Biochemistry Peptide Synthesis (Merrifield Process)

Hello and welcome back to Educator.com, and welcome back to Biochemistry.0000

In our last lesson, we closed off by talking about breaking a protein down to elucidate its amino acid sequence.0004

Now, we want to go on reverse.0012

What if there is a particular peptide or protein that I want to build.0014

Today, that is what we are going to be talking about- peptide synthesis.0019

And, the process that we are going to be talking about is something called the Merrifield process, after Bruce Merrifield who created this back in the late 60's.0022

OK.0032

Let's just jump right on in.0033

OK.0036

Now, let's use blue.0038

We can synthesize small peptides with a sequence of our choosing.0049

Now, if there is a particular sequence that I need, I need the peptide, I can actually do it- the sequence of our choosing.0069

And by small, we generally mean up to about 100 amino acids.0081

We actually do that pretty well.0091

And, we also have methods for attaching these fragments to create larger proteins.0093

Before, we took a large protein, we fragmented it, we sequenced the fragments.0122

Now, we are just going in reverse.0126

We are creating sequences up to about 100 amino acids long, and then, we can take those, let's say, 5 fragments, stick those 5 fragments together, and create a really large protein.0128

This is really, really fantastic.0140

OK.0142

So, what we are going to do, we will describe the synthesis of a peptide on a solid support; and I'll tell you what that means in just a second.0143

And, this is called the Merrifield peptide synthesis.0170

OK.0183

The Merrifield peptide synthesis, so here is the general scheme.0184

OK.0194

Now, this thing that we call a solid support, all these fancy terms, I tell you, you are going to want to cross all kinds of fancy terms in the biology, physics, chemistry, and science in general, and well, I guess they are necessary.0195

I don't know; a part of me always wonders whether they actually are necessary.0212

Why don't they just call things for what they are, but I guess, you know how it is; you have to give things certain names.0216

OK.0221

Solid support, it refers to insoluble polymer beads.0222

Just think of really, really, really tiny, small pieces of plastic- that's all it is.0237

Solid support refers to insoluble polymer beads that have reactive chemical groups attached to them, and they look like this.0241

I guess I can do this in blue; it's not a problem.0270

So, I have this bead, and then in this particular case, the group that I have attached to it.0272

That's fine, I'll do it down here, CH₂Cl.0285

This is our polymer bead.0294

This is the bead, and it is insoluble, so it doesn't dissolve.0297

You can actually see it like sand at the bottom of a glass of water, and this our reactive chemical group right here.0299

In this particular case, it happens to be an alkyl halide.0309

Alkyl halide, chlorine, alkyl halide, this chloride, this chlorine is going to be a good leaving group, chloride.0312

So, you can imagine, the chemistry is going to take place right here.0318

Some nucleophile is going to come in, and it's going to displace the chloride.0320

This is the reactive chemical group.0325

Basically, the chemistry takes place here; and basically, this solid support, because we can see it, always see it and keep track of it, we are just going to build the molecule in a long chain all along there, and when we are done, we are just going to pull the beads out, and they are going to have these hairs of peptides.0329

That is all that's happening here.0346

A solid support refers to the insoluble polymer beads that have reactive chemical groups attached to them, really, really tiny pieces of plastic that have these chemical groups attached to them, and the reaction can take place.0347

That is it.0359

And, because they are insoluble, we can keep track of them; we can filter them.0360

OK.0365

So, what we do is we start with the bead, and just add amino acids one at a time; and just add amino acids extending from the bead.0369

That is it.0408

Basically, let's just take our...I only have some bead like that.0409

Well, we add an amino acid, that's one, and then we another amino acid, and then we add another amino acid.0417

We add another amino acid; we add another amino acid.0423

We go as long as we want, and we know what sequence we are going in because it's the sequence of our choosing.0425

When we are done, we split this, we break that bond.0430

There we go.0434

We have the protein that we wanted, and because we are actually just adding one at a time, we can always keep track of this because it is insoluble.0435

We see it; we know exactly where it is, and it is very, very easy to deal with.0440

It's a fantastic, fantastic procedure, and in fact, it is fully automated.0445

You just put the reagents in, and you let the machine do your work for you.0449

You come back a day or two later, depending on how long you want your protein to be.0452

