Biosynthesis lectures by Dr. Refaat Hamed

Dr. Refaat Hamed
Tuesday, 30 December 2014
Biosynthesis of Natural Products
(Secondary Metabolites)

Biosynthesis = Biogenesis = Anabolism
The main goals of these lectures are :
- To provide a mechanistic understanding of how the different
classes of complex natural products are constructed in Nature.
- Recognize the major building blocks used by nature to assemble
secondary metabolites.
- To illustrate the methods and techniques that are used to
study biosynthetic pathways.
- Rationalize the structures of novel secondary metabolites in terms
of a plausible biogenesis and be able to apply the knowledge gained
together with other modern tools (e.g. Bioinformatics) to
discuss the heterologous production of new “unnatural” natural
products.

Reading material and slides will be available at:
- “Applied Pharmacognosy_4th year” Facebook group
(closed group)
- https://www.facebook.com/groups/1562751973962122/
- To be a member, send a request with a scan of your
University ID.
- Medicinal Natural Products - A Biosynthetic
Approach, 3rd edition, P. M. Dewick, Wiley, 2010.

Definition: A multi-step enzyme-catalyzed process where
simple substrates are converted into more complex products.
• Some biosynthetic pathways take place within a single
cellular organelle, while others involve enzymes that are
located within multiple cellular organelles.

The central Dogma of Molecular Biology
A hierarchical flow of information from DNA to other biooligomers.

The prerequisites for biosynthesis include:
1. Precursor compounds: Substrates = starting
material = reactants = building blocks
2. Chemical energy: High energy molecules (e.g. ATP)
required in the case of energetically unfavorable
reactions. The hydrolysis of high energy molecules drives
a reaction forward.
Adenosine triphosphate

The prerequisites for biosynthesis include:
1. Precursor compounds: Substrates = starting
material = reactants = building blocks
2. Chemical energy: High energy molecules (e.g. ATP)
required in the case of energetically unfavorable
reactions. The hydrolysis of high energy molecules drives
a reaction forward.
3. Catalytic enzymes: Special macromolecules (usually
proteins, and may require coenzymes, e.g. metal ions and
NADH) that can perform catalysis by increasing the rate
of a reaction via lowering its activation energy.

Primary # Secondary Metabolism
• Primary metabolic processes include the pathways for
generally modifying and synthesizing carbohydrates,
proteins, fats, and nucleic acids, which are essentially the
same in all organisms, with minor variations.
• Ex. 1: Degradation of sugars via glycolysis or Krebs/citric
acid/tricarboxylic acid cycle (TCA), to release energy from
organic compounds by oxidative reactions.
• Ex. 2: Oxidation of fatty acids via -oxidation.
• On the other hand, secondary metabolic pathways are
concerned with the metabolism of compounds that have a
much more limited distribution in nature (secondary
metabolites).

Primary # Secondary Metabolism
• Secondary metabolites are not necessarily produced under all
conditions, and in many cases the function of these
compounds and their benefit to the producing organism are
not yet known.
• Some secondary metabolites are undoubtedly produced for
obvious reasons, e.g. (1) toxins that provide defense
against predators, and (2) volatile attractants towards the
same or other species.
• Therefore, it is logical to assume that secondary metabolites
do play some vital role for the well-being of the producer.
• Secondary metabolism provides most of the
pharmacologically active natural products.

Summary of 2ry metabolic pathways in Nature

Building Blocks:
• Relatively few building blocks are routinely employed.
• The most important building blocks employed by Nature in the
biosynthesis of secondary metabolites are derived from
acetyl-coenzyme A (acetyl-CoA), shikimic acid, mevalonic
acid, and 1-deoxyxylulose 5-phosphate. These are utilized,
respectively, in the acetate, shikimate, mevalonate, and
deoxyxylulose phosphate pathways (as will be discussed in
the incoming lectures)

C1 Pool:
• The simplest building block is composed of a single carbon
atom, usually in the form of a methyl group, and most
frequently it is attached to oxygen or nitrogen, but
occasionally to carbon or sulfur.
• It is derived from the S-methyl of L-methionine (in the form of
S-adenosylmethionine (SAM, AdoMet) and is introduced via
an SN2-type nucleophilic substitution reaction.
• The methylenedioxy group (–OCH2O–) is another example of
a C1 unit.

C1 Pool: O- and N-alkylation using SAM

C1 Pool: C-alkylation using SAM
Ortho and para positions are
activated for methylation by
the –OH group.
Carbonyl groups increase
acidity and allow formation of
enolate anion.

O-alkylation using DMAPP
• A C5 isoprene unit in the form of dimethylallyl diphosphate
(DMAPP) may also act as an alkylating agent.
• The alkylation reaction here involves an SN1 process rather
than an SN2 nucleophilic substitution (in the case of SAM)!

Alkylation via Electrophilic Addition
• During the biosynthesis of terpenoids and steroids, two or
more C5 units can be joined together and the reactions are
rationalized in terms of electrophilic addition of carbocations
onto alkenes.

Building Blocks: C2 and C5
More reactive form of acetyl-CoA

Building Blocks: C6C3 and C6C2N

Building Blocks: C4N and C5N
• Ornithine supplies its -amino nitrogen. The carboxylic acid function and
-amino nitrogen are both lost.
Pyrrolidine alkaloids
Piperidine alkaloids

Catalysis
• An acceleration of the rate of a reaction brought about
by a catalyst that is usually present in small managed
quantities and unaffected at the end of the reaction.
•A catalyst permits reactions to take place more
effectively or under milder conditions than would
otherwise be possible.
Chemical catalysis
• Used in organic synthesis to accelerate the rate of a
reaction by combining with reactants in stoichiometric or
sub-stoichiometric amounts.
Biological catalysis
• The use of enzymes or entire cells to carry out defined
chemical reactions under controlled conditions, in order
to convert raw materials into commercially more
valuable products.
Chemical& Biological catalysis

Enzymes are wonderful Catalysts

Enzyme kcat/kuncat
Sweet potato -amylase 7.2 x 1017
Orotidine 5’-phosphate decarboxylase 1.4 x 1017
Fumarase 3.5 x 1015
Mandelate racemase 1.7 x 1015
Staphylococcal nuclease 5.6 x 1014
Carboxypeptidase B 1.3 x 1013
AMP nucleosidase 6.0 x 1012
Adenosine deaminase 2.1 x 1012
Ascites tumor dipeptidase 1.2 x 1012
Cytidine deaminase 1.2 x 1012
Ketosteroid isomerase 3.9 x 1011
Phosphotriesterase 2.8 x 1011
Triosephosphate isomerase 1.0 x 109
Carbonic anhydrase 7.7 x 109
Chorismate mutase 1.9 x 106
Cyclophilin (rotamase) 4.6 x 105
Reaction rate enhancement by enzymes

• The role of a catalyst is to decrease the energy of activation
of a reaction (i.e. the energy necessary to attain the
transition state).
E + S ES EP E + P
How do enzymes catalyze reactions?

Catalysis through proximity and orientation of reactants
General acid, general base catalysis
Catalysis by electrostatic effects
Covalent catalysis (nucleophilic or electrophilic)
metal ion catalysis
Catalysis by strain or distortion
catalysis by preferential transition state binding
• For most enzymes, more than one of these strategies are
used concomitantly.
Mechanisms of enzyme catalysis?

Beauty of enzyme catalysis!
Enzymes achieve speedy catalysis under very mild
conditions of pH, temperature, .... in aqueous media.
Enzymes have a superb selectivity for a certain substrate
or functional group as well as specificity of their product
(Chemo-, regio-, and stereoselective catalysis).
Enzymes are subject to metabolic control.
Enzymes are amenable to modification via protein
engineering approaches.

Beauty of enzyme catalysis!
Stereoselectivity: Resolution of enantiomers

Beauty of enzyme catalysis! Regio-selectivity

C1 Pool: O- and N-alkylation using SAM
• Genetic engineering is being used to obtain transgenic, low caffeine coffee
and tea that could be used to produce “natural” decaffeinated beverages.
• Tobacco plants genetically engineered to produce low levels of caffeine
do repel insects and exhibit resistance to viral and bacterial infection, which
offers safer and cheaper protection than treatment of crops with pesticides
and fungicides
• E. coli addicted to caffeine!!!!!

Some important biochemical transformations:
• C–C bond forming reactions: Claisen # Aldol
condensation.
• Carboxylation employing biotin.
• “Umpolung” Based Transformations.
• Transamination, Racemisation, and Decarboxylation
employing Pyridoxal and Pyridoxamine Chemistry.
• Glycosylation reaction.

Building Blocks: C2
Malonyl-CoA is a more reactive
form of acetyl-CoA as a C2 source

Carboxylation of acetyl-CoA to malonyl-CoA employing biotin
• Key step in fatty acid biosynthesis.
• Biocatalyst: acetyl-CoA carboxylase.
• Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme
that catalyses the irreversible carboxylation of acetyl-CoA to
malonyl-CoA.
• ACC is a multi-subunit enzyme in most prokaryotes and in
the chloroplasts of most plants and algae, whereas it is a
large, multi-domain enzyme in the endoplasmic reticulum of
most eukaryotes.

