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Example from Wang group. Work of Jiasheng Lu. Starting material. UV: Postulated Reaction. +. H-NMR: Mesitylene. H-NMR: Aliphatic region. Me-A. New compound. * : Starting material. Me-B. *. H-NMR: Aromatic region . * : Starting material. *. *. *. *. COSY: Full SW. Me. .
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Example from Wang group Work of Jiasheng Lu
H-NMR: Aliphatic region Me-A New compound *: Starting material Me-B *
H-NMR: Aromatic region *: Starting material * * * *
COSY: Full SW Me Aromatic
COSY expansion: Me vs aromatic Methyl groups have long range coupling with ortho aromatic protons Starting material H-C= H-C= H-C= Me Me Me Me New compound Me
Ms Ms Ms 1 A 3 2 4 D COSY: aromatic C B Most deshielded Py: 1 – 2 – 3 – 4 Other ring : A – B – C – D Not sure which proton is H-7 Start with a doublet (can be either H7 or H10) then go To the next proton to identify the suite. We will assign later with NOESY which one is H7
NOESY: Choosing the Mixing time • You cannot use too short mixing time: it takes time to develop NOE. It takes T1 • too short mixing time would not show any cross peaks between protons! • You cannot use too long mixing time. If you use too long mixing time, you can loose the labels of the chemical shifts that you record carefully during the evolution time (t1) • For small molecules, the best mixing time is ~ 80% of the T1 value. (this is where the signal goes to null intensity in inversion recovery
Prepare for NOESY: evaluate relaxation time Inversion recovery: Aromatic region A 4 HA relax fast at ~ same rate as H4: same type of neighbors: may be H7 ? D 3 C 2 1 Ms Why different rate? Dipole Relaxation depends on the distance between protons and on the mobility of the molecule Py: 1 – 2 – 3 – 4 Ms 12 ? Other: A – B – C – D In Pyridine ring, H4 has faster relaxation: it has one ortho neighbor (H3) and it’s close to H7on other ring. In fact, H7 must be very close to H4 because H3 and H2 (which has 2 ortho neighbors) relax slower than H4 Ms is the smallest molecule: fast motion, not efficient T1 longest relaxation time Ms
H-NMR Inversion recovery: Methyl region Starting material Ms Ms Ms Ms CH2-12 Newcompound Ms is the smallest molecule: fast motion, not efficient T1 longest relaxation time Ms
NOESY: Choosing the mixing time • You cannot use too short mixing time: it takes time to develop NOE. It takes T1 • too short mixing time would not show any cross peaks between protons! • You cannot use too long mixing time. If you use too long mixing time, you can loose the labels of the chemical shifts that you record carefully during the evolution time (t1) • For small molecules, the best mixing time is ~ 80% of the T1 value. (this is where the signal goes to null intensity in inversion recovery In our case, MIXING = 2 sec. will do good job according to T1
Ms Ms 12 ? Ms D A 3 C 4 2 NOESY : mix time 2sMethyl vs aromatic 1 Py B Starting material Me Me Me Me Py: 1 – 2 – 3 – 4 Other: A – B – C – D Me New compound
Ms Ms 12 Ms 3 4 A D 2 NOESY :mix time 2sAromatic 1 C B Py: 1 – 2 – 3 – 4 Other: A – B – C – D 7 – 8 – 9 – 10 D A 12 C D
What’s left? Assign C13 Use HSQC to assign carbons that are directly attached to protons 1JCH (HSQC : HeteronuclearSingle Quantum Correlation) Use HMBC to assign carbons that are further away from protons nJCH (HMBC: HeteronuclearMultiple Bond Correlation)
Ms Ms Ms HSQC: aromatic 12 3 4 7 10 2 1 8 9 Ms: 127.4 C12: 118.5 C7: 124.5 C1: 136.2 C8: 119.5 C2: 115.5 C9: 128.6 C3: 127.9 C10: 128.4 C4: 121.5
Ms Ms HSQC: Methyl Ms Ms Ms CH2 21.0 Starting material 20.6 25.2 New compound 21.1 22.5
Ms Ms HMBC: Methyl Ms Ms Ms CH2 21.0 Starting material 20.6 25.2 New compound 21.1 136.7 127.5 140.2 22.5 138.9
Ms Ms Ms HMBC: aromatic 12 4 7 10 3 2 1 8 9 C12: 118.5 2 6 C6: 118.8 C7: 124.5 C1: 136.2 C8: 119.5 C2: 115.5 9 10 C9: 128.6 C3: 127.9 C10: 128.4 C4: 121.5 C11: 145.6 C5: 142.3 11 11 21.1 136.7 127.5 140.2 22.5 138.9