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Analytical Chemistry

Absolute Configuration With Electrons

When I first wrote about small-molecule structures obtained by microED (electron diffraction), I wondered if there were some way to get absolute stereochemistry out of the data (as you can with X-ray diffraction under the right conditions). Several groups have been working on just that problem, and this new paper now shows that it can be done (commentary here at Science).

As usual, I Am Not a Crystallographer, and anyone who is already can dive right into the details of how this is done. I will no doubt make caveman mistakes in my explanations. But in the X-ray world, as even us synthetic-organic, med-chem, and chem-bio types already know, it depends on anomalous diffraction. You look for diffraction spots that are pairs related by an inversion symmetry (Bijvoet pairs). If your crystal has a center of symmetry, these will be identical, but if it’s in a non-centrosymmetric (chiral) space group, they can be subtly different. Unfortunately, these differences are determined partly by the size of the atoms involved in the diffraction, and the carbons, hydrogens, oxygens, and nitrogens we’re used to aren’t up to much. That’s why people are always trying to drop in a heavy (or at least heavier) atom to help solve such problems, to make the analysis of the Bijvoet pair differences more solid. (The anomalous diffraction effect is also dependent on wavelength, and adjusting your X-ray wavelength to the sort of heavy atoms you’re targeting, which you can do with a synchrotron source, gives you an edge as well. That’s a little X-ray humor in that last line.)

Anyway, that sort of breaks down in electron diffraction, at least if you treat it as a sort of souped-up X-ray diffraction. Doing it that way has jump-started the field, since you could use the (large) suite of software tools available to process the data, although I did hear one of its practitioners mention that the first time they tried it, the software crashed because the wavelength they entered was way out of what the program would accept as an X-ray value (!) Treating the data under simple X-ray rules means that you’re assuming that only one scattering event occurs (the “kinematic” assumption), and for X-rays you can simplify your life in that manner a lot of the time (although it was realized very early on that you could see multiple events as the X-rays made their way through the crystal). That’s called dynamical scattering.

Electrons interact with the atoms in a crystal far more strongly, which is what makes the microED technique able to work on such ridiculously small crystal samples in the first place. That makes dynamic scattering much more apparent; you get much more quickly out of the range where the kinematic assumption works. In fact, all the dynamic scattering was a nuisance earlier on in the electron-diffraction field, especially because crystals of inorganic compounds were being used where it was more of a factor than ever. Finding that the kinematic assumption held when you did ED on rotating protein crystals was actually a relief, from what I can see. (In fact, trying to measure electron diffraction on a too-thick protein crystal is inviting trouble as the kinematic assumption will start to break down on you). But that dynamic scattering can give you a basis for Bijvoet pair analysis and chirality determination, since the multiple scattering events are sensitive to violation of inversion symmetry, and that’s been done for inorganic crystals.

This new paper, though, charges right into dynamic electron scattering as a way to determine absolute structure for light-atom small molecules, and it’s the first time that’s ever been done.  The group had a very challenging situation: a cocrystal of sofosbuvir and L-proline, which is a very pharmaceutical crystal indeed. It had several disadvantages: the ribbon-like crystals were bent and twisted, and were very sensitive to radiation damage. The authors ended up scanning the electron beam along individual crystals, collecting diffraction patterns as they went, which put them into the same sort of regime as “serial electron crystallography“, where you hit a large number of individual nanocrystals and assemble a data set from those. The PEDT (precession electron diffraction tomography) technique (refined by some of the same authors earlier) proved to help out a lot in dealing with this situation. Rotating the sample/electron beam geometry gave more diffraction spots per shot and a better framework for matching them up in the data analysis.

So now that we know it’s possible with our new rotating electron diffractometers, we can expect even more use of microED. You’d expect it to be even less taxing on many crystals than it was here – long bent ribbon-shaped crystals are, frankly, one of the last things you’d want to deal with, given a choice. If we can (1) get both protein and small-molecule crystal structures by electron diffraction, (2) do it on microcrystals that previously would have been considered uselessly small, and (3) directly determine absolute configurations on the small molecule samples at the same time, what’s not to like?

14 comments on “Absolute Configuration With Electrons”

  1. HX says:

    Thank you for sharing this beautiful work. Electron crystallography is becoming a complementary technique to X-ray crystallography and neutron crystallography. It also offers new possibilities to biologists and chemists.

  2. Imaging guy says:

    When are we going to get X ray and Gamma ray microscope with high enough numerical aperture lens (mirrors) so that we can directly image cells and tissue sections and find out the structure of biomolecules?

    1. Nesprin says:

      We already have that, but it tends to take a cyclotron to work. See- soft electron tomography

  3. just asking says:

    What’s the advantage, if any, over VCD (vibrational circular dichroism)?

    1. Rhenium says:

      Thanks!

  4. anon the II says:

    I’m not a crystallographer, but I am married to one and I got my PhD in a lab that was half crystallographers and they always used the term “anomalous dispersion” rather than “anomalous diffraction”. Are they the same thing?

    1. Smarty pants says:

      Did you ask your wife?

      1. Not so smarty pants says:

        Or husband?

      2. anon the II says:

        On your suggestion, I did. She said she doesn’t remember the term “anomalous diffraction” being used. She retired a number of years ago, so maybe things changed.

  5. Natural chemist says:

    I get that microED and crystalline sponge and whatnot are pretty amazing but I just don’t see them (much to my chagrin) altering my life anytime soon. A combination of cost, availability, and the technical aspects of these methods are too much of a hurdle for me and my sparsely-funded research group. Plus I can interpret COSY/TOCSY/HSQC/HMBC/NOESY/ROESY NMR data of very complex natural products with deadly accuracy. Anyway, just rambling here…

    1. HX says:

      We are currently developing methods and open source software to spread 3D electron diffraction (or MicroED) based methods at Stockholm University. MicroED data can be collected on normal TEMs with normal CCD/CMOS detectors. You don’t need those sophisticated cryo-EMs.

      I could imagine that in the new future, tabletop electron diffractometer will be developed. I won’t say that ED is better than other techniques. Instead, I see it as a complementary technique to the powerful existing techniques, which provides new opportunities to structural related studies. At the end of the day, a method is only useful if it is simple and can be widely spread.

  6. downunder says:

    (also IANaC) “size of the atoms involved in the diffraction, and the carbons, hydrogens, oxygens, and nitrogens we’re used to aren’t up to much” certainly used to be the case (e.g. might derivatise with arylbromides etc), but at least for last 10 years or so N and O seem to be heavy enough for small molecule crystals? Certainly I’ve seen a number absolute Xray determinations without any ‘heavy’ atom. I don’t recall the R factors. I assumed that it was some combination of sequential advances in detectors and/or computing?

    1. Anonymous says:

      (also also IANAC) In grad school a few years ago I went to our X-ray crystallographer to get the absolute stereochemistry of a compound that I had coupled to an L-amino acid. He said something similar that if the compound has an O he could get the absolute stereochemistry assignment, but I never asked what the deal was with that.

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