Carboxylation of acetyl-CoA to malonyl-CoA employing biotin

Claisen condensation of malonyl-CoA and acetyl-CoA
• Decarboxylation of -keto acids is
usually assisted by the presence of
“oxyanion hole” (OAH) in the enzyme
active site as well as the
accommodation of the carboxylate
moiety in a hydrophobic pocket (e.g.
crotonase superfamily enzymes).

Decarboxylation of -keto acids: Umpolung via TPP
• Umpolung or polarity inversion is the chemical modification of
a functional group with the aim of reversal of polarity of that
group. This modification allows secondary reactions of this
functional group that would otherwise not be possible.
• Nature Umpolung reagent: Thiamine diphosphate (TPP).
TPP ylid

Decarboxylation of -keto acids: Umpolung via TPP
• Biocatalyst: Pyruvate decarboxylase
• Substrate: Pyruvate, Product: Acetaldehyde

Acetyl-CoA formation from pyruvate: Umpolung via TPP
• Biocatalyst: Pyruvate dehydrogenase
• Substrate(s): Pyruvate, CoASH Product: Acetyl-CoA
FAD: flavin adenine dinucleotide

Decarboxylation of -amino acids:
• Several of the basic building blocks (e.g.
C6C2N) are derived from an amino acid via
loss of the carboxyl group.
• Cofactor needed: Pyridoxal phosphate (PLP)
• The protonated nitrogen of PLP acts as an
electron sink that facilitates decarboxylation.

Transamination reaction: Reductive amination of -keto acids
• Cofactor needed: Pyridoxal phosphate (PLP)/ Pyridoxamine phosphate
• The reaction involves imine formation and the protonated nitrogen of
PLP acts as an electron sink. Stereoselective reduction then follows to
give a single enantiomer of amino acid.
• . The reverse reaction is also important in amino acid catabolism.
• The same mechanism can be employed for racemization of amino
acids!

Reduction of -keto acids: Dehydrogenases
• Coenzyme needed: nicotinamide adenine dinucleotide (NAD+) or
nicotinamide adenine dinucleotide phosphate (NADP+)
• Two hydrogens are removed from the substrate, one hydrogen is
transferred as hydride to the coenzyme, and the other as a proton is
passed to the medium. The reverse reaction is also possible.

NADPH
passed to the medium. The reverse reaction is also possible.

passed to the medium.
• The reverse reaction is also possible.

Reductive carboxylation

Glycosylation reaction: O-glycosylation
• (O-, S-, N- or C-) Glycoside = Aglycone + Sugar
• The sugar needs to be activated to react and give the glycoside.
• The activated sugar for glycosylation is usually uridine
diphosphoglucose (UDP-glucose), which is synthesized from
glucose 1-phosphate and uridine triphosphate (UTP).

Glycosylation reaction: O-glycosylation
• Since UDP-glucose has its leaving group in the -configuration,
the -configuration, as is most commonly found in
natural glucosides (SN2 reaction).

Glycosylation reaction: C-glycosylation
• In the case of C-glycosides, a suitable nucleophilic carbon is
required, e.g. aromatic systems activated by a phenolic group.

The role of vitamins as cofactors for certain enzymes in biosynthesis

Radioactive labeling studies as a tool in biosynthetic
investigations
• The idea here is to “feed” the plant/organism a
radioactively labeled precursor and then monitor what
happens to the radioactive atom(s) when they are taken in
“incorporated” by the plant/organism.

FEEDING METHODS FOR HIGHER PLANTS
NUTRIENTS
(1) HYDROPONIC
stem
bud
bulb
(4) DIRECT INJECTION
(2) PAINT LEAVES
(5) WICK METHOD
(3) SHALLOW TRAY
excised buds, leaves, roots in
tray of nutrients
compound
enters
root hairs
compound
enters leaf
stomata
compound
enters plant
through wicknutrients

*CO2
(6) SEALED ATMOSPHERE
Greenhouse or bag
(7) TISSUE OR CELL CULTURE
Shaker - Heater
NUTRIENT BROTH
supplies
products
(8) CELL-FREE CULTURE
like brewing or
fermentation of beer
heater
cell contents,
enzymes and
coenzymes and
nutrients
thin
slices
Cells are lysed
to release their contents.

1. Not all substances can cross the cell wall and cell membrane in
order for the label to be incorporated.
*C A
B C Z
Even though compound C is a
part of the biosynthetic sequence
that leads to natural product Z
it will not be incorporated if it
cannot cross the cell wall.compound C with a
radioactive atom,
perhaps 14C instead
of 12C.
cell nucleuscytosol
2. Most biosynthetic processes take place in the cytosol (fluid
content of the cell, or cytoplasm). However, sometimes
biosynthesis takes place by enzymes that are bound to a
membrane or are located in a certain organelle, which poses
additional problems for incorporation.
other “organelles”
(cellular structures that have membranes)
cell wall
Issue encountered during labeling studies in plants

A B C D E Z
M M N O
3. False or no incorporation: Sometimes a substance will be
incorporated, but is not actually in the targeted biosynthetic
pathway (ABCDEZ). It can be first converted in the cell to a
compound that is subsequently merged into the targeted
biosynthetic pathway.
A B C
COOH
COOH
COOH
COOH
COOH
COOH
Z
COOEt
COOEt
COOEt
COOEt
Succinic acid is in the pathway
to Z but cannot cross the cell
wall barrier - incorporation
studies fail!
Diethyl succinate is not in the
pathway to Z but can cross the
cell wall barrier. After
hydrolysis, it is incorporated.
One may incorrectly conclude
that diethyl succinate is a
precursor to Z!
EXAMPLE

4. GENERAL INCORPORATION - TOTAL DISPERSAL: Certain
substances, like glucose or acetyl-CoA, can be considered as
potential precursors to almost everything in the plant/living
organism. If labeled glucose was to be fed to the plant, almost all
the resultant metabolites would be labeled, but with low
percentage incorporation.
A
B C D E Z
W X Y
Q R S
L M N
A

Research approaches for elucidating biosynthetic pathways
• Biosynthetic methodology can be divided into labeling and enzymatic
experiments, or a mix of both.
• In labeling studies, a precursor (e.g. AA) is labelled with a stable or
radioactive isotope, and the fate of this precursor is determined when it is
incubated with the living system/isolated enzyme(s).
• In order to establish a sequence of events, the label from A must appear
sequentially in B, C and D. Furthermore, for the pathway A to D to be a true
biosynthesis and not a degradation and re-synthesis, the label must appear
in the metabolites of A specifically at the predicted place. If the site of the
label is randomized, this is an indication of degradation and re-synthesis.
• With radioisotopes (I4C and 3H) this has meant a careful chemical
degradation of the biosynthetic product to establish the specificity of
labelling (disadvantage).

• With II3C and 2H, the specificity may be established much more easily by
NMR methods (appearance of enhanced signals in the 13C-NMR
spectrum).rum).
13C-NMR spectrum of labeled thienamycin

• Mass spectrometry may also be used to establish the incorporation and
location of isotopes such as 118O.
Massspectrum

• The use of double-labeling techniques can establish the structural
integrity of a unit. For example, if a precursor is labeled at two or more
centres with 33H and 14C and the ratio of tritium to carbon-14 remains
constant between the precursor and the product, it is unlikely that the 3H
and 14C labels have parted company during the biosynthesis.
• The doubly-labelled (13C) acetate has been used extensively in determining
biosynthetic pathways. In this case, all the acetate-derived carbon atoms
are labeled and adjacent nuclei will exhibit coupling in the 13C-NMR
spectrum. Adjacent nuclei can therefore be identified through their 13C-13C
coupling constants.

• When consecutive acetate units are joined (in the form of acetyl-
CoA/malonyl-CoA), this will give a sequence of 4 carbon atoms (C1 to C4).
Since the incorporation of labelled isotopes in experiments is usually quite
low, statistically it is unlikely that these labelled units will be incorporated
sequentially and hence coupling is not observed between C2 and C3.
Thus, for the sequence C1-C2-C3-C4, we would expect to see two coupling
constants J1,2 and J3,4 from the two component acetyl-CoA/malonyl-CoA
precursors. Any acetate precursor that is cleaved during biosynthesis will
end up enhancing a natural abundance signal, and not show any coupling.

• Often, the fine details of the pathway can only be established via isolation
and detailed study of the individual enzymes in a certain biosynthetic
pathways.
• Enzyme isolation and fractionation used to be tedious, and the amount of
protein obtained used to be very small indeed, limiting subsequent studies.
Recent rapid progress in enzyme production is the result of significant
advances in genetic (molecular biology) techniques.
• A gene is a segment of DNA that contains the information necessary for
the synthesis of a particular protein/enzyme.
• Now, it is possible to search for likely genes in DNA sequences, produce
them synthetically, and to express them in a suitable bacterium or yeast; to
avoid complications with the normal biosynthetic machinery in the source
organism.
Modern research approaches for elucidating biosynthetic pathways

• Recombinant proteins can then be tested for the predicted enzyme
activity. The prediction is facilitated by comparison to published gene
sequences for similar enzymes, or by characteristic sequences that can
be assigned to a particular class of enzyme, usually by the need to bind a
specific cofactor, e.g. NADPH or SAM (Bioinformatics!).
• In some organisms, especially bacteria and fungi, a group of genes
involved in secondary metabolite biosynthesis may lie in close proximity as
“gene clusters”. This makes prediction easier and can provide further
information to the roles of the individual genes in the cluster.

