WEBVTT

00:00:00.500 --> 00:00:02.810
The following content is
provided under a Creative

00:00:02.810 --> 00:00:04.380
Commons license.

00:00:04.380 --> 00:00:06.670
Your support will help
MIT OpenCourseWare

00:00:06.670 --> 00:00:11.010
continue to offer high-quality
educational resources for free.

00:00:11.010 --> 00:00:13.670
To make a donation or
view additional materials

00:00:13.670 --> 00:00:17.600
from hundreds of MIT courses,
visit MIT OpenCourseWare

00:00:17.600 --> 00:00:18.800
at ocw.mit.edu.

00:00:25.800 --> 00:00:29.130
JOANNE STUBBE: So we're talking
about cholesterol homeostasis.

00:00:29.130 --> 00:00:31.770
And I said at the
very beginning,

00:00:31.770 --> 00:00:36.030
the first two lectures are going
to be focused on the terpenome

00:00:36.030 --> 00:00:38.430
and how you make cholesterol.

00:00:38.430 --> 00:00:44.100
And in the beginning
in Monday's lecture,

00:00:44.100 --> 00:00:46.170
we had gotten through
the first few steps

00:00:46.170 --> 00:00:50.490
in cholesterol biosynthesis,
starting with acetyl CoA.

00:00:50.490 --> 00:00:53.220
And we'd gotten up
to the position where

00:00:53.220 --> 00:00:57.240
we had condensed three
molecules of acetyl CoA using

00:00:57.240 --> 00:01:01.020
Claisen and aldol reactions
to form this molecule,

00:01:01.020 --> 00:01:03.090
hydroxymethyl glutaryl CoA.

00:01:03.090 --> 00:01:05.069
And at the end of
the last lecture,

00:01:05.069 --> 00:01:09.600
we were talking about
HMG CoA reductase, which

00:01:09.600 --> 00:01:12.150
is abbreviated in the
Brown and Goldstein papers

00:01:12.150 --> 00:01:16.170
like that, which
requires two molecules.

00:01:16.170 --> 00:01:17.910
This should be "NADPH."

00:01:17.910 --> 00:01:20.970
I announced that before
all of the notes.

00:01:20.970 --> 00:01:23.910
"NADH" should be
replaced with "NADPH."

00:01:23.910 --> 00:01:26.340
We're doing biosynthesis.

00:01:26.340 --> 00:01:33.630
We talked about the mechanism of
how you go from this CoA analog

00:01:33.630 --> 00:01:38.070
to a double reduction
to form an alcohol.

00:01:38.070 --> 00:01:41.993
And the question is, why is
that interesting and important?

00:01:41.993 --> 00:01:43.410
And it's interesting
and important

00:01:43.410 --> 00:01:49.930
because it's the major target of
$30 billion drugs, the statins,

00:01:49.930 --> 00:01:54.300
which specifically
target HMG CoA reductase.

00:01:54.300 --> 00:01:56.205
And so what I want to do--

00:01:56.205 --> 00:01:58.080
I'm not going to spend
a lot of time on this,

00:01:58.080 --> 00:01:59.830
but I want to say a
little bit about this,

00:01:59.830 --> 00:02:06.690
given its central role in
many people's health nowadays.

00:02:06.690 --> 00:02:12.360
So how do these
analogs end up working?

00:02:12.360 --> 00:02:16.680
And so we have an intermediate
in this process, which

00:02:16.680 --> 00:02:20.890
I drew out in detail last time.

00:02:20.890 --> 00:02:22.860
I'm not going to
draw it out again.

00:02:22.860 --> 00:02:25.050
So we're going to go
through two reductions.

00:02:25.050 --> 00:02:29.880
The first reduction
forms a thiohemiacetal,

00:02:29.880 --> 00:02:32.250
which then kicks out CoA
to form the aldehyde, which

00:02:32.250 --> 00:02:33.360
then gets reduced again.

00:02:33.360 --> 00:02:35.050
I'm not going to write that out.

00:02:35.050 --> 00:02:37.620
This is the first
intermediate you see.

00:02:37.620 --> 00:02:42.140
And what we'll see
is the inhibitors

00:02:42.140 --> 00:02:45.590
all look like this intermediate.

00:02:45.590 --> 00:02:47.750
So when they look like
the normal substrate

00:02:47.750 --> 00:02:50.900
or an intermediate
along the pathway,

00:02:50.900 --> 00:02:53.420
those are called
"competitive inhibitors."

00:02:53.420 --> 00:02:57.080
So these are
competitive inhibitors.

00:02:57.080 --> 00:02:59.510
If you don't remember what
a competitive inhibitor is

00:02:59.510 --> 00:03:01.120
or how it's described,
you might want

00:03:01.120 --> 00:03:03.940
to go back and
look it up in Voet

00:03:03.940 --> 00:03:08.000
or whatever your basic
biochemistry textbook is.

00:03:08.000 --> 00:03:14.090
And so what we'll see is all of
the compounds that are actually

00:03:14.090 --> 00:03:17.570
used clinically look like this.

00:03:20.540 --> 00:03:25.670
And so if you look at this,
they're not exactly the same.

00:03:25.670 --> 00:03:30.870
But it's proposed to be a model
of this particular intermediate

00:03:30.870 --> 00:03:31.710
in the reaction.

00:03:31.710 --> 00:03:35.000
So this is the thiohemiacetal.

00:03:35.000 --> 00:03:39.620
But here, I've drawn a
lactone rather than an acid.

00:03:39.620 --> 00:03:42.350
And in general-- whoops.

00:03:42.350 --> 00:03:45.560
In general, lactones
are actually

00:03:45.560 --> 00:03:48.830
given as the drug,
rather than the acid.

00:03:48.830 --> 00:03:52.550
And does anybody have any clue
as to why that might be true?

00:03:55.480 --> 00:03:59.830
Why would you want to use
this molecule, as opposed

00:03:59.830 --> 00:04:07.145
to the ring-opened species,
which would look like this?

00:04:07.145 --> 00:04:08.075
AUDIENCE: Uptake.

00:04:08.075 --> 00:04:10.387
JOANNE STUBBE: Pardon me?

00:04:10.387 --> 00:04:11.720
AUDIENCE: I said, uptake issues.

00:04:11.720 --> 00:04:11.960
JOANNE STUBBE: Yeah.

00:04:11.960 --> 00:04:13.190
So it's uptake issues.

00:04:13.190 --> 00:04:15.020
So what happens
is you give this--

00:04:15.020 --> 00:04:19.640
and you'll see this is true
also in the last section

00:04:19.640 --> 00:04:20.899
in purines and pyrimidines.

00:04:20.899 --> 00:04:23.030
How you deliver
the drug, it needs

00:04:23.030 --> 00:04:24.650
to be able to get
across the membrane

00:04:24.650 --> 00:04:26.720
in an efficient fashion.

00:04:26.720 --> 00:04:30.380
And so they give them lactone,
but the lactone rapidly

00:04:30.380 --> 00:04:34.120
ring-opens inside the cell.

00:04:34.120 --> 00:04:35.323
So that's the analog.

00:04:35.323 --> 00:04:36.990
I don't know if you
can see it, but it's

00:04:36.990 --> 00:04:38.550
the same analog
I've drawn up there

00:04:38.550 --> 00:04:41.280
and I drew last
time on the board.

00:04:41.280 --> 00:04:43.080
And then these are the drugs.

00:04:43.080 --> 00:04:44.910
And so the drug--

00:04:44.910 --> 00:04:47.920
I've written this part
looks like this part.

00:04:47.920 --> 00:04:51.000
So this is where the
competitive part comes from.

00:04:51.000 --> 00:04:52.410
And what's down here?

00:04:52.410 --> 00:04:56.040
Down here is, remember, the CoA.

00:04:56.040 --> 00:04:59.100
And down here in
the drugs is stuff.

00:04:59.100 --> 00:05:03.540
And the key to this
stuff is hydrophobicity.

00:05:03.540 --> 00:05:09.330
And so the key to almost
all drugs is hydrophobicity.

00:05:09.330 --> 00:05:12.450
So it can't be so hydrophobic
that it's insoluble.

00:05:12.450 --> 00:05:14.982
But lots of times,
you have pockets

00:05:14.982 --> 00:05:16.690
that you can't see in
protein structures.

00:05:16.690 --> 00:05:20.190
It inserts itself in, and you
get a lot of binding energy.

00:05:20.190 --> 00:05:22.290
So if you look at
these, these, again,

00:05:22.290 --> 00:05:24.750
are on the PowerPoint
presentation.

00:05:24.750 --> 00:05:27.390
What you see here, these
are all the ring-opened here

00:05:27.390 --> 00:05:29.100
and ring-closed.

00:05:29.100 --> 00:05:32.990
And then if you look down here,
what you see is all the stuff.

00:05:32.990 --> 00:05:36.630
And you see the stuff is
dramatically different.

00:05:36.630 --> 00:05:38.970
You can look at it,
but each company

00:05:38.970 --> 00:05:45.040
has tried to get its cut
of the $30 billion market.

00:05:45.040 --> 00:05:47.040
And what I'm going to
show you on the next slide

00:05:47.040 --> 00:05:48.810
is this molecule.

00:05:48.810 --> 00:05:50.400
So again, the key
thing is what's

00:05:50.400 --> 00:05:57.480
common to all of these
inhibitors is this moiety.

00:05:57.480 --> 00:05:59.640
And when you look
at the structures,

00:05:59.640 --> 00:06:02.100
you can see this guy.

00:06:02.100 --> 00:06:05.580
And then this guy will have
slightly different orientations

00:06:05.580 --> 00:06:07.650
within the structures.

00:06:07.650 --> 00:06:12.270
So if you look at the
structures of HMG CoA reductase,

00:06:12.270 --> 00:06:15.115
what you can see-- and if you
pull this up and stare at it,

00:06:15.115 --> 00:06:15.990
it looks like a mess.

00:06:15.990 --> 00:06:18.870
But it really isn't a mess.

00:06:18.870 --> 00:06:20.730
If you look at
this, you're going

00:06:20.730 --> 00:06:23.130
to see in all the structures,
here's the carboxylate.

00:06:23.130 --> 00:06:24.900
Here's a carboxylate.

00:06:24.900 --> 00:06:26.910
And this is the hydroxyl group.

