1. Chiral Recognition detected by Mass Spectrometry CHEN Ping 2013.12.06 1

2. Outline I.Introduction II. Hyphenated Mass Spectrometric Techniques for Chiral Analysis III. Mass Spectrometric Chiral Recognition Mechanisms 1.Host-Guest (H-G) Associations 2.Guest Exchange Ion-Molecule Reactions 3.Chiral Recognition Based on Complex Dissociation IV. Application of Chiral Recognition Organocatalytic Asymmetric Conjungate Addition of Aldehydes to Nitroolefins V. Summary 2

3. More than half of the currently approved drugs are chiral molecules. Develop single enantiomer drugs Reducing the required dose Increasing the potency Improving the safety profile Asymmetric synthesis (Catalysts screening) Chiral analysis (Quality control) Chiral Recognition Mass Spectrometry Introduction Ranking of the top 10 best-selling US pharmaceutical products in 2011 was obtained from webpage: http://www.imshealth.com/. 7 of the 10 best-selling US pharmaceutical products are single enantiomer. 3

4. Traditionally, MS has been considered a “chiral-blind” technique Enantiomers: Same mass and show identical mass spectra Two strategies to differentiate a pair of enantiomers with MS 1. Coupling of chiral sensitive analytical tools with MS Liquid Chromatography-Mass Spectrometry (LC-MS) Gas Chromatography-Mass Spectrometry (GC-MS) … Introduction 2. MS is used solely in chiral analysis based on different methods of chiral recognition Host-Guest (H-G) Associations Guest Exchange Ion-Molecule Reactions Chiral Recognition Based on Complex Dissociation H. Awad, A. EI-Aneed, Mass Spectrom Rev, 2013, 32, 466–483 4

5. Liquid Chromatography-Mass Spectrometry (LC-MS) Gas Chromatography-Mass Spectrometry (GC-MS) Capillary Electrophoresis-Mass Spectrometry (CE-MS) Capillary Electrochromatography-Mass Spectrometry (CEC-MS) Supercritical Fluid Chromatography-Mass Spectrometry (SFC-MS) Coupling of chiral sensitive analytical tools with MS Hyphenated MS techniques New detector: Mass Spectrometer (MS)  Sensitive  Accurate  Speed  High throughput Advantages:Limitations:  Nonpolar solvents were incompatible with ESI or APCI  Salts and other nonvolatile compounds in the mobile phase were incompatible with ESI  Choosing chiral stationary phase is a daunting task H. Awad, A. EI-Aneed, Mass Spectrom Rev, 2013, 32, 466–483 5

6. Two options for chiral analysis using hyphenated MS techniques: Hyphenated MS techniques Indirect approach: Analysis of covalent diastereomeric complexes Separated by conventional methods CS: chiral derivatization reagent Direct approach: Analysis of noncovalent diastereomeric complexes CS: chiral mobile phase additives (CMPAs) chiral stationary phases (CSPs) Indirect approach: Need more time for the reaction step Direct approach is preferred H. Awad, A. EI-Aneed, Mass Spectrom Rev, 2013, 32, 466–483 6 Derivatized by the CS to form covalent complexes Form transient bond with CS

7. Hyphenated MS techniques The HPLC-MS chromatograms of (S,R) ifosfamide (IF) R. V. Oliveira, et al, J. Pharm. Biomed. Anal. 2007, 45, 295–303. 7

8. Two strategies to differentiate a pair of enantiomers with MS 1. Coupling of chiral sensitive analytical tools with MS Liquid Chromatography-Mass Spectrometry (LC-MS) Gas Chromatography-Mass Spectrometry (GC-MS) … 2. MS is used solely in chiral analysis based on different methods of chiral recognition Host-Guest (H-G) Associations Guest Exchange Ion-Molecule Reactions Chiral Recognition Based on Complex Dissociation Chiral Recognition Mechanisms 8

9. 1. Host-Guest (H-G) Associations One of the two enantiomers (guest) tagged with deuterium atoms CS (host) Ion abundance ratio: The affinity of each enantiomer towards the CS J. Kim, et al, Bull. Korean. Chem. Soc. 2008, 29, 1069-1072. 9

10. 2. Guest Exchange Ion-Molecule Reactions Unlabeled analyte enantiomers (guest) react with the CS (host) forming identical diastereomeric complexes Principle: Depends on the different exchange behavior of enantiomers with a foreign reagent R Chiral Recognition Mechanisms J. Ramirez, et al, J. Am. Chem. Soc. 1998, 120, 7387–7388. G. Grigorean, et al, Anal. Chem. 2001, 73, 1684–1691. 10 Can’t be separated in a single stage MS The complex ions are mass selected and allowed to react with a neutral gas-phase reagent R Different intensity ratio

11. Relative abundances based on two factors: the enantiomeric ratio of the used chiral analyte the time of the exchange reaction Solely varying the enantiomeric ratios of the chiral analytes Chiral Recognition Mechanisms 2. Guest Exchange Ion-Molecule Reactions J. Ramirez, et al, J. Am. Chem. Soc. 1998, 120, 7387–7388. G. Grigorean, et al, Anal. Chem. 2001, 73, 1684–1691. 11

12. Chiral Recognition Mechanisms 3. Chiral Recognition Based on Complex Dissociation The chiral analyte and chiral reference compound (ref*) are complexed with a transition-metal ion (M) to generate high-order metal ion-bound cluster ions W. A. Tao, R. G. Cooks, Anal. Chem. 2003, 25-31. 12

13. 3. Chiral Recognition Based on Complex Dissociation Chiral Recognition Mechanisms IRIR ISIS I ref*(1) I ref*(2) R chiral =1 : no chiral discrimination R chiral is more different from 1, the chiral recognition ability is higher W. A. Tao, R. G. Cooks, Anal. Chem. 2003, 25-31. R. Berkecz, et al, J. Mass. Spectrom. 2010,45, 1312–1319. 13 Chiral selectivity R chiral is defined as

