 Good morning. Good morning. Well, so I'm just looking at the quizzes, and Johnny and Kim Xerox, the wrong one. So don't worry. We're going to do them at the end. So just, oh, you want it sooner? I can give you the wrong quiz. I've got the wrong one, but it's harder. No. All right. So hang in there till the end. Save your adrenaline. Johnny and Kim are off running as fast as they can, and we'll be back with lots and lots of copies of the newer correct quiz. All right. So what I want to do today is to talk about chapter 28. And chapter 28 is on amino acids, peptides, and proteins. So beautiful chapter, beautiful topic, fantastically important, follows on the heels of carbohydrates, the main classes of biomolecules are sugars, proteins, nucleic acids, and then you throw a few lipids and secondary metabolites like alkaloids and so forth on top of that. And chapter 28 is a wonderful chapter in the sense it tries to cover everything. And the result, of course, is given the limitations of the course. If I don't cut it down, we will cover nothing. And so what I've decided to do is really focus on amino acids and particularly peptides from the point of view of synthetic chemistry and not even so much the synthetic chemistry of amino acids. There's some cool chemistry on the Strecker synthesis. There's some interesting stuff in the chapter that overlaps with your biochemistry class on isoelectric points and other aspects of amino acids and peptides. And some of this you'll get in your biochemistry class. But I really wanted to give you my own perspective on this topic. And what I see is really some of the beautiful and profound aspects. So let me start with a simple amino acid structure and then a simple peptide structure. And so maybe for regular amino acids, meaning sort of typical amino acids, maybe the amino acid alanine is sort of the archetype. It's an amino acid and people, because the amino group is at the alpha position, call these alpha amino acids. And as I said, this particular one is called alanine. It's the smallest amino acid with a side chain. Now amino acids of course have amino groups and they have carboxy groups and so they can be connected together end to end. And when you connect them together end to end, you get a peptide, small collections of amino acids containing up to about 40 amino acids end to end are typically referred to as peptides. Larger ones, often in nature, polypeptides have folded structures and are typically referred to as proteins. There's not a hard and fast definition of where the boundary is between peptides and proteins. But usually the emphasis would be on folded structures of biological origins for proteins. So I want to draw out a simple tripeptide and I've just arbitrarily taken three amino acids. The first one is phenylalanine. The second one is isoleucine. And the third amino acid is leucine in this chain. So we call this a tripeptide because it's composed of three amino acids, so peptide or a tripeptide. And the particular name of this tripeptide, if you want to get technical, would be phenylalaninele isoleucine, P-H-E-N-Y-L-A-L-A-N-Y-L isoleucine. All one word, not super important how you name polypeptides. Polypeptides have attracted the attention of chemists for organic chemists for so, so long because these are the molecules of life. And because organic chemistry really is the science of life and its molecules. And some of them are remarkably, remarkably powerful. So for example, the polypeptide oxytocin was first elucidated. The structure was first elucidated and then in a tour de force effort it was synthesized by Vincent de Veniode in 1953. And he immediately, I mean timescale of Nobel Prize has immediately won the Nobel Prize for it by 1955. So it's a NONAPeptide, that means nine amino acids and it contains a disulfide bond. I'll just give you a cartoon of the structure but I want to talk about it. So I'm not going to even draw out all the nine amino acids in it, the N-terminal amino acid, the back end, so this is called the N-terminus is assisting. Sistine is like alanine but with a sulfur instead of a CH3 group and then you run through another four amino acids and then the molecule snakes back on itself. Oops, let me get the structure right here. And again, I'm sort of giving an abbreviated cartoon of the molecule. We have another cysteine, so another CH2S and the sulfurs can link together to give disulfide. Sulfur linkages, if your hair gets frizzy or if you uncurl your hair with a curling iron or with straightening solution and then it locks back in with curls in it, it's sulfur linkages in your hair that actually lock in that structure because of course hair is protein. All right, and then we continue through another three amino acids and I'm not going to again draw it. I'll just say three AAs to the C-terminus of the structure and it happens to have an amide group here. And so I'll just write over here C-terminus. So this peptide, in addition to having this fantastic historic importance of being this sort of milestone in synthetic chemistry and a real mark of