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  • "Today is going to be another, sort of, special topic. It's actually really important [inaudible].

  • One of my favorite things because it is so useful. We're going to be talking about using

  • nuclear overhauser effect in structured stereochemistry determination, and I'll try to show you why

  • I think this is so useful with some examples, maybe related things to the exam.

  • So in terms of what the nuclear overhauser effect is; I've been talking about this in

  • C13 NMR but not why this is useful. So the nuclear overhauser effect, or NOE,

  • is a change in intensity of the resonance of a proton or another nucleus, I'll put this

  • in terms of protons but I'm really talking about any type of nucleus, in response to

  • or upon irradiation of a nearby proton. And, so what do I mean; so let's say from

  • the point of view of a molecule I mean that you have two protons, of course it could be

  • a proton and a C13 in a molecule like so, where they're near to each other in space,

  • not necessarily in connectivity, that are just a few angstrom apart in space.

  • And, we're going to do something that specifically irradiates one proton and obviously what you

  • are doing is not a spatial resolution but rather frequency resolution; in other words

  • each protons appears at a different frequency and so you're going to hit one of these specifically

  • at this frequency and what that does here is it responds -- now, when you're irradiating

  • what you're doing is equalizing the population of alpha and beta states. And, when you do

  • that, that in turn alters the equilibrium population of alpha and beta states of other

  • nuclei that are nearby. And the way it does this is it opens new relaxation pathways.

  • And since it is a relaxation process, it doesn't occur instantly. It takes time on the order

  • of relaxation time; in other words, hundreds of milliseconds, typically, to build up.

  • And so, what you see, well, you see in your C NMR -- there are two reasons in the C NMR

  • that your C-H peaks, and C-H2 peaks, are bigger than your [inaudible]: one of these reasons

  • is the nuclear overhauser effect, carbon that has hydrogens attached to it is nearby to

  • a hydrogen, so when you irradiate the hydrogens you actually affect the population of alpha

  • and beta states in the carbon; another reason is that relaxation, because your pulsing reasonably

  • fast and [inaudible] usually relax slowly you end up with lower intensity but one of

  • the reasons is the NOE. Ok, so what does that mean for that hypothetical

  • cartoon of a molecule where you have Ha and Hb that are next to each other in space? It

  • means that if you have a spectrum that looks like this where you have Ha and Hb, if I irradiate

  • Ha, now the spectrum changes and we don't see Ha anymore and the peak for Hb gets a

  • little bit bigger. Now, these effects are pretty small in proton

  • NMR. The theoretical maximum H-H NOE is only 50%. And normally you see values that are

  • a lot smaller than that, you might see typically 10%. So what I've drawn here is a cartoon

  • where Hb is appreciably bigger, is actually not recall the case, its just going to be

  • just a teeny tiny bit bigger. Now, I said there's this effect that affects

  • carbon and because of the difference in magnetogyric ratio with proton and carbon you actually

  • have a bigger effect. So with H1 to C13 NOE here the affect is actually

  • 200%. So there you can have a peak get substantially bigger.

  • Now, NOEs involve relaxation, this in turn involves motion of molecules so like tumbling

  • motion which means the NOW is sensitive to the size of the molecule and how fast it tumbles

  • because that is going to affect different types of relaxation in molecules. So high

  • molecular weight molecules, so we're talking typically over 2000, but these numbers aren't

  • carved in stone, but let's say greater than 2000 or if you slow down the tumbling by a

  • viscous solvent, then your NOE is actually negative and the theoretical maximum is -100%

  • for H-H NOE. And this is important in protein structure

  • determination rather than in small molecule chemistry, which is where we focus. Now these

  • days, if you look at some of the natural products tat are getting published in the Journal of

  • Organic Chemistry or Journal of natural Products or JACS, some of these small molecules are

  • pretty hair molecules; in other words, hey are small molecules but they are big small

  • molecules. And that big small molecule regime is actually a pain in the neck, because what

  • happens as you go from small molecules that have positive NOE to big molecules that have

  • a negative NOE is what?" "No NOE?"

