Structural Elucidation of a Diterpene Derivative from Stemodia Maritima

1
Structural Elucidation of a Diterpene Derivative
from Stemodia Maritima
Eugene E. Kwan and William F. Reynolds April 2003
2
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
3
History Structural elucidation of natural
products used to be very hard and take
forever. Strychnine alkaloid toxin Past H.
Leuchs worked on structure for 40 years until R.
Woodward beat him to it. Today lt1 mg
sample needed a weekend would be enough.
4

• 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.

5

• The NMR Experiment
• Modern NMR uses the FT pulse technique.

6
Outline
traditional treatment for venereal disease
7
Analysis
8
Preliminary Results Mass Spectrometry MW
444 bromine present 13C NMR (1D) 20
carbons ketone present (CO) 1H NMR (1D)
30 hydrogens HSQC NMR (2D) Deduced
Molecular Formula C20H30Br2O
9

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

10
Two Dimensional NMR 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
11
tROESY H-H spatial proximity
12

• Two Dimensional NMR
• B. HSQC (spectrally edited)
• connects each proton to its adjacent carbon
• separates overlapping peaks
• distinguishes between CH2, and CH3/CH
• C. HMBC
• shows C-H connections over more than one bond
• COSY
• shows H-H coupling mostly over one bond
• often shows peaks from coupling over multiple
  bonds
• interactions governed by coupling constants J

13
HSQC C-H connections
CH2
CH3
carbon axis
CH
projection on axes 1D spectrum
proton axis
14
Working Out Fragments I fragment part of
molecule C20 molecule from plant suggests
common diterpene fragment analysis of
data consistent with this structure
15
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 -
buttrouble! nothing fit!
16

• 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

17
The NOE Experiment How can we distinguish
between Nuclear Overhauser Effect (NOE)
Experiment
18
Accomplishing the Pulse In general, signals are
very close together, maybe 0.01 ppm!! How do we
only ping one proton?
19
The Answer
Connolly solvent accessible surface PM3 geometry
optimization
20
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.