Lecture 15. Glycobiology & Polyketides.
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KohlenhydrateChemische ForschungGolgi-ApparatSekretionsvesikelPlasmamembranGlättung <Oberflächenbehandlung>XyloseOligomereEukaryontische ZelleNatriumCobaltoxideNeotenieFunktionelle GruppeBlitzschlagsyndromPosttranslationale ÄnderungGlykogenFülle <Speise>GlucoseStratotypMembranproteineNucleinsäurenPolysaccharideWasserPhasengleichgewichtSulfateOberflächenchemiePotenz <Homöopathie>Elektronische ZigaretteGlykosylierungLeckageWursthülleKohlenhydrateDisposition <Medizin>TiermodellElektronentransferHydroxideHydrogensulfateThermoformenGolgi-ApparatUntereinheitSatelliten-DNSSäureGummi arabicumErdrutschSchwermetallOktanzahlKohlenstofffaserChemische ForschungFarbenindustrieMolekülKettenlänge <Makromolekül>CelluloseBukett <Wein>SymptomatologieGoldBiosyntheseEnzymIonenbindungPlasmamembranMultiproteinkomplexPolymereAntikörperHydrophobe WechselwirkungRückstandHydroxylgruppeStärkeSekundärstrukturFlussmittelEndoplasmatisches RetikulumAldehydeKetoneMonosaccharideGlykosideHydrateHexamereAnomerer EffektOxoniumXyloseAntigenitätSerinThreoninGlykoproteineBindungstheorie <Chemie>KluftflächeAlphaspektroskopieTrisaccharideProteoglykaneOligosaccharideGalactoseMucineSeitenkettePhosphate
09:56
HeparinChemische ForschungDisulfidePolysaccharideMembranproteineHalbedelsteinLactoseMultiproteinkomplexGalactoseDiole <1,2->MühleGlykosylierungElektronentransferCobaltoxideChemische StrukturStickstoffatomKohlenstofffaserFülle <Speise>OktanzahlEukaryontische ZelleHydrogensulfateKohlenhydrateCalciumWursthülleAusgangsgesteinKörpergewichtEinsames ElektronenpaarSeitenketteReaktionsmechanismusSubstrat <Chemie>LagerungAsparaginSäureSekundärstrukturMembranproteineCarboxylierungIonenbindungEnergiearmes LebensmittelHeparansulfatWasserChemische ForschungDoppelbindungÜbergangszustandSetzen <Verfahrenstechnik>HydroxideChemische ReaktionBukett <Wein>PlasmamembranSekretHydrocarboxylierungKluftflächeGlucoseTopizitätSubstituentDeprotonierungNeutralisation <Chemie>MedroxyprogesteronOberflächenchemieElektron <Legierung>HydroxylgruppeSymptomatologieAllmendeGesundheitsstörungFunktionelle GruppeBodenverdichtung <Bodenkunde>ReaktivitätHydrophobe WechselwirkungErythromycinElektronentransferKupfererzGlykosideGlykosylierungTransportGolgi-ApparatXyloseMonosaccharideAmideGalactoseMannaneMesomerieLysinErdrutschAmineProteoglykaneSerinEukaryotenAviditätDisulfidbrückeOligosaccharide
19:52
GlykosylierungChemische ForschungGlykosideOligosaccharideEinschlussMedroxyprogesteronGalactoseRoher SchinkenAlauneRedoxpotentialCocainSimulation <Medizin>SilberiodidDiatomics-in-molecules-MethodeHalbedelsteinHydroxybuttersäure <gamma->MembranproteineAmalgamOberflächenchemieTopizitätChemischer ProzessOrganische ChemieAntikörperEukaryontische ZelleFunktionelle GruppeMeeresspiegelReglersubstanzMolekülWasserstoffbrückenbindungChemische StrukturHydroxylgruppeBukett <Wein>KohlendioxidGlucoseChemische BiologieChemische SyntheseGlykosylierungIonenbindungGezeitenstromCobaltoxideMembranproteineKohlenhydrateAlkoholische