• Although enzymes from different sources may catalyse the same reaction
on the same/related substrate(s), the proteins may not have the same
amino acid sequence, though they are likely to be identical or similar for
most of sequence, especially the functional part (active site residues).
GGDFNEVKQLSRSEDIEEWIDRVIDLYQAVLNVNKPTIAAVDGYAIGMGFQFALMFDQRLMASTANFVMPELKHGI
GGDFNEVKNLSGGADVERWIDRVIDLYEAVLHINKPTVAAVDGYAIGMGFQFALMFDYRIMANGARFVMPELKHGI
GGDFSEVKNLS-GESVERWIDRVIDLYCAVLNVNKPTVAAVDGYAIGMGFQFSLMFDQRIVSSEAKFIMPELKHGI
GGDFNEVKFLSRTVEIENWIDRVIELYQSVLKVTKPTVAAIDGYAIGMGFQFAMMFDQRLMSADASLIMPELQHGI
GGDFHEVSEFTGGDEVNAWIDDITDLYTTVAAISKPVIAAIDGYAIGVGLQISLCCDYRLGSEQARLVMPEFRVGI
GGDFNEVSAFTGGDEVSDWIDDITDLYTAIAGISKPVVAAIDGYAIGIGLQIALCCDYRVAADTARLVMPELRVGI
CarB P. carotovorum
CarB D. zea
CarB P. luminescens
CarB Pantoea sp.
ThnE S. cattleya
ThnE S. flavogriseus
61
61
61
61
75
106
135
136
181
136
136
150
Sequence alignment of proteins based on primary amino acid sequence revealing the
identical and/or similar parts of their overall sequences.
Superposition of crystal structures of two proteins
revealing their close 3-D similarity.

• Modified/mutant genes can be synthesized (via a protocol known as site
directed mutagenesis) to produce new proteins with specific changes to
amino acid residues, thus shedding more light on the enzyme’s mechanism
of catalysis (or as part of a protein engineering approach).
• Specific genes can be damaged or deleted to prevent a particular enzyme
being expressed in the organism.
• Genes from different organisms can be combined and expressed together
so that an organism synthesizes abnormal combinations of enzyme
activities, allowing production of modified products (combinatorial
biosynthesis).
• Biosynthetic pathways are under regulatory control in which there may be
restricted availability or localization of enzymes and/or substrates.

MMedicine
Bioinformatics
Agriculture
Proteomics
Genomics
Drug
Discovery
Chemistry
Metabolomics
Biology
Short introduction to bioinformatics and genomics
• Bioinformatics can be broadly described as the application of information
technology to the field of biology.

GATTACAGATTACAGATTACAGATTACAGATTACAG
ATTACAGATTACAGATTACAGATTACAGATTACAGA
TTACAGATTACAGATTACAGATTACAGATTACAGAT
TACAGATTAGAGATTACAGATTACAGATTACAGATT
ACAGATTACAGATTACAGATTACAGATTACAGATTA
CAGATTACAGATTACAGATTACAGATTACAGATTAC
AGATTACAGATTACAGATTACAGATTACAGATTACA
GATTACAGATTACAGATTACAGATTACAGATTACAG
ATTACAGATTACAGATTACAGATTACAGATTACAGA
TTACAGATTACAGATTACAGATTACAGATTACAGAT
• DNA Sequencing
Recent advances in natural products discovery: connecting genes to molecules

T7 bacteriophage
completed in 1983
39,937 bp
59 coded proteins
Escherichia coli
completed in 1998
4,639,221 bp
4293 ORFs
Sacchoromyces cerevisae
completed in 1996
12,069,252 bp
5800 genes
• DNA Sequencing Success Stories!

Caenorhabditis elegans
completed in 1998
95,078,296 bp
19,099 genes
Drosophila melanogaster
completed in 2000
116,117,226 bp
13,601 genes
Homo sapiens
completed in 2003
3,201,762,515 bp
31,780 genes
Costed about 3 billion dollars!
• DNA Sequencing Success Stories!

• 5 vertebrates (human, mouse, rat, fugu, zebrafish)
• 2 plants (arabadopsis, rice)
• 2 insects (fruit fly, mosquito)
• 2 nematodes (C. elegans, C. briggsae)
• 1 sea squirt
• 4 parasites (plasmodium, guillardia)
• 4 fungi (S. cerevisae, S. pombe)
• 140 bacteria and archebacteria
• 1000+ viruses
• DNA Sequencing Success Stories to date!

Forward: ATGCTATCTGTACTATATGATCTA
Complement: TACGATAGACATGATATACTAGAT
5’ 3’
Reverse: TAGATCATATAGTACAGAGATCAT
5’ 3’
Complement
(Sense)
(Antisense)
+
_
• DNA Sequence Nomenclature

• The Genetic Code

• Gene Finding in Prokaryotes
• Simple gene structure.
• Small genomes (0.5 to 10 million bp) without introns (uninterrupted).
• Genes are called Open Reading Frames or “ORFs” (include start &
stop codon).
• Some genes overlap (nested) and Some genes are short (<60 bp).
• For ORF finding tool, visit: http://www.ncbi.nlm.nih.gov/gorf/gorf.html

ORF (open reading frame)
Start codon Stop codon
Promotor
ATGACAGATTACAGATTACAGATTACAGGATAG
Frame 1
Frame 2
Frame 3
• Prokaryotic gene structure
• An open reading frame (ORF) is the part of a reading frame that contains
no stop codons.

• Scan forward strand until a start codon
is found
• Staying in same frame scan in groups
of three until a stop codon is found
• If # of codons between start and end is
greater than 50, identify as gene and
go to last start codon and proceed with
step 1
• If # codons between start and end is
less than 50, go back to last start
codon and go to step 1
• At end of chromosome, repeat process
for reverse complement
• Gene Finding in Prokaryotes

metabolite
• Natural products have traditionally been identified from a top-down
perspective, but more recently genomics- and bioinformatics-guided
bottom-up approaches have provided powerful alternative strategies.
• Top-down approaches have traditionally been the primary means of
natural product discovery, as they do not rely on genome sequencing or
sophisticated genetic manipulation. They begin with the collection of
biological samples from diverse environments for either direct extraction
or laboratory cultivation. Extracts are then screened for a desired
bioactivity, with “hits” isolated for structural characterization.
• Bottom-up approaches, in contrast, are those that first identify a gene
cluster of interest and then utilize various gene manipulation techniques to
drive transcription, translation, and eventual synthesis of the
corresponding natural product.
Recent advances in natural products discovery: connecting
genes to molecules

metabolite
• While traditional top-down approaches have been successful in identifying
many bioactive natural products, the volume of genome sequence data
now available has revised our view of the biosynthetic potential and
metabolic capabilities of living organisms, sparking a renaissance in the
field of natural product discovery.
• Genomics-based bottom-up approaches have been developed to unveil
new natural products that were undetected under standard
growth/fermentation conditions. These strategies leverage powerful
functional genomics, bioinformatics, and genetic manipulation tools to
identify and activate gene clusters of interest either in the native host or in
a heterologous host.
genes to molecules

antiSMASH: antibiotic & Secondary Metabolite Analysis Shell
SMURF: Secondary Metabolite Unknown Regions Finder
genes to molecules
Genomic
databases

metabolite
• Bioinformatics tools are indispensible in the identification and
characterization of potential natural product gene clusters from
sequenced genomes and metagenomes.
• metagenomics can be defined as the application of modern genomics
techniques to the study of communities of microbial organisms directly in
their natural environments, bypassing the need for isolation and lab
cultivation of individual species (<1% of m.o. can be cultivated in lab!).
• Recently, two powerful bioinformatics tools have been developed:
Secondary Metabolite Unknown Regions Finder (SMURF) and antibiotics &
Secondary Metabolite Analysis Shell (AntiSMASH).
• SMURF can predict putative genes in fungal genomes with high accuracy,
and can identify gene clusters for nonribosomal peptides, polyketides,
nonribosomal peptide-polyketide hybrids, indole alkaloids, and terpenes.

• AntiSMASH expands the coverage beyond fungal genomes and natural
products to the whole range of biosynthetic loci of known natural product
classes from any input genome sequence. AntiSMASH is considered the
current state of the art for in silico natural product gene cluster analysis.

• Heterologous host expression: For the majority of organisms, genetic
manipulation is either difficult or yet-to-be established. Therefore,
heterologous expression of a single gene, a cassette of genes, or an entire
biosynthetic gene cluster is a promising alternative route for identifying
the corresponding natural product.
• This strategy may also enable the activation of silent gene clusters in the
genomes of certain organisms, resulting in the discovery of new natural
products. The successful production of natural products from silent gene
clusters is based on the assumption that the heterologous host could
recognize all the genetic elements involved in the pathway and removal of
the regulatory control within the original host.

