Metabonomics databases

Having access to NMR databases of most common metabolites is very important if you’re interested or involved in NMR-based metabonomomics/metabolomics studies. Here are two good sites worth checking out:

• BioMagResBank
• Human Metabolome Database

For a more comprehensive review, see this article

13C Chemical Shift prediction: Typos and NMR misinterpretations

It is well known that 13C Chemical Shifts are essential for structure verification and elucidation of organic molecules by NMR. For example, in the field of structure verification, one can compare the observed 13C chemical shifts with the calculated (predicted) values in such a way that the structure hypotheses can be assessed by means on some numerical matching factor.
What is less evident is the great potential that 13C chemical shift prediction has in order to reveal typos and misinterpretations of NMR data that often (much more than expected, I’m afraid) appear in the experimental sections of scientific literature.

Wolfgan Robien has recently created a very interesting Web page in which he shows how his famous CSEARCH program for 13C –NMR prediction can be used for automatic data-checking. According to his personal opinion, there are at least 3 scenarios in which such checks should be done:

• Daily routine during generation and interpretation of NMR-data
• Check again during preparation of a manuscript
• Check again during the peer-reviewing process ('robot-referee')

Mnova is honoured to include Wolfgang Robien’s CSEARCH algorithm within its NMRPredict Desktop plugin and includes simple yet very useful verification tools which can be used to easily identify errors in 13C-NMR data assignments.

Basic Misinterpretations, Typos and other Sad Events in NMR-Spectroscopy

Wavelet-based filtering applied to the diagonal suppression of 2D NMR data sets

Published in Stan's Library
Click here to read the full article
Permalink via DOI: 10.3247/SL2Nmr08.002

Why aren’t Bruker FIDs time corrected?

Most of NMR has finally gone totally digital and is now routinely resorting to high-frequency ADC sampling at rates of several tens of MHz, with a tendency to ever higher sampling frequencies. Such a drastic oversampling has clear advantages in simplifying the front-end electronics. It has also many side benefits due to the fact that the subsequent down-conversion to low frequency range is done digitally and therefore theoretically artefact free. This includes perfect, calibration-free quadrature detection and drastic reduction of quantization noise. By the way, such techniques are routine in other areas of electronics (military, astronomy, audio, …); NMR is just a latecomer to this world.

High-frequency (HF) oversampling, however, has also some problems of its own. The digital decimation from the HF range to the audio range and the contextual digital filtering (a combination of CIC and FIR filters) need to be properly implemented in the hardware in order to be completely transparent to the User.

However, in the case of Bruker spectra a death time or group delay can be observed in the FID: it starts with very small values and then, after some points (usually between 60-80 points) the normal FID starts.

If a plain FT is applied to this FID, we will get a spectrum with a lot of wiggles in the baseline analogous to the convolution with a sinc function centred in the middle of the spectral window. This can be explained by recalling the time shift theorem of the Fourier Transform which says that if the time domain signal is shifted by n points, the frequency domain spectrum corresponds to the standard spectrum (when the FID has not been shifted) multiplied by exp(-i2*pi*w*n). In other words, we have introduced a very large first order phase correction in the spectrum. For example, if the FID is right shifted by 60 points (death time = 60 points), f-spectrum will exhibit a first order phase distortion of 60 * 360 = 21600 degrees.

In order to work around this problem, most NMR software packages read the decimation factor (and the DSP firmware version) from Bruker files and calculate the required phase correction. So far, so good.
However, the fact remains that Bruker FID’s are not time corrected. Evidently, Varian also uses oversampling and digital filtering and their FIDs are time corrected, that is, they start at time = 0. If the digital filter is known in advance, which is always the case, the group delay should be compensated in the spectrometer, therefore, in my opinion, this death time or group delay is a bug in the spectrometer. For this reason, and going back to the title of this article, any input given as to why Bruker FID’s are not time corrected, would be greatly appreciated.

Mspin and Diasterotopic 1H NMR Assignment using 3J and RDCs

Scalar coupling constants, in particular vicinal (3J) couplings, are widely used in NMR for the determination of relative stereochemistry and preferred conformation of molecules. A number of different empiric equations to correlate the dihedral angle with the 3J values have been proposed, the Karplus equation being the most famous. The NOE (nuclear Overhauser effect) experiment is also extensively used, primarily, to define the stereochemistry within a molecule. Unlike scalar couplings, its mode of operation relies on the direct, through-space interaction between nuclei, and is independent of the presence of through-bond couplings.

