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.