Structural Elucidation of Novel Diterpene Derivative from xxx

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					Structural Elucidation of
 a Diterpene Derivative
from Stemodia Maritima
                                           Br
    O




                                  H




                              H
                                      Br

   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.

Strychnine alkaloid 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
      identify a promising plant:   traditional treatment
         stemodia maritima          for venereal disease



        extract the interesting
           organic material


         separate via HPLC


my
job     analyze the fractions
  Analysis
 preliminary analysis


detailed spectroscopic
     investigation


 work out fragments


  guess at structure


check for consistency;
    assign peaks
           Preliminary Results
Mass Spectrometry       MW = 444

                        bromine present

13C NMR (1D)            20 carbons

                        ketone present (C=O)

1H NMR (1D) &           30 hydrogens
HSQC NMR (2D)


Deduced Molecular Formula: C20H30Br2O
            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 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
tROESY: H-H spatial proximity




              “off-diagonal
               cross peak”


                 “diagonal
                   peak”

                         projection on axes =
                             1D spectrum
         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

D. COSY
     shows H-H coupling mostly over one bond

      often shows peaks from coupling over multiple bonds

      interactions governed by coupling constants J
HSQC: C-H connections
carbon axis




                    CH2
                                   CH3

              CH



                          projection on axes =
                              1D spectrum



                                proton axis
        Working Out Fragments I
“fragment” = part of molecule

C20 molecule from plant: suggests common diterpene fragment

             O       ?              CH3 probable
                            CH3

                 ?
                     H3C
                             H
                 common trans-decalin fragment

analysis of data: consistent with this structure
       Working Out Fragments II
                                O
- further analysis suggested:



- what was the rest? what are the possibilities?

- molecular formula and lack of double bonds in NMR suggests
four rings:
  O                                   O




- but…trouble! nothing fit!
      Clever Thinking – A Guess
- much thought + some help produced a complete structure:
         O                      Br

                                      stereochemistry
                                        ambiguous!

                             Br

- 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:
          H        R                     H       H
                             vs.                                 ?
      R'                 H         R'                    R


“Nuclear Overhauser Effect” (NOE) Experiment
 excite this           resonance
  proton               seen here                                 no
                                             H       R       resonance
           H     H
                             vs.
    R'               R              R'                       H
       Accomplishing the Pulse
In general, signals are very close together, maybe 0.01 ppm!!
How do we only “ping” one proton?
             pulse
                            Fourier

                           Transform

                 time                           frequency


          "shaped" pulse


                                      Fourier

                                  Transform

                        time                    frequency
                     The Answer




                                               Br
                            O
    Connolly solvent
   accessible surface;
PM3 geometry optimization             H




                                  H
                                          Br
           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                           Br
                         O


Jordan Dinglasan: T.A.
                                                   H




                                               H
                                                       Br

				
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