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NMR Continues to Bear Down on Structures

Determining the structure of a new molecule is one of those things that you’d think would be simple – at least, nonscientists often seem surprised at how much of our time we spend on such problems. (It doesn’t help that dramatic depictions involving chemistry almost invariably skip over this problem in the interest of moving the plot along). Most of the time during a new synthesis we pretty much know where we are, true – there’s only one carboxylic acid on the starting material, and we just did something routine to it, and there it is, by gosh. But if something unexpected happens, or some odd side product forms, or if you isolate a new compound from a natural product source, well, that’s a different matter.

The best news you can get in such a situation is that the compound forms a decent crystal, because then you have X-ray crystallography on your side. That’s not foolproof, but it’s a pretty damned strong technique, and a good X-ray structure will pretty much settle most arguments. First, though, grow your decent crystal. That gets harder and harder as your molecules get more complex and your available sample shrinks, as you’d figure, and growing good crystals in general is known far and wide (and correctly!) as a black art. Things have gotten easier as X-ray sources have gotten brighter and the available techniques to work up the data have improved, but there’s a good reason that people have been excited about the “crystalline sponge” technique, which can (in some cases) get rid of the crystal-growing step completely.

NMR can also solve your structure for you, but it has its own limitations. There’s a whole list of neat experiments that can be run on a modern NMR machine, limited mostly by your sample size, which has a direct correlation with your time and patience. You get a lot of connectivity data out of many of these (this is next to this, which is next to this, etc.), but there are many structures that can make the easy experiments less informative or more equivocal. So this paper, which proposes a new protocol entirely, is quite interesting. It’s a joint effort from Merck, Harvard, UConn, and Amsterdam, and it depends heavily on DFT (density function theory) calculations as applied to less-commonly-applied NMR data, specifically residual dipolar coupling (RDC) and residual chemical shift anisotropy (RCSA). Taking these measurements under what the rest of us organic chemists consider weird conditions (letting the molecules align themselves in a gel matrix) and comparing this with solution data allows for the range of possible structures to be narrowed down to the point that doing all the DFT calculations becomes feasible. (To give you an idea, those tend to run to the “several hours per molecule” range, so if you’re down to a dozen structure, you can crank those out in maybe three days.

RDCs and RCSAs, in fact, are only observed when a molecule is aligned, and not when it’s tumbling around freely in solution, which is the usual NMR experiment familiar to most chemists. Comparing these can give you RDCs that report on the relative orientation (for example) of all the C-H bonds in the molecule. The RCSAs, in turn, give information on the relative orientation of carbon atoms, including those that don’t have a hydrogen on them at all. The calculation values for these can be compared to the experimental ones, and it appears that the range of possible values for them is wide enough for a good match to tell you that you’ve hit the right structure. Shown at right is one of several examples from the paper, the natural product aquatolide, whose structure had recently been revised after a great deal of effort. At upper left is the currently accepted structure, and at upper right is the originally proposed one. The lower two structures are alternate ideas generated computationally. One of those isn’t bad, but the fit to the data is by far the best for the upper right structure, and is pretty poor for the original one next to it.

Here are the authors:

Once a reasonable 3D model associated with each candidate is generated, whether via computational methods or investigator deduc- tion, and chemical shielding tensors are calculated by DFT based on this 3D model, RDC and RCSA data can be employed as a sensitive critical measure to evaluate the validity of the structural assignment. The possibility of a false-positive determination—that is, agreement of RDC and RCSA data with an incorrect structure—is substantially lower than that in an analysis using only conventional NMR data, especially when both RDC and RCSA are jointly used. These data can serve as a convenient NMR litmus test of structure and stereochemical validity. As such, the method described in this work has considerable potential to be widely applied, which could help to quell the flow of incorrect structures appearing in the literature.

That would be a good thing, for sure, because there’s a lot of error out there. One problem will be how common such measurements become. Some of the mistake structure in the literature are completely honest, as with the original aquatolide structure, but some of it is the result of carelessness, and those are the ones that are least likely to go on to do a more rigorous technique like the one described. But it’s very good to have something like this to add to the list of structure-solving (and structure-confirming) techniques, because we really do need all the help that we can get. Natural products chemists and medicinal chemists will especially welcome the chance to try it out!