It's done; it's ready to go.0456

OK.0458

Now, OK, in the body, proteins are synthesized.0460

Proteins are built from nitrogen end towards the carbon end.0478

In other words, on a piece of paper, they are built from left to right, from nitrogen to carbon, nitrogen toward carbon.0488

OK.0496

In the Merrifield synthesis, it happens in reverse.0497

In the Merrifield process, synthesis is...I wrote the N and the C in the same place, but now, I reversed the arrow.0506

It goes from the carbon end to the nitrogen end.0521

So, when we build this protein, we are building it from the carbon; we are going to the left, right?0524

Because we are adding this first, and then we are adding something to the left of it, to the left of it, to the left of it, to the left of it, it is in reverse; but again, the final product is what's important, but you should know that in the body, it is actually built from N forward.0532

You are adding amino acids; here you are adding amino acids backward.0546

That is it.0549

You are starting from the carboxyl end, and you're going back that way.0550

OK.0553

Let's go ahead and illustrate the procedure with an example.0555

We are actually going to build a tripeptide.0559

So, let's illustrate the procedure with the synthesis of the tripeptide Ala, Gly, Ser- alanine, glycine, serine.0563

We want to make this tripeptide; how are we going to do it?0593

OK.0596

Well, let's take a look at this, just some things we want to observe here.0597

Alanine is the N-terminal amino acid.0603

That is the N-terminus.0608

Serine is the C-terminus, the carboxyl terminus.0611

So, we are actually going to be starting with serine, and then we are going to add glycine, and then we are going to add alanine; because again, in Merrifield synthesis, we move from the carboxyl end towards the nitrogen end- the opposite of what happens in the body.0615

OK.0628

What I'm going to do is I'm going to give a general schematic of all the reactions that take place, and then I'm going to go back, and I'm going to number these reactions, and then I'm going to go back and go through the details of each of these reactions including the mechanism.0631

Now, I have to warn you, there is going to be a lot of chemical structures flying around all over the page; but it is really, really important to be able to do this, to understand what is happening at the atomic level.0649

It is very, very important0661

It makes all the difference between a marginal understanding and a complete command of your material.0663

This is why I'm doing it by hand, and again, this is why I'm not showing you an actual illustration, and just going through the illustration, because you have the illustration in your book already, and they are wonderful illustrations in the book; and I absolutely encourage you to look at it, but that is passive.0668

You need to be able to reproduce this.0683

You need to be able to write what is happening.0685

Draw the structure, write the reagent, give the product- that is where full command comes in.0688

OK.0695

Hopefully, I can actually fit this all in in one page; if not, it's not a problem.0697

I'm going to give the general schematic, and again, I'm going to go back and go through it in detail.0702

Alright.0707

Here is where we start.0708

I'm going to start on the left here.0711

I'm going to start, remember we said serine?0712

Let me just...where can I put this?0716

I'll put her on top.0718

Again, our peptide we are trying to build is the alanine, glycine, serine.0719

We want Ala, Gly, Ser.0726

We are going to start with serine, add glycine, then add alanine.0728

OK.0731

Here is the process.0732

I'm going to take serine, and I'm going to react it.0734

Actually, that's fine; I can go a little bit further to the right here.0744

I take serine, and I'm going to react it with something called Fmoc chloride; and again, this is a general schematic.0748

I'm going to go back and go through all the details.0757

When I do that, I'm going to end up with: Fmoc Ser.0759

And then, I'm going to react this with the bead, and I'm going to end up getting Fmoc serine attached to the bead.0767

OK.0785

Then, I'm going to react this with trifluoroacetic acid or mild base - either one is fine - and I'm going to end up with serine, and a bead.0786

OK.0808

Now, over here, I'm going to do the reactions in red.0810

This is going to be reaction 1; this is going to be reaction 2.0818

This is going to be reaction 3.0822

Now, let me go back to blue.0824

OK.0826

Over here, now, I'm going to deal with the glycine.0827

I'm going to make it ready.0830

This is going to be coming in this way.0832

I've got glycine.0835

I'm going to react glycine with my Fmoc chloride in order to produce Fmoc-glycine, and then I'm going to react that with something called DCC; and I'm going to end up producing Fmoc-Gly-DCC.0838

Now, I'm going to take this, and this is going to be my reagent for this reaction.0862

It's going to come in here, and what is going to end up leaving is DCC.0869

This is going to react with this, and what I'm going to end up with is the following.0876