achievement of what organic chemists can do, also has remarkable biological properties. This peptide is produced by your pituitary gland. It's involved in uterine contractions, in pregnancy and in birth. It's involved in lactation. But incredibly, this is also involved in emotions and pair bonding, in other words love. You know, you say, oh, I'm in love with a person and it's that person and it's you. In the end, it's chemicals in your brain. I mean, this is really scary, you know. It's not you, it's the lack of oxytocin I feel for you. My God, what a deal breaker. There's fascinating literature. I don't want to get started out there. On differences in monogamous behavior among different species of voles, you know, these little creatures that run around on the ground like mice or shrews, and the prairie voles are monogamous. They participate in pair bonding. They hook up for life with a partner and it turns out that ties into oxytocin and a related hormone vasopressin and its receptor levels. So this is incredibly powerful stuff. I'll give you one more example because this is a cool example of the types of things that we can synthesize right now. So the virus HIV, or it's horrible, horrible stuff, the AIDS virus, and one of the enzymes that's essential for its life cycle is called HIV1 protease. And it's a 99 amino acid polypeptide protein. It cuts the other proteins that are needed for the life cycle of the AIDS virus. It's what these powerful drugs against AIDS, the HIV protease inhibitors, work against their molecules that fit into this enzyme and gum it up so it can't cut the proteins that need to. And this protein has been synthesized. It's a dimer, by the way, meaning two halves come together noncovalently, but this protein has been synthesized chemically in the laboratory and what's really cool. Okay, so you'd say, well, great, nature can make this thing too. What's really cool is chemists can make the mirror image of the protein and have used that to discover drugs which is really, really cool where you can go ahead and then use biological techniques to get a peptide that interacts with the mirror image and then you make the mirror image of the peptide that interacts and you've got something that's biologically is functional but doesn't break down. Anyway, incredibly cool stuff. I'm just going to give you a little bit of flavor of things. So there are 20 common naturally occurring amino acids. People often refer to these as 20 natural amino acids. Natural is a little bit of a fake word here because you can find others in nature, but there are 20 that are regularly coded for in the proteins in your body. I'll leave it to your classes in biology to insist that you memorize all of them or memorize the three-letter codes just like what we did with the sugars where I focused on a few sugars. I'd rather you know a few of them. I'll put up more than I think you should know but I want to show you some principles here and show you some of the variations. So a lot of the amino acids have nonpolar side chains. So for example, we already saw alanine with a methyl group and that's kind of the archetype for amino acids is to have a side chain. If you have an alkyl group as the side chain, you describe it as a nonpolar amino acid, right? You've got a greasy side chain. Grease and hydrophobic interactions, the desire to get away from water are a big factor in protein folding. So typically you have more of these hydrophobic amino acids on the inside of proteins. There are all sorts, not all sorts, but quite a number of different alkyl side chains. So for example, alanine is a methyl group, valine is an isopropyl group, phenylalanine is a benzoyl group, leucine is an isobutyl group. I'll just draw out a few of these, leucine. All right, so there are a couple of hydrophobic, a couple of greasy amino acids that are sort of odd balls in structure. Glycine is actually the smallest of amino acids. So glycine is the only one that doesn't have a side chain. So all of the other amino acids have a side chain. They all have the same direction of the side chain. Cysteine, because of the conningold pralogue rules, they're all by the way S stereochemistry. So I'll write S here with one exception. The amino acid cysteine, because of the higher conningold pralogue priority of sulfur ends up being R even though the chain points in the same direction, but that's kind of a technicality. Okay, so this is glycine. So glycine of course is achyro. We don't have a stereogenic center at the alpha position. The other sort of odd ball among the nonpolar amino acids is the amino acid proline. Technically proline gets called an amino acid, but that's not particularly important. What is important is that the amino group in proline is a secondary amine rather than a primary amine. This gives rise to some interesting structural features in proteins. Proline in particular has a tendency to turn back on itself, for example, because you can get cysts and transconformations about the amide group that it forms. All right, so that's kind of a selection of some