Different Isotopes Absorb at Different Frequencies 15N 2H 13C 31P 19F 1H 50 MHz 77 MHz 125 MHz 200 MHz 470 MHz 500 MHz low frequency high frequency
Resonance Frequencies Depends on Magnetic Field 1H 1H 1H 1H 1H 1H 200 MHz 400 MHz 600 MHz 700 MHz 800 MHz 950 MHz low field high field
Rapporto giromagnetico E= = -ħg m B DE= =ħg B La separazione in energia dipende dal valore del rapporto giromagnetico
Frequenza di precessione n0 = - g B0 /2π Se cosi fosse, ogni nucleo attivo entrerebbe in risonanza con il campo esterno alla sua frequenza e tutti gli isotopi uguali si comporterebbero allo stesso modo (un unico segnale). La frequenza di precessione di un determinato nucleo ad un determinato campo magnetico è detta FREQUENZA DI PRECESSIONE DI LARMOR Es: al campo magnetico di 11.7 T, La FREQUENZA DI PRECESSIONE DI LARMOR del nuclide 1H è 500 MHz.
La costante di schermo n= g/2p B0 (1-s) Dipende dall’intorno elettronico
Campi magnetici elevati determinano un aumento della risoluzione e della sensibilità
Chemical shift (n-nref/nref)*106 = d (ppm) Es: w1= 500.131 MHz w0=500.13 MHz w1-w0=1000 Hz = 1000/500.13x106 (ppm)= 2.0
TMS (Tetramethylsilane) chemical shift d d = 0
1H Chemical Shift Table stot= slocal + smagn+ src + sel + ssolv Pople, 1960
Fattori che influenzano il chemical shift Caratteristiche funzionali Effetti induttivi Effetti mesomeriEffetti attraverso lo spazioEffetti paramagnetici
Effetto della Sostituzione sul Chemical Shift CHCl3 CH2Cl2 CH3Cl 7.26 5.32 3.05 ppm -CH2-Br -CH2-CH2Br -CH2-CH2CH2Br 3.30 1.69 1.25 ppm
Shoolery Equation Il chemical shift dipende dalla sommatoria degli effetti di tutti I sostituenti
Fattori che influenzano il chemical shift Caratteristiche funzionaliEffetti attraverso lo spazio Correnti d’anello Anisotropia magnetica Effetti stericiEffetti paramagnetici Dd= Ddel +Ddanis+Ddst
Correnti d’anello Pople -Dipole model Modelling 1H NMR Spectra of Organic Compounds: Theory, Applications and NMR prediction Software Di Raymond Abraham,Mehdi Mobli
Equazione di Mc Connell Dc = c -c Dc C-H 90 Dc C-C 140 Dc C≡C -340 x 1036 m-3mol-1 Ds = Dc (1-3cos2q)/12pR3 Dd (Heq-Hax)= ca. 0.50 ppm
Legami a idrogeno CH3OH CH3OH Diluito in CDCl3 5.34 ppm 1.1 ppm C6H5OH C6H5OH Diluito in CDCl3 7.45 ppm 4.60 ppm 12.1 ppm
Water Solvent Shift (ppm) Benzene(d6) 0.5 CCl4 1.1 CDCl3 1.5 THF 2.5 Ac(d6) 2.8 DMSO 3.3 H2O 4.7 EtOD 5.3 Pyr(d5) 5.0
H2O Hg Hb CH3 NH sidechains Ha (helices) Ha (sheets) aromatic NH backbone
Simple shielding effects--electronegativity The amount of shielding the nucleus experiences will vary with the density of the surrounding electron cloud If a 1H nucleus is bound to a more electronegative atom e.g. N or O as opposed to C, the density of the electron cloud will be lower and it will be less shielded or “deshielded”. These considerations extend beyond what is directly bonded to the H atom as well. more electron withdrawing-- less shielded less electron withdrawing-- more shielded N C H H
Simple shielding effects-electronegativity less shielded higher resonance frequency more shielded lower resonance frequency aliphatic/alpha/beta etc.(HC) amides (HN) most HN nuclei come between 6-11 ppm while most HC nuclei come between -1 and 6 ppm.