00:06:26.910 --> 00:06:30.660
So that's the key part
over here, the carboxylate

00:06:30.660 --> 00:06:33.660
in the hydroxyl group.

00:06:33.660 --> 00:06:37.210
There in the hemiacetal, the
carboxylate's not there yet.

00:06:37.210 --> 00:06:40.380
So you can't put in the active
species, or they turn over.

00:06:40.380 --> 00:06:43.470
So what you're doing is
throwing in components

00:06:43.470 --> 00:06:45.300
to prevent turnover
but to give you

00:06:45.300 --> 00:06:48.650
a feeling for where the
different substrates bind.

00:06:48.650 --> 00:06:51.690
CoA is not attached
to this moiety,

00:06:51.690 --> 00:06:55.170
but CoA is attached here.

00:06:55.170 --> 00:06:57.060
And I think what's
unusual about this--

00:06:57.060 --> 00:06:59.430
I don't know if you
looked at any structures

00:06:59.430 --> 00:07:01.180
in the polyketide synthases.

00:07:01.180 --> 00:07:04.410
But you would think
at the end of CoA,

00:07:04.410 --> 00:07:07.470
if you look at the structure,
it has an adenine moiety on it.

00:07:07.470 --> 00:07:10.620
And you would think you would
get a lot of binding energy

00:07:10.620 --> 00:07:13.080
onto this hydrophobic
adenine moiety, which

00:07:13.080 --> 00:07:14.630
can also hydrogen-bond.

00:07:14.630 --> 00:07:16.950
And almost all structures
to that part of the molecule

00:07:16.950 --> 00:07:18.570
never bind to the protein.

00:07:18.570 --> 00:07:20.320
It's stuck out in solution.

00:07:20.320 --> 00:07:23.190
So this is sort of typical.

00:07:23.190 --> 00:07:25.740
Why nature designed things
this way, I don't know.

00:07:25.740 --> 00:07:26.970
She's got this huge--

00:07:26.970 --> 00:07:29.585
she could have used
a thiomethyl group,

00:07:29.585 --> 00:07:31.710
and the chemistry would
have been exactly the same.

00:07:31.710 --> 00:07:35.140
But she uses this
huge CoA moiety.

00:07:35.140 --> 00:07:37.560
And in most cases, you
see the chain extended

00:07:37.560 --> 00:07:40.530
and the adenine on the outside.

00:07:40.530 --> 00:07:42.360
So if you look at
that, the chemistry

00:07:42.360 --> 00:07:44.080
is going to come over here.

00:07:44.080 --> 00:07:46.980
And if you look at this
yellow, yellow is sulfur.

00:07:46.980 --> 00:07:50.010
So that's where the
sulfur would be connected

00:07:50.010 --> 00:07:52.440
to the hydroxymethyl glutarate.

00:07:52.440 --> 00:07:54.090
And then what do
you see over here?

00:07:54.090 --> 00:07:55.380
Hopefully, you can see this.

00:07:55.380 --> 00:07:57.600
But this is another
adenine ring.

00:07:57.600 --> 00:08:00.975
And then over here
is the pyridine ring.

00:08:00.975 --> 00:08:02.350
And we went through
the mechanism

00:08:02.350 --> 00:08:04.590
last time that
transfers the hydride.

00:08:04.590 --> 00:08:08.360
So this green part
is the redundant,

00:08:08.360 --> 00:08:15.090
and this part is a mimic of
the hydroxymethyl glutaryl CoA.

00:08:15.090 --> 00:08:16.680
And in all cases--

00:08:16.680 --> 00:08:19.260
we have hundreds
of structures now--

00:08:19.260 --> 00:08:22.890
what you will see if you
look at the inhibitor binding

00:08:22.890 --> 00:08:26.280
is you have changes in
conformation in this region.

00:08:26.280 --> 00:08:30.240
And the Km for binding of
hydroxymethyl glutaryl CoA

00:08:30.240 --> 00:08:31.770
is 4 micromolar.

00:08:31.770 --> 00:08:33.809
But the KD for binding
of the inhibitors

00:08:33.809 --> 00:08:36.480
is about a nanomolar.

00:08:36.480 --> 00:08:39.830
So you're gaining a lot by
sticking this hydrophobic mess

00:08:39.830 --> 00:08:40.919
on.

00:08:40.919 --> 00:08:45.785
And what happens-- so here's,
again, this hydrophobic mess.

00:08:45.785 --> 00:08:47.160
And what you can
see, this is one

00:08:47.160 --> 00:08:49.980
of the analogs I had before.

00:08:49.980 --> 00:08:54.360
Again, hydroxymethyl glutarate--
the glutarate and the two

00:08:54.360 --> 00:08:56.520
carboxylates are there.

00:08:56.520 --> 00:08:59.940
And then they stick on
something hydrophobic

00:08:59.940 --> 00:09:01.750
in this part of the molecule.

00:09:01.750 --> 00:09:04.440
And if you look at
the conformation

00:09:04.440 --> 00:09:06.480
of these helices
in this region--

00:09:06.480 --> 00:09:09.270
and you really have to look at
the three-dimensional structure

00:09:09.270 --> 00:09:11.190
to see something--
that's the region where

00:09:11.190 --> 00:09:12.440
you see changes in binding.

00:09:12.440 --> 00:09:16.650
So it's an induced fit
mechanism of binding.

00:09:16.650 --> 00:09:21.000
And in fact, this
induced fit occurs

00:09:21.000 --> 00:09:23.790
in all of the analogs
that are looked at.

00:09:23.790 --> 00:09:26.408
And so if you look here--

00:09:26.408 --> 00:09:27.450
and you can look at this.

00:09:27.450 --> 00:09:31.380
I would say take it home and
spend some time looking at it.

00:09:31.380 --> 00:09:36.000
Again, this part of the
molecule in all of these analogs

00:09:36.000 --> 00:09:37.380
is exactly the same.

00:09:37.380 --> 00:09:41.880
And what you had different
is this hydrophobic mess

00:09:41.880 --> 00:09:44.970
in this part of the
molecule and changes

00:09:44.970 --> 00:09:47.130
in this region of binding.

00:09:47.130 --> 00:09:50.010
And people are still working
on it, trying to make--

00:09:50.010 --> 00:09:52.020
usually, it's the
first couple of drugs

00:09:52.020 --> 00:09:53.130
that make all the money.

00:09:53.130 --> 00:09:55.830
And if you're third or fourth,
you don't make enough money.

00:09:55.830 --> 00:10:00.300
But there are lots of
problems that keep coming up.

00:10:00.300 --> 00:10:02.340
And so people are still
really heavily focused

00:10:02.340 --> 00:10:06.150
on trying to lower
cholesterol levels.

00:10:06.150 --> 00:10:10.010
So HMG CoA reductase--

00:10:10.010 --> 00:10:14.760
I told you last time, it's a
huge protein, 880 amino acids.

00:10:14.760 --> 00:10:17.730
Half of it's stuck in the ER.

00:10:17.730 --> 00:10:21.300
You can cut off the-- the
other half that is soluble

00:10:21.300 --> 00:10:23.580
is in the cytosol.

00:10:23.580 --> 00:10:25.110
And we're going to
come back to this

00:10:25.110 --> 00:10:29.800
because this rate-limiting step
plays a key role in sensing

00:10:29.800 --> 00:10:31.160
of cholesterol levels.

00:10:31.160 --> 00:10:35.280
So the end of lecture three
and into lecture four,

00:10:35.280 --> 00:10:39.750
we're going to come back to
HMG CoA reductase because

00:10:39.750 --> 00:10:43.410
of its central role in
cholesterol homeostasis.

00:10:43.410 --> 00:10:44.940
So we're not here yet.

00:10:44.940 --> 00:10:50.910
Remember, the goal
of the terpenome

00:10:50.910 --> 00:10:52.620
was to get to the
building blocks.

00:10:52.620 --> 00:10:54.780
We still haven't gotten to
the building blocks yet.

00:10:54.780 --> 00:10:56.650
What were the building blocks?

00:10:56.650 --> 00:11:00.300
Isopentenyl and
dimethylallyl pyrophosphate.

00:11:00.300 --> 00:11:04.560
Remember, the common building
block is an isoprene,

00:11:04.560 --> 00:11:07.200
but the isoprene is not
the reactive species

00:11:07.200 --> 00:11:09.980
we needed to get
it into some form

00:11:09.980 --> 00:11:11.730
where you can actually
do chemistry on it.

00:11:11.730 --> 00:11:18.210
So our goal has been to get
to IPP, and we are here.

00:11:18.210 --> 00:11:25.800
So the rest of the biosynthetic
pathway to get to IPP

00:11:25.800 --> 00:11:28.470
is pretty straightforward.

00:11:28.470 --> 00:11:31.620
I'm not going to draw
out the details at all.

00:11:31.620 --> 00:11:35.930
But we go from mevalonic acid.

00:11:35.930 --> 00:11:40.710
And so we use ATP and a kinase.

00:11:40.710 --> 00:11:47.760
And we use a second
ATP and a kinase.

00:11:47.760 --> 00:11:53.220
And then we use a third ATP.

00:11:53.220 --> 00:11:56.820
And so if you look at the
pathway here, what happens

00:11:56.820 --> 00:12:02.010
is you're phosphorylating
the alcohol that we just

00:12:02.010 --> 00:12:05.160
created with HMG CoA reductase.

00:12:05.160 --> 00:12:07.680
So we're sticking
a phosphate on.

00:12:07.680 --> 00:12:11.340
Another ATP sticks a
second phosphate on.

00:12:11.340 --> 00:12:16.660
And in the end, we need to get
to isopentenyl pyrophosphate.

00:12:16.660 --> 00:12:19.860
And so we have a
third enzyme that

00:12:19.860 --> 00:12:25.380
is going to phosphorylate to
facilitate, finally, conversion

00:12:25.380 --> 00:12:32.450
of the C6, three acetyl
CoAs, into the C5 isopentenyl

00:12:32.450 --> 00:12:34.410
pyrophosphate.

00:12:34.410 --> 00:12:37.710
So if you look at what's
going on in that reaction,

00:12:37.710 --> 00:12:39.330
we have a third ATP.

00:12:39.330 --> 00:12:53.250
And the ATP is used to
phosphorylate the alcohol.

