Reaction product analysis by HPLC-SPE

University of Innsbruck
Institute of Pharmacy / Pharmacognosy
Reaction product analysis by HPLC-SPE-NMR:
Application to the absolute configuration determination
of naturally occurring polyyne alcohols
Christoph Seger1, Markus Godejohann2,
Manfred Spraul2, Hermann Stuppner1, Franz Hadacek2
1Institute
of Pharmacy, Pharmacognosy, University of Innsbruck, Innsbruck, Austria
2Bruker-Biospin
3Department
GmbH, Rheinstetten, Germany
of Chemical Ecology and Ecosystem Research, University of Vienna, Austria
Polyacetylenes
The analytes
Mainly found in Asteraceae and Apiaceae
> 1000 derivatives known
-
rod like structures
highly lipophilic
sometimes toxic
instable (isomerization, oxidation)
most often poorly described
1
Cicuta virosa toxins
HO
Cicutoxin
Isocicutoxin
HO
HO
Z
E
OH
Ferdinand Bohlmann
Scope of the project
Absolute configuration
of secondary OH functions
-X-Ray diffraction
-Synthesis
-Circular dichroism (CD)
-Chiral lanthanide induced shift (LIS)
-Derivatization (=Acylation) & NMR
Mosher’s method
Hoereau’s method
Uwai et al. 1999
Otha et al. 1999
Mosher’s Method
Absolute configuration determination of
chiral secondary hydroxy functions
- Based on formation of diastereomeric esters.
- Empirical rules well understood, limitations known.
- Simple reaction setup.
- Applied to polyacetylenes.
Dale & Mosher 1969, 1973
Usual workflow
• react analyte with chiral MTPA-Cl in DCM or pyridine.
• workup by preparative chromatography.
• analysis by NMR spectroscopy (∆δ values).
• application of empirical rules (Kakisawa 1991)
Mosher reaction
NMR
Seco JM, Quinoa E, Riguera R Chem Rev (2004) 104: 17.
Mosher - Model
H
O
F3C
(S)-MTPA
O
Assuming co-planar arrangement
of CF3-Cα-C(O)-O-C(OH)
L1
L2
MeO
L2 shielded in (S)-MTPA Ester
L1 shielded in (R)-MTPA Ester
THUS:
δL2(S) < δL2(R) and δL1(S) > δL1(R)
shielding
shielding
(R)-MTPA
MeO
L2
L1
O
F3C
O
∆δSRL2 = δL2(S) - δL2(R) < 0
and
∆δSRL1 = δL1(S) - δL1(R) > 0
H
Kakisawa model
Experimental details
SAMPLES
7 samples + achiral control (cicudiol)
Established model system falcarinol (1)
Conventional structure elucidation
1
HO
OH
2
HO
REACTION
~2 mg/analyte
~10 mg (R) or (S)-MTPA-Cl, pyridine, r.t.
Evaporation of pyridine, reconst. in ACN
SEPARATION
3
HO
OH
4
OH
ACN/H2O (80->98%), HPLC-DAD and
HPLC-SPE-NMR (~20 µg/column)
TRAPPING / NMR
SPE trapping on GP phase, release in CDCl3
1H NMR for all esters (ns = 128, 500 MHz)
19F NMR for selected analytes
5
6
OH
OH
7
AcO
LC-SPE-NMR setup
HPLC-DAD (separation)
Agilent 1100
used as usual with regular solvents
SPE (transfer)
Spark; 96 well format
Solid phase extraction allows
analyte selection and solvent
change to NMR solvents
NMR (analysis)
allows all 1D and 2D NMR experiments.
Advantages of the LC-SPE-NMR setup
No analyte isolation needed
No contamination
No loss by adsorption, oxidative breakdown, or salt formation
Sample amount needed
Sub mg as present in HPLC peaks
Peak enrichment on SPE
Multiple trapping, narrow elution band
Solvent change
HPLC in HPLC grade solvent
NMR in deuterated solvent
Removal of solvent additives
Simplification of solvent suppression
Well defined NMR conditions = full data comparability
Saving of deuterated solvents
Limitations of using LC-SPE-NMR ?
HPLC knowledge needed
Only optimized chromatography will
lead to decent results
Little experience in average NMR lab
BUT:
… its easy and straightforward …
Trapping process in the SPE unit
Works for 95 % of all analytes
Need to optimize trap and release
on an analyte specific basis
BUT:
Tremendous knowledge from offline SPE available
Limitations are well known
HPLC-UV data
HPLC-DAD at 205 nm
Clear separation
Ester / Alcohols
Monitoring of
Reaction progress
Sample degradation
Solvent residues
Derived 1H and 19F NMR spectra
8-(S)-Ester
δS values
8-(R)-Ester
δR values
Application of empirical rules
Kakisawa model
¾ arrange residues in the model
¾ apply CIP rules
¾ assign stereodescriptor R/S
Configuration determination
Falcarinol (1)
R
III
R
II
=> (R)
Aethusanol A (4)
R
III
R
II
=> (R)
Results
OH
•LC-SPE-NMR:
- no sample workup
- CDCl3 for transfer
- <20 µg analyte
(R)
OH
4
1
(R)
(R)
HO
5
2
(S)
OH
(R)
(S)
HO
OH
OH
6
3
(R)
(R)
HO
•Mosher Ester:
7
AcO
RED Mosher derived
- most analytes stable
BLUE [α]D derived
- failed to get esters at C-8 in
falcarinol-type polyacetylenes
•Structural data:
- good agreement with literature data / optical rotations
- absolute configuration of rare analytes determined
Seger, Godejohann et al. J Chromatogr A (2006) in press
Thank you for your attention
Christoph.Seger@uibk.ac.at