3a-homo2

Chem-806Identification of organic and inorganic compounds by advance NMR techniques : 

Chem-806Identification of organic and inorganic compounds by advance NMR techniques Tool box 2D-NMR: Homonuclear 2D-NMR: Heteronuclear 3D-NMR

2D NMR : 

2D NMR

Homonuclear 2D correlation techniques : 

Homonuclear 2D correlation techniques Through Bond: nJHH (scalar coupling) COSY : COrrelated SpectroscopY Directly coupled neighbors Relay-COSY : RELAY-COrrelated SpectroscopY Directly coupled neighbors and protons coupled to the coupled neighbors (relay transfer) TOCSY : TOtal Correlation SpectroscopY Directly coupled neighbors and protons coupled to the coupled neighbors (More efficient than Relay) Through Space: Distance NOESY : NOE SpectroscopY ROESY : ROE SpectroscopY NOE in Rotating frame

COSY : 

COSY

Indirect chemical shift detection during t1 : 

Indirect chemical shift detection during t1

COSY: evolution during t1 : 

COSY: evolution during t1

Processing for Absolute value COSY: Sine-belland Pseudo-echo shaping : 

Processing for Absolute value COSY: Sine-belland Pseudo-echo shaping

COSY : explanation of cross peaks using Vector Model : 

COSY : explanation of cross peaks using Vector Model

1H NMR of glucose derivative : 

1H NMR of glucose derivative

2D-COSY of glucose derivative : 

2D-COSY of glucose derivative 1 2 3 4 5 5 6 a/b 4

C4H8O : 

C4H8O CH3 – CH2 – CH2 – OH

C4H8O: COSY : 

C4H8O: COSY CH3 – CH2 – CH2 – OH CH3 CH2 CH2 OH

C5H8O2 I= 5 – 8/2 +1 = 2 : 

C5H8O2 I= 5 – 8/2 +1 = 2 CH2 - O CH2 - O O-C=O

C5H8O2 : 

C5H8O2 CH2 - O a b c d a CH2 c CH2 b CH2

C8H16O : I = 8 – 16/2 + 1 = 1 : 

C8H16O : I = 8 – 16/2 + 1 = 1 C=O Ketone CH2 – CO – CH2

C8H16O : 

C8H16O Me-1 2 Me1 – CH22 3 – CH23 4 3 5 – CH24 CH25 Me8 5 C=O CH27 Me-8 CH27/5

C11H20O4 : 

C11H20O4 O-C=O 2H 2H 3H 3H O – CH2 – CH3 CH2 – CH3 O-C

C11H20O4 : 

C11H20O4 O – CH2 – CH3 CH2 – CH3 O = C – O – CH2 – CH3 O = C – O – CH2 – CH3 CH2 – CH3 CH3 – CH2 Only missing quaternary carbon C

COSY-90: disaccharide : 

COSY-90: disaccharide H1 H2 H3? H1 H2 H3 H4/2 H5a H5b

COSY-45 : 

COSY-45 J1,2 => negative J1,3 J2,3 => positive

COSY-45 : 

COSY-45 If we consider cross peak 3/1 Passive couplings – involving passive nuclei 2 - (J1,2 and J2,3 ) have different Sign (negative slant) If we consider cross peak 2/1 Passive couplings – involving passive nuclei 3 - (J1,3 and J2,3 ) have same Sign (positive slant)

COSY45: H2 and H4 overlap at ~ 4.7 ppm. Cross-peak in COSY-45 allow to assign H3 and H5a/b unambiguously as H4 is coupled to a geminal pair => different sign in J : 

COSY45: H2 and H4 overlap at ~ 4.7 ppm. Cross-peak in COSY-45 allow to assign H3 and H5a/b unambiguously as H4 is coupled to a geminal pair => different sign in J 2/4 1 2 3/5a/5b? 5a 5b

Slide 23: 

H2 H3 H4 H5 H6a/b

Slide 24: 

H2 H3 H4 H5 H6a/b Deshielded multiplet H5’ H6a’ H6b’ H4’ H4’ H3’ H2’ H1’ H6a H6b H5