00:12:53.250 --> 00:12:56.010
So what we're doing
basically is making it

00:12:56.010 --> 00:12:57.420
into a good leaving group.

00:13:00.060 --> 00:13:02.070
And we've got the two
phosphates on there

00:13:02.070 --> 00:13:04.740
by the first two kinases.

00:13:04.740 --> 00:13:06.220
And so now what we want to do--

00:13:06.220 --> 00:13:07.530
I forgot a methyl group here.

00:13:10.540 --> 00:13:15.400
And so now what we've done by
phosphorylating this is we've

00:13:15.400 --> 00:13:18.005
activated this for a
decarboxylative elimination

00:13:18.005 --> 00:13:18.505
reaction.

00:13:23.110 --> 00:13:24.910
And so now, where are we?

00:13:24.910 --> 00:13:32.710
We're now finally at
isopentenyl pyrophosphate.

00:13:32.710 --> 00:13:35.350
So we've gotten to our C5.

00:13:35.350 --> 00:13:39.760
And the key thing, remember,
is we started with acetyl CoA.

00:13:39.760 --> 00:13:44.440
During this reaction,
we lose CO2.

00:13:44.440 --> 00:13:48.670
And that's why we've
gone from a C6 to a C5

00:13:48.670 --> 00:13:49.630
during this reaction.

00:13:49.630 --> 00:13:51.010
We lose that.

00:13:51.010 --> 00:13:53.360
And again, we see ATP
used over and over again.

00:13:53.360 --> 00:13:55.510
And GTP, you see the same thing.

00:13:55.510 --> 00:13:59.110
It's used to make things
into better leaving groups

00:13:59.110 --> 00:14:02.140
and facilitate the
overall chemistry.

00:14:02.140 --> 00:14:05.200
So I'm not going to talk
again about the details of any

00:14:05.200 --> 00:14:07.123
of these steps.

00:14:07.123 --> 00:14:08.540
The steps are all
straightforward.

00:14:08.540 --> 00:14:10.630
You've seen these steps
in primary metabolism

00:14:10.630 --> 00:14:13.480
with the role of ATP
over and over again.

00:14:13.480 --> 00:14:18.040
But the key thing we want to
talk about is the terpenome.

00:14:18.040 --> 00:14:20.530
And to get to the
terpenome, we needed

00:14:20.530 --> 00:14:25.270
to get to isopentenyl
pyrophosphate and dimethylallyl

00:14:25.270 --> 00:14:29.680
pyrophosphate so
that we can look

00:14:29.680 --> 00:14:35.010
at the new way of forming
carbon-carbon bonds with C5

00:14:35.010 --> 00:14:37.030
units.

00:14:37.030 --> 00:14:40.840
So we've gotten through
the first few steps.

00:14:40.840 --> 00:14:43.090
That's what I call the
"initiation process."

00:14:43.090 --> 00:14:48.460
We started with acetyl
CoA and got to IPP,

00:14:48.460 --> 00:14:52.213
dimethylallyl pyrophosphate.

00:14:52.213 --> 00:14:54.130
If you look at these
hydrogens, hopefully, you

00:14:54.130 --> 00:14:56.860
know allylic hydrogens
are moderately acidic.

00:14:56.860 --> 00:15:01.870
And there's an isomerase that
can convert this molecule

00:15:01.870 --> 00:15:03.650
into this molecule.

00:15:03.650 --> 00:15:06.790
And so this is
dimethylallyl pyrophosphate.

00:15:06.790 --> 00:15:12.010
And we're into the second part
of the biosynthetic pathway

00:15:12.010 --> 00:15:12.950
for cholesterol.

00:15:12.950 --> 00:15:14.320
So we're through the initiation.

00:15:14.320 --> 00:15:16.210
We've got our building blocks.

00:15:16.210 --> 00:15:20.830
Now, what we want to do
is do the elongation step.

00:15:20.830 --> 00:15:22.870
And so now we're
going to use this.

00:15:22.870 --> 00:15:30.670
So IPP-- we're now going to look
at the elongation reactions.

00:15:30.670 --> 00:15:33.730
And I guess I'll use a
second board over here.

00:15:33.730 --> 00:15:36.370
And so let me do it over here,
and then I'll do the next one.

00:15:36.370 --> 00:15:45.783
And so what we want to do
then is take C5 plus C5.

00:15:49.850 --> 00:15:53.200
And we're going to look at
this reaction in detail.

00:15:53.200 --> 00:15:58.450
And the enzyme that's
going to do this

00:15:58.450 --> 00:16:04.180
is called FPP synthase,
farnesyl pyrophosphate synthase.

00:16:04.180 --> 00:16:06.190
I'll write that
down in a minute.

00:16:06.190 --> 00:16:08.440
And you form C10.

00:16:11.170 --> 00:16:15.770
And C10-- then this
is the same enzyme.

00:16:15.770 --> 00:16:18.790
So it's also FPP synthase.

00:16:18.790 --> 00:16:22.140
FPP is the product,
farnesyl pyrophosphate.

00:16:22.140 --> 00:16:27.970
And IPP gives us C15.

00:16:27.970 --> 00:16:31.000
So this is a major
elongation reaction.

00:16:31.000 --> 00:16:36.310
And what I want you to see is
that this C15, three C5s stuck

00:16:36.310 --> 00:16:38.500
together, is linear.

00:16:38.500 --> 00:16:43.900
And if you go back and you look
at your notes from last time,

00:16:43.900 --> 00:16:46.600
we talked about
isopernoids and terpenoids.

00:16:46.600 --> 00:16:48.850
And you can make
linear molecules

00:16:48.850 --> 00:16:51.940
that can go from
a couple of units,

00:16:51.940 --> 00:16:54.670
like geranyl, geranyl, C10--

00:16:54.670 --> 00:16:56.470
sorry-- geranyl pyrophosphate.

00:16:56.470 --> 00:16:57.700
That's C10.

00:16:57.700 --> 00:17:01.210
That's called a "monoterpene."

00:17:01.210 --> 00:17:06.490
And you can add another C5,
isopentenyl pyrophosphate.

00:17:06.490 --> 00:17:07.930
That's a C15.

00:17:07.930 --> 00:17:10.579
That's called a "sesquiterpene."

00:17:10.579 --> 00:17:12.430
"Sesqui" comes from 1 and 1/2.

00:17:12.430 --> 00:17:15.599
So what you'll see in the next
thing, if you add another five,

00:17:15.599 --> 00:17:16.839
you have a C20.

00:17:16.839 --> 00:17:18.400
That's a diterpene.

00:17:18.400 --> 00:17:21.880
And if you go to a C30,
that's a triterpene.

00:17:21.880 --> 00:17:24.710
You can google it, but the
nomenclature's complicated.

00:17:24.710 --> 00:17:28.930
But that's where they come
from is the different C5 units.

00:17:28.930 --> 00:17:35.740
So really, this chemistry is
the basis for all the reactions

00:17:35.740 --> 00:17:37.288
in the terpenome.

00:17:37.288 --> 00:17:39.580
So what we're going to do is
go through that chemistry.

00:17:39.580 --> 00:17:42.250
How do you form a new
carbon-carbon bond

00:17:42.250 --> 00:17:43.640
using these building blocks?

00:17:43.640 --> 00:17:45.550
What are the general principles?

00:17:45.550 --> 00:17:47.650
And I showed you the
hundreds of different kinds

00:17:47.650 --> 00:17:49.630
of natural products
that you can find

00:17:49.630 --> 00:17:52.350
in humans and plants and
bacteria all over the place.

00:17:52.350 --> 00:17:53.980
They play an incredibly
important role

00:17:53.980 --> 00:17:56.920
in primary and
secondary metabolism.

00:17:56.920 --> 00:17:59.680
And what we're going to
look at is the general way

00:17:59.680 --> 00:18:02.440
that these carbon-carbon
bonds are made.

00:18:06.030 --> 00:18:06.760
All right.

00:18:06.760 --> 00:18:11.770
So again, let me stress
that this is linear.

00:18:11.770 --> 00:18:14.800
And you'll see that when
we actually look at this.

00:18:14.800 --> 00:18:20.500
So FPP-- let me write this down.

00:18:20.500 --> 00:18:25.390
So it's farnesyl
PP, pyrophosphate.

00:18:25.390 --> 00:18:27.640
We talked about this last time.

00:18:27.640 --> 00:18:30.690
It's a central player in
many, many, many reactions,

00:18:30.690 --> 00:18:32.770
and it's a C15.

00:18:32.770 --> 00:18:37.750
So the farnesyl
pyrophosphate synthase

00:18:37.750 --> 00:18:41.650
was the first
enzyme characterized

00:18:41.650 --> 00:18:45.400
for parental transfer reactions,
for these C5-forming reactions.

00:18:45.400 --> 00:18:50.320
It's been studied extremely
extensively by Dale Poulter's

00:18:50.320 --> 00:18:53.050
lab at Utah, and it's
served as a paradigm,

00:18:53.050 --> 00:18:55.880
really, for thinking about
all of the biochemistry.

00:18:55.880 --> 00:18:59.336
Did any of you guys ever
hear of Saul Winstein?

00:18:59.336 --> 00:18:59.836
No.

00:18:59.836 --> 00:19:01.380
It shows how old I am.

00:19:01.380 --> 00:19:05.100
Anyhow, Saul Winstein
was a faculty member

00:19:05.100 --> 00:19:09.360
at UCLA many years ago,
probably in the 1970s.

00:19:09.360 --> 00:19:12.810
But if you've taken 5.43,
hopefully, they still

00:19:12.810 --> 00:19:13.920
talk about--

00:19:13.920 --> 00:19:17.310
or what's the advanced
physical organic chemistry

00:19:17.310 --> 00:19:18.360
course you guys take?

00:19:18.360 --> 00:19:19.980
Any of you'd had that?

00:19:19.980 --> 00:19:22.370
Anyhow, you've had-- have
you heard about Saul?

00:19:22.370 --> 00:19:24.780
You've never heard
of Saul-- bad, bad.

00:19:24.780 --> 00:19:27.590
Anyhow, he's the
one that figured out

00:19:27.590 --> 00:19:31.860
how to think about classical
and non-classical carbocations.

00:19:31.860 --> 00:19:33.370
And Dale Poulter
worked from him.

00:19:33.370 --> 00:19:36.420
Dale Poulter moved into
enzymatic reaction systems

00:19:36.420 --> 00:19:39.270
and really sort of unravelled
how these things work.

