Introduction to Chemical Biology 128. Lecture 15. Glycobiology & Polyketides.

>> So very briefly, let me just
review what we saw last time before we go on to
some new stuff. Last time we were saying
that in the human body, there's really only nine
monosaccharides that are found, that are used in carbohydrate
chemistry and this is kind of astonishing because
if you think about it, there really could be an
infinite number of ways of arraying carbons that are
hydrated or hydrates a carbon, which is the chemical
definition of carbohydrates, but there's only nine
of these that are found. And we talked very briefly
about the anomeric effect.

I don't think this
is such a big deal, so I'm not going to dwell on it. We talked more extensively,
and this is actually important, that carbohydrates interconvert
between a hemiacetal form and an aldehyde or ketone
form and that aldehyde or ketone form is reactive. That's the form that
has an electrophile that can start reacting with
surface proteins in your cells and then cause eventually
advanced glycosylation end products that we'll
talk about today. And then we also
talked about the oxonium and oxocarbenium ions.

These are key ionic
intermediates that are used that are observed
when you either form or break glycosidic bonds. And then we talked at the very
end about oligosaccharides, things like starch that are
long strings of carbohydrates that are strung together
by glycosidic bonds. Okay, now today I want to
talk to you about complexity and about polysaccharides that
are a good deal more complex. So oligosaccharides are,
we're going to define them as simply– let's say, sorry. Polysaccharides are going
to be just polymers of one or maybe two or three, you know,
some small number of subunits, okay, like glucose
strung together in a repeating chain could
give us cellulose, right? We talked about that.

We talked about how wood
could form, or wood if formed from cellulose which
is formed from glucose, so the polysaccharide of glucose
is called cellulose, okay, so that's one form and we
also talked about glycogen, the other form, depending on whether it's the
alpha or the beta anomer. Now today I want to get a
little more complicated and talk about sequences of
saccharides, of oligosaccharides where it's not always
the same carbohydrate, the same monosaccharide
strung together, and it turns out these have important
consequences for the cell.

We're going to see that they
can form epitopes for antibodies to react to that these
can form antigens that antibodies can react to it and they have other
consequences as well. So for this reason,
we're going to talk about oligosaccharides
consisting of carbohydrate oligomers
and more complicated things. Okay, so let's get started. I left off on this final slide
right here and I was showing you that N-linked glycosylation
takes place in the endoplasmic reticulum and
the Golgi complex during export of proteins after their
synthesis at the ribosome. Okay, so this is a very
simplified view of the cell but here in blue are
the expelled proteins or the proteins that are
going to appear on the surface of the cell and these
get modified as they're being transported
by the Golgi apparatus. This is, so this N-linked
glycosylation is important for proteins that are destined
for export to the cell surface but it's not found
at all for proteins that are found inside the cell. Okay, so proteins that hang out inside the cell
don't get modified. It's only the proteins on
the outside of the cell.

So the cell is sort of
this exterior of lots of carbohydrate stuff and an
interior that's sugar free. Okay, this is sort
of the gumdrop model for what a cell would
look like, right? The outside over here has all
the carbohydrates and sugars and the inside is chock full
of proteins and DNA and RNA. Okay, alright, okay, so
let's talk very briefly about how you assemble,
in this case, the O-linked glycoproteins,
okay, so an O-linked glycoprotein
has oxygen that will be the recipient
nucleophile for modification, okay, so in other words either
a serine or a threonine residue, where this beta hydroxyl becomes
modified by the carbohydrate.

So the hydroxyl is
the nucleophile, the electrophile then must be
the monosaccharide that's going to be adding to it and so
the way nature does this to convert a carbohydrate into
an effective electrophile is to attach it to UDP, okay. So this is being attached to UDP and this UDP is basically
a big leaving group. We've seen this strategy before. This is analogous to HEP, where the AMP was just
a big leaving group for a transfer of
a phosphate group. In this case, we want to
transfer this glycan subunit and so we attach it up to a UDP and then use an enzyme called
beta-D-xylosyltransferase, okay, so the key though is that this
is a big leaving group and so that in the end transfers xylose to this hydroxide giving
us a new glycosidic bond. And then over here in this case
over here, this UDP is attached to a different monosaccharide. This one N-acetylgalactose
GalNAc and it also can be used to modify either a serine
or threonine side chains. Okay, so in the end, these over
here that are modified first with xylose turn
into proteoglycans so those are the proteins on the
surface of the cell that are, you know, basically hanging
out as big shrubbery.

The other ones, the
ones that get modified by the N-acetylgalactose,
these GalNAc's then get turned into mucins so these mucins
are proteins that are secreted and are these sort of
water-loving proteins that are important at sort of the membrane interstices
between, you know, air and water phases and so
I'm being coy about this. This is the snot
of the cell, okay. This is the mucus,
mucins, mucus, derive from the same root. We'll take a look
closer in a moment to see all the other
stuff that gets added on. Both of these though
involve more glycosylation. So in all cases that
we're going to see today, we're going to start with a
common core and then we're going to do a lot of modification.

