Okay gang. Um, welcome back. This is part two to the chapter on amino acids and peptides. And we're going to uh pick up where we left off last. Uh, last time we talked about uh protecting the amino group and protecting the uh caroxy end or the caroxyic acid group of amino acids. Um shown on this slide is um how how to remove um the amine protecting group which is also an important step that um we'll talk about. You don't typically take it right off the amino acid. Um you actually take it off after you've made a peptide bond, but I'm going to show you how you take it off um right straight from the single amino acid just to illustrate the reagents. And the good news is the mechanism on these is complicated enough um that we will not be featuring the mechanism for this for the deep protection um on our upcoming exam. But uh you should know the uh reaction mix or what you treat the reagent with. Excuse me. So if we treat a buck protected amino acid with HCl or TFA that's an important one to know what it is. TFA stands for trio acetic acid and it looks like this. essentially trifor group and it is a acetic acid derivative and it has a pKa um of about -3 whereas the pKa of HCl um is about -9 - 8 or let's see wait a minute 8 sorry about that some textbooks say seven or minus somewhere in that ballpark. Very very strong acid. This also is very strong acid and these acidic conditioners will take off a B group. They will also take off a CBZ. I don't have that over this arrow, but it turns out that you can also cleave the CBZ. Um so I could say or HCl or TFA for taking off a CBZ. um mechanistically slightly different but both the Bach and the CBZ CBZ are acid sensitive. Uh the FMOK is base sensitive and therein lies its utility in that it can be removed um using conditions that will not will not touch the box. So for example that base which is called the paroditine or triethylamine both of these this is a um secondary amine triethylamine is a tertiary amine and either one of those is strong enough base to actually deproinate that proton um pull that off and I might need to walk back what I said about the mechanism it's been a while since I've um been since last summer. So, let's let's wait and see. I think maybe last summer we actually did um cover this mechanism. Um it makes a little more clear why a base can take off the CBZ. Turns out that proton is acidic enough. Um because once it's been deproinated, you leave behind an aromatic annion. An annion is aromatic. They have 2 4 6 8 10 12 14 pi electrons. Uh this is a cyclic system that looks planer pure on every atom in the ring on the on the conjugate base of this thing. The negative charge there and long story short that makes it so this base is strong enough to deproinate that proton. you can take cleave that proton uh under basic conditions to give this and a deprotected amino acid. Um so let's kind of hold that thought um when we come back and you can ask me in class but this mechanism usually is is one that have sometimes featured on exams. So this is a possible mechanism that you will probably want to be thinking about um for an exam. This mechanism probably not so much. Um know this one for sure. The hydrogenation of of CBZ is pretty easy. Um long pair attacks that proton that hydrogen. The electrons in that HH bond attack that carbon and that H becomes more of those. There were two H's there, CH2. Now there's a CH3. The third hydrogen here came from that guy. So maybe if I doctorred this light a little bit and get CH2 and then you know another H and let's choose maybe I guess we could use blue H and that H goes there and one of the H's on on the nitrogen ends up coming from that that that other H. Uh anyway, okay, this is a possible mechanism I would encourage you to know for an upcoming exam. But these are the three ways to remove the common protectant groups, either a Bach, an FMC or CBZ. Okay. All right. Um this is actually on in your peptides handout. It's on page five of the peptides handout. page five uh step or number eight. Um okay, on uh page six, interestingly enough, um we have this is page six uh of the gra of the peptides handout. We have the method for removing the methylester or removing the benzilester. Um and so to remove a methylester I'm just looking at and make sure I've got the right page that is page six and it is number nine removal of the methylster remove viaification which is essentially code talk for base promoted hydraysis of Okay, that's what sonification is. And so you can soponify the methylester or the benzil. Okay, so both benzil and methyl can be removed viaification. Okay, so again I was going to cut and paste this down here. Oops. Dang it. I'm meant to copy it and paste it. So this also is a base hydraysis on esester. Same recipe as up here. The added acid is to protetonate the sodium salt. So if you just use this step, you would have a sodium salt of the caroxyic acid. Um, same thing there. But if you add acid, it deproinates the sodium salt and you have your fully proteinated caroxilic acid. The other way you can take it off is via something called a hydrogenation. And in hydrogenation, as you can see here, um, you're adding the elements H2 across that OC bond and it gives you talline. So this piece here becomes tall toine and that becomes an O. Um, which um, yeah, there it is right there. Becomes a caroxilic acid. Sorry. Uh