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  • >> All right, well I think maybe we'll begin even

  • if the last few people come in.

  • So I want to talk about two related techniques today.

  • They are related in pulse sequence.

  • They're completely different in what they do.

  • One of them is TOCSY and one of them is ROESY.

  • So TOCSY is a correlation experiment.

  • It stands for Total Correlation Spectroscopy.

  • I'll put total in quotes because one doesn't get an infinite

  • number of cross peaks.

  • This technique was co-developed, developed at the same time

  • with another technique that's the same pulse sequence called

  • HOHAHA which always sounds good at Christmastime.

  • It stands for Homonuclear Hartmann-Hahn Spectroscopy

  • and they're the same technique but TOCSY has taken over,

  • so the idea is that you get cross peaks with all

  • and again I'm going to put this in quotes

  • because there are limits, other spins in the spin system

  • and so what this technique is like is it's like a super COSY.

  • I mean, I'll give you a really simple example here.

  • If we have propanol and that was sort of the sketch

  • of the molecule when I gave the first [inaudible] in 2D spectra

  • if you have propanol COSY will link your methyl group

  • to your central methylene and it will link the central methylene

  • to the next methylene

  • and assuming the OH is exchanging rapidly

  • that won't be linked in there.

  • What TOCSY will do since all of these groups are

  • in the same spin system is TOCSY will also link the methyl group

  • to the terminal methylene and where TOCSY really,

  • really shines, there are sort of two different situations

  • that TOCSY really shines.

  • In the small molecules realm where TOCSY really shines is

  • in situations of overlap and I've been pretty good

  • about giving you spectra where there's not too much overlap

  • and you can walk your way through COSY

  • but sometimes you'll end up with peaks overlapping on other peaks

  • and you simply stop being able to walk your way through

  • and so you look and you say, hey what's going

  • on here I can't figure out my way all the way through,

  • so I'll just give you a couple of hypothetical examples

  • that maybe illustrate my thinking on this of a case

  • where you might have overlap.

  • So like if you take a molecule of pentanol and we think

  • about where things typically show

  • up at it we'd say all right plain vanilla methyl is.4

  • and methylene that's next to an oxygen say is

  • at 3.5 parts per million and methylene that's beta

  • to an oxygen that's going to be I don't know that's going

  • to be somewhere around 1.9 parts per million

  • but by the time you get down to this methylene chain--

  • bless you-- you're going to have your methylenes pretty much

  • unperturbed both at about 1.4 ppm.

  • In other words, if you go into the COSY spectrum you're going

  • to get into a quagmire right here

  • where you have trouble tracing your way

  • through the COSY spectrum and so those types of issues,

  • if you've got multiple spin systems in the molecule

  • and you're really trying to figure

  • out what your fragments are those types

  • of issues can be problematic

  • and so again let me give you a molecule and just sort of talk

  • about typical chemical shifts, so if we'd say,

  • all right an ester of methylene next to an ester,

  • I usually think 4.1 ppm,

  • plane vanilla methylenes I think about, methyls I think

  • about 9 parts per million.

  • A methyl group that's beta to a carbonyl I think about,

  • say about 1.1 parts per million.

  • In other words it's beta to an electron withdrawing group

  • so it's at the plane position shifted down by a couple

  • of tenth of a ppm but then if you think

  • about a methine that's say beta to an oxygen, normally I think

  • about a methine maybe at 1.9 ppm but by being beta

  • to an oxygen maybe it will be about 2.4 ppm

  • and normally I'll think of say a methyl group as next to an ester

  • as being about 2 parts per million

  • but if it's a methylene group it will go

  • down by another about.4 parts per million

  • so you could easily how a molecule

  • like this might have two spin systems where when you try

  • to trace your way through you get caught up and so

  • if everything overlapped at 2.4 it would be really hard

  • in a COSY of a molecule like this

  • to distinguish this spin system from this one, in other words

  • to try to walk your way through and see if this methyl was part

  • of a different spin system than this methyl over here

  • and TOCSY's extremely good at doing this.

  • The other thing that TOCSY is good at,

  • there's a parameter that's very important

  • in TOCSY called the spin lock mixing time and so

  • when I say all other spins

  • in the spin system we're typically talking

  • about within a limit maybe through about 7 bonds.

  • If you vary the spin lock mixing time and you can use TOCSY

  • as sort of a super COSY experiment

  • where if you have a very short time you basically get just one

  • jump just like from here to here

  • but if you go a little longer two jumps starts to appear.

  • If you go a little longer

  • in your spin lock mixing time more and more go.

  • So you can do a series of TOCSY experiments

  • that will basically walk your way from one spin to the next

  • to the next and what's good is if you have a region

  • where there's overlap and then a region

  • where there isn't overlap, so like the overlap might be at 2.4

  • in this molecule and the region

  • that doesn't have overlap might be

  • at say 1.1 and.9 you can go ahead

  • and walk along those TOCSY tracks and see

  • who the coupling partner is for each and then do longer

  • and longer mixing times to pick them all out.

  • So that's one really good use for TOCSY is overlap.

  • The other really good use is I'll call it biopolymers

  • which sounds like something intimidating but any sort

  • of molecule that has spin systems that are units within,

  • so there are many types of macro lactams and many types

  • of antibiotic, cyclic esters that have unnatural amino acids

  • that have a series of spin systems.

  • I'll just show you some biopolymers for example,

  • peptides and proteins and so if you think

  • about it each amino acid in a peptide

  • or a protein is its own spin system and so forth

  • where each unit comprises a spin system and so you can pick

  • out all of these spin systems

  • and basically very quickly assign all of the resonances

  • in a polypeptide and we'll do an example of this

  • with a cyclic peptide.