However, both NOE and scalar coupling have limitations for measuring the structure information between atoms which are far apart. In addition, there are cases in which the 3J or NOE values of different stereoisomeric compounds are very similar so unambiguous assignment is not possible.

On the other hand, residual dipolar coupling (RDC) is able to provide global orientations between remote internuclear vectors, and thus gives a potential solution to these limitations. RDC’s have been widely used for the analysis of proteins and nucleic acids, but to a lesser extent in the small molecules area. Roberto Gil et al. have just published an article in which they propose the use of PMMA as a novel alignment media and discuss in a very nice way the unambiguous stereochemistry and assignment determination of the diastereotopic protons of Ludartin.

Stretched Poly(methyl methacrylate) Gel Aligns Small Organic Molecules in Chloroform. Stereochemical Analysis and Diastereotopic Proton NMR Assignment in Ludartin Using Residual Dipolar Couplings and 3J Coupling Constant Analysis
Gil, R. R.; Gayathri, C.; Tsarevsky, N. V.; Matyjaszewski, K.
J. Org. Chem.; (Article); 2008; ASAP Article; DOI: 10.1021/jo701871g

Roberto Gil and collaborators have used Mspin to calculate RDCs and 3J coupling constants for Ludartin. Below is an excerpt from the original article:



[...] The Mspin package includes modules for the calculations of 3J coupling constants, RDCs, and NOEs from 3D structures. Its usage is straightforward, and it can run on multiple platforms. For non-NMR experts, solving the structure using RDCs would be no longer a difficult task


Should you be involved in NMR structure elucidation, I believe you will find Mspin a very valuable tool, and we would very much appreciate your feedback on this application, which is currently in alpha version. It is available for download from here.

Induced Coupling & Chirality

As a sequel to my last entry, I’m glad to post here a letter from my colleague and friend Stan Sykora about induced chilarity and coupling.

_______________________________________

Dear Carlos,
I wonder whether you could put this as an entry on your blog. Originally, it ought to be a comment on your last entry, but then it grew …

Concerning the chains of CH2 groups with convergent chemical shifts, I would like to point out another mechanism which has similar NMR consequences and leads inevitably to strong couplings even at the highest fields (present and future):

Consider a chain of the -(CH2)n-R attached to a chiral carbon:

(S1S2S3)C-CHaHb-R
(S1S2S3)C-CHaHb-CHcHd-R
(S1S2S3)C-CHaHb-CHcHd-CHeHf-R

etc.,

with the substituents S1,S2 and S3 all distinct. The chirality of the first carbon is explicit, so I will say that it is first-order. Now, when R is not a proton, Ha and Hb have different chemical shifts. But this means that the two protons are not equivalent and therefore the second carbon is also chiral (let us call this the induced chirality of second-order). Which means that the protons Hc and Hd in the second formula are also chemically distinct, etc. This logic carries on iteratively, leading to induced chiralities of ever higher orders.

Naturally, the chemical shifts Ha-Hb, Hc-Hd, He-Hf, etc. decrease rapidly with induced chirality order, while their geminal couplings stay more or less fixed at about -20 Hz (geminal J's are always negative and quite large). Consequently, even in a relatively short chain of methylenes attached to a chiral carbon, the protons in the first one will be non-equivalent and well resolved (maybe even first order), but the shifts of the protons in the subsequent methylenes will be much closer (hence leading to strong coupling patterns), and finally each CH2 will approximate an equivalent A2 spin group. Again, moving to a higher field may help a bit, but not too much - at best it might push up by one the limit of induced chirality treatable by first-order approach.

This also brings to my mind another problem: the predictions of the chemical shifts due to the above cases of chirality. I have noticed that the Modgraph and ACD softwares both do some physically funny things there, so I do not trust them. ACD apparently just drops-in a fixed value (like 0.01 ppm) every time there is any chance of induced chirality, while Modgraph, more appropriately, makes the shifts 0 whenever they don't know better. Since I have a cute idea about what could/should be done, could you tell me (or ask an expert like Erno Pretsch) what is the current state-of-the art on this point? As often happens with my cute ideas, it might be well known since 50 years (in which case, I will keep my mouth shut). But if it is not, it might be a thesis assignment for a student.

