NMR Applications in Chemistry Chem Shift J Coupling

NMR Applications in Chemistry Chem. Shift J Coupling NMR spectrum Peak Int. Structure NOE Correlation H-H，C-H Applications: • Sample quality control for Synthetic works. • Elucidation of chemical structures. • Getting functional group, bonding, dynamics, kinetics and chemical exchange information of molecules. • 3 D structures of the molecules.

Some common NMR experiments: q Connections through bonds（spin-spin coupling） Homonuclear： 1 D、2 D 1 H-1 H COSY, DQF-COSY, TOCSY usage：spin system assignment Heteronuclear： Direct (detect 13 C): APT, DEPT, HETCOR Inverse (detect 1 H): HMQC, HSQC, HMBC, HSQC-COSY, HSQC-TOCSY, HMQC-TOCSY usage：assigning heteronuclei、connecting spin systems q Connections through space（dipolar coupling） 1 D、2 D NOESY，ROESY, HOESY(HSQC-NOESY) usage： connecting spin systems 、structure determination

2 D COSY — Homonuclear Shift COrrelation Spectroscop. Y x° 90° t 1 t 2 AQ The basic COSY (x=45° or 90°) pulse sequence COSY spectrum is used for determining the connectivities between protons on the basis of geminal and vicinal couplings. Disadvantage: bulky dispersive diagonal peaks.

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

2 D DQF－COSY(Double-Quantum Filtered COSY) 90 t 1 90 t 2 F allow J coupling constant measurements F observing cross-peaks close to diagonal F But lower sensitivity (~40%) of COSY expt.

2 D Gradient DQF-COSY

3 J=8. 00 Hz

TOCSY (TOtal Correlation Spectroscop. Y) or HOHAHA(Homonuclear Hartman-Hahn Spectroscopy) t 1 mixing time MLEV 17 t 2 AQ 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.

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

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

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

DEPT: Distortionless Enhancement by Polarization Transfer Heteronuclear expt. Detection: 13 C Distinguish CH, CH 2, CH 3 By suitable combination of =45, 90 & 135 spectra CH & CH 3 up CH 2 down Only CH All CH’s

HETCOR (Heteronuclear chemical shift correlation, 1 H - 13 C COSY) 13 C 1 H AQ t 1 1 2 t 2 1 H decoupling Removing JCH splittings The standard pulse sequence for 13 C-detected 1 H-13 C chemical shift correlation. *But Inverse experiment has the following Advantages: • increase sensitivity of detecting the less sensitive nuclei • 1 H is in the direct detection dimension => larger np => better resolution

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

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

HSQC-TOCSY mixing time SLy 1 H 13 C SLy MLEV 17 /2 t 1 /2 AQ GARP-1 Pulse sequence for 1 H-detected HSQC-TOCSY experiment through single-quantum coherence. Longer mixing time gives longer extend of spin-spin correlations.

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

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

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

HMBC (Heteronuclear Multiple-Bond Correlation Spectroscopy) 1 H t 1 AQ 13 C Pulse sequence for HMBC C 2, C 3 and C 4: Quaternary or protonated carbons X: O, N Long range connections or connections between spin systems

Gradient HMBC 3 1 H-dimension 876 5 4 C-dimension 3 2 1 5 2 3 -4 3 -5 2 -6 2 -7 1 -8

2 D NOESY (Nuclear Overhauser Enhancements Spectroscop. Y) t 1 t 2 m AQ The NOESY pulse sequence. —C — ~ —C — Ha Hb r 0. 5 nm V C*r -6, r 0. 5 nm For resonance assignment, chemical structure elucidation & 3 D structure determination

Gradient NOESY 3 -CH 3, 5 -H

3 1 2 21 3 2 3 1 NOESY gives sequential assignment of peptides

2 D ROESY pulse program 90° mixing time t 1 Spin-lock t 2 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 Ø 1 D 1 H & 13 C & DEPT (+MS、IR，basic chemical structure or functional groups information） ØEstablish 13 C-1 H connections by thru bond JCH couplings HMQC、HSQC-TOCSY experiments ØEstablish 1 H-1 H connection (spin systems or partial pieces） Decoupled 1 H, 1 D TOCSY, 2 D 1 H-1 H COSY, TOCSY expts. （usually starts with well-resolved 1 H signals） ØLong range connections（connecting spin systems & assigning quaternary carbon） 1 D NOESY & 2 D HMBC, NOESY, ROESY experiments Ø 3 D structure or conformation determination 1 D NOESY & 2 D NOESY, ROESY, (HSQC)-NOESY expts.