Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
42912_25_eCh25_0877-0904b.indd 877 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
878 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 878 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
• 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
Unless otherwise noted, all content on this page is © Cengage Learning.
25-2 bioSyntheSiSofpyridoxalphoSphate 879
42912_25_eCh25_0877-0904b.indd 879 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
880 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 880 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
25-2 bioSyntheSiSofpyridoxalphoSphate 881
42912_25_eCh25_0877-0904b.indd 881 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
(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
McM
urry
Unless otherwise noted, all content on this page is © Cengage Learning.
882 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 882 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
© J
ohn
McM
urry
Unless otherwise noted, all content on this page is © Cengage Learning.
25-2 bioSyntheSiSofpyridoxalphoSphate 883
42912_25_eCh25_0877-0904b.indd 883 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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
Unless otherwise noted, all content on this page is © Cengage Learning.
884 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 884 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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
Unless otherwise noted, all content on this page is © Cengage Learning.
25-3 bioSyntheSiSofMorphine 885
42912_25_eCh25_0877-0904b.indd 885 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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).
Unless otherwise noted, all content on this page is © Cengage Learning.
886 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 886 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
25-3 bioSyntheSiSofMorphine 887
42912_25_eCh25_0877-0904b.indd 887 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
888 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 888 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
25-3 bioSyntheSiSofMorphine 889
42912_25_eCh25_0877-0904b.indd 889 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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).
Unless otherwise noted, all content on this page is © Cengage Learning.
890 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 890 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
25-3 bioSyntheSiSofMorphine 891
42912_25_eCh25_0877-0904b.indd 891 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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).
Unless otherwise noted, all content on this page is © Cengage Learning.
892 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 892 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
25-3 bioSyntheSiSofMorphine 893
42912_25_eCh25_0877-0904b.indd 893 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
894 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 894 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
25-4 bioSyntheSiSoferythroMycin 895
42912_25_eCh25_0877-0904b.indd 895 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
896 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 896 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
25-4 bioSyntheSiSoferythroMycin 897
42912_25_eCh25_0877-0904b.indd 897 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
898 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 898 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
25-4 bioSyntheSiSoferythroMycin 899
42912_25_eCh25_0877-0904b.indd 899 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
900 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 900 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
25-4 bioSyntheSiSoferythroMycin 901
42912_25_eCh25_0877-0904b.indd 901 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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
Unless otherwise noted, all content on this page is © Cengage Learning.
902 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 902 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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).
Unless otherwise noted, all content on this page is © Cengage Learning.
SoMethinGextra 903
42912_25_eCh25_0877-0904b.indd 903 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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
Unless otherwise noted, all content on this page is © Cengage Learning.
904 chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 904 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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
–
Unless otherwise noted, all content on this page is © Cengage Learning.
exerciSeS 904a
42912_25_eCh25_0877-0904b.indd 1 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.
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.
Unless otherwise noted, all content on this page is © Cengage Learning.
904b chaptere25 SecondaryMetaboliteS:anintroductiontonaturalproductScheMiStry
42912_25_eCh25_0877-0904b.indd 2 1/15/14 4:43 PM
Not For Sale
© 2
014
Cen
gage
Lea
rnin
g. A
ll R
ight
s Res
erve
d. T
his c
onte
nt is
not
yet
fina
l and
Cen
gage
Lea
rnin
g do
es n
ot g
uara
ntee
this
pag
e w
ill c
onta
in c
urre
nt m
ater
ial o
r mat
ch th
e pu
blis
hed
prod
uct.