3...let me label some reactions here.0883

This is reaction 4; I'm going to call this one 4a, and I'm going to call this one reaction no. 5; and let me go back to blue.0885

So, what I end up is Fmoc-Gly-Ser, and bead.0895

OK.0906

And again, I subject this to my trifluoroacetic acid or base, mild base, and what I end up with is Gly - I really hope I can do this in one page - Ser and bead; and let's go ahead and call this one reaction no. 6.0908

I should be able to go, yes, that's not a problem...oops.0940

OK.0948

Now, let me go back over here and prepare my alanine.0949

I'm going to take alanine, and I'm going to react it with this thing called Fmoc chloride in order to get Fmoc-Ala, and I'm going to react it with this thing called DCC, in order to get Fmoc-Ala-DCC.0952

I'm going to take that.0977

That becomes the reagent for this one.0980

DCC leaves the procedure.0985

This is going to react with this, and what I'm going to end up with is the following.0989

I end up with...I have to write it over here: Fmoc-Ala-Gly-Ser with a bead, and then I'm going to subject this to trifluoroacetic acid or mild base; and I'm going to end up with, let me see, so I've got Ala, Gly, Serine, bead, and then of course I subject this one to hydrofluoric acid, and I'm left with Ala, Gly and Serine- what it is that I wanted.0994

This is my general schematic.1051

Let me go through this again, and then I'll go ahead and go through the details.1052

Let me label my reactions.1056

That is reaction 6; this is reaction 7.1058

This is reaction 7a; this is reaction 8.1063

This is reaction 9, and this is reaction 10.1068

I start with my serine; I react it with something called Fmoc chloride to create this Fmoc protected serine.1073

I react that with a bead; I attach it to the bead, so now, I have the bead, a serine residue and Fmoc.1080

React it with trifluoroacetic acid, I take off the Fmoc because I need to attach the glycine directly to serine.1086

I prepare my glycine reagent; I take glycine.1094

I protect it with Fmoc, and then I add DCC to the right side of it.1097

I take that; I react it with my bead and serine.1101

Now, I have my bead, my serine, my glycine, and I have my Fmoc, DCC has gone away.1104

I subject that to trifluoroacetic acid.1111

I break off the Fmoc, so now, I have the glycine that is free to react.1114

I prepare my alanine residue - alanine, Fmoc chloride, this is Cl - to produce Fmoc-Ala.1117

I take the Fmoc-Ala; I react it with DCC to create Fmoc-Ala-DCC.1127

I use this reagent to react with the bead, serine, glycine.1131

Now, I have bead, serine, glycine, alanine, Fmoc.1134

I do TFA to get rid of the Fmoc, and then I use hydrofluoric acid to break this thing off the bead.1137

I'm left with my tripeptide.1145

This is the procedure.1146

Notice, it goes in cycles.1147

This is why anything that goes in cycles like this, it's perfect for automated procedures.1149

OK.1153

Now, we are going to go through each one of these reactions in detail and mechanisms.1154

Alright, so, here we go.1160

Here is where the structures start to fly around all over the page.1164

Let's hope to God that we can keep this straight.1167

It's actually not that bad.1169

If you can follow this, believe me, you can follow anything.1170

OK.1175

Our first reaction, let me see, what should I do this in?1176

You know what, I think I'll do these reactions in red.1179

Let me go ahead and do.1181

So, we said we started with our serine.1183

OK.1187

We have H₂N, and we had C, C.1188

We had that, and we had O^-.1197

Now, our serine is going to be CH₂OH.1201

I hope that I got my R-group right; I hope that is correct.1204

If not, I hope you'll correct me.1207

Now, our first reaction, reaction no. 1, we reacted this with something called Fmoc chloride.1210

This was reaction no. 1, and I'll go ahead and write the reaction number in blue.1219

Let me go back to the red, and what we ended up with was Fmoc, N, H, C, C, O^-, CH₂, and OH.1227

OK.1244

Let me say what happened here.1246

We are reacting it with this reagent called Fmoc chloride in order to protect the amino group, and here is the reason why we need to protect the amino group.1248

Well, if I take serine, let me do this in black, if I have H₂N, or if I had just any random amino acid, notice, I have electrons here.1258

