Using Iron Porphyrins as Models for Hemoglobin

The system:

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

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?

Sample for Evans’ Magnetic Susceptibility Method

Inside capillary: sample in CHCl3, 1) with imidazole 2) without imidazole

Outside capillary: CDCl3 and CHCl3

NMR tube

NMR Spectrum from Evans’ Method

Inside capillary: sample in CHCl3,

produces broad singlet for paramagnetically shifted CHCl3 below 7.3 ppm

Outside capillary: CDCl3 and CHCl3

produces usual sharp singlet for 0.5% CHCl3 at 7.3 ppm

Why is H resonance in CHCl3 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?

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 106 Hz)

Concentration of sample,in g/mL

Magnetic field lines of flux

Magnetic field linesaffected by a paramagneticsubstance: attractsSusceptibility, X > 0

Magnetic field linesaffected by a diamagneticsubstance: repelsSusceptibility, X < 0

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 susceptibility Molar susceptibility

Diamagnetic Corrections (cgs units)

Xo (CHCl3) = - 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

The Role of Axial Ligation and the Allosteric Effect in Hemoglobin O2 Binding

3d orbitalson 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

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 13C NMR

Use of Paramagnetic NMR in Bioinorganic Systems

v

v

m

m

m

p p

One big mess of piled up H’son protein!!

NMR Paramagnetic Shift Reagents

Ground state electron configuration: [Xe] 4f7 6s2

Term Symbol:   8S7/2 how many unpaired e-?

EuFOD :also called Eu(fod)3.

Eu(OCC(CH3)3CHCOC3F7)3

NMR Paramagnetic Shift Reagents: Eu vs Pr

Using Eu(fod)3

Using Pr(fod)3

With NOShift rgt

Hmmm, not so pretty

oooh! Lovely!!

Huh? – signals shifted upfield with Pr

Gadoteric acid

Effect of contrast agent on images: Defect of the blood–brain barrier after stroke shown in MRI. T1-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.

• MRI contrast agents are used as oral or intravenous administration.