this also is called hydrogenation taking off the CBZ. In the CBZ you're taking off the N protecting group, the amunoprotecting group. Uh but here you're taking off the C protecting group or the esther. You're cleaving an esther here. There you're cleaving this is not an esther. It's a carbine. It's related to an esther. Looks like this part looks like an esther. It's true. what you have with having that nitrogen there um makes that whole function look actually called a urethane. I won't test you on that name, but that is a urethane. An N carbonel O is a urethane. Here we're cleaving similar group, a benzil group off of this is a straight up est regular estester, not a urethane. If we had a nitrogen there, that would be a urethane. Okay. All right. Let us talk about solution phase peptide synthesis. Okay. I've been building the whole time talking about protecting groups, putting them on, removing them, building towards this goal. Okay, solution phase peptide synthesis. And what this is is a synthesis of peptides in solution. Okay, it's in some sort of solvent. Typical solvents usually are DMF and um there are some other solvents but that that's the most common one DMF. It's an aproic solvent we learned about in 351. Okay. So there are a couple ways to do a solution phase peptide synthesis and again this is solution phase that is a a poorly written word phase. PH A S E that's what that's saying solution phase is a reaction that's done in liquid it's done in DMF so it's a it's a solution of reagents two ways to do it one to start from the N terminal end so we're going to start with an N protected amino acid I'll write that down there N protected Okay. Protected as a urethane. Again, I mentioned the point of protecting this nitrogen is to make it so it is no longer an amine. This is no longer an amine. It's a urethane. This functional group with an N lond O that's called a urethane. No longer an amine. Now it's a urethane. Okay. Urethanes um do have lone pairs on them but that lone pair deoizes into the carbonale enough that it is not nucleophilic. Okay, I make that point that's not nucleophilic and therefore cannot participate in the amid bond forming reaction. All right. So we start with an N terminal protected amino acid. This guy is protected on the amino end protected. It has a free caroxile group. Okay. And the sky is the limit as to what amino what side chain you have here. This could be a alanine or a glycine or phenol alanine or a serarene or methionine. There's all sorts of different things the other could be. you bring in another amino acid. This one is protected on the C terminal end. The caroxilic acid is is protected as an esther. That me stands for methyl. That's the methylester of amino acid. I think we saw saw them here. That's a methylester. So here here we're protecting deep here I'm trying to deprotect pull off the methylester. Um here we're bringing in a a methylester protected amino acid with a free amine. Okay, got a free amino end and that end will react with that carbon to make a peptide bond between that end and that carbon make a bond. The reagent you use is dcc. And you go back and look at the last slides and I told you what dcc is. I told you that you should know a structure and I also mentioned that you know should you should know what it does and why we use it. But this is this is a structure that has uh two cycllohexal groups two nitrogens. Okay, that is dcc and it stands for di cycllohexyl carbo diamimid. That's a dimeid and that piece activates the free acid here and makes this an active isolating agent and will catalyze formation of an NC bond. You're going to make a peptide bond. So the bond you're going to make is this morning. That's the bond you make from steps one and two. In step three, you cleave the methylester off of. So before you get to this, you're going to have a methylester here. Before you do steps three and four, there will be a methylester deposition. May have been more clear if I hadn't had three and four on the same arrow. So um before you soponify you have a methylester here. When you soponify that hydrayzees a methylester and um when you acidify it proteinates the sodium salt. Okay. So these two steps one through two were needed to make that bond. Steps three and four were needed to um hydrayze the methylster to give you a free acid. Okay. You then repeat steps one through four with new amino acids. Okay. So bring in looks like I'm naming this first R group R1 and that's R2. with the new amino acid we brought in over here and kind of highlight. Let's make it blow through it, shall we? Okay. Or she was that's the new one we brought in. And the old one we had that's a little bit orange feels fun of it. One we started with was this one. Okay. And we've made that red bond between the end of the blue piece and the carbon of the orange piece. And H2O came out uh in this reaction. It's called a condensation action where you lose water DCC. Most that reaction and D these two steps make that bond. these two steps cleave the methylester and generate a caroxilic acid. If you do steps one through four with new amino acids and I'm talking um we have a third one with a third R group or fourth one with a fourth R group or a fifth one with a fifth R group all the way up to N. Okay. And it turns out um I'm not going to hold you this nor test you on it, but usually you can do