BTW, I wonder whether the adjective "induced" might not be more proper also in the context of virtual coupling you were writing about in your last entry. Maybe "induced coupling" would be a bit less controversial. Though I am not sure. You could propose it for an e-poll.
Ciao, Stan

1H NMR Analysis: Common Myths and Misconceptions

The analysis of NMR spectra, in particular 1H-NMR, is certainly an exciting and challenging area of research and considerable effort has been made in the past 50 years or so to overcome many of the difficulties that this analysis faces. Actually, most of the work on this subject was done in the early 50’s and computer programs for the analysis of complex spin systems appeared in the 60’s and 70’s (see for example, references [1-4]). However, even though the analysis of 1H NMR spectra is for sure one of the most frequent tasks carried out by chemists, almost on a daily basis, I have found that there are several very popular misconceptions and fallacies amongst chemists which I would like to bring to your attention. In fact, some of these incorrect explanations can be found in books and articles. By doing this, I could run into the risk of introducing some inaccuracies in my comments (which is very likely). I’d like to take advantage of the interactive nature of this blog so, if you see any errors, please, just post them as a comment below.

This post will be somewhat lengthy, I tried to cover these points with all the detail I could think of, including illustrative figures and potential pitfalls, so grab your favourite caffeinated drink before you start reading!

Myth #1 All NMR signals are symmetric

When a chemist is first introduced into 1H NMR analysis, he/she is trained with the basis of the simple and well known first order multiplet patterns which, by definition, are always symmetric. It would be nice if all multiplets would behave following these simple rules. However, lines in any real-life spectrum are highly composite (more on this below), so no experimental line is "pure"; generally, experimental lines are a superposition of a large number of transitions. For example, let’s take the very simple case of the NMR spectrum of ethanol at 500 MHz:

The multiplets in this synthetic spectrum show indeed a large degree of symmetry and, in principle, as expected from the first order rules, we observe 4 lines for the CH2 group (triplet) and 3 lines for the CH3 group (assuming that coupling with OH does not occur).
This is certainly what one would observe in a modern high field spectrometer, but the truth is that there are 12 distinct main transitions in the CH2 group and 13 in the CH3 group, but all of them are never resolved. However, this more complex than expected structure of the CH3 and CH2 groups can be appreciated if the spectrum is recorded at lower field as illustrated in the CW experimental spectrum (from reference [2]).


This does not mean that all the 12+13 transitions only exist at low field. It’s just that the distance between the different transitions depends on the magnetic field in such a way that at higher fields they are too close to be observed and several transitions often contribute to the same peak. For example, in the following two figures, all these transitions are displayed as sticks superimposed on the observed multiplets which, apparently, look like a first order quadruplet and triplet respectively. As you can see, the inner structure is much more complex.




By the way, do not take too lightly the powerful capability of Mnova to classify and display graphically all the transitions corresponding to a given nucleus. If only first order rules were used, this would be a trivial task, however, when a rigorous quantum mechanics treatment is carried out to calculate all the transitions, this classification is far from being trivial. Combination lines, that is, transitions which correspond to the simultaneous change of spin of several nuclei, are particularly challenging on this classification system. They are forbidden in the first order limit but will have a small, non-zero intensity in the general case. These lines, when their intensity is large enough, are also taken into account in the classification system of Mnova. To the best of my knowledge, this capability is available in Mnova only, but if you know any other application which allows you to select one proton with the mouse and display the entire individual transitions corresponding to that nucleus (and the other way round, that is, selecting one transition in the spectrum and highlighting the nucleus causing such transition), please let me know.

Well, you may argue that even though the fine structure of the ethanol spectrum is somewhat complex, the truth is that the multiplets look symmetric and first order compliant (please note however that even in the simulated 500 MHz spectrum, the multiplets are STILL not symmetric, look at the sticks: The asymmetry is about 15% which is not little!) . But let us take, as a new example, a spectrum comprising an ABX spin system (as per Pople notation) with the couplings depicted in this figure:

In this spectrum we can observe the expected doublet for the X nucleus as a result of its coupling with B. However, if the chemical shift difference between A and B is reduced, the splitting pattern will become more complex. For example let’s now take a look at the same spin system when |A-B| = 15 Hz.

