1. Using Iron Porphyrins as Models for Hemoglobin The system:

2. Key Features of Hemes Fe oxidation state Fe spin state porphyrin oxidation state porphyrin hydrophobicity

3. Low Spin S = 1/2 n = 1 High Spin S = 5/2 n = 5 Intermediate Spin S = 3/2 n = 3 How will the spin state of Fe(porphyrin) complexes change on binding imidazole?

4. Sample for Evans’ Magnetic Susceptibility Method Inside capillary: sample in CHCl 3, 1) with imidazole 2) without imidazole Outside capillary: CDCl 3 and CHCl 3 NMR tube

5. NMR Spectrum from Evans’ Method Inside capillary: sample in CHCl 3, produces broad singlet for paramagnetically shifted CHCl 3 below 7.3 ppm Outside capillary: CDCl 3 and CHCl 3 produces usual sharp singlet for 0.5% CHCl 3 at 7.3 ppm 

6. Why is H resonance in CHCl 3 shifted downfield and broadened? pseudocontact and contact terms addition of new small magnetic field to local magnetic fields of neighboring nuclei is used in NMR Shift Reagents to “de-tangle” complicated spectra

7. How does shift, , relate to a magnetization of paramagnetic sample?  g = 3  0     c Mass susceptibility (+) Shift of signal, in Hz mass susceptibility of solvent -a diamagnetic contribution, a (-) value Magnetic field (400 MHz, or 400 x 10 6 Hz) Concentration of sample, in g/mL

8. Magnetic field lines of flux Magnetic field lines affected by a paramagnetic substance: attracts Susceptibility, X > 0 Magnetic field lines affected by a diamagnetic substance: repels Susceptibility, X < 0

9. How does mass susceptibility,  g, relate to unpaired electrons in a paramagnetic sample?  g x (Mol. Wt.) =  M  corr =  M -  diamagnetic corrections where  diamagnetic corrections for Fe, porphyrin, Cl, imidazole, a negative number!  eff = 3 R T  corr 1/2 = 2.828 ( T  corr ) 1/2 N  2  eff = (n(n+2)) 1/2 Mass susceptibilityMolar susceptibility

10. Diamagnetic Corrections (cgs units) X o (CHCl 3 ) = - 4.97 x 10 -7 cgs Porphyrin: TPP= -700 x 10 -6 cgs TTP= -753 x 10 -6 cgs TClPP= -760 x 10 -6 cgs Fe = -13 x 10 -6 cgs Cl = -20 x 10 -6 cgs Imidazole = -38 x 10 -6 cgs

11. The Role of Axial Ligation and the Allosteric Effect in Hemoglobin O 2 Binding

12. 3d orbitals on Fe Spin State of Fe affects size of ion

13. Large, high spin Fe(2+): In T state, transmitted by His on protein helix Small, low spin Fe(2+): In R state, transmitted to His on protein helix

14. How Magnetic Nuclei Benefit NMR Experiments

15. Use of Cr(acac) 3 as a Paramagnetic Relaxation Agent

16. With Cr(acac) 3 (note: does not affect chemical shifts) With d1=6.0s (d1: relaxation time) Use of Cr(acac) 3 as a Paramagnetic Relaxation Agent in 13 C NMR

17. Use of Paramagnetic NMR in Bioinorganic Systems v v m m m pp One big mess of piled up H’s on protein!!

18. NMR Paramagnetic Shift Reagents Ground state electron configuration: [Xe] 4f 7 6s 2 Term Symbol: 8 S 7/2 how many unpaired e-? EuFOD :also called Eu(fod) 3. Eu(OCC(CH 3 ) 3 CHCOC 3 F 7 ) 3

19. NMR Paramagnetic Shift Reagents: Eu vs Pr Using Eu(fod) 3  Using Pr(fod) 3  With NO Shift rgt  Hmmm, not so pretty oooh! Lovely!! Huh? – signals shifted upfield with Pr

20. Gadoteric acid Effect of contrast agent on images: Defect of the blood–brain barrier after stroke shown in MRI. T 1 -weighted images, left image without, right image with contrast medium administration. MRI Contrast agents: same principles, applied to medicine MRI Contrast Agents: observes differential magnetization of protons in different types of molecules that predominate in different tissues. The different magnetization signal intensities produce the contrast between tissues. The nuclear magnetization is produced by the pulse sequence applied, by the density of nuclear spins sub- fractions (water vs fat protons) and by the spin-lattice relaxation time T1 and phase relaxation time T2 in each nuclear spin sub-fraction. T1 and T2 depend on tissues type. MRI Contrast Agents interact with one sub-fraction type (usually that easily exchangeable protons, like water) to increase the T1 spin-lattice relaxation times. The most commonly used compounds for contrast enhancement are gadolinium-based.gadolinium MRI contrast agents are used as oral or intravenous administration.intravenous administration