BIOC 2200 Practical One:

NMR of Macromolecules

Convener: Prof. S. W. Homans (Tel: 33125, Email: s.w.homans@leeds.ac.uk)
Demonstrator: Neil Syme

Contents:
Introduction Concepts NMR spectra of Amino Acids Assignment of glutathione ¹H - ¹H COSY Spectrum of Glutathione

Introduction
The purpose of this practical is to enable you to gain experience in the interpretation of NMR spectra of simple amino-acids. The concepts involved build directly on the three NMR lectures in BIOC 2200. These may appear to be rather daunting at first, but in reality are not too difficult. We will be available to help if things get too difficult.

Concepts

In order to understand this practical, you need to be aware of two concepts:

Chemical Shift
Spin Coupling

These can best be understood by reference to the NMR spectrum of e.g. the amino-acid alanine. At low resolution this amino acid has an NMR spectrum comprised of two resonance lines, as shown in figure 1 below:

NMR Spectrum of the amino-acid Alanine

Figure 1: Low-resolution NMR spectrum of the amino-acid Alanine

Chemical Shift:

Figure 1 is a proton NMR spectrum, so each peak in the spectrum corresponds with a proton or group of protons in the molecule. The frequency at which each peak resonates is called the Chemical Shift, and is measured in parts per million (ppm). The chemical shift arises because each type of nucleus in the amino-acid experiences a slightly different value of the static magnetic field that is applied during the NMR experiment. This difference arises in turn from the shielding or deshielding effects of chemical groups in the molecule (hence the name chemical shift). In the case of alanine (or indeed any other molecule), the chemical shift is a good indicator of the chemical group that gives rise to the peak.

For example, methyl groups resonate at high field, so the large peak furthest to the right in the above spectrum corresponds to the Alanine's methyl group (β-protons), which has three protons.

Question 21: Why do the three protons in the methyl-group resonate at the same frequency?
(Click on the radio-button at the left of the correct answer below.)

	Because all protons resonate at the same frequency, regardless of the chemical group they are attached to.
	Because the nearby Nitrogen atom donates an electron to the methyl group.
	Because two of the methyl groups protons (hydrogen atoms) are replaced by Deuterium (²H)
	Because the methyl group can rotate freely about the carbon-carbon bond.
	The reason has not yet been discovered.

Your Answer: YourAnswer Mark

Correct Answer: Because the methyl group can rotate freely about the carbon-carbon bond..

Each β-methyl group can rotate freely about the carbon-carbon bond, so each proton in the methyl group experiences the same environment, on average. Thus we see a single peak, whose relative intensity is three units because it corresponds to three protons.

The α-proton (the hydrogen atom attached directly to the central alpha-carbon atom) of alanine resonates at lower field in the spectrum (further to the left), and has a relative intensity of one unit, since it corresponds to a single proton.

At the far left of the spectrum, there is a very large peak (labelled 'HOD'). This does not derive from alanine, but from solvent water. If we were to acquire NMR spectra in pure water, then this peak would be ~100,000 times bigger than the peaks from alanine (the sample contains 1mM alanine, whereas the concentration of protons in water is 110 M). Therefore we acquire spectra in deuterium oxide (heavy water, D₂O). This reduces the solvent peak dramatically, but there is still sufficient residual H₂O (and H₂O + D₂O tends to become 2 HOD, ie: Hydrogen-Oxygen-Deuterium) to give rise to a significant peak.

Another manifestation of the use of heavy water as solvent is that exchangeable protons such as -OH, -NH and -NH₂ will exchange for deuterons (ie. these H atoms exchange with D atoms from the heavy water solvent) and thus will be 'invisible' to proton NMR. This is why there are no peaks for the -COOH and -NH₂ protons of alanine.

Spin Coupling:

The allocation of a peak in the NMR spectrum to a given proton in the molecule corresponds to the important process of resonance assignment in NMR. In more complicated molecules, it is not possible to assign peaks in the spectrum by consideration of chemical shifts alone, and we additionally need to make use of spin-spin couplings. In order to explain the latter let's now look at a high-resolution NMR spectrum of alanine:

High-resolution NMR spectrum of Alanine

Figure 2: High-resolution NMR spectrum of the amino-acid Alanine

Although the peaks are in the same place as in the low resolution spectrum, and have the same relative intensities, each peak is now split into two or more 'sub-peaks' (we call these multiplets). These multiplets arise from the fact that each nuclear spin is spin-spin coupled to its neighbours that are connected by up to three covalent bonds. For convenience, let's look at the β-protons. You can see from the above spectrum that the β-proton peak is split into two peaks (also known as a doublet).

