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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:

Download the poster here

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.