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2D NMR ORGANIC SPECTROSCOPY by DR ANTHONY CRASTO

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2 DIMENSIONAL NMR ORGANIC SPECTROSCOPY by DR ANTHONY CRASTO

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2D NMR ORGANIC SPECTROSCOPY by DR ANTHONY CRASTO

  1. 1. 2 D N M R O R G A N I C S P E C T R O S C O P Y DR ANTHONY MELVIN CRASTO PRESENTS
  2. 2. HELLO! I AM DR ANTHONY MELVIN CRASTO WORLDDRUGTRACKER HELPING MILLIONS
  3. 3. Agenda 1 2D NMR Basics. 2 2D COSY 3 HETCOR 4 TOCSY 5 DEPT 6 NOESY 7 ROESY
  4. 4. Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) • Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) is a set of nuclear magnetic resonance spectroscopy (NMR) methods which give data plotted in a space defined by two frequency axes rather than one. Types of 2D NMR include correlation spectroscopy (COSY), J-spectroscopy, exchange spectroscopy (EXSY), and nuclear Overhauser effect spectroscopy (NOESY). Two-dimensional NMR spectra provide more information about a molecule than one-dimensional NMR spectra and are especially useful in determining the structure of a molecule, particularly for molecules that are too complicated to work with using one-dimensional NMR. • The first two-dimensional experiment, COSY, was proposed by Jean Jeener, a professor at the Université Libre de Bruxelles, in 1971. This experiment was later implemented by Walter P. Aue, Enrico Bartholdi and Richard R. Ernst, who published their work in 1976
  5. 5. Structure Determination Procedures 1D 1H & 13C & DEPT (+MS 、 IR , basic chemical structure or functional groups information) Establish 13C-1H connections by thru bond JCH couplings HMQC、HSQC、HSQC-TOCSY experiments Establish 1H-1H connection (spin systems or partial pieces) Decoupled 1H, 1D TOCSY, 2D 1H-1H COSY, TOCSY expts. (usually starts with well-resolved 1H signals) Long range connections ( connecting spin systems & assigning quaternary carbon) 1D NOESY & 2D HMBC, NOESY, ROESY experiments 3D structure or conformation determination 1D NOESY & 2D NOESY, ROESY, (HSQC)-NOESY expts.
  6. 6. MY BLOGS ORGANIC SPECTROSCOPY INTERNATIONAL LINK….. http://orgspectroscopyint.blogspot.in/ ORGANIC SPECTROSCOPY INTERNATIONAL Organic Chemists from Industry and academics to Interact on Spectroscopy Techniques for Organic Compounds ie NMR, MASS, IR, UV Etc. Starters, Learners, advanced, all alike, contains content which is basic or advanced, by Dr Anthony Melvin Crasto, Worlddrugtracker, email me ........... amcrasto@gmail.com, call +91 9323115463
  7. 7. LIONEL MY SON He was only in first standard in school when I was hit by a deadly one in a million spine stroke called acute transverse mylitis, it made me 90% paralysed and bound to a wheel chair, Now I keep him as my source of inspiration and helping millions, thanks to millions of my readers who keep me going and help me to keep my son happy
  8. 8. 2D NMR BASICS1
  9. 9. PROTON-PROTON CORRELATION THROUGH J- COUPLING 2D NMR Basics. • In actuality, the techniques we have already covered 1H, 13C, and DEPT are 2-D (frequency vs. intensity) however, by tradition the intensity component is dropped when discussing dimensionality • In 2-D techniques, many FIDs (proto-NMR spectra) are taken one after another, with some acquisition variable or pulse sequenced varied by small increments • Since each FID is a collection of digitized data points in the first dimension (say 10 points to make a spectrum) if 10 spectra are accumulated with an incremental change in variable, an FT can be performed in the other dimension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-D FID 1-D spectra, each with an incremental variable change FTs can be performed on the vertical data sets