this about without with about 100 amino acids. Okay? So you can make what they call a 100 mer. And so um you repeat those those same four steps over and over and over again. the incoming amino acid plus dcc then you subonify then acidify and you extend the growing peptide chain this direction from from the starting one all the way out. So if we use the same color color scheme, this is our B protected amino acid one. Okay, that's this guy. And then let's bring in blue. This is amino acid two. That's that guy. Amino acid three would be some other. We just use whatever color. Okay. But it would basically look like this but had a third a different R group here in the lesson four would be kind of similar u in a sense that it would be identical to this but you'd have a different R group. Okay. And you're building and going from the internal end you're going towards the right each time making a new amid bond. Okay. So the next time we added one, you'd make a new amid bond at that position. Finally, when you're done, um, after repeating those four steps over and over and over again, you treat with HCl or TFA. I can write that right here or TFA. and TFA uh will hydraize um that well not it's not hydrayzeed but will cleave that that group off that carbounding off that urethane group off and convert that NH into an NH2. Okay. So you end up with a fully deprotected peptide with amino acid. I'm just going to go ahead and copy these. got both faster and placed them here. So, we've got amino acid 2 attached amino acid 3 attached amino acid four all the way out to however many amino acids we want to put into the peptide. Right? So, we could use a different color. Let's use maybe brown. Okay. And so, that is one way to make a peptides in solution. from the N terminal end. Okay, that's intern terminal solution phase synthesis. I got to take a little bit and drink water second. All right. Um let's take a look at the second way you can do this. And this is starting from the C terminal end. Okay. So, from the C terminal, you start with an esther protected amino acid with your R1 and we'll color um um him. Let's just use a let's use brown, I guess. Okay. And then we bring in a new amino acid which I'm going to use. These colors are arbitrary. Don't try to correlate the two the two approaches to different colors. I'm just doing it whatever color I feel like but it's to illustrate the principle. Okay. So this carbon is going to form an amid with that NH free caroxyic acid and a free amino group. The DCC will catalyze the coupling of that NH2 to that carbon. Okay. Um and we'll make this bond red here. It's going to make the peptide bond right there. So using the same color scheme. Guess I can keep doing math. There's our brown amino acid, the C terminal end, and we're introducing our blue amino acid like so. Okay, we've made now a peptide bond between the blue carbonil and the brown NH2. We repeat those three steps with new amino acids. Okay? And each time we do, we make a new amit bond. So there's our first amino acid. Here's our second amino acid. Here's our third. I'm just choosing whatever color I want to kind of highlight the point. What the colors mean is these are different amino acids, different from each other. Okay, it's a darker blue. I hope you can tell that. Okay. So, working from the C terminal end, which has a free amine, you're going to bring in successively boach or CBZ or FOC protected amino acids with a free caroxile group. And that free caroxile group will react with the amine. So each time you make a peptide bond you will have a N protecting group you need to remove which can be removed by HCl. Okay is here and then we're in the next one and so forth. Okay. Um that's that. When you're done, uh, you need to remove all the protectant groups. And at this point, um, if you treat with HCL as your as your step three, that will have taken off the B group and the growing peptide. So now all you have to do is deprotect the sodium hydroxide, which hydrayzees the into caroxyic acid. Okay. So I think my next slide gives you a chance to practice this. So I'd encourage you to go ahead and turn these slides off. this voice over recording and try to prepare the following tripeptide using solution phase chemistry. We're going to make the alibli and so uh you can turn your computer off or pause it. But basically what you're trying to make is this guy NH2. This is the N terminal end. So we have a 3 NH2. This is a C terminal end. So we have a entry roll in over here teach there. And so basically what we're making I don't care about showing the kirality at this point. um you could do it and it is important but it just complicates things and so I'm okay with you showing um the chyro amino acids like this even though this doesn't really communicate their actual completely I kind of slotted slanted that up because I'm I'm looking at my iPad from a bit of an angle It's got just kind of tilted in my desk. Hopefully that's a little straighter. How's that? Yeah. Okay. So, this is phenol alanine bound to alanine to glycepect. There's two ways we can make it. We can make them the internal end or the C terminal end. We just talked about C terminal right here, right? And we also talked about uh internal and all of these are in solution in DMX. Okay. So let's do the internal first. Okay. I think I've already ought to keyed out. But if we do the