of the hydrophobic amino acids. I want to show you some of the polar and charged amino acids. So the amino acid cyrene, for example, has a hydroxy group. One of the reasons that we're doing this right now and why you're writing these out is that unless you go ahead and really see things, unless you actually write and think about them, all of this just ends up structures in black and white. So cyrene, if you can't read my writing, S-E-R-I-N-E, is just like alanine, but it has an alcohol group. It has a hydroxy group off of the methyl position. There's threonine as well, which has an extra methyl on there, but I won't write it out for you. So I talked about cross-links from sulfur, and they're one of the important things in protein structure. So the amino acid cysteine is just like cyrene, but it has a thiol group. It has a sulfhydro group. And thiol groups oxidize very well. They oxidize very easily and form disulfide cross-links. And so this ends up, as I mentioned before, with hair and so forth, giving structure to proteins. Not all of the amino acids have side chains that are neutral in water at physiological pH. And again, I will leave it to you and your biochemistry classes to discuss more the charge of amino acids at various conditions. But this amino acid is glutamic acid. You may have heard of it in terms of monosodium glutamate, MSG, and your food in Chinese restaurants. It's the salt of it, gives a meaty flavor to things. And of course, in water, at neutral pH, the carboxyl group, right, carboxylic acids have a pK of 4 or 5. So in water at neutral pH, you're going to have the carboxyl group deprotonated, even if the amino acid is part of a polypeptide, where the amino group and the carboxyl group of what's called the mean chain are part of the polypeptide. The side chain is going to be deprotonated. So glutamic acid is an acidic amino acid. There are basic amino acids. But I want to show you its sister. Its ill-named sister, I might add, another polar amino acid. So we have a primary amide group here. This is called glutamine. Glutamine is just like glutamic acid but with an NH2 group instead of a carboxyl group with an amide group. Now to me, that doesn't make any sense. Glutamine isn't an amine. An amide group isn't an amino group. It's not basic. It doesn't behave like an amino group. But that's its name, and you call it that. There are amino acids with real amine groups here, lysine being one of them, another basic amino acid. And I'll give you one last amino acid. As I said, I'd say this is more than I would expect you to know unless you're a practitioner of the art. But you've seen now examples of all of these functional groups here. And so I think this is a really, really nice introduction to amino acid structure in polypeptide. So lysine, of course, at physiological pH, remember your pKa's, side chain of lysine, it's an amine, pKa's going to be about 10 or 11 so it'll be protonated at physiological pH. Arginine is the last one I'll show you. Also has a basic side chain. It's got a guanidine group. Arginine is even, arginine, arginine, arginine, the side chain's even more basic than an amino group. And so it's going to really be protonated at physiological pH at just about any pH you can imagine. All right, now as I was saying at the beginning what I think is just fantastic is the progress that chemists have made in the chemical synthesis of peptides and proteins. And if you think about it, the way in which one can investigate biological function, for example, the function of oxytocin or the function of vasopressin is to make molecules that have similar structures and say what does this group do? What does the cysteine group do? What happens if we rearrange the structure? And so the chemical synthesis, in addition to being a way of initially proving structure, also ends up being a tremendously powerful exercise in investigating biology. Now the conundrum, if you look, let's take a simple dipeptide. And so if I draw out just a simple dipeptide, I'll say R2 for just a generic side chain and R1 for just a generic side chain. And again, that could be anything. It could be phenylalanine. It could be isoleucine. It could be leucine. So if we look at this basic structure, you say, okay, well it's pretty obvious that the main bond in a polypeptide is an amide bond. You can say, okay, it makes sense if we were to think about how to make this molecule. We'd look at that bond and say we've learned a heck about making amide bonds. We've been talking about carboxylic acid since the beginning of the course in chapter 19 and then some more about it in chapter 22. We'd look and say from a strategic point of view, this is really simple. You'd go and say, all right, we just have to envision making that amide bond. And you know in the abstract in the view at 20,000 feet that we can make an amide bond by the reaction of a carboxylic acid and an amine. And now the problem begins. Well, how do you actually, how do you do this? We've learned lots of chemical reactions. We've learned that under the right conditions, if