C O N T E N T S

25-1 ClassifyingNaturalProducts

25-2 BiosynthesisofPyridoxalPhosphate

25-3 BiosynthesisofMorphine

25-4 BiosynthesisofErythromycin

SOMETHINGEXTRABioprospecting:HuntingforNaturalProducts

SecondaryMetabolites:AnIntroductiontoNaturalProductsChemistry e25

WHyTHISCHAPTER? In the past six chapters, we’ve looked at the chemistry and

metabolism of the four major classes of biomolecules—proteins, carbohydrates, lipids, and nucleic acids. But there

is far more to do, for all living organisms also contain a vast diversity of substances usually grouped under the heading natural products. The term natural product really refers to any naturally occurring substance but is generally taken to mean a secondary metabolite—a small molecule that is not essential to the growth and development of the producing organism and is not classified by structure. In this chapter, we’ll look at some familiar natural products and see how they are biosynthesized.

It has been estimated that well over 300,000 secondary metabolites exist, and it’s thought that their primary function is to increase the likelihood of an organism’s survival by repelling or attracting other organisms. Alkaloids, such as morphine; eicosanoids, such as prostaglandin E1; and antibiotics, such as erythromycin and the penicillins, are examples.

Prostaglandin E1

BenzylpenicillinErythromycin A

Morphine

CO2HH

HH OHHOH

H H

OCH3O

H

H

H H

O

HO

HO

HO

O

CH3

CH3

CH3

CO2–

N

OO

N

H

H

O

OH

OH

OH OH

O O

H3C

H3C CH3

H3CH3C

CH3

O

CH3

CH3

CH3

CH3

N(CH3)2

O

O

S

N

877

Norcoclaurinesynthasecatalyzesthecouplingofdopaminewithp-hydroxy-phenylacetaldehyde,astepinmorphinebiosynthesis.

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Prostaglandin E1

BenzylpenicillinErythromycin A

Morphine

CO2HH

HH OHHOH

H H

OCH3O

H

H

H H

O

HO

HO

HO

O

CH3

CH3

CH3

CO2–

N

OO

N

H

H

O

OH

OH

OH OH

O O

H3C

H3C CH3

H3CH3C

CH3

O

CH3

CH3

CH3

CH3

N(CH3)2

O

O

S

N

25-1 ClassifyingNaturalProductsThere is no rigid scheme for classifying natural products—their immense diver­sity in structure, function, and biosynthesis is too great to allow them to fit neatly into a few simple categories. In practice, however, workers in the field often speak of five main classes of natural products: terpenoids and steroids, fatty acid–derived substances and polyketides, alkaloids, nonribosomal poly­peptides, and enzyme cofactors.

Terpenoids,Steroids Alkaloids

Natural Products(secondary metabolites)

Fatty acids,Polyketides

Nonribosomalpolypeptides

Enzymecofactors

• Terpenoids and steroids, as discussed previously in Chapter 23, are a vast group of substances—more than 35,000 are known—derived biosyntheti­cally from isopentenyl diphosphate. Terpenoids have an immense variety of apparently unrelated structures, while steroids have a common tetra­cyclic carbon skeleton and are modified terpenoids that are biosynthe­sized from the triterpene lanosterol. We looked at terpenoid and steroid biosynthesis in Sections 23­8–23­10.

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• Alkaloids, like terpenoids, are a large and diverse class of compounds, with more than 12,000 examples known at present. They contain a basic amine group in their structure and are derived biosynthetically from amino acids. We’ll look at morphine biosynthesis as an example in Sec­tion 25­3.

• Fatty acid–derived substances and polyketides, of which more than 10,000 are known, are biosynthesized from simple acyl precursors such as acetyl CoA, propionyl CoA, and methylmalonyl CoA. Natural products derived from fatty acids generally have most of the oxygen atoms removed, but polyketides, such as the antibiotic erythromycin A, often have many oxygen substituents remaining. We’ll look at erythromycin biosynthesis in Section 25­4.

• Nonribosomal polypeptides are peptidelike compounds that are bio­synthesized from amino acids by a multifunctional enzyme complex without direct RNA transcription. The penicillins are good examples, but their chemistry is a bit complicated and we’ll not discuss their bio­synthesis.

• Enzyme cofactors don’t fit one of the other general categories of natural products and are usually classed separately. We’ve seen numerous exam­ples of coenzymes in past chapters (see the list in Table 19.3) and will look at the biosynthesis of pyridoxal phosphate (PLP) in Section 25­2.

As you might imagine, unraveling the biosynthetic pathways by which specific natural products are made is difficult and time­consuming work. Small precursor molecules have to be identified, guesses about likely routes made, and individual enzymes that catalyze each step isolated, characterized, and mechanistically studied. The payoff for all this painstaking work is a fun­damental understanding of how organisms function at the molecular level, an understanding that can be used to design new pharmaceutical agents.

25-2 BiosynthesisofPyridoxalPhosphateLet’s begin this quick tour of natural­products chemistry by looking at the biosynthesis of pyridoxal 5′­phosphate (PLP), a relatively simple but enor­mously important enzyme cofactor we’ve encountered several times in dif­ferent metabolic pathways. An overview of PLP biosynthesis is shown in FIGURE25.1.

STEPS 1 – 2 OF FIGURE 25.1: OXIDATION Pyridoxal phosphate biosyn­thesis begins with oxidation of the aldehyde group in d­erythrose 4­phosphate to give the corresponding carboxylic acid, d­erythronate 4­phosphate. The oxidation requires NAD1 as cofactor and occurs by a mechanism similar to that of step 6 in glycolysis, in which glyceraldehyde 3­phosphate is oxidized to the corresponding acid (Figure 22.6; page 780). A cysteine –SH group in the enzyme adds to the aldehyde carbonyl group of d­erythrose 4­phosphate to give an intermediate hemithioacetal, which is then oxidized by NAD1 to a

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thioester. Hydrolysis of the thioester yields erythronate 4­phosphate, and a further oxidation of the –OH group at C2 by NAD1 gives 3­hydroxy­4­phos­phohydroxy­2­ketobutyrate (FIGURE25.2).

STEPS 3 – 4 OF FIGURE 25.1: TRANSAMINATION AND OXIDATION–DECARBOXYLATION 3­Hydroxy­4­phosphohydroxy­2­ketobutyrate undergoes a transamination in step 3 on reaction with a­ketoglutarate by the usual PLP­dependent mechanism, shown previously in Figure 20.2 on page 720. The product, 4­phosphohydroxythreonine, is then oxidized by NAD1 to give an intermediate b­keto ester, which undergoes concurrent decarboxylation and yields 1­amino­3­hydroxyacetone 3­phosphate. The reactions are shown in FIGURE25.3.