00:19:39.270 --> 00:19:41.880
And the paradigm I'm going
to give you-- every enzyme's

00:19:41.880 --> 00:19:42.493
different.

00:19:42.493 --> 00:19:44.160
But the paradigm I'm
going to give you I

00:19:44.160 --> 00:19:46.740
really think came
partially founded

00:19:46.740 --> 00:19:50.520
on the physical organic
chemistry and from Dale's lab.

00:19:50.520 --> 00:19:53.170
So these are pretty
important contributions.

00:19:53.170 --> 00:20:00.510
And what we'll see is this is
called a "type I synthase."

00:20:00.510 --> 00:20:03.400
And if you read the
assigned reading,

00:20:03.400 --> 00:20:05.610
you'll see there are
type II synthases.

00:20:05.610 --> 00:20:09.900
So there's more
than one structure

00:20:09.900 --> 00:20:12.060
of the enzymes involved
in these systems.

00:20:12.060 --> 00:20:16.050
We're going to
specifically focus in class

00:20:16.050 --> 00:20:17.820
on the type I synthase.

00:20:17.820 --> 00:20:20.685
And it's basically an
alpha helical bundle.

00:20:26.490 --> 00:20:30.110
And I think on the next-- if
I could remember what I have.

00:20:30.110 --> 00:20:30.630
Yeah.

00:20:30.630 --> 00:20:32.490
So this was taken
out of the article

00:20:32.490 --> 00:20:35.100
you are supposed to read.

00:20:35.100 --> 00:20:37.260
And so this is FPP synthase.

00:20:37.260 --> 00:20:39.630
This is a monomer,
but it's a dimer.

00:20:39.630 --> 00:20:46.350
And all I want you to see if
you take the 30,000-foot view,

00:20:46.350 --> 00:20:48.150
there are five helices here.

00:20:48.150 --> 00:20:50.130
They're in red.

00:20:50.130 --> 00:20:53.740
If you look at this long
helix, it's everywhere.

00:20:53.740 --> 00:20:56.820
Everything's a little
bit juggled around.

00:20:56.820 --> 00:20:58.695
You can see you have
a couple blues here

00:20:58.695 --> 00:20:59.820
and a couple of blues here.

00:20:59.820 --> 00:21:02.920
So they're structurally
homologous to each other.

00:21:02.920 --> 00:21:05.490
And I think what's most
remarkable about this--

00:21:05.490 --> 00:21:11.760
so FPP synthase takes two C5s,
makes a C10-- this is a C15.

00:21:11.760 --> 00:21:14.010
So it's a linear.

00:21:14.010 --> 00:21:16.830
Squalene synthase, which
we'll look at in a minute,

00:21:16.830 --> 00:21:20.670
takes two C15s and makes
a C30, the precursor

00:21:20.670 --> 00:21:23.310
to making the ring
structure for cholesterol.

00:21:23.310 --> 00:21:26.640
But these two guys in
the middle, which look--

00:21:26.640 --> 00:21:28.440
and this is linear, as well.

00:21:28.440 --> 00:21:31.290
These two guys in the middle,
which look remarkably similar--

00:21:31.290 --> 00:21:36.605
actually, structurally, if you
superimpose the structures,

00:21:36.605 --> 00:21:37.605
they're really similar--

00:21:40.200 --> 00:21:41.940
form cyclic terpenes.

00:21:41.940 --> 00:21:43.830
So they form cyclic
sesquiterpenes.

00:21:43.830 --> 00:21:45.750
I'll show you this in a minute.

00:21:45.750 --> 00:21:47.940
And they use FPP.

00:21:47.940 --> 00:21:51.780
So here, all of these
enzymes use FPP.

00:21:51.780 --> 00:21:57.220
And they all look alike sort
of from the 30,000-foot point

00:21:57.220 --> 00:21:57.720
of view.

00:21:57.720 --> 00:21:59.220
And the question
is then, how do you

00:21:59.220 --> 00:22:03.870
control what the chemistry
is in the active site?

00:22:03.870 --> 00:22:06.340
So they have
homologous structure.

00:22:06.340 --> 00:22:08.850
So these are all
structurally homologous.

00:22:08.850 --> 00:22:13.140
Another thing that you need to
remember about these systems

00:22:13.140 --> 00:22:20.970
is that they have similar
metal binding motifs.

00:22:25.710 --> 00:22:30.270
Now, if you look at
the reaction of IPP,

00:22:30.270 --> 00:22:34.060
if you think about this, this
isn't what PP looks like.

00:22:34.060 --> 00:22:36.100
Does everybody know
what PP looks like?

00:22:36.100 --> 00:22:38.100
Hopefully, you've seen
this over and over again.

00:22:38.100 --> 00:22:41.180
What would the
metal be or metals?

00:22:41.180 --> 00:22:42.160
AUDIENCE: Magnesium.

00:22:42.160 --> 00:22:42.993
JOANNE STUBBE: Yeah.

00:22:42.993 --> 00:22:46.470
So whenever you have
pyrophosphates or ATPs or GTPs,

00:22:46.470 --> 00:22:47.730
you always have magnesium.

00:22:47.730 --> 00:22:50.940
Magnesium plays a central
role in everything in biology.

00:22:50.940 --> 00:22:53.280
And we can never
look at magnesium

00:22:53.280 --> 00:22:55.470
because the ligands
are fast-changing.

00:22:55.470 --> 00:22:57.250
It moves around
all over the place.

00:22:57.250 --> 00:23:00.060
So it's hard to freeze
out and understand

00:23:00.060 --> 00:23:01.380
the function of magnesium.

00:23:01.380 --> 00:23:06.000
But it turns out most
of these proteins

00:23:06.000 --> 00:23:08.550
require three magnesiums.

00:23:08.550 --> 00:23:11.490
And as with many
metal-based reactions,

00:23:11.490 --> 00:23:14.220
if you line things
up, you really

00:23:14.220 --> 00:23:16.080
don't find that much
sequence homology.

00:23:16.080 --> 00:23:18.510
But if you know where to look,
you find sequence homology

00:23:18.510 --> 00:23:20.160
around where the metals bind.

00:23:20.160 --> 00:23:25.260
So what you see in the
case of the linear farnesyl

00:23:25.260 --> 00:23:30.750
pyrophosphate, you
see a DDXXD motif.

00:23:34.440 --> 00:23:37.950
And you find that in almost
all of these enzymes.

00:23:37.950 --> 00:23:39.560
And if you go to
the terpenoids--

00:23:39.560 --> 00:23:41.730
so the non-linear ones--

00:23:41.730 --> 00:23:43.950
you see a D.

00:23:43.950 --> 00:23:46.470
Again, I don't expect you to
remember something like this.

00:23:46.470 --> 00:23:50.400
But I do expect you to
remember that these metal

00:23:50.400 --> 00:23:54.480
motifs, once you know how
to think about something,

00:23:54.480 --> 00:23:56.850
are actually very
helpful in trying

00:23:56.850 --> 00:24:00.170
to define the function of an
unknown open reading frame,

00:24:00.170 --> 00:24:01.750
if you know how to look.

00:24:01.750 --> 00:24:07.580
And more than 50% of
all annotated genes

00:24:07.580 --> 00:24:10.340
code for proteins we have
no idea what they do.

00:24:10.340 --> 00:24:13.190
So looking at these
kinds of motifs

00:24:13.190 --> 00:24:15.598
can actually be
quite informative.

00:24:15.598 --> 00:24:18.140
So then what you need to do to
really understand what they're

00:24:18.140 --> 00:24:20.415
doing is dive in
and look at where

00:24:20.415 --> 00:24:22.040
the metals bind, if
you're lucky enough

00:24:22.040 --> 00:24:26.940
to be able to get a structure
with the metals bound.

00:24:26.940 --> 00:24:32.240
So we have alpha helical motifs
and metal binding motifs.

00:24:32.240 --> 00:24:34.670
And then the other kind
of motif that I think

00:24:34.670 --> 00:24:38.900
is really interesting
for the linear system--

00:24:38.900 --> 00:24:41.900
so that's farnesyl
pyrophosphate--

00:24:41.900 --> 00:24:43.720
is how you control chain length.

00:24:46.250 --> 00:24:51.120
So FPP synthase-- that's
what we're talking about--

00:24:51.120 --> 00:24:52.640
is a dimer.

00:24:52.640 --> 00:24:57.560
And the metal binding
motifs sit up there.

00:24:57.560 --> 00:25:00.530
So the metal binding motifs
sit in the top, the way

00:25:00.530 --> 00:25:01.430
I've drawn this here.

00:25:01.430 --> 00:25:03.713
So you have metals.

00:25:03.713 --> 00:25:05.255
There are thought
to be three metals.

00:25:08.270 --> 00:25:11.360
And then we're
building C5, C5, C5.

00:25:11.360 --> 00:25:12.440
Where does the chain go?

00:25:15.110 --> 00:25:17.270
And what you'll see
is there is a cone

00:25:17.270 --> 00:25:20.990
shape which migrates towards
the bottom of the structure.

00:25:20.990 --> 00:25:23.880
I'll show you a picture
of this in a minute.

00:25:23.880 --> 00:25:27.320
And so then the question is,
what controls chain length?

00:25:27.320 --> 00:25:32.930
So if you end up looking at
the structure, what you see

00:25:32.930 --> 00:25:34.900
is a phenylalanine.

00:25:34.900 --> 00:25:37.070
And the phenylalanine
is a molecular doorstop.

00:25:42.410 --> 00:25:46.430
So the chain is
extending, because we're

00:25:46.430 --> 00:25:50.120
going from C5 to C10 to C15.

00:25:50.120 --> 00:25:52.280
Why don't we go to C50?

00:25:52.280 --> 00:25:55.370
And I showed you in the
first lecture dolichol

00:25:55.370 --> 00:25:59.330
and lipid II have C20s, C55s.

00:25:59.330 --> 00:26:01.190
How do you control
the chain length?

00:26:01.190 --> 00:26:02.690
So that's an
interesting question

00:26:02.690 --> 00:26:05.630
in polymer biochemistry.

00:26:05.630 --> 00:26:08.660
Here, we control it by
a molecular doorstop.

