1H NMR is for sure the most powerful technique for structure elucidation, especially for small organic molecules. Typically, an organic chemist uses the chemical shift, coupling constants and integration information contained in an 1H-NMR spectrum to either verify or elucidate an unknown compound. Of course, it’s quite common that a simple 1H-NMR spectrum is not enough to unambiguously confirm a structure and thus other NMR experiments (e.g. 13C-NMR, HSQC, COSY, etc) are used to get more structural information.
Nevertheless, I have often found that many organic chemists do not always try to get the most out of 1H-NMR spectra (which is the cheapest experiment), in particular when some multiplets are complex to interpret (strong coupling) or when peaks overlap prevents valuable information to be detected in some multiplets. Overlapping peaks and new ways to get around it will be the subject of this post.
As it is well known, there are two principal factors limiting the resolution power in a spectrum. First, we have the natural line width limitation imposed by the T2 (spin-spin relaxation). For example, if T2 is about 1 second, the peak linewidth at half height cannot be less than 0.32 Hz (remember, line width at half height = 1 / (pi * T2) = 1 / 3.1415 = 0.32) no matter how powerful is our NMR instrument or the field homogeneity. On the other hand, there are instrumental shortcomings (e.g. spatial uniformity of the applied magnetic field, etc).
Nonetheless, there is an additional limiting factor, and whose importance is generally underestimated which has to do with the generally large number of transitions in 1H-NMR spectra. In short, the peaks we can observe in a 1H-NMR are just a small fraction of the actual transition resonances which are not observable because of the limited digital resolution. In fact, every peak in an 1H-NMR spectrum is basically an envelope of a large number of transitions and its shape is dominated by the coupling pattern of the spin system. Even in molecules of modest size the number of distinct peaks is tens to thousands times smaller than that of quantum transitions. As a very simple example, consider an A3B2 spin system. Depending on the second order interaction and on the available digital resolution, we might observe the expected triplet / quadruplet multiplet patterns. This is illustrated in the figure below.
However, if we use Mnova capabilities to display all main transitions of any coupled spin system by simply hovering with the mouse over the particle of interest, we can appreciate the additional number of resonances (see below):
Furthermore, I can easily increase the digital resolution of the A3B2 spectrum above by just reducing the line width used in the spin simulation module of Mnova. As a result, it’s now possible to observe more resonances in this particular A3B2 spin system (although not all of them, of course):
Of course, this way of increasing the digital resolution is only possible with synthetic spectra and cannot be applied to experimental data. Obviously there are many resolution enhancement techniques being Resolution Booster one of the most powerful ones. As a nice example of the application of this technique, let me tell you this story:
A couple of weeks ago, a very good friend of mine, a professor of organic chemist, came to me with an interesting structural problem. His research group had carried out a reaction which resulted in one single product whose 1H-NMR spectrum was, in principle, compatible with two potential structures. In order to ambiguously find the right structure, they acquired more NMR spectra (DEPT, HSQC, HMBC, COSY) which allowed them to find the correct molecule However, while discussing the problem having a few beers at a bar in Santiago, we found that just the 1H spectrum was more than enough in order to discard one of the two structures and completely assign the correct one without the necessity to acquire any other NMR experiment. The key was the ability to resolve a long range coupling (homo-allylic) with the assistance of Resolution Booster. Basically, the 1H-NMR showed a clean double doublet which was compatible with both structures (I’m sorry, but I cannot reveal those structures). This multiplet is shown below:
After appling Resolution Booster, we could clearly appreciate a further splitting which we could assign to the expected homo-allylic coupling with a value of 1.76 Hz. This coupling was also found in its corresponding multiplet partner confirming the structure:
At this point, it’s worth mentioning that Resolution Booster is a very powerful method to resolve overlapped peaks, but it cannot be used for integration as the area of the peaks get distorted by this process. The good news is that we have developed a new method which in addition to taking advantage of the power of resolution booster, it yields accurate integrals.
This method has been named as Global Spectral Deconvolution (GSD) and as its name says, it automatically deconvolves all the peaks in a spectrum. In short, this method first recognizes all significant peaks in a spectrum, then assigns a realistic a-priori bounds to all peak parameters (chemical shift, heights, line widths, etc) and finally fits all these parameters in a very reasonable time.
Following with the example above, if we apply GSD, we get a multiplet with all the individual peaks clearly resolved and this time, with accurate integrals.
It’s important to mention that we haven’t just fitted the multiplet above, but we have actually fitted the whole spectrum!
We are confident that GSD will open new avenues in NMR data interpretation and quantitative analysis (qNMR). I will blog about these points in future posts.