internal, we're going to start with a nitrogen protected uh free acid here. So our final alanine will be nitrogen protected and a free acid. Um I think I just used the I haven't done it yet. Okay. And this is basically what I just wrote there. Okay. Same structure. Yeah. I just better move the NH2 down like that. Doesn't matter. Told you we're not worried about the corality for this problem. Okay. Let's see how to make it. Okay. Yeah. We're going to start with a nitrogen protected phenol alanine. And I'm using this symbol because there are three different ways you could do this. This could be a Bach group, an F mock group or a CBZ. Okay, those are three nitrous protective groups. But you would have a free caroxilic acid here and bring in a esther protected alanine. That's our second amino acid we want to install is that that's alanine. So bring it in. uh this will be caroxil protected that makes it so the only thing that's going to happen okay only thing that's going to happen is that that nitrogen will react with that carbon to make that amid bond okay period it okay period okay and we use DCC to promote the formation of that I need to say a few words about why we use DCC and I need to Say that a couple more times. Um, kind of forgot to do that. The reason you use dcc is to minimize rasmization at that center. I just got through saying I don't care that you show the actual kirality there, but it does matter. It matters enormously. I mean uh whatever the configuration is there has a huge impact on the biological properties of the peptide that you make from an amino acid and the naturally occurring ones are like 100% inatumericically pure disposition but many of the commercial synthetic methods for making peptide bonds like this one many of those will rasomize this center and introduce a a new ananter at that center and that's bad is we don't want that. Okay. DCC suppresses rasmization. Okay. So DCC the beauty of DCC I'm going to write this down. I hadn't done it before. It suppresses or minimizes rationalization which is problematic. in peptide synthesis. That's why you use dcc. Okay? But it promotes coupling between that NH2 and that carbonal and we lose water. HO and H. HO and H. That's water. H. HO. That's water. We all water. We make that amid bond. And boom, we've made our first. Okay. But we need to make one more. We're making a tripeptide. This is a deptide. We're what we're going to do next is cleave that methylester. And as you recall, the way you cleave a methylester is to do asoponification, right? So that would be an N A, which will basically cleave that an Omethyl and replace it with an O NA. Okay. Now when I um imported this, this is not editable. is coming that came in as a formula. Oops. See, got that. That's right. Okay. So, can't draw on top of it and have it have a copy. Uh, so I don't want to I don't want to take the time to to redraw that whole thing this whole thing. But the bottom line is that hydroxide will will hydraize that methylester and convert it into a sodium salt. Okay? which then would require um after uh base hydraysis you need to add some H+ to protetonate the sodium salt to make the caroxylic acid and I think I show that um right here. Okay. Um we're sonifying that. Looks like I I probably should have added some H+ there. Forgot to do that. H+ is going to be um the first step is going to be quonify to then we acidify to put a H on the sodium salt is formed when we hydrayzeed the metalester. Okay, but now we have a dipeptide. Let me show the bond that we made the DCC coupling. Okay, we're now ready to bring in the next amino acid. Okay. And here we go. The next one is supposed to be a glycine. So we bring in glycine protected on the caroxy end. That makes it so this end cannot react with that. We only want the N to react with that carbonal. And with the protecting group, the methyl protecting group, that lone pair on that nitrogen is the only thing that will react with that carbon. And we use dcc. Why? Why do we use dcc? to suppress rasmization of these two kyality centers. Okay. So the purpose of these two steps is to generate that peptide bond one we made before. I'm going to go ahead and highlight the one we made. Maybe I should use those letters in red. The other one in red. We had done that one in red before. Let's make this one different color just for fun. Cool. Okay. This is now a fully protected tripeptide. We have phenol alanine right there. This phenol alanine. Okay. This is alanine. This is glycine. It's fully protected. We need to get rid of the protective groups. Generally speaking, peptides are biologically active only when they do not have protectant groups. technically just mess up the biological activity. So you need to get rid of these. Okay, we can use the methods we discussed earlier in the earlier part um of the slides I was talking about methods for deprotecting and the best method for hydraizing methylster is to do a spawnation. Okay, I really ought to include this as a two-step with H+ as a second step now because this will will leave you with an O Na. Okay, the H+ will fully probate that it it bottom line is it cleaves off that methylster and makes a caroxyic acid. Okay, so the caroxy end now is deprotected. Now all we need to do is detect the amino end. get rid of that that protecting group. And those um amop protecting groups are listed on page five of