you go ahead and mix a carboxylic acid and an amine, maybe with a coupling agent, you learned about DCC. For example, you say we've learned ways of making amide bonds. You look at this and you say, yeah, okay, maybe somehow we could envision putting this together. But obviously you've got fantastic problems here. You've got two different carboxo groups, two different amino groups. Even if I threw together one amino acid and the other amino acid with something to make the carboxyl group react with the amino group, you've got a molecule could react with itself. You could get a molecule with two R1s in it or two R2s in it or an R2 at the C terminus and an R1 at the N terminus or the molecule could continue to build a polymer and you get three or four. In other words, you'd get an unholy mess no matter what you do. And we've been talking for the entire class about how organic chemistry is all about control and how organic chemists seek to control how reactions occur and that really boils down to being the essence of the synthesis of peptides and proteins. So imagine for a moment, I'll show you the answer and then we can talk about sort of the whys and so forth. Imagine for a moment we took one of our components and we were to tie up, we were to protect the amino group here. We've already learned about the concept of protecting groups. You've learned for example when you have an alcohol that you don't want to react as an alcohol, you can mask it. You can protect it as a TBDMS ether, a tert-butyl dimethylsilate ether group. And you've learned carboxylic acids don't react like carboxylic acids when they're part of an ester group. So you can envision protecting the carboxyl group as well. And so by being able to selectively protect, to tie up the amino acids, you can achieve this element of control. The groups that are widely used for these types of protection reactions are various esters including benzyl-like esters, benzyl and benzyl-related esters for the carboxyl group. And the T-Boc, tert-butoxy-oxy carbamil and other carbamate groups for the amino group. Let me show you a specific example of just a standard way of putting together a peptide. We'll make valoalanine by this. So imagine for a moment now that we put on valine, your textbook will talk in more detail about how these protecting groups can be introduced. I think in the time we have, I'll simply say it's very easy to take the amino acid valine and react it with a reagent like di-tert-butyl-dicarbonate to go ahead and put on a Boc group on the N-terminus of it. And by various esterification processes, one can go ahead and put on a benzyl ether or a related group to a benzyl ether on the C-terminal group. And I think I said we're going to use valoalanine here. So I will just go ahead and point out that this is, BN is used to abbreviate benzyl group. And we've learned about reagents that can react with a carboxyl group and render it more reactive. So for example, the reagent DCC, I'll show you it in just one second, can go ahead and can activate this carboxyl group and then allow it to react with this amino group. So the overall product of this reaction, and I will now start to use shorthand for these very big molecules, the overall product for this reaction then is the Boc amino valine on the N-terminus, O-benzyl ester on the C-terminus, and I haven't drawn the byproduct of DCC. All right, so just to reduce things, we're going to have to reduce things to a lot of our groups. Let's imagine for a moment that this carboxyl group is the carboxyl group on that carboxylic acid, that Boc valine carboxylic acid. DCC is a diametry agent. We talked about it briefly. Your textbook gives a mechanism for its reaction. It's really not the best way of writing the mechanism, but I think many students would think of the mechanism in the way your textbook writes it. Suffice it to say, cyclo-DCC-dicyclohexyl-carbodi-imid can react with a carboxyl group to give an activated carboxyl group. And the structure that I'm drawing here looks awfully fancy until you think about it. I'll abbreviate those cyclohexyls as CY. This structure looks awfully fancy until you think about it, and then you say, wait a second, for the longest time, he's been saying that a double bond to nitrogen is very similar to a double bond to oxygen. So this structure, this intermediate, this oacylurea intermediate, I'll just abbreviate it as int, is basically like an anhydride. In other words, we have a carbonyl with an electron with drawing group on the oxygen, and so the amine partner, which in this case I will just abbreviate as R prime NH2, now can act that, of course, being the CBC ester of alanine there, or rather the benzyl ester of alanine, alanine-benzyl ester, can react. I won't take us through all of the steps of the mechanism, but suffice to say the first step of the mechanism is the amine, a nucleophile, can attack the carboxyl group of the oacylurea intermediate. Electrons kick up on the oxygen, electrons kick down, they kick out what ultimately is dicyclohexylurea after some proton shuffling, and