LösungChemieingenieurinFülle <Speise>Chemische ForschungPosttranslationale ÄnderungT-LymphozytProteinfaltungPulverMalerfarbeKrebsforschungCytochromeGesundheitsstörungSerumAzokupplungChemische ReaktionKunstlederChemische VerbindungenSäureZunderbeständigkeitBiotechnologieWursthülleAntigenEnzymSolubilisationWerkzeugstahlEisflächeErythromycinPharmazieLöslichkeitSymptomatologieThreoninLactitolSubstrat <Chemie>LagerungQuellgebietAktives ZentrumMultiproteinkomplexHydrophobe WechselwirkungFunkeSetzen <Verfahrenstechnik>Chemisches ElementThermoformenWasserAntigenitätStereoselektivitätGlykosideAlkoholische GärungSerinCytochrom P-450AcylneuraminsäurenHeck-ReaktionGlykosidasenOligosaccharideVorlesung/Konferenz
29:48
GlucoseAder <Geologie>InsulinEukaryontische ZelleChemische ForschungDiole <1,2->EnzymGlykosylierungHydroglimmerChemische ReaktionACEEntzündungFructoseRübenzuckerAspartamGezeitenküsteSüßstoffOktanzahlSucralosePyridinAldehydeKohlenhydrateSüßkraftEukaryontische ZelleMembranproteineAder <Geologie>Chemische ReaktionFülle <Speise>Aktives ZentrumRübenzuckerUmlagerungGlucoseBukett <Wein>AlphaspektroskopieHydrocarboxylierungAlkoholfreies GetränkAlpha-1-RezeptorSucraloseInsulinKörperfettCarboxylterminusChemieanlageDipeptideZuckerErythromycinChemische VerbindungenFunktionelle GruppeRöstkaffeeSubstitutionsreaktionPharmakokinetikWaldhonigTopizitätEntzündungVernetzungsmittelChlorHydroxylgruppeAspartamAktionspotenzialSeitenketteFructoseBleierzThermoformenSystemische Therapie <Pharmakologie>Chemischer ProzessKohlenstofffaserPharmazieLysinGesundheitsstörungGlykosylierungOberflächenchemieEnergiearmes LebensmittelSerumKonzentratWeibliche ToteReaktionsmechanismusAmineElektronentransferChemische StrukturElektronische ZigarettePosttranslationale ÄnderungReglersubstanzLimonadeOxygenierungBeta-FaltblattKörpertemperaturWursthülleMolvolumenSäureDeprotonierungAstra <Firma>Gangart <Erzlagerstätte>OktanzahlSetzen <Verfahrenstechnik>Akkretion <Geologie>PolymorphismusGoldZuchtzielSchwermetallKartoffelstärkePentapeptideAusschwitzenKlinischer TodInsolationSirupVorlesung/Konferenz
39:43
AspartamChemische ForschungEtherSüßstoffProteineMembranproteineMolekülBiosyntheseDNS-SyntheseMedroxyprogesteronGezeitenküsteMetabolitPolyketideMagmaModul <Membranverfahren>UntereinheitHydroxyaldehydeChemische ReaktionAcetylgruppeFunktionelle GruppeMetalloenzymAcetyl-CoAHalbedelsteinStromschnelleSäurePentapeptideChemische VerbindungenIonenbindungWursthülleKohlenstofffaserOrganische ChemieModul <Membranverfahren>StockfischMeeresströmungSäuref-ElementBukett <Wein>Chemische ReaktionEukaryontische ZelleAlkeneMembranproteineEnergiearmes LebensmittelElektronische ZigaretteMetallKörpertemperaturSchelfeisGezeitenküsteElektron <Legierung>ErdrutschCobaltoxideChemische ForschungEsterAsparaginsäureAzokupplungStromschnelleQuerprofilSeitenketteAktives ZentrumFunktionelle GruppeEnzymReaktionsmechanismusAlphaspektroskopieFülle <Speise>DeprotonierungBaseUntereinheitMolekülMethylgruppeTriterpeneBiosyntheseAktivität <Konzentration>AromatizitätOmega-6-FettsäurenChromosomenkondensationGangart <Erzlagerstätte>PolymorphismusErfrischungsgetränkThermoformenLokalantibiotikumSensePolypeptideCarboxylierungChemische StrukturSetzen <Verfahrenstechnik>Gummi arabicumErythromycinMilchproduktCobaltAspartamSucraloseCysteinClaisen-UmlagerungPolyketideDesacetylierungAcetyl-CoAFettAldolreaktionThiolgruppeEnoleSingle electron transferThioesterLactoneClaisen-KondensationKetonePropionsäureIsoprenOligosaccharideKohlendioxidCarboxylateDecarboxylierungHydroxyaldehydeComputeranimation