14. 14 Mass Spectrometric Chiral Recognition Mechanisms 3. Chiral Recognition Based on Complex Dissociation Chiral Recognition Mechanisms 1. Host-Guest (H-G) Associations2. Guest Exchange Ion-Molecule Reactions

15. Application of Chiral Recognition 15 B. Florian, et al, Angew. Chem. Int. Ed. 2013, 52,1–6

16. Enamine mechanism Widely accepted Not been validated experimentally Application of Chiral Recognition 16 Possible mechanisms of amine catalyzed reaction of aldehyde with electrophiles Z. G. Hajos, D. R. Parrish, J. Org. Chem. 1974, 39, 1615 – 1621.

17. Addition reaction between aldehydes and nitroolefins catalyzed by H-d-Pro-Pro-Glu-NH 2 Excellent yields and stereoselectivities Catalyst loadings lower than 1 mol % Proposed catalytic cycle Problem: Enamine mechanism not been validated experimentally Experimental proof of enamine mechanism: Detect an enamine intermediate by ESI-MS Application of Chiral Recognition 17 Enamine mechanism

18. Methodology: ESI-MS back-reaction screening A pair of mass-labeled quasienantiomeric conjugate addition products Concept: Host-Guest (H-G) Associations Host (Chiral Selector) Guests Application of Chiral Recognition 18

19. The stereoselectivity 2/ent-2’(= k 1 /k 2 ) is determined by ΔΔG ≠ of the transition state. If En/En’ ratio (back reaction) = 2/ent-2’ ratio (forward reaction), it will provides strong evidence to enamine mechanism. Application of Chiral Recognition 19 R=k 1 /k 2 = I En /I En’ = e ΔΔG≠/RT ent-2’ 2 Im Im’ En En’ ΔΔG ≠ Back reaction

20. Back-reaction screening and enantioselectivity of the forward reaction in DMSO Application of Chiral Recognition 20 En/En’ (back reaction) = 2/ent-2’ (forward reaction): Enamine mechanism Stereomeric determining step is En to Im.

21. Additional organocatalysts investigated in this study Application of Chiral Recognition Catalyst Screening 21

22. I. Chirality is significant Summary 22 II. Concepts of hyphenated MS techniques III. Mass Spectrometric Chiral Recognition Mechanisms Host-Guest (H-G) Associations Guest Exchange Ion-Molecule Reactions Chiral Recognition Based on Complex Dissociation Hyphenated MS techniques IV. An example that using chiral recognition to solve mechanistic problem

23. What can we do by using MS? Mass Spectrometry 23

24. Studying Reaction Mechanism 24 Interesting reaction systems Propose reaction mechanism Combine MS with DFT calculation

25. Catalysts Screening by Mass Spectrometry Simultaneous screening of a mixture of five catalysts C. Markert, A. Pfaltz, Angew. Chem. Int. Ed. 2004, 116, 2552-2554 25

26. Thanks for your attention! 26

27. Mass Spectrometry to study reaction mechanism ESI-MS to capture reaction intermediates Propose reaction mechnism Combined with DFT calculation 27 H. Guo, et al, J. Am. Chem. Soc. 2005, 127, 13060-13064

28. Chiral Recognition Mechanisms 3. Chiral Recognition Based on Complex Dissociation W. A. Tao, R. G. Cooks, Anal. Chem. 2003, 25-31. 28 Metal: Cu 2+ Ref: two L-Trp Analytes: (+)-ephedrine (–)-ephedrine Cu 2+ (L-Trp) 2 (+)-ephedrine Cu 2+ (L-Trp) 2 (-)-ephedrine Chiral selectivity R chiral : I + /I ref*(1) = 3.8 I - /I ref*(2) = 0.91 R chiral = 4.7 -A -Ref The interaction between (+)-ephedrine and ref* is stronger

29. Chiral Drug Asymmetric Synthesis Chiral Analysis Chiral Resolution Chiral Recognition Mass Spectrometry Introduction PPT from Xinhao Catalyst screening Quality control Chromatography 29

30. Catalysts Screening by Mass Spectrometry Screening Methodology Mass Spectrometric Screening of Their Racemic Forms 30

31. Conformation Analysis by Ion Mobility Spectrometry-Mass Spectrometry Drift time versus m/z plot measured by Mass Spectrometer Conformers produced for cyclic peptide from Molecular Dynamics simulations Plot of Normalized MD energy versus collision cross-section from the simulated annealing T. R. Brandon, J. Am. Soc. Mass. Spectrom. 2004, 15, 870-878 31

32. Structural Characterization of Oligomer-Aggregates of β-Amyloid Polypeptide ESI-mass spectra (LC-MS) of Aß(1–40) 32

33. 3. Chiral Recognition Based on Complex Dissociation Chiral Recognition Mechanisms Quantitative chiral analysis The relative rates of the two competitive dissociations (k A and k ref ) can be expressed as the relative abundance ratio: Calibration curves for chiral analysis Different ratio of A R and A S W. A. Tao, R. G. Cooks, J. Am. Chem. Soc., 2000, 122, 10598-10609 △ [Cu II (Pro) 2 (Tyr)-H]+ complex, Pro as the analyte [Cu II (Phe) 2 (Ile)-H]+ complex, Phe as the analyte  [Cu II (Trp) 2 (Met)-H]+ complex, Trp as the analyte 33

34. 3. Chiral Recognition Based on Complex Dissociation Chiral Recognition Mechanisms Quantitative chiral analysis W. A. Tao, R. G. Cooks, J. Am. Chem. Soc., 2000, 122, 10598-10609 34