Update: for another application of this approach, from another group entirely, see this paper from February. There are many other reports in the last few years on the use of RDCs and RCSAs, for those wanting to dig into the subject.

21 comments on “NMR Continues to Bear Down on Structures”

  1. enantiostereomer says:

    Derek, I think that you meant that the fit is the best for the upper-left (the revised structure), not the upper-right (the original)

    1. Gary says:

      It was indeed the upper right panel.

      1. Gary says:

        brain dead, I can’t type this afternoon… UPPER LEFT panel is the correct structure.

  2. Alan Goldhammer says:

    Every time I read about new advances in identification of complex organic molecules I take a step back and marvel at what the chemists of 100 years ago had to do. They didn’t have the instruments we have today yet they still solved some very tough molecular structures. I once had to translate a Willstatter paper from German as a final project for my German class. It was amazing to see the path to structure identification.

    1. NJBiologist says:

      I think that’s one (of many) good argument for reading old literature. You gain some appreciation for the people who came before you, but you also you value your own tools more.

  3. RBW says:

    At the end of the day, structure determination depends on human interpretation of the data. And there are plenty of examples where humans will ignore what doesn’t fit their notion. Patchoulene is a classic example and more recently diazonamide is another.
    Then there are structures where NMR is insufficient. Take a look at breitfussin in Angew Chem, they used a mix of AFM and calculations.

    1. Algirdas says:

      Actually, this emerging RDC + RCSA technique has a good chance of working on breitfussin:

      i) Flat aromatic fragments = fewer rotating bonds in DFT minimization of candidate structures;

      ii) Aromatic carbons have CSA values significantly larger than aliphatic;

      iii) Multiple heteroatom substitutions should further increase spread of CSA (and thus RCSA) values.

      Once you get your molecule aligned, measurement of RCSA values is trivial; and while I personally have no experience in computing these things, I suspect predicted RCSA values should be very different for plausible breitfussin structures.

      Also, looking at Angew Chem breitfussin paper I think your point wrt “structure determination depends on human interpretation of the data.” is very salient: you have to squint really hard to make the AFM image in their figure 1 panel a to overlay with molecular model like they show in panel b.

      1. RBW says:

        The breitfussin paper, like a lot of science nowadays, was hyped as that’s the only way to get into the high impact journals.
        Yes, AFM was helpful but in reality if you thought about the biosynthesis and looked up similar natural products the connectivity was obvious.

  4. LiqC says:

    Back when I was young and pretty I tried to used CSA to study viscosity of dispersed phase in an emulsion… didn’t get very far.

    But I saw a triplet of triplet of triplets in 13C NMR!

  5. milkshaken says:

    the problem with gel-aligned samples is that if you have one milligram of a precious compound isolated from some sponge that was collected 150 feet below (and the sponge may not contain the same material the next time, due to microbial symbionts), you may want to get your compound back from the sample.

    I have an idea for a potentially better alternative: benzene or C6D6 forms 1:1 complex with hexafluorobenzene which has the same volatility as starting solvents (80C b.p.) but it melts at around 30C and is composed of infinite columns of pi-stacked alternating benzene-C6F6 molecules. The 1:1 complex is also an excellent solvent mixture able to solubilize graphene. If you were to melt this complex, dissolve your molecule in it, put it as warm liquid into magnet and let the sample solidify in there within the strong magnetic field, it is quite likely the arene ring columns would align themselves along the magnetic field axis and orient the dissolved samples the same way the gel does, except that the sample recovery would be much easier

    1. Algirdas says:

      RE: benzene-C6F6 idea

      But now you are talking about solid state NMR spectrum. If you try this in regular liquids NMR tube you’ll get no signal. Actually, solids NMR is making large advances, so putting your precious 1 mg into a magic-angle-spinning rotor and recording 100 kHz MAS-based spectra is a perfectly good option. See: Solid State Nucl Magn Reson. 2015 Apr-May;66-67:56-61. doi: 10.1016/j.ssnmr.2015.02.001. “Studies of minute quantities of natural abundance molecules using 2D heteronuclear correlation spectroscopy under 100 kHz MAS” PMID 25773137

      And if you are measuring solids spectra, there are pulse sequences to reintroduce dipolar couplings into your MAS spectra (don’t know about CSA), so you access the same information as in aligned liquids.