49:39
SäureChemische ForschungOmega-3-FettsäurenMetAmmoniumverbindungenGalactosePolyketideMethyliodidMolekularstrahlAdvanced glycosylation end productsFunktionelle GruppeMetalloenzymTransaminasenHydrideNADPHAcepromazinPasteIonenbindungEukaryontische ZelleOktanzahlLammfleischChemische ReaktionExplosionFreies ElektronKohlenstofffaserAlkohole <tertiär->ThermoformenMembranproteineFunktionelle GruppeChemische StrukturOmega-6-FettsäurenGesundheitsstörungCarboxylierungKonzentratChemische ForschungFettWursthülleQuellgebietChromosomenkondensationMolekülbibliothekEnzymDomäne <Biochemie>Aktives ZentrumChemische EigenschaftEnergiearmes LebensmittelErdrutschBiochemieKettenlänge <Makromolekül>MolekülSpanbarkeitBiosyntheseFeuerAllmendeGangart <Erzlagerstätte>AlkeneSäurePhenobarbitalZellwandHydrateChemischer ProzessOberflächenchemieSingulettzustandPlasmamembranIsoliergasOmega-3-FettsäurenGezeitenstromFließverhaltenFülle <Speise>ReduktionsmittelMaiskeimölSojaölHydrideCupcakeReaktionsmechanismusÖlFremdstoffMischanlageEliminierungsreaktionElektronische ZigaretteBiodieselThiolgruppeKetoneThioesterFettsäure-SynthaseAcylgruppePhosphorEtomidatHydroxideCarboxylateRückstandSerinEnoleDecarboxylierungAktivität <Konzentration>Claisen-KondensationClaisen-UmlagerungAcetylgruppeDesacetylierungGesättigte KohlenwasserstoffeRapsölLaichgewässerDoppelbindungComputeranimation
59:35
BiosyntheseStockfischMultiproteinkomplexPolyketideVorlesung/Konferenz
Transkript: English(automatisch erzeugt)
00:08
So, very briefly, let me just review what we saw last time before we go on to some new stuff. Last time we were seeing that in the human body, there's really only nine monosaccharides that are found
00:22
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 of carbon which is the chemical definition of carbohydrates.
00:40
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.
01:03
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 talked very, we also talked about the oxonium and oxocarbenium ions.
01:21
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.
01:42
OK. Now, today, I want to talk to you about complexity and about oligosaccharides, about polysaccharides that are a good deal more complex. So oligosaccharides are, we're going to define them as simply or let's say, sorry,
02:01
polysaccharides are going to be just polymers of one or maybe two or three, you know, some small number of subunits. OK. So like glucose strung together in a repeating chain can give us cellulose, right? We talked about that. We talked about how wood could form cellulose or wood is formed from cellulose which is formed from glucose.