Okay? So here's one
example of this. So after we start
with this common core that I've already shown you,
this was the xylose that we saw on the previous slide. There's a series of
galactocele transferases that use UDP galactose and
then transfer on one galactose at a time, so we start
again with xylose, we add on one galactose in
black, a second galactose in black, and this gives
us the trisaccharide core that the proteoglycan
will use then as sort of its starting unit. Okay? So this becomes
sort of the– it's kind of like the spool that the thread is
going to be wound onto. Many different colors of thread
can wound onto the same spool but in all cases we're going
to use a spool that starts off as this trisaccharide. Okay, and so this strategy here that I'm showing
you can also be used for making repeating
disaccharides as well.

Alright, let's take a look
at some examples of this. Do you remember on Thursday and I'm hoping you all watched
the Thursday video tape, that's why I gave you the quiz, if you watched the
Thursday video tape, the second to the
last slide dealt with those knee joint
polysaccharides, okay or knee joint
oligosaccharides and we talked a little bit
about how these were things that were heavily
sulfated, right? So the sulfates were nice. They're highly negatively
charged. The negative charge attracts
water and it also repels them from each other so
that pushes them apart. This makes a gel that's
pretty cushioning, right? Because all the molecules
are kind of forced apart from each other and there's
plenty of water in between.

Okay, so this is
the start over here, so here's the protein that's
going to be modified, a serine and then as usual it also
starts with the xylose and then [inaudible]
galactose gal-gal, gal-gal and then there's a series
of other modifications that are appended to this. These are things like
N-acetylgalactose that also has a sulfate group,
okay, or N-acetylgalactose that doesn't have
a sulfate group but it still has N-Acetyl
portion, so in the end, we can get, sorry, this
is actually glucose, N-acetyl-glucose that
is, you know, attached, so this gives us
heparin sulfate, which plays important
roles also on just kind of the cell surfaces
and for cell signaling.

We'll see that later. And then over here these
chondroitin sulfates, dermatan sulfates, these
are things that are going to be shuffled off into the
interstices between joints. Okay? Notice that these are
pretty heavily sulfated as well. So these are negatively
charged hygroscopic, meaning they attract water. Okay, so snot and mucus,
I mean everyone's puzzled about what this stuff
could possibly be. I can finally tell you. So these are highly-glycosylated
proteins that are held together by disulfide bonds
and then they have– the counter ions are calcium. Okay, so when these things are
synthesized, they're synthesized with these calcium ions,
okay, and that has the effect of making them really compact
when they're synthesized, right? You have the positive
charge on the calcium, you have the negative
charges on the sulfates, the whole thing kind of curls
up really, really tightly.

Okay? Now, what happens
is when they get excreted, the calcium ions
are stripped off. They're grabbed by machinery,
a transport machinery that exchanges them off, so
the calcium ions are pulled off and then what ends up happening
is, this stuff over here, all this negative charge then
soaks up water like, you know, to a tremendous degree. So the water comes
rushing in over here and that has the effect of tremendously expanding
the volume of these mucins, so they're super
compact when they're in the calcium neutralized
state, the calcium gets pulled away,
and now they expand hugely so this is how your
little mucous, you know, membranes can secrete enormous
quantities of stuff from such, you know, small little cells.

And so for example, snails
leave behind these tracks. This is more or less
the oligosaccharide that depicted here and a
very similar strategy is used in disposable diapers, which
have these polyacrylates, okay, so again you have this
negative charge that's feeling unsatisfied and it's looking for
water and when it finds water, it grabs onto it with
tremendous avidity and grabs onto lots of water by weight. These disposable diapers
are actually this kind of miracle of modern chemistry. Right? These things soak
up enormous quantities of water relative
to their weights. Okay and similarly, the
snot in your nose is soaking up enormous quantities of water
as well and being secreted, okay and that makes it
very effective, right, as a way of forming a transition
barrier between gas phase and then liquid phase.

Okay, one year I got
asked by a dieting student if it would be a good idea for
her to blow her nose more often as a way of secreting
carbohydrates to lose weight and I thought that was
a really novel idea. The truth is though the
quantity of carbohydrate in snot is actually very,
very low because a lot of that stuff is
just water, okay, because of the sulfates, right. This thing is so highly sulfated that there's really very
little carbohydrate there. It's mainly just water. Okay, so let's talk next
about the N-linked glycosides. Now things are going to
get more complex here. We've already seen– Oh, okay, so first of all there's three
major types that are found in eukaryotes; however,
they all have a common core. This is comforting to us, right? This is similar to what
we saw when we looked at the O-linked glycosides, the O-linked proteoglycans
on a previous slide. They all had that
common xylose and GalNAc or GalNAc/GalNAc core. In this case, we're seeing
a very similar structure where we have these
two GlcNAc's in a row, so these are glucose N-Acetyl
glucosamines in a row, so one, two, and then there's
these mannoses over here and then things start
to get a little crazy.

Okay? But you can see again
the strategy here is start with a fairly common core,
not counting the [inaudible] but for the most part, you know,
if things are fairly common and then modify and customize
depending upon the needs. Okay? So everything is
starting off pretty normal but then things start to get
more wild as we go down here. Okay, one last thought. I've switched nomenclatures. Earlier I was showing you
nomenclature like this. This starts to become
increasingly less useful to us. Okay? I mean in this case we
have an amine that's appended to a glucose and it's sulfated,
you know, these things start to get increasingly broke
and so rather than trying to depict these structures and
spending time thinking about oh, is that is a glucose,
is that is a galactose.

