>> Okay, we're back.
Today, we're going to be
talking about polyketides,
and then we'll be
on to terpenes.
So actually, I expect to cover
a little bit of terpenes today
and then finish off the
discussion on Tuesday and then
on Tuesday, we're on to chapter
nine to discuss cell signaling
and small molecules
and cell signaling.
And actually, I'm going to
touch on that topic today.
Last time, we saw
that oligosaccharides are quite
heterogeneous and quite diverse.
That there is no one
oligosaccharide component per
protein that's encoded
by DNA in some way.
Rather, we saw there was a
sort of a random distribution
of different structures really
on the surface of the cell.
And this randomness
is a key attribute
that affects their function.
All right, we discussed
for example,
how oligosaccharides
cause proteins to fold.
And so having, you might
imagine that having a diversity
of different structures
might also give you a range
of different confirmations
accessed by the proteins.
Now that latter remark is
a little bit speculative
but that's one of the ways
that people are thinking
about these things.
Okay, so we also discussed how
sulfated polysaccharides avidly
take up water and if you're
blowing your nose on the way
into work this morning or the
way into class this morning,
you definitely know about that.
We talked about mucus
and [inaudible].
We talked a little bit about how
O-linked glycans are added one
at a time.
Actually, this is good.
This is making a couple of
points that I meant to make
but might have lost over.
So the O-linked glycans are
added one monosaccharide
at a time to build
up to big structures
and the N-link glycans are added
as complex branched
polysaccharides,
sometimes in segments
of three which we saw
and sometimes even more complex.
After the synthesis of these
N and O-linked glycans,
there's lots of changes
that take place
through trimming
by glucosidases.
These are little
scissors that come along
and snip apart glycosidic bonds.
And they do it in sort
of semi-random fashion
and that introduces
additional diversity
that further complicates
our lives if we want
to study these things.
We also talked about
how these oligo-
and polysaccharides are
exclusively found in the surface
of the cell and are very
heterogeneous in composition.
So there are really no
glycosylated proteins found
inside the cell although
I'm looking for someone
to prove me wrong on that one.
I know it'll be someone
in this class
or someone watching this video.
And that would be great.
For now, our dogma is that
there are no polysaccharides
that are attached to proteins
found inside the cell.
Rather, this is sort
of an exterior attribute that's
grafted onto proteins found
on the outside of the cell.
And we described this as a
gumball model for the cell
where the outside of the cell
is sweet with carbohydrates
and then the inside is
chockfull of proteins and DNA
where gumballs, of course,
have gelatin as the protein.
Then we discussed how too much
glucose, too many gumballs leads
to protein crosslinking and
inflammation and how this leads
to diseases like
diabetes, et cetera.
And how these are really
unavoidable consequences
of a western diet that we
eat such high concentrations
of carbohydrates that it's
kind of inevitable that we end
up with some inflammation
resulting
from this sort of thing.
And so this is one of the
major challenges, I think,
going forward is now
to apply this knowledge
about chemical biology, about
the underlying mechanisms
that are taking place at the
level of atoms and bonds and try
to convince other
people in the country
to change their lifestyle
really.
That's a nontrivial challenge,
that challenge that sort
of next era which is
going from atoms and bonds
and then going to society.
That's really a major link
that I leave to you to take up.
All right, are there
any questions
about what we saw last time,
about carbohydrates,
about glycosides?
Anything you want to
know about carbohydrates,
I will endeavor to answer.
I don't know everything
but I'll do my best.
Stump the chump, yup.
>> Is that like we
use the substitutes
for sugar, instead of sugars?
>> Yeah.
>> But I'm [inaudible] that
substitutes are bad for us
like they cause cancer
essentially?
>> Okay, so -- okay
so the question,
let me just repeat the question
in case you didn't hear it.
The question was you
discussed the sugar substitutes
and aren't some of those
sugar substitutes linked
to other diseases?
Okay so, I'll answer your
question in a moment.
But before I do, I think you
raised a really good point.
The sugar substitutes that
I presented towards the end,
compounds like the thing
isolated from stevia, sucralose,
et cetera, those
aren't a panacea.
They are not completely benign
and one of the astounding things
that we find is that those sugar
substitutes even though they're
not digested, even though they
don't directly produce calories,
do seem to result in
caloric intake by animals
who are fed the sugar
substitute.
And our theory, our best
guess at what's going on is
that there's actually
sweet receptors not just
on your tongue but also that are
in your stomach that are kind
of gauging what kind
of food is present.
And when those sweet
receptors get stimulated
by sugar substitutes, they
sort of actively start taking
up a lot more calories
than they otherwise would.
And so, you know, even though
your soft drink might be zero
calories, it has the effect
of stimulating you to take
up more calories than
you'd otherwise take
up because your body function
is expecting something sweet
and sugary.
Okay, so that's one
aspect of what's going on.
You know, this is no
free lunch basically.
Okay now, the second aspect
is the linkage to cancer
and I'll be honest, I don't
know very much about that,
about the -- you know,
the latest research
on that sort of thing.
I think there was -- there's
kind of a history looking
at certain sweeteners
in that context.
And some of those
are kind of early,
I'm talking about like 1970's
kind of trials were not done
with the kind of rigor
that we would use today.
So I think it's time to go back
and reevaluate our thinking
about that sort of thing
using different standards
that we now apply.
So for example, a lot of
those involve really high
concentrations of
sugar substitutes
where it's just physiologically
unrealistic.
Although I don't know.
Some people drink liters upon
liters of diet soft drinks
so maybe that is
actually kind of less.
So anyway, someone who could
design better experiments needs
to go back and look
at it more closely.
That's all I have to
say about that topic.
But thanks for asking.
Other questions?
Anything you want to
know about carbohydrates,
monosaccharides,
polysaccharides?
Okay, in the back.
>> Talking about the glycans
you added them one at a time,
what kind of time scale
are we talking about?
>> Oh, okay.
Thanks for asking.
That's a great question.
And I actually don't
know your name.