O HC

CH2OPO32–

OHH

OHH

D-Erythrose4-phosphate

Pyruvate

D-Glyceraldehyde3-phosphate

1 2 3C O

C

C O

CH3

O O–C

CH2OPO32–

OHH

OHH

O O–C

CH2OPO32–

OPO32–

OHH

HH3N+

H3N+

D-Erythronate4-phosphate

CH2OPO32–

OHH

HHO

C O

CH3

+CO2

–

1-Deoxyxylulose5-phosphate

Pyridoxine5′-phosphate

Pyridoxal5′-phosphate (PLP)

O O–

CH2OPO32–

OHH

CO H

CH2OPO32–

OHH

3-Hydroxy-4-phospho-hydroxy-2-ketobutyrate

4-Phospho-hydroxythreonine

1-Amino-3-hydroxy-acetone 3-phosphate

CO2CO2

4

5

6

O

CH2OPO32–

CH3

CH2OH

OHH+N

CH2OPO32–

CH3

CHO

OHH+N

7

FIGURE25.1 Anoverviewofthepathwayforpyridoxal5′-phosphatebiosynthesis.Individualstepsareexplainedinthetext.

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O HC

CH2OPO32–

OHH

OHH

D-Erythrose4-phosphate

CH2OPO32–

NAD+

OHH

OHH

Hemithioacetal

H

HA

O SC

CH2OPO32–

OHH

OHH

Thioester

S

B

B

O O–C

CH2OPO32–

OHH

OHH

D-Erythronate4-phosphate

CO HH

S CONH2

N+

NADH/H+

NADH/H+

NAD+

C O

CO O–

CH2OPO32–

OHH

3-Hydroxy-4-phospho-hydroxy-2-ketobutyrate

H2O SH

EnzEnz

Enz Enz

FIGURE25.2 Mechanismofsteps1and2inPLPbiosynthesis.Oxidationofd-erythrose4-phosphategives3-hydroxy-4-phosphohydroxy-2-ketobutyrate.

O O–C

CH2OPO32–

OHH

H

4-Phosphohydroxy-threonine

NADH/H+

NAD+

C O

CO O–

CH2OPO32–

OHH

3-Hydroxy-4-phospho-hydroxy-2-ketobutyrate

C O

CO O–

CH2OPO32–

H

A �-keto ester

H3N+Glutamate

�-Ketoglutarate

CO2

OPO32–H3N

+H3N

+

1-Amino-3-hydroxy-acetone 3-phosphate

O

H A

STEP 5 OF FIGURE 25.1: FORMATION OF 1-DEOXYXYLULOSE 5-PHOS-PHATE The 1­amino­3­hydroxyacetone 3­phosphate formed in step 4 of PLP biosynthesis reacts in step 6 with 1­deoxyxylulose 5­phosphate (DXP). DXP arises in step 5 by an aldol­like condensation of d­glyceraldehyde 3­phosphate with pyruvate in a thiamin­dependent reaction catalyzed by DXP synthase.

You might recall from Figure 22.7 on page 784 that pyruvate is converted to acetyl CoA by a process that begins with addition of thiamin diphosphate

FIGURE25.3 Mechanismofsteps3and4inPLPbiosynthesis.

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(TPP) ylide to the ketone carbonyl group, followed by decarboxylation to give hydroxyethylthiamin diphosphate (HETPP). Exactly the same reaction occurs in DXP biosynthesis, but instead of reacting with lipoamide to give a thioester, as in the formation of acetyl CoA, HETPP adds to glyceraldehyde 3­phosphate in an aldol­like reaction. The tetrahedral intermediate that results expels TPP ylide as leaving group and yields DXP. The mechanism is shown in FIGURE25.4.

Thiamin diphosphate ylide adds to theketone carbonyl group of pyruvate toyield an alcohol addition product.

1

The addition product contains a C=Nbond two carbons away from thecarboxylate and is structurally similarto a �-keto acid. It therefore loses CO2,giving the enamine HETPP.

2

The enamine adds to glyceraldehyde3-phosphate in an aldol-like reaction.

3

Cleavage of the adduct in a retro-aldolreaction gives 1-deoxy-D-xylulose5-phosphate and regenerates TPP ylide.

4

TPP ylide Pyruvate

H3C

H3C

CH3

R′

–

S

+

R

N

H3C

H3C

R′ S

+

+

R

N

C

C

–O

–O

O

O

O

OH

H A

1

2

3

CO2

R′

R

S

N

H3C

H3C

H3C

R′

R

S

N

+

H3C

R′

R+

S

N

OH

CH3 H

H

OH

OPO32–

OPO32–

H OH

HHO

O

O

H

H

A

HETPP

Glyceraldehyde3-phosphate

OPO32–

H OH

HHO

B

4

–

TPP ylide 1-Deoxy-D-xylulose5-phosphate

O

STEP 6 OF FIGURE 25.1: CONDENSATION AND CYCLIZATION 1­Deoxy­d­xylulose 5­phosphate is dephosphorylated and then condenses with 1­amino­

FIGURE25.4Mechanismofstep5inpyridoxalphosphatebiosynthesis.Thethiamin-dependentaldolreactionofd-glycer-aldehyde3-phosphatewithpyruvategives1-deoxyxylulose5-phosphate.

© J

ohn

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urry

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3­hydroxyacetone 3­phosphate in step 6 to give pyridoxine 5′­phosphate. The reaction begins with formation of an enamine, followed by loss of water to form an enol that also contains a ketone group six atoms away. The enol adds to the ketone in an intramolecular aldol reaction (Section 17­8) to form a six­membered ring, which then loses water. Tautomerization of the resultant unsaturated ketone gives an aromatic pyridine ring. Note that a loss of phosphate ion occurs at some point in the process, although the exact point at which this happens is not known. The mechanism is shown in FIGURE25.5.

Nucleophilic addition of the amineto 1-deoxy-D-xylulose gives anenamine . . .

1

. . . which loses water to form anenol that also contains a ketonegroup six atoms away.

2

The enol undergoes anintramolecular aldol reactionwith the ketone . . .

3

. . . and the aldol intermediate thenloses water. Tautomerization of thecarbonyl group yields pyridoxine5′-phosphate.

4

1-Deoxy-D-xylulose5-phosphate

1-Amino-3-hydroxy-acetone 3-phosphate

Enamine

Enol

CH2OPO32–

CH2OPO32–

CH2OPO32–

2–O3PO

CH3

CH3

H2N

O O

HO

HO

+

+ H2O

O

OH

OH

1

N

H

H

CH2OPO32–

CH2OPO32–

2–O3PO

2–O3PO

CH3

O

O HN

H

H

H

A

2

+

HA

HA

B

B

CH3

ON+

HO

3

4

CH2OPO32–

CH3

CH2OH

OHHN+

+ H2O + Pi

Pyridoxine5′-phosphate

FIGURE25.5 Mecha-nismofstep6inPLPbiosynthesis.Thereactionof1-amino-3-hydroxy-acetone3-phosphatewith1-deoxy-d-xylulose5-phosphategivespyri-doxine5′-phosphate.