Instead, we're going
to transition to a much simpler type of nomenclature that's
based upon three letters, so glucose would be GLC,
galactose would be GAL and mannose would
be MAN, et cetera. So from that, from these three
letters, you can kind of figure out what structures
you're looking at. Okay, now admittedly,
this would be a challenge for anyone in this classroom. Okay? And I'm not
asking you to do that.

Okay, so I'm not asking you to
memorize the nine structures of monosaccharides that
are found in humans, okay, but I just want you to be
comfortable with the idea that these are carbohydrates and
these designate carbohydrates. You never know when this
information is going to useful. Who knows, maybe some
pharmacology class that you take many
years from now. Okay, let's talk a little
bit about the mechanism for glycosyl transfer. It turns out this is
actually not a very straightforward mechanism. I didn't dwell on this in the
case of the O-linked glycosides because those work so well. Oxygen is a fantastic
nucleophile.

Oxygen doesn't have
a carbonyl nearby but for the N-linked glycosides, things are a little bit
more complicated and that's because we're going to attach
things, not to nitrogen found on lysine side chains but instead nitrogen
that's found largely on asparagine side
chains and so the nitrogen over here is next to a carbonyl. It's a carboxamide, right, and this carboxamide
functionality is not nearly as nucleophilic as just
a free-floating nitrogen with its lone pair hanging out. Remember earlier in the class,
I told you that the lone pair on this nitrogen spends a
good 40% of its time hanging out as a resin instructor
forming a nitrogen and carbon double
bond right here.

Okay and because that nitrogen-carbon double
bond is there 40% of the time, the lone pair on the
nitrogen isn't so reactive. It doesn't have some moral
imperative that makes it want to run out the door in the
morning and start looking around for electrophiles. It's extremely unreactive and so instead what happens is
there are specific sequences that present asparagine in a way that allows this
reaction to take place. Okay, so this is an example of
substrate-assisted catalysis. I'll explain in a moment. But note in the structure of
these glycosyl transferases, there's a base, the base can
deprotonate this nitrogen and the electrons
then bounce their way to form the nitrogen-carbon
double bond but then on a nearby hydroxyl-bearing
side chain either a serine or a threonine, there's a proton
that can protonate this carbonyl and so the net result
is an imidate tautomer, this structure here, which
has an unmasked lone pair; otherwise, the lone
pair is hidden away.

It's not so available
really for doing reactivity but after this neat sort of
bendy side chain gets into place and gives you the
perfect proton nearby, then all of the sudden
the lone pair is uncovered and ready for reactivity. Okay and notice and it
doesn't have anywhere to go. It like naked out there
and it's trying to figure out what it should do next and so it will more readily
attack this activated glycan. Note that X over here
is some leaving group. So we already saw earlier today
one good leaving group was UDP and we don't have to
dwell on this structure of the leaving group but
suffice it to say it's something that likes to take off, okay,
it's effective leaving group. Note too, the structural
requirements for this reaction to take place, there has to be
this bend, this 180-degree turn that places the hydroxide in
close proximity to the carbonyl of this asparagine side chain.

If you don't get
that bendy structure, the reaction doesn't take place. That's absolutely
mandatory for this reaction. Okay? So I call this example
of substrate-assisted catalysis because this is catalysis
that's assisted by the substrate itself. The substrate, the starting
material for the reaction, is this asparagine bearing
motif and that participates in this case by providing
acid, Lewis, sorry, Bronsted acid catalysis to
potontate this carbonyl. Okay? So again, certain
sequences are required if R is not correct here. If a serine or threonine
is not available at this position,
it's game over. Okay? So this only works with
certain substrates. Okay? And that actually gives
you a degree of selectivity. Alright, brace yourself. Now things are going
to really complicated. What happens is very complicated
structures get synthesized as N-linked glycans and
then they get trimmed back by scissors, by glycosidases,
the class of enzymes that we saw last Thursday.

So what happens is you get these
very complicated structures coming out and then they're
kind of randomly chopped apart by a series of different,
in this case, glucosidases or mannosidases. These are just simply
glycosidases. These are enzymes that
cleave apart glycosidic bonds and we saw a good
example of that last time when we talked about lysozyme. I think actually
you've seen it now for a couple of weeks running. So here's the thing though,
because these glucosidases and these glycosidases in
general aren't programmed to be really specific about
this bond versus this bond, this has the effect of
introducing randomness onto the surface of your cells.

So really the ultimate glycan
that gets appended and appears out on the surface of the cells
is sort of a little bit random. It's not exactly programmed in. This element of random
dramatically increases the structural diversity of the
chemical compounds found on the surface of your
cells and note too that this diversity is
not encoded by the genome. Okay, this is diversity
that's kind of, this is post-translational
modification diversity that just adds like a whole
new element of complexity to thinking about the chemical
environment of the cell. And I'll be honest,
this is daunting. Okay, this kind of stuff of
randomness and, you know, different structures
scares the heck out of me.

Okay, I don't know how to even
think about this sort of thing. It is very, very
intimidating in a way. The idea that I cannot determine
exactly what the structures are on the surface of the
cell and furthermore that the analytical tools
that have available to me as a chemist in 2013 are not
good enough for me to go in and tell you exactly
what the structures are on the surface of a cell. I find that really annoying
and very, very intimidating. Okay, and so this is a
very important frontier in chemical biology and I
encourage you to think about it. Okay, if you want to develop
an A plus proposal topic, come up with a way of figuring
out what these structures are on the surface of the cell.