00:26:08.660 --> 00:26:13.580
So if I replace the
phenylalanine with an alanine,

00:26:13.580 --> 00:26:14.360
what might happen?

00:26:19.750 --> 00:26:20.250
Go.

00:26:20.250 --> 00:26:21.300
AUDIENCE: You'd have longer.

00:26:21.300 --> 00:26:21.640
JOANNE STUBBE: Yeah.

00:26:21.640 --> 00:26:22.770
So they made up to--

00:26:22.770 --> 00:26:23.580
I can't remember.

00:26:23.580 --> 00:26:25.247
I haven't read the
paper in a long time.

00:26:25.247 --> 00:26:27.540
But they can make C50-mers.

00:26:27.540 --> 00:26:30.883
So they can actually see,
and they're not uniform.

00:26:30.883 --> 00:26:31.800
So that's a key thing.

00:26:31.800 --> 00:26:34.180
You want them to be uniform.

00:26:34.180 --> 00:26:35.520
So if you look--

00:26:35.520 --> 00:26:37.650
I think in the next
has a picture of this.

00:26:37.650 --> 00:26:41.730
Again, this is graphics
from really quite some time

00:26:41.730 --> 00:26:43.200
ago now, 1996.

00:26:43.200 --> 00:26:44.850
So the picture's not very good.

00:26:44.850 --> 00:26:47.310
But you can see
sort of the tunnel,

00:26:47.310 --> 00:26:50.165
and the metal binding sites
are actually up there.

00:26:50.165 --> 00:26:51.540
And if you look
at the structure,

00:26:51.540 --> 00:26:54.210
you can see the phenylalanine.

00:26:54.210 --> 00:26:59.280
So that's what we know sort
of about the type I synthases.

00:26:59.280 --> 00:27:06.000
They're involved in making the
C15 farnesyl pyrophosphate.

00:27:06.000 --> 00:27:08.217
So now we want to look
at the chemistry, what's

00:27:08.217 --> 00:27:09.300
going on in the chemistry.

00:27:09.300 --> 00:27:12.480
And can we make a
generalization about how

00:27:12.480 --> 00:27:17.370
this chemistry is used to
put all C5 units together?

00:27:17.370 --> 00:27:20.860
So that's what I want
to focus on next.

00:27:24.120 --> 00:27:24.620
Whoops.

00:27:31.460 --> 00:27:34.750
So what I'm going to now look
at are the proposed mechanisms.

00:27:39.330 --> 00:27:42.540
And I'm not really going
to go into much detail.

00:27:42.540 --> 00:27:45.480
I'm going to give you
a generic overview

00:27:45.480 --> 00:27:48.120
of the things you need to
remember if you encounter

00:27:48.120 --> 00:27:50.220
something like this.

00:27:50.220 --> 00:27:52.110
The first guess
would be a mechanism

00:27:52.110 --> 00:27:54.330
similar to the one
I'm proposing now,

00:27:54.330 --> 00:27:56.370
but then you have to
look at it in more detail

00:27:56.370 --> 00:27:59.030
to figure out what's
really going on.

00:27:59.030 --> 00:28:00.030
So what do we have?

00:28:03.470 --> 00:28:06.760
We have dimethylallyl
pyrophosphate.

00:28:06.760 --> 00:28:08.938
And I have a cartoon for
you to look at there,

00:28:08.938 --> 00:28:11.230
but I'm going to draw it
differently than this cartoon.

00:28:11.230 --> 00:28:14.560
But you can just watch me
because, again, the key thing

00:28:14.560 --> 00:28:17.050
is thinking about how you
form the carbon-carbon bond

00:28:17.050 --> 00:28:19.780
and what's going on
in these reactions.

00:28:19.780 --> 00:28:23.350
So we just looked at
the pyrophosphate.

00:28:23.350 --> 00:28:27.760
And if you look over
there, what do you need?

00:28:27.760 --> 00:28:31.330
You need to have a
bunch of metals bound.

00:28:31.330 --> 00:28:34.390
And recently-- this
is a fairly old paper.

00:28:34.390 --> 00:28:35.530
They have better papers.

00:28:35.530 --> 00:28:37.892
I think I took all
the pictures out,

00:28:37.892 --> 00:28:39.850
because it's hard to see
things without looking

00:28:39.850 --> 00:28:41.260
at it in detail.

00:28:41.260 --> 00:28:44.080
But in fact, the
magnesiums are interacting

00:28:44.080 --> 00:28:47.470
with the pyrophosphate and
adjacent to the pyrophosphate.

00:28:47.470 --> 00:28:50.500
And it's clear they play
a key role in catalysis.

00:28:50.500 --> 00:28:52.660
But whether they move
during the transformation,

00:28:52.660 --> 00:28:55.690
again, I think we just don't
know that much at this stage.

00:28:55.690 --> 00:29:00.010
It's hard to trap it in
an informative state,

00:29:00.010 --> 00:29:03.400
like it is with all
crystallography.

00:29:03.400 --> 00:29:06.590
So here's dimethylallyl
pyrophosphate.

00:29:06.590 --> 00:29:08.860
Here's isopentenyl
pyrophosphate,

00:29:08.860 --> 00:29:11.380
the two guys we were after.

00:29:11.380 --> 00:29:16.480
And the first step in all of
these reactions is ionization.

00:29:16.480 --> 00:29:19.960
So this is an unusual
reaction in biochemistry.

00:29:19.960 --> 00:29:22.960
There are almost no
examples of carbocation

00:29:22.960 --> 00:29:25.220
in biological transformations.

00:29:25.220 --> 00:29:28.570
This is one of the few
places where you see this.

00:29:28.570 --> 00:29:34.270
So this is the ionization step.

00:29:34.270 --> 00:29:35.890
And all of the
reactions we are going

00:29:35.890 --> 00:29:39.710
to be looking at
involve ionization,

00:29:39.710 --> 00:29:42.490
but other kinds of
chemistry can also

00:29:42.490 --> 00:29:46.500
happen that we're
not going to discuss.

00:29:46.500 --> 00:29:47.680
So what have we generated?

00:29:47.680 --> 00:29:51.040
We generated an allylic cation.

00:29:51.040 --> 00:29:54.180
And what we also have is
we lost pyrophosphate.

00:29:58.120 --> 00:29:59.620
And I'm being sloppy.

00:29:59.620 --> 00:30:05.080
I'm not drawing out how these
are interacting with metals,

00:30:05.080 --> 00:30:07.903
but the charges are pretty
much neutralized in some form

00:30:07.903 --> 00:30:09.320
that we don't know
the details of.

00:30:09.320 --> 00:30:12.470
So you can't forget
about the charges.

00:30:12.470 --> 00:30:18.790
And so we can just
put down magnesium 3+.

00:30:18.790 --> 00:30:20.740
And then what we
want to do, we want

00:30:20.740 --> 00:30:22.375
to make a carbon-carbon bond.

00:30:24.900 --> 00:30:25.400
Whoops.

00:30:25.400 --> 00:30:26.590
Let me get this right.

00:30:29.160 --> 00:30:31.390
If I make a mistake on the
board-- like sometimes,

00:30:31.390 --> 00:30:33.790
I always get mixed up with
four or five carbons--

00:30:33.790 --> 00:30:35.248
raise your hand
and say, you've got

00:30:35.248 --> 00:30:36.760
the wrong number of carbons.

00:30:36.760 --> 00:30:37.360
You tell me.

00:30:37.360 --> 00:30:39.880
You be the cops.

00:30:39.880 --> 00:30:41.830
So what we're going
to do now is we're

00:30:41.830 --> 00:30:44.440
ready to form a
carbon-carbon bond.

00:30:44.440 --> 00:30:47.530
And we're going to be
forming a new carbocation.

00:30:47.530 --> 00:30:49.960
Hopefully, you remember
from introductory chemistry

00:30:49.960 --> 00:30:53.320
that carbocations that are
tertiary are more stable.

00:30:53.320 --> 00:30:55.810
And when you look at
terpene types of chemistry,

00:30:55.810 --> 00:30:58.210
you see tertiary
carbocations used

00:30:58.210 --> 00:30:59.920
over and over and over again.

00:30:59.920 --> 00:31:05.920
That being said, I'm
putting brackets around this

00:31:05.920 --> 00:31:08.830
because despite the fact that
I draw this intermediate,

00:31:08.830 --> 00:31:11.590
no one's ever seen it in
the enzymatic reaction using

00:31:11.590 --> 00:31:12.810
the normal substrates.

00:31:12.810 --> 00:31:15.280
So you have to play
games to study mechanism,

00:31:15.280 --> 00:31:18.470
just like you have to
do in organic chemistry.

00:31:18.470 --> 00:31:21.580
So what happens now
is you're set up

00:31:21.580 --> 00:31:23.440
to form the
carbon-carbon bond, which

00:31:23.440 --> 00:31:25.900
has been the goal
of what we've been

00:31:25.900 --> 00:31:30.280
trying to do in the
first couple of lectures.

00:31:30.280 --> 00:31:32.200
And so what do you generate?

00:31:32.200 --> 00:31:37.150
You generate the new
carbon-carbon bond,

00:31:37.150 --> 00:31:41.770
which is the skeleton for
geranyl pyrophosphate.

00:31:41.770 --> 00:31:45.760
You generated a new
carbocation, and it's

00:31:45.760 --> 00:31:47.638
a tertiary carbocation.

00:31:52.840 --> 00:31:56.950
And our pyrophosphate is still
sitting in the active site.

00:32:04.810 --> 00:32:07.090
And so now what
we're ready to do

00:32:07.090 --> 00:32:11.718
is we're going to form our
C10, geranyl pyrophosphate.

00:32:11.718 --> 00:32:13.510
And we'll see one of
the types of reactions

00:32:13.510 --> 00:32:16.390
that you see over
and over again when

00:32:16.390 --> 00:32:20.140
you make carbon-carbon
bonds is loss of a proton.

00:32:20.140 --> 00:32:22.420
And that gives
you the C10, which

00:32:22.420 --> 00:32:24.160
is these two things
stuck together, which

00:32:24.160 --> 00:32:28.360
is a monoterpene, which is
called "geranyl pyrophosphate."

00:32:28.360 --> 00:32:30.160
So what's interesting
about this--

00:32:30.160 --> 00:32:32.890
and I think this is sort
of something that's pretty

00:32:32.890 --> 00:32:33.490
general--

00:32:33.490 --> 00:32:35.470
is the pyrophosphate
in the active site.