so the product of this reaction is the amide plus the byproduct dicyclohexylurea. And as I've been emphasizing in this class, organic chemists are very bad about writing byproducts of reaction because they're typically focused on the main product. The byproduct is called DCU dicyclohexylurea. So dicyc, by the way, was the first major coupling reagent that was invented, but it wasn't the last. It's actually terrible stuff to work with. It reacts with the proteins in your body to functionalize them, which sets your immune system on guard against them, which means that after working with it for a while, you'll start to develop terrible, terrible rashes, which is why Johnny and Kim, even though they will synthesize peptides in the laboratory as part of their research on cancer and Alzheimer's disease, never worked with DCC. So here we are at our peptide, and I want to draw out the structure again and talk about what can happen to this because now we have a peptide, we've synthesized a peptide in which our C and N terminus are protected with protecting groups, the benzyl group on the benzyl ester group on the C terminus, the Bok group on the N terminus. The Bok group is labile to acid. That means that a strong acid can take off the Bok protecting group and return to you in a mean, or technically in a mean salt that can be deprotonated. The benzyl group is labile to, among other things, hydrogenalysis. It's also labile to very, very strong acid like hydrogen bromide and anhydrous acetic acid. So if we take our dipeptide and we carry out two deep protection steps on it, a hydrogenal, we'll start with a trifluoroacetic acid deep protection step. This is TFA, I'll write this in parenthesis. Trifluoroacetic acid is a pretty strong acid. It's right on that cusp between weak acid and strong acid, not as strong as HCl, not as strong as sulfuric acid. It's PKA is about 0.23. So it's way, way stronger than a regular carboxylic acid due to the inductive effect of the trifluoro methyl group. And then if we carry out a hydrogenation with palladium on carbon, we can remove both of the protecting groups. And so I'll just remind us, I'll say Bok is able to be removed with strong acid, e.g. for example TFA. And the benzyl group is able to be removed by hydrogenalysis or very strong acid. And as I said, by very strong acid I mean, for example, hydrochloric acid, HCl and dioxane, hydrogen chloride in dioxane or HBr and acetic acid. All right, so the overall result of this is that we have now removed both of the protecting groups to give us just the free peptide like so, in other words, we have hidden one carboxyl group, hidden one amino group, brought together the other carboxyl group and the other amino group and coupled them together. Now I'm lying to you just a hair about this because I haven't used a base. Technically, we'll still be the TFA salt. I'll say it's still the TFA salt at this point. But honestly, if you wanted the free base you could just go ahead and add a base anyway. So technically still the TFA salt. All right, I want to show one thing. We're kind of taking a traipse through mechanism here but sort of mechanism light and getting kind of a summary of how things work. We've taken certain mechanisms in the course and worked them over in gruesome detail like acetyl formation and acetyl hydrolysis which came back to us in sugars. We're getting others where we're getting the general gist of it. Let's talk about the Bokk deprotection mechanism. So okay, so here you have your Bokk group like so. Now in a in strong acid like TFA you can protonate all the different positions in the Bokk group. And the easiest way to think about taking off the Bokk group is if we protonate on this nitrogen here. So I'll write this sort of as a transient intermediate. If we protonate on this nitrogen here now we're all set up to lose a tert-butyl carbocation. And so if we just go ahead and kick out electrons we're going to fragment this molecule into carbon dioxide. Carbon dioxide is very stable and tert-butyl carbocation and an amine like so. And then of course as I was hinting at over here your amine, I'll just draw this down here with more TFA we'll go to RN8, we'll protonate to give the ammonium salt like so. So I want to point out a couple of amino acids that are special. We've already seen them. So we saw for example with lysine you have an extra amino group and we saw with glutamic acid that you have an extra carboxyl group. And so in these amino acids and in several of the other functionalized amino acids, amino acids with acidic and basic and polar side chains, additional protecting groups are needed. And suffice it to say there's chemistry to put on these protecting groups. And I want to stop at this point talking about the details of protecting groups and come on a little bit more to the theory of what we do. So okay, so the general gist of things is we're going to build our peptide amino acid by amino acid starting at the C-terminus and working to the N-terminus. In other words, you could imagine with the valolalanine that we made if