I guarantee you that
will rock the world. Okay, because we know
that these are important in various diseases, yet
we don't have a good way of characterizing what they are. It's one of the last frontiers
of analytical chemistry, really. Okay and let's talk
about what they do. Alright, so one thing
that was thought early on is maybe they're hanging
out on the surface of the cell to grab on to passing other
cells and a great example of this would be a
T-cell communicating with an antigen-presenting
cell down here and we definitely see
the carbohydrates.

They're highlighted in these
sort of brown structures that are hanging out and you
can see they're even, you know, making contact and they're
doing stuff but the truth is when we cut them off the
surface of the cell or we set up a cell line that doesn't
produce those, the cells talk to each other just fine. Okay, so it doesn't
seem to be required for every communication. It only seems to be required
for some communications, some molecular recognition
between cells. Oh and recall that we discussed
earlier how communication between cells and communication
between proteins is like a Braille process,
where the proteins and the molecules feel
each other and look for complementary surfaces,
complementary functionalities in different structures,
that's the sort of thing that we're talking about here.

So in this case, we can
remove the carbohydrates and the two cells talk
to each other just fine. Okay? But here's one thing
that they do seem to do, so for example they
can play a key role in antibody recognition,
so the glycosides that modify antibodies tend
to be pretty heterogeneous. Again, that's the
whole randomness that we discussed earlier. On the other hand, they
do seem to be required for antibody function. Okay, so here's the
oligosaccharides down here, here's the structure of the
antibody that I introduced to you way back I think
on week one of this class, and then recall that they're
going to be recognizing antigens up here, they're modified as
N-linked glycans and again as N-linked glycans,
they have the common core that we discussed earlier today. And then there's a bunch of
sialic acids and others types of modifications that
are appended to this and even though they
are quite heterogeneous, they do seem to be important.

If you produce your antibodies
without the carbohydrates, they tend not to fold as well. They tend not to function
as well at recognition. So the carbohydrates seem
to play important roles in protein folding,
okay that's one role. Number two, they seem
to play important roles in solubilizing proteins, and
number three, they also seem to be important for
protecting structures that otherwise might
be recognized by the immune response. They seem to be good at
kind of providing shielding like a force field or something that keeps back immune
molecules. A big challenge for
us and a big challenge for the biotechnology industry
in general is that the proteins that we produce aren't being
produced largely by human cells.

Okay? And so in the
biotechnology industry, we sold something like
25 to 30 billion dollars, billion with a b, worth
of antibodies last year. Okay? So this structure here, that's a 25 billion dollar plus
industry in the United States. Okay and these are used for
everything from treating cancer to treating autoimmune diseases. But we rely very heavily on
Chinese hamster ovary cells to produce the antibodies
for us. And I know what you're thinking. Why Chinese hamster ovaries, why cells from that
particular organism. It's mainly historical. These are cells that
grow really robustly. They really whip out a huge
quantity of antibodies, and you can grow these cells
in 10,000-liter fermenters.

Okay? I mean the size of this, the scale of this
production boggles the mind. Okay? Just imagine this whole
room here that we're in filled with fetal calf serum, which
is what these guys like to eat, you know, or something
else that's kind of like the serum found
out of blood, you know, but it's artificial, just
this whole room filled with this stuff and
cells sloshing around and then you have a bunch
of chemical engineers that are carefully
controlling the oxygen content, the carbon dioxide content,
and the pH of the solution. The level of control is
pretty amazing too but all that is necessary to produce
this 25 billion dollar product. And here's an issue
that comes up. When we look carefully
at the identity of the carbohydrates found on
the surface of the antibodies, there's divergence
between what's found on human antibodies shown in
this column versus what's found in these Chinese hamster ovary
cells found in this column.

That divergence though
doesn't seem to have very much
functional consequence. We seem to be okay with that. So antibodies that are
produced in this way are given to patients on a daily basis and
seem to be perfectly functional. Okay, even over long,
long terms. Okay now along the lines of
giving stuff to patients, modification by glycans is a
very important side reaction that takes place almost as soon as pharmaceuticals are
taken by the patient. This is one that I know someone
in this class is going to end up spending their
lifetime studying. Okay, anyone that goes into
pharmaceuticals, you know, some small percentage
of you will be concerned about what happens
to the pharmaceutical after it gets taken up
by the patient and one of the first reactions
that takes place in the body is the body tries
to solubilize the thing.

Oftentimes pharmaceuticals
are pretty insoluble and we already talked
earlier this quarter about cytochrome P450 that has
a strategy of introducing oxygen to solubilize say benzopyrenes, things that otherwise
would be insoluble. In this case though the
strategy for solubilization is to transfer this glucose, this
glucuronide molecule to it, so starting with
UDP glucuronide, you can basically transfer this to give us a glucuronidated
molecule, so there's a hydroxyl in the compound that's
being given to the patient. That then becomes
the nucleophile to attack this activated glycan to give us a modified
product over here. And this thing is going
to be a lot more soluble. Right? It is negative charge. It has lots of hydroxyls that
could hydrogen bonds with water and so this has the effect of taking something
that's pretty insoluble and converting it into
something that's really soluble. Okay and this is
a good strategy. This works a lot. This is one of the very
first breakdown products that are found when we look at
what happens to pharmaceuticals after they're ingested
by patients. Okay, last topic that I
want to talk to you about.