  • "No NOE. So the medium-sized molecules often end up have 0 NOE. And I really don't want

  • to be putting hard numbers on this because it depends on what solvent you're using, it

  • depends on the field strength of the spectrometer, it depends on the shape of the molecule, it

  • depends on the temperature, but I'm going to step out on a limb here and say molecular

  • weight range of say 1000-1500, which is a big small molecule, is often 0 NOE. Or close

  • to 0. And there is a related technique called a

  • rotating overhauser effect that's often used to bring out those NOE in that intermediate

  • range. Alright, I want to show you an example of a traditional NOE experiment; and I'll

  • show you an old example and then I'll show you an example that is sort of relevant to

  • organic chemistry research. I just want to show you the general gist of it; it's on the

  • first page on the handout. Alright, so this is an example in the book by Derome which

  • is a nice, a nice book, it's actually a precursor to Claridge, which is a book I've given you

  • some readings from that's sort of a second edition because Derome had passed away. So

  • here's a molecule, the particular molecule isn't important, buy you'll see the issues.

  • So this is an H1 NMR spectrum of the molecule and this is an NMR spectrum in which we've

  • irradiated one of the protons; specifically we irradiated this proton. And, in doing this,

  • the authors didn't quite equalize the population -- you still see a little bit of the peak.

  • The peak has gone from this over to the smaller version. And it's hard to tell if anything

  • has gotten bigger, it looks like that one has gotten bigger. But the way in which one

  • historically does this, because the spectrum involves such small changes, the way one historically

  • does this is called a difference NOE spectrum. And, difference NOE spectrum, one is literally

  • subtracting one spectrum from another spectrum; you're subtracting the unradiated spectrum

  • from irradiated spectrum so I'll put a minus sign, or actually I guess your technically

  • subtracting the [inaudible] and going ahead and coordinate transforming. And so I want

  • to point out the features that we see: so the fist thing we see of course is the now

  • irradiated peak is negative; after all, if we had something where we've almost equalized

  • the population or alpha and beta states and we take away something where we have a positive

  • peak you get a negative peak, but I want to show you the features.

  • Ok, the thing that's glaring you in the face in this literally textbook example is that

  • we see a nice NOE over to the peak here. So there's some spatial proximity between this

  • proton and this proton in the molecule. I want to show you some of the other features

  • in the spectrum: now, one of the things about the traditional NOE experiment is the conditions

  • of doing the irradiation, create little perturbations in the spectrum, and actually to do it right

  • what you do is you do one spectrum where you irradiate here in this case about 1.15 ppm

  • and this one in order to minimize the subtraction artifacts you actually go ahead and irradiate

  • somewhere else where nothing is, like over here or over here; but, even with that, there

  • are some perturbations. So this is what you would call a subtraction artifact. In other

  • words, it's not an NOE, we don't have a particular peak up or down, what's happened is there's

  • been an infinitesimally, a teeny tiny shift in the position of this peak because o f the

  • irradiation which gives us the positive character on one side and the negative character on

  • the other side. If I were looking at this I'd know just from recognition that there's

  • no NOE, but another way, a very good way, and something you really should do, is slap

  • an integral on it; and if you slap an integral on it of course the integral would go up as

  • the area registered and then go back down, and you'd end up, this would be an integral

  • here, so you'd end up with no net rise, no net area.

  • Now, one of the challenges, in any sort of conventional one-dimension NOE experiment

  • is selectively hitting this peak here. 'Cause you're trying to hit all of the lines here

  • in this peak, without hitting this peak; when the peaks are maybe 1/10 of a ppm apart, it's

  • hard to do that. It's easier if you have singlets; I usually tend to go to singlets if I can,

  • harder if you have multiplets because you have to apply a band of radiation that's wide

  • enough to hit this, without hitting this. And as you can see, we've got incomplete selectivity

  • here. So, to put it another way if I was testing a hypothesis that this proton is spatially

  • close to this one, and I hit this one and I see this one get bigger, but I also hit

  • this one a teeny tiny bit, there's this worry in the back of my mind: Oh maybe my hypothesis

  • wasn't being tested completely, because maybe I'm hitting this one as well, and maybe it's

  • this one enhancing this one. So what kind of experiment could you do to corroborate

  • this result; what kind of NOE experiment could you do to corroborate the result from this

  • experiment?" "Could you try to hit the number 1 peak?"