• Gene(s) of interest is (are) first
synthesized or cloned from native host
then cut out with restriction enzymes
(REs).
• Heterologous host plasmid/ expression
vector (circular piece of DNA with an
origin of replication) is cut with same REs.
• Gene is ligated into plasmid with a ligase,
• New (engineered) plasmid inserted into a
host cell.
• Host cells are grown under conditions
ideal for protein expression.
• Purified protein is used for activity assays.
• Heterologous host expression

• Heterologous host expression

• Heterologous host expression: Refactoring a pathway
• Top: the chosen gene cluster in its
native form. Arrows above the
genes represent transcriptional
units, and horizontal bars
represent regions where genes
overlap each other.
• Middle shows how the parts
chosen for the refactoring
comprise all genes for which a
(putative) function in biosynthesis
can be assigned. Native regulatory
genes are deleted/removed.
• Bottom shows how the genes are
subsequently embedded in a
template of standardized parts,
with promoters (arrows with
purple boxes that signify their
strength), ribosome binding sites
(filled half circles), and insulators
(double squares) and terminators
(bold T's).
• The final construct is assembled
and transferred to the chosen host
for heterologous expression.

THE ACETATE PATHWAY: Biosynthesis of POLYKETIDES
• 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- -keto chains, formed by coupling of acetic acid
(C2) units via condensation reactions, i.e.
• Polyketides include fatty acids, polyacetylenes, prostaglandins,
macrolide antibiotics and many aromatic compounds, e.g. anthraquinones,
flavonoids and tetracyclines.

• Fatty acid biosynthesis involves initial carboxylation of acetyl-CoA to
malonyl-CoA.
• The conversion of acetyl-CoA into malonyl-CoA increases the acidity of the
-hydrogen atoms, thus providing a better nucleophile for the Claisen
condensation.
• An alternative rationalization is that decarboxylation of the malonyl
thioester is used to generate the acetyl enolate anion without any
requirement for a strong base.

Claisen condensation of malonyl-CoA and acetyl-CoA (lecture 2!)
Acetyl-CoA: Starter unit
Malonyl-CoA: Extender unit

• The pathways to fatty acids, macrolides, and aromatic polyketides branch
early.
• The chain extension process continues for aromatics, generating a highly
reactive poly- -keto chain that is stabilized by association with groups on the
enzyme surface until chain assembly is complete and cyclization/aromatization
reactions occur.
• However, for fatty acids, the carbonyl groups are fully reduced (to methylenes)
before attachment of the next acetate group.
• Partial reduction processes, leading to a mixture of methylenes,
hydroxymethines, and carbonyls, are characteristic of macrolides.

Fatty acids
• Fatty acids are mainly found in ester combination with glycerol in the
form of triglycerides. These materials are called fats or oils,
depending on whether they are solid or liquid at room temperature.
• If all three esterifying acids are the same, then the triglyceride is
termed simple, whereas a mixed triglyceride is produced if two or
more of the fatty acids are different.
• Animal fats contain a high proportion of glycerides of saturated fatty
acids and tend to be solids, whilst those from plants and fish contain
predominantly unsaturated fatty acid esters and tend to be liquids.
• Fatty acid biosynthesis is the opposite of -oxidation process.

Classes of Fatty acid synthases
• The group of enzymes involved in the overall process is called fatty acid
synthase (FAS).
• FASs from various organisms show significant structural differences?
• Type I FAS: In animals, The FAS is a large multifunctional protein with seven
discrete functional domains, providing all of the catalytic activities required.
All domains are on a single polypeptide, encoded by a single gene, though the
enzyme exists as a homodimer and requires both units for activity.
• Type II FAS: The FAS in bacteria and plants consists of an assembly of
separable enzymes, encoded by seven different genes, that interact only
transiently during acyl-chain growth.
• Because of the fundamental differences in mammalian FAS I and bacterial
FAS II, there is considerable potential for exploiting these differences to
develop selective inhibitors of fatty acid synthesis as antibacterial agents!

Fatty acids assembly line (in Type I FAS)
• The seven functional domains of type I FAS are:
1. KS: Keto-acyl synthase
2. MAT: Malonyl/acetyltransferase
3. DH: Dehydratase
4. ER: Enoyl-CoA reductase
5. KR: Keto-acyl reductase
6. ACP: Acyl carrier protein
7. TE: Thioesterase
TThe head to tail model of type I FAS
• Note that isoniazid is an
inhibitor of bacterial ER
activity

Stepwise biosynthesis of saturated fatty acids as catalysed by FAS
1. Acetyl-CoA and malonyl-CoA are converted into ACP-bound thioesters (MAT).
2. The Claisen reaction follows giving acetoacetyl-ACP (KS), which is then
reduced stereospecifically to the corresponding -hydroxy ester, consuming
NADPH in the reaction (KR).
3. Elimination of water giving the E (trans)- , -unsaturated-ACP (DH).
4. Reduction of the double bond (ER) again utilizes NADPH and generates a
saturated acyl-ACP (fatty acyl-ACP), which is two carbon atoms longer than
the starting material.
5. This product can condense again with malonyl-ACP (KS) and go through the
successive reduction (KR), dehydration (DH) and reduction (ER) steps,
gradually increasing the chain length by two carbon atoms for each cycle
until the required chain length is obtained.
6. The free fatty acid can be released by the action of a thioesterase (TE).

Stepwise biosynthesis of saturated fatty acids as catalysed by FAS
• The ACP carries a phospho-pantetheine group, analogous to that in
coenzyme A, and this provides a long flexible arm, enabling the growing fatty
acid chain to reach the active site of each domain in the multi-enzyme
complex, allowing the different chemical reactions to be performed without
releasing intermediates from the multi-enzyme complex.

Biosynthesis of saturated fatty acids

Biosynthesis of saturated fatty acids
CE = KS
• There is an active-site cysteine
residue in the keto-acyl
synthase (KS = CE) domain.

Some of the naturally occurring saturated fatty acids
• The majority of naturally-occurring fatty acids have straight-chains
possessing an even number of carbon atoms.

Rare naturally occurring saturated fatty acids
• The rarer fatty acids contain an odd number of carbon atoms, and typically
originate from incorporation of a different starter unit, e.g. propionyl-CoA
(instead of acetyl-CoA).
• Other structures (e.g. branched FA) can arise by utilizing different starter
units (e.g. isobutyryl-CoA) and/or different extender units (e.g. methylmalonyl-
CoA).
Example for branched saturated
fatty acid: tuberculostearic acid

Unsaturated fatty acids
• Animal fats contain a high proportion of glycerides of saturated fatty acids
and tend to be solids, whilst those from plants and fish contain predominantly
unsaturated fatty acid esters and tend to be liquids.
• Double bonds at position 9 are common, but unsaturation can occur at other
positions in the chain.
• The stereochemistry of the double bond is Z (cis)-geometry, thus introducing
a ‘bend’ into the alkyl chain, which interferes with the close association and
aggregation of molecules that is possible in saturated structures and helps to
maintain the fluidity in oils and cellular membranes.
• Plants growing in colder climates possess a high proportion of
polyunsaturated fatty acids so that they can maintain the fluidity of their
storage fats and membranes.

• A characteristic feature of olive oil is its very high of oleic acid (18:1)
content. Typical fatty acids in fish oils have high unsaturation and also long
chain lengths, e.g. eicosapentaenoic acid (20:5) in cod liver oil.
• Polyunsaturated fatty acids tend to have their double bonds in a non-
conjugated manner.
• A range of metabolites necessary for good health, including prostaglandins,
leukotrienes, are produced from the plant fatty acid linoleic acid and its
derivatives, which have to be obtained in the diet. Accordingly, these plant
fatty acids are referred to as ‘essential fatty acids’.

• Since beneficial fatty acids, including eicosapentaenoic acid, have a double
bond three carbon atoms from the end of the carbon chain, they are grouped
together under the term –3 fatty acids (omega-3 fatty acids).
• Omega (lowercase: ) is the last letter of the Greek alphabet.
• Regular consumption of cold water oily fish is claimed to reduce the risk of
heart attacks and atherosclerosis.

Biosynthesis of unsaturated fatty acids
• Desaturation of stearic acid (as its coenzyme A thioester) to oleic acid is
catalysed by stearoyl-CoA desaturase (SCD), which is an iron-containing
protein that uses a cytochrome-b5-dependent monoxygenase mechanism.
• Monooxygenase means an enzyme using one atom of O2 to oxidize substrate,
while the second O atom is reduced to H2O by a reductant, in this case NADPH.
• Polyunsaturated fatty acids are produced by further oxidative desaturation of
unsaturated precursors.unsaturated precursors.

Biosynthesis of unsaturated fatty acids
• Mice with whole-body knockout of the SCD gene isoforms are remarkably
protected from obesity and insulin resistance!!
• Stearoyl-CoA desaturase (SCD) enzymes catalyze the conversion of
palmitate and stearate into the corresponding monounsaturated fatty acids
palmitoleate and oleate, respectively, which are the major fatty acids found in
triglycerides.
Photos of obese control mice and lean SCD-deficient mice

Oxylipins
• Oxylipins = Oxygenated fatty acids = Prostanoids
• In mammals, oxylipins are derived principally from arachidonic acid (C20
polyunsaturated fatty acid ).
• Examples include leukotrienes, prostaglandins and thromboxanes.
• These classes of compounds exert a diverse range of pharmacological
effects in mammals including regulation of blood pressure, control of blood
platelet aggregation, allergic responses and inflammation processes.

