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High-Field NMR Experiments in the Upper-Level Laboratory Courses at Furman University

High-Field NMR Experiments in the Upper-Level Laboratory Courses at Furman University . Tim Hanks, Moses Lee and Larry Trzupek Dept. of Chemistry, Furman University. Current NMR Instrumentation. Varian EM-360A (1981; $31,000) CW; H-1 only

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High-Field NMR Experiments in the Upper-Level Laboratory Courses at Furman University

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  1. High-Field NMR Experiments in the Upper-Level Laboratory Courses at Furman University Tim Hanks, Moses Lee and Larry Trzupek Dept. of Chemistry, Furman University

  2. Current NMR Instrumentation Varian EM-360A (1981; $31,000) CW; H-1 only Varian VXR-300S (1988; $329,000) FT; H-1/C-13 and multinuclear; VT; computer system updated to Sun SPARCstation 5 in 1996 Varian Inova 500 (1996; $454,000) FT; indirect detection probe, H-1 {N-15/P-31}; VT; Sun SPARC station 5 computer system.

  3. Calendar/Chemistry Curriculum at Furman

  4. Enantioselective Epoxidation (Hanks; Chem 23) NMR Techniques: - H-1 NMR analysis of fructose-based intermediates, -methylstyrene, and epoxide product - C-13 NMR analysis of the chiral catalyst and epoxide product - use of chiral lanthanide shift reagent for determination of enantiomeric purity

  5. Enantioselective Epoxidation (Hanks; Chem 23)

  6. Enantioselective Epoxidation (Hanks; Chem 23) (33%) (86%)

  7. Enantioselective Epoxidation (Hanks; Chem 23)

  8. Enantioselective Epoxidation (Hanks; Chem 23) H-1 NMR analysis, trans--methylstyrene, vinyl region 6.6 6.4 6.2

  9. Enantioselective Epoxidation (Hanks; Chem 23) C-13 NMR analysis, chiral oxirane precursor 180 140 100 60 20 ppm

  10. Enantioselective Epoxidation (Hanks; Chem 23) NMR determination of enantiomeric purity using Eu(hfc)3 4.8 4.4 4.0 3.6 3.2 ppm

  11. Enantioselective Epoxidation (Hanks; Chem 23) NMR determination of enantiomeric purity; results Typical yield of epoxide product: 60% Typical enantiomeric excess: 84%ee Reference: “Catalytic Asymmetric Epoxidation Using a Fructose-Derived Catalyst”; Andy Burke, Patrick Dillon, Kyle Martin and Tim Hanks, J. Chem. Ed., accepted for publication, 1999.

  12. Features: - Microscale preparation - Multi-step reaction sequence - Use of basic 2-D NMR to establish structure Initial Reactants: cyclopentadiene, maleic anhydride Target Compound: endo-9-methoxycarbonyl-3-oxatricyclo[4,2,1,0]-2-nonane Structure of a Tricyclic Compound (Lee; Chem 23)

  13. Structure of a Tricyclic Compound (Lee; Chem 23) (yield: 40 - 70%) References: W. J. Shepard, J. Chem. Ed., 40, 40-41 (1963); L. F. Fieser and K. L. Williamson, Organic Experiments, 7th ed., D. C. Heath, pp. 283-294 (1992)

  14. Structure of a Tricyclic Compound (Lee; Chem 23) COSY spectrum of tricyclic product Reference: “The Microscale Synthesis and the Structure Determination of Endo-9-methoxycar- bonyl-3-oxatricyclo[4,2,1,0]-2-nonane”; M. Lee, J. Chem. Ed., 69, A172-A173 (1992)

  15. Goals: to develop - a basic understanding of 2D NMR methods - the ability to carry out 2D experiments independently - the ability to process 2D data productively - a facility with the interpretation of such data Requirements: assignment of - all H-1 resonances (chemical shift, mulitplet pattern) - all C-13 resonances (chemical shift) - all H-H coupling constant values - all C-H coupling constant values Detailed NMR Characterization (Trzupek; Chem 34)

  16. NMR Techniques Available: - simple H-1 spectrum - resolution-enhanced H-1 spectrum - proton decoupled H-1 spectrum - use of lanthanide shift reagents - relay COSY - multiple quantum filtered COSY - homonuclear 2D-J - simple C-13 spectrum - heteronuclear 2D-J - HETCOR -spectral simulation (H-1) Detailed NMR Characterization (Trzupek; Chem 34)

  17. Sample requirements: - ready availability (commercial or easily prepared) - good purity, solubility - overlapping proton resonances - complex splitting patterns - manageable molecular size (5 to 8 types of H’s) Typical candidates: Detailed NMR Characterization (Trzupek; Chem 34) (5-hexen-2-one) (3,4-pentadien-1-ol) (8-hydroxyquinoline)

  18. Results, 3,4-pentadien-1-ol: COSY spectrum Detailed NMR Characterization (Trzupek; Chem 34) HE HD HC HB HA D E C D B A

  19. Results, 8-hydroxyquinoline Detailed NMR Characterization (Trzupek; Chem 34) 9 8 7 ppm

  20. Results, 8-hydroxyquinoline: homonuclear 2D-J Detailed NMR Characterization (Trzupek; Chem 34) HF HE HB HD HC HA

  21. Results, 5-hexene-2-one: heteronuclear 2D-J Detailed NMR Characterization (Trzupek; Chem 34) Hd Hf He c a e d g f (CDCl3) Ha Hc