This nitrogen is nucleophilic.1271

Well, I have this O^- here.1273

This carboxyl group is also nucleophilic.1274

The next step I'm going to take is I'm going to react this with a bead.1278

Well, the bead is an alkyl halide.1281

Well, since this is a nucleophile and this is a nucleophile, I don't want this to attach to it by accident.1284

I want strictly the carboxyl group to react with the bead end, to react with the bead.1290

I don't want this to react, so I have to protect it.1296

I have to cover it up, keep it from being nucleophilic, so that the only nucleophile on this molecule is that.1298

This is why I'm actually reacting this Fmoc chloride.1305

I'm attaching a protective group, so nothing reacts with the nitrogen, right, because this is nucleophilic, in other words, carrying a negative charge, and this is nucleophilic.1308

I need just that to react with my bead.1325

I don't want this to just randomly react with it, so I have to protect it.1329

That is what's going on.1332

I hope that made sense.1338

OK.1340

Now, let's go ahead and draw the structure for Fmoc chloride just so you see what it looks like, but I'm just going to refer to it as Fmoc chloride, and it looks like this.1341

We have that, and then we have another one, and then, of course, we have this, then we have CH₂COCl.1356

OK.1369

This is an acid chloride; it's a carbonyl chloride.1370

OK.1375

This chloride is going to be your leaving group.1376

Sorry, let me go ahead and put my aromatic, because this is definitely aromatic.1377

OK.1384

This right here, that's the Fmoc part, and, of course, this is the chloride.1385

Just so you know, this stands for 9 fluorenylmethoxycarbonyl, all these names, methoxycarbonyl chloride.1395

Wait, am I...I think I have my structure wrong here.1418

Oh well, it doesn't matter; we are only concerned with the Fmoc chloride.1430

This is what we are concerned with right here.1433

I think I'm missing an oxygen here somewhere, but that's OK.1435

Again, Fmoc chloride, it is the chloride part that we are concerned with.1440

This is where the reaction is going to take place.1444

We are just going to call this Fmoc; it's just a protecting group, and here is how it happens.1445

The mechanism is, these electrons, they attack that carbon, and they kick off a chloride, so now, you have this Fmoc group attached to the nitrogen.1450

That is how it ends up attached to the nitrogen, and this is our final product.1470

OK.1474

Here we go.1479

Great.1482

Now, we have an N-protected serine.1485

OK.1499

Yes, that's fine; let me go ahead and do this in red.1503

Let's take our Fmoc, and let's take our serine, N, C, C.1506

So, that's our serine.1517

Now, let's go ahead and do reaction no. 2.1523

Should I do it vertically, or should I do it...that's fine, I guess I can do it this way.1527

Actually, let me go ahead and do it this way.1534

Now, I'm going to react this with the bead, and I'm going to actually draw out the bead here.1538

Boom, boom, boom, boom, boom here.1544

That is that; we have CH₂.1549

We have Cl, and here is what happens.1554

This is going to be reaction no. 2.1556

Let me erase something here and do this over.1569

Alright.1583

Let me put my H₂s down here, and then let me go ahead and do my blue.1584

The mechanism is this nucleophilic oxygen attacks that carbon, and it kicks off that.1591

What ends up leaving here is our chloride.1600

This reagent attacks the bead, and now, it's going to be attached to the bead.1604

Now, what we have is the following.1611

Let me go back to red.1614

I have Fmoc; I have N, C, C.1616

This is carbonyl; this is O, and this is, I'm just going to write bead.1630

OK.1636

And this is our serine, so this is CH₂.1637

This is OH, and let me go ahead and put my H there.1642

N-protected serine, I react it with the bead; now, I have bead, serine, and I have Fmoc.1647

There we go.1653

Now, I'm going to go ahead and react this with...this is going to be reaction no. 3.1655

Go back to red.1666

This is going to be our trifluoroacetic acid or our mild base, and this is to take off the Fmoc group, to deprotect the nitrogen.1668

So, what I end up with is the following.1679

I end up with, let me just write it as H₂N, C, C.1683

There is that; there is that...oops.1691

There is no minus there.1696

I'm actually attached to the bead.1698

Let me see, yes, attached to the bead.1705

Then, of course, I have my CH₂, and I have my OH.1708

There you go.1714

Now, I have the bead, and I have my serine.1715

So, this is my serine residue, and that is the third reaction.1718

OK.1726

Now, let's go ahead and deal with our fourth reaction and our 4a reaction- the preparation of glycine.1728

