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>> All right, well I think maybe we'll begin even
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if the last few people come in.
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So I want to talk about two related techniques today.
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They are related in pulse sequence.
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They're completely different in what they do.
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One of them is TOCSY and one of them is ROESY.
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So TOCSY is a correlation experiment.
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It stands for Total Correlation Spectroscopy.
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I'll put total in quotes because one doesn't get an infinite
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number of cross peaks.
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This technique was co-developed, developed at the same time
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with another technique that's the same pulse sequence called
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HOHAHA which always sounds good at Christmastime.
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It stands for Homonuclear Hartmann-Hahn Spectroscopy
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and they're the same technique but TOCSY has taken over,
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so the idea is that you get cross peaks with all
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and again I'm going to put this in quotes
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because there are limits, other spins in the spin system
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and so what this technique is like is it's like a super COSY.
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I mean, I'll give you a really simple example here.
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If we have propanol and that was sort of the sketch
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of the molecule when I gave the first [inaudible] in 2D spectra
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if you have propanol COSY will link your methyl group
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to your central methylene and it will link the central methylene
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to the next methylene
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and assuming the OH is exchanging rapidly
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that won't be linked in there.
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What TOCSY will do since all of these groups are
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in the same spin system is TOCSY will also link the methyl group
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to the terminal methylene and where TOCSY really,
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really shines, there are sort of two different situations
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that TOCSY really shines.
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In the small molecules realm where TOCSY really shines is
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in situations of overlap and I've been pretty good
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about giving you spectra where there's not too much overlap
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and you can walk your way through COSY
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but sometimes you'll end up with peaks overlapping on other peaks
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and you simply stop being able to walk your way through
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and so you look and you say, hey what's going
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on here I can't figure out my way all the way through,
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so I'll just give you a couple of hypothetical examples
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that maybe illustrate my thinking on this of a case
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where you might have overlap.
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So like if you take a molecule of pentanol and we think
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about where things typically show
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up at it we'd say all right plain vanilla methyl is.4
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and methylene that's next to an oxygen say is
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at 3.5 parts per million and methylene that's beta
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to an oxygen that's going to be I don't know that's going
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to be somewhere around 1.9 parts per million
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but by the time you get down to this methylene chain--
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bless you-- you're going to have your methylenes pretty much
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unperturbed both at about 1.4 ppm.
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In other words, if you go into the COSY spectrum you're going
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to get into a quagmire right here
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where you have trouble tracing your way
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through the COSY spectrum and so those types of issues,
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if you've got multiple spin systems in the molecule
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and you're really trying to figure
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out what your fragments are those types
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of issues can be problematic
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and so again let me give you a molecule and just sort of talk
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about typical chemical shifts, so if we'd say,
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all right an ester of methylene next to an ester,
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I usually think 4.1 ppm,
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plane vanilla methylenes I think about, methyls I think
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about 9 parts per million.
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A methyl group that's beta to a carbonyl I think about,
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say about 1.1 parts per million.
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In other words it's beta to an electron withdrawing group
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so it's at the plane position shifted down by a couple
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of tenth of a ppm but then if you think
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about a methine that's say beta to an oxygen, normally I think
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about a methine maybe at 1.9 ppm but by being beta
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to an oxygen maybe it will be about 2.4 ppm
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and normally I'll think of say a methyl group as next to an ester
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as being about 2 parts per million
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but if it's a methylene group it will go
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down by another about.4 parts per million
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so you could easily how a molecule
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like this might have two spin systems where when you try
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to trace your way through you get caught up and so
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if everything overlapped at 2.4 it would be really hard
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in a COSY of a molecule like this
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to distinguish this spin system from this one, in other words
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to try to walk your way through and see if this methyl was part
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of a different spin system than this methyl over here
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and TOCSY's extremely good at doing this.
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The other thing that TOCSY is good at,
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there's a parameter that's very important
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in TOCSY called the spin lock mixing time and so
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when I say all other spins
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in the spin system we're typically talking
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about within a limit maybe through about 7 bonds.
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If you vary the spin lock mixing time and you can use TOCSY
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as sort of a super COSY experiment
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where if you have a very short time you basically get just one
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jump just like from here to here
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but if you go a little longer two jumps starts to appear.
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If you go a little longer
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in your spin lock mixing time more and more go.
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So you can do a series of TOCSY experiments
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that will basically walk your way from one spin to the next
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to the next and what's good is if you have a region
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where there's overlap and then a region
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where there isn't overlap, so like the overlap might be at 2.4
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in this molecule and the region
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that doesn't have overlap might be
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at say 1.1 and.9 you can go ahead
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and walk along those TOCSY tracks and see
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who the coupling partner is for each and then do longer
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and longer mixing times to pick them all out.
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So that's one really good use for TOCSY is overlap.
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The other really good use is I'll call it biopolymers
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which sounds like something intimidating but any sort
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of molecule that has spin systems that are units within,
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so there are many types of macro lactams and many types
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of antibiotic, cyclic esters that have unnatural amino acids
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that have a series of spin systems.
