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Introduction to 2D NMR Multipulse techniques Organic Structure Analysis, Crews, Rodriguez and Jaspars

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ONE-PULSE SEQUENCE

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Organic Structure Analysis, Crews, Rodriguez and Jaspars ONE-PULSE SEQUENCE 1H1H (90 o ) x PreparationDetection

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Organic Structure Analysis, Crews, Rodriguez and Jaspars BASIC LAYOUT OF A 2D NMR EXPERIMENT Preparation Evolution t 1 Mixing Detection t 2

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Organic Structure Analysis, Crews, Rodriguez and Jaspars INVERSION-RECOVERY PULSE SEQUENCE (180 o ) x (90 o ) x t1t1 PreparationEvolutionDetection 1H1H t2t2

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Organic Structure Analysis, Crews, Rodriguez and Jaspars INVERSION-RECOVERY PULSE SEQUENCE t1t1 t1t1

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Organic Structure Analysis, Crews, Rodriguez and Jaspars SPIN-ECHO PULSE SEQUENCE (90 o ) x (180 o ) x t1t1 Prep. EvolutionDetection 13 C t1t1 t2t2

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Organic Structure Analysis, Crews, Rodriguez and Jaspars SPIN-ECHO PULSE SEQUENCE FT gives null signal

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Organic Structure Analysis, Crews, Rodriguez and Jaspars 1 H- 1 H COSY (COrrelated SpectroscopY) (90 o ) x t1t1 PreparationEvolutionDetection 1H1H t2t2

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Organic Structure Analysis, Crews, Rodriguez and Jaspars PROCESSING 2D DATA n is the number of increments

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TYPES OF 2D NMR EXPERIMENTS AUTOCORRELATED –Homonuclear J resolved – 1 H- 1 H COSY –TOCSY –NOESY –ROESY –INADEQUATE CROSS-CORRELATED –Heteronuclear J resolved – 1 H- 13 C COSY –HMQC –HSQC –HMBC –HSQC-TOCSY Organic Structure Analysis, Crews, Rodriguez and Jaspars

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AUTOCORRELATED EXPERIMENTS – 1 H- 1 H COSY f 1 =f 2 =diagonal Gives:

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Organic Structure Analysis, Crews, Rodriguez and Jaspars AUTOCORRELATED EXPERIMENTS – 1 H- 1 H COSY

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REQUIREMENTS FOR 1 H- 1 H COSY Number of transients required is half that needed to give decent 1D 1 H NMR spectrum Most of the time we use a double quantum filtered COSY (DQF-COSY): –Same information as COSY but removes single quantum transitions (large singlet peaks from Me groups), meaning we can see things closer to the diagonal. Solves problems in case where there is a dynamic range problem (very large and very small peaks in same spectrum) –It is phase sensitive, we acquire 2 x number of increments (real and imaginary). Get coupling information from phases of correlation peaks. Organic Structure Analysis, Crews, Rodriguez and Jaspars

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PEAK PICKING FOR 1 H- 1 H COSY COSYDQF-COSY

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Organic Structure Analysis, Crews, Rodriguez and Jaspars PEAK PICKING FOR DQF-COSY

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Organic Structure Analysis, Crews, Rodriguez and Jaspars TOtal Correlation SpectroscopY (TOCSY) HOmonuclear HArtman-HAhn spectroscopy (HOHAHA) Increasing the mixing time (30 – 180 ms):

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Organic Structure Analysis, Crews, Rodriguez and Jaspars TOtal Correlation SpectroscopY (TOCSY) HOmonuclear HArtman-HAhn spectroscopy (HOHAHA) H H

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TOtal Correlation SpectroscopY (TOCSY) HOmonuclear HArtman-HAhn spectroscopy (HOHAHA) Like COSY in appearance Relies on relayed coherence during spin-lock mixing time The longer t mix, the longer the correlations (30 – 180 ms gives bonds) Relays can occur only across protonated carbons – not across quaternary carbons (spin systems) Very useful for systems containing discrete units eg proteins and polysaccharides Organic Structure Analysis, Crews, Rodriguez and Jaspars

