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Miscellaneous topics

Miscellaneous topics. Heteronuclear NOE : HOESY Shift reagent Chemical exchange Measuring pH Diffusion : DOSY. t 1. t m. Dec. 1 H. X. X-detected HOESY experiment. H eteronuclear O verhauser E ffect S pectroscop Y. The first 90 o pulse create transverse H magnetization

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Miscellaneous topics

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  1. Miscellaneous topics Heteronuclear NOE : HOESY Shift reagent Chemical exchange Measuring pHDiffusion : DOSY

  2. t1 tm Dec 1H X X-detected HOESY experiment Heteronuclear Overhauser Effect SpectroscopY • The first 90opulse create transverse H magnetization • During the evolution time t1 H chemical shift is characterized (coupling with X nuclei is removed with 180o (X) applied at mid evolution) • The second 90opulse on H returns magnetization to Z axis where it is left to exchange with NOE during the mixing period • At the end of the mixing, a 90o pulse on X-nuclei create the NOE induce X-magnetization which is then detected during t2

  3. thymolphthalein monophosphate hydrate HOESY Walter Bauer, MAGNETIC RESONANCE IN CHEMISTRY, VOL. 34, 532-537 (1996)

  4. HOESY Me-8/9 H7 H2O H6 Figure 4. 'Normal' (heteronucleus detected) '' P,'H HOESY spectrum of 1, 0.17 M solution in CD,OD, pulse sequence as in Fig. 1 (I = ' H, S = 31 P). The one-dimensional spectra are separately measured single pulse-acquire data. Standard phase cycling22 was employed. S =solvent. For spectrum parameters, see Experimental

  5. PFG - HOESY experiment Inverse Heteronuclear Overhauser Effect SpectroscopY t1 tm 1H D2 D2 Dec D1 D1 X G • The first 90opulse create transverse H magnetization. During the evolution time t1 H chemical shift is characterized. [The first gradient encode H-shift] • The second 90opulse on H returns magnetization to Z axis For exchange with NOE • The second gradient destroy transverse magnetization • At the end of the mixing, a 90o pulse on X-nuclei create the NOE induce X-magnetization which is then detected during t2. • The third gradient rephase magnetization. G1/G3 = gx/gH

  6. HOESY- H-detected Figure 6. 'Inverse' ('H detected) PFG-enhanced 31P,1H HOESY spectrum of 1, 0.17 M solution in CD,OD, pulse sequence as in Fig. 2 (I = ,'P, S = 'H). The one-dimensional spectra are separately measured single pulse-acquire data. For 'H spectral parameters, see Fig. 4. For spectrum parameters, see Experimental.

  7. Butyl-Lithium: HOESY Li->H a b a b Figure 7. 'Normal' (heteronucleus detected) 7Li,'H HOESY spectrum of n-BuLi, 0.8 M solution in hexane-CEDE (1 3, v/v),

  8. Butyl-Lithium: HOESY Li->H a b b a Figure 9. 'Inverse' ('H detected) PFG-enhanced 7Li,'H HOESY spectrum of n-BuLi, 0.8 M solution in hexane-C,D, (1 :1, v/v),

  9. Shift reagents Paramagnetic ions (Ni, Co, Fe..) usually brings broadening of the peaks But it was discovered that paramagnetic Lanthanide ionsdo not broaden the spectra significantly and produce large shift in the signals + CH3-CH2-CH2-CH2-CH2-CH2-OH Eu(DPM)3 • Addition of Eu(DPM)3 causes: • Deshielding of all protons shift (lower field) • Amount of shift increase with the proximity of the oxygen

  10. 3 cos2 - 1 Dd = K r3 r Shift reagents  L + Eu(dpm)3 L-Eu(dpm)3 d(exp) = p(L) * dL + p(L-Eu)* dL-Eu

  11. Chiral Shift reagents + Eu(TFC)3 Pure L • Magnitude of the shift depends on: • Molar ratio of shift reagent added • Complex strength : • NH2 > OH > C=O > COOR > C=N

  12. r - s ee = r + s Chiral Lanthanide Shift reagents Enantiomers are not distinguishable in NMR (unless diastereomer are formed) M(+) + M (-) + LSH(-) M(+) / LSH(-) + M (-) / LSH(-) ee=enantiomeric excess

  13. Chiral solvent

  14. Chemical exchange vs temperature calculation of a two-site exchange system for the ratio between the chemical shift difference ∆δand the rate constant 1/τ varying between 40 and 0.1 daverage = pAdA + pBdB Fast Exchange Coalescence ∆δ Slow exchange