Biosynthesis of oxylipins
• The biosynthetic pathway to oxylipins often involves the addition of
molecular oxygen to an unsaturated fatty acid to give a hydroperoxide,
catalysed by lipoxygenases, which are iron-containing dioxygenase
enzymes that introduce both oxygen atoms from molecular oxygen into the
product. The hydroperoxide can then undergo a series of reactions to give
the final oxylipin.
• Note that molecular oxygen is a diradical (it has two unpaired electrons in
its electronic ground state, triplet oxygen).

Biosynthesis of oxylipins
• Leukotrienes are a
series of fatty acid
derivatives with a
conjugated triene
functionality and first
isolated from leukocytes.
• The unstable allylic
epoxide leukotriene A4
may hydrolyse by
conjugate addition to
give LTB4 or may be
attacked directly by the
nucleophilic sulfhydryl-
moiety of the tripeptide
glutathione to give LTC4.

Summary
• Even-numbered straight-chain saturated fatty acids are biosynthesized by
elongation of an acetyl starter unit with malonyl units.
• Odd-numbered straight-chain saturated fatty acids are biosynthesized by
elongation of a propionyl starter unit with malonyl units.
• Branched starter units or substituted malonyl units give branched saturated
fatty acids.
• Mono-unsaturated fatty acids are produced from saturated precursors by
oxidative desaturation, generally with production of a cis-double bond
between carbon atoms 9 and 10.
• Polyunsaturated fatty acids are produced by further oxidative desaturation of
unsaturated precursors.
• The addition of oxygen to unsaturated fatty acids gives hydroperoxides which
are the precursors of many oxygenated fatty acid derivatives (oxylipins).

• Polyketides are derived from poly- -keto chains, formed by coupling of
acetic acid (C2) units via condensation reactions, i.e.
• Today we will mainly discuss macrolide antibiotics and aromatic
polyketides, e.g. anthraquinones, flavonoids and tetracyclines.
• The pathways to fatty acids, macrolides, and aromatic polyketides
branch early.
Acetyl-CoA: Starter unit
Malonyl-CoA: Extender unit

1. For fatty acids, the carbonyl groups are fully reduced before attachment of
the next acetate group.
2. For aromatics, a highly reactive poly- -keto chain is generated by chain
extension and stabilized by association with groups on/within the enzyme until
chain assembly is complete and cyclization/aromatization reactions occur.
3. For macrolides, partial reduction occurs leading to a mixture of methylenes,
hydroxymethines, and carbonyls.
4. In the case of 22 and 33, these processes are catalysed by enzymes known as
polyketide synthases (PKSs).
• Type I FAS consists of 7 functional domains: KS, MAT (AAT), DH, ER, KR, ACP
(Thiolation = TT) and TE.

• This means that the enzyme activities KR, DH, and ER, in polyketide synthases
are not all active/present during a particular extension cycle. The order in
which the modifications (e.g. reduction) occur (or do not occur) is closely
controlled by the type of PKS.
• Detailed studies of the genes, protein amino acid sequences, and mechanistic
similarities in various PKSs has led to three general types being distinguished.
• Type I PKS: very large multifunctional proteins with individual functional
domains. They are responsible for macrolide biosynthesis.
• Type II PKS: are composed of a complex of individual monofunctional
proteins.
• Type III PKS = chalcone synthase-like PKS: homodimeric proteins that utilize
coenzyme A esters rather than ACPs, and they employ a single active site to
perform a series of decarboxylation, condensation, cyclization, and
aromatization reactions.
Biosynthesis of aromatic polyketides and macrolides: classes of PKSs

• Type I PKSs are found in bacteria and fungi, type II PKSs are restricted to
bacteria, whilst type III PKSs are found in plants, bacteria, and fungi.
• Type I PKSs can also be subdivided into ‘iterative’ (i.e. repeating) and
‘noniterative’ categories. Iterative systems (like the FASs) use their functional
domains repeatedly to produce a particular polyketide.
• Non-iterative systems possess a distinct active site for every enzyme-
catalysed step. Type II PKSs are of the iterative type.
• Many Aromatic compounds can be produced by type II PKSs employing
malonyl-CoA as an extender unit.
• Type III PKSs use cinnamoyl-CoA (or a derivative of) as a starter unit to
produce flavonoids and stilbenes.
• FASs and PKSs probably share a common evolutionary ancestor.
Biosynthesis of aromatic polyketides and macrolides: classes of PKSs

• The macrolides are a large family of compounds, many with antibiotic activity,
characterized by a macrocyclic lactone (sometimes lactam) ring. Rings are
commonly 12-, 14-, or 16-membered.
• The starter unit can be either acetyl-CoA or propionyl-CoA.
• Macrolide assembly is most often accomplished by non-iterative type I PKSs.
Biosynthesis of macrolides
• Example for macrolides: Erythromycin A from
Saccharopolyspora erythraea is a valuable
antibacterial drug that contains a 14-membered
macrocycle, and biosynthesized employing
propionyl-CoA as a starter unit and
methylmalonyl-CoA as an extender unit.
• Sugar units (L-cladinose and D-desosamine) are
attached through glycosidic linkages to the
hydroxyls at C-3 and C-5).

Retro-biosynthesis of Erythromycin A
• Erythromycin A is biosynthesized employing propionyl-CoA (x1) as a starter
unit and methylmalonyl-CoA (x6) as an extender unit.
1. Identify starter and extender units starting by breaking the lactone ring.
2. Deduce the fate of carbonyls (reduced or not).
3. Deduce the other modifications (post-PKS changes) required to get the target molecule
(e.g. hydroxylation at C-6 and C-12 (asterisked) and O-glycosylation at C-3 and C-5).

The assembly line for 6-Deoxyerythronolide B
• 6-Deoxyerythronolide B synthase (DDEBS) is a modular type I PKS involved in
erythromycin biosynthesis. DEBS complex (10283 amino acid) consists of 3
large multi-functional proteins, DEBS 1,2, and 3 that each exist as a dimer of two
modules (overall 6 modules). Module 1 has a loading domain.
• Module 3 lacks any -carbon-modifying domains.
• Modules 1, 2, 5, and 6 contain KR domains and
are responsible for production of hydroxy
substituents.
• Module 4 contains the complete KR, DH, and ER
set, and results in complete reduction to a
methylene.
• The chain is finally terminated by a thioesterase
(TE) activity, which releases the polyketide from
the enzyme and allows cyclization.

• The starter unit used is determined by the specificity of the AT in the loading
domain.
• The AT specificity and the catalytic domains on each module determine the
structure and stereochemistry of the extension unit.
• The order of the modules specifies the sequence of the units, and the number
of modules determines the size of the resultant polyketide chain.
• The vast sstructural diversity of natural polyketides arises from
1. Combinatorial possibilities of arranging modules containing the various
catalytic domains.
2. The sequence and number of modules.
3. The stereochemistry of associated side-chains.
4. The post-PKS enzymes which subsequently modify the first-formed product
(e.g. oxidation and glycosylation of 6-deoxyerythronolide B to erythromycin
A).

Post assembly line modification of 6-Deoxyerythronolide B
• 6-deoxyerythronolide B released by the PKS assembly line undergoes stereo-
and regio-specific hydroxylation at C-6 by a cytochrome P450 enzyme.
• This product is then a substrate for the two glycosyltransferases that attach
deoxysugars to the 3-hydroxyl and 5-hydroxyl of the macrolactone scaffold.
• Then a second cytochrome P450 monooxygenase acts to hydroxylate C-12.
L-cladinose
D-desosamine

Biosynthesis of Aromatic POLYKETIDES
• In the absence of any reduction processes, the growing poly- -keto chain
needs to be stabilized on the enzyme surface until the chain length is
appropriate, at which point cyclization or other reactions can occur.
• A poly- -keto ester is very reactive, and there are various possibilities for
undergoing intramolecular Claisen or aldol reactions, controlled by the nature
of the enzyme and how the substrate is folded.
• Methylenes flanked by two carbonyls are activated, allowing formation of
carbanions/enolates and subsequent reaction with ketone or ester carbonyl
groups, with a natural tendency to form strain-free six-membered rings.
• Aromatic compounds are mainly typical products from type II and type III
PKSs.
1,3-dicarbonyl condensation
with aldehyde/ketone:
Knoevenagel reaction
(modified aldol
condensation)

Major cyclization pathways for a tetraketide followed by aromatization
A: Electrophilic carbon
a: Nucleophile
• Ionization of the -methylene (a or b) allows nucleophilic addition onto the carbonyl
group that is six carbon atoms distant along the chain (A or B, respectively).

Other (minor) possible cyclization pathways for a tetraketide

Major cyclization pathway for a pentaketide followed by aromatization

Biosynthesis of 6-Methylsalicylic acid
• The enzyme 6-methylsalicylic acid synthase is one of the smallest type I PKSs
known.