02:22
So the polysaccharide of glucose is called cellulose. OK. That's one form. 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,
02:43
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
03:01
that antibodies can react with 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. OK. So let's get started. I left off on this final slide right here and I was showing you that N-linked glycosylation takes
03:24
place in the endoplasmic reticulum and the Golgi complex during export of proteins after their synthesis at the ribosome. OK. So this is a very simplified view of the cell. But here in blue are the expelled proteins
03:41
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
04:01
that are found inside the cell. OK. 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. OK. This is sort of the gumdrop model
04:22
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. OK. All right. OK. So let's talk very briefly about how you assemble,
04:41
in this case, the O-linked glycoproteins. OK. So an O-linked glycoprotein has an oxygen that will be the recipient nucleophile for modification. OK. So in other words, either a serine or a threonine residue where this beta hydroxyl becomes modified
05:04
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.
05:25
OK. 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 transfer of a phosphate group.
05:41
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. OK. So the key though is that this is a big leaving group and so that in the end transfer xylose to this hydroxide giving us a new glycosidic bond.
06:06
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 serine or threonine side chains.
06:23
OK. 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 and that are, you know, basically hanging out as big shrubbery. The other ones, the ones that get modified by the N-acetylgalactose,
06:42
these GalNacs 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.
07:02
OK. And so I'm being coy about this. This is the snot of the cell. OK. This is the mucus. Mucins, mucus, derived 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
07:21
and then we're going to do a lot of modification. OK. So here's one example of this. So after we start with this common core that I've already showed you, this is the galacto, this was the xylose that we saw in the previous slide. There's a series of galactocele transferases that use UDP galactose and then transfer
07:44
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
08:00
of its starting unit. OK. So this becomes sort of the, it's kind of like the spool that the thread is going to be wound on to. Many different colors of thread can be wound on to the same spool, but in all cases we're going to use a spool that starts off as this trisaccharide. OK. And so the strategy here that I'm showing you can also
08:23
be used for making repeating disaccharides as well. All right, let's take a look at some examples of this. Do you remember on Thursday, and I'm hoping you all watched the Thursday videotape, that's why I gave you the quiz, if you watched the Thursday videotape, the second to last slide dealt with those knee joint polysaccharides,
08:45
OK, 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.
09:00
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. OK, so this is the start over here. So here's the protein that's going to be modified, a serate, and then as usual it always starts with a xylose
09:22
and then two galactose, gal-gal, gal-gal. And then there's a series of other modifications that are appended to this. These are things like N-acetyl-glucose-galactose that also has a sulfate group, OK, or N-acetyl-galactose that doesn't have a sulfate group but it has,
09:46
it still has N-acetyl portion. So in the end, this, we can get, we can get, or sorry, this is actually glucose, N-acetyl-glucose that is, you know, attached. So this gives us heparin sulfate which plays important roles also
10:02
on just kind of the cell surfaces and for cell signaling, we'll see that later. And then over here, these chondroitin sulfates, dermatin sulfates, these are things that are going to be shuffled off into the inner stichies between joints. OK, and notice that these are pretty heavily sulfated as well.
10:20
So these are negatively charged, hygroscopic meaning they attract water. OK. So snot and mucus, I mean, everyone is 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
10:41
and then they have, the counter ions are calcium, OK? So when these things are synthesized, they're synthesized at these calcium ions, OK? 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
11:00
up really, really tightly, OK? Now, what happens is when they get excreted, the calcium ions are stripped off. They're grabbed by a 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,
11:23
just in a tremendous 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.
11:42
So this is how your little mucus, 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's depicted here. And a very similar strategy is used in disposable diapers
12:03
which have these polyacrylates, OK? So again, you have this negative charge that's feeling unsatisfied and is looking for water. And when it finds water, it grabs onto a tremendous avidity and grabs onto lots of water by weight. These disposable diapers are actually kind of this miracle of modern chemistry, right?
12:22
These things soak up enormous quantities of water relative to their weights, OK? And similarly, the snot in your nose is soaking up enormous quantities of water as well and being secreted, OK? And that makes it very effective, right, as a way of forming a transition barrier
12:41
between gas phase and then liquid phase. OK. 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.
13:02
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, OK, because of the sulfates, right? This thing is so highly sulfated that there's really very little carbohydrate there. It's mainly just water.
13:22
OK. So, let's talk next about the N-linked glycosides. Now, things are going to get more complex here. We've already seen, oh, OK. 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?