>> It's Ty.
>> Ty. Okay, so Ty's
question is,
how fast do the monosaccharides
get at it?
And the answer is really fast.
Like these are being added
on the subsecond time scale.
So on the order of
like milliseconds.
These reactions are
happening very quickly.
They are nicely enzyme catalyzed
and their reactions
are not slow.
These are really fast reactions
and I think your question
would be really interesting
when you take it up here
against say DNA being added
to a DNA [inaudible] and I don't
have the exact numbers for that
and I think that's a great
food for thought question.
So thanks for asking.
Other questions?
All right, let's push on.
When we last left off
last time, we were talking
about polysaccharides or sorry,
not polysaccharides, terpenes
and just give me a moment
to find where we left off.
And then we'll start again.
All right, one moment.
All right, so I showed you the
fatty acid synthase as kind
of our final thought and I think
maybe what I'll do is I'll start
there, okay?
So we saw that the fatty acid
synthase was this really complex
machine that allows
the synthesis of fats.
And it does this by
having this robot arm
that cycles around, okay?
And we saw a key
reaction in here
which was this Claisen
condensation reaction
where it's basically an aldol
that involves a thioester.
Okay now, take a look at this
reaction really closely, okay?
I want to give a quiz about
this and I want you to be able
to redo this reaction
for me, okay?
So please look at this.
All right, moving on.
At the very last step, I kind
of glossed over this reaction
but it's a really
interesting one and I want
to take a moment
to appreciate this.
So we discussed how
the robot arm
that moved the intermediate
amongst the various enzyme
domains was linked
to the intermediates
through a thioester.
And that was
that phosphopantetheinyl
arm that I showed you.
And so the very last step
is this bioesterase that has
to hydrolyze this bioester and
it's really absolutely notable.
It's absolutely fascinating
and a great,
great place of converted
evolution or perhaps borrow,
evolutionary borrowing
that the mechanism
for this hydrolysis
borrows very heavily
from the serine proteases
down to the point
where it has an active site
serine, an active site histidine
and an active site
aspartic acid.
And that's really notable
and I think really fun
because in the end, that
gives us a very rapid way
of hydrolyzing these
bioesters over here all the way
down to a carboxylic acid.
And what's great is that
we don't really have
to learn some new mechanism.
This is just something that's
totally familiar to us.
Okay, so I want to talk to
you about what happens next.
So I've shown you how these
things are synthesized.
I want to talk to you next about
how they're applied in the cell
and how they're applied
in cell signaling.
So let's start with what
they're used for primarily.
So these fatty acids form the
barrier on the cell surface.
They are what's composing
that plasma membrane.
And I want you to recall
that the plasma membrane
is this layer
on that divides the
exterior cellular,
extracellular [inaudible]
up here
from the cytoplasm down here.
And this barrier is
pretty impermeable.
It's really super
hydrophobic over here.
There are some hydrophilic
functionalities that interface
between the water and the
hydrophobic stuff over here
and we'll look at the
structure in a moment.
But the fact here is that the
cell very tightly controls
what's going to be allowed to
pass through into its interior,
into its cytoplasm with
one major exception.
That major exception is, of
course, pharmaceuticals, right?
So small molecule drugs that
are hydrophobic are going
to be readily able to slip
through this semi-impermeable
barrier,
this otherwise impermeable
barrier.
Okay, so all of these compounds
in here, all of these chains
in here are fatty acids.
Let's zoom in and
take a closer look.
Okay, so when we look
at the composition
of the plasma membranes found
in human cells, what we find is,
no surprise, chemical
heterogeneity.
Okay, we find diversity.
We find that there are a
number of different molecules
and I can just give
you some percentages
of those molecules over here.
Okay, so these are average
percentages over here
and you can see that the
plasma membrane is composed
of a diverse array of these
different lipids, okay?
And the effect here is different
changes in the viscosity
of the plasma membrane and
we find that there are areas
of the plasma membrane,
they're a good deal less viscous
than others, that are
less fluid than others,
that are more viscous
than other areas.
These are regions that are
sometimes called lipid rafts
where there's even areas
where the lipids
themselves are insoluble
in solvents like acetone.
You know, you could take
out the cell and usually all
that stuff goes neatly into
organic solvents like acetone.
There are some regions that
refuse that kind of treatment.
Okay, so anyway, notice
that these are ranges
of different percentages and
they range pretty broadly.
And those ranges have
important consequences in terms
of the fluidity of the membrane.
Okay and the model that
you might have learned back
in high school, the fluid
mosaic model of the cell is true
to a certain extent but we're
finding is that it's less fluid,
that there's actually regions
of real density on
the cell surface.
Okay now, here's the thing.
In order to change to signal
things inside the cell,
in order to affect cell
function, there's a series
of enzymes called phospholipases
that play a key role in the cell
by hydrolyzing the lipids that
I showed on the previous slide.
So they go through and
they hydrolyze these esters
or these phosphodiesters
in particular spaces,
where in particular places
where each enzyme has a
particular specificity
for a specific ester,
for example.
And the consequences here
are really fascinating.
This has the effect of
altering cell function
by providing molecules that can
then bind to proteins and also
by altering very local, to a
local extent the composition
of the membranes that are close
to membrane-bound proteins.
Okay, so we're starting to see
that lipids play important roles
in cell signaling and I
also want to talk to you
about cell-to-cell signaling.
Okay, on this slide over
here, this is an example
of lipids playing a role
of the cells signaling
with inside the cell.
And now, I want to talk to you
about how cells talk
to each other.
Okay, so there are
many different modes
of cell-cell communication.
Many of them involve the
[Inaudible] communication,
the touch, the cell-to-cell
contact
that we discussed
earlier in this class.
But there are also
ways for cells
to secrete lipid-based
molecules that then go off
and signal other cells to
get in on the activity, okay,
to get in on the action.
And let me show you
an example of that.
So platelet cells
release this TXA2.
This should be a subscript,
that's released by platelets
and that amplifies
clot formation.