  • "Beautiful! Exactly! And, so we would try a corroboratory experiment as well

  • where you irradiate here. And usually NOE experiments usually end up being done in sets.

  • So you're going to do some 1-D NOE experiments on [inaudible], and this was part of the course

  • that everyone hated so I've cut it down; I've had you go ahead and hit every peak that could

  • be hit selectively in [inaudible]. It honestly doesn't take that long, imagine if this were

  • your thesis molecule it would be no big deal to spend three or four hours on the NMR spectrometer

  • for an important problem; but, we're 22 people here, you've got other things to do. So I've

  • cut it down to 1 or 2 nice, 1-D experiments where I basically preselected the key experiment

  • and we'll also do a [inaudible] experiment. But the 1-D NOE experiment is a beautiful

  • experiment because you can probe very specific questions.

  • So this is kind of a textbook example: I want to talk -- I'll give you a real example in

  • just a second and show you something I think is cool, if I can find my eraser that I've

  • seem to have misplaced here, but fortunately I have the emergency backup eraser -- anyway,

  • before I give you a real example and we look a [inaudible] spectra, I just want to show

  • you one other point of this that actually ties in sort of to thinking about problems

  • that you might encounter. So I just want to point out one sort f thing here, and that's

  • a three-spin system. So, sometimes observing an NOE doesn't necessarily mean proximity

  • and I'll show you an example. So a three-spin system wit h coupling, and again I'll give

  • you my little [inaudible] cartoon for things. So imagine that Hc is J-coupled with Hb, but

  • it's not spatially close to Ha, whereas Ha and Hb are close to each other. What can happen

  • is if I irradiate Ha, of course we'll see a NOE to Hb, in this case a positive NOE.

  • And remember, this is occurring because by leveling the populations of alpha and beta

  • states of Ha, I'm setting up new relaxation pathways that are perturbing the alpha and

  • beta states of Hb. But that perturbation then ends up altering the populations Hc, and in

  • this particular alignment, we end up with an NOE over here, a negative NOE. So, it's

  • usually going to be smaller, you usually can tell, but let me give you a real example;

  • and I think this was taken from the Derome book. So the molecule in this particular case

  • was a trichloro-toluene derivative, like so, and in this particular real experiment we

  • have an ortho proton and we have a meta proton. In this particular experiment they irradiated

  • the chloromethyl group over here and observed a 19.2% NOE over here to the ortho proton

  • and a -2.6% NOE over here to the meta proton. Now, I guess, looking at this particular molecule

  • it reminds me of your exam problem [inaudible], so on the first part of your midterm exam

  • remember the nitro-toluene problem and you were there just using a combination of understanding

  • coupling patterns and the inductive effect of a nitro group, the electron-withdrawing

  • effect, the resonance effect of the nitro group and the effect of a methoxy group, most

  • of you were able to assign your resonances and figure out among the 2,4-disubstituted

  • isomers and the 2,5-disubstituted isomers. Here, of course, the effects with chlorine

  • aren't as pronounce, just imagine in our minds eye that you had a molecule and you were trying

  • to tell whether it was the 2,4 or the 2,5 compound, and of course maybe in this particular

  • case, you wouldn't have as clear a differentiation in chemical shift, but if you look at this

  • here you can imagine, if we irradiated in this case you would see an enhancement in

  • this ortho proton here which would be a doublet with only meta coupling, a tight doublet;

  • if you irradiated over here in this molecule, you would see the ortho proton enhanced which

  • would be a doublet with ortho coupling. So in other words, even if the spectra of these

  • two molecules, the 2,4 and 2,5 isomers, were very similar in chemical shift, you would

  • be able to tell, from an NOE experiment, which isomer you had by telling whether it was a

  • doublet of 8 Hz being enhanced or a doublet of 3 Hz being enhanced. So that's an example

  • immediately that I can hand you of the utility of an NOE experiment. Now, another example

  • that I can give you, and again I'll harken back to the exam to a problem that I guess

  • about 2/3 of you did, and that was the beta-lactone problem and there we weren't trying to tell

  • stereoisomers apart, but imagine for a moment, I'll get [inaudible] the beta-lactone case

  • as an example. Imagine for a moment we have a beta-lactone and imagine instead of just

  • having a methyl group at this position, imagine that we had methyl group and an ethyl group

  • at these two positions, and now we had a methyl group over here of unknown stereochemistry.