  10. 10. PROTON-PROTON CORRELATION THROUGH J- COUPLING 2D NMR Basics. • The first perturbation of the system (pulse) is called the preparation of the spin system. • The effects of this pulse are allowed to coalesce; this is known as the evolution time, t1 (NOT T1 – the relaxation time) • During this time, a mixing event, in which information from one part of the spin system is relayed to other parts, occurs • Finally, an acquisition period (t2) as with all 1-D experiments. Preparation Evolution Acquisition t1 t2 Mixing
  11. 11. NMR spectrum Structure Chem. Shift J Coupling Peak Int. NOE Correlation H-H,C-H O O OH O CH3 O H OH OH OH CH2OH COOCH2 1 2 3 4 5 6 7 8 9 10 1'' 2''3'' 4'' 5'' 6'' 1' 2'3' 4' 5' 6' 7'' Applications: • Sample quality control for Synthetic works. • Elucidation of chemical structures. • Getting functional group, bonding, dynamics, kinetics and chemical exchange information of molecules. • 3D structures of the molecules. NMR Applications in Chemistry
  12. 12.  Connections through space(dipolar coupling) 1D、2D NOESY,ROESY, HOESY(HSQC-NOESY) usage: connecting spin systems 、structure determination  Connections through bonds(spin-spin coupling) Homonuclear: 1D、2D 1H-1H COSY, DQF-COSY, TOCSY usage:spin system assignment Heteronuclear: Direct (detect 13C): APT, DEPT, HETCOR Inverse (detect1H): HMQC, HSQC, HMBC, HSQC-COSY, HSQC-TOCSY, HMQC-TOCSY usage:assigning heteronuclei、connecting spin systems Some common NMR experiments:
  13. 13. COSY spectrum is used for determining the connectivities between protons on the basis of geminal and vicinal couplings. Disadvantage: bulky dispersive diagonal peaks. C C H HH Vicinal Geminal 2D COSY — Homonuclear Shift COrrelation SpectroscopY t1 AQ The basic COSY (x=45° or 90°) pulse sequence 90° x° t2
  14. 14. ppm 1.01.52.02.53.03.5 ppm 1.0 1.5 2.0 2.5 3.0 3.5 Current Data Parameters NAME butcosy EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001102 Time 8.04 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG cosygp TD 1024 SOLVENT CDCl3 NS 1 DS 16 SWH 2185.315 Hz FIDRES 2.134096 Hz AQ 0.2345700 sec RG 40.3 DW 228.800 usec DE 6.00 usec TE 288.0 K D0 0.00000300 sec D1 1.60000002 sec D13 0.00000300 sec D16 0.00010000 sec IN0 0.00045765 sec ============ CHANNEL f1 ============= NUC1 1H P0 3.00 usec P1 6.00 usec PL1 -4.00 dB SFO1 500.1310815 MHz ============ GRADIENT CHANNEL ======== GPNAM1 sine.100 GPNAM2 sine.100 GPX1 0.00 % GPX2 0.00 % GPY1 0.00 % GPY2 0.00 % GPZ1 10.00 % GPZ2 10.00 % P16 1000.00 usec F1 - Acquisition parameters ND0 1 TD 256 SFO1 500.1311 MHz FIDRES 8.535453 Hz SW 4.369 ppm FnMODE undefined F2 - Processing parameters SI 2048 SF 500.1300144 MHz WDW QSINE SSB 0 LB 0.00 Hz GB 0 PC 0.20 F1 - Processing parameters SI 1024 MC2 QF SF 500.1300144 MHz WDW QSINE SSB 0 LB 0.00 Hz GB 0 1 2 3 41 234 HO C H1 H C C C H2 H H3 H H4 H H 1-2 2-3 3-4 2D Gradient COSY-45
  15. 15. 2D COSY NMR2
  16. 16. Correlated Spectroscopy
  17. 17. FT of the t1 domain, acetone
  18. 18. October 20, 2004 Joanna R. Long 15 2D Exchange NMR A. S. Edison University of Florida t1 t2       FT of t2 FTint1willgive2Dfrequencyspectrum
  19. 19. PROTON-PROTON CORRELATION THROUGH J- COUPLING 2D COSY. • H-H COrrelation SpectroscopY (COSY): • The pulse sequence for COSY is as follows: • A 90o pulse in the x-direction is what we used for 1-D 1H NMR • Here, after a variable “mixing” period, a second 90o pulse is performed, followed by acquisition of a spectrum 19 90x90x t1 t2
  20. 20. October 20, 2004 Joanna R. Long 16 15 l N•Ndimethylacetamide in 700 l d-chloroform at 29° C
  21. 21. A-B coupling in COSY Spectrum
  22. 22. CH3CH2CCH3 O
  23. 23. CH2CH3
  24. 24. Isobutyl Alcohol OH
  25. 25. O O
  26. 26. 1H NMR Spectrum of Ipsenol
  27. 27. COSY Spectra of Ipsenol
  28. 28. Share a big idea or quote here.
