We’ve been seeing more macrocycle compounds in drug discovery and other areas of chemistry over the years – well, macrocycles that we’ve made deliberately, that is. There have long been natural products of this sort and their derivatives in the pharmacopeia, things like erythromycin, cyclosporin, amphotericin B, azithromycin, rapamycin, rifampicin, eribulin. . .those are a few that come to mind, and there are plenty more. Letting bacterial, fungi, and other creatures make and optimize these things for us has long been the rule, since synthetically they can be very challenging, not least in simply closing rings of this size from acyclic percursors. A great deal of 20th-century total synthesis work took place in this area for just that reason, and it’s not like all those synthetic problems have been solved yet, either.
Why do we care about large rings – what’s special about them? It turns out that they can have unusual properties, in several ways. They can bind smaller species inside the perimeter of their rings, for one thing – everything from ions all the way up to substantial molecules in host/guest complexes. They can cover interesting binding sites on protein targets in cells (thus all those natural products), things that often you can’t achieve with acyclic compounds. That’s partly because of entropic factors – with a macrocycle you have “pre-ordered” the molecule and placed its various functional groups and regions in a constrained space compared to what they’d be exploring in an acyclic compound. That means that cyclization (of all sizes) is often a sort of “death or glory” move in medicinal chemistry. If you can get things arranged into the right places and hold them there with a cyclic structure, you can get really remarkable boosts in activity and selectivity. On the other hand, your new cyclic structure could also tie things back so that there’s absolutely no way for these groups to get into the right positions any more, and your activity might almost completely disappear.
There’s another effect that we’ve all been trying to exploit as well. If you look at many of these natural product macrocycles and start counting up polar groups and the like, the good ol’ rule-of-five heuristic or the like, you quickly realize that your rules don’t seem to apply too well in this space. Some of these things look (from that approach) as if they shouldn’t get absorbed from the gut as well as they do and shouldn’t penetrate cell membranes as well as they do, either. There’s some benefit to macrocyclic space that the “count up the molecular features” approach misses, and a great deal of work has gone into figuring out how that works and how we can made it happen on demand. One of the answers is surely that there are interactions within these molecules (across the interior of the ring space) that stabilize their conformations and arrange the groups on the outside of the ring in particular ways. But nailing these down is a challenge for analytical chemistry techniques.
Here’s a new paper from a group at Tennessee looking at cyclosporin A, which is a good prototype for this kind of thing. The structure’s at right (or at least it should be; I’ve been having some hiccups on putting in images around here). It’s a cyclic peptide at heart, with modifications (specifically a lot of N-methylation). Like most of these structures it’s not a lot of fun to draw. Especially if you want to draw it in a way where you don’t have to stretch bonds and jam things together!
That’s a 33-membered ring you’re looking at (I think), so the one thing you can be sure of is that it doesn’t actually look like any reasonable flat structure you can draw. But just what it does look like in aqueous solution has been a longstanding problem. CycA is not too soluble in water, which is a hint that a lot of those greasy alkyl side chains are exposed to solvent. And you would have to think that there are a number of closely-spaced low-energy conformations accessible, so you’re going to have to deal with that equilibrium when you take measurements, too. In this work the authors used three main techniques to work out the details: X-ray diffraction, neutron diffraction, and NMR. Those first two will give you a static view of a crystalline form (which the team obtained in the presence of added KCl), and the NMR is for watching what’s going on in solution. This same group has already noted at least eleven different CycA conformers in methanol/water solution, to give you the idea.
The authors mention that a similar X-ray structure of crystalline cyclosporin A has been determined, but for some reason the data were never deposited in the Cambridge Crystallographic Database, the worldwide repository for such things. That linked paper will also take you to references for some other X-ray structures that have been determined over the years, many of which are hydrates (with water molecules as an integral part of the crystalline form). That was the case here as well. Seeing a hydrate gives you some hope that you’re also getting insight into the aqueous solution structure as well, but there’s no guarantee of that, since bulk water is its own thing entirely. For example, in a crystal like this one most of each CycA molecule is probably being packed against other CycA molecules, but that’s not going to be the case in dilute solution, where they’re going to have to make their living shouldering networks of hydrogen-bonded water molecules out of the way without the assistance of their own kind.