Let me go back to red.1736

Let me go ahead and draw out the...so, we have H₂N.1737

This is C, C; this is O^-, and, of course, glycine has just a hydrogen there.1745

This is glycine.1753

Now, what I'm doing is I'm reacting this again with my Fmoc chloride to create my N-protected glycine.1760

This is going to produce Fmoc, N, C, C.1769

I have that; this is glycine.1779

There is an H there.1783

OK.1785

This is my N-protected glycine residue.1786

Now, I'm going to subject that to my DCC, dicyclohexylcarbodiimide, and I'll discuss this in just a minute.1790

Let me go ahead and draw the structure.1799

When I react it with my DCC, I end up with the following.1800

I end up with Fmoc on the left; I end up with my amino acid on the right.1804

O, D, C, C, and I end up with the DCC on the right attached to the amino acid.1815

This is glycine.1821

I'll go ahead and put an H here.1823

I have Fmoc; I have my glycine, and I have my DCC.1825

I've prepared my reagent to react with what's on the bead.1829

Let me go ahead and write the fact that this is glycine, and now, let me start talking about what is going on here.1833

Let me draw the structure of DCC, and then I'll show you the mechanism going from this step to this step.1843

The mechanism from here to here is, again, this nitrogen, it attacks the Fmoc, and it kicks off the chloride, so chloride actually goes away.1849

This is reaction no. 4.1865

This is reaction 4a.1868

Now, what happens is this.1871

Now, let me go ahead and draw dicyclohexylcarbodiimide.1872

That's fine; I guess I can draw it here.1878

This is N, double bonded with a C, double bonded with an N, and another cyclohexyl group, something like that.1885

OK.1894

I'm going to go ahead and put just a floating H...not yet...OK.1895

So, this is DCC.1902

This is called dicyclohexylcarbodiimide.1907

We will just call it DCC.1918

Once I've created my N-protected glycine, now, this is no longer nucleophilic that's why I protected it.1919

This is going to be nucleophilic.1927

Let me do my mechanisms in blue actually.1933

This is going to attack that carbon, and, of course, we are pushing electrons, so it's going to push this out towards grabbing some H, and what you end up with, now, I'm going to draw this structure with this thing attached.1942

Let me do it in red just so you see it.1958

I'm actually just going to be writing Fmoc amino acid and DCC, but I wanted you to see the picture because again, this is biochemistry, and it's all about the chemistry, the organic chemistry, the structures.1961

N, C, C, this goes there; this is O.1973

This is going to be C; this is going to be NH-cyclohexyl.1980

This is going to be N, and this is going to be a cyclohexyl.1989

So, this DCC; that is this thing right here.1992

That is that.2000

That's it.2001

Now, we have this reagent.2002

Now, we are ready to go through and do reaction no. 5.2003

OK.2008

We have 4, 4a; everything is good.2013

Yes, everything is good.2017

OK.2019

Now, let's see what we've got.2020

We have our serine attached to the bead.2022

Let's go ahead and do that.2025

Let's go to red because that is what we are doing our reaction in.2027

We have H₂N, C, C; that is a carbonyl.2030

That's O; this is CH₂.2040

I'm just going to write bead.2044

O, and this is the bead; this is CH₂.2048

This is OH.2057

OK.2059

So, this reaction that takes place is going to be the following.2060

Should I go straight down?2064

Yes, that's fine; I can go straight down.2065

Now, I'm going to react it with the reagent that I just made.2069

I'm going to react it with the Fmoc, N, C, C, O, DCC, our glycine, right?2076

So, here is how this one works.2098

Now, mechanisms we do in blue.2101

These electrons, that is nucleophilic, that is going to come around.2106

It's going to attack this carbonyl because it is electrophilic.2115

These electrons are going to pop up here.2120

It is going to form a tetrahedral intermediate.2122

Then the electrons are going to pop back down here, and they are going to kick of this DCCO group.2124

Our final product, what you end up with is the following2130

You end up with this thing on the left of this thing.2133

So, our final product is going to be Fmoc, N, C, C, and then, N, C, C, carbonyl, carbonyl, O.2138

This is attached to the bead.2159

This is there; this is our glycine.2163

This is there; this is our serine, CH₂OH.2168

This is what I've made.2174

Now, I have my serine, and I have my glycine to the left of it.2175

That is what I've done.2185

OK.2187

Now, let's stop and take a look at where we are just to make sure we've got everything.2189