Who had glucose with their
cereal this morning or who had like sugary cereals
for breakfast? Okay. I did too. I love sugar in the mornings. Okay, so chances are
that that glucose, the sucrose that you
took has now been broken down into glucose and fructose
and that stuff is now running around in your bloodstream
as we speak. And in response to this, your body has evolved
this really effective way of coaxing this glucose to be
either taken up or taken down and you probably
even know about this. This is a system
that's controlled by the hormone insulin. Okay, so the way this
works is the goal is to have a steady stay
concentration of glucose out here in the blood vessels and glucose is constantly being
either expelled or pumped in.

Insulin triggers glucose
uptake by the cells. So after you eat, insulin is
released, glucose gets taken up by the muscle or
fat cells and they do with it what they will. Okay? So when you are
sweating, when we took that little quiz earlier, that
was your glucose going to work. Okay? The problem though is when
the cells become less sensitive to insulin and this part over
here shuts down, when that shuts down the concentration
of glucose in the blood vessels skyrockets and the problem is this glucose
stuff is not totally benign. Okay? Recall earlier, we discussed how it can
form the hemiacetal form and it can form an
aldehyde form. The aldehyde form is a
very effective electrophile and if you have a high enough
concentration, you have lots and lots of these aldehyde
forms running around, looking for some
nucleophile to react with and that can't be good. Okay and so what happens is you
end up with random modifications of proteins on the
surface of the cell.

So this happens spontaneously
to all proteins found in serum in the blood and
this causes problems. Okay, so here's a structure of a protein that's been
heavily glycosylated and these modifications
are spontaneous. These are non-enzymatically
controlled. They just happen spontaneously. Let me show you the
mechanism for this reaction. Okay, so here's glucose
over here. Here's lysine on
some surface cell; let's just call it
serum albumin.

Okay? So if human serum
albumin is present at millimolar concentration
in your blood and there will be a lysine
side chain that can then react with the electrophilic
anomeric carbon of this glucose. Okay? This is a reaction
called an Amadori reaction. Okay? It happens
pretty spontaneously. Key intermediate here, what
do you guys think the key intermediate it is? How does this Amadori
reaction go? [ Pause ] Sergio? [ Inaudible Student Comment ] Okay so we have nucleophile.

What's electrophile? Carl? [ Inaudible Student Comment ] What's that? [ Inaudible Student Comment ] Yes! Alright. In the clutch. Nice job. Okay so this
anomeric carbon can tautomerize into an aldehyde. That can then react with this
immune to give you a shift base and through some other, you
know, proton transfer steps, you get to this product here. Okay? This doesn't
look so bad, right? The problem though is this sets
you up though for something that is a lot less benign.

Okay, we have a carbonyl over here that's a
new electrophilic site and this can rearrange
to give us an alpha beta unsaturated carbonyl. Another lysine either
on the same protein or neighboring protein
can then react with this and the net effect here is
to crosslink two proteins. Okay, so you take these
two free-floating proteins that are normally just kind
of swimming around and happy as can be in your serum and now
you're tethering them together or even worse, you're tethering
to the surface of the cell. So the cell doesn't know
what to do about this. The immune system doesn't
know what to do about this and the immune system really
is kind of the sledgehammer. It responds the way
it likes to respond, which is to increase
inflammation and so the net effect of this is
you get an inflammation response which, you know, kind of
spirals out of control. Okay? So you get these things
that are tethering carbohydrates to the surface of the cell
and then they get more and more complicated
and more and more broke as more reactions take
place and you just start to accumulate these advanced
glycosylation end products that, you know, lead to
inflammation and disease.

Okay, so this is why too much
glucose is a really bad thing and our American diets
seem to be ideally suited for maximizing concentrations
of glucose, which is a really
particularly bad thing, pernicious thing, really. Okay, advanced glycosylation end
products lead to inflammation. Okay, it's like of
like accretion, right? It's sort of getting in the
way of the immune system. Okay, so naturally
being chemists and being innovative people,
we like to invent stuff that would offer us that same
wonderful taste of sugar sweet but not offer the same
glucose potentialities. So, we've been doing things
for years that involve trying to have the same
amount of sweetness but just lower concentrations
of glucose. So for example, fructose is
like 2x sweeter than sucrose, but because it's just
half of the sucrose, it's actually half the calories and it doesn't have the glucose
that's going to be floating around looking for
reactions to do. Okay? So you can get fructose
pretty readily out of honey. Honey is twice as
sweet for the calories. It doesn't taste the same, I
know, but it's pretty effective.

This is another one trehalulose
is also used pretty extensively. So we do things like
this all the time. We'll substitute one
thing for another. Some things are sweeter than
others, offering less calories. This has been done for,
you know, hundred years or so, maybe even longer. Okay, the other thing that happens is we've also
invented a series of compounds that don't look anything
like carbohydrates but activate the
same carbohydrate or activate the same
receptors for sweet taste. So for example, aspartame is
a dipeptide that's methylated at the C terminus that is
180 times sweeter than sugar. Okay? You can eat this stuff and
it is insanely, insanely sweet.

Okay, I mean it leaves your
lips going [lip smacking] like that for hours. I mean it's really,
really that sweet. Okay, you don't want to like
stick your tongue in this stuff. Okay? The problem with
this though is that it can, it has a rearrangement that
forms dichetopiperazine; this is a dichetopiperazine,
and Neotame, its more modern variant,
avoid the dichetopiperazine by having this big, you know,
functional group on the side and it's also way, way
sweeter than sucrose. Okay, sucrose is kind of
the gold standard here.

