Using Iron Porphyrins as Models for Hemoglobin The

Using Iron Porphyrins as Models for Hemoglobin The system:

Key Features of Hemes • • Fe oxidation state Fe spin state porphyrin oxidation state porphyrin hydrophobicity

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

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

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

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

How does shift, , relate to a magnetization of paramagnetic sample? Shift of signal, in Hz g = 3 0 c mass susceptibility of solvent Mass susceptibility (+) -a diamagnetic contribution, a (-) value Magnetic field (400 MHz, or 400 x 106 Hz) Concentration of sample, in g/m. L

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

How does mass susceptibility, g , relate to unpaired electrons in a paramagnetic sample? Mass susceptibility g x (Mol. Wt. ) = M Molar susceptibility corr = M - diamagnetic corrections where diamagnetic corrections for Fe, porphyrin, Cl, imidazole, a negative number! eff = 3 R T corr N 2 eff = (n(n+2))1/2 = 2. 828 (T corr ) 1/2

Diamagnetic Corrections (cgs units) Xo (CHCl 3) = - 4. 97 x 10 -7 cgs Porphyrin: TPP= -700 x 10 -6 cgs TTP= -753 x 10 -6 cgs TCl. PP= -760 x 10 -6 cgs Fe = -13 x 10 -6 cgs Cl = -20 x 10 -6 cgs Imidazole = -38 x 10 -6 cgs

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

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

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

How Magnetic Nuclei Benefit NMR Experiments

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

Use of Cr(acac)3 as a Paramagnetic Relaxation Agent in With Cr(acac)3 (note: does not affect chemical shifts) With d 1=6. 0 s (d 1: relaxation time)

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

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

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

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 subfractions (water vs fat protons) and by the spin-lattice relaxation time T 1 and phase relaxation time T 2 in each nuclear spin sub-fraction. T 1 and T 2 depend on tissues type. MRI Contrast Agents interact with one sub-fraction type (usually that easily exchangeable protons, like water) to increase the T 1 spin-lattice relaxation times. The most commonly used compounds for contrast enhancement are gadolinium-based. MRI contrast agents are used as oral or intravenous administration. 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.