  15. Fast chemical exchange and binding study • “fast exchange” protein ligand titration by NMR. • The top left: start situation where no ligand has been added. The NMR signal is at δf. • The middle line : enough ligand has been added to saturate half of the binding sites in the protein. • The bottom line: ligand has been added to occupy all binding sites and the NMR signal is now at δb. daverage = pFdF + pBdB

  16. Slow chemical exchange and binding studies “slow exchange” protein ligand titration. Left: change in spectrum with increasing ligand concentration. Right:change in signal intensities with ligand concentration

  17. intermediate rate for chemical exchange and binding studies

  18. -OOC-CH-CH3 HOOC-CH-CH3 NH2 NH2 dmax - d pH = pKa + log d - dmin Chemical shift and pH dmax  shift under acidic conditions d= p1·d1 + p2·d2 dmin  shift under basic conditions d Observed shift

  19. dmax - d pH = pKa + log d - dmin Chemical shift and pH d= p1* d1 + p2* d2 p1 + p2 = 1 dmax  shift under acidic conditions dmin  shift under basic conditions d Observed shift p78

  20. Measuring Hydrogen exchange rate in amides The decrease of the NH NMR signal as a function of time as the amide proton is being exchanged with deuteron, The amide hydrogen atoms are engaged in hydrogen bond formation. By recording the hydrogen exchange rates the stability of the individual hydrogen bonds can be measured for every single peptide group of a protein

  21. Tyrosine : with TSPA reference (pH=2.3)

  22. http://asnmr4.la.asu.edu/nmr/labs.html Tyrosine vs pH (aromatic) pK ~ 10

  23. Tyrosine vs pH (aliphatic)

  24. Diffusion Studies: T2 relaxation .

  25. Gradient Echo Without diffusion 2tdelay : signal attenuation due to T2 relaxation only

  26. Diffusion

  27. Stejskal and Tanner PGSEPulse Gradient Spin Echo -D g2 g2d2 (D – d/3) I = I0 e -D q2 (D – d/3) I = I0 e D : diffusion Coefficient

  28. Diffusion of water at different temperature

  29. Diffusion of water at different temperature

  30. STE – Stimulated Echo t2 t1 t1 d d gz D - d • First pulse and gradient establish labeled phase signals (according to “z” position ) • Second pulse store this information along z into longitudinal magnetization • During D diffusion and relaxation (known attenuation) occur • The last pulse and gradient restore magnetization (attenuated by relaxation and diffusion). Diffusion attenuation is dependant on gradient strenght. Advantages over Spin-Echo: During diffusion, magnetization evolve along “Z” axis no problem with J-modulation method of choice when T1 >> T2 (large molecule)

  31. LED –Longitudinal Eddy Current Delay t2 t1 t1 STE d d gz D - d t2 t1 t1 te d d LED gz D - d • The delay t1 is chosen as small as possible – long enough to contain gradient pulse (d =1 ms and a short delay to eliminate eddy current generated by gradient) • In LED after the last gradient, an extra pulse store magnetization along Z and an extra delay te allow for the decay of eddy current

  32. BPPLED –bipolar pulses LED t2 t1 t1 te t1 t1 d/2 gz gz -gz D Very efficient to eliminate Eddy current • Gradient d is replaced by two shorter gradient d/2 separated by 180` pulse • The 180` pulse negate the sign of the phase change generated by first gradient • The second gradient is therefore reverse in sign. This correspond to applying a a total gradient pulse d. • As shorter gradient are used less Eddy current is generated and the second gradient is also self compensating for eddy current created by first gradient. • The delay te at the end can be shorten by large factor (up to 20)

  33. Mixture of compounds 4 3 1 Caffeine, Glycol and HOD 2 Glycol 4 2 3 HOD 1

  34. DOSY Diffusion-Ordered Spectroscopy Mixture of Caffeine, Glycol and D2O Diffusion

  35. Gurpreet S. Kapur,y Eurico J. Cabrita and Stefan Berger* Tetrahedron Letters 41 (2000) 7181±7185 Diffusion : DOSY Figure 1. (a) The 1H-DOSY spectrum of a mixture containing phenol (1) and cyclohexanol (2) in CDCl3 containing TMS as a reference; and (b) The 1H-DOSY spectrum of the same mixture containing DMSO-d6 (3) as an additional component. The spectra were recorded on a Bruker DRX-400 instrument using the stimulated echo sequence incorporating bipolar gradients with an longitudinal eddy delay (BPPLED). The gradient strengths of 1 ms duration were incremented in 32 steps, with diffusion times of 50 ms

  36. DOSYAnd hydrogen bound Magn. Reson. Chem. 2001; 39: S142–S148 Both components have almost similar effective sizes, and as expected, cyclohexanol, being slightly heavier and having probably the more extended hydrodynamic surface due to its chair conformation, is associated with a lower diffusion coefficient when compared to phenol. diffusion profile of the mixture is also a reflection of the molecular association between the two components, since they will be involved in H-bonding both among themselves and with each other.