Question 22: Why are the β-protons split into a doublet?

Answer: First we need to find out how many protons are within three covalent bonds of the β-proton. Since the -NH₂ and -COOH protons are rendered invisible in D₂O, these can be ignored. Also, there is an important rule in NMR that tells us that protons with the same chemical shift do not couple to each other, and thus do not give rise to splittings. Therefore the three methyl protons of alanine do not split each other. Inspection of the structure of alanine therefore reveals that only one proton needs to be considered that splits the β-methyl peak, namely the α-proton:

Structure of Alanine, showing α and β protons

Now, all protons can exist in one of two spin states, which we can simply call 'up' or 'down' ( ↑ or ↓ ). On average a given proton (e.g. the α-proton) will spend 50% of its time in the 'up' state and 50% of its time in the 'down' state. The α-proton spin state is transmitted through the bonding electrons to the β-protons, and causes very small changes in the chemical environment, thus giving rise to a small shift, and splitting of the β-proton peak into two peaks (a doublet) with the same relative intensities:

α-proton spin states:

Now let's look at the α-methyl proton. This peak looks like it has been split into four peaks (quadruplet) with different intensities.

Four peaks (quadruplet) from α-Methyl-Proton, due to four states of the β-protons in the Methyl group

Question 23: Why is the α-proton split into a quadruplet?

Answer: Using the same reasoning as above, we can see that the three β-methyl protons can split the α-proton. Now, since each β-proton can exist in two states ( ↑ or ↓ ), and there are three β-protons, we have the following combinations:

We might therefore expect the α-proton to be split into 8 peaks because the combination of β-proton spins offers 8 environments. However, the β-proton spin states with two spins up and one spin down (b, c, and d) give rise to the same given environment of the α-proton. Similarly, the β-proton spin states with two spins down and one spin up (e, f, and g) give rise to a second given environment of the α-proton. The net effect is that there are four possible environments (some more probable than others), and the α-proton is thus split into four lines of unequal intensity:

β-protons spin states:

NMR spectra of Amino Acids

Why is all this important? The answer is that it is possible to determine the structures of more complex molecules by analysis of spin-spin coupling patterns. To give you some experience in doing this, you will find below the NMR spectra of three other amino-acids. By analyzing the multiplets in the spectra, try to determine which amino-acid corresponds to each spectrum. Don't hesitate to ask Prof. Homans or a demonstrator if necessary.

Question 24: Which amino-acid does this NMR spectrum represent:

(The numbers above peaks are the relative intensities)

All Amino Acid Structures

Select your answer from the ammino-acids listed below. (The correct answer will then appear):

Alanine

Arginine

Asparagine

Aspartate

Cysteine

Glutamate

Glutamine

Glycine

Histidine

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Proline

Serine

Threonine

Tryptophan

Tyrosine

Valine

Your Answer:

Correct Answer: Valine - as its R-group has two methyl groups each with 3 protons (which match the two intensity "3" peaks at 0.95 and 1.05 ppm), and a single proton (which matches the intensity "1" peak at 2.23 ppm, which will be a multiplet as it experiences spin-spin coupling from the six protons on the two methyl groups)

The tall left-most peak (at 4.75ppm) is due to the presence of some water as HOD molecules, and the intensity "1" peak at 3.55ppm is due to the α proton which is present on all amino acids.

(Note that the intensity is given by the area under the peak, so a low wide peak can have same intensity as a tall narrow peak as illustrated by the two intensity "1" peaks here.)

Question 25: Which amino-acid does this NMR spectrum represent:

NMR Spectrum B

All Amino Acid Structures

Select your answer from the ammino-acids listed below. (The correct answer will then appear):

Alanine

Arginine

Asparagine

Aspartate

Cysteine

Glutamate

Glutamine

Glycine

Histidine

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Proline

Serine

Threonine

Tryptophan

Tyrosine

Valine

Your Answer:

Correct Answer: Lysine - as its R-group has a four carbons each with two protons, which matches the four peaks each of intensity "2" at 1.5, 1.7, 1.9 and 3.0 ppm.