  29. 29. HETCOR3
  30. 30. Heteronuclear through-bond correlation methods • Heteronuclear correlation spectroscopy gives signal based upon coupling between nuclei between two different types. Often the two nuclei are protons and another nucleus (called a "heteronucleus"). For historical reasons, experiments which record the proton rather than the heteronucleus spectrum during the detection period are called "inverse" experiments. • This is because the low natural abundance of most heteronuclei would result in the proton spectrum being overwhelmed with signals from molecules with no active heteronuclei, making it useless for observing the desired, coupled signals. • With the advent of techniques for suppressing these undesired signals, inverse correlation experiments such as HSQC, HMQC, and HMBC are actually much more common today. "Normal" heteronuclear correlation spectroscopy, in which the hetronucleus spectrum is recorded, is known as HETCOR
  31. 31. Heteronuclear multiple-bond correlation spectroscopy (HMBC) • HMBC detects heteronuclear correlations over longer ranges of about 2–4 bonds. The difficulty of detecting multiple-bond correlations is that the HSQC and HMQC sequences contain a specific delay time between pulses which allows detection only of a range around a specific coupling constant. This is not a problem for the single-bond methods since the coupling constants tend to lie in a narrow range, but multiple-bond coupling constants cover a much wider range and cannot all be captured in a single HSQC or HMQC experiment. • In HMBC, this difficulty is overcome by omitting one of these delays from an HMQC sequence. This increases the range of coupling constants that can be detected, and also reduces signal loss from relaxation. The cost is that this eliminates the possibility of decoupling the spectrum, and introduces phase distortions into the signal. There is a modification of the HMBC method which suppresses one-bond signals, leaving only the multiple-bond signals
  32. 32. Heteronuclear single-quantum correlation spectroscopy (HSQC) • HSQC detects correlations between nuclei of two different types which are separated by one bond. This method gives one peak per pair of coupled nuclei, whose two coordinates are the chemical shifts of the two coupled atoms. • HSQC works by transferring magnetization from the I nucleus (usually the proton) to the S nucleus (usually the heteroatom) using the INEPT pulse sequence; this first step is done because the proton has a greater equilibrium magnetization and thus this step creates a stronger signal. The magnetization then evolves and then is transferred back to the I nucleus for observation. An extra spin echo step can then optionally be used to decouple the signal, simplifying the spectrum by collapsing multiplets to a single peak. The undesired uncoupled signals are removed by running the experiment twice with the phase of one specific pulse reversed; this reverses the signs of the desired but not the undesired peaks, so subtracting the two spectra will give only the desired peaks.Heteronuclear multiple-quantum correlation spectroscopy (HMQC) gives an identical spectrum as HSQC, but using a different method. The two methods give similar quality results for small to medium-sized molecules, but HSQC is considered to be superior for larger molecules
  33. 33. HETCOR (Heteronuclear chemical shift correlation, 1H - 13C COSY) 13C 1H t1 1 2 The standard pulse sequence for 13C-detected 1H-13C chemical shift correlation. AQ t2 1H decoupling Removing JCH splittings *But Inverse experiment has the following Advantages: •increase sensitivity of detecting the less sensitive nuclei •1H is in the direct detection dimension => larger np => better resolution
  34. 34. HMBC (Heteronuclear Multiple-Bond Correlation Spectroscopy) 13C 1H t1 AQ C2, C3 and C4: Quaternary or protonated carbons X: O, N C1 C2 C3 C4 H1 C1 X C2 C3 H1 Pulse sequence for HMBC Long range connections or connections between spin systems
  35. 35. HETCOR Spctrum of Ipsenol
  36. 36. N COCH2CH3 O H H H H Exercise 5.6, p. 291
  37. 37. N COCH2CH3 O H H H H Exercise 5.6, p. 292
  38. 38. O CH3
