Topic 10 – Part 2: The Polyketide Pathway – Phenols

Contents
Learning Objectives: .................................................................................................................. 2
Introduction ................................................................................................................................ 3
Acetate/Malonyl Synthesis ........................................................................................................ 4
Reaction Between Malonyl CoA and Acetyl CoA................................................................. 5
Polyketide .................................................................................................................................. 5
Naturally Occurring Compound Conforming to the Acetate Hypothesis. ................................. 7
Examples of Some Secondary Modifications ........................................................................ 8
Example 1. 6-Methylsalicyclic Acid .................................................................................. 8
Example 2: The Lactone Mellein ....................................................................................... 9
Examples 3: Ustic Acid .................................................................................................... 10
Examples 4: ...................................................................................................................... 10
Labeling Studies in Biosynthesis ............................................................................................. 10
Single and Double-labelled Precursors ................................................................................ 11
Single-labelling: ................................................................................................................... 11
Double-labelling ................................................................................................................... 11
Example: ........................................................................................................................... 11
Example 2. ........................................................................................................................ 12
Carbon Carbon Bond Formation: ..................................................................................... 13
Carbon Oxygen Bond Formation: .................................................................................... 14
Self-Test Problems ........................................................................................................... 15
Self-Test Problems ........................................................................................................... 15

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Learning Objectives:
At the end of this topic you should be able to:

 Be able to write pathway from acetyl CoA to linear polyketide chains. Recognize
Aldol and Claisen types of cyclizations in the formation of aromatic compounds.
 Be able to recognise the structural affinities of polyketide secondary metabolites and
appreciate the biological and chemical origins of these compounds
 Offer rational mechanistic explanations for some of the key transformations.
 Phenol oxidative coupling in griseofulvin biosynthesis.
 Be able to understand how isotope labelling can be used as a tool to determine
biosynthetic pathways and identification of starter unit and folding patterns
(especially [13C] acetate.

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Introduction
Polyketides constitute a large class of natural products grouped together on purely
biosynthetic grounds. Their diverse structures can be explained as being derived from poly-
β-keto chains, formed by coupling of acetic acid (C2) units via condensation reactions.
Many natural products (usually aromatic compounds, fatty acids, polyacetylenes and
macrolide antibiotics) can be constructed biosynthetically through the linear combination of
acetic (C2) units i.e. acetic acid or its biosynthetic equivalent, acetyl CoA.
The form in which acetate is used in most of its important biochemical reactions is acetyl
coenzyme A (acetyl-CoA). Acetyl-CoA is formed by oxidative decarboxylation of pyruvate as
shown below
Pyruvate + NAD+ CoA---------> Acetyl CoA + CO2 + NADH
The formation of the poly-β-keto chain could be envisaged as a series of Claisen reactions.
Two molecules of acetyl- CoA, through Claisen condensation will give acetoacetyl-CoA, and a
repeat of this reaction will generate a poly-β-keto ester of appropriate chain length. See
figure 1)

Figure 1: Formation of the poly-β-keto chain

For example: Orsellinic acid – 4 acetate units give rise to a polyketide of eight (8) carbon
atoms which cyclises to give Orsellinic acid.

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Acetate/Malonyl Synthesis
A study of the enzymes involved in polyketide biosynthesis showed that this simple
rationalization could not be correct, and a more complex series of reactions was operating.
It is now known that polyketide biosynthesis involves initial carboxylation of acetyl-CoA to
malonyl-CoA, a reaction involving ATP, CO2 (as bicarbonate, HCO− 3 ), and the coenzyme
biotin as the carrier of CO2. Malonyl CoA is formed in the presence of ATP and the enzyme
biotin as shown below.

Figure 2: Malonyl CoA is formed in the presence of ATP and the enzyme biotin

In the next step, acety CoA and malonyl CoA undergo a Claisen condensation reaction to
give acetoacetyl-CoA. A series of repeating Claisen condensation reactions give rise to a
polyketide chain as shown below.

Figure 3: Formation of the poly-β-keto chain.

Aromatic compounds and fatty acids are formed from this pathway. The number of
aromatic rings formed depends on the size of the polyketide chain. A general biosynthetic
outline for the formation of a polyketide chain is shown below. Following a Claisen
condensation reaction between acety CoA and malonyl CoA, acetoacetyl-CoA is formed and
undego another Claisen condensation reaction with malonyl CoA, elongating the chain by
another C2 unit until the required number of polyketide chain is formed. Phenolic
compounds and Fatty Acids are compounds that are formed from polyketides.

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Reaction Between Malonyl CoA and Acetyl CoA

Figure 4: Formation of polyketide chain

Polyketide
The presence of doubly-activated methylene (-CH2-) groups and carbonyl groups make
possible 2 types of reactions leading to cyclization (reactions): Aldol reaction and Claisen
reaction.
1. Intramolecular Aldol reaction

e.g. Orsellinic acid

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 2. Claisen Condensation - intramolecular carbon acylation
e.g phloracetophenone

There are 2 types of Phenolics (aromatics) formed by the two cyclisation reactions. Note
that both have a 1,3 oxygenation pattern which arises from the carbonyl groups within the
polyketide chain.