So TXA2 is synthesized
from a compound called
the arachidonic acid.
I'll show you the structure of
arachidonic acid in a moment.
Long story short, you get this
compound here that is derived
from fatty acids and
this has the effect
of signaling other
platelets to start clotting,
to initiated clotting
and also to initiate,
to get smooth muscle
to relax in the area
to encourage blood
flow into the region.
Okay so, small molecules are
providing important signaling
molecules for cell function.
It turns out this is
actually very complex.
Okay and I'm going to
just gloss over it.
I encourage you to read
more about it in the book.
It's an absolutely
fascinating topic and in fact,
I could devote a whole
lecture to nothing but this.
This sort of cell-to-cell
communication involving these
leukotrienes and eicosanoids
is actually remarkable, okay.
So what I'm showing you here are
a series of different compounds
that are synthesized by
various enzymes shown in blue.
So each one of these blue
arrows is an enzyme-catalyzed
transformation and
we'll take a look just
at one example of this.
So for example, this
leukotriene A4 has an epoxide.
An epoxide, of course, is
a terrific electrophile
and in concert with glutathione,
this epoxide can be
nucleophilically attacked
by the thiol from
the glutathione
to give you this conjugate
that now has a thioether bond.
This thioether, this compound
here keeps going until it gets
to this leukotriene
D4 which is then used
to stimulate leukocytes,
neuronal cells, et cetera
and most importantly, bronchial
and vascular smooth
muscle in the lung.
And so what we're
seeing, what I'm trying
to show you is a cell signaling
cascade that eventually leads
to inflammation in the lung
and asthmatic response.
This is what happens when you
feel your lungs tightening
up as you can't breathe anymore
when you're getting an allergic
attack or an asthmatic attack.
What's happening is all
of the signaling
molecules are implicated
and so this coordinates
by having lots
of signaling molecules
running around.
This coordinates the response of
very diverse cell types ranging
from neurons to macrophages
and it's actually essential
that you coordinate
these things, right?
You wouldn't want, for example,
the bronchial cells contracting
and you know, constricting
unless you had other cells
that can, you know, redouble
efforts to try to deal
with whatever it is that's
causing their pain, right?
You want the macrophages to be
on in the action to chew apart
and recognize whatever it is
that's causing that, you know,
the cells to contract.
Okay so, there are
many different classes
of these eicosanoids.
They all share a common core,
this arachidonic acid over here.
And note that this is
simply a fatty acid.
This can be synthesized using
exactly the polyketide synthase
that I showed earlier,
the fatty acid synthase
that I showed earlier,
the one with the robot arm
that moves back and forth
between the different
subdomains.
Okay, let's take a closer
look at arachidonic acid.
So the first step
in the modification
of arachidonic acid is
absolutely fascinating.
There is a class of enzymes
called cyclooxygenase
that add oxygen across
these two double bonds
to give you this
prostaglandin molecule.
Okay and this is the very first
step in all of the cascades
that I've been discussing
with you.
The very first step here is
this oxygen being added by,
catalyzed by two
different enzymes,
cyclooxygenase1, and COX-2.
They're abbreviated,
C-O-X or COX.
And all of these, so once
this thing is synthesized,
there's a whole bunch of
other enzymes that then lead
to inflammation, okay?
So pathways like these go down
to lots of different cell types
down here and start
stimulating inflammation.
So if we can block this step
up here then we have
the very effective way
of dealing with inflammation.
And by the way, by
inflammation, I mean fever,
I mean you know, swelling.
I mean like you know, the
immune system starting
to spiral out of control.
Okay, just you know, walloping
on whatever is nearby
including itself, okay?
And that's a bad thing.
So for over a century, humans
have been using aspirin as a way
of combating this pathway.
Aspirin, it turns out
is a covalent inhibitor
of cyclooxygenase1 and 2.
It's fairly nonspecific.
And if we zoom in on the
cyclooxygenase active site,
here's the active
site over here.
This is arachidonic acid
bound to the active site
and in this red stick model
right here is a serene hydroxyl
that's found in the active site.
This serene hydroxyl is
acetylated by aspirin.
So that aspirin transacetylates,
it transfers the ester
from the aspirin ester to
the cyclooxygenase ester
from this side to this side.
You can imagine this is a very
easy reaction, very facile,
very low energy, analogous to
all the transesterase reactions
that we saw on Tuesday, right?
On Tuesday, I was showing you
all the thioester exchange
reactions, right?
This is totally analogous
to that.
You eat the aspirin; the
aspirin readily flows
through your whole body
in literally minutes,
flowing readily through
your cell membranes
because it's hydrophobic enough.
And then when it
reaches this active site,
it starts attacking
this hydroxyl
and covalently acetylates it.
That has the effect of shutting
down this pathway over here
and doing so, prevents
the cells from responding
by inflammation symptoms.
Okay, so that shuts down
very quickly the fever
or whatever it is that
you're dealing with.
Okay, make sense?
Headaches, fever,
et cetera gone.
All right, so this notion
that aspirin is actually
a covalent inhibitor is a
relatively new one.
I think it was discovered maybe
two decades ago or something
like that and so --
oh, actually sorry,
the molecular mechanism
underlying its covalence
relatively new and
absolutely fascinating.
Okay, it's actually --
I'm pointing this out because
it's actually very hard
to get molecules that
covalently modify targets
in the cell approved by the
Food and Drug Administration.
Medicinal chemists in
general are leery of compounds
that form covalent
bonds with targets.
And the reason is
some misplaced fear
of allerginicity,
of antigenicity.
There's a worry that you're
going to modify cell proteins
and that in turn will
stimulate the immune response.
And to those who say that,
I always point to aspirin
as a prime example of why
that's not such a big concern.
Now having said that,
there is a small percentage
of the population that has
something I think called
Reye's syndrome.
There must be one
person in here.
We have about a hundred
people in this class.
Is there anyone who's
allergic to aspirin?
All right, it would be
about 1% of the population.