the peptides handout. They're the Bach, the FMOK, and the CBZ. And um all two of them Bach and CBZ can be removed with HCl and or triositic acid and FMO can be removed the base. Okay. Okay. So, we take the tripeptide that has been deprotected under the boxy end and we treat it with HCl or TFA that will cleave Bach or CBZ. So, if this is a Bach, these reagents will cleave it. Or if it's a CBZ, these reagents will cleave it. Okay, that's what I'm trying to say here. So, we could have chosen to use a Bach protected amino acid on N terminal. or a CBZ either one can be cleaved with HCl or TFA but an FM mock if we used FMO that will require triathlamine or pipodine that's the name of that that's pirodine I didn't write it down it's a common solid pepine secondary mean this is a tertiary mood and both secondary and tertiary means are strong enough bases to cleave the F mark group. So take the FM mark group off and make long story short by treating this tripeptide which is still protected on the N terminal end. We want to get rid of that protectant group. Any of these reagents will remove either the well as I said this is what's required to take off the block and CBZ. This or this will take off the mo FM and there is your fully deprotected tripeptide. No protective groups at all. This is felonine. This is alanine. That's glycine. Cool. Okay. Let's take it from the C terminal. Okay. And there's the same peptide. We want to build it from this end. So, we're going to start with the methylester here, the free amine. And we're going to bring in in its place a three caroxylic acid protected with this nitrogen protected with the boach florz to make that bond. Okay, that'll be the first step. Let's take a look at it. Okay, so there it is. There is the methyl estster protected glycine. We bring in an amine of the alanine protected either with CBZ, ethmok or boach. Use dcc to make the amid between this carbon and that nitrogen. And um this is the bond we make between this nitrogen and that carbon. Okay, that's good. That's the one we just made right there. Okay, we'll choose colors just for the fun of it. Color we use back here. Remember used orange on the glycine and green for alanine and pink for felon fen fennel alanine. So the same color scheme. So there is your um methylester of glycine or she knows right there. Okay. We've brought in what color was it? Was it green for the Yes. for the alanine. Alanine is green that color scheme. So bring this in again. We have made the amid bond. We've locked out an O and an H. O 2 H's O, excuse me. O2 H's O2 or H2O. H2O. lpping out water to make a nitrogen carbon bond. Okay, cool. All right. Now, all we have to do, think about it, we're starting from this end and going that direction. Working from the C terminal end to the N terminal end. Now, I didn't do I didn't start this like this kind of backwards to confuse you. I wanted to illustrate that this is the C terminal end. Yes, we're building it towards the right, but when we're done, okay, the peptide actually will be between the N of the glycine, the C of the alanine and the C of the phenyl alanine. I want you to think about that as we go along that face value kind of looks like I'm doing it backward, right? But I imagine me taking this and flipping the whole dang thing 180 degrees, starting with this over there and building that direction. That's exactly what I'm doing. I just chose to draw the star material on the left. I could have I could have, you know, taken that and dragged it over here and and gone that direction. Okay. So we are still starting with a C protected amino acid, a free amine. We're bringing in an N protected amino acid making a peptide. You're going to cleave off that protecting group using um one of two different sets of conditions. If this is Bach or CBZ, we can use HCl or TFA. If it is an FM mock, we have to use a base like triamine or piperine. Okay. So, oh, you may be feeling this way. I know sometimes in the past students have felt a little bit this way. Mr. Osborne, may I excuse my brain is full here. Sometimes I feel that way like I know way more than I wanted to. Oh, look at this. So, their their class is at 10 in the morning. Not dogs. Um, okay. So, continuing on. This is what we just got through talking about. I use the same color um color scheme. This was the green one we were bringing in, right? The alanine and we were connecting it to the um caroxy protected glycine. There it is the caroxy protected glycine and we have made the end bond. weight there between the N of glycine and the C of aline. Okay. All right. So, repeat those columns again. This would have been easier if um this were editable, but drawings or not. They were imported from PDF. So, they're kind of static. I can do is comb on top of them start. So, what I did, I just redrew this whole shmear down here. I'm going to cleave off the protecting group either with HCl and TFA with a takeoff bulk or CBZ or I'll cleave it with trig. And what this is determines which of these sets of conditions I use to take it off. Okay, that'll get me to here where I now have a fully deprotected dot. Well, not fully. This end is deprotected. That C terminal is still protected. So, I'm just going to go ahead and I think I can copy these colors easily enough and drag them down. So, I don't have to redo that every time. Look at that. Cool. So, but we we cleaved