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ohn

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urry

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STEP 7 OF FIGURE 25.1: OXIDATION The final step in PLP biosynthesis is oxidation of the primary alcohol group in pyridoxine 5′­phosphate to the cor­responding aldehyde. Typically, as we’ve seen on numerous occasions, alco­hol oxidations are carried out by either NAD1 or NADP1. In this instance, however, flavin mononucleotide (FMN) is involved as the oxidizing coen­zyme and reduced flavin mononucleotide (FMNH2) is the by­product. The details of the reaction are not clear, but evidence suggests that a hydride trans­fer is involved, just as in NAD1 oxidations.

Pyridoxine5′-phosphate

Flavin mono-nucleotide (FMN)

2–O3POCH2

CH3

O

OHH

H

+N

H

H

H B

H3C

H3C

O

O

N

N N

N

H A

Pyridoxal5′-phosphate (PLP)

Reduced �avin mono-nucleotide (FMNH2)

2–O3POCH2

CH3

O

OHH+N

H

H3C

H3C

O

O

N

H

N N

H

N

C

H

P R O B L E M 2 5 . 1

In the addition of HETPP to glyceraldehyde 3­phosphate shown in Figure 25.4, does the reaction take place on the Re face or the Si face of the glyceraldehyde carbonyl group?

P R O B L E M 2 5 . 2

Show a likely mechanism for the final tautomerization in the reaction of 1­amino­3­hydroxyacetone 3­phosphate with 1­deoxy­d­xylulose to give pyri­doxine 5′­phosphate (Figure 25.5).

25-3 BiosynthesisofMorphineHaving looked at the biosynthesis of pyridoxal 5′­phosphate in the previous section, let’s now go up a level in complexity by looking at morphine biosyn­thesis. Morphine, perhaps the oldest and best known of all alkaloids, is obtained from the opium poppy, Papaver somniferum, which has been culti­vated for more than 6000 years. Medical uses of the poppy have been known since the early 1500s, when crude extracts, called opium, were used for the relief of pain. Morphine was the first pure compound to be isolated from opium, but its close relative codeine also occurs naturally. Codeine, which is

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simply the methyl ether of morphine and is converted to morphine in the body, is used in prescription cough medicines and as an analgesic. Heroin, another close relative of morphine, does not occur naturally but is synthe­sized in the laboratory by diacetylation of morphine.

Morphine

H

H

H H H H

HO

HO

O

CH3N

Codeine

H

H

H

CH3O

HO

O

CH3N

Heroin

H

H

H

CH3CO

CH3CO

O

CH3N

O

O

Chemical investigations into the structure of morphine occupied some of the finest chemical minds of the 19th and early 20th centuries, and it was not until 1924 that the puzzle was finally solved by Robert Robinson, who received the 1947 Nobel Prize in Chemistry for this and other work with alkaloids.

Morphine and its relatives are extremely useful pharmaceutical agents, yet they also pose an enormous social problem because of their addictive properties. Much effort has therefore gone into understanding how morphine works and into developing modified morphine analogs that retain the analge­sic activity but don’t cause physical dependence. Our present understanding is that morphine functions by binding to so­called mu opioid receptor sites in both the spinal cord, where it interferes with the transmission of pain signals, and brain neurons, where it changes the brain’s reception of the signal.

Hundreds of morphine­like molecules have been synthesized and tested for their analgesic properties. Research has shown that not all the complex framework of morphine is necessary for biological activity. According to the “morphine rule,” biological activity requires (1) an aromatic ring attached to (2) a quaternary carbon atom, followed by (3) two more carbon atoms and (4) a tertiary amine. Meperidine (Demerol), a widely used analgesic, and methadone, a substance used in the treatment of heroin addiction, are two compounds that fit the morphine rule.

The morphine rule

H

H

H H

HO

HO

O

CH3N

Methadone

CH3

H3C

An aromatic ringattached to a quaternary carbon ( )followed by two more carbons ( )and a tertiary amine (N)

OCH2CH3

CH3

C6H5 N CH3NO

Meperidine

O

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Morphine is biosynthesized from two molecules of the amino acid tyro­sine. One tyrosine is converted into dopamine, the second is converted into p­hydroxyphenylacetaldehyde, and the two are coupled to give morphine. The entire pathway is a bit complex at several points, but an abbreviated scheme is given in FIGURE25.6.

Morphine

H

H

H H

HO

HO

O

CH3N

Codeine

H

H

H H

CH3O

HO

O

CH3N

Thebaine

HH

CH3O

CH3O

O

CH3N

Salutaridine(R)-Reticuline

H

CH3O

CH3O

HO

CH3N

O

H

CH3O

CH3O

HO

CH3N

OH

CH3O

CH3O

HO

=HO

CH3H

N

HO

HO

HOH

NH

6

(S)-Norcoclaurine

Dopamine

Tyrosine

4

3

2

1

7 8

5

HO

HO

HO

HO

CHO

NH2

p-Hydroxyphenyl-acetaldehyde

CO2–

H3N+ H

FIGURE25.6 Anabbreviatedpathwayforthebiosynthesisofmorphinefromtwomoleculesoftyrosine.Individualstepsareexplainedinthetext.

STEP 1 OF FIGURE 25.6: DOPAMINE BIOSYNTHESIS Dopamine is formed from tyrosine in two steps: an initial hydroxylation of the aromatic ring, followed by decarboxylation. The hydroxylation is catalyzed by tyrosine 3­monooxygenase, requires a cofactor called tetrahydrobiopterin, and occurs through a somewhat complex pathway that involves an iron–oxo (Fe=O) com­plex analogous to that involved in prostaglandin biosynthesis (Figure 8.11).

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The decarboxylation is catalyzed by the PLP­dependent enzyme aromatic l­amino acid decarboxylase.

L-Dopa

HO

HO CO2–

H

Dopamine

HO

HO NH3

CO2H2OO2

+

NH3+

Tyrosine

HO

CO2–

H NH3+

Recall from Section 20­2 that pyridoxal 5′­phosphate reacts with the a amino group of an a­amino acid to form an imine, or Schiff base. When l­dopa reacts with PLP, the resultant imine undergoes decarboxylation, with the pyridinium ion of PLP acting as the electron acceptor. Hydrolysis then gives dopamine and regenerated PLP. The mechanism is shown in FIGURE25.7.

H

Pyridoxalphosphate (PLP)

L-Dopa–PLP imineL-Dopa

2–O3PO

CH3

O

OH+N

C

H

H

HO

HO

+

2–O3PO

CH3

OH+N

N

H

H

H3N+ O–

C

O

H

HO

HO

O–C

O

2–O3PO

CH3

OHN

N

H

H

OH

OH

OH

OH

H

2–O3PO

CH3

OH+N

N

H

H H

HO

HO

H

CO2

H A

H2O

PLP Dopamine

2–O3PO

CH3

O

OH+N

C

H

H+

H3N+

FIGURE25.7 Mechanismofstep1inmorphinebiosynthesis.ThePLP-dependentdecarboxylationofl-dopagivesdopamine.