That's table sugar, 10,000
times sweeter for the weight. That's kind of amazing. The other thing is we've also
come up with things that look like carbohydrates but cannot
be hydrolyzed and digested. So for example, these
[inaudible] substituted versions of sucrose, okay, so
this is like sucrose over here except now instead of
hydroxyls, we have chlorines. This is a compound
called sucralose. You can also isolate
from plants, from the sweet-leaf plant shown
here, you can isolate Stevia. To me this one tasted
a little bit bitter. I don't know if anyone does
Stevia with their coffee but I can definitely taste it. That one just doesn't
taste the same. It gets even wilder than that. There's, you know,
amazingly sweet compounds that you can extract out of
bushes and plants that are so sweet that they kind of
overwhelm your sweet receptors and leave you with this
permanent sweet taste that affects the flavor of
everything you eat afterwards. Okay, so I mean you can
eat these compounds. One of them is called like
the miracle berry or something like that and you eat this
stuff and then, you know, for 10 minutes afterwards
you can eat like lemon juice and you know drink lemon
juice or eat olives and stuff like that and everything
taste sweet.

I mean it taste really
good sweet. It also tastes a little weird. Okay? But this stuff is
just amazingly effective. Okay, any questions
about carbohydrates? Ask now. Yeah, Chelsey? [ Inaudible Student Question ] Yeah, dichetopiperazine. >> What is the [inaudible]
of that? Is that– >> Oh, okay. Yeah, that is an issue because
this dichetopiperazine thing no longer tastes sweet. Okay and the problem is when
you cook with aspartame, the high temperatures
encourage this to form.

Okay and that's a problem
because you want sweet over here and suddenly you have something
that's not sweet and so that why we find aspartame in
like Coca-Cola, like Diet Coke, and or actually I
shouldn't say that. I don't know what's actually
in Diet Coke, but you find it in like diet soft drinks
but you don't really find it in say diet doughnuts. Okay, right? Anything that encounters
high temperature, aspartame is not going to work
for, so instead we tend to turn to things like sucralose
and other things.

Okay? Thanks for asking. Over here? [ Inaudible Student Question ] I have not tried it. Have you? Okay, I'd
like to try it. I like trying things that
taste weird that are, you know, fully edible and–
Okay, let's move on. I want to talk to you next
about polyketides and earlier in the class, I told
you that we're going to organize everything according to the central dogma
of modern biology. We're now down here. We've talked about
oligosaccharides. We're now at the point
that we're going to talk about polyketides and terpenes
in the next couple of days. And this is a fascinating
class of compounds that really gets
under-played in biology classes but really deserves
the spotlight because they do so much for us. These are found– Oh, let me
talk to you about the structure and then I'll show you
where they're found.

They're found in all kinds
of antibiotics and fats. So for example, this polyketide
is a very nice fatty acid and all polyketides
and terpenes are formed from repeating subunits,
which I have highlighted here. So in black, these two carbon
units, subunits are going to be introduced
into modular fashion such that the red bonds can
be synthesized the same way every time.

Similarly, terpenes are
modules of five-carbon units, isoprene units that
are strung together and connected by
these red bonds. Okay? So we're going to be
talking about, okay, so again, these are composed of repeating
subunits of modular bonds. Okay, so here's some
examples of polyketides and I think this illustrates
their tremendous structural diversity and dare it
say it, their beauty. If molecules can have beauty,
these are beautiful because look at this erythronolide over here. It's just so kid and cute to me.

It has a lactone
structure, lots and lots of functionality
sticking off of it. It's got a ketone over here
and it's perfectly evolved to the point which they're
very effective antibiotics so this is erythromycin
antibiotic that many of you have probably encountered
at some point in your lifetime. These also extend to the
fatty acids and fats as well. So you can get really
complicated polyketides like this one. You can also have the
aromatic compounds. These aromatic compounds
are basically folded up fatty acid type
things that have a key set of carbon-carbon double
bonds that then cyclize to give you these
aromatic rings. So if you wonder when we talked about how those aromatic
rings form, they're being formed along
the same polyketide synthases.

Okay, so earlier in the quarter,
I showed you [inaudible] and rebeccamycin, a variant
of the compound shown here, that was synthesized by
polyketides synthases, okay, synthesizes by cells. Okay, so all polyketides
are built by a straightforward
aldol reaction but because this aldol
involves an ester, it's called by the name Claisen,
it's a Claisen reaction. Okay, and this is why I
love the aldol reaction. This is how the majority of carbon-carbon bonds
are formed in nature. Okay, the vast majority are
formed using this reaction and so I want to
take a moment just to appreciate how this works. Okay, and we're going to start with the variant that's
found in the laboratory. Okay so in the laboratory,
an aldol reaction or a Claisen reaction, you
would start with an ester and then you'd add some
sort of strong base that would then deprotonate
this alpha proton over here, give you an enolate and the
enolate can attack electrons, switch down here,
switch over here, and then kick all the
way up to the oxygen.

The net effect is we have a new
carbon-carbon bond right here. Okay, this works really well. This is a great way to
make carbon-carbon bonds. This is really how the experts
build carbon-carbon bonds and then in the end, as tetrahedral intermediate
collapses and that gives us
this new compound that has a new carbon-carbon
bond on it. Okay, the problem for nature is
that nature doesn't have access to strong bases like this one.