OK.2192

Let's go ahead and label some reactions.2193

This is reaction no. 5.2196

And, of course, the thing that ends up leaving from this reaction is DCC, because that is our leaving group.2199

Now, what we do, we are going to subject this to trifluoroacetic acid or mild base in order to deprotect, to take off this Fmoc group.2206

So, what I'm left with now, is my glycine residue, my serine residue, attached to my bead.2217

I've got H₂N, C, C, N, C, C.2225

I have a carbonyl on the second carbon, carbonyl on the second carbon.2233

This is attached to our bead covalently.2236

I'm going to go ahead and put that there.2240

I'm going to go ahead and put an H there.2243

Now, I'll go ahead and do my R-groups.2245

This is glycine, and this is CH₂OH.2247

That is my serine.2251

I have my bead, my serine, my glycine.2252

Now, I'm ready to start the next process.2256

This reaction was reaction no. 6 in our general scheme.2258

OK, we are almost there.2265

We are more than halfway through.2266

Now, we have to prepare the alanine in order to have it react with this thing, and attach it to the bead; and that is going to be our last amino acid.2268

So, let's go ahead and do that.2276

Let's take alanine.2278

Let's go ahead and do it in black actually.2280

We have got H₂N, C, C.2285

We take our alanine which is CH₃.2292

We react it with Fmoc chloride, and again, let's go ahead and do the mechanism in blue.2299

This nitrogen is nucleophilic.2305

It will attach that; it will kick that off, chloride leaves.2310

This is reaction 7, and what we end up forming - let me go back to black - is our Fmoc-protected alanine residue.2316

N, C, C, O^-, I'll go ahead and attach an H, and this is alanine, so this is CH₃.2326

We go ahead and activate the carboxyl by reacting it with our DCC.2336

Now, I go this way, and I react it with DCC.2341

This is reaction 7a, and when I do this, what I end up with is the following reagents: Fmoc, N, C, C.2348

This is H; this is carbonyl.2364

This is O; this is our DCC, and this is alanine.2367

So, this is CH₃, we have this alanine.2372

Fmoc-alanine-DCC- this is ready for the next step.2376

Now, let's go to reaction no. 8.2382

OK, reaction no. 8.2387

Now, we have our bead, our bead has a serine and then a glycine.2391

We are going to react that with the reagent we just produced- the alanine reagent.2395

Let me go to red.2399

I've got H₂N, C, C, N, C, C, N, C, C.2403

There is a carbonyl here; there is a carbonyl here.2415

There is an O, and there is a bead.2418

Our first residue is serine, that is there.2422

Our second residue is glycine, and I have these electrons on the nitrogen.2427

What I'm going to do now, I'm going to react this with my - let me write this in black - the reagent that I just produced, which is...no, I said we are going to do this in black.2432

Let's hope this cooperates.2450

Yes, there we go.2452

I have Fmoc; I have N.2455

I have C; I have C, O, DCC.2459

This is H; this is my alanine reagent, right?2466

This is my alanine reagent.2472

I'm going to stop writing this down.2474

When it reacts with that, DCC actually leaves.2477

This reaction is reaction no. 8, and here is the mechanism.2484

Let me see, where are we?2491

Yes, yes, here we go.2496

I have blue ink.2503

I'm telling you, this gets kind of crazy trying to keep track of everything, but this is actually the nice part, taking the time to keep track of everything to be able to know where you are, to search through all of these chemical jungle that we are looking through to actually make sure you know exactly which electrons are going where.2504

This is where you have a complete understanding of the material.2521

These electrons come here; they attack that.2524

These electrons go there; they fall back down.2530

They kick off the DCC leaving group, and what you end up with is the following.2532

Let me write this in red.2538

You end up with Fmoc, N, C, C, N, C, C, N, C, C, carbonyl, carbonyl.2540

Those are my points of reference, my carbonyls.2556

I have my O; I have my bead.2560

My first residue on the bead is my serine, CH₂OH.2563

My second is my glycine, which is just H.2570

My third is my alanine, which is CH₃.2572

There is a hydrogen attached to the nitrogen, a hydrogen attached to the nitrogen, a hydrogen attached to the nitrogen.2576

There you go.2582

I've got my serine.2583

Sorry, I'm not going to actually do this.2587

I've got my bead, serine residue, glycine residue, alanine residue, and now, it is protected.2590

