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EnantiodiscriminationEnantiodiscrimination Studies by Studies by 1313C DNPC DNP‐‐NMR SpectroscopyNMR Spectroscopy
Eva Monteagudo Eva Monteagudo , , MíriamMíriam PérezPérez‐‐Trujillo, Trujillo, TeodorTeodor ParellaParella
Servei de Ressonància Magnètica Nuclear, Servei de Ressonància Magnètica Nuclear, UniversitatUniversitat AutònomaAutònoma de Barcelona, de Barcelona, BellaterraBellaterra, , CataloniaCatalonia, , SpainSpain
Why differentiating enantiomeric molecules?
SyntheticSynthetic Chiral CompoundsChiral CompoundsNatural Natural Chiral CompoundsChiral Compounds
PharmaceuticalsPharmaceuticals
DrugsDrugs Reactants …Reactants …
SugarsSugars Amino acidsAmino acids
TerpensTerpens Enzymes …Enzymes …
Different pharmacological activity, toxicity, reactivity
Different biological activity &functionality
ProteinsProteinsDNADNA
RR ++ SS
CSA*CSA* RR
SS
CSA*CSA*
CSA*CSA*
++
CSA & 1H NMR Spectroscopy for Enantiodifferentiation Applications
Organic synthesis
Pharmacology
Chiral Metabonomics[1]
Natural Products
Toxicity Studies
RR SS
Diastereoisomeric complexes
RR + + SS
Example of enantiodifferentiation of a racemic mixture by 1H NMR using a chiral solvating agent (CSA)
…
1H Simple signals (singlets)
Larger chemical shift range
Poor sensitivity
Large acquisition times
13C Easy and fast enantiomeric excess measurement through
signal integration
Signal complexity (multiplets)
Signal overlappingHamper the
enantiodifferentiation study
RR SS
How to avoid How to avoid 11H NMR H NMR drawbacks? drawbacks?
How to avoid How to avoid 11H NMR H NMR drawbacks? drawbacks?
How to avoid How to avoid 1313C NMR C NMR drawbacks? drawbacks?
How to avoid How to avoid 1313C NMR C NMR drawbacks? drawbacks?
Enantiodifferentiation by dissolution 13C DNP‐NMR
RR SS
RR SS RR SS
Enhanced signals
Single scan 13C NMR
enantiodifferentiation study
Download the
Transfer solvent +
CSA*CSA*
CSA*CSA*
SS
CSA*CSA*
++
RR
Trityl radical
Chiral analyte
Glassing agent
R R SS
Sample:
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HyperSense® (Oxford Instruments) Bruker 600 UltraShieldTMsample cup
Insert sampleInsert sampleInsert sampleInsert sample Sample dissolutionSample dissolutionSample dissolutionSample dissolution AcquisitionAcquisitionAcquisitionAcquisition
RR SS
C3 = 33 Hz
C4 = 16 Hz
C5C2
Sample (50 l) Polarization Dissolution
Chiral analyte
RadicalGlassing Agent
MW freq.Polarization
time Transf. solvent
(5 ml)Transf.time
Methionine OX63 H2O:glycerol 94.078 4h D O 3
13C All (FIVE) carbon signals enantiodifferentiated Dissolution 13C DNP‐NMR Enhancement of all (FIVE) carbon signals
RS‐methionine (‐)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid(18C6H4)
Chiral analyte CSA
2.41 mM RS‐methionineexpt. time = 17 h
1a)
1b)
H5 = 4.2 Hz
18C6H4
H2 = 48 Hz
= 5 Hz
TSP
C2 = 12 HzC1
= 10 Hz
**
No enantiodiff. No
enantiodiff.
2a)
2b)
glycerol
C5
C1
C2 C3 C4
(232 mM) (15 mM)2 g y(1:1) GHz
≈ 4h D2O 3 s
H5
H3
H4H2
18C6H4
C1
C2C3
C4C5
With CSA
No CSA
Only TWO proton signals enantiodifferentiated1H
With CSA
No CSA No CSA
2.32 mM hyperpolarized RS‐methionineexpt. time = 1 s
1a)
Figure 1. a) 1H NMR 250 mM racemic methionine in D2O; b) 1H NMR 2.41 mMRS‐methionine, 46 mM (19 eq.) 18C6H4 in D2O. Experiments performed in a 500MHz spectrometer equipped with TCI cryoprobe and using TSP as externalreference.
2a)
Figure 2. a) 13C NMR 250 mM racemic methionine in D2O; b) 13C NMR 2.41 mMRS‐methionine, 46 mM (19 eq.) 18C6H4 in D2O. Experiments performed in a 500MHz spectrometer equipped with TCI cryoprobe and using TSP as externalreference.
← Figure 4. C1 hyperpolarized signal of RS‐methionine (2.32 mM, nat. abu.) throughSSFT/FLASH method[2] a) experiments withCSA (10 eq. 18C6H4) b)without CSA
→ Figure 5. Apparent T1 relaxation time ofC1 hyperpolarized signal of RS‐methioninefrom Fig 4 The T for the other protonated
Figure 3. 13C DNP‐NMR 600 MHz spectrum of a hyperpolarized dissolution of2.32mM natural abundance RS‐methionine.
C1
No CSA No CSA No CSA
SUMMARYSUMMARY::
4b)No CSA
0,3
0,5
0,7
0,9
1,1
sign
al in
tensity [a.u.]
methionine methionine + 18H6H4
T1 = 12 s
T1 = 5.8 s
So far, no studies of chiral discrimination have been performed usingdissolution 13C DNP‐NMR although this methodology overcomes the maindrawbacks of both 1H and 13C NMR experiments.
• The RS‐methionine sample preparation, polarization, dissolution andNMR experiment have been optimized in order to obtain its enhancedsignals in a single scan 13C DNP‐NMR experiment.
• The formation of RS methioine/18C6H complex has been
No CSA
With CSA
44thth International DNP Symposium. 28International DNP Symposium. 28thth ‐‐ 3030thth August, Technical University of August, Technical University of Denmark Denmark SeRMNSeRMN –– UAB blog: http://sermn.uab.cat UAB blog: http://sermn.uab.cat 44thth International DNP Symposium. 28International DNP Symposium. 28thth ‐‐ 3030thth August, Technical University of August, Technical University of Denmark Denmark SeRMNSeRMN –– UAB blog: http://sermn.uab.cat UAB blog: http://sermn.uab.cat
[1] Pérez‐Trujillo, M. Lindon, J.C., Parella, T., Keun, H., Nicholson, J.K., Athersuch, T.J. Anal. Chem. 2012, 84, 2868‐2874.
[2] Day, I.J. Mitchell, J.C. Snowden, M.J. Davis, A.L. J. Magn. Reson. 2007, 187, 216‐224.
Financial support for this research provided by MICINN (project CTQ2012‐32436) and Bruker Española S.A. are gratefully acknowledged. We also thank to the SeRMN, Universitat Autònoma de Barcelona, for allocating instrument time to this project.
Acknowledgements
from Fig 4. The T1 for the other protonatedcarbons were too short to be accuratelymeasured.
4a)With CSA
ns
‐0,1
0,1
0 20 40 60 80 100 120 140time [s]
• The formation of RS‐methioine/18C6H4 complex has beendemonstrated by the decrease of the T1 relaxation time value.
• Further work is being done on the resolution optimization ofhyperpolarized racemic methionine + 18C6H4.