So those people allergic to
aspirin actually have you know,
stimulated immune response
to this covalent adduct
or perhaps another
covalent adduct.
Okay so lipids and fats,
of course, play a key role
in our everyday lives as soaps.
So all the lipids and
fats that I showed you
on the plasma membrane slide
can be hydrolyzed quite readily
using catalytic amounts
of sodium hydroxide.
This is typically lye, L-Y-E,
and this quantification
reaction is something
that humans have been
doing for millennia really.
Most notably, of course,
by the protagonists
of Fight Club
pro/antiantagonists
of Fight Club who use excess
lipids from liposuction
to make really high-end
soaps for boutiques.
I happen to love that movie.
If you see the movie, check out
the microscopy at the very end
of the opening credits.
It's pretty awesome.
If you haven't seen this
movie, you should see it just
for the saponification
scene alone [laughter].
Okay, here's an old tiny
picture showing, you know,
how historically this
process took place.
So when you do this,
you can isolate
out depending on how you do it.
If you do this in the presence
of ethanol, you can isolate
out esters, fatty acid esters
that basically are nothing
more than biodiesel.
This is stuff that you
could put in the tank
of your biodiesel
Mercedes Benz and use it
to drive around the campus.
The other thing that's
isolated from this is glycerin
which is a very valuable
commodity item.
It's used pretty extensively
as moisturizing soap.
And actually the glycerin
soaps are slightly better
than the fatty acid soaps
so when you hydrolyze this,
the study of hydroxide
and no ethanol,
you end up with fatty acids.
You end up with two
kinds of soap really
and the glycerin soaps wash
away a little bit better
than the fatty acid soaps
and they're slightly
more moisturizing.
All right, let's see.
I think that's all I have to
say about fats and lipids.
And if you have any
questions about fats
or lipids, I'll take them now.
Anything you ever
wanted to know about fat
but were afraid to ask?
All right, what about cellulite?
An inevitable consequence
of being human.
Don't panic.
All right, diversification.
So I want to switch gears
and I want to talk to you
about other kinds
of polyketides.
I've been showing you
the simple ones, okay.
But it turns out, this is
an amazingly rich class
of compounds.
This class of molecules
extends to all kinds
of different antibiotics
and all kinds
of different circumstances.
And some of these have
nice bitter flavors
like this hop constituent.
Others, you know, we just don't
even know really what they do
but we could find all
kinds of examples.
So one way of diversifying
these is to have hydroxides
in the you know, these sort of
these enolates in the structure,
these enols in the structure
and that sets you up very neatly
for lactonization,
this molecule here.
So this is the way
that we can start
with straight chain precursors
and get to rings, okay?
This is a very simple way
of getting to a ring, right?
We have an electrophile which
is the thioester up here.
We have a nucleophile
which is this hydroxide.
Note that this even
though it's an enol,
it's not attacking like an enol.
It's actually attacking -- it's
going to form an enol ester.
And note too, the
compound that results.
Oh, okay. So this sets you
up very neatly for the attack
because this is perfectly
poised in terms of distances
of nucleophile and
electrophile up here, okay?
Beautiful chemistry.
So this is going to set us
up to make really
complex things, okay.
You're ready for complexity?
Brace yourself.
Here's what can happen.
What can happen is you can get
to these aromatic compounds
that I alluded to at
the very beginning
of this discussion
of polyketides.
And all you do is
you simply start
with a straight chain precursors
that have carbonyls on them.
Recall that this is one of
the products from the sequence
that I showed when we talked
about fatty acid synthases.
And I showed that
it was very quickly,
this intermediate was very
quickly reduced by NADPH.
But here, it doesn't go
on to immediate reduction.
Instead this ketone hangs
around and it sets you
up for an aldol condensation.
My all-time favorite
reaction, the beautiful aldol,
object of wonder, a reaction
of wonder because it does
such an effective job at
making carbon-carbon bonds.
Okay, so this is a
really classic reaction.
A major challenge however is
these polyketide things are
ridiculously reactive.
I mean, you can really readily
isolate them so well in the lab.
You could freeze them in
benzene and work with them.
And I actually have a
colleague who does that.
They're really nontrivial
to work with.
The polyketides are so desperate
to do this aldol reaction.
Everything is so nicely
set up because it's going
to form this beautiful
six-member ring
in intramolecular fashion
but these are far too
reactive to really work with.
And it's remarkable that that
cell has evolved mechanisms
to work with these
intermediates.
So a key attribute of
this class of enzymes is
that they can't have these
intermediates kind of bumping
around the cell and looking
for the next active site.
Rather, everything has to
be tightly held in place.
The intermediate has to move
from one spot to the next spot
to the next spot and it can't
have time to flap around
and start making random things.
Okay so here's what can
result and this is the thing
that really blows me away.
The number of molecules that
result is really astonishing
in its diversity,
in its complexity
and its sheer chemical beauty.
These things are really
wondrous molecules.
Tetracycline is a very
effective antibiotic.
I believe we talked about
it, that in the context
of ribosomal protein synthesis.
These amphotericin is
another effective antibiotic.
This one forms little
pores in the cell membrane.
And in fact, there's
greater than 50 of the 2000
or so approved pharmaceuticals
are directly polyketides
and probably another
couple of hundred
or so are derived
from polyketides.
So these are synthesized
on massive scale,
not by organic synthesis.
Rather, they are isolated
through fermentation
by microorganisms and
the microorganisms are
like little factories that
whip these things out.
And they're incredibly
valuable, right?
Something like tetracycline
is sold probably
in the ton level per year and
it's used quite extensively.
Okay and I've even,
in the little italics,
that actually tells you
what organism this can be
isolated from.
So each one of these
compounds is synthesized
by a different microorganism.
Now, you can imagine it takes
the microorganism a lot of time
and energy to make
these compounds.
So why do you think it is that
the microorganism is doing this?
Why would a microorganism
want to kill,
why would the microorganism
want to synthesize compounds
that are going to
kill microorganisms?