That strong base is totally
unique just to this particular, you know, what's
found in nature. Okay, so it doesn't
work so well for cells. Okay? Just don't have access
to bases that are going to be strong enough to readily
deprotonate an alpha proton and so instead what nature
tends to do is a little trick that I'll show you a
couple of slides from now. Okay? So first let me
just set the stage. What we're going to see is we're
going to see instead of esters, we're going to see thioesters but it's also classed
a Claisen as well. Okay and we're going
to see thioesters between either acetyl-CoA's like
this guy or propionyl-CoA's, so if the compound needs
an extra methyl group, you start with the shelf
that has the propionyl. If you just want two carbons,
then you start with the acetyl, okay but the idea is the same. We get more or less
the same reaction. The problem is these two and three-carbon building
blocks are small and slippery. It would be very hard for
the cell to kind of like grab onto these things if they were
just two or three carbons, so instead the strategy
that the cells applies is to attach a big old
handle to the two and three-carbon building block
and that handle is this molecule down here called coenzyme
A.

So from now on, we're going to leave
off this part. We're going to simply
it as just Co-A. Okay, that's all
of this over here. That's the handle. Okay. So enzyme grabs onto
this part and those down there, that's the two or
three-carbon part down there. Okay? Make sense? Okay. Let's get back
to the strategy that the cell uses
now to do its Claisen. Okay, and I've already told you
the cell doesn't have a strong enough base to make the Claisen
that we use in the lab work. So instead what the cell does
is a de-carboxylation reaction, okay, where it actually
does, oh actually, shoot. This isn't correct. It's going to do this
de-carboxylation, loss of carbon dioxide
to give us an enolate.

Okay, this structure over
here is missing a carboxylate. I will have to fix that. Okay, so again, the strategy
allows it to assess the enolate without having a really
strong base available. Okay? And in practice, things
get even more complicated. In practice, the enzyme that catalyses the Claisen
condensation simultaneously protonates the recipient ester,
thioester at the same time that it holds in place
this enol or enolate. Okay? And this reaction
works for both two-carbon as shown here or
three-carbon subunits, where these two methyl
groups just become like little spectators. Stereochemistry, of course, can be tightly controlled
in an active site. Okay, everyone still with me? We're good on the Claisen? Okay. I'm just going to
invoke the Claisen from now on as though it's understood. Okay, so we don't have to do
mechanism of Claisen anymore. But here's a mechanism that
we do have to talk about, one more that also kind of
going to be in our toolkit and we'll see quite a bit.

It turns out that these
thioesters can very readily do rapid exchange so you
can go from an S-acetyl, an S-CoA thioester to say
a cysteine thioester simply by the nucleophilic thiolate
attacking the carbonyl, kicking up, forming a
tetrahedral intermediate, which then collapses to give us
now this acetyl unit attached to the thiolate of the
cysteine side chain. This happens really readily. Okay? And this is going to be
important because earlier I said that we have these two
carbon things attached to this big CoA handle
but eventually we're going to want stuff that's sort
of in exactly the right spot at the right time and this
gives a way for the enzymes to have a cysteine in their
active site and then grab on to a two-carbon
piece very specifically.

Okay? So what we're going to
see in a moment is one piece of the reaction is going to
still have the [inaudible] CoA and the other piece will
be attached covalently to the enzyme's active site. Okay? Sound good? Simple reaction,
nothing too special. Okay, now from those simple
reactions that I showed you on the previous slide,
all kinds of, you know, chemical craziness can emerge. For example, you can
very readily form all of these fatty acids,
okay, so these fatty acids, these are all, you know, two
carbon units have been built up, these can basically be
synthesized using exactly the same Claisen reaction that I
showed on a previous slide. Okay? This is kind of wild. Before I get too far, I want to introduce you to
some nomenclature. First, the real aficionados
memorize the structures; don't memorize the
structures of these. Okay? Instead, what I want you to know is this omega
nomenclature. Okay, so the omega nomenclature
counts from the last carbon of the fatty acid tail.

So over here on this side,
this is the carboxylate. You can number these carbons
one, two, three, four, five, but it turns out
actually the key to controlling their structure and their properties is the
carbon-carbon double bonds from the tail of the fatty acid. Okay? So you've probably heard of omega-3 fatty acids being
important in your diet. Omega-3 fatty acids refers to have a carbon-carbon double
bond that's three carbons from the tail. Okay? So this one would
be an omega-369 fatty acid because that positions
there, six, and nine counted from the tail, you have a
carbon-carbon double bond. That carbon-carbon double
bond crucially sets a lot of the properties of
these fatty acids. First of all, notice that all of these carbon-carbon double
bonds are cis carbon-carbon double bonds, in other words
they have the alkyl groups on the same side. Check this out. All the ones on the
previous slide also cis.