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NOESY (Nuclear Overhauser Effect SpectroscopY) ROESY (Rotating Overhauser Effect SpectroscopY) Through-space correlations Up to 5 Å

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Organic Structure Analysis, Crews, Rodriguez and Jaspars NOESY (Nuclear Overhauser Effect SpectroscopY) H H MW = 300 Da t mix = 800 ms

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Organic Structure Analysis, Crews, Rodriguez and Jaspars ROESY (Rotating Overhauser Effect SpectroscopY) H H MW = 800 Da t mix = 300 ms

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Give through-space correlations up to 5 Å The effect relies on molecular size. The NOE effect ~ 0 at 1000 Da. It works well for small molecules (t mix ~ 800 ms) and macromolecules (t mix ~ 100 ms). In the intermediate range use ROESY with t mix ~ ms Both NOESY and ROESY need long relaxation delays (2 s) True NOE and ROE peaks are negative. In NOESY can get COSY peaks showing (positive). In ROESY can get TOCSY peaks showing (antiphase). To determine mixing time do inversion-recovery experiment to find average T 1. As a rule of thumb, NOESY t mix = T 1 /0.7, ROESY t mix = T 1 /1.4 NOESY (Nuclear Overhauser Effect SpectroscopY) ROESY (Rotating Overhauser Effect SpectroscopY) Organic Structure Analysis, Crews, Rodriguez and Jaspars

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INADEQUATE – Incredible Natural Abundance DoublE QUAntum Transfer Experiment 13 C- 13 C

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Organic Structure Analysis, Crews, Rodriguez and Jaspars INADEQUATE – Incredible Natural Abundance DoublE QUAntum Transfer Experiment C C

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C-C correlation experiment Relies on two 13 C being adjacent. Chance of 13 C- 13 C = 1/ Works by suppressing 13 C single quantum signal (hence DQ) Needs signal/noise of 25/1 with 1 transient 13 C NMR experiment to get spectrum in 24 h For compound of 150 Da, need 700 mg in 0.7 mL CDCl 3 (~ 6M) With low volume probes and image recognition software can get away with much smaller samples and poorer signal/noise INADEQUATE – Incredible Natural Abundance DoublE QUAntum Transfer Experiment Organic Structure Analysis, Crews, Rodriguez and Jaspars

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HETERO CORRELATED EXPERIMENTS ( 13 C- 1 H) 13 C DETECTED 1 H- 13 C COSY (also called HETCOR). Two types: –Direct correlations ( 1 J CH = 140 Hz) C-H –Indirect (long-range) correlations ( 2-3 J CH = 9 Hz) C-C-H and C-C-C-H Very insensitive For J = 140 Hz take 1/3 number of transients needed to get 13 C NMR spectrum with S/N = 20/1. If 300 transients for 13 C NMR, 2D with 256 increments takes 14 h. For J = 9 Hz take 1/2 number of transients needed to get 13 C NMR spectrum with S/N = 20/1. Needs longer relaxation time (2s). If 300 transients for 13 C NMR, 2D with 256 increments takes 32 h. Outdated Organic Structure Analysis, Crews, Rodriguez and Jaspars

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HETERO CORRELATED EXPERIMENTS ( 13 C- 1 H) 1 H (INVERSE) DETECTED Direct correlations (C-H, 1 J CH = 140 Hz) obtained from HMQC or HSQC experiment (Heteronuclear Multiple/Single Quantum Coherence) Indirect (long-range) correlations (C-C-H, C-C-C-H, 2-3 J CH = 9Hz) obtained from HMBC experiment (Heteronuclear Multiple Bond Correlation). Set J CH to other values for certain systems. These experiments are 1 H detected and have inherent sensitivity advantage ( H = 4 C ) Chance of 13 C- 1 H is 1/100 With pulsed field gradients (PFG), it is possible to run 2D heterocorrelated experiments with single transients and 256 increments in 8-15 minutes! Without PFG need to phase cycle to remove artefacts. (4 transients minimum: t = 30 min; but 64 for full phase cycle: t = 9h). Organic Structure Analysis, Crews, Rodriguez and Jaspars