13:41
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 N-galnac or galnac-galnac core. In this case, we're seeing a very similar structure
14:00
where we have these two glycnacs in a row. So, these are glucose N-acetylglucosamines in a row. So, one, two, and then there's these mannoses over here and then things start to get a little crazy, OK? But you could see, again, the strategy here is start with a fairly common core, not counting this Fucose,
14:20
but for the most part, you know, things are fairly common and then modify and customize depending upon the needs, OK? So, everything is starting off pretty normal but then things start to get more wild as we go down here. OK. One last thought, I've switched nomenclatures. Earlier, I was showing you nomenclature
14:41
like this, this starts to become increasingly less useful to us, OK? I mean, in this case, we have an N, we have an amine that's appended to a glucose and that's sulfated, you know, these things start to get increasingly broke. And so, rather than trying to depict these structures
15:00
and then spending time thinking about, oh, is that a glucose, is that 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 could kind
15:21
of figure out what structures you're looking at, OK? Now, admittedly, this would be a challenge for anyone in this classroom, OK? And I'm not asking you to do that, OK? So, I'm not asking you to memorize the nine structures of monosaccharides that are found in these, found in humans, OK? But I just want you to be comfortable with the idea
15:42
that these are carbohydrates and these designate carbohydrates. You never know when this information is going to be useful. Who knows, maybe some pharmacology class that you take many years from now. OK, 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
16:01
of the O-linked glycosides because those work so well. Oxygen is a fantastic nucleophile. The oxygen doesn't have a carbonyl nearby. But for the N-linked glycosides, for the things that are a little bit more complicated and that's because we're going to attach things not
16:22
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
16:40
as nucleophilic as just a free-floating nitrogen with its lone pair hanging out. You remember earlier in the class, I told you that the lone pair on this nitrogen spends a good 40 percent of its time hanging out as the resonance structure forming a nitrogen carbon double bond right here, OK? And because that nitrogen carbon double bond is there 40 percent
17:02
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
17:21
that present asparagine in a way that allows this reaction to take place, OK? So this is an example of substrate-assisted catalysis. I'll explain in a moment. But note in the structure of these glycosyltransferases, there's a base. The base can deprotonate this nitrogen and the electrons
17:42
that are 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,
18:01
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,
18:21
then all of a sudden the lone pair is uncovered and ready for reactivity, OK? And notice that it doesn't have anywhere to go. It's 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.
18:41
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 the structure of the leaving group. But suffice it to say, it's something that likes to take off, OK? It's effective leaving group. Note too, the structural requirements for this reaction to take place.
19:02
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.
19:20
For this to, for this reaction, OK? So, I call this an 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,
19:44
or sorry, Bronsted acid catalysis to protonate this carbonyl. OK? 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, OK?
20:00
So, this only works with certain substrates, OK? And that actually gives you a degree of selectivity. All right, brace yourself. Now, things are going to get really complicated. What happens is very complicated structures get synthesized as N-linked glycans and then they get trimmed back
20:22
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 mannocidases.
20:43
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
21:02
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
21:23
of a little bit random. It's not exactly programmed in. This element of random last 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
21:40
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?
22:02
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 I 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
22:22
on the surface of the 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.
22:42
I guarantee it to 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. All right, so one thing that was thought early
23:02
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 could see they're even,
23:21
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,
23:41
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 and different structures. That's the sort of thing that we're talking about here.
24:02
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
24:23
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.
24:41
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've discussed earlier today. And then there's a bunch of sialic acids and other types of modifications that are appended to this. And even though they're quite heterogeneous,
25:01
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.
25:21
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
25:41
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,
26:01
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 in, we rely very heavily on Chinese hamster ovary cells to produce the antibodies for us.
26:23
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.
26:43
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,
27:00
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.