00:32:35.470 --> 00:32:39.100
If you look in the active sites,
they're amazingly hydrophobic.

00:32:39.100 --> 00:32:43.860
And the pyrophosphate in
some way stereospecifically--

00:32:43.860 --> 00:32:45.610
I haven't drawn the
stereochemistry here--

00:32:45.610 --> 00:32:50.530
removes the HR proton
to generate the olefin.

00:32:50.530 --> 00:32:54.460
So what you've now generated
is geranyl pyrophosphate.

00:33:04.490 --> 00:33:10.430
So here's C10, and this
is geranyl pyrophosphate.

00:33:10.430 --> 00:33:15.740
Let me also put brackets
around this intermediate.

00:33:15.740 --> 00:33:18.830
Again, we haven't seen
this intermediate.

00:33:18.830 --> 00:33:20.540
And how do we know this is true?

00:33:20.540 --> 00:33:22.850
Because we know a lot
from Winstein and Brown

00:33:22.850 --> 00:33:24.440
about carbocation chemistry.

00:33:24.440 --> 00:33:27.020
And people have been really
creative in figuring out

00:33:27.020 --> 00:33:30.290
how to show that this
model is in fact correct.

00:33:30.290 --> 00:33:34.880
Hopefully, I have C10 there.

00:33:34.880 --> 00:33:38.030
And so this is an intermediate
because we're still

00:33:38.030 --> 00:33:38.720
going to go on.

00:33:38.720 --> 00:33:40.340
The enzyme doesn't stop at C10.

00:33:40.340 --> 00:33:43.820
It adds another
isopentenyl pyrophosphate.

00:33:43.820 --> 00:33:47.420
So if you want to think about
how nature might design that,

00:33:47.420 --> 00:33:50.150
if you look at this
molecule and you

00:33:50.150 --> 00:33:53.420
look at this part
of the molecule

00:33:53.420 --> 00:33:56.330
and replace it with an R group--

00:33:56.330 --> 00:33:57.320
so we have an R here.

00:33:57.320 --> 00:33:58.850
What does this look like?

00:33:58.850 --> 00:34:04.270
It looks just like
dimethylallyl pyrophosphate.

00:34:04.270 --> 00:34:06.180
But we need to put
the R group somewhere.

00:34:06.180 --> 00:34:09.980
So in the case of FPP synthase,
we're going down the tunnel.

00:34:09.980 --> 00:34:12.010
So we're getting
it out of the way.

00:34:12.010 --> 00:34:14.719
But we're going to do the
same chemistry that we just

00:34:14.719 --> 00:34:19.280
did over again, and we
just replaced a methyl

00:34:19.280 --> 00:34:21.710
with an R group.

00:34:21.710 --> 00:34:24.139
So that's the basic chemistry.

00:34:24.139 --> 00:34:26.960
It's pretty straightforward,
the only chemistry

00:34:26.960 --> 00:34:29.800
that I'm aware of in
biological systems that

00:34:29.800 --> 00:34:31.760
involves carbocations.

00:34:31.760 --> 00:34:35.210
These are special carbocations.

00:34:35.210 --> 00:34:37.239
That is, they're, in
general, stabilized.

00:34:37.239 --> 00:34:38.510
They're allylic.

00:34:38.510 --> 00:34:42.500
Or in many cases,
they can be tertiary.

00:34:42.500 --> 00:34:44.233
So let's emphasize that.

00:34:44.233 --> 00:34:46.400
Again, if you don't remember
your organic chemistry,

00:34:46.400 --> 00:34:51.230
you should go back and look up
the sections on carbocations.

00:34:51.230 --> 00:34:54.139
So I told you the
farnesyl pyrophosphate is

00:34:54.139 --> 00:34:57.410
sort of central to many things.

00:34:57.410 --> 00:34:59.930
And farnesyl, in this case--

00:34:59.930 --> 00:35:02.588
I'm not going to draw out
farnesyl pyrophosphate.

00:35:02.588 --> 00:35:03.630
The chemistry's the same.

00:35:03.630 --> 00:35:06.360
You can repeat it yourself.

00:35:06.360 --> 00:35:09.170
But here is our
farnesyl pyrophosphate,

00:35:09.170 --> 00:35:12.340
but look what it can form.

00:35:12.340 --> 00:35:14.410
Remember, you saw
all those smells.

00:35:14.410 --> 00:35:18.760
If you break a pine
needle, you have pinene.

00:35:18.760 --> 00:35:22.990
What you see is this
one intermediate

00:35:22.990 --> 00:35:26.840
can form all of these compounds.

00:35:26.840 --> 00:35:28.660
So the question
is-- with an enzyme

00:35:28.660 --> 00:35:30.790
that looks just like
farnesyl pyrophosphate

00:35:30.790 --> 00:35:32.710
in three-dimensional structure.

00:35:32.710 --> 00:35:34.780
So that's sort of amazing.

00:35:34.780 --> 00:35:38.680
And what you're doing here
is taking a linear molecule.

00:35:38.680 --> 00:35:41.770
And in this particular case--
and I'm not going to talk about

00:35:41.770 --> 00:35:47.170
this slide in detail, but I will
talk about one case in detail--

00:35:47.170 --> 00:35:50.980
what you're now doing is getting
it to do alternative chemistry.

00:35:50.980 --> 00:35:55.660
So how would you design the
active site of your enzyme

00:35:55.660 --> 00:36:00.370
to end up doing that, to use
the same chemistry, ionization?

00:36:00.370 --> 00:36:03.220
And then you have to
do cyclization and loss

00:36:03.220 --> 00:36:05.410
of a proton or whatever.

00:36:05.410 --> 00:36:07.430
How does nature
design all of this?

00:36:07.430 --> 00:36:10.420
So once we get through
this set of lectures,

00:36:10.420 --> 00:36:12.910
I would suggest this
would be something you

00:36:12.910 --> 00:36:14.440
could go back and practice on.

00:36:14.440 --> 00:36:16.050
How do we get to all these guys?

00:36:16.050 --> 00:36:17.800
I'm going to show you
one example of that.

00:36:17.800 --> 00:36:19.425
I'm not going to go
through this slide.

00:36:19.425 --> 00:36:21.190
It's way too
complicated, but I think

00:36:21.190 --> 00:36:25.420
it shows you sort of the amazing
diversity of the terpenome,

00:36:25.420 --> 00:36:28.710
using farnesyl pyrophosphate.

00:36:28.710 --> 00:36:34.300
So what I want to do is give
you an overview of the rules.

00:36:34.300 --> 00:36:37.060
And then I'll go through
one specific example.

00:36:37.060 --> 00:36:47.030
So let's make general
mechanistic comments.

00:36:47.030 --> 00:36:51.140
And in the original,
version of the PowerPoint,

00:36:51.140 --> 00:36:53.240
this slide wasn't in there.

00:36:53.240 --> 00:36:58.610
Anyhow, the first thing is
you've already seen up here,

00:36:58.610 --> 00:37:02.830
and this is going to be
common, is you lose a proton.

00:37:02.830 --> 00:37:06.080
So the first step is ionization.

00:37:06.080 --> 00:37:10.200
So ionization happens in
almost all these reactions.

00:37:10.200 --> 00:37:13.790
There are exceptions to
this, but most first steps

00:37:13.790 --> 00:37:16.040
are ionization.

00:37:16.040 --> 00:37:19.670
The second step can
involve proton loss.

00:37:22.452 --> 00:37:24.410
And I'm going to write
down what the steps are,

00:37:24.410 --> 00:37:28.760
and then we'll come back and
look at a specific example.

00:37:28.760 --> 00:37:31.340
And we're going to see
this in cholesterol.

00:37:31.340 --> 00:37:33.680
One can have with
carbocations-- if you go back

00:37:33.680 --> 00:37:36.170
and you think about what
you learned if you've had

00:37:36.170 --> 00:37:38.900
the second semester of organic.

00:37:38.900 --> 00:37:42.260
With carbocations, you
can do hydride transfers.

00:37:42.260 --> 00:37:47.970
So that's a hydrogen
with a pair of electrons.

00:37:47.970 --> 00:37:51.260
We can have hydride transfers.

00:37:51.260 --> 00:37:52.600
We're also going to see--

00:37:52.600 --> 00:37:57.440
and both of these are key
in cholesterol biosynthesis.

00:37:57.440 --> 00:38:00.440
We can have methyl
anion transfers.

00:38:07.090 --> 00:38:09.800
And the other thing
is these reactions

00:38:09.800 --> 00:38:11.900
all go stereospecifically.

00:38:15.410 --> 00:38:16.790
And that's one thing.

00:38:16.790 --> 00:38:19.720
If you become an enzymologist,
you realize that's what's cool.

00:38:19.720 --> 00:38:21.470
That's why you have
such big huge enzymes,

00:38:21.470 --> 00:38:23.870
so they can control the
stereochemistry of everything.

00:38:23.870 --> 00:38:26.055
So they do everything
with 100% EE.

00:38:26.055 --> 00:38:28.430
And they don't have to worry
about it like chemists worry

00:38:28.430 --> 00:38:30.950
about it, but they pay a price.

00:38:30.950 --> 00:38:34.130
They have a big huge protein.

00:38:34.130 --> 00:38:36.620
The third thing-- and this
is going to become important.

00:38:36.620 --> 00:38:40.430
It was just important in the
slide I showed you previously--

00:38:40.430 --> 00:38:42.065
we're going to see cyclizations.

00:38:44.600 --> 00:38:51.600
And cyclizations
require, in general,

00:38:51.600 --> 00:38:56.040
protonation of an olefin--

00:38:56.040 --> 00:38:58.200
I'll give you an
example of that--

00:38:58.200 --> 00:39:05.460
or protonation of an epoxide.

00:39:05.460 --> 00:39:08.520
So in some way, you're going
to have to do some more

00:39:08.520 --> 00:39:11.160
chemistry to get your olefin.

00:39:11.160 --> 00:39:14.130
Everybody know
what an epoxide is?

00:39:14.130 --> 00:39:16.380
So we're converting an
olefin into an epoxide.

00:39:16.380 --> 00:39:18.172
We're going to protonate
it, and then we're

00:39:18.172 --> 00:39:20.550
going to do cyclizations.