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HSQC versus HMQC HMQC –Absolute value –Half the resolution of an HSQC –Can alter pulse sequence to get HMBC HSQC –Phase sensitive –Double the resolution of an HMQC –Can edit to get positive peaks for CH, CH 3 and negative peaks for CH 2. Organic Structure Analysis, Crews, Rodriguez and Jaspars

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HSQC – Heteronuclear Single Quantum Coherence 1 J CH = 140 Hz; C-H direct correlations (1 bond)

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Organic Structure Analysis, Crews, Rodriguez and Jaspars HSQC – Heteronuclear Single Quantum Coherence

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Organic Structure Analysis, Crews, Rodriguez and Jaspars Edited HSQC – Heteronuclear Single Quantum Coherence CH 3 CH CH 2

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Organic Structure Analysis, Crews, Rodriguez and Jaspars HMBC – Heteronuclear Multiple Bond Correlation 2-3 J CH = 9 Hz; C-H indirect (long range) correlations (2-3 bonds) C-C-H & C-C-C-H

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Organic Structure Analysis, Crews, Rodriguez and Jaspars HMBC – Heteronuclear Multiple Bond Correlation

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Organic Structure Analysis, Crews, Rodriguez and Jaspars 3D Experiments – HSQC-TOCSY Mixing time ms 3-7 bonds

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Organic Structure Analysis, Crews, Rodriguez and Jaspars 3D Experiments – HSQC-TOCSY

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3D experiment condensed into 2D. Concatenation of HSQC and TOCSY pulse sequences Sorts TOCSY correlations in spin system according to carbon chemical shift – increases resolution of TOCSY by adding 13 C dimension See direct (C-H) correlations as in HSQC, and long range correlations within spin systems depending on mixing time (30 – 180 ms, 3 – 7 bonds). Cant go across quaternary C or heteroatom as it the TOCSY effect needs protons. Very effective for modular systems with separate spin systems such as polysaccharides and peptides. Organic Structure Analysis, Crews, Rodriguez and Jaspars

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General procedure for running 2D spectra 1.Insert sample, tune 1 H and 13 C channels 2.Lock and shim (determine 90 o pulse width) 3.Acquire 1 H NMR spectrum 4.Change spectral window to ± 1 ppm of spectrum 5.Re-acquire 1 H spectrum 6.Phase spectrum, apply baseline correction 7.Acquire 13 C spectrum in optimum spectral window 8.Call up macro for 2D experiment. Use 1 H and 13 C parameters for 2D experiments 9.Alter number of transients, number of increments to fit the time available 10.Repeat steps 8 & 9 for other 2D experiments required 11.Set experiments running Organic Structure Analysis, Crews, Rodriguez and Jaspars

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Processing 2D spectra – Phase sensitive experiments (DQF-COSY, TOCSY, NOESY, ROESY, HSQC, HSQC-TOCSY) 1.Fourier transform the first increment 2.Apodise t 2 using shifted sine bell squared 3.Fourier transform t 2 f 2 using apodisation function in 2. 4.Apodise t 1 using shifted sine bell squared 5.Fourier transform t 1 f 1 using apodisation function in 4. 6.Phase spectrum in both dimensions if necessary Organic Structure Analysis, Crews, Rodriguez and Jaspars

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Processing 2D spectra – Absolute value experiments (COSY, HMBC) 1.Fourier transform the first increment 2.Apodise t 2 using sine bell 3.Fourier transform t 2 f 2 using apodisation function in 2. 4.Apodise t 1 using sine bell 5.Fourier transform t 1 f 1 using apodisation function in 4. 6.No phasing necessary Organic Structure Analysis, Crews, Rodriguez and Jaspars

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APODISATION - Phase sensitive experiments APODISATION - Absolute value experiments

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