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25-3 bioSyntheSiSofMorphine 887

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STEP 2 OF FIGURE 25.6: P-HYDROXYPHENYLACETALDEHYDE BIO-SYNTHESIS p­Hydroxyphenylacetaldehyde, the second tyrosine­derived precursor of morphine, is also formed in two steps: an initial PLP­dependent transamination with a­ketoglutarate to give p­hydroxyphenylpyruvate, fol­lowed by decarboxylation of the a keto acid. The transamination occurs by the mechanism previously shown in Figure 20.2 on page 720. The decarboxyl­ation requires thiamin diphosphate as coenzyme and occurs by a slight vari­ant of the mechanism described previously in Figure 22.7 on page 784, for the formation of acetyl CoA from pyruvate.

Decarboxylation of p­hydroxyphenylpyruvate begins with nucleophilic addition of TPP ylide to the ketone carbonyl group, followed by loss of CO2 to give an enamine in the usual way. But whereas the enamine formed from pyruvate decarboxylation reacts with lipoamide to give a thioester and regen­erated TPP ylide, the enamine from p­hydroxyphenylpyruvate decarboxyl­ation is simply protonated to give an aldehyde plus TPP ylide. The mechanism is shown in FIGURE25.8.

CO2

Tyrosine

HO

CO2–

H NH3+

p-Hydroxyphenyl-pyruvate

p-Hydroxyphenyl-acetaldehyde

Enamine

HO

CO2–

O–

CH3

Glutamate

�-Ketoglutarate

O

O

HOHOHO OH

HATPP ylide

–

S

+

R

N

CH3

R′

S

R

N

H

H

A

C O

CH3S

+

R

N

+

HOO

HO

TPP ylide+

H

H

CH3

R′

S

R

N

B

R′

R′

STEP 3 OF FIGURE 25.6: COUPLING The coupling of dopamine and p­hydroxyphenylacetaldehyde is catalyzed by (S)­norcoclaurine synthase and is relatively straightforward. The reaction proceeds through initial for­mation of an intermediate iminium ion, followed by intramolecular electro­philic aromatic substitution at a position para to one of the hydroxyl groups (FIGURE25.9).

FIGURE25.8Mechanismofstep2inmorphinebio-synthesis.TPP-depen-dentdecarboxylationofp-hydroxyphenyl-pyruvategivesp-hydroxyphenylacet-aldehyde.

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888 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry

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HO+

HO

HOH

NH

Dopamine

HO

HO

HOH

NH

(S)-Norcoclaurine

HO

HO

HO

CHO H

HO

NH2

HO

HO

p-Hydroxyphenyl-acetaldehyde

Iminium ion

H2O NH+

B

STEP 4 OF FIGURE 25.6: METHYLATION, HYDROXYLATION, AND EPI-MERIZATION (S)­Norcoclaurine next undergoes two methylations and a hydroxylation to give (S)­3′­hydroxy­N­methylcoclaurine, which is methyl­ated a third time to produce (S)­reticuline. Epimerization of (S)­reticuline then yields (R)­reticuline (FIGURE25.10).

HO

HO

HOH

NH

(S)-Norcoclaurine

CH3O

HO

HOH

NH

(S)-Coclaurine

SAHSAM SAHSAM O2 H2O

CH3O

HO

HOH

N

(S)-N-Methylcoclaurine

CH3

SAHSAM

CH3O

HO

HO

HOH

N

(S)-3′-Hydroxy-N-methylcoclaurine

CH3

CH3O

CH3O

HO

HOH

N

(S)-Reticuline

CH3

CH3O

CH3O

HO

HON

(R)-Reticuline

CH3H

FIGURE25.10 Anoverviewofthereactionsinstep4ofmorphinebiosynthesis.(S)-Norcoclaurineisconvertedto(R)-reticuline.

FIGURE25.9 Mecha-nismofstep3inmor-phinebiosynthesis.Couplingofdopamineandp-hydroxyphenyl-acetaldehydegives(S)-norcoclaurine.

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25-3 bioSyntheSiSofMorphine 889

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Both initial methylations use S­adenosylmethionine (SAM) as the methyl donor, as discussed in Section 12­11. S­Adenosylhomocysteine (SAH) is the by­product in each case, and the reactions occur by the usual SN2 substitution pathway. The first methylation occurs on a phenol oxygen, and the second takes place on the amine nitrogen.

The hydroxylation of (S)­N­methylcoclaurine to give (S)­3′­hydroxy­N­methylcoclaurine is superficially similar to the hydroxylation of tyrosine in step 1 in that both involve an iron–oxo complex as the active hydroxylating agent. Unlike the enzyme in the tyrosine hydroxylation, however, that respon­sible for hydroxylation of N­methylcoclaurine is a so­called cytochrome P450 enzyme. These enzymes, of which more than 500 are known, contain an iron–heme cofactor ligated to the sulfur atom of a cysteine residue in the enzyme. The details of the hydroxylation itself are not clear, although it may well occur through a straightforward electrophilic aromatic substitution mechanism.

CH3

CH3

H3C

H3C

Heme

HO2C CO2H

Fe(II)

N N

N N

CH3

CH3

H3C

H3C

Heme iron–oxo complex

HO2C CO2H

Fe(V)

N N

N N

O2

O

S

Cys

Enz

Methylation of a phenolic –OH group in (S)­3′­hydroxy­N­methylco­claurine by SAM gives (S)­reticuline through the usual SN2 pathway, and epimerization of the chirality center forms (R)­reticuline. The epimerization is a two­step process, the first an oxidation of the tertiary amine to an inter­mediate iminium ion, and the second a hydride reduction of the iminium ion. The mechanism of the oxidation step is not yet known, but the reduction of the iminium ion requires NADPH as cofactor (FIGURE25.11).

Why does morphine biosynthesis proceed through initial formation of (S)­reticuline as an intermediate, followed by epimerization, rather than through (R)­reticuline directly? There is no obvious answer other than to say that many metabolic pathways contain such small inefficiencies, probably as a result of the evolutionary development of the responsible enzymes—what some people have called “unintelligent design.”

STEP 5 OF FIGURE 25.6: OXIDATIVE COUPLING (R)­Reticuline is con­verted into salutaridine in step 5 by an oxidative coupling between the ortho position of one phenol ring and the para position of the other. The reaction is catalyzed by another cytochrome P450 enzyme like that involved in the hydroxylation of (S)­N­methylcoclaurine in step 4. Formation of the phen­oxide ions and abstraction of a nonbonding electron from each oxygen atom to give radicals occurs, followed by radical coupling and a keto–enol tautomer­ization to yield salutaridine (FIGURE25.12).

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890 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry

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NADP+

NADPH/H+

CH3O

CH3O

HO

HOH

N

(S)-Reticuline

CH3

CH3O

CH3O

HO

HON+

Iminium ion

CH3

(R)-Reticuline

H

CH3O

CH3O

HO

CH3N

OH

CH3O

CH3O

HO

=HO

CH3H

N

FIGURE25.11 Mechanismoftheepimerizationof(S)-reticulineto(R)-reticulineinstep4ofmorphinebiosynthesis.