27:21
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 diversity, though, doesn't seem, divergence, though,
27:41
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
28:04
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 who goes into pharmaceuticals, you know, some small percentage of you will be concerned
28:21
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 a 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,
28:44
things that otherwise would be insoluble. In this case, though, the strategy for solubilization is to transfer this glucose, this glucuronidide molecule to it. So, starting with UDP glucuronidide, you can basically transfer this
29:02
to give us a glucuronidide, glucuronidated molecule. So, there's a hydroxyl in the compound that's being given to the patient that then gets, becomes the nucleophile to attack this activated glycan to give us a modified product over here.
29:24
And this thing is going to be a lot more soluble, right? It has negative charge. It has lots of hydroxyls that could form hydrogen bonds with water. And so, this takes, 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.
29:40
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?
30:01
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 your bloodstream as we speak. And in response to this, your body has evolved this really effective way of coaxing this glucose
30:22
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 state concentration of glucose out here in the blood vessels. And glucose is constantly being either expelled or pumped in.
30:44
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 were sweating, when we took that little quiz earlier, that was your glucose going to work. Okay. The problem though is
31:04
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 sky rockets. And the problem is this glucose stuff is not totally benign. Okay. Recall earlier,
31:21
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
31:41
up with the random modifications of proteins on the surface of the cell. So, this happens spontaneously to all proteins found in serum and in the blood and this causes problems. Okay. So, here's the structure of a protein that's been heavily glycosylated.
32:00
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.
32:21
Okay. So, 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 amidori reaction. Okay. It happens pretty spontaneously.
32:43
Key intermediate here, what do you guys think of key intermediate it is? How does this amidori reaction go? Sergio? Okay.
33:03
So, you have a nucleophile. What's electrophile? Carl? What's that? Yes. All right. In the clutch. Nice job. Okay. So, this anomeric carbon can tautomerize
33:24
into an aldehyde that can then react with this amine 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
33:44
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
34:00
or neighboring protein can then react with this. And the net effect here is to cross link 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.
34:21
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 a massive, you get an inflammation response which, you know,
34:40
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.
35:02
Okay. So, this is why on too much glucose is a really bad thing. And our American diets are seen to be ideally suited for maximizing concentrations of glucose which is a really particularly bad thing, a pernicious thing really. Okay. So, advanced glycosylation and products lead to inflammation.
35:21
Okay. It's kind 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
35:40
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
36:04
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.
36:20
Trihalulous 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, 100 years or so, maybe even longer. Okay. The other thing that happens is we also have invented a series of compounds that don't look anything like carbohydrates
36:43
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
37:01
and it is insanely, insanely sweet. Okay. I mean it leaves your lips going 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 a diketopiprazine.
37:20
This is a diketopiprazine. And neotame, it's more modern variant, avoids the diketopiprazine 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.
37:41
Ten thousand 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 chlorine substituted versions of sucrose.
38:01
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 tastes a little bit bitter. I don't know if anyone does stevia with their coffee.
38:22
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
38:42
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 the stuff and then, you know, for ten minutes afterwards you can eat like lemon juice and, you know, drink lemon juice
39:00
or eat olives and stuff like that. And everything tastes sweet. I mean, it tastes a really good sweet. It also tastes a little weird. Okay. But the stuff is just amazingly effective. Okay. Any questions about carbohydrates? Ask now. Yeah. Chelsea.
39:23
Yeah. Yeah. Diketopiprazine. Okay. Yeah, that is an issue. Because this diketopiprazine thing no longer tastes sweet. Okay. And the problem is when you cook with aspartame, the high temperatures encourages this to form.
39:42
Okay. And that's a problem because you want sweet over here and suddenly you have something that's not sweet. And so that's why we find aspartame in like Coca-Cola, like Diet Coke, 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 donuts.
40:02
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. I have not tried it. Have you? Okay. I'd like to try it.
40:21
I like trying things that taste weird. Better, you know, fully edible and healthy. 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.
40:41
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 then terpenes in the next couple of days. And this is a fascinating class of compounds that really gets underplayed in biology classes but really deserves the spotlight because they do so much for us.