The vast majority of
carbon-carbon double bonds found in fats, found in
nature are cis olefins, okay, not trans olefins. Transfats are found in
artificial fat sources that have been partially
hydrogenated. Those transfats are difficult
to digest and tend to do things like clog arteries
and things like that, which is why they're
associated with heart disease. Okay, so naturally
occurring cis olefins counted from the omega side over here, fish and canola oils have
a very high concentration of these omega-3 fatty
acids and it's crucial to maintain the correct
viscosity of your cells to have a certain ratio of
these omega-3 fatty acids versus omega-6 fatty acids, so
omega-6 fatty acids are found in things like corn oil and sort
of cheap soybean oils, you know, inexpensive forms of oil
like safflower oil, you know, things like that that are
found in processed foods. The problem though is that
when your ratios of omega-6 to omega-3 get off from
where they should be ideally, the viscosity of your cell
walls, of your plasma membrane, not the walls but the membrane,
changes and that viscosity seems to be a crucial characteristic
of brain function and other functions and
so it's really important that in your diet you have
enough omega-3 fatty acids, again this is omega-3
fatty acid, to replace these
omega-6 fatty acids.

It's simply an equilibrium
depending on your diet. The more omega-6 you eat,
the more omega-6s that appear on the surfaces of your cells. Okay, how are these things made? This is the machine of dreams. This little machine over
here synthesizes these fats in a truly wondrous cycle, okay, and I absolutely
love this chemistry because it's totally
easy to understand, yet it's so incredibly powerful. Okay, so what we're looking
at here is a schematic diagram for a fatty acid synthase, okay. So this is the thing
that's going to be synthesizing a molecule
like this and it turns out that the enzyme
is built of one chain. Okay, so it has a single
continuous amid bond linked protein. Okay, it happens to be
a very large protein. This thing is a monster in terms
of size and different domains of the protein are folded up into different
enzyme active sites.

Each one of these enzyme
active sites is labeled with a little code that
I'll decipher for you next. Okay? So we're going to
have in the very center for example a domain called
an acyl carrier protein. This is going to act as
a robot arm that's going to be carrying the
intermediates between each of these active sites and the
whole thing is going to go around a bunch of times as it's
acted upon during the synthesis of the fat. Okay? Everyone still with me? Great. Let's get
started with step one. This is the loading of
the starting material onto this acyl carrier protein. Acyl carrier protein
looks like this. There happens to be a
serine residue over here. That's going to be where
this thing is going to get loaded on to. Okay? Between the
starting piece over here, there's also this
phosphopantetheinyl group that just gives it a
little bit more space. Okay? It extends it out
a little bit further. Okay, so the acyl carrier
protein is over here.

Again, we have a
thiol over here. The thiol is perfect
for the thiol exchange, the thioester exchange that
I showed on a previous slide. Okay? Thiol then can exchange
with acetyl Co-A to set you up to start this process. Okay, so here's how it works. Here's that thiol on the phosphopantetheinyl
group over here. Here's the thiolate. It attacks acetyl-Co-A. You get a nice transacylation
reaction. Okay? So, you can either
start off with two carbons or you can start off
with three carbons.

Three carbons is nice,
right, because that sets you up for forming an enolate so it
kind of depends on if you want to start off by being the
enolate, being the nucleophile or start off as being
the electrophile. Okay? Alright, the next step
is acyl carrier protein, the robot arm, the phosphopantetheinyl
moves the acetyl, sorry, moves the acetyl group over to
the keto-synthase active site, abbreviated KS. KS then does a thioester
exchange, grabbing onto the acetyl
functionality and setting you up for a Claisen condensation. Here's first the decarboxylation
to form the enolate and then here's our
Claisen reaction that we've seen previously that gives us the new
carbon-carbon bond right here. And in the end, the acyl
carrier protein comes back and then picks up
this product again. Okay, so basically the acyl
carrier protein delivers the thing to one of these active
sites, the reaction takes place, in this case a Claisen, and
then it picks up back up and moves to the next site. That's really elegant stuff. Okay, the next reaction that can
take place is a keto reductase.

Notice that the product
over here is a ketone and so you can obviously
ketones don't appear in these fatty acids and so we
have to get rid of the ketone, so the first step is to do
a reduction of the ketone, nature's hydrides choice is
NADPH, this is analogous to NEDH that we saw earlier
in the quarter. Hydride gets kicked out,
reduces the carbonyl and that gives us an alcohol. This alcohol then
can be eliminated by a dehydratase, okay. So dehydratase protonates
the hydroxide and then we do a straightforward
either E2 elimination or E1CB, the jury is still a little
bit mixed on this one. In the end though we get a
carbon-carbon double bond and then this carbon-carbon
double bond can be reduced using again NADPH, nature's reductant
using exactly the same reaction more or less.

Okay? And in the end, and then
the very last reaction will be simplify hydrolyzing off from acyl carrier protein using
a mechanism that's analogous to a serine protease. Okay, so let's put
all of this together. Okay, so here we are. This is our schematic diagram. Acyl carrier protein
starts off first. It gets loaded up and then
it brings the acetyl group to the keto, or sorry, it's going to bring the acetyl
group to the keto synthase. This then does its
Claisen reaction. It comes over here
to the keto reductase and then the dehydratase
over here and then the enol reductase.

I know these things don't
seem like they're in order but it's a schematic diagram. It seems like it's kind
of jerking back and forth. But that's more or
less what's happening. And then, after the first
two carbons are added, then you get back
to the keto synthase and you add another two
carbons or three carbons. Go to this one, go to
this one, go to that one, and repeat the process
multiple times until the fatty acid
can no longer fit in the fatty acid synthase at which point then this
thioesterase comes along and hydrolyzes off the thioester
from the acyl carrier protein.

Okay, let's stop here. When we come back,
we'll be looking at even more complex
polyketides and their synthesis.
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