we had for example only taken off the buck protecting group off of the amino terminus, we could then go ahead and couple in another amino acid, say phenylalanine, another amino acid say phenylalanine to make a tripeptide. We would introduce that phenylalanine by having a buck group on the amino terminus, on the amino group and a free carboxyl group and again carrying out a DCC coupling reaction. And so this is the type of technology that allowed Vincent de Veniode to build up to synthesize the Nonapeptide oxytocin back in the 1950s and ultimately really create the beginning of a revolution. Now another huge advance came along with an idea that Bruce Merrifield introduced in the 1960s and his colleagues thought he was crazy for this but he won the Nobel Prize in 1984 for what he did. And his idea was to go ahead and connect the polypeptide to a solid support, in other words to make the C terminus of the polypeptide instead of just a protecting group, a big plastic bead. And so let me show you the gist of modern solid phase peptide synthesis and then we can run through a few of the details. So imagine for a moment that we have a polymer bead, polymer plastic polystyrene, I'll show you the structure in a moment and it's not exactly what Merrifield initially used but imagine for a moment that the polymer is basically a big benzoyl alcohol and that you have an OH group on there and then in the first step we couple one amino acid, I'll call it sort of in the abstract, we'll just call it R1 for our side chain and we'll say that we have a protecting group on the nitrogen. So this would be like a buck protected amino acid. And so now we have our amino acid with our protecting group, I'm just calling that PG onto the resin and I'll abbreviate the polymer with this big ball here. All right, now imagine for a moment that we deprotect, in other words we carry out a reaction like the TFA reaction to remove that protecting group for example to remove the buck group that was introduced. At this point you're going to get a, you're going to have the amino group here, the free amino group connected to the first amino acid, connected to the resin which I'm abbreviating as a circle. Now imagine for a moment that I couple in the next amino acid and so I'll say I'll couple and we'll call this R2 on an amino acid again with a protecting group which I will call PG and I think I'll move down to the blackboard here to show you what we've now got. So now we have R2 connected and now we have our protecting group. And imagine for a moment that we again deprotect and now I'm going to shorten things and write deprotect this step one in couple and I'll show you our third amino acid. You notice that we're starting at the C terminus and we're building up toward the N terminus so I'll call this R3 and our structure is getting so big that I now have to go to the other blackboard and at this point we're at a tripeptide now attached and I've gone from the N terminus to the C terminus here with our third residue, our second residue and our first residue. And finally imagine if in the abstract we cleave from the resin and we deprotect, I'll say cleave and deprotect because they're sometimes done at the same time and the result of all of that now is that we have synthesized a tripeptide and the operation is so simple once you work out the chemistry that it can be done on a machine repeating these operations one after another after another to build up polypeptides and even very small proteins purely through chemical synthesis. And this was what got Merrifield the Nobel Prize. It was this recognition that you could do this and the achievement of doing it and so while the work of synthesizing an oxytocin, anonopeptide was a fantastic tour to force, this opened the job to make things trivial to make molecules of that size and one of the beauties of this is you really need your chemistry to work cleanly and so you can use a huge excess of reagents for equivalents of each amino acid to drive the chemistry. Great question. Great question. Why are we going from C to N? And that question is particularly profound when it is counterintuitive when on the ribosome you synthesize it or the ribosome synthesizes the protein from N to C. It turns out that there are reactions that occur. If I try to activate, let's even assume I had this protecting group and I tried to activate this carboxyl group with DCC, there are reactions that occur that will epimerize this stereocenter in chemical environments with vigorous activating agents like DCC so you will get a mixture of diastereomers. So in general, like all the time with certain very special exceptions including something called native chemical ligation that may give rise to the next Nobel Prize in this area. With certain exceptions, with virtually no exceptions I should say, we always go from C to N. The opposite of the way of the ribosome. All right, I want to show you a couple of last bits of the genius that was involved in all of this chemistry. So one of the pieces of genius is really very simple. It's the polystyrene. So