  • Sugars are another example.

  • We just saw Professor Peng Wu's seminar and he was working

  • with various types of oligosaccharides

  • and so each oligosaccharide,

  • each monosaccharide unit is an isolated unit

  • and they're often very heavily overlapped

  • and so I'm just giving sort of a generic cartoon

  • of an oligosaccharide structure.

  • And so each sugar unit is its own spin system

  • and TOCSY they are very crowded together.

  • They all end up having similar chemical shifts

  • and TOCSY really shines at working with oligosaccharides.

  • And the other area that works out very well

  • with TOCSY is nucleic acids,

  • DNA and RNA where again the basic unit is sugars and bases

  • and each sugar is its own little spin system.

  • And so you have a nuclear base and depending on if it's DNA

  • or RNA you'll have an OH at these positions

  • so I'll just put this in brackets.

  • So again, all of these are representing kind of pieces

  • of a biopolymer structure that might be useful for elucidating.

  • [ Silence ]

  • So as I was hinting at before, one of the limits

  • of TOCSY is it doesn't go on forever

  • and so the limits are I'm going to say, I always hate

  • to put a hard number, about 7 bonds

  • so for example, what do I mean?

  • I mean let's say we look at the molecule lysine,

  • so lysine is an amino acid with a four carbon chain.

  • I'll put this as part of a biopolymer.

  • So if you're talking about tracing your way

  • from the epsilon carbon to the NH group you're going

  • through one, two, three, four, five, six, seven bonds.

  • That's about as far as you would go and so

  • in other words you'd end up having this hydrogen,

  • if you do it right, crossing with all

  • of the methylenes along the way giving cross peaks as well

  • as if you do right the NH group, unless you do the experiment

  • in D2O in which case the NH group will have exchanged.

  • So as I said the parameter is,

  • the key parameter is a mixing time and typically this is one

  • of the experiments, the experiments downstairs that are

  • like a COSY experiment or an HMQC experiment

  • or an HMBC experiment the parameters John Greaves gives

  • you, if you take this default 10 hertz HMBC experiment that's

  • going to be sort of one size fits all.

  • In the case of a TOCSY experiment you actually have

  • to think intelligently about the experiment.

  • Typical values are about 75

  • to 100 millisecond spin lock mixing time and you'd obviously,

  • you'd want to go to the high end to pick

  • up longer correlations you might go up to say 200 milliseconds.

  • If you go shorter, particularity if you're down sort of in the 25

  • to 75 range you'll be using the experiment as kind

  • of a super COSY but one where you can walk your way

  • from one bond to the next to the next.

  • Now one of the implications for your own project is

  • that with strychnine is because strychnine has some really

  • extended spin systems you may not be able to trace your way

  • through all of the spin systems

  • but you'll be able to get part way.

  • The other limitation, so obviously,

  • so one limit is the number of bonds.

  • The other limit is your coupling basically proceeds directly

  • depending on how strongly things are coupled, in other words

  • if you have a very small J that can lead to an absence

  • of cross peaks so it's not so much an issue

  • with a flexible chain but if you come down to strychnine

  • and you're tracing your way through a spin system

  • where one dihedral is close to 90 degrees.

  • Your coupling constant is very small,

  • a hertz or two or zero hertz.

  • If you have a really small coupling constant TOCSY may not

  • take you through.

  • Basically, you need to have some reasonably large couplings,

  • so you may see things behaving like as

  • if they're isolated spin systems

  • or nearly isolated spin systems so, now the nice thing

  • about TOCSY as I said is it's very good at dealing

  • with overlap and I'll show you in just a moment an example

  • where you just would be struggling like crazy by COSY

  • and we're going to assign a zillion different protons

  • in one fell swoop.

  • There's an alternative that's extremely powerful

  • and we'll talk about it in the last week of class

  • and that's the HMQC TOCSY.

  • So TOCSY works as long as you can find some regions

  • where there aren't overlap and you can get one resonance

  • that isn't overlapping

  • but if you've got really bad overlap you may even have

  • trouble tracing your way through a TOCSY.

  • HMQC TOCSY is a variant that's like TOCSY

  • but it has the dispersion of the C 13 dimension.

  • Remember C 13 resonance is because you have 200 ppm end

  • up having very little overlap

  • and so the dispersion can be very, very powerful.

  • That's too much for us to assimilate at this point

  • so let me just say I'll show you that in the future.

  • All right, what I'd like to do now is to talk about,

  • give us an example of one molecule.

  • We're going to assign every resonance in this molecule

  • and the molecule is gramicidin S. It's an antibiotic

  • and it's a non-ribosomal peptide.

  • What that means is that it's not synthesized

  • by the traditional T RNA or DNA or messenger RNA,

  • T RNA mechanism and its structure consists

  • of five amino acids that are repeated trice and so I'm going

  • to draw the structure of the molecule and I'll draw it kind

  • of in a stylized fashion because I think that's actually useful

  • for reflecting the confirmation of the molecule.

  • So the molecule starts with a proline and we next continue

  • with a valine and we next, it's a non-ribosomal polypeptide

  • so we next have an, call it unnatural amino acid

  • but it would be better to say non-proteinogenic

  • or non-ribosomal amino acid

  • and so the next one I'll label this as valine.

  • The next one is ornithine.

  • Ornithine is like lysine except instead

  • of having a four carbon chain it has a three carbon chain,

  • so one, two, three.

  • The next amino acid is in the molecule is leucine

  • and the final amino acid before we repeat ourselves is the

  • unnatural enantiomer of phenylalanine

  • so that's D-phenylalanine and so that's half the molecule

  • and then the molecule repeats itself.