Now, I need to deprotect it and break it off from the bead.2600

That's it- reactions 9 and 10.2603

Now, I'm going to do these as single arrows.2606

Let me go ahead.2611

This first step is going to be the trifluoroacetic acid.2614

This is reaction 9 or mild base- to remove the Fmoc.2619

Once I remove the Fmoc, now, my no. 10 reaction, I'm going to react it with hydrofluoric acid to remove the bead, or actually, because that is the solid support, I'm going to remove the peptide from the bead; and what I end up with is exactly what I wanted, and this one I'm going to do in blue.2631

I end up with alanine, glycine - I never, ever, ever get that right, OK - and serine.2659

Serine is my C-terminus.2672

Alanine is my N-terminus.2678

That is it.2683

I just run through the Merrifield process with mechanism, with structure.2685

I've kept it as Fmoc; I've kept it as bead in order to concentrate just on the structure of this, but running through the same thing.2689

You protect the nitrogen group.2699

You add an amino acid to the bead.2702

You prepare the next group.2704

You protect the nitrogen group.2706

You activate the carboxyl group with the DCC.2708

You react these two.2711

Now, you have something else on the bead.2714

You deprotect the nitrogen group, prepare it.2716

You prepare your third amino acid, react it, protect the nitrogen group, activate the carboxyl group, react it.2718

Now, you've added one more chain, deprotect the nitrogen, prepare it, go to your fourth amino acid- over and over and over again.2725

That is the Merrifield synthesis; it is an automated procedure.2734

Now, what I would like to do is I'd like to actually go ahead and show you how this takes place physically, as far as the reaction vessel that it actually takes place in.2738

So, let me go ahead and just go forward to my picture here.2748

OK.2757

This is a chromatography column.2758

Now, you can do this on a chromatography column, or you can also do it by hand by just having it...well, you know what, let's just deal with the chromatography column.2761

This stationary phase right here, these are our beads.2771

Basically, what you are looking at is you are looking at, just imagine a bunch of really, really, really, really tiny beads- that's it.2778

You've got hundreds and hundreds and hundreds of thousands of beads- that's it.2786

It's like sand basically.2791

It's just like really, really, finely ground sand is what it is.2792

So, you take this column, this chromatography column, which is just a hollow tube that comes in different sizes.2796

It could be really, really thin; it could be really, really thick.2803

Generally, they tend to be pretty small for the Merrifield synthesis, and you just fill it up with a bunch of beads.2806

Well, you remember the process that you went through- the schematic.2814

Let me go back to the schematic.2817

Now, because you are actually constructing this molecule, this peptide, on a solid support, all of your solutions, you just pour them in.2819

The reaction takes places as it filters through and passes through the beads, and then it exits when you want it to exit; and, of course, you can control the flow down here.2829

That's it.2838

So, for example, you create your serine and you react it with Fmoc chloride, so you create your N-protected serine residue, well, now, you're going to react it with the bead but the bead is already in the column, so what you do is you take that solution and you just pour through.2839

That is going to be reaction no. 2, and it is going to go ahead and react, and the serine is going to stick to the bead, and everything else is just going to wash through.2857

That is what makes this great.2867

You don't have to purify anything.2868

All the excess reagents, everything that you don't need, just washes through, you can throw it away.2870

Well, now you go ahead and you pour in your trifluoroacetic acid or your mild base to deprotect again.2876

The reaction will take place as it passes through, passes over all of the beads; and any excess will run off.2882

Now, you go ahead and you create your glycine reagent.2890

You pour it in here, it will react as it passes over the bead, any excess will run off.2894

You put your trifluoroacetic acid, it will run through, run over the beads, and that is what you are doing- stationary phase, it doesn't move.2899

You are literally just washing all of the beads with all of the reagents that you need.2907

The beads just stay there; the reaction takes place on the bead.2913

When you are done, you take the column; you empty out the beads.2917

Actually, no, you don't even have to do that.2920

In the final step, the hydrofluoric acid, you just pour it in, the hydrofluoric acid runs over the bead.2922

It breaks the bond between the bead and the first peptide, and then, of course, you just wash it into a flask; and now, you have your peptide in a flask down here for further processing.2931

That is the Merrifield synthesis.2945

Thank you very much for joining us here at Educator.com.2948

We'll see you next time, bye-bye.2950