Phase sensitive COSY : 

Phase sensitive COSY

GE-DQCOSY : 

GE-DQCOSY

DQCOSY: Active coupling is antiphase and there is no intensity on central peak of a triplet (as positive and negative peaks cancel out) : 

DQCOSY: Active coupling is antiphase and there is no intensity on central peak of a triplet (as positive and negative peaks cancel out)

Soft-COSY : 

Soft-COSY

Multiple Relay-COSY:As we have longer relay sequence => relaxation during transfer step attenuate the signal and Magnetization get weaker : 

Multiple Relay-COSY:As we have longer relay sequence => relaxation during transfer step attenuate the signal and Magnetization get weaker

Relay-1 and Relay-2 COSY on disaccharide : 

Relay-1 and Relay-2 COSY on disaccharide

TOCSY or HOHAHA : 

TOCSY or HOHAHA

1D-HOHAHA : 

1D-HOHAHA

NOE and distance : 

NOE is a consequence of cross-relaxation between 2 spins close to each other in space. NOE is a consequence of modulation of the Dipole-Dipole coupling by motion of the molecule in solution. The NOE intensity is related to the internuclear distance r and is a function of the correlation time tc NOE and distance

Relaxation and tumbling rate : 

Relaxation and tumbling rate Relaxation is caused by fluctuating magnetic field generated by neighboring dipole. If the rate at which the fluctuation occur in the transverse plane matches the frequency of double quantum transition, positive NOE will be observed. If the fluctuation is slower, zero quantum transition will produce negative NOE.

NOE is related to dipole relaxation : 

NOE is related to dipole relaxation For small molecules, translation and rotational motion occur at high frequency. The vector linking two nuclei rij change orientation more frequently in small molecule than in larger molecule (Small molecules tumble at rates around 1011 Hz, Larger molecules such as proteins tumble at rates around 107 Hz), dissipating the energy between different spin states In small molecules the frequency of motion can occur frequently at Larmor frequency o and twice 2x o (W2) dissipating energy between single quantum and double quantum state. (this produce Positive NOE) In Large molecule, only low frequency transition like Zero quantum (W0) can dissipate energy (aibj  biaj ) (Negative NOE)

NOE: applying gB2 to the A of an AX spin system : 

NOE: applying gB2 to the A of an AX spin system X1 X2 A2 A1 {A} Immediately after irradiation, there is NO change in the intensity of X Turning on the Decoupler do not change population of the X transition

NOE: relaxation with double quantum pathway W2 probability (positive NOE) : 

NOE: relaxation with double quantum pathway W2 probability (positive NOE) After W2 relaxation, there is a net increase in the intensity of X (50%) … T1 Dec. continue Relaxation takes time to establish a new equilibrium: T1 process delay W2

NOE: Relaxation with zero quantum pathway W0 probability (negative NOE) : 

NOE: Relaxation with zero quantum pathway W0 probability (negative NOE) After W0 relaxation, there is a net decrease in the intensity of X (50%)  negative NOE … T1 Dec. continue Relaxation takes time to establish a new equilibrium: T1 process delay W0 W0

NOE: summary of relaxation pathways : 

NOE: summary of relaxation pathways W1: probability of single quantum relaxation do not create nOe A new population ditribution is generated by relaxation through dipole-dipole relaxation : double quantum and zero quantum pathway W2 and W0 If W2 is efficient (small molecule – fast motion  large frequency ) Level  increase  level  increase also with decoupler continuing W2 W0 W2 pathway yield positive nOe If W0 is efficient (large molecule – slow motion small freq. Diff.) Level  increase  level  increase also with decoupler continuing W0 pathway yield negative nOe

Maximum NOE vs correlation time (nuclei interacting with 1H) : 

Fast tumbling maximum NOE : 0.5 Line intensity : Ii = 1 + hi {H} Large molecule Small molecule w tc ~1 Maximum NOE vs correlation time (nuclei interacting with 1H) hC = 4/2 = 2 hN = 10/-2 = -5