O
O O O
O O
SEnz
O O
O
O O
OH
O O
OH
OH O
OH
O OH
O
OH
OH O OH
O
OH O OH
O
MeO
emodinphysicon
Senna
Casacra
Rhubarb
Aloes
Frangula
OH O OH
O
OH
aloe-emodin
OH O OH
O
OH
O
rhein
O OH
OH
Glu O
OH Glu
cascaroside
[O]
SAM
Acetyl-CoA + 7x malonyl-CoA
Biosynthesis of anthraquinones
-CO2

O
SCoA
OH
O
OH
O
O
O
SCoA
4-hydroxycinnamoyl-CoA
O
OH
O
OH
OH
H
OH
OH O
O
OH
naringenin
A FLAVONOID
(flavanone)
3x malonyl-CoA
Michael
addition
comes from the shikimic acid pathway
STARTER UNIT
The starter unit originates from the shikimate pathway (next lecture). So, one
aromatic ring originates from shikimate while the other from aromatic
polyketide biosynthesis pathway!
Naringenin is found in grapefruit flowers, fruit and rind as the 7-rhamnogylcoside
A CHALCONE
Biosynthesis of Flavonoids
chalcone synthase
chalcone
isomerase

O
SCoA
OH
4-hydroxycinnamoyl-CoA
3x malonyl-CoA
comes from the shikimic acid pathway
STARTER UNIT
Biosynthesis of Stilbenes
stilbene synthase
• The way the precursor polyketide is
folded is different from the case of
flavonoids.

THE SHIKIMATE PATHWAY: Aromatic amino acids and Phenylpropanoids
• The shikimate pathway (named after the central intermediate in the
pathway, shikimic acid) provides an alternative route to aromatic
compounds, particularly the aromatic amino acids L-phenylalanine, L-
tyrosine, and L-tryptophan.
• This pathway is employed by microorganisms and plants, but not by
animals; accordingly, aromatic amino acids feature among the essential
amino acids for man and have to be obtained in our diets.
• Shikimic acid is the raw chiral material for synthesis of the
antiviral oseltamivir (Tamiflu®, against avian influenza).
• The main source of shikimic acid is star anise fruits;
however, cultures of genetically engineered E. coli are
considered as an alternative (~84 g/L through inhibition of
feedback regulatory mechanisms), particularly as fears of
potential pandemic are leading to a production shortage of
Tamiflu.

THE SHIKIMATE PATHWAY: Aromatic amino acids and Phenylpropanoids
• Phenylalanine and tyrosine form the basis of C6C3 phenylpropane units
found in many natural products, e.g. cinnamic acids, coumarins, lignans,
and flavonoids.
• Phenylalanine, tyrosine and tryptophan are precursors for many alkaloids.
• The shikimate pathway begins with a coupling of phosphoenolpyruvate
from the glycolytic pathway and D-erythrose-4-phosphate from the
pentose phosphate cycle.
• Benzoic acid derivatives (e.g. gallic and protocatechuic acids, components
of many tannins) can be produced via branchpoints in the shikimate
pathway.
• Reduction of 3-dehydroquinic acid leads to quinic acid, a fairly common
natural product found in the free form, as ester, or in combination with
alkaloids such as quinine.

Biosynthesis of Chorismate
• A very important branchpoint compound in the shikimate pathway is
chorismic acid. The name derives from a Greek word meaning "to separate"
because the compound acts as a branch-point in the biosynthesis of many
2ry metabolites.

Metabolism of chorismate
Chorismate is It is a precursor for:
1. 4-Hydroxybenzoic acid (note that p-hydroxylation and the O-hydroxylation
patterns contrast with the typical m-hydroxylation patterns characteristic
of phenols derived via the acetate pathway, and in many cases allow the
biosynthetic origin (acetate or shikimate) of an aromatic ring to be
deduced).
2. 2,3-Dihydroxybenzoic acid (precursor in enterobactin biosynthesis;
enterobactin is a powerful iron chelator) and the plant hormone salicylic
acid (2-hydroxybenzoic acid), via its isomer isochorismic acid.
3. The folate precursor p-aminobenzoate.
4. 2-Aminobenzoic (anthranilic) acid, an intermediate in the biosynthesis of
the indole-containing aromatic amino acid L-tryptophan.
5. 4-Amino-4-deoxychorismic acid, the precursor for chloramphenicol.
6. The aromatic amino acids phenylalanine and tyrosine, via prephenic acid.
The Claisen rearrangement of chorismate to prephanate is catalysed by
the enzyme chorismate mutase.

Metabolism of chorismate into L-phenylalanine and L-tyrosine
Although plants can convert phenylalanine
to tyrosine using hydroxylases, this
conversion is a minor pathway.

Major Shikimic acid pathway metabolites
1. Tannins – in green tea, red raspberry & witch hazel.
2. Lignans – Anti-cancer drugs e.g. Podophyllotxin.
3. C6-C1 Compounds e.g. vanillin
4. Coumarins – Anticoagulants e.g. warfarin.
5. Flavonoids – water soluble yellow plant pigments.
6. Anthocyanins and Anthocyanidins – water soluble
plant pigments.

1- Biosynthesis of Hydrolysable Tannins
• Tannin a general name for a large group of complex phenolic substances
that are capable of tanning animal hides into leather because of their ability
to cross-link protein molecules.
• They are found in almost every plant part - abundant in unripe fruit. They
deter herbivores due to their astringent properties - bind saliva and other
digestive proteins. They have antioxidant and Antimicrobial properties.
• hydrolysable tannin is a type of tannin that, on heating with acids, yields
gallic or ellagic acids.
• Gallotannins are esters of gallic acid with a polyalcohol, typically glucose
(e.g. pentagalloylglucose).
• Ellagitannins contain one or more hexahydroxydiphenic acid function (e.g.
tellimagrandin II). Hexahydroxydiphenic acid is formed by phenolic
oxidative coupling of 2 galloyl functions catalysed by a phenol oxidase
enzyme.

1- Biosynthesis of Hydrolysable Tannins

• L-Phenylalanine and L-tyrosine, as C6C3 building blocks, are
precursors for a wide range of natural products. The elimination of
ammonia from from the side-chain of L-phenylalanine generates trans
(E)-cinnamic acid. The deamination reaction is catalysed via the
enzyme phenylalanine ammonia lyase (PAL).
• Other phenylpropanoids are obtained by further hydroxylation and
methylation of (E)-cinnamic acid derivatives.
• Lignans are typically found as dimeric phenylpropanoids derivatives,
mostly in the free form, seldom as glycosides.
• They have antitumor, antiviral, liver protective, etc. activities.
• The two phenylpropanoid units mostly linked through the -C atom of
the C3 side chains as a result of one-electron oxidation of the
phenolic groups as catalysed by a peroxidase enzyme.
2- Biosynthesis of Lignans and lignins (C6-C3 compounds)

sinapic acid
HSCoA NADPH NADPH
sinapyl alcoholsinapyl aldehyde
REDUCTION OF PHENYL PROPANOIDS
activation
Sinapyl-CoA
*
dehydrogenase dehydrogenase

:
vanillic acid
reverse
Claisen
H2O
H2O
ferulic acid
HSCoA NADP+
3- Biosynthesis of C6-C1 Compounds
activation Michael
addition
Thioester
hydrolysis
dehydrogenase

umbelliferoneaesculetin
trans–cis
isomerization Lactonization
After activation
2-hydroxylase
coumarin
4- Biosynthesis of COUMARINS
cinnamic acid 2-coumaric acid
• The hydroxylation of cinnamic acids ortho to the side-chain is a crucial
step in the formation of coumarins, cinnamic acid lactone derivatives.
• Both the trans–cis isomerization and the lactonization steps are enzyme
mediated.

• One of the largest classes of natural phenolics.
• Carbon skeleton has 15 carbons with two benzene rings connected by a 3-C
bridge (i.e. C6-C3-C6). They are classified into different groups based on the
degree of oxidation of the C3 bridge: anthocyanins, flavones, flavonols, and
isoflavones. Majority of flavonoids exist as glycosides.
• Isoflavonoids: Some have insecticidal activity (e.g. rotenoids), some have
estrogenic/anti-estrogenic activity, many are phytoalexins - antimicrobial
compounds produced in response to bacteria and fungi.
5- Biosynthesis of Flavonoids (see Lecture 5, aromatic polyketides)

• Isoflavonoids: a distinct subclass of flavonoids because the shikimate-
derived aromatic ring has migrated to the adjacent carbon of the heterocycle.
The rearrangement is catalysed by a cytochrome P-450-dependent enzyme
requiring NADPH and O2 cofactors.
5- Biosynthesis of Isoflavonoids

naringenin (R=H)
NADPH
+
- 2 H2O
O2, 2-oxoglutarate
pelargonidin (R=H, blue)
cyanidin (R=OH, pink)
6- Biosynthesis of ANTHOCYANINS and ANTHOCYANIDINS
reductase
• Anthocyanins (Greek: anthos = flower, kyanos = blue) are water-soluble
pigments that may appear red, purple, or blue depending on the pH.
• Anthocyanidins are aglycones (Flavylium cations), anthocyanins are the
glycosidic forms.
flavone synthase I
Anthocyanidin
synthase
O2, 2-oxoglutarate

CHORISMIC ACID
SHIKIMIC ACID
PREPHENIC
ACID
TYROSINE
PHENYLALANINE
Lignans&
lignins
ALKALOIDS ALKALOIDS
C6-C3 Phenyl-
propanoids
ANTHRANILIC
ACID
TRYPTOPHAN
ALKALOIDS
C6-C1
compounds
FLAVANOIDS&
Anthocyanins
(+ acetogenin)
Summary of Shikimate Pathway

Terpenoids Biosynthesis: THE Mevalonate and Nonmevalonate pathways
• Terpenoids form a large and structurally diverse family of natural products
derived from C5 isoprene units. Typical structures contain carbon skeletons
represented by (C5)n, and are classified as hemiterpenes (C5),
monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes
(C25), triterpenes (C30), and tetraterpenes (C40).
• The biochemically active isoprene units are the diphosphate
(pyrophosphate) esters dimethylallyl diphosphate (DMAPP) and isopentenyl
diphosphate (IPP) (good leaving groups!), and they can be derived from two
intermediates: mevalonic acid (MVA) or methylerythritol phosphate (MEP,
nonmevalonate pathway, in bacteria and plants).