  22. Results, 3,4-pentadien-1-ol: H-1 simulation Detailed NMR Characterization (Trzupek; Chem 34) HC HA D E C D B A HD HE HB (simulated) (actual) 5 4 3 2 ppm

  23. Results, 5-hexene-2-one: HETCOR Detailed NMR Characterization (Trzupek; Chem 34) Ce Cf Cd Cc B C Ca c a e E A D d F g f HC HA,B HE HF

  24. Results, 5-hexene-2-one: student report sheet Detailed NMR Characterization (Trzupek; Chem 34) Hchemshift multiplet J(x,y) J(x,y) J(x,y) A 5.04 tdd AB, 2.2 AC, 17.0 AD, 1.1 B 4.97 tdd AB, 2.2 BC, 10.4 BD, 1.4 C 5.82 ddt AC, 17.0 BC, 10.4 CD, 6.6 D 2.33 tddd BD, 1.4 CD, 6.6 DE, 7.4 E 2.55 t DE, 7.4 F 2.16 s Cchem shiftJ(H) a 114.9 160 (A,B) c 136.7 156 (C) d 27.5 122 (D,D) e 42.5 124 (E,E) f 29.7 128 (F,F,F) g 207.8 --- (all chemical shifts in ppm; all J values in Hz) B C c a e E A d D F g f

  25. Background: - bioactivation of AZT: AZT ---1---> AZTMP ---2----> AZTDP ---3---> AZTTP - reaction rate of step 2 (thymidylate kinase) - v. slow - consequence: build-up of AZTMP; imbalance in the nucleoside pool (the basis of AZT toxicity) Goal: - to determine if the solution conformation of AZTMP is significantly different from that of AMP and if that dif- ference might be the basis for the sluggish kinase reaction 3D structure of AZTMP by NMR (Lee; Chem 44)

  26. NMR techniques employed: - H-1 spectrum - P-31 spectrum - COSY analysis - homonuclear decoupling (use of the above to assign proton chemical shifts and ob- tain H-H coupling constants throughout the molecule) - determination of T1 relaxation time values for each H - acquisition of NOE difference spectra (use of the above to obtain non-bonded distances between selected protons in the molecule) 3D structure of AZTMP by NMR (Lee; Chem 44)

  27. 3D structure of AZTMP by NMR (Lee; Chem 44) AZTMP; H-1 spectrum in buffer (20:1 D2O/DMSO-D6) (DMSO-d5) 5’ 1’ 4’ 3’ 2’ (HOD) T-H6 H1’ T-CH3 8 6 4 2 ppm

  28. Peak assignments; COSY spectrum of AZTMP 3D structure of AZTMP by NMR (Lee; Chem 44) H-1’ 5’ 1’ 4’ 3’ 2’

  29. 3D structure of AZTMP by NMR (Lee; Chem 44) J-values by homonuclear decoupling 5’ H-2’ 1’ 4’ 3’ H-2” 2’ H-2” H-2’ Decoouple At H-3’ ========> 2.40 2.36 // 2.24 2.20 ppm 2.40 2.36 // 2.24 2.20 ppm

  30. 3D structure of AZTMP by NMR (Lee; Chem 44) Dihedral angles from the Karplus relationship H-H couplingJ (Hz)degrees 1’-2’ 7.0 136 1’-2” 7.0 20 2’-3’ 5.5 30 2”-3’ 5.5 130 3’-4’ 3.7 128 4’-5’ 2.2 57 4’-5” 2.9 53 5’-5” 14.0 --- 5’-P 6.1 --- 5”-P 4.6 -- 5’ 1’ 4’ 3’ 2’

  31. 3D structure of AZTMP by NMR (Lee; Chem 44) Additional conformational features from the J-values 5’ 1’ 4’ 3’ 2’

  32. Inversion-recovery method for the determination of T1’s 3D structure of AZTMP by NMR (Lee; Chem 44) 5’ 1’ 4’ 3’ * 2’ 0.9 (* = H-1’) 0.7 * 0.5 * 0.3 * 0.1 *

  33. Graphical use of inversion-recovery data to get T1 values 3D structure of AZTMP by NMR (Lee; Chem 44) * 0.9 (* = H-1’) 0.7 * 0.5 * 0.3 * 0.1 *

  34. Determination of the glycosidic torsional angle,  3D structure of AZTMP by NMR (Lee; Chem 44) - obtain NOE’s for irradiation at thymine H-6 - use known H-6/CH3 distance, NOE, and T1 to obtain molecular correla- tion time, c - use c thus determined, other NOE’s, and other T1’s to get other distances

  35. Results: solution-phase conformational structure of AZTMP 3D structure of AZTMP by NMR (Lee; Chem 44) Comparison to structure of TMP: very similar; conclusion: some other factor (steric bulk of azido group) responsible for poor interaction with the thymidylate kinase. M. Lee, J. Chem. Ed., 73, 184-187 (1996)

  36. Acknowledgments - National Science Foundation - Keck Foundation - Milliken Foundation - Furman Chemistry Alumni - Furman University

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