(R)-Reticuline

Salutaridine

H

CH3O

CH3O

HO

CH3N

OH

H

H

CH3O

CH3O

•O

CH3N

O•

•

•

H

CH3O

CH3O

O

O

CH3N

H

H

CH3O

CH3O

O

O

CH3NH

CH3O

O

CH3N

CH3O

HO

FIGURE25.12 Mechanismofstep5inmorphinebiosynthesis.Oxidativephenolcouplingof(R)-reticulinegivessalutaridine.

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25-3 bioSyntheSiSofMorphine 891

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STEP 6 OF FIGURE 25.6: REDUCTION AND CYCLIZATION Reduction of salutaridine to salutaridinol is catalyzed by salutaridine reductase, with NADPH as cofactor. This alcohol then undergoes a nucleophilic acyl substitu­tion reaction with acetyl CoA to give a doubly allylic acetate, which spontane­ously eliminates acetate ion in an SN1­like process and cyclizes to thebaine (FIGURE25.13).

Salutaridine

H

CH3O

O

CH3O

HO

Salutaridinol

H

CH3O

CH3CSCoA

HSCoA

CH3O

HONADP+

NADPH/H+

H OH

H

CH3O

CH3O

HO

H OCOCH3

O

H

H

+CH3O

CH3O

OHCH3CO2–

B

Thebaine

HH

CH3O

CH3O

O

NCH3

NCH3

NCH3

NCH3

NCH3

FIGURE25.13 Mechanismofstep6inmorphinebiosynthesis.Thebaineisformedfromsalutaridine.

STEPS 7 – 8 OF FIGURE 25.6: DEMETHYLATION AND REDUCTION The remaining steps in the biosynthesis of morphine involve two demethyl­ation reactions and a reduction. The first demethylation is catalyzed by a cytochrome P450 enzyme, which hydroxylates the –OCH3 group of the­baine to form –OCH2OH, a hemiacetal. Loss of formaldehyde then gives an enol that tautomerizes to codeinone. Reduction of the resultant ketone by NADPH yields codeine, and demethylation by a P450 enzyme produces morphine (FIGURE25.14).

P R O B L E M 2 5 . 3

Show the mechanism of the reaction of (S)­norcoclaurine with S­adenosyl­methionine to give (S)­coclaurine (Figure 25.10).

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892 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry

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Morphine

H

H

H

HO

HO

O

CH3N

Codeine

H

H

H

CH3O

HO

O

CH3N

Codeinone

HH

CH3O

O

CH3N

Thebaine

HH

CH3O

CH3

O

O

CH3N HH

CH3O

CH2•

O

O

CH3N

NADP+

NADPH/H+

HH

CH3O

CH2

O

O

CH3N

H

OH A

B

CH2O

O

O2H2OCH2O,

O2H2O

H

HH

P R O B L E M 2 5 . 4

Convince yourself that the following two structures both represent (R)­reticu­line. Which carbon atoms in the structure on the right correspond to the two carbons indicated in the structure on the left?

H

CH3O

CH3O

HO

CH3N

OH

CH3O

CH3O

HO

=HO

CH3H

N

FIGURE25.14 Mechanismofstep7inmorphinebiosynthesis.DemethylationofthebainetogivecodeinoneiscatalyzedbyaP450enzyme.ReductionofcodeinonewithNADPHthenyieldscodeine,andafinaldemethylationproducesmorphine.

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25-3 bioSyntheSiSofMorphine 893

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25-4 BiosynthesisofErythromycinHaving discussed the biosynthesis of pyridoxal phosphate and morphine in the preceding two sections, we’ll end this chapter on natural­products chemis­try by going up yet one more level in complexity and looking at polyketide biosynthesis. Unlike what happens in many metabolic pathways, where each separate step is catalyzed by a separate, relatively small enzyme, erythromycin

H

H

H

OH

OH

O

O O

O

O

CH3

NHH

OHOH

OH

Tetracycline(antibiotic)

OH

O O O

NH2

H3C OHCH3H3C

OCH3

CH3 CH3

CH3

HO

OH

HO

O

O

OH

OH

Doxorubicin(anticancer)

Rapamycin(immunosuppressant)

Lovastatin(cholesterol lowering)

Amphotericin B(antifungal)

O

O O

CH2OH

H3C

NH2

O

OCH3CH3

H3C

H3C

H3C

CH3O

OH

CH3O

N

O

O

H

CH3

CH3

H

H

OHOH

OH

H3C

H3C

H3C

O

O

O OHHO OH

O

OH OH

O

HONH2

OHO CH3

OCO2H

H

FIGURE25.15 Structuresofsomepolyketidesusedaspharmaceuticalagents.

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894 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry

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and other polyketides are assembled by a single massive synthase. The syn­thase contains many enzyme domains linked together, with each domain cata­lyzing a specific biosynthetic step in sequence.

Polyketides are an extraordinarily valuable class of natural products, numbering over 10,000 compounds. Commercially important polyketides include antibiotics (erythromycin A, tetracycline) and immunosuppressants (rapamycin), as well as anticancer (doxorubicin), antifungal (amphotericin B), and cholesterol­lowering (lovastatin) agents (FIGURE 25.15). It has been esti­mated that the sales of these and other polyketide pharmaceuticals total more than $15 billion per year.

Polyketides are biosynthesized by the joining together of the simple acyl CoA’s acetyl CoA, propionyl CoA, methylmalonyl CoA, and (less frequently) butyryl CoA. The key carbon–carbon bond­forming step in each joining is a Claisen condensation (Section 17­9). Once the carbon chain is assembled and released from the enzyme, further transformations take place to give the final product. Erythromycin A, for instance, is prepared from one propionate and six methylmalonate units by the pathway outlined in FIGURE25.16. Following initial assembly of the acyl units into the macrocyclic lactone 6­deoxyerythronolide B, two hydroxylations, two glycosylations, and a final methylation complete the biosynthesis.

The initial assembly of seven acyl CoA precursors to build a polyketide carbon chain is carried out by a multienzyme complex called a polyketide synthase, or PKS. The 6­deoxyerythronolide B synthase (DEBS) is a massive structure of greater than 2 million molecular weight and containing more than 20,000 amino acids. Furthermore, it is a homodimer, meaning that it consists of two identical protein chains held together by noncovalent interactions, with each chain containing all the enzymes necessary for constructing the polyketide.

Each separate enzyme domain in the erythromycin synthase is a folded, globular region within a huge protein chain that catalyzes a specific biosyn­thetic step. The domains are grouped into modules, where each module car­ries out the sequential addition and processing of an acyl CoA to the growing polyketide. In addition, adjacent modules form three larger groups (DEBS 1, DEBS 2, and DEBS 3) that are linked by peptide spacers. As shown in FIGURE25.17, the erythromycin PKS consists of an initial loading module to attach the first acyl group, six extension modules to add six further acyl groups, and an ending module to cleave the thioester bond and release the polyketide. The ending module also catalyzes cyclization to give a macro­cyclic lactone.

