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Structural Elucidation of a Diterpene Derivative from
Stemodia MaritimaBr
H
BrH
O
Eugene E. Kwan and William F. Reynolds
April 2003
BackgroundPurpose: 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
HistoryStructural elucidation of natural products used to be very hard and take forever.
Strychnine alkaloid toxin Past: H. Leuchs workedon structure for 40 yearsuntil R. Woodward beathim to it.
Today: <1 mg sampleneeded; a weekend wouldbe 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.
Outlineidentify a promising plant:
stemodia maritima
extract the interestingorganic material
separate via HPLC
analyze the fractionsmyjob
traditional treatment for venereal disease
Analysispreliminary analysis
detailed spectroscopicinvestigation
work out fragments
guess at structure
check for consistency;assign peaks
Preliminary ResultsMass Spectrometry MW = 444
bromine present
13C NMR (1D) 20 carbons
ketone present (C=O)
1H NMR (1D) & 30 hydrogensHSQC NMR (2D)
Deduced Molecular Formula: C20H30Br2O
1H Proton SpectrumOne 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 NMRTwo Dimensional NMR
- two dimensional = two frequency domains- can show H-H or H-C interactions through space or through bonds
Key ExperimentsA. 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 NMRB. HSQC (spectrally edited)
connects each proton to its adjacent carbon
separates overlapping peaks
distinguishes between CH2, and CH3/CH
C. HMBCshows C-H connections over more than one bond
D. COSYshows 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
CH2
CH3
proton axis
carb
on a
xis
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
CH3
H3C
CH3
H
O ?
common trans-decalin fragment
?
probable
Working Out Fragments II
- further analysis suggested:
- what was the rest? what are the possibilities?
- molecular formula and lack of double bonds in NMR suggestsfour rings:
- but…trouble! nothing fit!
O
O O
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
O Br
Br
stereochemistryambiguous!
The NOE ExperimentHow can we distinguish between:
“Nuclear Overhauser Effect” (NOE) Experiment
H
H R
R' R
H H
R'vs. ?
H
H R
R'R
H H
R'
excite thisproton
resonanceseen here
vs.
noresonance
time
pulse
Fourier
Transform
frequency
time
"shaped" pulse
Fourier
Transform
frequency
Accomplishing the PulseIn general, signals are very close together, maybe 0.01 ppm!!How do we only “ping” one proton?
The Answer
Br
H
BrH
OConnolly solvent
accessible surface;PM3 geometry optimization
AcknowledgementsProf. 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.
Br
H
BrH
O