41:00
These are found, oh, let me talk to you about their 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've highlighted here. So in black, these two carbon subunits are going
41:23
to be introduced in 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
41:42
by these red bonds, OK? So we're going to be talking about, OK, so again, these are composed of repeating subunits and modular bonds. OK. So here's some examples of polyketides. And I think this illustrates their tremendous structural
42:02
diversity and dare I 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.
42:20
It's got a ketone over here and it's perfectly evolved to the point where it's a very effective antibiotic. So this is the erythromycin antibiotic that many of you have probably encountered at some point in your lifetimes. These are also extend to the fatty acids and fats as well.
42:42
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 cyclized to give you these aromatic rings.
43:01
So if you wonder, when we talked about how those aromatic rings form, they're being formed along the same polyketide synthases. OK. So earlier in the quarter, I showed you donomycin, rebecomycin, a variant of the compound shown here. That was synthesized by polyketide synthases, OK, synthesizes by cells.
43:22
OK. So all polyketides are built by a straightforward aldol reaction. But because this aldol involves an ester, it's called by the name clazin. It's a clazin reaction. OK. And this is why I love the aldol reaction. This is how the majority
43:42
of carbon-carbon bonds are formed in nature. OK. The vast majority are formed using this reaction. And so, I want to take a moment just to appreciate how this works. OK. And we're going to start with the variant that's found in the laboratory. OK. So in the laboratory, an aldol reaction,
44:01
you would start a clazin reaction. You'd 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, swish down here, swish over here and then kick all the way up to the oxygen.
44:24
The net effect is we have a new carbon-carbon bond right here. OK. 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, this tetrahedral intermediate collapses and that gives us this new compound
44:43
that has a new carbon-carbon bond on it. OK. 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. OK. So it doesn't work so well for cells.
45:04
OK. 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. OK. So first, let me just set the stage.
45:21
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 called a clazen as well. OK. And we're going to see thioesters between either acetyl-CoAs like this guy or propionyl-CoAs. So, if the compound needs an extra methyl group,
45:42
you start with the shelf that has the propionyl. If you just want two carbons, then you start with the acetyl, OK? 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
46:02
on to these things if they were just two or three carbons. So instead, the strategy that the cell 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.
46:20
We're going to simplify it as just CoA, OK? That's all of this over here. That's the handle, OK? So enzyme grabs on to this part and knows down here, that's the two or three carbon part down there, OK? Make sense? OK. Let's get back to the strategy that the cell uses now to do its Claisen, OK?
46:40
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 decarboxylation reaction, OK, where it actually does, oh, actually, shoot, this is incorrect. It's going to do this decarboxylation loss
47:00
of carbon dioxide to give us an enolate, OK? This structure over here is missing a carboxylate. I will have to fix that. OK. So again, the strategy allows it to access this enolate without having a really strong base available, OK? And in practice, things get even more complicated.
47:20
In practice, the enzyme that catalyzes the Claisen condensation simultaneously protonates the recipient ester, thioester, at the same time that it holds in place this enol or enolate, OK? And this reaction works for both two carbon as shown here
47:42
or three carbon subunits, where these two methyl groups just become like little spectators. Stereochemistry, of course, can be tightly controlled in the active site. OK. Everyone still with me? We're good on the Claisen. OK. I'm just going to invoke the Claisen from now
48:00
on as though it's understood. OK. 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's 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.
48:21
So you can go from an S-Acetyl and 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
48:41
to the thiolate of the cysteine side chain. This happens really readily. OK. 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.
49:00
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. OK. So what we're going to see in a moment is one piece of the reaction is going to still have the S-CoA and the other piece will have be attached covalently
49:21
to the enzymes active site. OK. Sound good? Simple reaction, nothing too special. OK. 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
49:41
of these fatty acids. OK. So these fatty acids, these are all, you know, carbon unit, 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. OK. And this is kind of wild. Before I get too far, I want to introduce you
50:00
to some nomenclature. First, the real aficionados memorize the structures, don't memorize the structures of these. OK. Instead, what I want you to know is this omega nomenclature. OK. So the omega nomenclature counts from the last carbon of the fatty acid tail. So over here on this side, this is the carboxylate.