    2. Algirdas says:

      More on aligned samples.

      Isolating macromolecules from gel slabs is pain in the butt (even when it simply melts, like agarose DNA gels), but I bet if you could isolate 1 mg of your whateverene from 10 kg of sponge, dealing with 0.5 gram of organic gel is much less of a challenge.

      Certainly your C6D6-C6F6 idea is neat because one would simply evaporate the solvent.

      But you don’t have to stick to gels for alignment medium anyways. Most liquid crystals align in the field, so many lyotropic liquid crystals would work (in protein NMR we had this period about 10 years ago where everyone and their dog was publishing new alignment medium every month.) For instance, I’ve used lyotropic medium based on C12E5 surfactant. It would not be surprising at all, if there are lyotropic liquid crystal media suitable for organic compounds, may be even based benzene and(or) C6F6.

  6. I just hope that NMR remembers to breathe between contractions.

  7. tangent says:

    How well can the IR spectrum of a molecule be computed from its structure? I mean that’s just springs and stuff, right? 🙂 That seems like it could in principle be used as a check on a postulated structure for a sample in hand. If you’re cool with using Grampa’s spectrograph.

  8. michael says:

    Lets not forget this technique is at least 15 years old!!! Come on guys read the literature.

  9. Professor Electron says:

    Residual dipolar couplings (D) give long-range information about relative orientation, which you can’t get from any other solution NMR method. This makes them a useful add-on to comparing observed and predicted 13C shifts, which is the main current method for validating structures. However, the same natural product was studied by Pauli et al (DOI: 10.1021/acs.joc.5b02456) and they claim that the diasteroisomers can be distinguished by complete analysis of 1H NMR spectra. I wonder whether complete analysis of NOE build-up curves would also distinguish them. What is needed are easy ways of doing all these things – pyDP4 from the Goodman group is an excellent example of what is needed (DOI: 10.1039/C6OB00015K).

    The partial alignment technique has been around for a while, and my experience is that it needs mg of substance and it is hard to reproduce. I’m sure it gets easier the more you do it.

    Note: haven’t been able to read the paper (paywall)

  10. Red Fiona says:

    For once I can comment with experience. I’ve used RDCs to help with protein structures, and the main problem is aligning the damn things (I love phage alignment media, and loathe bicelle media). I do wonder if the same problem will also apply to small molecules.

    1. ANV says:

      This was certainly the case for a long time. However there are now several media with very good behavior for small molecules. In my experience the most practical aligning media by far are compressible polymethacrylate, for CDCl3, ( DOI: 10.1002/chem.200903378 ) or poly-HEMA gels for DMSO (10.1002/chem.201503449).
      For studies in water, of small molecules disodium cromoglycate/NaCl medium, a liquid crystal, is extremely easy to prepare ( DOI: 10.1039/c3ob42338g ) and provides very clean spectra.

    2. tangent says:

      “Phage alignment media” just sounds like a badass concept invented by a science fiction writer to sound totally badass.

      1. Algirdas says:


        when some biologist-turned-novelist like Peter Watts needs to throw in some really deep insight (TM), “tesseract” is their go-to thing.

        But come on, “lyotropic liquid crystal” got as much technobable potential as “Phage alignment media”.

  11. First says:

    Yan J1, Delaglio F, Kaerner A, Kline AD, Mo H, Shapiro MJ, Smitka TA, Stephenson GA, Zartler ER. J Am Chem Soc. 2004 Apr 21;126(15):5008-17.
    Complete relative stereochemistry of multiple stereocenters using only residual dipolar couplings

    Jiangli Yan , Allen D. Kline , Huaping Mo , Michael J. Shapiro ,* and Edward R. Zartler
    J. Org. Chem., 2003, 68 (5), pp 1786–1795
    A Novel Method for the Determination of Stereochemistry in Six-Membered Chairlike Rings Using Residual Dipolar Couplings

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