Terpenoids Biosynthesis: THE Nonmevalonate pathway
• In plants and most bacteria, IPP/DMAPP is synthesized from the
condensation of glyceraldehyde-3-phosphate (3 carbons) and pyruvate (3
carbons) to form a 5 carbon intermediate (deoxyxylulose-5-phosphate,
lecture 1) catalysed by a thiamin diphosphate-dependent enzyme, which
can then be reduced to MEP.
• Occurs in chloroplast of plants. Involved in synthesis of chlorophyll,
carotenoids, Vitamins A, E and K.
• The MEP pathway is a favorable target for antimicrobial drug development?

JOINING ISOPRENE UNITS
• Isoprene units are
usually joined in a
head-to-tail fashion.
• Squalene (C30)
displays a tail-to-tail
linkage at the centre of
the molecule.
• Most terpenoids are
modified further by
cyclization reactions,
though the head-to-tail
arrangement of the
units can usually still
be recognized, e.g.
menthol.
tail-tail
mmonoterpenes
Sesquiterpenes
Diterpenes
Triterpenes

CH3
CH3
CH CH3
CH3
guaiazulene
CH3
CH3
CH3
caryophyllene camphor
O
-pinene

All steroids are triterpenes but their skeletons
have been rearranged and/or modified so that
they can not be analyzed into isoprene units.
For Example, cholesterol has 27, and not 30,
carbons, and it is biosynthesized from the
triterpene lanosterol. Lanosterol is a
tetracyclic triterpenoid from which all steroids
are derived.
rearrangements
in this area
20 steps

THE Mevalonate pathway: Generation of MVA, DMAPP and IPP

• The biosynthesis of terpenes can often be rationalized using carbocation
chemistry.
• Many of the carbocations required as intermediates in terpene
biosynthesis are commonly generated by heterolysis of pyrophosphates
(the pyrophosphate is a good leaving group) with the two electrons of the
cleaved bond reside on the leaving group resulting in a positively charged
carbon atom. The resulting carbocation intermediate is often converted
into an isomeric carbocation leading to considerable structural diversity in
terpene biosynthesis.
Carbocations as intermediates in terpene biosynthesis

• Three particularly important types of reactions of carbocations in terpene
chemistry are: hydride shifts, alkyl shifts and cyclizations.
• If a hydrogen atom migrates from one carbon to a vicinal carbon, this is
known as a 1,2-hydride shift. Since the hydrogen atom migrates with the
pair of electrons, it is formally hydride (H-) that is migrating.
• A 1,2-alkyl shift is mechanistically similar to the 1,2-hydride shift, and is
termed Wagner-Meerwein rearrangements.
• The intramolecular cyclization of carbocationic intermediates and alkene
groups is responsible for further structural diversity in terpene
biosynthesis.
Reactions of the carbocation intermediates

• There are ttwo major routes
through which carbocations
are converted into terpene
products. These routes are:
1. loss of a proton: usually
occurs from the carbon atom
adjacent to the carbocationic
centre, giving an alkene
derivative.
2. addition of water followed by
loss of a proton: A
carbocation can often
undergo nucleophilic
addition of water giving,
after loss of a proton, an
alcohol derivative.
Termination of carbocation intermediates

1. DMAP ionizes to form eelectrophilic carbocation.
2. Nucleophilic attack by the alkene of IPP forms geranyl-PP.
3. Stereospecific loss of HR, forming a new double bond.
4. Geranyl-PP ionizes, rearranges to form the carbocation intermediate
that we studies its reactions in the last two slides. Geranyl-PP is the
precursor to most monoterpenes.
Biosynthesis of Monoterpenes (C10)

(+)-carvone = caraway seed
(-)-carvone = spearmint
(++)-limonene = oranges
(--)- limonene = lemons
• Note that most cyclic monoterpenes have a distinctive odor- basis of
perfume & flavor industries.
• Stereoisomers have different characteristic smells, which
demonstrates that smell receptors are 3D-proteins, i.e. have chiral
active sites that can distinguish enantiomers
Biosynthesis of Monoterpenes (C10)

OPP
+
(+)
+
H
H
+
H+
taxadiene
OOOO
OH
O
O O OH
CH3
O
H
O
OH
NHO
CH3
O
paclitaxel (taxol)
many steps:
oxidations,
esterification
Phe
Phe
Phe
acetylCoA
acetylCoA
Pacific Yew (bark)
GGPP
Biosynthesis of Diterpenes (C20): Taxol Biosynthesis

Biosynthesis of triterpenes and steroids
• Squalene Biosynthesis: Farnesyl pyrophosphate (FPP) undergoes
dimerization. Loss of a proton from the resulting carbocation generates a
cyclopropane derivative, which then affords a cyclobutyl-carbocation by a
1,2-alkyl migration. Ring-opening of the cyclobutyl-carbocation followed by
reaction of the resulting linear carbocation with NADPH gives squalene.

• The biosynthesis of steroids and triterpenoids is initiated either by
epoxidation or protonation of the terminal alkene group. The addition
of an electrophile (e.g. a proton) at one of the alkene groups
generates a (tertiary?) carbocation, which can then undergo ring
formation (cyclization) reactions.
• Cyclization of squalene is via the intermediate squalene-2,3-oxide,
which is produced in a reaction catalysed by squalene epoxidase, a
flavoprotein requiring O2 and NADPH cofactors.
• The conformation in which squalene-2,3-oxide cyclizes is important in
determining the stereochemistry of the final product. Squalene oxide
is the precursor to steroids.
• Triterpenes form flexible rings (chair, boat conformations) with many
chiral centers.

Biosynthesis of Alkaloids: Introduction
• In 1819, Carl Meissner, a pioneering German chemist, coined the term
“alkaloid” which referred then to “any natural product with the
characteristic presence of a basic nitrogen atom, excluding amino acids
and peptides”.
HHistorical uses of alkaloids
• In the middle east-the latex of opium poppy (Papaver) was
already used at 1200 B.C.
• Queen Cleopatra used extracts of Hyoscymus to expand her
pupils and appear more attractive.
• Theriak, a mixture of opium, dried snake meat and wine was
used as antidote.
• Morphine named for Morpheus, the god of dreams in the Greek
mythology.

Biosynthesis of Alkaloids
DDefinition
Low molecular weight, naturally occurring chemical compounds with
pharmacologically activity and characterized by the presence of a (basic)
nitrogen atom. Some alkaloids where the nitrogen is part of an amide
function are neutral.
Classification:
1. True alkaloids: contain nitrogen in an heterocyclic ring and originate
from amino acids (e.g. atropine, morphine, ..). The primary precursors of
true alkaloids include L-ornithine, L-lysine, L-tyrosine, L-tryptophan and L-
histidine.
2. Protoalkaloids: They originate from amino acids (e.g. mescaline and
adrenaline) but the nitrogen atom is not part of an heterocycle.
3. Pseudalkaloids: alkaloids that do not originate from amino acids (e.g.
ephedrine, capsaicin, coniine, caffeine). They usually result from a
transamination reaction.

• Alkaloids are often classified according to the nature of the nitrogen-
containing heterocycle/structure (e.g. pyrrolidine, piperidine, quinoline,
isoquinoline, indole, …).
• The nitrogen atom in many alkaloids originate from an L-amino acid, and,
in general, the carbon skeleton of the particular amino acid precursor is
largely retained intact in the alkaloid structure, though the carboxylic acid
carbon is often lost through decarboxylation.
• Relatively few amino acid precursors are actually involved in alkaloid
biosynthesis (the principal ones are ornithine, lysine, tyrosine,
tryptophan, and histidine). Nicotinic acid (biosynthesized from
tryptophan) and anthranilic acid (biosynthesied from chorismate) can
also provide the nitrogen atom of some true alkaloids.
• Building blocks from the acetate (polyketide), shikimate or
methylerythritol phosphate pathways are also frequently incorporated
into the alkaloid structures.
Biosynthesis of Alkaloids

• Ornithine contains both - and -amino groups, and it is the -
nitrogen which is incorporated into alkaloid structures along with the
carbon chain, except for the carboxyl group. Thus, ornithine supplies
a C4N building block as a pyrrolidine ring system.
Biosynthesis of Alkaloids derived from ornithine and lysine
• The reactions of ornithine are almost
exactly paralleled by those of L-lysine,
which provides a C5N unit containing its -
amino group (as a piperidine ring system).
• Most of the other amino acid alkaloid
precursors supply nitrogen from their
solitary -amino group.

