Structural Elucidation of a Diterpene Derivative from Stemodia Maritima Eugene E. Kwan and William F. Reynolds April 2003

Background Purpose: determine the structure of a natural product via NMR and MS. Q: Why study natural products? A: New pharmaceuticals. e.g., quinine, digitalis, cyclosporin, penicillin… - very difficult to design pharmaceuticals - takes advantage of traditional folk knowledge - even animals known to use plants for medicine

History Structural elucidation of natural products used to be very hard and take forever. Strychninealkaloid toxin Past: H. Leuchs worked on structure for 40 years until R. Woodward beat him to it. Today: <1 mg sample needed; a weekend would be enough.

Nuclear Magnetic Resonance - Modern structural elucidation relies on NMR. - Nuclear spin energies: quantized. - Can observe transitions in magnetic field. - Transition energy depends on field strength. - Each nucleus experiences a local magnetic field, which is slightly different from the bulk magnetic field. This difference reveals different chemical environments. -A molecule has many different nuclei, each with different magnetic resonance frequencies.

The NMR Experiment -Modern NMR uses the “FT pulse” technique.

Outline traditional treatment for venereal disease

Analysis

Preliminary Results Mass SpectrometryMW = 444 bromine present 13C NMR (1D)20 carbons ketone present (C=O) 1H NMR (1D) &30 hydrogens HSQC NMR (2D) Deduced Molecular Formula: C 20 H 30 Br 2 O

1H Proton Spectrum One Dimensional NMR - one dimensional = one frequency domain - complicated spectrum; many overlapping peaks (1)Each proton has a peak. (2)Each peak has a splitting pattern. (3)Splitting pattern = H-H spin coupling.

Two Dimensional NMR - two dimensional = two frequency domains - can show H-H or H-C interactions through space or through bonds Key Experiments A. tROESY tells if two protons are close in space 1D proton spectra appear on x and y axes (“f1, f2”) if two protons are near each other, “cross peak” helps determine absolute stereochemistry

“diagonal peak” “off-diagonal cross peak” projection on axes = 1D spectrum tROESY: H-H spatial proximity

Two Dimensional NMR B. HSQC (spectrally edited) connects each proton to its adjacent carbon separates overlapping peaks distinguishes between CH 2, and CH 3 /CH C. HMBC shows C-H connections over more than one bond D.COSY shows H-H coupling mostly over one bond often shows peaks from coupling over multiple bonds interactions governed by coupling constants J

projection on axes = 1D spectrum CH CH 2 CH 3 proton axis carbon axis HSQC: C-H connections

Working Out Fragments I “fragment” = part of molecule C20 molecule from plant: suggests common diterpene fragment analysis of data: consistent with this structure

Working Out Fragments II - further analysis suggested: - what was the rest? what are the possibilities? - molecular formula and lack of double bonds in NMR suggests four rings: - but…trouble! nothing fit!

Clever Thinking – A Guess - much thought + some help produced a complete structure: - funny: ring system too complicated to be named by computer - stereochemistry ambiguous at bromines - parts of tROESY “fuzzy”, needed better technique: 1D NOE

The NOE Experiment How can we distinguish between: “Nuclear Overhauser Effect” (NOE) Experiment

Accomplishing the Pulse In general, signals are very close together, maybe 0.01 ppm!! How do we only “ping” one proton?

The Answer Connolly solvent accessible surface; PM3 geometry optimization

Acknowledgements Prof. Reynolds: product sample, spectral acquisition & processing, help with structural elucidation Prof. Reese, University of the West Indies: sample acquisition and extraction; preliminary analyses Tim Burrow: NMR spectrometer help Jordan Dinglasan: T.A.