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