off darn it. Oops. Okay. All right. But we cleaved off um the protecting group. Yeah. Off the end. Now, all we have to do is see what are we trying to do. This a dipeptide. We're trying to do what? Make a what? I think we're trying to make a tripeptide, aren't we? Let's go back and look. Yeah, we're trying to make glycine bound to alanine bound to phenol alanine. So, next thing we're going to do is add phenol alanine also protected at the end terminal end. And go ahead and copy our colors here. We're going to use this as a starting material. Next slide. We got red bond and green. It doesn't overlap perfectly, but you get the idea, right? And we're going to bring in a nitrogen protected phenol alanine. Okay. So, the only end that can work can react here is this one with that. Okay. So, we're going to have that lone pair reacting. So that mean react to that carbon and we'll make a new amid bond to that carbon using DCC to promote the solution. Okay. So the new bond that we make the color of the with kind of purple that's the new new bond you make. Okay. In terms of color of the amino acids that was present alanine that's alanine. Um, glycine is that one. Okay. And then last but not least is pretty good. Okay, that's unprotected. Maybe that Oh, I forgot to color in the first we made, which is this one. Okay. So essentially what we've now made is a gly l okay written backwards. Okay. This is why I say you can do it this way because we've got the this the c terminal end doesn't have to be h yet. This is the c terminal end. We've got Y l NH2 on that end and a C terminus on that. Um this is backward from what the convention requires. Convention requires that we flip it 180 degrees. So you should actually have have it written as F l. When when you write the name with the threelet acronym, it should be written with the neo terminus on the left hand side and the C terminus. So that's the N terminus. This is the C terminus by convention. Okay. But that doesn't mean you can't take an object um that's supposed to be written this direction. There's nothing that says you can take can't take it and and flip it 180. That's what we've done. We flipped the actual molecule 180 degrees. So that what we've really got is glafy um in the backwards order based on the convention that the writers convention say you know what every time you write it you should write the threeletter acronyms in an N terminus to C terminus direction we're breaking that convention when we write it like this which is okay as long as you can see the structure and know that it's backwards and then flip Okay. And if I if I labeled his NH2 and that is a C2 H, um, it's arguable that this thing means the same as that. But you darn well better put the NH2 out there and the CO2 out there to illustrate that you know that you've written it backward because the convention is if you just see a fe conventionally okay that um means you must be written with the internal end on this side and C on that. Okay. So this is backward to compared to that convention. Okay. All right. So how do you make the fully protected tripeptide? Um you take the okay we haven't we haven't uh forgot we haven't deprotected the internal end yet I think. Okay. So we're going to take off the C terminal protecting group which you do by subonification. And then we take off the internal protective groups either using uh HCl or TFA to take off a buck or a CBZ or triathlamine or preparing take off an FM. So again here's a review of the deep protecting groups or methods. Take off a bachelta take off fmok repair. take off CDZ, H2 and platium or you could also use HCl and TFA to take off the CBZ. Okay. All right. Oh, and how do you take off the metal? You bonify you. This will modification will hydrayze this to the sodium salt and you add acid to to proteinate the sodium salt and you get your free acid. And again the benzil estester can be soponified or it can be removed via hydrogenation and I don't show the the radius. I don't know why if I cut and paste that wrong I can go ahead and and uh copy this and paste it here. The hydrogenation you just add H2 And it does require platium on carbon as a catalyst. Um the H2 will one H will add there one H will add there you know for short um that will do convert the benzoester into a free acid. We still have an amid bond intact to the rest of the peptide. That's what this is representing is the rest of the peptide. The rest of the peptide. Yeah, we're just clearing off the C terminal end either via sonification or hydrogenation of the exhaust. Okay. And so there it is. We subonify the C terone and we cleave that group using one of these these two mixes. I think we're kind of getting it right a little bit redundant. Okay, so here we go. This is phenol alanine. That's alamine. I forgotten about this. Maybe that's okay. I did a drawing slide still in there. that that's got a typo in it. Okay, my bad. I vaguely remember this now. Yeah, this has I think I fixed it in the uh the other version of this. So, my bad I hadn't caught that. Um, when did I introduce the extra amino acid? Okay. Um Oopsie. Okay. So let's lop that out. Think I inadvertently introduced a second elean. So this is a this is a tetropeptide. So that shouldn't have that there. Okay. Now should that be there? hazards of cutting and pasting. So, we never introduced a fourth amino acid put in put in in three. And