Peak height versus peak volume
Given a standard NOESY-based protein structure determination: Does anyone have any information on the benefits of measuring peak intensity by a volume integration method rather than simply measuring the peak height.
Obviously integration is theoretically more accurate, but does it make any difference to the quality of the structures produced? especially if peak lineshapes are comparable?
I was hoping to find some study comparing structures produced by both methods.....
I'm also curious about the benefits of distance-calbrating NOEs to a curve rather than simply putting restraints...
paul
NMR Questions and Answers
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09-15-2015 07:48 PM
[NMR software blog] Fighting the Pirate
Fighting the Pirate
Six years ago I took the decision of investing my efforts into the creation of a software application. It was a complex project that potentially required thousands of hours of work. I had to make it financially viable. No sponsor was in sight, so the only solution was to make a commercial program and sell it. I was extremely lucky, because the facilities to sell it were readily available and convenient too.
The difference between a normal application and a commercial one is that the latter must be copy-protected in some way.
I had complete freedom about how which...
nmrlearner
News from NMR blogs
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04-11-2011 01:00 PM
Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins
Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins
Abstract The solution NMR resonance assignment of the protein backbone is most commonly carried out using triple resonance experiments that involve 15N and 1HN resonances. The assignment becomes problematic when there is resonance overlap of 15Nâ??1HN cross peaks. For such residues, one cannot unambiguously link the â??leftâ?? side of the NH root to the â??rightâ?? side, and the residues associated with such overlapping HN resonances remain often unassigned. Here we present a...
nmrlearner
Journal club
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12-31-2010 08:38 PM
[NMR paper] Addressing the overlap problem in the quantitative analysis of two dimensional NMR sp
Addressing the overlap problem in the quantitative analysis of two dimensional NMR spectra: application to (15)N relaxation measurements.
Related Articles Addressing the overlap problem in the quantitative analysis of two dimensional NMR spectra: application to (15)N relaxation measurements.
J Biomol NMR. 2004 Nov;30(3):347-52
Authors: Tugarinov V, Choy WY, Kupce E, Kay LE
A quantitative analysis of 2D (1)H-(15)N spectra is often complicated by resonance overlap. Here a simple method is presented for resolving overlapped correlations by...
nmrlearner
Journal club
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11-24-2010 10:03 PM
[NMR paper] Elimination of 13Calpha splitting in protein NMR spectra by deconvolution with maximu
Elimination of 13Calpha splitting in protein NMR spectra by deconvolution with maximum entropy reconstruction.
Related Articles Elimination of 13Calpha splitting in protein NMR spectra by deconvolution with maximum entropy reconstruction.
J Am Chem Soc. 2003 Mar 5;125(9):2382-3
Authors: Shimba N, Stern AS, Craik CS, Hoch JC, Dötsch V
Homonuclear 13C-13C couplings can significantly reduce the sensitivity and resolution of multidimensional NMR experiments. The most important of these couplings is the 13Calpha-13Cbeta coupling, and several...
nmrlearner
Journal club
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11-24-2010 09:01 PM
[NMR paper] Automated peak picking and peak integration in macromolecular NMR spectra using AUTOP
Automated peak picking and peak integration in macromolecular NMR spectra using AUTOPSY.
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J Magn Reson. 1998 Dec;135(2):288-97
Authors: Koradi R, Billeter M, Engeli M, Güntert P, Wüthrich K
A new approach for automated peak picking of multidimensional protein NMR spectra with strong overlap is introduced, which makes use of the program AUTOPSY (automated peak picking for NMR spectroscopy). The main elements of this program are a novel...
nmrlearner
Journal club
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11-17-2010 11:15 PM
Conformations of stevastelin C3 analogs: computational deconvolution of NMR data reve
Conformations of stevastelin C3 analogs: computational deconvolution of NMR data reveals conformational heterogeneity and novel motifs.
Related Articles Conformations of stevastelin C3 analogs: computational deconvolution of NMR data reveals conformational heterogeneity and novel motifs.
Biopolymers. 2010 Nov;93(11):968-76
Authors: Jogalekar AS
The stevastelins are depsipeptide natural products that show valuable immunomodulatory and phosphatase inhibitory activity. A previous report described the synthesis, conformational analysis, and biological...
nmrlearner
Journal club
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11-06-2010 11:02 AM
[CNS Yahoo group] Introducing one-click access to PDB data at PDBe
Introducing one-click access to PDB data at PDBe
Introducing one-click access to PDB data at PDBe ... When you visit the newly redesigned home page of the Protein Data Bank in Europe (PDBe; http://pdbe.org/),
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