NMR Applications in Chemistry Chem. Shift J Coupling NMR spectrum Structure Peak Int. Correlation H-H，C-H NOE 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.

Some common NMR experiments: 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 Connections through space（dipolar coupling） 1D、2D NOESY，ROESY, HOESY(HSQC-NOESY) usage： connecting spin systems 、structure determination

2D COSY — Homonuclear Shift COrrelation SpectroscopY AQ The basic COSY (x=45° or 90°) pulse sequence 90° x° t2 COSY spectrum is used for determining the connectivities between protons on the basis of geminal and vicinal couplings. Disadvantage: bulky dispersive diagonal peaks.

2D Gradient COSY-45 1 2 3 4 4 3-4 2-3 3 1-2 2 1

2D DQF－COSY(Double-Quantum Filtered COSY) 90 t1 t2 allow J coupling constant measurements observing cross-peaks close to diagonal But lower sensitivity (~40%) of COSY expt.

2D Gradient DQF-COSY

3J=8.00Hz

TOCSY (TOtal Correlation SpectroscopY) or HOHAHA(Homonuclear Hartman-Hahn Spectroscopy) mixing time AQ t2 t1   MLEV17 Pulse sequence for a TOCSY spectrum. COSY RL-COSY TOCSY 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.

2D Gradient TOCSY, mixing time=10ms 3 4 4 3-4 2-3 3 1-2 2 Same as COSY spectrum 1

2D Gradient TOCSY, mixing time=20ms 1 2 3 4 4 2-4 3-4 1-3 2-3 3 2 1-2 Same as RL－COSY 1

2D Gradient TOCSY, mixing time=50ms 1 2 3 4 4 2-4 3-4 1-4 1-3 2-3 3 2 1-2 Total Correlation 1

By suitable combination of DEPT: Distortionless Enhancement by Polarization Transfer Heteronuclear expt. Detection: 13C Distinguish CH, CH2, CH3 By suitable combination of =45, 90 & 135 spectra CH & CH3up CH2 down Only CH All CH’s

HETCOR (Heteronuclear chemical shift correlation, 1H - 13C COSY) AQ 1H t1 1 2 1H decoupling Removing JCH splittings The standard pulse sequence for 13C-detected 1H-13C chemical shift correlation. *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

Pulse sequence for 1H-detected 1H-13C HMQC (Heteronuclear Multiple Quantum Coherence) HSQC (Heteronuclear Single Quantum Coherence) t2 1H AQ 13C /2 /2 t1 /2 /2 GARP-1 13C decoupling Removing JCH splittings Pulse sequence for 1H-detected 1H-13C correlation through single-quantum coherence • HSQC can give considerably better 13C resolution and sensitivity than HMQC for CH2 groups of natural products • HMQC has simpler pulse sequence and it is more robust and easier to perform.

(HMQC and HETCOR spectra look similar) HSQC spectrum: H-C correlated 1 2 3 4 H-dimension 4 3 2 C-dimension 1 (HMQC and HETCOR spectra look similar)

HSQC-TOCSY Pulse sequence for 1H-detected HSQC-TOCSY experiment mixing time SLy SLy 1H AQ MLEV17 13C /2 /2 t1 /2 /2 GARP-1 Pulse sequence for 1H-detected HSQC-TOCSY experiment through single-quantum coherence. Longer mixing time gives longer extend of spin-spin correlations.

Gradient HSQC-TOCSY(mixing time=10ms) 2 3 4 H-dimension 4 3 2 1-2 C-dimension Same as HSQC－COSY spectrum 1 1-1 2-1

Gradient HSQC-TOCSY(mixing time=20ms) 1 2 3 4 H-dimension 4 3 1-3 2 1-2 C-dimension 1 1-1 2-1 3-1

Gradient HSQC-TOCSY(mixing time=80ms) 1 2 3 4 H-dimension 4 1-4 3 1-3 2 1-2 C-dimension 1 1-1 2-1 3-1 4-1

HMBC (Heteronuclear Multiple-Bond Correlation Spectroscopy) AQ 13C Pulse sequence for HMBC C2, C3 and C4: Quaternary or protonated carbons X: O, N Long range connections or connections between spin systems

Gradient HMBC H-dimension C-dimension 2 3 1 5 1 2 3 3-4 4 3-5 5 2-6 1-7 7 8 2-7 1-8

2D NOESY (Nuclear Overhauser Enhancements SpectroscopY) AQ The NOESY pulse sequence. —C — ~ —C — VC*r -6, r0.5nm Ha Hb r0.5nm For resonance assignment, chemical structure elucidation & 3D structure determination

Gradient NOESY 3-CH3, 5-H

assignment of peptides 2 3 1 1 2 1 3 3 NOESY gives sequential assignment of peptides 2

2D ROESY pulse program 90° t1 Spin-lock t2 mixing time t1 Spin-lock t2 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.

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.