You are on page 1of 16

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 GmbH, Rheinstetten, Germany
3Department 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 Cicutoxin


HO Isocicutoxin

HO

HO

E
Ferdinand Bohlmann
OH
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
O H Assuming co-planar arrangement
of CF3-Cα-C(O)-O-C(OH)
F3C
O L1 L2 shielded in (S)-MTPA Ester
(S)-MTPA L1 shielded in (R)-MTPA Ester
MeO L2
THUS:

δL2(S) < δL2(R) and δL1(S) > δL1(R)

shielding
∆δSRL2 = δL2(S) - δL2(R) < 0

and
shielding
∆δSRL1 = δL1(S) - δL1(R) > 0
(R)-MTPA MeO L2
O L1
F3C
O H

Kakisawa model
Experimental details
SAMPLES
7 samples + achiral control (cicudiol) 1
HO
Established model system falcarinol (1)
Conventional structure elucidation OH
2
HO
REACTION
~2 mg/analyte 3
~10 mg (R) or (S)-MTPA-Cl, pyridine, r.t. HO

Evaporation of pyridine, reconst. in ACN OH

4
SEPARATION
OH
ACN/H2O (80->98%), HPLC-DAD and
HPLC-SPE-NMR (~20 µg/column) 5

TRAPPING / NMR 6 OH
SPE trapping on GP phase, release in CDCl3 OH
1H NMR for all esters (ns = 128, 500 MHz)

19F NMR for selected analytes 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) III


R R II => (R)

III
R
II
=> (R)
R
Aethusanol A (4)
Results
OH

•LC-SPE-NMR: (R)

4 OH

- no sample workup
1 (R)
(R)
HO

- CDCl3 for transfer


5
(S)
2 (R) OH

(S) OH
HO
- <20 µg analyte 3
6 OH

(R) (R)
HO
AcO

•Mosher Ester:
7

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

You might also like