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Hint:
1. Look for the polyketide chain in the final aromatic compound by locating the tail-end
and ester head of the “polyketide”.
2. Distinct feature of aromatics derived from acetate pathway: several of the carbonyl
oxygen survive (alternate carbons) – meta oxygenation pattern!

Naturally Occurring Compound Conforming to the Acetate Hypothesis.

A great variety of metabolites are formed depending upon several factors:
1. the number of acetyl CoA units involved
2. the mode of cyclization
3. the condensation of separately synthesized polyketides
4. the secondary processes such as halogenation, alkylation, reduction, oxidation,
rearrangement, etc. (this is also true for other types of metabolites that will be
discussed in the following sections).
A variety of phenolic compounds can form (Figure 5).

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 Figure 5: Phenolic compounds

Examples of Some Secondary Modifications

Example 1. 6-Methylsalicyclic Acid
 Produced by Penicillium patulum.
 Note reduced number of OH groups – missing O’s functions are removed during biosynthesis
of this metabolite i.e. Orsellinic acid cannot be deoxygenated to 6- methylsalicyclic acid.
 Formed from four acetate units as above.

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Example 2: The Lactone Mellein
Isolated from the fungus Aspergillus
The growing polyketide chains are held bound to the biosynthetic enzyme, passed from one
active site to the next. Following this, different active sites carry out the various cyclizations
and reductions.

The lactone ring is formed by reaction of end-of-chain thioester with the OH group of starter
unit.
o Reduction of OH of starter occurs after two Claisen reactions.
o Methylation is the final step in the process.

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Examples 3: Ustic Acid
The product is formed following oxidation, cyclisation (aldol reaction), dehydration,
enolisation and methylation to give the product.

Examples 4: Eugenetin. Cyclisation (claisen condensation), methylation, reduction,

enolisation, cyclisation and dehydration gives rise to the product.

Labeling Studies in Biosynthesis

Using isotopically enriched precursors, it is possible to trace the incorporation of building
blocks into complex metabolites. By doing so, one can follow the label and see which atoms
in the natural product end up labeled.

Originally done with radioactive labels (14C, 3H) where cells are fed a precursor with one 14C.
Tracing of label was done using chemical degradation where the product is broken apart to
see which pieces contain the radioactivity. The drawback is that this is sloppy and it was
usually not possible to completely determine biosynthetic route.

Now most labeling studies are done using NMR where non-radioactive isotope 13C is used.
This is because:
- common carbon isotope 12C is invisible to NMR
- rare 13C has nuclear spin, so is detectable by NMR

Cells are therefore fed a precursor with a position 13C labeled; then the NMR of the natural
products formed is obtained to see which positions show exaggerated peaks in the carbon
NMR spectrum.

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Since only 1% of carbons are naturally 13C, the positions that incorporate the label will give
much higher peaks. Precursors can now be synthesized with > 99% enriched with 13C.

Single and Double-labelled Precursors

[1-13C] acetate means C1 carbon is >99% 13C (Single-labelling)
[1,2-13C2] acetate means that each carbon atom is >99% 13C (double-labelling).

Single-labelling:
Singly-labelled precursor (say acetate) incorporated into a polyketide (Figure 6)

Figure 6: Label is easily detectable through enhancement over natural abundance signal.

Double-labelling
Assume [1,2-13C2] acetate is incorporated intact in a metabolite (Figure 7)
The labeled C2 unit can be detected because of spin-spin coupling between the two
adjacent labeled atoms

Figure 7: acetate is incorporated intact in a metabolite

Example: It has been established through labeling experiments that the following
compound has a polyketide origin. Suggest a possible biosynthetic pathway of the
metabolites and indicate the carbon atoms that would be labelled if the organism producing
this metabolite was fed with:

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Solution: First identify the C2 units and construct polyketide chain

Cyclisation, reduction, dehydration and enolisation gives rise to the product as shown
below. Using the labelled 13C the carbonyl carbons were all traced and the location
identified.

Example 2. Using labelling experiments to confirm acetate hypothesis. If a labelled acetate

unit is incorporated into the biosynthesis, it is possible to locate the position of the labelled
carbon within the final structure of a compound: Orsellinic acid and 6- methylsalicyclic acids
as examples (Figure 8).

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 Figure 8: final structure of a compound: Orsellinic acid and 6- methylsalicyclic

An important reaction often encountered when dealing with aromatic compounds is

Phenolic Coupling (radical coupling) formed through coupling of phenolic, ortho and para
radicals.

Figure 9: Phenolic Coupling (radical coupling)

Carbon Carbon Bond Formation: e. g any otho or para combination would give the
following structure. Can also have para – ortho or Para para combination

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Carbon Oxygen Bond Formation: Any otho or para combination, can also have Ortho or
para position

Example: Griseofulvin Biosynthesis

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 Self-Test Problems
Self-Test Problems

Show the mechanism and biosynthesis of the compounds shown below using the
appropriate polyketide chain.

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