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Two-dimensional hetero correlation experiments HSQC, HMQC, HMBC, H2BC

Two-dimensional hetero correlation experiments HSQC, HMQC, HMBC, H2BC. Nils Nyberg NPR, Department of Drug Design and Pharmacology. Outline. Two dimensional correlation experiments Short review of experiments Example of HSQC, HMBC and H2BC Second dimension and how to optimize the results

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Two-dimensional hetero correlation experiments HSQC, HMQC, HMBC, H2BC

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  1. NTDR, 2012 Two-dimensional hetero correlation experimentsHSQC, HMQC, HMBC, H2BC Nils Nyberg NPR, Department of Drug Design and Pharmacology

  2. NTDR, 2012 Outline • Two dimensional correlation experiments • Short review of experiments • Example of HSQC, HMBC and H2BC • Second dimension and how to optimize the results • Choosing delays in HMQC/HSQC and HMBC • H2BC

  3. NTDR, 2012 Two-dimensional correlation experiments • Experiment review and type of information

  4. NTDR, 2012 Two-dimensional correlation experiments • Mark expected correlations (but not all HMBC-correlations) COSY, 3JH,H NOESY HSQC, 1JH,C HMBC, 2JH,C HMBC, 3JH,C H2BC, 2-bond H-C correlations

  5. NTDR, 2012 Two-dimensional correlation experiments • Mark expected correlations COSY, 3JH,H NOESY HSQC, 1JH,C HMBC, 2JH,C HMBC, 3JH,C H2BC, 2-bond H-C correlations

  6. NTDR, 2012 HSQC

  7. NTDR, 2012 HSQC + H2BC

  8. NTDR, 2012 HSQC + H2BC + HMBC

  9. NTDR, 2012 HSQC + H2BC + HMBC

  10. NTDR, 2012 The second dimension in homonuclear experiments • Prepare – evolution – mix – detect t1 t2 Fouriertransform f1 f2

  11. NTDR, 2012 The second dimension in heteronuclear experiments • Prepare – evolution – mix – detect t1 t2 Fouriertransform C f1 H f2

  12. NTDR, 2012 Design of experiments • Double resonance experiment (with two different nuclei) • Several channels on the spectrometer (at least two) • 1H and 13C • 1H and 15N • 1H and 31P • Probe with coils for all nuclei frequencies • Single coil, doubly tuned • Dual probes dedicated to e.g., 1H + 13C • Broadband probes, 1H + (109Ag – 31P) • Inverse configuration (inner coil 1H) • Normal configuration (inner coil X-nuclei) • http://chemnmr.colorado.edu/moreinfo/instruments/probe_whatis.html

  13. NTDR, 2012 Design of experiments • The most sensitive experiments start with 1H and detects the 1H-signal. • ”Inverse experiments” • 1H – 13C: 32 times more sensitive • 1H – 15N: 300 times more sensitive

  14. NTDR, 2012 Resolution and time of 1H-detected experiments • Direct dimension, 1H (= High resolution) • Double number of scans => Double time • Indirectdimension, 13C (= Low resolution) • Number of increments in second dimension • Double resolution => Double time 13C f1 512 rows240 ppm (36 kHz @ 150 MHz) 70 Hz/row High dispersion, singlets, minimal overlap 1H f2 1024 points (4 scans)8 ppm (4.8 kHz @ 600 MHz) ~5 Hz/point

  15. NTDR, 2012 Optimal use of experiment time • Sample preparation • High concentration (but not too high...) • Particle free, easy to shim • Optimize spectrometer • Tune/match/pulse calibration • Shimming • Minimize spectral width in indirect dimension • Favor high S/N instead of high resolution • Post processing • Window function (QSINE, ssb 2) • Linear prediction (better version of zero filling)

  16. NTDR, 2012 Post processing of HMBC • QSINE ssb 2 in both directions • Linear prediction (128 -> 1024) • XFB (pk in f1) + XF2m

  17. NTDR, 2012 Post processing of HMBC • QSINE ssb 2 in both directions • No linear prediction (zero filling, 128 -> 1024) • XFB (pk in f1) + XF2m

  18. NTDR, 2012 Post processing of HMBC • QSINE ssb 1 in both directions • Zero-filling, No linear prediction • XFB (mc in f1)

  19. NTDR, 2012 HMQC and HSQC • Correlates 1H to 13C (or 31P, 15N, ...) • Identifies diasterotopic pairs • Dispersion of signals in crowded regions in a second dimension • HMQC • Few pulses, robust, broadening in f1 due to passive couplings (H-H couplings) • HSQC • Less robust, need carfuly calibrated pulses, narrow peaks

  20. NTDR, 2012 HMQC and HSQC • Optimal delay, Δ = (2 x 145)-1 ~3.3 ms • Decoupling during acquistion HMQC HSQC

  21. NTDR, 2012 Removal of 1H signals connected to 12C • A major obstacle of 1H detected heteronuclear experiments is to ”filter” all signals from 1H bound to 13C (1%) from 1H bound to 12C (99%) • Phase cycling • Pulsed field gradients (PFG) • Coherence selection 13C-satelites Difference Inverted byphase cycling

  22. NTDR, 2012 Choosing delays in HMBC • HMBC is similar to HMQC, but with a longer delay in the preparation part to be more sensitive to long-range H-C couplings. • Tune delay to expected coupling size [1/(2 x nJC,H)]. • But too long delay => less signal due to relaxation... • 65 ms ~7-8 Hz HMQC HMBC

  23. NTDR, 2012 Choosing delays in HMBC Green: 200 ms [2.5 Hz] Red: 65 ms [7.7 Hz]

  24. NTDR, 2012 H2BC – two bond correlation • HMBC = Heteronuclear Multiple Bond Correlation • H2BC = Heteronuclear Two Bond Correlation • Constant time experiment • H,H coupling during T (COSY), H-C transfer during Tau (HMQC)

  25. NTDR, 2012 H2BC example • Edited version – different signals for CH2 and CH/CH3 • Prednisolon

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