  • You can now imagine that you irradiate this methyl group, this is going to be your methyl

  • doublet [inaudible], and now you ask is it enhancing the CH2 group of the ethyl group

  • or is it enhancing the CH3 singlet of the methyl group and you can again address the

  • question of whether your diastereomer the cis or the trans relationship between the

  • two methyl groups; in other words, whether we had this diastereomer or this diastereomer."

  • "And, it would enhance the one that is one the same side as the [inaudible]?"

  • "Well, it would enhance it more, and you're asking a very, very good question. So, the

  • question that you're asking is basically: Is the NOE a litmus test? And the answer is

  • no; this is why comparison is so important. And now I didn't happen to include these in

  • the handout for the class but I have a very similar example, and I'm going to show you

  • exactly what it means and then we are going to talk about some distances. And actually,

  • you know I've been harping on the value of molecular models, molecular models become

  • really, really, really useful when you want to ask questions about distances.

  • Alright, so let's take a look at a real example, this is just one that I pulled from my own

  • experience with the use of NOEs to determine stereochemistry. The example that I'm going

  • to [inaudible] is actually a cool reaction, it's a named reaction that probably nobody

  • in this room has heard of, it's the McCoy reaction and if the professor Van Vranken

  • asks you for a mechanism for it I bet you would all get it. So, the substrate for the

  • reaction is an alpha-halo carbonyl compound. This happened to be a silo ketone or what's

  • called an acylsilane and it's a TBDMS [inaudible] but you can also do this for an ester. And

  • when you take this compound, an alpha-halo carbonyl compound and you treat it with LDA

  • and then you treat it with and alpha-beta unsaturated carbonyl compound, in this case

  • I used [inaudible], you get a cyclopropane product, and I'll draw that for you. And the

  • great thing about reactions invented early on in the century is that every new reaction

  • that you invent can get you a name [inaudible], so this is the McCoy reaction. Alright, so

  • the product that we get is a cyclopropane of undetermined stereochemistry, and unfortunately

  • in this particular example we have a 3:1 mixture of diastereomers. Alright, let me pull down

  • the screen and give us a chance to take a look at the spectrum of these two diastereomers.

  • Alright, so we have these two diastereomers and we'll call them isomer B and isomer A.

  • And, just to get our bearings straight the region around 7ppm is the aromatic region

  • in each of these. The region over here is the methyl groups on our silicon. Here is

  • our tert-butyl on the silicone. Here is our isolated methyl group, and these are the ring

  • protons on the cyclopropane ring. And so on isomer B we see a similar thing, we see our

  • aromatic resonances, we see our ring CHs, we see our methyl, our tert-butyl, and now

  • we see our two methyls on silicon. Alright, why do we get two peaks for two methyls on

  • silicon?" [Inaudible]

  • "Diastereo-what?" [Inaudible]

  • "They are diastereotopic! Now, remember I said when you have any sort of stereocenter

  • in a molecule and now you have a methylene group with two hydrogens on it or a carbon

  • with two methyls on it, those are diastereotopic. They're topologically different from each

  • other. In one case, they show up at pretty similar chemical shifts; in other case, which

  • is interesting, they have a high degree of what we call magnetic [inaudible], in other

  • words difference in chemical shifts. Now this is an example of where the NOE just shines

  • as an experiment because we have a testable hypothesis built into the molecule. In the

  • diastereomer on the left, the methyl group is going to be relatively close to those hydrogens

  • on the ring; in the diastereomer on the right, the methyl group is relatively far away, and

  • we're fortunate enough to have both of them. So, let's start with our NOE experiment on

  • isomer B. So in this particular NOE experiment, we irradiate that methyl group and this is

  • the difference NOE so this is the spectrum of isomer B, and this is the difference NOE.

  • And we don't see a heck of a lot, you can see this is an example of a subtraction artifact

  • here, but you look and you see very, very clearly those two ring protons have nice NOEs.

  • I want to know how big an NOE: I can slap my ruler on this integral, I've already done

  • this, this distance here was 74 millimeters, and that's for 3 hydrogens. And over here