The protons on the NH3⁺ group would be replaced by deuterium from the surrounding heavy water, so would not appear on the NMR spectrum.

(Note that the intensity is given by the area under the peak, so a low wide peak can have same intensity as a tall narrow peak as illustrated by the three intensity "2" peaks here.)

Question 26: Which amino-acid does this NMR spectrum represent:

NMR Spectrum C

All Amino Acid Structures

Select your answer from the ammino-acids listed below. (The correct answer will then appear):

Alanine

Arginine

Asparagine

Aspartate

Cysteine

Glutamate

Glutamine

Glycine

Histidine

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Proline

Serine

Threonine

Tryptophan

Tyrosine

Valine

Your Answer:

Correct Answer: Glutamine - as its R-group has two carbons each with two protons, which matches the two intensity "2" peaks at 2.1 and 2.4 ppm.

However, Glutamate (Glutamic acid) has a similar spectrum as shown below, the difference being the increased spin-spin coupling on the right-most peak.

Glutamic Acid H-NMR Spectrum, is similar to Glutamine
1H-NMR Spectrum for Glutamine (Glutamic acid)
(Image from: Biological Magnetic Resonance Data Bank)

(Note that the intensity is given by the area under the peak, so although the intensity "1" peak here is slightly taller than the intensity "2" peaks, it is not as wide.)

Assignment of glutathione

As molecules become even more complicated, analysis of spin-couplings alone cannot yield unambiguous assignments. We must therefore resort to alternative methods. One possibility is to exploit the fact that the chemical nature of inonisable groups in the molecule will change as the pH of the solvent is altered. As the chemical nature of these groups changes, then so will the chemical shifts of protons that are spatially close to these groups, since their chemical environment will change. By measuring the chemical shifts of protons in a given molecule versus pH, it is thus often possible to make assignments based on the responsiveness (titration) of these chemical shifts to pH change. As an example, we will examine the pH dependence of 1H chemical shifts of the tripeptide glutathione.

Question 27: Shown below are a table of chemical shifts versus pH from proton NMR spectra, together with the structure of glutathione. Using Excel, or other software, plot a graph of chemical shift versus pH for each peak, then from the pH-dependence and multiplicity of the peaks, try to assign them to the protons of glutathione. You can download this data as either a tab-deliminated Text file or as an Excel spreadsheet file (In Internet Explorer, be sure to click Save in the download dialog box, otherwise will open file in the Internet Explorer window.).

pH Dependence of ¹H Chemical Shifts of Glutathione:

pH	peak 'a'	peak 'b'	peak 'c'	peak 'd'	peak 'e'	peak 'f'
0.4	4.5	4.2	4.05	2.86	2.55	2.25
1.1	4.5	4.2	4.05	2.86	2.55	2.25
2.0	4.5	4.2	4.05	2.86	2.55	2.25
3.01	4.5	4.15	4.0	2.86	2.53	2.24
4.03	4.5	3.95	3.8	2.86	2.47	2.15
5.03	4.5	3.92	3.76	2.86	2.46	2.12
6.08	4.5	3.91	3.76	2.85	2.46	2.12
7.08	4.5	3.90	3.75	2.85	2.45	2.10
8.02	4.5	3.90	3.75	2.81	2.44	2.05
9.03	4.45	3.90	3.45	2.65	2.42	2.00
10.04	4.4	3.90	3.4	2.58	2.36	1.95
11.28	4.4	3.90	3.39	2.57	2.35	1.94

The peaks 'a' to 'f' are identified in the spectrum shown below. The chemical shifts (centres of multiplets) at various pH values are given in ppm from external DSS (2,2-Dimethyl-2-silapentane-5-sulfonic acid, which is used as a NMR reference peak ?????).