00:39:20.550 --> 00:39:25.050
And the third general type of
reactions is water addition.

00:39:28.560 --> 00:39:30.480
So if you have a
carbocation sitting around.

00:39:30.480 --> 00:39:34.635
You add water, bang, you have
a reaction and form an alcohol.

00:39:40.200 --> 00:39:42.293
So the other
generalizations I want

00:39:42.293 --> 00:39:43.710
to make-- so that's
the chemistry.

00:39:43.710 --> 00:39:46.380
We're going to see
this chemistry play out

00:39:46.380 --> 00:39:49.530
over and over again because
I've selected examples

00:39:49.530 --> 00:39:51.790
of this for you to look at.

00:39:51.790 --> 00:39:53.640
But it's quite common.

00:39:53.640 --> 00:39:56.580
The second thing besides
these mechanistic issues

00:39:56.580 --> 00:40:01.800
is, how do you distinguish
between linear versus cyclic?

00:40:04.390 --> 00:40:06.570
And you've already
seen the strategy

00:40:06.570 --> 00:40:08.790
with farnesyl pyrophosphate.

00:40:08.790 --> 00:40:10.890
You really sort of have
a tiny little cavity

00:40:10.890 --> 00:40:13.260
where the IPP and
the dimethylallyl

00:40:13.260 --> 00:40:17.280
pyrophosphate bind, and
then you have a long tunnel.

00:40:17.280 --> 00:40:21.810
What do you have in the case
of cyclic terpenes, which

00:40:21.810 --> 00:40:25.770
you saw in the previous
slide to this one?

00:40:25.770 --> 00:40:32.170
And the key thing is the
shape of the active site.

00:40:34.970 --> 00:40:36.680
And what you will
see if you look

00:40:36.680 --> 00:40:39.680
at a lot of these active
sites is, in general,

00:40:39.680 --> 00:40:43.430
they're very hydrophobic.

00:40:43.430 --> 00:40:44.933
Why is that true?

00:40:44.933 --> 00:40:47.350
So somehow, you've got to take
care of the pyrophosphates.

00:40:47.350 --> 00:40:49.100
But they're very
hydrophobic because we're

00:40:49.100 --> 00:40:53.150
dealing with these hydrocarbons,
which are hydrophobic.

00:40:53.150 --> 00:40:54.830
So the question then
is, can you take

00:40:54.830 --> 00:40:58.310
this farnesyl
pyrophosphate and fold it?

00:40:58.310 --> 00:41:00.730
And folding it in
different ways--

00:41:00.730 --> 00:41:03.540
if we go back to the last--

00:41:03.540 --> 00:41:04.110
whoops.

00:41:04.110 --> 00:41:07.050
If we go back to the last
slide, if you look at it here,

00:41:07.050 --> 00:41:12.180
for example, and you ionize
here to form a carbocation,

00:41:12.180 --> 00:41:15.180
you can have a cis or
a trans carbocation.

00:41:15.180 --> 00:41:18.570
And that then can lead to
further types of chemistry,

00:41:18.570 --> 00:41:21.240
where you form different
kinds of ring structures.

00:41:21.240 --> 00:41:25.600
So it really is all about
folding in the active site

00:41:25.600 --> 00:41:26.340
of the enzyme.

00:41:26.340 --> 00:41:29.310
So the active site is
the key to determine

00:41:29.310 --> 00:41:31.200
which of these many
kinds of things that

00:41:31.200 --> 00:41:33.600
can happen that if you
did this in solution,

00:41:33.600 --> 00:41:36.360
you might actually
get a mixture of all

00:41:36.360 --> 00:41:38.610
of these kinds of things.

00:41:38.610 --> 00:41:43.050
So the key then is
hydrophobic and the shape

00:41:43.050 --> 00:41:44.190
of the active site.

00:41:44.190 --> 00:41:48.660
And then another key thing
is I'm going to show you that

00:41:48.660 --> 00:41:50.730
in many of these
reactions, you go through--

00:41:50.730 --> 00:41:52.170
like we saw up there--

00:41:52.170 --> 00:41:54.930
these carbocation intermediates.

00:41:54.930 --> 00:41:56.940
Well, there might be three
different carbocation

00:41:56.940 --> 00:41:58.560
intermediates you
could go through.

00:41:58.560 --> 00:42:00.240
How do you decide?

00:42:00.240 --> 00:42:01.740
How do you decide--

00:42:01.740 --> 00:42:06.750
how did enzymes evolve to
give you specific carbocation

00:42:06.750 --> 00:42:07.380
intermediates?

00:42:07.380 --> 00:42:10.800
How might you stabilize a
carbocation intermediate?

00:42:10.800 --> 00:42:13.440
Anybody got any ideas?

00:42:13.440 --> 00:42:15.690
What would you expect to
find in the active site then?

00:42:15.690 --> 00:42:18.480
I'm going to show you
on the next slide, which

00:42:18.480 --> 00:42:23.470
is sort of a generic active site
of a terpene that can cyclize.

00:42:26.350 --> 00:42:27.280
Any guesses?

00:42:27.280 --> 00:42:29.101
How would you stabilize
a carbocation?

00:42:34.392 --> 00:42:35.840
AUDIENCE: Negative mixtures.

00:42:35.840 --> 00:42:36.673
JOANNE STUBBE: Yeah.

00:42:36.673 --> 00:42:38.920
So one way-- you might
have an aspartate.

00:42:38.920 --> 00:42:40.710
Nature doesn't do that.

00:42:40.710 --> 00:42:43.300
So that might-- well, the
problem is if you do that

00:42:43.300 --> 00:42:45.010
and you form a
covalent bond, that's

00:42:45.010 --> 00:42:46.790
the end of your reaction.

00:42:46.790 --> 00:42:47.290
So

00:42:47.290 --> 00:42:50.037
So how you do this
is I don't think

00:42:50.037 --> 00:42:51.370
we really totally understand it.

00:42:51.370 --> 00:42:53.320
But how else could
you stabilize it?

00:42:53.320 --> 00:42:54.430
Anybody else?

00:42:54.430 --> 00:42:56.920
What did you learn about weak
non-covalent interactions

00:42:56.920 --> 00:42:59.950
in biochemistry
that could help us?

00:42:59.950 --> 00:43:02.530
Everybody hates waiting on
covalent interactions, the key

00:43:02.530 --> 00:43:03.760
to everything--

00:43:03.760 --> 00:43:06.015
key to everything in
how enzymes function.

00:43:06.015 --> 00:43:08.140
AUDIENCE: You could just
have something [INAUDIBLE]

00:43:08.140 --> 00:43:09.090
in general.

00:43:09.090 --> 00:43:10.720
JOANNE STUBBE: But
electron-rich--

00:43:10.720 --> 00:43:13.095
but that would be doing--
that's what she was suggesting.

00:43:13.095 --> 00:43:16.210
You have a carboxylate, an
aspartate or a glutamate.

00:43:16.210 --> 00:43:19.540
Then you would form a bond,
and then you would be stuck.

00:43:19.540 --> 00:43:24.020
So the way nature actually does
this is she uses aromatics.

00:43:27.740 --> 00:43:31.430
And it was discovered
maybe about 15 years ago

00:43:31.430 --> 00:43:35.530
that you can have an
aromatic whatever.

00:43:35.530 --> 00:43:38.960
And you have some
kind of a cation.

00:43:38.960 --> 00:43:44.750
So this is called a "pi
cation interaction."

00:43:44.750 --> 00:43:47.780
Usually, the pi cation
interactions are with metals.

00:43:47.780 --> 00:43:49.610
But here, we have a carbocation.

00:43:49.610 --> 00:43:52.280
So the model is
that you might find

00:43:52.280 --> 00:43:58.070
in the active site tryptophans,
tyrosines, phenylalanines.

00:43:58.070 --> 00:44:00.710
And so these become really key.

00:44:00.710 --> 00:44:04.370
And in fact, if you
look at an active site--

00:44:04.370 --> 00:44:07.610
so I don't even remember
which enzyme this is.

00:44:07.610 --> 00:44:09.470
And somebody was trying
to study something,

00:44:09.470 --> 00:44:13.340
and they have a small
inhibitor in the active site.

00:44:13.340 --> 00:44:16.220
But you notice you don't
have a long site where

00:44:16.220 --> 00:44:18.140
this chain can extend.

00:44:18.140 --> 00:44:21.440
What you've done is constrained
the active site much more,

00:44:21.440 --> 00:44:24.740
and that shape is going to
be key to the many different

00:44:24.740 --> 00:44:26.090
reactions you could have.

00:44:26.090 --> 00:44:27.710
And then if you
look carefully, you

00:44:27.710 --> 00:44:29.030
can't really think about this.

00:44:29.030 --> 00:44:31.970
But you have phenylalanine,
tyrosine, tryptophan,

00:44:31.970 --> 00:44:33.830
and another tyrosine
in the active site.

00:44:33.830 --> 00:44:37.730
And that's what you see in many
of these protein structures

00:44:37.730 --> 00:44:38.240
all over.

00:44:38.240 --> 00:44:41.390
Again, we have FPP synthase,
which has this thing.

00:44:41.390 --> 00:44:43.490
And then we have these
terpene cyclases,

00:44:43.490 --> 00:44:44.960
which have this thing.

00:44:44.960 --> 00:44:46.820
And each one of
them is different.

00:44:46.820 --> 00:44:51.590
And so the difference
is related to the shape.

00:44:51.590 --> 00:44:54.710
And it's proposed that this
stabilizes this interaction.

00:44:54.710 --> 00:44:57.350
It's been challenging
to show this chemically,

00:44:57.350 --> 00:45:00.230
but these interactions
are worth quite a bit.

00:45:00.230 --> 00:45:02.300
These are also hard
to measure, but it's

00:45:02.300 --> 00:45:06.050
something that was discovered
and now has been actually

00:45:06.050 --> 00:45:07.923
widely observed.

00:45:07.923 --> 00:45:10.340
And the other thing I want to
mention about these enzymes,

00:45:10.340 --> 00:45:14.720
which I think is interesting
and distinct from other enzymes

00:45:14.720 --> 00:45:18.470
that you've encountered, is
that, in general, they're

00:45:18.470 --> 00:45:20.760
really not very specific.

00:45:20.760 --> 00:45:23.630
So if you start
looking at these--

00:45:23.630 --> 00:45:24.230
look at this.

