CHEM 4126 / Topic 1: Tracer Experiments

What does the brief history of Natural Products Chemistry look like?

• Pre-history to early 19th century: What plant/microbe produces what response?

• 1800s to 1950s: Identify structure of biologically active natural products.

• 1950s to 1990s: Identify steps involved in biosynthesis of natural products & feeding of isotopically labeled precursors.

What are natural products, and why are they produced?

Compounds made by living organisms, mostly from secondary metabolism, produced due to interspecies competition and communication.

What’s the difference between primary and secondary metabolism?

• Primary metabolism: Essential network of all chemical reactions in a cell to carry out processes of life.

• Secondary metabolism: non-essential network of chemical reactions in a cell that produces compounds not required for basic life processes but that provide ecological/competitive advantages.

What does “non-producing organisms survive” mean in the context of secondary metabolism?

> Non-producing = no secondary metabolites.

> Still alive → growth + reproduction.

> Life functions intact.

Why study natural products?

• Major advances in civilization.

• Novel and complex structures.

• Diverse biological activities and biosynthetic pathways.

• Synthetic methodology.

• Drug discovery.

What does “biogenic origin” mean?

Source of a substance or material that is derived directly from biological organisms.

What are the biogenic origins of the following?

• Fatty acids and polyketides.

• Monoterpenes, diterpenes, and steroids.

• Polyphenols and aromatic amino acids.

• Non-ribosomal peptides.

• Carbohydrate-derived natural products.

*

What carries out reactions in natural product biosynthesis?

Enzymes catalyze all biosynthetic reactions.

• Why are enzymes essential in biosynthesis?

• Under what conditions do biosynthetic reactions occur?

• What are the key advantages of enzymatic catalysis in cells?

• Enzymes: ↓ E_a ↑ rate.

• General conditions: Physiological pH ≈ 7, room temperature, aqueous solution.

• *Advantages: High reaction rates, (chemio-, regio-, and stereo-) selectivity, and control under gentle conditions.

How are reactions in cells different and same from reactions carried out in labs?

• *Labs: Harsh reagents and conditions with slow rates.

• Cells: Requires cofactors and gentle conditions with faster rates.

• Both: Obey the same organic mechanisms and rules; enzymes may alter rate and selectivity but not chemical possibility.

• What does treating enzymes as “black boxes” imply?

• What does [O] represent in biosynthetic schemes?

• What does [H] represent in biosynthetic schemes?

• *Mechanistic details are ignored; only overall transformations matter.

• A generic oxidation catalyzed by an enzyme.

• A generic reduction catalyzed by an enzyme.

What is NADH analogous to in organic synthesis?

*NADH acts as a biological hydride donor, analogous to NaBH₄ in organic synthesis, enabling enzymatic reduction reactions.

What is cytochrome P₄₅₀ oxidase analogous to in organic synthesis?

*Cytochrome P450 oxidase acts as a biological C–H oxidation and hydroxylation system, analogous to strong chemical oxidants in organic synthesis, enabling selective oxygen insertion into organic substrates.

What are CoA thioesters analogous to in organic synthesis?

CoA thioesters act as biological activated acyl donors, analogous to activated esters or acyl halides in organic synthesis, enabling efficient acyl transfer and hydrolysis reactions under mild conditions.

How do radioactive atoms assist in isotope tracing for structure elucidation and therefore mechanism tracking in natural products? What’s the major downside?

• Emits β radiation → Sens det of where ¹⁴C is incorporated.

• Not direct NMR-based positional or connectivity info.

What info does feeding [1-¹³C]-labeled substrates provide, such as in polyketide synthesis?

It reveals which product carbons originate from a specific position of the precursor molecule.

What information does feeding [2-¹³C]-labeled substrates provide, such as in polyketide synthesis?

It identifies complementary carbon positions derived from the alternate carbon of the precursor.

Why is doubly labeled [1,2-¹³C] substrate especially powerful, such as in polyketide synthesis?

It allows detection of adjacent ¹³C-¹³C coupling, proving that two carbons are incorporated together from the same precursor unit.

• What does ¹³C–¹³C coupling in NMR indicate?