  37. addition of a third component with the ability to be involved in H-bonds acting as H-bond acceptor alcohol mixture, containing 1 molar equivalent of DMSO-d6 as an additional component. Both components are capable of forming hydrogen bonds with DMSO Diffusion contours has reversed with the addition of DMSO-d6. The phenol, which was moving faster, has now a lower value of the diffusion coefficient, moving slower This means that phenol is forming much stronger hydrogen bonds with DMSO.

  38. (a) The 31P-DOSY spectrum of a mixture containing 4 phosphorous components viz. trimethyl phosphate (4), dibutyl phosphite (5), triphenylphosphine oxide (6), and triethylphosphine oxide (7); (b) The 31P-DOSY spectrum of the same mixture containing triethanol amine (8) as an additional component. triethanol amine (8) (MW=152; TEA) the additional component is H-bond donor.

  39. 31P NMR spectrum in the projection shows a small downfield shift in the resonances of TEPO and TPPO, due to their interactions with TEA. The DOSY plots provide insight about the strength of interactions which are clearly manifested in the diffusion dimension. There is a decrease in the value of diffusion coefficient of all the components due to increase of viscosity resulted due to addition of viscous TEA. However, the order of the components in the diffusion dimension has also changed, where the diffusion coefficient of TEPO has decreased considerably compared with the components TMP and DBP. This decrease in the diffusion coefficient of TEPO is attributed to its complexation with TEA through hydrogen bonds, resulting in an overall increase in the effective size.

  40. Relative H-bond acidity probed by diffusionAlcohol mixture–dimethyl sulfoxide (DMSO) ) Figure 2. (a) Expansion of the aromatic region of the 1H-DOSY plot of an equimolar mixture of phenol (1) and benzyl alcohol (4) in CCl4 containing TMS as a reference and (b) 1H-DOSY plot of the same mixture after the addition of DMSO. In both cases an expansion of the TMS region is shown.

  41. Relative H-bond acidity probed by diffusion Alcohol mixture+ DMSO slow Alcohol mixture • initial mixture, the two alcohols have identical diffusion coefficients • DOSY plot shows immediately that the two compounds experience different interactions with the H-bond acceptor DMSO since they no longer have the same diffusion coefficient. Compound 1 becomes slower than 4as a result of a stronger association with DMSO,

  42. Hindered alcohol/non-hindered alcohol–HMPA equimolar mixture of 2,6-di-tert-butylphenol (5) and 2,4-di-tert-buthylphenol (6) (both with MW D 206.3) (0.1 M in CDCl3 containing 1% TMS) The two isomeric alcohols were chosen in order to have two H-bond donors sites with very similar acidity but different steric environments,

  43. Due to steric hindrance, compound 6 interact more with H-acceptor Slower

  44. DOSY and chemical exchange Some OH exchange faster with H2O And appear at intermediate Diffusion DOSY in DMSO DOSY in DMSO + H2O Eurico J. Cabrita, and Stefan Berger, Magn. Reson. Chem. 2002; 40: S122–S127

  45. Host-guest Tetrahedron Letters 40 (1999) 3661-3664 J. Org. Chem. 2002, 67, 2639-2644

  46. Non-equivalence of CH2-N caused by CD Proof: the 2 triplet diffuse at same rate than CD n=5 + CD then HCl n=5 + HCl then CD n=5 + CD n=5 Liat Avram and Yoram Cohen*, J. Org. Chem. 2002, 67, 2639-2644

  47. Diffusion and Host-Guest CD CD n=4+CD n=2+CD n=4 n=2 CD ~ no interaction with CD n=1 + CD n=2, partial interaction with CD n=4, complete interaction with CD

  48. Host-Guest at different temperature n=4 n=5 F=Free state B=Bond state Coalescence B B F

  49. Diffusion, Host-Guest and organometallic The affinity of the cobaltocenium cation 32 for a host is at least 5 orders of magnitude larger than that of ferrocene, 33. The DOSY spectrum nicely demonstrates the different interactions and, specifically, that the ferrocene is moving much faster. Host Figure 8. The DOSY spectrum86 (400 MHz, 298 K) of a solution containing ferrocene and cobaltocenium cation in C2D4Cl2. The signal at ä ) 2.72 ppm represents the encapsulated cobaltocenium cation, which has the same diffusion coefficient as all the signals of the tetraureacalix- [4]arene dimer. The signals of the free ferrocene and cobaltocenium cation at ä ) 4.06 and 5.61 ppm, respectively, were found to have much higher diffusion coefficients. Hetero 2D Chemical Reviews, 2005, Vol. 105, No. 8

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