00:45:24.230 --> 00:45:28.910
How could you make one cation
here versus the three others?

00:45:28.910 --> 00:45:32.240
If you start looking at how to
get to these cyclized products,

00:45:32.240 --> 00:45:34.550
you say, how the heck
did nature ever do that?

00:45:34.550 --> 00:45:37.320
There's no way you could guess
at what the product would be,

00:45:37.320 --> 00:45:38.580
in my opinion.

00:45:38.580 --> 00:45:41.987
So what happens is
these enzymes actually

00:45:41.987 --> 00:45:44.570
when you start looking-- we have
good analytical methods-- are

00:45:44.570 --> 00:45:46.460
really promiscuous.

00:45:46.460 --> 00:45:48.560
So they might produce
a predominant product,

00:45:48.560 --> 00:45:51.110
but they always
produce a bunch--

00:45:51.110 --> 00:45:54.860
1%, 5%, sometimes even more--

00:45:54.860 --> 00:45:57.420
of other products.

00:45:57.420 --> 00:45:59.570
And I think if you
look at the chemistry

00:45:59.570 --> 00:46:02.330
that we've been talking
about, basically, all

00:46:02.330 --> 00:46:05.900
of this sort of makes sense.

00:46:05.900 --> 00:46:08.540
So what I want to
do now is give you

00:46:08.540 --> 00:46:13.040
an example of all of these
reactions in one case.

00:46:13.040 --> 00:46:16.040
And this case, I guess I didn't
write down the references.

00:46:16.040 --> 00:46:18.050
But I took it out
of the literature.

00:46:18.050 --> 00:46:20.490
It's from David
Christianson's lab.

00:46:20.490 --> 00:46:22.882
So here, we have
farnesyl pyrophosphate,

00:46:22.882 --> 00:46:24.590
and here's the product
we want to get to.

00:46:24.590 --> 00:46:28.100
So you'll have
something like this

00:46:28.100 --> 00:46:31.040
on a problem set that
I'm going to ask you,

00:46:31.040 --> 00:46:32.180
and it will be simple.

00:46:32.180 --> 00:46:34.222
I won't give you something
that's so hard to see.

00:46:34.222 --> 00:46:35.840
But for me, lots
of times, when you

00:46:35.840 --> 00:46:38.538
look at these rearrangements,
it's easier if you make models.

00:46:38.538 --> 00:46:40.580
I don't know if anybody
ever uses models anymore.

00:46:40.580 --> 00:46:43.430
I still use models, because
you have to bend things

00:46:43.430 --> 00:46:45.230
in the right way to
see what's possible

00:46:45.230 --> 00:46:47.480
and if the orbital's
overlapping in the right way.

00:46:47.480 --> 00:46:50.600
You've got to really think
about the stereochemistry.

00:46:50.600 --> 00:46:52.440
So what do we have here?

00:46:52.440 --> 00:46:54.470
So the first step is ionization.

00:46:54.470 --> 00:46:56.930
So we would form an
allylic cation here.

00:46:56.930 --> 00:47:02.220
That's what we just did
over here, which I hid.

00:47:02.220 --> 00:47:05.300
So that's what we
just did over here.

00:47:05.300 --> 00:47:06.690
Oh, we didn't do it over here.

00:47:06.690 --> 00:47:11.990
Here-- over here, we
formed this allylic cation.

00:47:11.990 --> 00:47:14.750
And once you do this,
then they didn't

00:47:14.750 --> 00:47:15.920
show you that intermediate.

00:47:15.920 --> 00:47:17.250
They went on to the next step.

00:47:17.250 --> 00:47:19.940
So once you generate
a cation there,

00:47:19.940 --> 00:47:24.710
they drew the conformation such
that this thing could cyclize.

00:47:24.710 --> 00:47:29.450
But when you cyclize, you
have electron deficiency

00:47:29.450 --> 00:47:30.740
at this carbon.

00:47:30.740 --> 00:47:33.200
So you have a
second carbocation.

00:47:33.200 --> 00:47:37.680
This is not allylic, but
it's a tertiary carbocation.

00:47:37.680 --> 00:47:40.610
So now the question
is, what can happen?

00:47:40.610 --> 00:47:43.790
And again, you've got to keep
your eye on what your goal is

00:47:43.790 --> 00:47:44.870
way down at the end.

00:47:44.870 --> 00:47:47.780
And you could probably draw
more than one mechanism

00:47:47.780 --> 00:47:49.370
to get from A to B.
And then you have

00:47:49.370 --> 00:47:51.500
to figure out experiments
of how you would test it

00:47:51.500 --> 00:47:54.320
if you really care about that.

00:47:54.320 --> 00:47:55.970
So what happens here?

00:47:55.970 --> 00:47:58.100
You're losing a proton.

00:47:58.100 --> 00:48:01.170
And again, the pyrophosphate
is acting as a general base

00:48:01.170 --> 00:48:01.670
catalyst.

00:48:01.670 --> 00:48:04.070
So that's exactly what
happens in the case

00:48:04.070 --> 00:48:05.900
or what's proposed
to happen in the case

00:48:05.900 --> 00:48:08.120
of farnesyl pyrophosphate.

00:48:08.120 --> 00:48:09.920
So you generate this species.

00:48:09.920 --> 00:48:11.120
Well, this might be stable.

00:48:11.120 --> 00:48:12.710
You might actually
be able to isolate

00:48:12.710 --> 00:48:15.230
that as an intermediate
along the reaction pathway.

00:48:15.230 --> 00:48:17.600
But we know in
the end, we end up

00:48:17.600 --> 00:48:21.350
with two six-membered rings
with this stereochemistry

00:48:21.350 --> 00:48:23.750
and with methyl groups
in certain places.

00:48:23.750 --> 00:48:28.070
And so then you have to think
about how can we get there.

00:48:28.070 --> 00:48:32.720
So remember that I told you
that terpenoids do cyclizations.

00:48:32.720 --> 00:48:36.230
And one way they can do it
is to protonate the olefin.

00:48:36.230 --> 00:48:40.130
So here, there might be a
group in the active site.

00:48:40.130 --> 00:48:42.920
Maybe it's the phosphate
that would help facilitate.

00:48:42.920 --> 00:48:45.710
You've just used it as
a general base catalyst.

00:48:45.710 --> 00:48:47.270
Now, it's got a proton.

00:48:47.270 --> 00:48:50.600
It could now function as
a general acid catalyst.

00:48:50.600 --> 00:48:53.780
You could protonate
this position

00:48:53.780 --> 00:49:00.050
and now form two six-membered
rings and a new carbocation.

00:49:00.050 --> 00:49:04.280
So in general, the nomenclature
is when you draw these things,

00:49:04.280 --> 00:49:07.310
if you have a stick
like that, that means

00:49:07.310 --> 00:49:09.920
you've got a methyl group.

00:49:09.920 --> 00:49:11.420
If you want to put
a hydrogen there,

00:49:11.420 --> 00:49:12.890
you put a hydrogen on it.

00:49:12.890 --> 00:49:15.860
So if there's nothing there
because CH3 takes up more room

00:49:15.860 --> 00:49:17.890
and they become very
complicated to draw,

00:49:17.890 --> 00:49:19.530
the methyl group has methane.

00:49:19.530 --> 00:49:21.410
And the hydrogen, you put on.

00:49:21.410 --> 00:49:24.020
So you can distinguish
one from the other.

00:49:24.020 --> 00:49:28.190
So now what happens is
remember, one of the mechanisms

00:49:28.190 --> 00:49:30.510
I told you is hydride transfer.

00:49:30.510 --> 00:49:33.110
And again, I think looking
at the stereochemistry

00:49:33.110 --> 00:49:35.580
of these systems helps
see how this could happen.

00:49:35.580 --> 00:49:37.620
But these are all
stereospecific.

00:49:37.620 --> 00:49:41.030
So you have hydride
transfer from this position

00:49:41.030 --> 00:49:42.680
to this position.

00:49:42.680 --> 00:49:44.300
And when you have
hydride, a hydrogen

00:49:44.300 --> 00:49:46.940
with a pair of electrons,
what you have left

00:49:46.940 --> 00:49:50.240
is a new tertiary carbocation.

00:49:50.240 --> 00:49:52.340
And this new tertiary
carbocation--

00:49:52.340 --> 00:49:55.190
let me see what's
going on-- is now--

00:49:55.190 --> 00:49:56.870
in the end, we get
a methyl group here.

00:49:56.870 --> 00:49:58.550
We have no methyl group there.

00:49:58.550 --> 00:50:03.080
Now, we have a CH3-
group migrating.

00:50:03.080 --> 00:50:08.070
And that's the third
method that I described.

00:50:08.070 --> 00:50:13.760
So the CH3- group migrates,
giving you a new carbocation.

00:50:13.760 --> 00:50:17.210
And then the last step is,
again, loss of a proton.

00:50:17.210 --> 00:50:18.570
So here's an example.

00:50:18.570 --> 00:50:24.230
This is a complex example, but
there are 70,000 of these guys.

00:50:24.230 --> 00:50:27.650
So these are sort of
the general rules.

00:50:27.650 --> 00:50:30.740
Nature has figured out how to
make all these different kinds

00:50:30.740 --> 00:50:34.150
of natural products.

00:50:34.150 --> 00:50:36.710
So what I want to
do now-- so those

00:50:36.710 --> 00:50:39.940
are the general overview
of how these systems work.

00:50:39.940 --> 00:50:40.730
What am I doing?

00:50:40.730 --> 00:50:42.500
Oh, I'm sorry.

00:50:42.500 --> 00:50:43.460
I get so lost.

00:50:43.460 --> 00:50:45.440
Anyhow, I wanted to get
through cholesterol.

00:50:45.440 --> 00:50:46.732
But next time, we'll come back.

00:50:46.732 --> 00:50:48.320
And in the very
beginning, we're going

00:50:48.320 --> 00:50:53.060
to see how we take
C15s to go to C30s

00:50:53.060 --> 00:50:56.060
and then how you cyclize this
in the most, in my opinion,

00:50:56.060 --> 00:50:58.160
amazing reaction in biology--

00:50:58.160 --> 00:51:02.030
other than ribonucleotide
reductase, anyhow.

00:51:02.030 --> 00:51:04.630
See you next-- see you Friday.