The loading module has two domains: an acyl transfer (AT) domain and an acyl carrier protein (ACP) domain. The AT selects the first acyl CoA (propionyl CoA in the case of erythromycin) and transfers it to the adjacent ACP, which binds it through a thioester linkage and holds it for further reac­tion. Each extension module has a minimum of three domains: an AT, an ACP, and a ketosynthase (KS), which catalyzes the Claisen condensation reaction that builds the polyketide chain. In addition to the three minimum domains, some extension modules also contain a ketoreductase (KR) to reduce a ketone carbonyl group and produce an alcohol, a dehydratase (DH) to dehydrate the alcohol and produce a C=C bond, and an enoyl reductase (ER) to reduce the C=C bond. Finally, the ending domain is a thioesterase (TE), which releases the product by catalyzing a lactonization.

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O

CH3

CH3

Erythronolide B6-Deoxyerythronolide BMethylmalonyl CoA

Propionyl CoA

CH3

H3C

H3C

OH

OH

H3C

OH

H3C

OH

O

O

O

CH3

CH3

CH3

H3C

H3C

OHH3C

OH

H3C

OH

O

OO

CoAS

+

CO2–

6

O

CoAS

O

CH3

CH3

3-O-Mycarosyl-erythronolide B

CH3

H3C

H3C

OH

OH

H3C

OH

H3C

O

O

O

OH

OH

O

CH3

CH3

O

CH3

CH3

Erythromycin D

CH3

H3C

H3COH

H3C

OH

H3C

O

O

O

OH

OH

O

CH3

CH3

HOO

N(CH3)2

O CH3

O

CH3

CH3

Erythromycin C

CH3

H3C

H3COH

H3C

OH

OH

H3C

O

O

O

OH

OH

O

CH3

CH3

HOO

N(CH3)2

O CH3

O

CH3

CH3

Erythromycin A

CH3

H3C

H3COH

H3C

OH

OH

H3C

O

O

O

OH

OCH3

O

CH3

CH3

HOO

N(CH3)2

O CH3

FIGURE25.16 AnoutlineofthepathwayforthebiosynthesisoferythromycinA.Onepropionateandsixmethylmalonateunitsarefirstassembledintothemacrocycliclactone6-deoxyerythronolideB,whichisthenhydroxylated,glycosylatedbytwodifferentsugars,hydroxylatedagain,andfinallymethylated.

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ACP KSKS ATAT TE

End

KR

ExtensionModule 6

DEBS 3 (3179 aa)

ExtensionModule 5

KR

O

O

OH

OH

S

OH

OH

Heptaketide

Hexaketide

O

O

OH

OH

S

OH

ACPKS ATKS DHAT ACPER KR

ExtensionModule 4

DEBS 2 (3568 aa)

ExtensionModule 3

ACP

O

O

OH

S

OH

Pentaketide

O

O

OH

S

OH

Tetraketide

KRACP KSKS ACP - -AT KR

ExtensionModule 2

DEBS 1 (~3174 aa)

ExtensionModule 1Load

ATAT

O

S

OH

Triketide

OH

ACP

O

S

OH

Diketide

O

S

6-Deoxyerythronolide B

DEBS—6-Deoxyerythronolide B synthaseAT—AcyltransferaseACP—Acyl carrier proteinKS—Ketoacyl synthaseKR—Ketoacyl reductaseDH—DehydrataseER—Enoyl reductaseTE—Thioesterase

O

CH3

CH3

CH3

H3C

H3C

OHH3C

OH

H3C

OH

O

O

FIGURE25.17 Aschematicviewofthe6-deoxyerythronolideBsynthase(DEBS).Locationsoftheenzymedomainswithintheloadingmoduleandthesixextensionmodulesareshown.Thefigureisexplainedindetailinthetext.

Polyketide chain extension occurs when an extension module AT selects a new acyl CoA, transfers it to the ACP, and the KS then catalyzes a Claisen condensation reaction between the newly bonded acyl group and the acyl group of the previous module. FIGURE25.18 shows the steps occurring in the first extension cycle; other extension cycles take place similarly.

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Loadingmodule

Extensionmodule 1

O

S

AT

HS

AT

HS

KS

HS

ACP KR

HS

ACP

Loadingmodule

Extensionmodule 1

HS

O

S

AT

HS

AT

HS

KSACP KR

HS

ACP

HS

O

AT

HS

AT

S

KS

HS

ACP KR

HS

ACP

HS

O

AT

HS

AT

S

KS

HS

ACP KR ACP

HS

AT

HS

AT

HS

KS

HS

ACP KR

S

ACP

HS

AT

HS

AT

HS

KS

HS

ACP KR ACP

O

R

O

S

H3C

HS

AT

HS

AT

HS

KS

HS

ACP KR

S

ACP

O

S

O

H3C

S

O

S

O–O

S

O

OH

1 2

3 4

65

FIGURE25.18 Theinitialloadingandfirstchain-extensioncyclecatalyzedbytheerythromycinPKS.Individualstepsareexplainedinthetext.

STEP 1 OF FIGURE 25.18: LOADING The loading AT domain begins the erythromycin biosynthesis by binding a propionyl CoA through a thioester bond to the –SH of a cysteine residue. The AT then transfers the propionyl group to the adjacent ACP. Each ACP in the synthase contains a phosphopante­theine bonded to the hydroxyl of a serine residue, and bonding of the acyl group to the enzyme occurs by thioester formation with the phosphopante­theine –SH (FIGURE25.19). The phosphopantetheine effectively acts as a long, flexible arm to allow movement of the acyl group from one catalytic domain to another.

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Abbreviatedmechanism

AT

ACP

O

S

AT

SH

B

Phosphopantetheine

Acyl ACP

O

CH2CH2NHCCH2CH2NHCCHCCH2OP

O

OH S

CH3 O–

OCH3

HO

ACP

O

CH2CH2NHCCH2CH2NHCCHCCH2OP

O

O=

S

CH3 O–

OCH3

HO

ACP

O

S

O

STEPS 2 – 4 OF FIGURE 25.18: CHAIN EXTENSION Polyketide chain extension begins (step 2) when the acyl ACP of the loading module transfers the propionyl group to the ketosynthase of module 1 (KS1), again forming a thioester bond to a cysteine residue. At the same time (step 3), the AT and ACP of module 1 load a (2S)­methylmalonyl CoA onto the thiol terminus of the ACP1 phosphopantetheine. The key carbon–carbon bond formation occurs in step 4 when KS1 catalyzes a Claisen condensation and decarboxylation to form an enzyme­bound b­keto thioester. It’s likely that the decarboxylation occurs simultaneously with the Claisen condensation, giving the enolate ion necessary for nucleophilic addition to the second thioester.