More complex shielding effects:Aromatic protons aromatic region (6-8 ppm) amide region (7-10 ppm) One consequence of these effects is that aromatic protons, which are attached to aromatic rings, are deshielded relative to other HC protons. In fact, aromatic ring protons overlap with the amide (HN) region. Questo lo hai già visto nella descrizione delle molecole organiche
Example: shielding by aromatic side chains in folded proteins Picture shows the side chain packing in the hydrophobic core of a protein--the side chains are packed in a very specific manner, somewhat like a jigsaw puzzle + + a consequence of this packing is that some protons may be positioned within the shielding cone of an aromatic ring such as Phe 51. Such protons will exhibit unusually low resonance frequencies (see picture at left). Note that such effects depend upon precise positioning of side chains within folded proteins shielded methyl group methyl region of protein spectrum
Amino acid structures and chemical shifts note: the shifts are somewhat different from the previous page because they are measured on the free amino acids, not on amino acids within peptides
“Average” or “random coil” chemical shifts in proteins It should now be apparent to you that different types of proton in a protein will resonate at different frequencies based on simple chemical considerations. For instance, Ha protons will resonate in a region centered around the relatively high shift of 4.4 ppm, based on the fact that they are adjacent to a carbonyl and an amine group, both of which withdraw electron density. But not all Ha protons resonate at 4.4 ppm: They are dispersed as low as ~3 and as high as ~5.5. Why? “Ha region”
“Average” or “random coil” chemical shifts in proteins One reason for this dispersion is that the side chains of the 20 amino acids are different, and these differences will have some effect on the Ha shift. The table at right shows “typical” values observed for different protons in the 20 amino acids. These were measured in unstructured peptides to mimic the environment experienced by the proton averaged over essentially all possible conformations. These are sometimes called “random coil” shift values. Note that the Ha shifts range from ~4-4.8, but Ha shifts in proteins range from ~3 to 5.5. So this cannot entirely explain the observed dispersion.
“Average” or “random coil” chemical shifts in proteins A simple reason for the increased shift dispersion is that the environment experienced by 1H nuclei in a folded protein (B) is not the same as in a unfolded, extended protein or “random coil” (A). shift of particular proton in unfolded protein is averaged over many fluctuating structures will be near random coil value shift of particular proton in folded protein influenced by groups nearby in space, conformation of the backbone, etc. Not averaged among many structures because there is only one folded structure. So, some protons in folded proteins will experience very particular environments and will stray far from the average.
You can tell if your protein is folded or not by looking at the 1D spectrum... poorly dispersed methyls poorly dispersed amides poorly dispersed alphas poorly dispersed aromatics unfolded ubiquitin very shielded methyl folded ubiquitin
What specifically to look for in a nicely folded protein notice aromatic/amide protons with shifts above 9 and below 7 notice alpha protons with shifts above 5 notice all these methyl peaks with chemical shifts around zero or even negative
Linewidths in 1D spectra: aggregation and conformational flexibility Linewidths get broader with larger particle size, due to faster transverse relaxation rates. We’ll learn the physical basis for the faster relaxation later. Broader than expected linewidths can indicate that the protein is aggregated. It can also indicate that the protein has conformational flexibility, i.e. that its structure is fluctuating between several slightly different forms. We’ll learn why this is when we cover the effect of protein dynamics on NMR spectra. Conformational flexibility also tends to reduce dispersion by averaging the environment experienced by a nucleus.
An example of analyzing linewidths and dispersion: Hill & DeGrado used measurements of chemical shift dispersion and line broadening in the methyl region of 1D spectra to gauge the effect of mutations at position 7 on the conformational flexibility of a2D protein leucine and valine mutants have poor dispersion and broad lines, despite being very stably folded and not aggregated (circular dichroism and analytical ultra- centrifugation measurements). These mutants are folded but flexible. Hill & DeGrado (2000) Structure 8: 471-9.
13C NMR The rules discussed for 1H spins, (shielding and deshielding effects) hold also for 13C spins. Some general features of 13C should be pointed out: Unlike 1H atoms, 13C atoms may form a different number and type of chemical bonds. Therefore, the so called paramagnetic contributions (see later) are much more effective for deshielding. The chemical shift range of 13C spins spans more than 200 ppm