• Many aliphatic alkaloids are formed by modification of ornithine and
lysine via a cyclic iminium ion or an N-methylated cyclic iminium ion. N-
methylation occurs as catalysed by a methyltransferase in the presence
of S-adenosyl methionine (SAM).

• Examples of alkaloids derived from ornithine include:
tropane alkaloids (e.g. hyoscyamine and cocaine),
pyrrolizidine alkaloids and nicotine.
• Examples of alkaloids derived from lysine include:
piperidine alkaloid (e.g. pelletierine and lobeline), and
quinolizidine alkaloids.

Biosynthesis of Tropane Alkaloids: Cocaine and hyoscyamine

Biosynthesis of Tropane Alkaloids: Hyoscyamine

Littorine conversion to hyoscyamine
• Littorine undergoes a radical rearrangement initiated with a P450 enzyme
forming hyoscyamine aldehyde. A dehydrogenase then reduces the
aldehyde to a primary alcohol making (-)-hyoscamine, which upon
racemization (occur during acid/base extraction) forms atropine.

Biosynthesis of Nicotine: from ornithine and nicotinic acid
• Nicotine originates from a cyclic iminium ion and nicotinic acid (vitamin
B3). Note that nicotinic acid must be reduced and decarboxylated to yield
an electron-rich enamine that can then react with the iminium ion.

3- Biosynthesis of Piperidine alkaloids from lysine
• Ex: Pelletierine alkaloids, which are constituents of the bark of
pomegranate (Punica granatum; Punicacae).

Biosynthesis of alkaloids derived from tyrosine
Ex: Phenylethylamine alkaloids (e.g. adrenaline) and tetrahydroisoquinoline
alkaloids (e.g. morphine)
PPictet-Spengler reaction: Decarboxylation of tryptophan, phenylalanine
and tyrosine yields 2-arylethylamines. In Pictet-Spengler reaction, a 2-
arylethylamine condenses with an aldehyde in the presence of acid
(catalyst) to construct a new ring.
• The tetrahydroisoquinoline ring and related heterocycles are found in
many alkaloids. The first step in its formation is the condensation of an
amine with the aldehyde to form an imine. The protonated imine is
electron-deficient and subsequently acts as an electrophile in an
intramolecular electrophilic substitution reaction.

Biosynthesis of Morphine: formation of (S)-norcoclaurine
• Tyrosine is the precursor to an immense number of alkaloids.
• The formation of nnorcoclaurine is a typical example of how alkaloids
possessing a 1,2,3,4-tetrahydroisoquinoline ring are biosynthesized. The
amine an aldehyde functionalities, required for the Pictet-SSpengler
reaction, are derived from tyrosine by hydroxylation/decarboxylation and
transamination/decarboxylation, respectively.

• The trihydroxy-alkaloid (S)-norcoclaurine, is then, respectively, methylated
(x2), hydroxylated, then methylated (x1) to give (S)-reticuline, a pivotal
intermediate to many alkaloids. (S)-reticuline can be epimerized to (R)-
reticuline in two enzyme-catalysed steps (NADP/NADPH-dependent).
Biosynthesis of Morphine: Formation of (R)-reticuline

• Opium alkaloids (morphine, codeine, and thebaine) are elaborated from
(R)-reticuline. (R)-reticuline is phenolic. We have seen previously how
phenol oxidation (one electron oxidation) gives phenoxy radicals.
Similarly, (R)-reticuline (and other phenol-containing alkaloids) can yield
phenoxy radicals, which can subsequently dimerize leading to
structurally more complex natural products.
• This concept is exemplified in the formation of thebaine, which is the
precursor to morphine. Note that in the transformation of thebaine into
morphine, two methoxy groups are removed (converted to hydroxy
groups).
Biosynthesis of Morphine

• Morphine: 5 rings, 5 contiguous stereocentres(*).
• Codeine is the 3-O-methyl ether of morphine.
• Heroin is the diacetate of morphine (synthetic).

• Morphine: 5 rings, 5 contiguous stereocentres(*).

NH2
N
OH
H
O
N
OH
H
N
O
H
H
N
O
SCH3
HOOC
HH
NCH3
O
OH
OH
NH2
N
OH
H
O
N
OH
H
N
O
H
H
N
O
HOOC
HH
morphine
Leu-enkephalin
Met-enkephalin
Tyr-Gly-Gly-Phe-Met
Tyr-Gly-Gly-Phe-Leu
ENKEPHALINS
• From larger peptide structures
found in the brain called endorphins.
They bind to a pain-reducing
receptor in the brain.
• The enkephalins are rapidly
degraded in the body and are
therefore not good for use as drugs.

Biosynthesis of alkaloids derived from tryptophan: Indole alkaloids
Ex: Indole alkaloids (e.g. vinblastine and ergotamine) and Quinoline
alkaloids (e.g. quinine).
• Decarboxylation of tryptophan yields tryptamine.
• Many indole alkaloids are of mixed-origin (e.g. using geraniol, a terpene,
as a co-precursor in vinca alkaloids, e.g. vinblastine).
• A suggested pathway to physostigmine is by C-3 methylation of
tryptamine, followed by ring formation involving attack of the primary
amine function onto the iminium ion.
• Physostigmine is a reversible inhibitor of acetylcholinesterase.

Biosynthesis of Ergot alkaloids: Lysergic acid
• Ergot alkaloids are produced by the fungus Claviceps purpurea and
includes the hallucinogen lysergic acid. The building blocks for lysergic
acid are tryptophan (less the carboxyl group) and an isoprene unit (mixed
origin).

Biosynthesis of Pilocarpine
• Pilocarpine is an imidazole alkaloid that originates from L-histidine.

ALKALOIDS DERIVED BY AMINATION REACTIONS: Pseudo-alkaloids
• The majority of alkaloids are derived from amino acid precursors by
processes which incorporate into the final structure the nitrogen atom
together with the amino acid carbon skeleton or a large proportion of it
(True alkaloids).
• Many alkaloids do not conform to this description and are synthesized
primarily from non-amino acid precursors, with the nitrogen atom being
inserted into the structure at a relatively late stage. The term
‘ppseudoalkaloid’ is sometimes used to distinguish this group. Such
structures are frequently based on terpenoid or steroidal skeletons, though
some relatively simple alkaloids also appear to be derived by similar late
amination processes.
• In most of the examples studied, the nitrogen atom is donated from an
amino acid source through a transamination reaction with a suitable
aldehyde or ketone.

1- Acetate derived alkaloids: Coniine
• Coniine is a simple piperidine alkaloid (but not originating from lysine!) that
is produced by poisonous hemlock (Conium maculatum; F. Umbelliferae).
• Coniine biosynthesis starts from a fatty acid precursor, octanoic acid, and
this is transformed to 5-oxo-octanal by successive oxidation and reduction
steps. The ketoaldehyde is then subjected to a transamination reaction, the
amino group originates from L-alanine. Subsequent transformations are
imine formation, giving the heterocyclic ring of -coniceine and reduction
to coniine.

2- Phenylalanine-derived Alkaloids: Ephedrine
• While the aromatic amino acid L-tyrosine is a common and extremely
important precursor of alkaloids, L-phenylalanine is less frequently
utilized; usually it contributes only carbon atoms, e.g. C6C3, C6C2 or C6C1
units, without providing a nitrogen atom from its amino group.
• Ephedrine, the main alkaloid in species of Ephedra (Ephedraceae) and a
valuable nasal decongestant and broncho-dilator, is a prime example,
where only the C6C1 fragment of ephedrine originates from L-
phenylalanine.
• The C6C1 fragment is acylated with pyruvate, with decarboxylation of
pyruvate occurring during the acylation, through a TPP-mediated
mechanism (lecture 2). This reaction yields a diketone, and a
transamination reaction would then give cathinone.
• Reduction of the carbonyl group from either face provides the
diastereomeric norephedrine or norpseudoephedrine. Finally, N-
methylation would provide ephedrine and pseudoephedrine.

2- Phenylalanine-derived Alkaloids: Ephedrine

2- Phenylalanine-derived Alkaloids: Capsaicin
• The amide capsaicin constitutes the pungent principal in chilli peppers
(Capsicum annuum; F. Solanaceae).
• It is used medicinally in topical pain-relieving preparations. The initial
burning effect of capsaicin is found to affect the pain receptors, making
them less sensitive.
• The aromatic portion of capsaicin is derived from L-phenylalanine
through ferulic acid and vanillin, this aldehyde being the substrate for
transamination to give vanillylamine.
• The acid portion of capsaicin is of polyketide origin, with a branched-
chain fatty acid being produced by chain extension of isobutyryl-CoA.
This starter unit is valine derived.

2- Phenylalanine-derived Alkaloids: Capsaicin???

2- Purine-derived Alkaloids: Caffeine
The purine ring is constructed by piecing together small components from
primary metabolism (i.e. glycine (C2N), formate (x2), bicarbonate, glutamine
and aspartic acid).

Biosynthesis lectures by Dr. Refaat Hamed

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