polystyrene is a polymer in which you have a zigzag chain of polyethylene with benzene rings attached, like so. You make it by polymerizing the alkene styrene of linking the molecules together one after the next after the next. Now, styrene is styrofoam. If you've ever taken styrofoam peanuts and put them in any organic solvent like acetone, they form a big group. If you do this with a styrofoam cup, then you bring in to lab and you put a little acetone in there, it just melts. It just dissolves technically. So Merrifield's bright idea, one of his bright ideas was to link together two chains with a little bit of divinyl benzene to crosslink the polystyrene into a big network like so so that the chains all link together and the molecule would swell up but it wouldn't dissolve. And then he used the benzene ring and functionalized just a few of the benzene rings on the polystyrene to put on the protected amino acid with the protecting group. So that was one real piece of genius here. And so the chemistry that I've sort of been outlining here really can be thought of as, I'll write this as Bok, let's say for the one that we, or one that we could synthesize, Bok O polystyrene and then we can follow with a series of deprotection and coupling steps where we use TFA, trifluoroacetic acid to deprotect the amino group. And remember I said, well, your amino group technically is still protonated so you can add a base like diisopropyl ethylamine or triethylamine to deprotonate, IPR2NET to deprotonate the amino group that's now liberated and three will couple in the next amino acid. And so I'll say Bok, Phi, Phi is the abbreviation for phenylalanine so I'll say Bok, Phi, OH in DCC. And now we can get Bok, Vail, Bok, Phi, Vail, OPS and we can repeat and repeat and repeat to build up the polypeptide chain, one amino acid after the next, after the next. So I will, I think leave you with, well, let me go ahead and I'll just say repeat and let's say I did this this time with alanine so again it's, we shorthand a lot of stuff here. So let's say I go ahead and do this with, let's say, leucine. So repeat and now we go Bok, Leu, Phi, Vail, OPS and now if you imagine we use a very, very strong acid now we can get a tripeptide and if we had continued on the resin we could have gone up to a tetrapeptide. All right, the final innovation that I want to show you and this is realized, this is standing on the shoulders of giants for many years who made this chemistry possible to a point where it is now routinely possible to make peptides and small proteins. So one of the last innovations was Merrifield's initial chemistry was kind of nasty and by nasty I mean that that last step of deep protecting, of getting rather cleaving from the resin, getting the amino acid off of the resin, getting the polypeptide I should say off of the resin, required super, super strong acid, HBR, hydrogen bromide and acetic acid is one of the reagents used, it's just nasty stuff, anhydrous hydrofluoric acid, anhydrous HF eats through glass, burns through skin, burns to your bone, does horrible, horrible stuff and so the last innovation that I'll introduce was a base labial protecting group, the F-Moc group and the F-Moc group is a big, big, big aromatic unit. It stands for it's in the same family as the Boc group. It's a fluorino methyl, that means CH2, oxy carbonyl and the F-Moc group is cleaved with a base such as papyridine so I'll say a mild amine base and so the F-Moc group if you treat it with papyridine, whoops the base can pull off a proton here, kick out an alkene over here, kick out an oxygen and ultimately kick out carbon dioxide so when this occurs now you end up with the BH plus, the protonated base plus the group that came from the fluorino group which eventually reacts with some more base plus CO2 plus the amine and so putting all of this together I'll just show you an example of a modern solid base peptide synthesis and then we'll wrap things up. So in a modern peptide synthesis what's done is the polystyrene is linked to a much more acid labile linker and this might be a good final exam question to go ahead and explain why this linker here is much more labile to acid and now you have your first amino acid whoops that's a wedge here and so now we go ahead and I'll call this R1 and so we go ahead and we deprotect with papyridine, I'll just abbreviate that as PIP and then we couple and as I said typically nowadays people don't use DCC but I will write DCC so here's our F-mock protected second amino acid R2 and so now you build and build and build and this linker here is called a Wang linker and so I will write O Wang PS and finally after building and building and building you'll cleave with papyridine and so I'll just say repeat, repeat, repeat and then you finally cleave with papyridine, I'll write that as PIP and 2 TFA which cleaves the peptide off the Wang linker to give your peptide. All right well on that note we're going to take a moment to pass around the quizzes so take one minute to finish up writing and then you can put away your notebook.