 cofactors for plasma proteins O O essential for blood clotting O HO O CO2 CO ArC1 isochorismate 2 CO2 O Primary Metabolism - Overview

Primary metabolism Primary metabolites Secondary metabolites

CO2 + H2O

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

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