Recent Question/Assignment

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Question 3: Analytical NMR
Look carefully at compound X below:

Figure 4: Compound X
(3a) Using letters (a, b, c, d etc.) label each unique carbon chemical environment on the structure above with a different letter.
(3b) Sketch the 1D 1H NMR spectrum you would expect for the compound X (including spin-spin couplings). On your spectrum, label each 1H peak with the correct letter from part (3a) that defines the carbon to which each 1H is attached.
(3c) Sketch the 1D 13C NMR spectrum you would expect for compound X above (assume it is 1H decoupled). On your spectrum, label each 13C peak with the correct letter from part (3a) that defines the carbon in the spectrum.

(3d) Draw the DEPT-90 and DEPT-135 13C spectra for compound X. Label each carbon peak using your notation from (3a).
(3e) Sketch what you would think the 13C, 1H HSQC spectrum of compound X would look like. Label each carbon peak using your notation from (3a).

Question 4: Binding and protein NMR
Look at the 15N, 1H HSQC spectra shown below. Both spectra were obtained two samples of the same 14.5 kDa protein at 25°C. SDS-PAGE of samples A and B show only pure protein that run at 14.5 kDa on the gel. Samples A and B samples were also analysed by protein sequencing and electrospray mass spectrometry and both techniques confirm the same protein present by weight and sequence.

(4a) Ring (or highlight) all the NH2 side-chain groups you can see on spectrum (A) and spectrum (B).
(4b) What might have caused the differences in the NMR spectra in A and B?

Compound Y is a potential drug candidate that is designed to inhibit the activity of protein in sample B above. In an attempt to determine the affinity of the binding interaction of compound Y to the protein in B, a series of 15N, 1H HSQC spectra were collected at a protein concentration of 0.1 mM, and compound Y concentrations up to 5 mM. Some of the peaks are seen to shift as function of compound Y concentration as show below:

The overall changes in the chemical shift of one of the peaks is shown in the table below:
Concentration of compound Y / mM NMR chemical shift change / 103 • ppm
0.0 0.0
0.2 6.1
0.4 14.7
0.6 18.6
0.8 23.8
1.0 23.5
1.2 28.0
1.4 30.2
1.6 28.5
1.8 32.0
2.0 36.2
2.2 34.3
2.4 34.4
2.6 36.7
2.8 36.0
3.0 38.1
3.2 37.6
3.4 38.4
3.6 39.9
3.8 42.1
4.0 40.7
4.2 43.5
4.4 36.5
4.6 40.0
4.8 41.7
5.0 39.9
(4c) Draw the experimental binding isotherm data in a labelled figure showing the NMR chemical shift change as function of the concentration of the compound Y added.
(4d) Based on the above experimental results, what is the likely affinity (Kd) of compound Y binding to the protein in sample B? Show in the same graph how the binding curve will look like with your estimated Kd value in a graph, and calculate how much of the protein is saturated with compound Y at 5 mM addition of Y.
You can use the following points to guide you:
i. Using all experimental information available, guess a possible value of Kd as well as a possible scaling factor linking the chemical shift change and the concentration of the complex or % saturation.
ii. Plot how such a binding curve would look like in the same graph as the data and compare. The use of graphing software such as Excel will simplify this task.
iii. Refine your guess until the binding curve represent the data as closely as possible.
(4e) What is the standard free energy of association (?G°) for the above binding reaction? Show how you have calculated the ?G° value.
(4f) Is compound Y a good drug candidate? Explain why or why not.
End of assessment