Interestingly, the X nucleus appears as a double doublet (disregarding the two small combination lines) even though it’s only coupled to nucleus B (JAX = 0). This kind of situation, where a nucleus appears to be coupled to another nucleus, even though the actual coupling constant is zero, is called Virtual Coupling. In general, if one nucleus is coupled to another nucleus which forms part of a strongly coupled group, the former nucleus will behave as if it were coupled to all the members of the spin system.
Do you still believe that 1H NMR spectra are always symmetric? Let’s make the chemical shift difference between A and B even smaller (5 Hz).



Clearly, an analysis of the X nucleus based on first order rules will yield incorrect values for the coupling constants. Obviously, the AB spin system can only by interpreted by means of a rigorous quantum mechanics treatment. From a symmetry standpoint, the X nucleus is symmetric (which could mislead one to assume it is a first order multiplet) but the AB multiplet is not.
Typical examples of ABX systems are pro-R and pro-S protons of methylenes in pro-chiral molecules.

So as a conclusion, in general multiplets are never really perfectly symmetrical, even disregarding the fact that they often overlap. In addition, relaxation effects in coupled systems are nontrivial and affect each transition in a different way, and there are often other effects such as exchange processes, co-presence of isomeric forms, impurities, etc.

Myth #2 High magnetic fields make the analysis of 1H NMR spectra by means of first order rules possible

Well, this is not completely wrong as it’s true that by increasing the spectrometer frequency, most of the 1H spin systems may become suitable for First-Order Analysis at least as a rough or grosso modo approach. For example, if we were to go back to the previous ABX spin system, and increase the magnetic field, the chemical shifts between A and B will become larger in such a way that we can arrive to a point in which the 3 individual multiplets can be perfectly handled with simple first order rules (see figure below).




However, there are many situations in which higher magnetic fields will be of little or no help. Let me present just a couple of examples:

1. Saturated fatty acids
   
2. AA’BB’ spin system

Consider the 1H NMR spectra of fatty acids with long saturated chains. In the figure below I have simulated the 1H NMR spectra of 3 fatty acids of different lengths. The first thing to notice is that the CH3 does not appear as a simple triplet. This is because it is (weakly) coupled to a CH2 group which is strongly coupled to the next CH2 group in the chain, leading the CH3 resonance to show virtual coupling with the CH2 protons in the chain. Even at higher fields, the pack of CH2 groups is very strongly coupled, though such higher fields may make it possible to resolve the resonances of some of the CH2 groups. However, as the number of methylene groups increases, the "newly resolved" methylenes will have quite small relative chemical shifts and they will always couple to their neighbors by J's of the same order of magnitude. So, whilst the CH2s at the extremes of the chain become 1st order when increasing the magnetic field, there are always others which are strongly coupled, and still others which are insufficiently resolved.

Another, more striking example, occurs in spin systems of the type AA’XX’ (or AA’BB’ if the chemical shifts are close) as is the case, for example, in spectra of o-dichlorobenzene (ODCB). This compound is often used to calibrate instrument resolution.
In an AA’BB’/AA’XX’ system there are 2 pairs of magnetically non-equivalent protons with the same chemical shift, that is, they are chemically equivalent. In general, when groups of chemically equivalent nuclei exist, second order effects are expected. As the chemical shift difference separating the nuclei in the molecule is always zero because of the symmetry, second order phenomena will always exist regardless of the magnetic field applied. If the magnetic field is increased, it will be possible to get a larger chemical shift difference between the AA’ and the BB’ groups, but not between A and A’ or B and B’, so that the highest simplification one can achieve by increasing the magnetic field is to move from an AA’BB’ group to an AA’XX’ group which is a second order spin system too.
There are a total of 12 transitions for each spin (the AA’ part or the BB’ part).
As an example, the figure below depicts the spectrum of ODCB at two magnetic field strengths, 60 MHz and 400 MHz.. At 60 MHz the inner lines are more intense than the outer lines and there is an evident lack of symmetry. These peculiarities are easily recognizable as second order effects and they are caused by the small chemical shift difference between AA’ and BB’. If the spectrometer field strength is increased (e.g. 400 MHz), these effects are reduced as the chemical shift difference between the two groups is now larger and we can now see two more symmetric multiplets. However, each multiplet does not follow the first order rules (e.g. they are not simple doublets of doublets of doublets) because A and A’ and B and B’ will always be strongly coupled regardless of the magnetic field strength. Once again, the only way to accurately analyze these systems is by means of quantum mechanics calculations.


