The document discusses various techniques used to trace the biosynthesis of primary and secondary metabolites using radioactive isotopes, including precursor-product analysis, competitive feeding, and sequential analysis. It provides examples of how these techniques have been applied since 1934 to further understand biogenesis pathways and establish biosynthetic relationships between compounds.

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Dr. Amit Gangwal
Associate Professor
Shri Vile Parle Kelavani Mandal’s
Institute of Pharmacy, Dhule
Third Year B. Pharm. (Semester- V)
[Pharmacognosy & Phytochemistry II- (BP504T)]
Study of utilization of radioactive isotopes
in the investigation of biogenetic studies
Precursor-Product Analysis Competitive Feeding Sequential Analysis
Since 1934
To trace the journey of biogenesis of
primary & secondary metabolites

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Glycolysis
Glucose
Hexose Kinase
ATP
ADP
Glucose 6 Phosphate
Phospho glucose isomerase
Fructose 6 Phosphate
Phospho Fructose kinase
ATP
ADP
Fructose 1, 6 Bis Phosphate
GLAP DHAP
Trios PO4 isomerase
1,3 Bisphospho Glycerate
2 NAD+ + 2Pi
2 NADH
3 Phosphate Glycerate
Phospho glycerate kinase
2 ATP
2 ADP
2 Phosphate Glycerate
Phospho glycerate mutase
Phospho enol pyruvate
enolase
Pyruvate
Pyruvate kinase
2 ATP
2 ADP
aldolase
GLAP hydrogenase
DHAP: di hydroxy acetone phosphate
GLAP: Glyceraldehyde 3 Phospahte

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• Sugar or glucose alternatively enters pentose phosphate pathway
(HMP)
• It helps in formation of NADPH from fatty acids, synthesis of ribose
for nucleotide & nucleic acid (oxidative pathway)
• Helps in formation of erythrose 4 phosphate, used in synthesis of
aromatic amino acids through shikimic acid pathway.
(non oxidative)
↓ precursor for lignin, & other biosynthetic pathways.
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HMP SHUNT PATHWAY
Glucose 6 Phosphate
NADP+
NADPH + H+
Mg+2 Glucose 6 P
dehydrogenase
6 Phosphoglucanolactone
Glucanolactone
hydrolase
6 Phosphogluconate
NADP+
Mg+2
CO2, NADPH + H+
Phosphogluconate
dehydrogenase
Ribulose 5 phosphate

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Acetate Pathway
Glycolysis
Pyruvate
End product
Acetyl Co-A
TCA
Acetate mevalonate pathway Acetate melonate pathway-
mevalonic acid Malonyl Co-A
Higher terpenoids,
rubber etc.
Long chain fatty acids &
polyketides
Isoprenoids
Squalene
Steroids
Lipids, fats , waxes

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Iso Pentenyl Pyrophosphate + Di Methyl Allyl Pyro Phosphate (active isoprene units)
CONDENSE
Geranyl pyropyrophosphate C10 Monoterpenes e. g. Menthol, limonene,
geraniol, linalool, citral
Farnesyl PP C15
Sesquiterpenes e.g. termerone,
zingiberene, abscisic acid, santalone
IPP
C30
Triterpenoids
Squalene
Steroids, cholesterol
Solanine, diosgenin
IPP
Diterpenes Taxol
Polyterpenes Rubber
C40 Tetraterpenes
Carotenoids, lycopene, β carotene

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Methods in Tracer
Technique

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Incorporation of doubly-labelled lysine into anabasine
Precursor-Product Analysis

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Competitive Feeding

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Tyrosine
DOPA
3, 4 dihydroxy phenyl pyruvic acid
Similarly it was considered that 3, 4 dihydroxy phenyl pyruvic acid would
also be synthesized through DOPA but by labeling experiments and
competitive feeding it is confirmed that tyrosine directly gives 3, 4
dihydroxy phenyl pyruvic acid and there is no roll of DOPA in
biosynthesis of 3, 4 dihydroxy phenyl pyruvic acid.
correct

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• The principle of this method of investigation is to grow plant in
atmosphere of 14CO2 and then analyze the plant metabolites at a given
time intervals to obtain the sequence in which various related compounds
become labeled.
• Example: Mentha piperita 14 CO2 for about 5 mins provided the evidence
of probable biosynthetic sequences.
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O O OH
Piperitone Menthone Menthol
Sequential analysis method

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Thanks