>> Maybe you want to compete?
>> What's that?
>> Maybe you came up
to compete [inaudible]?
>> Ah, very good.
So it's the competition
for scarce resources.
These microorganisms are
fighting chemical warfare
against each other.
And they're in this
constant arms race to try
to develop better
chemical compounds
that will kill off their
competitors and if they succeed,
they wipe out all
of their competitors
and have the field free
to themselves to chew
on all the tasty
stuff that's around.
The problem though is that their
competitors are also developing
ways to nullify and
neutralize these compounds.
And so, these competitors
are really, really good.
And I think we've seen
one example of this.
Tetracycline can be readily
pumped out of the cell
and so the competitor
microorganisms develop these big
pumps that sit at the
cell surface and burn ATP
and operate night and day
to grab onto tetracycline
and other molecules like this
and simply expel those molecules
from the inside to the
outside of the cell.
And there's other strategies
that we've seen earlier in terms
of antibiotic resistance.
But it's a constant
cat-and-mouse game
between the microorganism
that's trying to compete
and the other microorganisms
that are trying not
to get killed.
So we chemists are spectators
watching from the sidelines
of this ongoing multimillion
year war.
This is an endless war.
It's a war that's
been going on longer
than humans have
been on this planet.
And this fascinates us
and it gives us all kinds
of opportunities to
control the pestilent bugs
that inflict us as well.
And so we do things like grow up
these things in large quantities
and then use them to
kill off the bacteria
that are infecting us yet
at the same time, of course,
we're knowing full well
that we're highly
dependent upon other bacteria
that have a symbiotic
relationship with us
and are affected by
these compounds equally.
All right, I'm a
little off topic.
I love this topic so forgive me.
I want to get back to what
we want to talk about today.
I want to talk to you
about this synthesis
of complex molecules like this.
And good news, a lot of what
we've already seen in terms
of fatty acid synthase applies
to much more complicated
polyketide synthases.
And so let's talk about how
erythromycin is synthesized.
So erythromycin is a
very common antibiotic.
It's used, I believe,
sometimes for treating acne.
You might have encountered
it at some point.
It's used for all
kinds of things.
It's a pretty effective
antibiotic.
How is it synthesized?
It's synthesized in large --
it's synthesized by three large
macromolecular machines, okay.
And these machines are
organized in the order
of the action along the assembly
line for this molecule, okay.
So again, there's three
open reading frames
where an open reading
frame refers
to a single gene that's encoded
at the DNA level, translated
or sorry, transcribed into
MRNA and then translated
as one contiguous protein.
So you have three of
these are that lined up.
Each one of these letters
tells us about the identity
of a different active
site domain.
So a different enzyme
active site domain up here.
Okay and so a lot of
these are familiar to us.
This is acyl carrier
protein, ACP.
This is which attaches
the, in this case,
[inaudible] starting
material to a thioester,
the phosphopantetheinyl
robot arm.
And then the next
step is a ketosynthase
so that does a Claisen
condensation.
Next step over here is
an acyl transferase.
That's going to then
transfer the intermediate
to some other thiol,
a ketoreductase.
We've seen ketoreductase
then this gets transferred
over to the acyl
carrier protein.
And then again, another Claisen,
another acyl transferase,
a ketoreductase, et cetera.
And so the product from
each of these, so over here,
here's the ketosynthase.
Here's the product
of the Claisen.
Okay, oh. After the
reduction step takes place.
Next step, another ketosynthase,
et cetera to the
acyl carrier protein.
And so each one of these
ketosynthases elongates the
thing by three carbons.
It introduces a new
propynyl fragment.
And then each ketone reductase
gives us a new hydroxyl and note
as usual, the stereochemistry
of that hydroxyl is
rigidly controlled.
Okay, and long story short,
at the very end over here,
the last step is carried
out by this thioesterase
that's actually rather
than doing a conventional
hydrolysis of this thioester,
it has the hydroxide, the
terminal hydroxide come looping
in and winging over here
to attack this carbonyl,
this electrophilic
carbonyl thioester.
And you can imagine this active
site bending the intermediate
structure to position this
hydroxide right up close
to this electrophilic carbonyl.
Beautiful chemistry,
I'm a big fan.
After this is synthesized by
these polyketide synthases,
things are passed off to a
series of tailoring enzymes
that then get in on the action.
These include P450 [inaudible].
This is similar to
cytochrome P450 that we talked
about earlier in the class.
This is an oxidase
and an effective enzyme
for oxidizing things.
And in this case, it's going
to be oxidizing this
tertiary carbon right here
to give us a tertiary alcohol.
And then there's a bunch of
other enzymes that append
on some carbohydrate
functionality.
And then the whole thing is
quickly pumped out of the cell
because the cell doesn't want
this very dangerous antibiotic
hanging around.
Okay, so the cell has
a complicated machine
and at the end of it, there's
a mechanism that's actually not
that well defined that kind
of sweeps up the product
and scoots it out the
door before it has time
to wreak havoc on the
interior of the cell.
Let's take a look at the
actual polyketide synthase.
This is from a structure that
was solved by my colleague,
Sheryl Tsai in the Chemistry
and Molecular Biology
and Biochemistry
Departments here at UC Irvine.
And she did this when she was
a post doc and it's really one
of my favorite structures
of all time.
And I have a lot of
favorite structures
but this is a good one.
Okay, so in the very center,
this is the acyl
carrier protein.
Oh okay, big picture first.
What we're looking at
is we're looking at one
of these polyketide
synthase machines
that I showed earlier
on a previous slide.
So over here, recall that I
showed that there are three
of these and I'll show you how
they're all linked together
in a moment.
But each one of these
is arranged
in this structure of a donut.
Okay and in the very center
is the acyl carrier protein
with the phosphopantetheinyl
arm that's then going to swivel
between each one
of these subunits.
Okay, we now don't think it's
entirely a swivel action.
It's a little more complicated
but I think that's a
good model for us to use.
