Rotamers- assigned by a simple NMR experiment


Rotamers are conformational isomers where interconversion by rotation around a single bond is restricted and an energy barrier has to be overcome in order to convert one conformer to another.  When this rotation strain barrier is high enough to allow for the isolation of the conformers then the isomers become atropoisomers. Rotamers are however not separable and their existence normally complicates the 1H NMR interpretation. Variable temperature (VT) NMR is the generally preferred method for  studying the equilibration of the rotamers and at low temperatures the spectrum is assigned to the frozen equilibrium and multiple peaks are observed while at higher temperatures the spectrum simplifies as the equivalent peaks are averaged out. Other methods for NMR simplification include the introduction of a complexing agent and solvent switching. All these techniques are inconvenient to the organic synthetic chemist when working on small scale. To overcome this problem, Steve Ley and co-workers have identified that chemical-exchange NMR experiments, such as 1D NOE, can be used to identified resonances corresponding to those protons in chemical exchange processes, therefore distinguishes rotamers form other impurities or even stereoisomers, in a non intrusive way.

 

In a 1D gradient NOE experiment, a selected peak is irradiated leading to a negative peak at the site of irradiation while those protons connected to the targeted frequency region through space appear as positive peaks (or in the opposite phase to the irradiated peak). On the other hands those protons undergoing chemical exchange with the irradiated protons will appear as negative peaks, (or in the same phase as the irradiated peak). Figure 1 illustrates this, where a chemical-exchange experiment can be used to distinguish sets of rotamers in the presence of diastereoisomers.

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Figure 1. (a) 1H NMR spectrum of a sample containing 3 and 4. Four NMR resonances (I, II, III, and IV) are observed corresponding to protons HA and HB in 3, 4, and their respective rotamers. (b) 1D gradient NOE spectrum after selective excitation of the resonance at 4.59 ppm (I) produces a single downfield resonance in the same phase at 4.28 ppm (III), indicating that resonances I and III belong to two rotamers of the same diastereomer (3) and that resonances due to one diastereomer do not transfer spin information via chemical exchange to the other. (c) 1D gradient NOE spectrum after selective excitation of the diastereomeric peak at 4.52 ppm (II) also produces a single downfield peak in the same phase (IV), indicating that resonances II and IV belong to two rotamers of the same diastereomer (4). Only the 5.2−4.1 ppm region is shown for clarity

Recently this technique has been applied by Proksch et al. to unambiguously determine the presence of four rotamers in two new depsipeptides. In this publication, they have also used 2D ROESY or NOESY experiments to find the same results.

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Fragment screen: Have you identified the right hits?


One of the problems faced by drug discovery scientists running a screening programme is the correlation of hits (or the lack of correlation) from one assay technique compared to another, run against the same drug target. Most often this occurs as part of screening cascade, when you move from one primary screening method to a secondary confirmation method and a vast majority of hits don’t repeat or have different ranked orders of potency. This can be for good reasons, and why the secondary technique is being used as a counter screen to remove false positives.

This process can also be used to validate a new screening methodology by comparing the correlation of actives from screening file when it is run with two differing techniques against the same target. This latter example was described in a recent publication

This paper has also been described on the Practical Fragments blog. Please read for a far more detailed description and many other interesting articles.

In this publication the authors from two separate groups ran a NMR and SPR fragment screen on the same target (HIV-1 integrase core domain) with the same fragment file (about 500 compounds). Once primary actives had been identified, the authors used X-ray crystallography as further evidence of binding. The results from this experiment showed that the NMR technique identified 62 primary hits of which 15 generated protein/fragment complexes. The SPR generated 16 primary hits which led to six clear protein/fragment complexes. When the fragments/crystal complexes were compared to one another there was no correlation between the different assay methods.

There were some differences between the two techniques which might account for the differences, for example the total amount of DMSO present varied in each technique, which could affect solubility of compounds, and the final screening concentration of the SPR (500µM) was less than that of the NMR (1mM) which would suggest some of the weaker compounds would be more difficult to find in the SPR. In addition to this, the conditions used for crystallisation are bespoke for each protein. This would favour a certain set of physiochemical properties which might assist the formation of a crystal complex for a certain sub-population of fragments. All these factors could affect the amount of hits identified for each method.

Another critical difference it seems between the two techniques is that the SPR method used a reference protein (AMA1) as control. Hits were identified from SPR, only if the binding response was twice the level with HIV-1 integrase compared to the AMA1. When comparing the NMR actives, 8 compounds which gave crystal structures did not show this fold binding difference in the SPR (even though they bound to the HIV-1 integrase and therefore were eliminated as potential hits from the SPR hit list.

When the 5 SPR hits which gave crystal structures were run as singletons with NMR, 4 of them gave a weak response and it was suggested that the pooling of the fragments (10 compounds per sample) made the identification of these compounds indistinguishable from the background signal.

SPR vs NMR

So overall both methods did find active fragments, however importantly each method did not find all the active fragments from the collection.

The different methods of analysis and screening had an impact on the population of the hits that were found for each of the techniques. Maybe once a reasonable subset of the active fragments has been identified, the use of structural similarities searches could be of assistance to locating other fragments from the file, to ensure that as few as possible active fragments are not missed.

From this review it does not look like one method has any greater performance than another in identifying active fragments, however it seems care needs to be taken with the analysis.