  39. 39. ppm 1.01.52.02.53.03.5 ppm 10 15 20 25 30 35 40 45 50 55 60 65 Current Data Parameters NAME buthsqc EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001102 Time 10.31 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG invietgpsi TD 1024 SOLVENT CDCl3 NS 1 DS 16 SWH 2185.315 Hz FIDRES 2.134096 Hz AQ 0.2345700 sec RG 2298.8 DW 228.800 usec DE 6.00 usec TE 300.0 K D0 0.00000300 sec D1 2.00000000 sec D4 0.00170000 sec D11 0.03000000 sec D13 0.00000300 sec D16 0.00010000 sec D24 0.00090000 sec DELTA 0.00116720 sec DELTA1 0.00110700 sec IN0 0.00003600 sec l3 256 ============ CHANNEL f1 ============= NUC1 1H P1 5.60 usec P2 11.20 usec P28 2500.00 usec PL1 -4.00 dB SFO1 500.1310815 MHz ============ CHANNEL f2 ============= CPDPRG2 garp NUC2 13C P3 17.70 usec P4 35.40 usec PCPD2 89.00 usec PL2 -1.00 dB PL12 13.00 dB SFO2 125.7633722 MHz ============ GRADIENT CHANNEL ======== GPNAM1 sine.100 GPNAM2 sine.100 GPX1 0.00 % GPX2 0.00 % GPY1 0.00 % GPY2 0.00 % GPZ1 80.00 % GPZ2 20.10 % P16 1000.00 usec F1 - Acquisition parameters ND0 2 TD 512 SFO1 125.7634 MHz FIDRES 27.126736 Hz SW 110.437 ppm FnMODE undefined F2 - Processing parameters SI 1024 SF 500.1300144 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0 PC 0.20 F1 - Processing parameters SI 1024 MC2 echo-antiecho SF 125.7577969 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0 HO C H1 H C C C H2 H H3 H H4 H H 1 2 3 4 1234 HSQC spectrum: H-C correlatedC- dimensio n H-dimension
  40. 40. TOCSY4
  41. 41. Total correlation spectroscopy (TOCSY) • The TOCSY experiment is similar to the COSY experiment, in that cross peaks of coupled protons are observed. However, cross peaks are observed not only for nuclei which are directly coupled, but also between nuclei which are connected by a chain of couplings. This makes it useful for identifying the larger interconnected networks of spin couplings. This ability is achieved by inserting a repetitive series of pulses which cause isotropic mixing during the mixing period. Longer isotropic mixing times cause the polarization to spread out through an increasing number of bonds. • In the case of oligosaccharides, each sugar residue is an isolated spin system, so it is possible to differentiate all the protons of a specific sugar residue. A 1D version of TOCSY is also available and by irradiating a single proton the rest of the spin system can be revealed. Recent advances in this technique include the 1D-CSSF-TOCSY (Chemical Shift Selective Filter - TOCSY) experiment, which produces higher quality spectra and allows coupling constants to be reliably extracted and used to help determine stereochemistry. • TOCSY is sometimes called "homonuclear Hartmann–Hahn spectroscopy" (HOHAHA)
  42. 42. A
  43. 43. TOCSY (TOtal Correlation SpectroscopY) or HOHAHA(Homonuclear Hartman-Hahn Spectroscopy) t1  MLEV17 AQ Pulse sequence for a TOCSY spectrum. Different mixing time gives different degree of relay of correlation. At small mixing time, TOCSY spectrum is similar to COSY spectrum. At long mixing time, gives total correlation. HO C H1 H C C C H2 H H3 H H4 H H HO C H1 H C C C H2 H H3 H H4 H H HO C H1 H C C C H2 H H3 H H4 H HCOSY RL-COSY TOCSY mixing time t2
  44. 44. DEPT5
  45. 45. DEPT-90, DEPT-135 Distortionless Enhancement by Polarization Transfer • Preferred procedure for determining # protons attached to carbons • Variable proton pulse angle q is set at 90o and 135o • In DEPT-90, only CH shows. In DEPT-135, CH2’s are phased down, CH and CH3 are phased up
  46. 46. DEPT: Distortionless Enhancement by Polarization Transfer Heteronuclear expt. Detection: 13C Distinguish CH, CH2, CH3 By suitable combination of q=45, 90 & 135 spectra All CH’s Only CH CH & CH3up CH2 down
  47. 47. Adjustment of 1H pulse angle Avoids overlapping multiplets CH CH2 CH3
  48. 48. 2-Heptanone, CPD and DEPT-35
  49. 49. 6-Methyl-5-hepten-2-ol Standard CPD Spectrum
  50. 50. DEPT-90 DEPT-135 OH
  51. 51. O CH3 DEPT-135 DEPT-90
  52. 52. < >
  53. 53. Ipsenol CPD, DEPT-135, DEPT 90 7 6 2 5 3 4 1 9 8,10
  54. 54. 2D NOESY6
  55. 55. Nuclear Overhauser effect spectroscopy (NOESY)In NOESY, the Nuclear Overhauser cross relaxation between nuclear spins during the mixing period is used to establish the correlations. The spectrum obtained is similar to COSY, with diagonal peaks and cross peaks, however the cross peaks connect resonances from nuclei that are spatially close rather than those that are through-bond coupled to each other. NOESY spectra also contain extra axial peaks which do not provide extra information and can be eliminated through a different experiment by reversing the phase of the first pulse. One application of NOESY is in the study of large biomolecules such as in protein NMR, which can often be assigned using sequential walking. The NOESY experiment can also be performed in a one-dimensional fashion by pre-selecting individual resonances. The spectra are read with the pre-selected nuclei giving a large, negative signal while neighboring nuclei are identified by weaker, positive signals. This only reveals which peaks have measurable NOEs to the resonance of interest but takes much less time than the full 2D experiment. In addition, if a pre-selected nucleus changes environment within the time scale of the experiment, multiple negative signals may be observed. This offers exchange information similar to the EXSY (exchange spectroscopy) NMR method. NOESY experiment is important tool to identify stereochemistry of a molecule in solvent whereas single crystal XRD used to identify stereochemistry of a molecule in solid form.