So the first intermediate,
the robot arm comes over here
to the ketone reductase
over here.
The Claisen condensation
reaction is catalyzed
and then it goes up to the
acyl transferase and then
over to the ketone reductase.
Oh sorry, I got this wrong.
Ketone synthase at the
top, acyl transferase
and then ketone reductase
and then thioesterase, okay?
So these big machines
are arranged kind
of the way you would arrange
the perfect assembly line
so that everything
is right there
and the intermediate
could just hop
between those different
active sites.
This is a thing of
beauty, isn't it?
Check this out.
This is the fatty acid
synthase that I showed earlier.
And it turns out that
these form dimers, okay.
And I'm showing you this
because I want to talk to you
about how the arrangement
of these donut-shaped things
into bigger assemblies, okay.
So over here, I said that there
were three of these donuts.
It turns out that these
donuts are actually dimers.
Before I show you that,
I need to show you
that in fact actually
I need to show you
that this dimerization
is precedented.
So this is the fatty acid
synthase that I showed earlier
at the very first slide
of today's lecture
and what I neglected
to tell you was
that actually this
forms a neat dimer
where one fatty acid is
synthesized on this side
and a different fatty acid is
synthesized on the other side.
So in similar fashion,
oh and by the way,
this structure was
solved by an added bond.
In similar fashion, oh
and it kind of looks
like a gingerbread man, right?
It kind of looks like
a gingerbread man.
You see the feet down
here, the arms, okay?
And this is the narrow waist.
Anyway, in a similar
fashion, the arrangement
of these donuts is
also in dimer form.
Okay, so check this out.
Okay so, here's one
donut over here.
This is module one up here.
This is module two,
module three.
And so what's actually happening
is this is actually a dimer
of two assembly lines
that are wrapped together
in double helix fashion.
And this one in blue is
synthesizing one polyketide
and this one in red is
synthesizing the same polyketide
but in parallel.
Okay so in other words,
the assembly lines
that make these complex chemical
structures are actually running
all the time and there's
multiple assembly lines
that are identical that
are running right alongside
each other.
Okay, so this kind of like
going to, I don't know,
a Mazda factory or something
and finding it's not just one
assembly line on the floor
but two assembly
lines that are moving
in parallel to each other.
And both producing the
same car at the very end.
Okay so, again this is
the -- oh okay, so again,
both polyketide synthases and
fatty acid synthases are dimers.
This one being a circular dimer
and this one being a
head-to-tail linear dimer.
When things move through
the polyketide synthase,
the intermediates precede down
this in unidirectional fashion.
So they're starting up
here, moving down here,
moving down here, moving down
here and they're handed off
by all those acyl
transferases, okay,
which then transfers
the intermediate
to the next acyl carrier
protein which then takes it
down to the next donut, okay?
And then in the end, you end
up with this complicated
erythromycin over here
or erythromycin precursor
over here.
The fatty acid synthases
as we discussed however,
rather than ending things off,
instead the same intermediate
keeps bumping between each one
of those different active sites.
And in fact, the intermediate
cycles through seven times
until you end up with this
long chain of fatty acid.
And at the very end,
evidently it falls off
because there's no more
room for it to expand.
Okay, it's run out of
place in active site.
Okay, make sense?
So you can all draw diagrams
to describe how this works
and even make some predictions
about that sort of thing?
You could see how that would
be pretty powerful, right?
Okay. I want to switch
gears now.
Okay, oh, oh before I do, any
questions about polyketides?
Anything you want to know
about polyketides, that topic?
Okay, yeah, Chelsea.
>> So this is your [inaudible]?
>> Yeah.
>> Two molecules that can
result in this diagram?
>> Yes, two molecules of
this erythromycin precursor.
>> Okay.
>> Yeah. You know,
okay, I can't resist.
There's so much more
to tell you about this.
One of the really
cool areas in this,
of research in this
area is to start moving
around these active site
domains so that in the end,
you end up programming
a different structure.
You might imagine setting
this thing up so that instead
of doing the reduction,
maybe you end
up with a ketone
over here instead.
And so this is a really active
and very exciting
area of research.
So okay. All right,
let's move on.
I want to talk to you
about a different class
of small molecules that
are synthesized by cells.
And these are molecules
called terpenes.
These are all built from
five carbon precursors, okay.
And these are familiar things.
Geraniol, for example,
has that kind of spicy,
wonderful flavor
of ginger, right?
You've all tried
that at some point.
These are things that are
really familiar to us.
Polyisoprene is natural
rubber, right.
That's actually isolated
from these rubber plants.
And all of these are built
up from five carbon
isoprene precursors.
Let's just go ahead and
count some carbons here.
Okay, so we're going to
have one, two, three, four,
five and then notice that
there is a red bond right here.
That red bond is going
to be joining together these
five carbon building blocks.
And in the end, these
could be strung together
in very long polymers, right.
so in the end, you have
isoprene, isoprene, isoprene,
and then a very, very
long polymer that has
that wonderful stretchy feel
that natural rubber has, okay?
So let's see, I have
to introduce you
to some important nomenclature.
We're going to call the C10
precursors geranols or geraniols
or geraniol and then we're going
to call the C15 precursors,
that's three isoprenes, farnesyl
or farnesol if it's an alcohol
and then the C20 precursors,
we're going to call
geranylgeranyl alcohol, okay?
So C20 is like two C10's
put together hence the
name geranylgeranyl.
Okay, and I'm going to be
using that because it turns
out that these are precursors
to much more complex compounds.
So totally analogous to
what I was discussing
when I was talking about
polyketides where we start
with these kind of
simple linear compounds,
terpenes start linear,
start simple and then
like origami become
amazingly complex.
And that complexity is going
to be a really great
topic for us to discuss.
All right, let me show you
where we're going
with that complexity.
So up at the top, these are
straight chain precursors
that I showed on the previous
slide, geranyl pyrophosphate,
farnesyl pyrophosphate, and
geranylgeranyl pyrophosphate.