The neutron diffraction analysis is a much more rare technique, because there are a limited number of places in the world that produce suitable beams of neutrons. This effort used the Spallation Neutron Source at Oak Ridge, conveniently located down Highway 62 from Knoxville and producing what I believe are the strongest neutron beams in the world at this point. It’s also the newest such source in the world, coming on line around 2006 or so. Those neutrons are obtained, if you’re wondering, by whipping negatively charged hydrogen ions up to high speed, running them through a foil target to leave you with a beam of protons, and then sending those into an accumulator ring. The bunches of high-energy protons from that are blasted into a mercury target (other heavy metals like lead, tantalum, and tungsten have been used as well), which causes those massive nuclei to cough up large numbers of energetic neutrons in the “spallation” process.
Those are slowed down to useful energies by passage through a moderator (like liquid methane, since neutrons interact pretty strongly with all those H atoms). As a side note, if you’re ever worried about being exposed to neutron flux (and if you are, you should worry), you can help yourself by taking refuge behind a stockpile of wax, lard, or vegetable oil for the same reasons. I hope that this is useless advice. It’s that interaction with the hydrogens that make neutron diffraction so interesting to organic chemists. It’s very difficult to get the real positions of hydrogens with X-ray studies, but neutron diffraction will nail them all down for you, including any bound water molecules in the crystal. So it’s just the thing for a hydrate of a complex molecule like this one.
Anyway, the use of the word “beam” back there is a bit of a stretch, because since neutrons have no charge, they (hah-hah!) cannot actually be focused, damn it all. If you figure out a way to do that, arrange a demonstration and wait for your Nobel Prize the next October. So to get useful information by passing neutrons through samples, you need a *lot* of neutrons produced at the source, since they’re going to be flying out in all directions. And that’s what the folks at Oak Ridge (and a few other locations around the world) are good at. If we ever get inertial confinement fusion to work, by the way, we will not only have a wonderful source of power, but a wonderful source of more neutrons than anyone’s ever had to deal with. That’ll be great for neutron diffraction studies, but it’s not so great for the integrity of the solid walls of the fusion device itself, to be honest, which is one of the (several) engineering problems that have kept everyone in that field busy.
There have been some past neutron diffraction studies of CycA, but these were years ago with lesser sources. And there has been plenty of X-ray and NMR work as well, which has shown that in organic (nonaqueous) solvents like chloroform, the molecule exists in a “closed” state with four intramolecule hydrogen bonds and a cis amide conformation at methyleucine 9/methyleucine 10. That’s the structure the earlier neutron work was done one. CycA is also seen in complexes with proteins in an “open” state, where that cis amide has flipped back to trans. For a long time, that open form has been thought to be the likely one in aqueous solution, with a lot of hypotheses generated about why it penetrates cell membranes more easily, etc. But that doesn’t seem to be the case, as witness the number of different conformers in real solutions and some strange differences between various cyclosporin analogs.
The crystal structure from this work has two water molecules that are tied up in the internal hydrogen bonding network, and a completely different cis amide between resides 1 and 11. That’s in equilibrium (via NMR studies) with another minor conformer, but it’s the major one of all by far in 10:90 methanol/water. There’s another minor conformer that’s in equilibrium with the “closed” form mentioned above, and three others off doing their own thing, one of which has two cis amides in it, weirdly. So those structures that show the closed form in CycA complexes indicate that its binding partners are actually picking a minor conformer out of solution, and this may be what’s happening with all of the various protein binding partners of the cyclosporins in general. They’re involved in complex equilibria, and only some of the species available are actually able to bind productively. Any hypotheses about solubility, membrane penetration, binding constants and so on are going to have to take this wooly situation into account!