Myth #3 Protons within a CH3 group do not couple with each other

I have found very frequently that chemists assume that CH3 protons are not coupled because only a singlet is observed in the spectrum. This is simply not true! The correct explanation is that couplings within a magnetically equivalent group such as a methyl group do not affect the spectrum appearance, but coupling definitely exists (it is known that geminal couplings are usually large and negative). In other words, if we synthesize a spectrum with and without couplings within a magnetically equivalent group, the spectrum will look exactly the same, so for calculation purposes, these couplings can be neglected.


That is all for now I hope you find these comments of some use and/or interest and if there are any points where I have failed to make myself dear, please do not hesitate to post a comment here. I’d also like to thank Stan Sykora for several useful discussion about this work.


References

[1] Pople J.A., Schneider W.G., Bernstein H.J., High-resolution Nuclear Magnetic Resonance, McGraw-Hill, New York 1959
[2] Roberts J.D., Nuclear Magnetic Resonance: Applications to Organic Chemistry, McGraw-Hill, New York 1959
[3] Early History of Nuclear Magnetic Resonance
[4] Automatic Analysis of NMR Spectra: An Alternative Approach Diehl P., Sykora S., Vogt J., J.Magn.Reson. 19, 67-82 (1975) [click here]

Proctor, Yu and Dickinson

NMR is by far the most powerful and widely used tool for the determination of organic structures by chemists. Of the wealth of information that this technique provides, the chemical shift is what makes NMR so attractive to chemists as it allows them to distinguish among the different protons within a molecule.

However, despite the fact that chemical shift is what makes NMR useful in chemistry, I’m amazed about how little is known in the chemistry community about their discoverers. I have run a quick survey among 10 chemists colleagues of mine and none of them was able to cite one single name! Maybe the words of M.E. Packard had a higher impact than what he expected when he said "chemists got the point very
quickly, thanked the physicists, and took over"
I think that all chemists are in debt with the pioneering work carried out by W.G.Proctor, F.C.Yu and W.C.Dickinson and I would like to take advantage of my blog to give more recognition to these scientists. Now that we live in a world in which self-promotion and ‘rock star’ popularity are so valued, I find it necessary to acknowledge the value of what they have contributed to humankind in such a quiet way.
For a recent, worth reading, post about Chemical Shift and W. G. Proctor, do not miss Stan’s blog and Reminiscences of the Early Days of Nuclear Magnetic Resonance at Stanford University

Glenn Facey’s blog

In case you’re interested in experimental aspects of NMR, do not miss Glenn Facey’s blog at University of Ottawa. Whether you are an NMR facility manager or a scientist using NMR routinely, I’m sure you will find it a very useful resource.

http://u-of-o-nmr-facility.blogspot.com/

Introducing 2D Resolution Booster ™ (RB)

In recent entries I presented Resolution Booster as a simple but robust method to obtain highly resolved NMR spectra and showed some of its properties. Today I want to show the preliminary results we are getting by applying an extension of this method to 2D NMR spectra.

In order to illustrate the performance of the algorithm I have simulated a 2D spectrum of an A2B3 spin system with the following parameters:

• Spectrometer Frequency = 500 MHz
• Shift A = 4 ppm (2000 Hz)
• Shift B = 8 ppm (4000 Hz)
• JAB = 30 Hz
• Line Width = 30 Hz
• Data points = 2048 x 2048

As the coupling constant is very close to the line width (they are actually exactly the same, 30 Hz), the multiplets are not resolved (2D spectrum at the left). After applying 2D RB, the spectrum achieved has a higher resolution along both dimensions, where all multiplets are now clearly well resolved.

We are still working on this method but the results we are currently getting are certainly very promising and we are confident that it will soon become a very valuable tool for automated 2D NMR processing. It is not available in the current version of Mnova but it will be included in the new release scheduled for the end of January 2008. Together with my friend Stan Sykora, we will be presenting a poster on RB in ENC 2008 at Asilomar. Should you be attending ENC, please stop by to see us. We will be delighted to discuss this (or any other) topic with you.