NOE vs Distance and motion : 

NOE vs Distance and motion If B is irradiated, nucleus A should show the largest NOE (closest to nucleus B) (the relative distances are shown as A to B = 1, B to C and C to D = 2). Nucleus C is relaxed by nucleus D as well as B, so it shows a smaller NOE. Nucleus D has an indirect NOE from nucleus B. Indirect effects usually give rise to negative NOEs. Note that as the tumbling rate decreases all other parameters become irrelevant and the NOEs tend towards -100%. The notation fA{B} means the NOE enhancement of spin A when spin B is saturated.

NOE difference: nOe-d : 

NOE is a kinetic effect: need delay ~ T1  It take time to develop  It takes time to decay NOE difference: nOe-d difference control nOe

Choosing a structure by nOe : 

H6 H5 Choosing a structure by nOe H3 {OMe} {OH}

Assigning the relative stereochemistry : 

Assigning the relative stereochemistry

NOEd (NOE difference) vs distance : 

C HB HA C HC HA HB HC HA HB HC - + hAB hAC hAB  rAB-6 hAC  rAC-6 rAC = rAB * (hAB / hAC)-1/6 NOEd (NOE difference) vs distance

NOE on small ligand : 

NOE on small ligand Irradiation of free- ligand resonance A result in NOE's slowly building up on HB

TR-NOE: Small molecule interacting with a large protein : 

Transfer of the spin saturation information via chemical exchange to the bound state results in a much more rapid build-up of NOE's on protons HC. This NOE is transferred back to the free resonance via a chemical exchange, where it decays relatively slowly. The net result is that the changes in intensity in free- ligand signals reflect predominantly bound ligand NOE's, provided certain kinetic conditions are met. TR-NOE: Small molecule interacting with a large protein

NOE experiments : 

NOE experiments Steady state NOE: D1 (5 * T1) Used for NOE-d) Truncated NOE (TOE): apply various short delay D1 (tau) to study NOE buildup rate Transient NOE: single resonance inverted. During tau delay transient NOE buildup(similar to NOESY)

NOE build-up : 

For different tm values, we get ideal NOE build-up curves, which in the case of two isolated protons are exponentials that grow until they reach hmax. tm tm hmax hmax NOE build-up If we take into account the T1/T2 relaxation, the NOE grows and then falls to zero (all the magnetization returns to the <z> axis…): Steady State Transient NOE

Transient NOE: Build-up rate : 

K. Wutrich Nobel lecture (03) Transient NOE: Build-up rate

NOE build-up rate : 

NOE build-up rate In transient experiments there are two competing processes 1- cross-relaxation from the perturbed spins which causes NOE enhancements. 2- T1 relaxation restores all intensities destroying NOE. The initial buildup rate in a 1D transient NOE experiment is twice the rate for NOESY and TOE because the spin S is inverted rather than being flipped through 90° (NOESY) or being saturated (TOE). In steady state and TOE experiments, As spin S is saturated continuously, the relaxation rate irrelevant.

Direct cross relaxation and spin diffusion : 

Direct cross relaxation and spin diffusion 1 2 3 Direct cross relxation between 1 and 2 And between 1 and 3 Spin diffusion between spin 2 and 3 : The NOE on 3 is no more representing The distance between 1 and 3

Transient NOE and NOESY : 

Transient NOE and NOESY If we had more spins and spin diffusion, the curve (for three spins, A, B, and C) would look like this hmax Diffusion to C Mixing time If we want to measure distances accurately, we have to find a compromise between the mixing time tm and the spin-diffusion we may have.

NOE example : 

NOE example

Other experiments to measure nOe : 

Other experiments to measure nOe Selective NOESY DPFGSE-NOE

DPFGSE-NOE : 

DPFGSE-NOE Double Pulse Field Gradient Spin Echo

DPFGSE-ROE : 

DPFGSE-ROE

Comparing : 

DPFGSE-ROE Positive ROE Comparing DPFGSE-NOE Negative NOE NOE-d

Which isomer is responsible of that spectra? : 

Which isomer is responsible of that spectra?

