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