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I'll just show you some biopolymers for example,
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peptides and proteins and so if you think
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about it each amino acid in a peptide
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or a protein is its own spin system and so forth
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where each unit comprises a spin system and so you can pick
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out all of these spin systems
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and basically very quickly assign all of the resonances
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in a polypeptide and we'll do an example of this
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with a cyclic peptide.
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Sugars are another example.
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We just saw Professor Peng Wu's seminar and he was working
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with various types of oligosaccharides
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and so each oligosaccharide,
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each monosaccharide unit is an isolated unit
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and they're often very heavily overlapped
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and so I'm just giving sort of a generic cartoon
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of an oligosaccharide structure.
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And so each sugar unit is its own spin system
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and TOCSY they are very crowded together.
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They all end up having similar chemical shifts
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and TOCSY really shines at working with oligosaccharides.
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And the other area that works out very well
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with TOCSY is nucleic acids,
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DNA and RNA where again the basic unit is sugars and bases
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and each sugar is its own little spin system.
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And so you have a nuclear base and depending on if it's DNA
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or RNA you'll have an OH at these positions
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so I'll just put this in brackets.
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So again, all of these are representing kind of pieces
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of a biopolymer structure that might be useful for elucidating.
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[ Silence ]
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So as I was hinting at before, one of the limits
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of TOCSY is it doesn't go on forever
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and so the limits are I'm going to say, I always hate
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to put a hard number, about 7 bonds
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so for example, what do I mean?
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I mean let's say we look at the molecule lysine,
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so lysine is an amino acid with a four carbon chain.
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I'll put this as part of a biopolymer.
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So if you're talking about tracing your way
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from the epsilon carbon to the NH group you're going
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through one, two, three, four, five, six, seven bonds.
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That's about as far as you would go and so
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in other words you'd end up having this hydrogen,
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if you do it right, crossing with all
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of the methylenes along the way giving cross peaks as well
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as if you do right the NH group, unless you do the experiment
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in D2O in which case the NH group will have exchanged.
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So as I said the parameter is,
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the key parameter is a mixing time and typically this is one
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of the experiments, the experiments downstairs that are
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like a COSY experiment or an HMQC experiment
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or an HMBC experiment the parameters John Greaves gives
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you, if you take this default 10 hertz HMBC experiment that's
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going to be sort of one size fits all.
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In the case of a TOCSY experiment you actually have
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to think intelligently about the experiment.
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Typical values are about 75
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to 100 millisecond spin lock mixing time and you'd obviously,
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you'd want to go to the high end to pick
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up longer correlations you might go up to say 200 milliseconds.
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If you go shorter, particularity if you're down sort of in the 25
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to 75 range you'll be using the experiment as kind
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of a super COSY but one where you can walk your way
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from one bond to the next to the next.
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Now one of the implications for your own project is
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that with strychnine is because strychnine has some really
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extended spin systems you may not be able to trace your way
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through all of the spin systems
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but you'll be able to get part way.
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The other limitation, so obviously,
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so one limit is the number of bonds.
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The other limit is your coupling basically proceeds directly
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depending on how strongly things are coupled, in other words
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if you have a very small J that can lead to an absence
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of cross peaks so it's not so much an issue
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with a flexible chain but if you come down to strychnine
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and you're tracing your way through a spin system
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where one dihedral is close to 90 degrees.
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Your coupling constant is very small,
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a hertz or two or zero hertz.
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If you have a really small coupling constant TOCSY may not
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take you through.
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Basically, you need to have some reasonably large couplings,
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so you may see things behaving like as
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if they're isolated spin systems
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or nearly isolated spin systems so, now the nice thing
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about TOCSY as I said is it's very good at dealing
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with overlap and I'll show you in just a moment an example
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where you just would be struggling like crazy by COSY
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and we're going to assign a zillion different protons
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in one fell swoop.
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There's an alternative that's extremely powerful
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and we'll talk about it in the last week of class
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and that's the HMQC TOCSY.
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So TOCSY works as long as you can find some regions
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where there aren't overlap and you can get one resonance
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that isn't overlapping
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but if you've got really bad overlap you may even have
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trouble tracing your way through a TOCSY.
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HMQC TOCSY is a variant that's like TOCSY
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but it has the dispersion of the C 13 dimension.
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Remember C 13 resonance is because you have 200 ppm end
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up having very little overlap
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and so the dispersion can be very, very powerful.
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That's too much for us to assimilate at this point
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so let me just say I'll show you that in the future.
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All right, what I'd like to do now is to talk about,
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give us an example of one molecule.
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We're going to assign every resonance in this molecule
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and the molecule is gramicidin S. It's an antibiotic
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and it's a non-ribosomal peptide.
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What that means is that it's not synthesized
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by the traditional T RNA or DNA or messenger RNA,
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T RNA mechanism and its structure consists
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of five amino acids that are repeated trice and so I'm going
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to draw the structure of the molecule and I'll draw it kind
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of in a stylized fashion because I think that's actually useful
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for reflecting the confirmation of the molecule.
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So the molecule starts with a proline and we next continue
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with a valine and we next, it's a non-ribosomal polypeptide
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so we next have an, call it unnatural amino acid
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but it would be better to say non-proteinogenic
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or non-ribosomal amino acid
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and so the next one I'll label this as valine.