O

HS

AT

S

KS KR ACP

S

O

S

O–O

HS

AT

HS

KS KR ACP

S

O

R

O

CO2

Abbreviatedmechanism

STEPS 5 – 6 OF FIGURE 25.18: EPIMERIZATION AND REDUCTION Inter­estingly, the Claisen condensation occurs with inversion of configuration at the methyl­bearing chirality center so that the initially formed diketide has (R) stereochemistry. Base catalyzed epimerization of the (R) product, an acidic b­diketone, occurs in step 5, however, so the product that goes on to the next step regains the (S) configuration. Finally, KR1 reduces the ketone to a b­hydroxy thioester in step 6 by transfer of the pro-S hydrogen from NADPH

FIGURE25.19 FormationofanacylACPduringpolyketidebiosynthesis.Phosphopante-theine,symbolizedbyazigzaglinebetweenSandACP,actsasalong,flexiblearmtoallowtheacylgrouptomovefromonecatalyticdomaintoanother.

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as cofactor. Module 1 is now finished, so the diketide is transferred to KS2 for another chain extension.

HS

AT

HS

KS KR

S

ACP

HS

AT

HS

KS KR ACP

O

R

O

SH3C

HS

AT

HS

KS KR

S

ACP

O

S

O

H3C

S

O

OH

The reactions catalyzed by extension modules 2, 5, and 6 are similar to those of module 1, although the stereochemistries of the Claisen condensation and reduction steps may differ. The reactions in modules 3 and 4, however, are different. Module 3 lacks a KR domain, so no reduction occurs and the tetraketide product contains a ketone carbonyl group (Figure 25.17). Module 4 contains a KR and two additional enzyme domains, so it catalyzes a ketone reduction plus two additional reactions. Following the reduction by KR4 of the pentaketide, a dehydratase (DH) dehydrates the pentaketide alcohol to an a,b­unsaturated thioester and the double bond is then reduced by an enoyl reductase (ER) domain (FIGURE25.20).

Note that the complete sequence of reactions carried out by module 4—Claisen condensation, ketone reduction, dehydration, and double­bond reduction—is identical to the series of reactions found in fatty­acid biosynthe­sis (Figure 23.6; page 822). In fact, all fatty­acid synthases have the same set of AT, ACP, KS, KR, DH, and ER domains as the polyketide synthases.

OHOH

A pentaketide

O

O

OH

OH

S

ACP

KR

OH

O

O

O

OH

S

ACP

O

O

OH

S

ACP

OH

O

O

OH

S

ACP

DH ER

Release of 6­deoxyerythronolide B from the PKS is catalyzed by the ending thioesterase module. A serine residue on the TE module first carries out a nucleophilic acyl substitution on the ACP­bound heptaketide, and the acyl enzyme that results undergoes lactonization. A histidine residue in the TE acts as base to catalyze nucleophilic acyl substitution of the serine ester by the terminal –OH group in the heptaketide (FIGURE25.21).

Following its release from the PKS, 6­deoxyerythronolide B is hydroxyl­ated at C6 with retention of configuration to give erythronolide B. The reaction

FIGURE25.20 Additionalprocessingofthepentaketideintermediateinmodule4.Acarbonylgroupisremovedbyareduction–dehydration–reductionsequence.

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is catalyzed by a P450 hydroxylase analogous to that involved in morphine biosynthesis (Section 25­3, Figure 25.14). l­Mycarose is then attached to the C3 hydroxyl group by reaction with thymidyl diphosphomycarose through an SN1­like process that proceeds by initial formation of the mycarosyl carbocation (FIGURE25.22).

Thymidine

6-Deoxyerythronolide B

O

CH3

CH3

CH3

H3C

H3C

OHH3C

OH

H3C

OH

O

O

O

CH3

CH3

Erythronolide B

CH3

H3C

H3C

OH

OH

H3C

OH

H3C

OH

O

O

O

CH3

CH3

3-O-Mycarosyl-erythronolide B

CH3

H3C

H3C

OH

OH

H3C

OH

H3C

O

O

O

OH

OH

O

CH3

CH3

O26

3

O P O P

O–

O

O–

O

O

TDP

OH

OH

O

CH3

CH3

FIGURE25.22 Hydroxylationandglycosylationof6-deoxyerythronolideBtogive3-O-mycarosylerythronolideB.

O

O

OH

OH

OH

OH

Heptaketide

6-Deoxyerythronolide B

O

CH3

CH3

CH3

H3C

H3C

OHH3C

OH

H3C

OH

O

O

S

O

O

O H

OH

OH

OH

O

OH

SerACP H A

TE

Ser TE

His

FIGURE25.21 Releaseof6-deoxyerythronolidefromthePKS.Thereactionoccursbylactonizationofanacylenzyme,formedbyreactionofaserineresidueintheTEmodulewiththeheptaketide.

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The final steps in erythromycin A biosynthesis are a further glycosylation, a further hydroxylation, and a methylation (FIGURE25.23). As in the attachment of mycarose, the attachment of the amino sugar d­desosamine also takes place by transfer from a thymidyl diphosphosugar. C12 hydroxylation by another P450 enzyme occurs with retention of configuration to give erythromycin C, and methylation of the C3′ hydroxyl group of the mycarose unit by reaction with S­adenosylmethionine gives erythromycin A.

O

CH3

CH3

3-O-Mycarosyl-erythronolide B

CH3

H3C

H3C

OH

OH

H3C

OH

H3C

O

O

O

OH

OH

O

CH3

CH3

OTDP

TDP

O

CH3

CH3

Erythromycin D

CH3

H3C

H3COH

H3C

OH

H3C

O

O

O

OH

OH

O

CH3

CH3

HOO

N(CH3)2

O CH3

O

CH3

CH3

Erythromycin C

CH3

H3C

H3COH

H3C

OH

OH

H3C

O

O

O

OH

OH

O

CH3

CH3

HOO

N(CH3)2

O CH3

O

CH3

CH3

Erythromycin A

CH3

H3C

H3COH

H3C

OH

OH

H3C

O

O

O

OH

OCH3

O

CH3

CH3

HOO

N(CH3)2

O CH3

HO

N(CH3)2

O CH3

SAHSAM

O2

FIGURE25.23 FinalstepsinthebiosynthesisoferythromycinA.

P R O B L E M 2 5 . 5

Show a likely mechanism for the epimer­ization that occurs in step 5 of Fig­ure 25.18.

S

O

R

O

H3C

S

O

S

O

H3C

ACP ACP

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SOMETHINGEXTRA

bacteriumculturedfromaPhilippinesoilsample,andbenzylpenicillin from Penicillium notatum. Still otherexamplesincluderapamycin(Figure25.15),animmuno-suppressantisolatedfromaStreptomyces hygroscopicusbacteriumfirstfoundinasoilsamplefromEasterIsland(RapaNui),andpaclitaxel(Taxol),ananticancerdrugisolatedfromthebarkofthePacificyewtreefoundintheAmericanNorthwest.

With less than 1% of living organisms yet investi-gated,bioprospectorshavealotofworktodo.Butthereisaracegoingon.Rainforeststhroughouttheworldarebeingdestroyedatanalarmingrate,causingmanyspe-ciesofbothplantsandanimalstobecomeextinctbeforetheycanevenbeexamined.Fortunately,thegovernmentsinmanycountriesseemawareoftheproblem,butthereisasyetnointerna-tional treaty on biodiversity that couldhelppreservevanishingspecies.