Proton NMR Spectrum of Glutathione

+	γ-Glu	Cys	Gly
H₃N

	CH-CH₂-CH₂-CO-NH-	CH-CO-NH	-CH₂-COO -

OOC		CH₂

		SH
Chemical formula for Glutathione

Use the drop-down menus, to assign each of the six peaks (a to f) to the protons (Hydrogen atoms) on the following diagram of Glutathione. (There will be four hydrogens with no assignment - ie. blank on the drow-down menus):

+
H₃N

	CH −CH₂ −CH₂ −CO−NH −	CH −CO−NH	−CH₂ −COO -

OOC		CH₂

		SH
Chemical formula for Glutathione

After specifying the peaks on the above diagram, then

Your score: yourScore correct out of 6 possible assignments.

Correct Answer: The correct assignment of peaks is:

+
H₃N

	CHc −CH₂f −CH₂e −CO−NH −	CHa −CO−NH	−CH₂b −COO -

OOC		CH₂d

		SH
Chemical formula for Glutathione with peaks a to f assigned.

Graph plotted of Chemical shift versus pH for Glutathione, plotted by Excel spreadsheet

Protons in -OH, -NH₂, SH have no characteristic chemical shift, and their peaks disappear in D₂O, as deuterium will replace a proton.

The intensity (area under the peak, not just their height) of peaks 'a' and 'c' appear to be half that of the other peaks, suggesting that peaks 'a' and 'c' likely each correspond to one proton, and the other peaks to two protons.

From the above graph of Chemical shift versus pH:

in alkaline conditions (pH > 7.0): peak 'c' has the greatest chemical shift decrease, followed by peak 'd', then by peaks 'a', 'f' and 'e'.
in acidic conditions (pH < 7.0): peaks 'b' and 'c' have the greatest shift increase, followed by peaks 'e' and 'f'
the chemical shifts of peaks 'a' and 'd' do not change in acidic conditions.

An acidic (pH < 7.0) solution donates Hydrogen ions (H+) to, in this case Glutathione, so the COO^- groups will accept H⁺ ions, which will attract electrons from neighbouring groups, hence deshielding these neighbouring groups, increasing their chemical shift. Peaks 'b' and 'c' experience increased chemical shift in acidic conditions, so must be beside the two COO^- groups.

Peak 'c' is most affected by reduced chemical shift (increased shielding by electrons) in alkaline conditions, so must be beside the NH₂⁺ group.

Peak ' ? ' is ....... Need to finish this explanation....

Please ask Prof. Homans or a demonstrator about this.

¹H - ¹H COSY Spectrum of Glutathione

While useful for illustrative purposes, pH titration is a rather old-fashioned method for obtaining assignments. Present day assignment techniques include two-dimensional NMR methods (described in the NMR lectures) such as correlated spectroscopy (abbreviated COSY).

(In 2D NMR, two pulses are applied separated by a time, t₁. In Correlated Spectroscopy (COSY), scalar (J) coupled spins can be correlated.)

A typical COSY spectrum for glutathione is shown below. This two-dimensional experiment permits rapid assignment of all the peaks in the spectrum by inspection.

¹H - ¹H COSY Spectrum of Glutathione

Question 28: Can you identify which peaks are correlated in the above COSY spectrum ?

Answer: As shown in the image below:

the 1D NMR spectrum peaks correspond to the lower-left to top-right diagonal of the 2D spectrum.
Peak 'a' correlates with peak 'd' (pink dashed-line).
Peak 'c' correlates with peak 'f' (green dashed-line).
Peak 'e' also correlates with peak 'f' (blue dashed-line).
So peak 'c' scaler-couples to peak 'e' ?

These correlations can be seen in the labelled structure of Glutathione shown below, where:

'c' is beside 'f' which is beside 'e'.
'a' is beside 'd'.
'b' has no immediate neighbours.

Need to finish this explanation....

+
H₃N

	CHc −CH₂f −CH₂e −CO−NH −	CHa −CO−NH	−CH₂b −COO -

OOC		CH₂d

		SH
Chemical formula for Glutathione with peaks a to f assigned.

Please ask Prof. Homans or a demonstrator to explain in more detail how assignments are obtained from this spectrum.

Your total score =

out of

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BIOC 2200 Practical One:

NMR of Macromolecules

Contents:

Introduction

Concepts

Chemical Shift:

Spin Coupling:

NMR spectra of Amino Acids

Assignment of glutathione

1H - 1H COSY Spectrum of Glutathione

Your questions and comments

¹H - ¹H COSY Spectrum of Glutathione