These again are synthesized from
five carbon building blocks.
Two building blocks,
isopentenyl pyrophosphate
and dimethylallyl pyrophosphate.
I'll show you those two in
closer detail in a moment.
So let's suspend for a
moment where they come from.
These guys over here,
these straight chain precursors
can be folded up by a class
of enzymes known as terpene
synthases or terpene cyclases
and the net effect is you get
out these very complex skeletons
of really complicated molecules.
Okay, so for example,
this is the skeleton
that eventually will
become taxol down here.
Taxol results from a
series of tailoring enzymes
that oxidize various carbons
in this taxodiene framework
and then append on things
like this little short
peptide fragment,
this dipeptide fragment.
Okay, so you start off simple.
You start off linear.
And then you get increasingly,
you fold things up.
You cyclize and then you
modify after it cyclized.
Okay, and what's great
about this is this follows the
same formula that I gave you
when we talked about
the fatty acids.
You start off simple.
You start two carbon, three
carbon building blocks.
You build linear things
and then you cyclize them
and then you get
more complicated.
Okay, exact same formula
here except now we're going
to build everything
out of hydrocarbons.
There's going to be
very few oxygen's.
Oh, and by the way, these are
familiar compounds as well.
Limonene, this is isolated.
I think you guys do
this, isolation, right?
From like orange peel?
You did this back
in Chem 51A lab
or something like
that or 51B lab?
Yeah, so this is the
compound that you isolated.
You've learned how to
synthesize by cells.
It can also be oxidized to give
menthol a nice wintergreen kind
of taste, a nice minty taste.
On this amorphadiene is
modified to make artemisinin.
This is the compound that
has important antimalarial
properties and is extremely
useful therapeutically.
All right, this is kind
of the big picture.
Everyone with me so far?
All right, let's move
down and start looking
at the nuts and bolts.
Oh, more big picture, sorry.
The enzymes that make the --
that do the cyclization
has remarkable,
remarkable chemical
specificities.
Okay, so starting from
the same precursor,
different terpene synthases
can direct the synthesis
of different stereoisomers,
these bisect the compound
and then other terpene
synthases can direct whether
or not you get spiro-fused
rings.
This is a spiro-fused ring
when two rings are joined
at a [inaudible] carbon,
that's called spiro-fused.
These are called, you
know, scissor transfused
and you can even
get monster rings.
So whether or not you get a
five-seven or a six-six ring
or a larger ring,
it's all controlled
by these terpene synthases
and they're really,
really good at what they do.
Okay, so they are
controlling stereochemistry
and regiochemistry in
water at room temperature.
Okay, I've already talked
about other, there's lots
of other complex
terpenes I can show you.
This is actually a
fascinating class of molecules
and they're targets for
synthesis, et cetera.
Let's talk about how
they're put together starting
at the most basic
level down here.
So I told you that all
of these are synthesized
from five carbon building blocks
that are going to be isoprenes
in the finished structure.
Okay, where an isoprene
is this compound here
of the five carbons with the
carbon-carbon double bond
in the middle.
Okay, what happens is
there's one starting material.
It's this dimethylallyl
pyrophosphate.
This has an allelic
pyrophosphate
as its leaving group.
And no surprise, the
active site that's going
to catalyze this reaction has,
of course, a magnesium available
to act as a Lewis acid
and stabilize the pyrophosphate
making it a good leaving group.
Okay and the product
here is going
to be the ultra-stable
allelic carbocation, okay?
So what happens is the
allelic carbocation forms,
this can form resident
structures hence its stability.
The residence structure will
be at the cation, sorry,
the tertiary carbocation.
But it's set up in a way,
the tertiary carbocation
is held away
from the incoming nucleophilic
isopentenyl pyrophosphate.
This compound here and this sets
you up for a nucleophilic attack
which then gives you a
new tertiary carbocation.
And then in the final step, a
beta elimination step gives you
in the end this geranyl
diphosphate, okay?
And then you can imagine this
could be the starting material
for additional IPP's to be added
on using exactly
the same chemistry
that I'm showing you here.
Okay, make sense?
All right, let's
take a closer look.
All right, so it turns out that
these things are synthesized
using reactions that are, you
know, completely understandable.
So now I'm stepping back
and I'm showing you how
to synthesize these
compounds down here,
IPP and dimethylallyl
pyrophosphate.
First, these two can result from
a simple isomerization, right?
That's this carbon-carbon
double bond
over here getting isomerized
to form the more stable,
more substituted [inaudible]
of dimethylallyl
pyrophosphate, okay?
And there's an enzyme that
catalyzes that reaction, okay.
And active site looks like this.
It has a manganese --
it has a manganese ion
that can form a Lewis acid
relationship with a glutamate
in the active site and this can
act as a base to catalyze this
or to do this deprotonation
over here.
And then there's a nearby
thiol that acts as an acid
to protonate the
resulting carbanion
as this thing gets deprotonated.
Okay, let's start
at the beginning.
Okay, so everyone, does this
reaction down here make sense?
Okay, so let's talk about how --
so if we can get to here
then we can get to here.
How do we get to this IPP?
Okay, IPP is synthesized using
more or less the same sort
of chemistry that I've shown
you previously in the context
of fatty acid synthesis.
Okay, it's basically going to be
set up using a series of Claisen
and aldol condensations.
Okay, so all carbon-carbon
forming events are going
to use the aldol reaction
and there's an enzyme called
HMG COA synthetase that's going
to synthesize this HMG
COA molecule over here.
Okay, from HMG COA, you
can then readily get
to this compound here using
phosphorylation from ATP
and then so you hydrolyze
this ester, reduce the --
or sorry, hydrolyze this
ester and then reduce it
down to the alcohol and then ATP
then transfers pyrophosphates
to the primary alcohol
and then you can do an
elimination reaction.
Okay, so this is
a decarboxylation
and then elimination of alcohol.
Does anyone remember the context
where we saw a reaction
like this on Tuesday?
We've seen the mechanism
of this reaction before.
