structural elucidation of a diterpene derivative from stemodia maritima eugene e. kwan and william...

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