• Why is background ¹³C–¹³C coupling negligible in natural samples?

• Indicates that two adjacent carbons originate from an intact precursor fragment rather than random metabolic scrambling

• Because natural ¹³C abundance is ~1%, making adjacent ¹³C pairs extremely rare (~1 in 10,000).

What’s the core takeaway for isotope tracer experiments?

Radioactive labels track where atoms go, while stable isotope NMR reveals how atoms are connected in biosynthetic pathways

What does the first labelling experiment show about the source of oxygen atoms in prostaglandin (PG) synthesis?

→ Precursor reaction w/ ¹⁸O₂ + H₂¹⁶O.

→ COX₁ catalyzes conversion to PG = catalyzed for ^xO isotope incorporation.

→ O-11 and O-15 psitions contain ¹⁸O.

→ Confirms these oxygen atoms derive from molecular O₂.

In the PG tracer experiments, what limitation exists in interpreting the oxygen origin at the ketone position in the first experiment?

→ Ketone oxygen exchangeable w/ H₂O.

→ Exchange obscures original isotope source.

→ O-9 ≠ conclusively assigned but experimentally valid for O-11 and O-15.

In the PG tracer experiments, how does the second tracer experiment determine whether the two oxygen atoms come from the same O₂ molecule and its isotope source?

→ Precursor reaction under 50% ¹⁶O–¹⁶O and 50% ¹⁸O–¹⁸O.

→ Product immediately reduced with NaBH₄.

→ Locks oxygen positions thus preventing exchange.

→ Prepared for isotope analysis.

In the PG tracer experiments, how does the oxygen isotope ratios support the conclusion that both oxygen atoms originate from the same O₂?

Oxygen ratios measured by GC–MS = ¹⁶O–¹⁶O ≈ 40% ; ¹⁸O–¹⁸O ≈ 60% ; ¹⁶O–¹⁸O ≈ 1%.

∴ Both O atoms inserted together.

In the PG tracer experiments, explain the experimental design of the third tracer experiment used to study PG biosynthesis.

→ PG precursor dual-labelled w/ ³H on specific hydrogens and ¹⁴C on carbon backbone (doesn’t synthetically change).

→ Labelled substrate introduced into intact PG biosynthetic pathway.

→ Detection relies on β-particle emission → ³H and ¹⁴C distinguishable due to different β-particle energies.

∴ Allows monitoring of hydrogen vs. carbon fate.

In the PG tracer experiments, explain how the results of the third tracer experiment support a mechanism for PG biosynthesis.

→ ¹⁴C signal remains constant, confirming C backbone retention and confirms no degradation or loss of precursor bit.

→ ³H signal also remains constant, indicating retention.

∴ Biosynthesis = without proton abstraction or addition at labelled position.

In the PG tracer experiments, explain the experimental design of the fourth tracer experiment used to study PG biosynthesis.

→ Precursor labelled with (a) ³H placed stereospecifically on pro-R / pro-R hydrogen and (b) ¹⁴C placed as internal control.

→ Substrates fed into PG biosynthesis pathway and tested separately (pro-S vs. pro-R).

→ Products isolated.

∴ ³H/¹⁴C ratio measured to track hydrogen fate.

In the PG tracer experiments, explain how the results of the fourth tracer experiment support a mechanism for PG biosynthesis.

→ Pro-S labelled substrate has ³H/¹⁴C drops to ≈ 0% → loss of pro-S hydrogen during reaction.

→ Pro-R labelled substrate has ³H/¹⁴C largely retained → pro-R hydrogen retained / not removed.

→ ¹⁴C retained → C backbone intact.

∴ Mechanism confirms stereospecific hydrogen elimination of pro-S only.

What role did labelling experiments historically play in biosynthetic studies?

→ Isotope labelling → track atom fate.

→ Propose sequence of reactions in biosynthesis.

→ Prepare for more advanced experiments.

What’s the role of labelling experiments in modern biosynthesis research?

→ Rarely used to establish full pathways but applied to specific mechanistic steps.

→ Current focus is to link genes to enzymes to natural product biosynthesis.