so, what I've what I've crossed out is that I added a second um alanine in there. So, this actually is um reading from the internal end. It's phenol alanine all ali. Okay. So, it's not a tripeptide, but I just fixed it. So, it is. Delete that. Delete that. Delete that. My bad. Delete that. Um, and wow, we've got quite a few repeats on there. Um, anyway, I feel I feel sheepish. My one of the older used to say when he'd make a mistake and then he'd bleed like a lamb. Cut that out. Cut that out. Okay. So, when everything's said and done, we were trying to make the tripeptide from the C terminal. We go back and look at the inter that one. That one does not have a trip in it. This is the one we were trying to make. And based on the reagents I gave you, it is the one we made. Um I just inadvertently uh drew in a second alanine and then it became a cut and paste error from that point on. Okay. So that is what we made using the same color scheme. Oh, what was it? It was I believe orange for lysine and I think it was green for alanine and pink for okay now And this is shown backwards from how the convention wants you to show it. When you write the the sequence, you would write this as f l. Okay, which implies that um the actual what this this molecule has been rotated 180 degrees. Okay, this is the way it should be according to the convention of the alagy femal first, Cternal last. Okay, but there is you. This is kind of hard to explain, but you can synthesize it like this. This is still that same peptide. As long as you show the structure and you can clearly see, okay, there's the internal end, there's the C terminal end. All you've done is taken the molecule. There's nothing that says that you cannot take the molecule and flip it 180 degrees. That's totally fine. Okay. Writing the name like this has a very specific meaning. And that means that you have FE on the N terminal end and glide on the C terminal end. And that's true on the on the on the end and glide on the C terminal end. But you can draw it like this. Flipped 180 degrees. As long as you're showing the structure, that's okay to do. But if you write the name K ally B. So that name um represents an entirely different tripeptide. That name represents an entirely different car type in there. What that one represents is an end terminal. Have an element in the middle to be sure that that's cool. a C on. Okay. So that name right here does not represent that peptide. This one does. This is this is drawn backward to the convention. Okay. But this is that peptide flipped 180 degrees. It is not that peptide because by convention if you write glyph that means you have glycine is the M terminal end and alanine as a C terminal end. These two are two very different tripeptides. Okay, you can use the same color scheme, but don't want to don't want you to spend too much time on this. I just um it is one of the aspects of peptides is kind of confusing. Uh the fact that you can take the actual object, the actual peptide itself, and flip it 180 degrees without changing the molecule. The fact that you can do that, you can't do that with the name. As soon as you invert the order of of gl and v. Oops, I'm going to do that. As soon as you invert the order of gly, you have changed the structure. But with those those those words, those words don't do not mean the same as those. Okay, that's because you're not dealing with the structure. You're dealing with the name. And so anyway, yeah. load this up and grab the orange which you didn't want to do it. Okay. So again, what did I Okay, let's go ahead and call that one a day. Sorry, that one went a little bit south with I hadn't realized what I what I did. I cut and paste it old. I used an older version of the slides. I think I fixed this in a in a in a different set that I hadn't caught. So, I didn't realize that this was in real time real time making this recording. So again, I accidentally introduced a second alanine in this structure, which I should not have done. Should have been three instead of four. So everything else I said about this is right except for the fact I accidentally included a fourth one. I've now erased it. Once you cleave off the internal end, that's protecting group and the C terminal end where that was taken off right here. that with sodium hydroxide in misoponification. Okay, you end up with desired tripeptide written and because of how I chose to build it. I build it from the C terminal end building towards the right. We have C terminal on left going to end to end and to the right which is okay with the chemical structure but it's not okay with the main. Okay. You've got to you've got to write the N terminal in first. I know I'm being a dead horse in the name. You must write the N terminal in first, then the next one, then the C terminal. That is the correct name for that trieptide. If you write it in the order it looks like uh up here. So if you call GL and then Alla and then V and write it like that what you are representing is this. Okay. And this is not the same as that. Okay. So again this is flipped 180 degrees but you need to write the name from the internal end. Okay. To the C terminal internal end cural end. Okay. All right, that's confusing. Join me in office hours and I will try to clarify it. I apologize. That was not as clear as it might. Okay, let's go ahead and pause recording and see you next