Do you recall when we saw it?
On Tuesday.
Decarboxylation giving
us an enol or enolate?
Do you remember that?
What [laughter] was the
-- what was the sequence?
Why do we do that?
>> Why did we do?
>> Yes, so we did this
decarboxylation, right?
And it gave us an enolate
and then what was
the enolate used for?
Do you remember?
>> When we [inaudible]?
>> It was the Claisen, right?
So this is being done
by ketosynthase, right.
So this is a totally
analogous chemistry.
Nothing new here, okay?
Thanks, Carl.
You rescued us.
Okay, then the thing is,
this reaction over here
with mevalonic acid
is synthesized.
Oh, here's the reduction
here by HMG COA reductase.
This mevalonic acid
then becomes a precursor
for the eventual
synthesis of cholesterol.
So if you can shut down
this step or this step,
you'd have a way of preventing
formation of cholesterol
and that actually is
pharmaceutically a very
important reaction.
This is a reaction that is
very extensively inhibited.
So HMG COA reductase
inhibitors include compounds
like Lipitor, Mevacor,
et cetera.
And many of these start
to look a little bit
like the mevalonic acid, right?
So this compound here is kind of
like a folded-up mevalonic acid
and then up here, you
can imagine hydrolysis
of this lactone will
give you something
that looks a little bit
like mevalonic acid as well.
So these compounds are
very widely prescribed.
It's literally billions upon
billions of dollars a year
of this stuff is sold.
Probably everyone you know
over the age of 50 is on one
of these compounds because
they're so effective
at suppressing formation of
cholesterol and that seems
to have such positive benefits
for cardiovascular health
and also even some
anticancer abilities as well.
So you know, these are
remarkable compounds.
They've changed how
we treat patients.
And they're sold in
enormous volumes.
Okay, and again, these
things look like the product.
So they work as product mimics.
All right, so I've already
shown you how to connect
up the isoprene units and one
thing I need to point out is
that this is an example
of a very rare non-aldol
carbon-carbon bond
forming reaction.
This is not very typical
and that makes it kind
of special, I think.
Okay, how much time do I have?
All right, last mechanism
of the day.
This one blows me away.
There's a class of it.
The next step here is to take
this straight chain precursor
and then fold it up into
some semblance of the rings
and then it cyclized things.
So the class of enzymes
that does this, again,
are called terpene synthases.
And here's one example of this,
a protein called
aristolochene synthase.
This starts with
farnesyl pyrophosphate.
The way this works is that
the enzyme has a very deep
active site.
It's kind of like
a deep cave, okay?
So deep, deep active
site that's shown here.
right here at the
mouth of the cave,
this is actually a transition
state analog, a product analog
that -- oh, sorry,
substrate analog
that the enzyme cannot act upon.
So when the crystallization
took place, this shows us
where the active site is.
And again, the active site
is this really deep cave.
Now, at the tips of my fingers,
over here, there's a series
of carboxylate bearing
side chains
that can then chelate
magnesium ions.
Okay, those magnesium
ions, as you might expect,
form a Lewis acid relationship
with the pyrophosphate.
Analogous to what we saw a
couple of slides ago, okay?
Similar chemistry.
The effect though is
that this draws the mouth
of the cave shut.
The magnesium's come down
and the pyrophosphate gets
coordinated to those magnesium's
and that draws the cave shut.
Now, the interior of the cave,
the kind of the back side
in here is highly hydrophobic.
This makes sense, right?
The molecule that it has to
bind is very hydrophobic.
It has 15 carbons in a row.
So this side over here that's
going to be at the mouth
of the cave, binding
to the magnesium ions is
really nicely hydrophilic
and then this side over
here is hydrophobic.
So what happens is as the
cave shuts, all of the water
in the cave gets squeezed out.
Okay, so all of the
water gets pushed out.
This will be critical in a
moment and I'll tell you why
that is in a second, okay?
Now, okay -- so we've
seen the binding
by the farnesyl pyrophosphate.
The second step here
is in the active site.
The active site is
shaped in such a way
that the C15 piece is
pushed into some semblance
of the correct product
so that the one enzyme
over here that's going
to be synthesizing aristolochene
will have an active site that's
shaped to push this
straight chain precursor
into these two rings.
But the one that's going
to be synthesizing say this
spiro-fused ring is going
to start with the same starting
material but in this case,
it's going to be pushing
the farnesyl into something
that looks like this, okay?
Next step here is again, to
clamp down the diphosphate
with the two magnesium ions
and push out the water.
The magnesium ions,
again, act as Lewis acids
and they trigger the
dephosphorylation reaction.
They stabilize the pyrophosphate
leaving group allowing it
to take off.
And that has the effect
of triggering a carbocation
cyclization
that eventually leads
you down to one
and only one product
like this one.
Now, the complicated
thing here is
that this carbocation
cyclization is not some simple
one stepper.
This is a really
complicated reaction.
I'll show what it
looks on upcoming slide
but it involves multiple steps
and it involves a requirement
to stabilize carbocations
preferentially carbocation
intermediates preferentially.
And so the enzyme has evolved
this active site that has,
for example, aromatic
residues positioned neatly
over carbocations to
stabilize as carbocations
by cation pi interactions,
the same interactions
that we saw very early in
this course when we talked
about noncovalent binding.
In addition, many of these
compounds are synthesized
through other intermediates
and the active site has ways
of actually then
catalyzing reactions
on those intermediate
compounds as well.
Oh, and then the final step, of
course, is the beta elimination
to produce the product.
And this is analogous
to the beta elimination
that we saw earlier where the
farnesyl pyrophosphate was
synthesized through an
elimination reaction.
This is that same step.
Okay, so these are truly
wondrous reactions.
And these are things that we
can talk about quite a bit.
I would love to talk to
you about them some more
but I actually want to reward
all of you who have shown
up today with a very,
very short and easy quiz.
So I'm going to stop
here actually.
So, please take out
a half sheet of paper
and get ready for a quiz.
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