50:21
You can number these carbons 1, 2, 3, 4, 5. 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. OK. So you probably have heard omega-3 fatty acids being
50:41
important in your diet. Omega-3 fatty acids refers to having a carbon-carbon double bond that's three carbons from the tail. OK. So this one would be an omega-3, 6, 9 fatty acid. OK. Because that positions 3, 6, and 9 counted from the tail,
51:01
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.
51:23
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, OK, not trans-olefins. Trans fats are found in artificial fat sources
51:42
that have been partially hydrogenated. Those trans fats 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. OK. So, naturally occurring cis-olefins counted from the omega side over here,
52:00
fish and canola oils have a very high concentration of these omega-3 fatty acids. And it's crucial to maintain the correct viscosity or 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
52:21
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,
52:40
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,
53:01
to replace these omega-6 fatty acids. It's simply an equilibrium depending on your diet. The more omega-6s you eat, the more omega-6s that appear on the surfaces of your cells. OK. How are these things made? This is the machine of dreams. This little machine over here synthesizes these fats
53:21
in a truly wondrous cycle, OK? And I absolutely love this chemistry because it's totally easy to understand, yet it's so incredibly powerful. OK. So, what we're looking at here is a schematic diagram for a fatty acid synthase, OK? So, this is the thing that's going to be synthesizing a molecule like this.
53:41
And it turns out that the enzyme is built of one chain, OK, so it has a single continuous amide bond linked protein, OK? 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
54:02
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, OK? 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
54:22
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, OK? Everyone still with me? Great. Let's get started with step one.
54:40
This is the loading of the starting material on to 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, OK? Between the serine, between the starting piece over here,
55:04
there's also this phospho pentathienyl group that just gives it a little bit more space, OK, extends it out a little bit further. OK. 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,
55:23
OK? Thiol then can exchange with acetyl-CoA to set you up to start this process. OK. So, here's how it works. Here's that thiol on the phospho pentathienyl group
55:40
over here. Here's the thiolate. It attacks acetyl-CoA. You get a nice transacylation reaction, OK? 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
56:01
or start off as being the electrophile, OK? All right. So, the next step is acyl carrier protein, the robot arm, the phospho pentathienyl moves the acetyl-CoA, sorry, it moves the acetyl group over to the ketosynthase active site,
56:21
abbreviated KS. KS then does a thioester exchange, grabbing on to 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
56:41
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, OK? 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 it back up
57:01
and moves to the next site. That's really elegant stuff. OK. The next reaction that can take place is a ketoreductase. 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.
57:25
So, the first step is to do a reduction of the ketone. Nature's hydrides choice is NADPH. This is analogous to NADH that we saw earlier in the quarter. Hydride gets kicked out, reduces the carbonyl and that gives us an alcohol.
57:43
This alcohol then could be eliminated by a dehydratase, OK? 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
58:02
and then this carbon-carbon double bond can be reduced using again NADPH, nature's reductant, using exactly the same reaction more or less, OK? And in the end, and then the very last reaction will be simply hydrolyzing off
58:21
from acyl carrier protein using a mechanism that's analogous to serine protease. OK. So, let's put all this together. OK. So, here we are. This is our schematic diagram. Acyl carrier protein starts off first and it gets loaded up and then it brings the acetyl group to the keto, or sorry,
58:44
it's going to bring the acetyl group to the ketosynthase. This then does its Claisen reaction. It comes over here to the keto reductase and then the, you know, and then the dehydratase over here and then the enoyl reductase. I know these things don't seem like they're in order, OK?
59:01
But schematic diagram, it seems like it's kind of jerking back and forth. But that's more or less what's happening. And then, when you get to after the first two carbons are added, then you get back to the ketosynthase and you add another two carbons or three carbons. Get to this, go to this one, go to this one, go to that one and repeat the process multiple times
59:21
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. OK. When we stop here, when we come back, we'll be looking at even more complex polyketides and their synthesis.