Rapamycin,animmunosuppressantnaturalproductusedduringorgantransplants,wasoriginallyisolatedfromasoilsamplefoundonEasterIsland,orRapaNui,anisland2200milesoffthecoastofChileknownforitsgiantMoaistatues.

Corb

is

Bioprospecting:HuntingforNaturalProductsMostchemistsandbiologistsspendthemajorityoftheir time in the laboratory. A few, however, spendtheir days scuba diving on South Pacific islands ortrekkingthroughtherainforestsofSouthAmericaandSoutheast Asia. They aren’t on vacation, though;they’reatworkasbioprospectors,andtheirjobistohuntfornewandunusualnaturalproductsthatmightbeusefulasdrugs.

As noted in the Chapter 6 Something Extra, morethanhalfofallnewdrugcandidatescomeeitherdirectlyor indirectly from natural products. All four naturalproducts shown in the introduction to this chapter,for instance, are used as drugs: morphine from theopium poppy, prostaglandin E1 from sheep prostateglands,erythromycinA fromaStreptomyces erythreus

O

O

O

O

Paclitaxel (Taxol)

OO

O

OO

N

H

H

OH CH3

OH

OH

O

H3C

H

O

P R O B L E M 2 5 . 6

Propose a mechanism for the reaction of erythronolide B with thymidyl diphosphomycarose to give 3­O­mycarosylerythronolide B (Figure 25.22).

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SUMMARy

In this brief chapter, we’ve just tickled the surface of natural­products chem­istry, looking at the pathways by which several well­known natural products are synthesized in living organisms.

The term natural product is generally taken to mean a secondary metabolite—a small molecule that is not essential to the growth and develop­ment of the producing organism and is not classified by structure. Well over 300,000 secondary metabolites probably exist, generally classified into five categories: terpenoids and steroids, fatty acid–derived substances and polyketides, alkaloids, nonribosomal polypeptides, and enzyme cofactors.

Unraveling the biosynthetic pathways by which natural products are made is difficult and time­consuming work, but the payoff is a fundamental understanding of how organisms function at the molecular level. The mol­ecules are sometimes complex, but the individual chemical steps by which they are made are familiar.

EXERCISES(Problems 25.1–25.6 appear within the chapter.)

25.7 Which hydrogen, pro-R or pro-S is removed from pyridoxine 5′­phos­phate in the final step of PLP biosynthesis?

Pyridoxine5′-phosphate

2–O3POCH2

CH3

OH

OHH+N

HH

Pyridoxal5′-phosphate (PLP)

2–O3POCH2

CH3

O

OHH

H

+N

FMNH2FMN

25.8 Does the ketone reduction step catalyzed by KR1 in erythromycin bio­synthesis occur on the Re or the Si face of the substrate carbonyl group? (See Figure 25.18.)

25.9 When the enoyl reductase domain (ER4) in the erythromycin PKS is deactivated by gene mutation, all further steps still occur normally. What is the structure of the lactone that results?

25.10 One of the steps in the biosynthesis of the alkaloid berbamunine is an epimerization of (S)­N­methylcoclaurine. Review the morphine bio synthesis in Figure 25.6, and propose a mechanism for the epimerization.

CH3O

HO

HOH

N

(S)-N-Methylcoclaurine

CH3

CH3O

HO

HON

(R)-N-Methylcoclaurine

CH3H

K E Y W O R D S

fatty­acid derived substance, 879

natural product, 877

nonribosomal polypeptide, 879

polyketide, 879

secondary metabolite, 877

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25.11 The final step in the biosynthesis of berbamunine is a coupling reaction of (S)­N­methylcoclaurine with (R)­N­methylcoclaurine (Problem 25.10). Propose a mechanism.

CH3O

OH

O

HOH

NCH3

Berbamunine

CH3O

HON

CH3H

25.12 5­Aminolevulinate is the precursor from which the large class of alka­loids called tetrapyrroles are biosynthesized. It arises by a PLP­dependent reaction of glycine and succinyl CoA. Review the mechanism of the for­mation of dopamine from l­dopa in Figure 25.7, and propose a mecha­nism for 5­aminolevulinate biosynthesis.

5-AminolevulinateSuccinyl CoAGlycine

H3N CO2–

CO2–CoAS

++H2N

+O

CO2–

O(PLP)

25.13 One of the steps in the biosynthesis of penicillins is a PLP­dependent epimerization of isopenicillin N to penicillin N.

Penicillin N

H H

CH3

CH3

CO2–

–O2C

OO

N

H

H

S

NHH3N+

(PLP)

Isopenicillin N

H H

CH3

CH3

CO2–

–O2C

OO

N

H

H

S

NH NH3+

The reaction occurs by initial formation of an imine, followed by a base­catalyzed isomerization. Propose a mechanism.

25.14 Propose a mechanism for the following biosynthetic conversion. What cofactors are likely to be involved?

HN+

O

ONH

CO2–

CO2CH3CO2

–

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25.15 The enzyme acetolactate syn­thase catalyzes the thiamin diphosphate­dependent con­version of two molecules of pyruvate to acetolactate. Pro­pose a mechanism.

H3C

CH3HO

CO2–

O

H3C2

CO2–

O CO2

25.16 1­Deoxy­d­xylulose 5­phosphate (DXP), in addition to being a precursor to PLP, is also a precursor to isopentenyl diphosphate in terpenoid bio­synthesis. The initial step in the pathway is a base­catalyzed rearrange­ment, followed by reduction with NADPH to give 2C­methyl­d­erythritol 4­phosphate. Show the structure of the rearranged intermediate, and propose a mechanism for its formation.

O

H3C

H

HO

HO

H

OPO32–

H3C OH

HO

HO

HH

1-Deoxy-D-xylulose5-phosphate

2C-Methyl-D-erythritol4-phosphate

H

OPO32–[ ? ]

NADPH/H+ NADP+

25.17 Biosynthesis of the b­lactam antibiotic clavulanic acid begins with a TPP­dependent reaction between d­glyceraldehyde 3­phosphate and arginine.

H CO2–

Arginine

Clavulanic acid

HO

OHC

H

D-Glyceraldehyde3-phosphate

OPO32– H2N+

N

H

NH2+

NH2

H CO2–

–O2C N

H

(TPP)

N

H

H

CO2–

CH2OH

H

NO

NH2+

NH2

(a) The first step is the reaction of d­glyceraldehyde 3­phosphate with TPP ylide, followed by dehydration to give an enol. Show the mechanism, and draw the structure of the product.

(b) The second step is loss of hydrogen phosphate from the enol to give an unsaturated carbonyl compound. Show the mechanism, and draw the structure of the product.

(c) The third step is a conjugate addition of arginine to the unsaturated carbonyl compound. Show the mechanism, and draw the structure of the product.

(d) The final step is a base­catalyzed hydrolysis to give the final prod­uct and regenerate TPP ylide. Show the mechanism.

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