NOESY : 

H3 H4 H5 OMe NOESY

NOESY and EXSY : 

NOESY and EXSY NOE SpectroscopY EXchange SpectroscopY

Dynamic NMR : 

Dynamic NMR A.D. Bain / Progress in Nuclear Magnetic Resonance Spectroscopy 43 (2003) 63–103 Rate of exchange at Coalescence temperature NOESY can only be acquired on Slow exchange regime Mixing time should be selected based on relaxation time and kc kc > (tm)-1 > 1 / T1 Dn Tc = 263K 273K 253K 243K 223K

NOESY: Vector Model : 

NOESY: Vector Model

NOESY : 

NOESY C HB HA C HC HA HB HC Measuring an unknown distances is possible using the peak intensities and the distance of a proton pair with a fixed distance as reference rref rAC = rAB * (VAB / VAC) 1/6 geminal proton-proton distance: 1.78 Å;

EXSY :Exchange Spectroscopy : 

EXSY :Exchange Spectroscopy A.D. Bain / Progress in Nuclear Magnetic Resonance Spectroscopy 43 (2003) 63–103 Very short mixing time (0.01 s): no exchange cross-peaks are visible. longer mixing time (1.0 s): shows the cross-peaks.

Example of EXSYExchange of heptamethyl Benzonium ion : 

Example of EXSYExchange of heptamethyl Benzonium ion 1 2 2 3 3 4 1  3 2  4 Ref. Meier & Ernst, JACS, 101, 6441 (1979)

heptamethyl benzenonium ion : 

heptamethyl benzenonium ion Two-dimensional exchange spectrum. of the protons in heptamethyl benzenonium ion in 9.4 M H2SO4 R. R. Ernst, G. Bodenhausen, and A. Wokaun, “Principles of Nuclear Magnetic Resonance in One and Two Dimensions”, Oxford University Press (1987).

EXSY example of 11B : 

EXSY example of 11B

NOESY gives information on 3D structure : 

NOESY gives information on 3D structure To get that information, NOESY need to be run using a mixing time characteristic of the sample’s relaxation time. For small molecule, use MIX~80% of T1 NOE

Relaxation Time : 

Relaxation Time 0.3 0.6 1.0

NOESY with small Mixing Time : 

NOESY with small Mixing Time

NOESY with longer Mixing Time : 

NOESY with longer Mixing Time

Intensity of cross peak and diagonal signal vs Mixing Time : 

Intensity of cross peak and diagonal signal vs Mixing Time

Evaluation of Distance in NOESY : 

Evaluation of Distance in NOESY

Using NOESY to assign DNA structure : 

Using NOESY to assign DNA structure

DNA: Proton-NMR : 

DNA: Proton-NMR

COSY Cytosine : 

COSY Cytosine 5/6 1’/2’ 3’ 3’/4’ 5’5”

NOESY-DNA : 

NOESY-DNA H1’ -> Base H2’/2” -> Base H2’/2” -> H1’

Slide 86: 

H1’-8/G8 G8 H1’-7 H1’-7/C7 C7-H6 C7-H5/6 H1’-6 C7-H5->G6 G6 G6 G6 H1’-5 A5 A5 H1’-4 T4 T4 C3 C3-H5/6 C3-H5->G2 H1’-3/2 G2 H1’-1 C1 C1 C1-H5/6

NOE Walk : 

NOE Walk

Slide 88: 

G8 C7 H1-8 H2’ H2” C7-H5 H1-7 H1-6 A5 G6 H1-5

11B : 

11B

COSY : 11B : 

COSY : 11B 6/9 2/4 5,7,8,10 1/3

Next Chapters : 

Next Chapters Example of homonuclear 2D technique On Cyclic peptide 1D and 2D heteronuclear NMR A few examples: ipsenol etc… More examples: Unknown example Miscellaneous topics: HOESY, DOSY, etc... 3D-NMR : protein assignment Example from Snieckus lab COSY and NOESY