  56. 56. 2D NOESY (Nuclear Overhauser Enhancements SpectroscopY) t1 m AQ The NOESY pulse sequence. —C — ~ —C — Ha Hb VC*r -6, r0.5nm r0.5nm For resonance assignment, chemical structure elucidation & 3D structure determination t2
  57. 57. ppm 3.54.04.55.05.56.06.57.07.5 ppm 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Current Data Parameters NAME cfynoesy EXPNO 1 PROCNO 1 F2 - Acquisition Parameters Date_ 20001103 Time 8.53 INSTRUM DRX500 PROBHD 5 mm TBI 1H/ PULPROG noesygptp TD 1024 SOLVENT CDCl3 NS 8 DS 16 SWH 3443.526 Hz FIDRES 3.362818 Hz AQ 0.1488800 sec RG 64 DW 145.200 usec DE 6.00 usec TE 288.0 K D0 0.00000300 sec D1 1.60000002 sec D8 0.40000001 sec D16 0.00010000 sec d20 0.19890000 sec IN0 0.00014520 sec ============ CHANNEL f1 ============= NUC1 1H P1 6.00 usec P2 12.00 usec PL1 -4.00 dB SFO1 500.1326379 MHz ============ GRADIENT CHANNEL ======== GPNAM1 sine.100 GPNAM2 sine.100 GPX1 0.00 % GPX2 0.00 % GPY1 0.00 % GPY2 0.00 % GPZ1 40.00 % GPZ2 -40.00 % P16 1000.00 usec F1 - Acquisition parameters ND0 2 TD 256 SFO1 500.1326 MHz FIDRES 13.451274 Hz SW 6.885 ppm FnMODE undefined F2 - Processing parameters SI 1024 SF 500.1300144 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0 PC 0.20 F1 - Processing parameters SI 1024 MC2 TPPI SF 500.1300144 MHz WDW SINE SSB 2 LB 0.00 Hz GB 0 N N N N O O CH3 CH3 H3C H 1 3 5 2 8 7 6 4 3-CH3, 5-H Gradient NOESY
  58. 58. NOESY gives sequential assignment of peptides 21 2 3 CH2 CH2N C H N O H C C H CH2O N H C CH2 C H OH O 1 321
  59. 59. 2D ROESY7
  60. 60. Rotating frame nuclear Overhauser effect spectroscopy (ROESY) • ROESY is similar to NOESY, except that the initial state is different. Instead of observing cross relaxation from an initial state of z-magnetization, the equilibrium magnetization is rotated onto the x axis and then spin-locked by an external magnetic field so that it cannot precess. This method is useful for certain molecules whose rotational correlation time falls in a range where the Nuclear Overhauser effect is too weak to be detectable, usually molecules with a molecular weight around 1000 daltons, because ROESY has a different dependence between the correlation time and the cross-relaxation rate constant. In NOESY the cross-relaxation rate constant goes from positive to negative as the correlation time increases, giving a range where it is near zero, whereas in ROESY the cross-relaxation rate constant is always positive. • ROESY is sometimes called "cross relaxation appropriate for minimolecules emulated by locked spins" (CAMELSPIN)
  61. 61. Spin-lock 90° t1 t2 2D ROESY pulse program For small molecule NOE can be very small or zero, ROESY can be used in place of NOESY experiment. ROE intensity is also related to the H-H distances. mixing time
  62. 62. 2D ROESY spectrum of ethylbenzene
  63. 63. Structure of 12,14- ditbutylbenzo[g]chrysene showing color conding of rings
  64. 64. Aromatic region of the 2D ROESY spectrum of 12,14- ditbutylbenzo[g]chrysene showing connectivity and separation into four color-coded proton groups
  65. 65. CASE STUDY ..ASPIRIN1
  66. 66. 1 H-NMR spectrum of acetylsalicylic acid
  67. 67. H, H-COSY spectrum of acetylsalicylic acid
  68. 68. THANK YOU!

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