00:00:00 At a Federation meeting in the spring of 1962, Bruce Merrifield reported the general principle

00:00:23 of carrying out peptide synthesis on a solid support.

00:00:27 I was making these small kind of peptides, and even though you could make them, it took

00:00:34 a very long time.

00:00:35 The yields were very low, even for a small peptide, and very time consuming.

00:00:42 So as an amateur in the field, I thought, well, there really ought to be some way to

00:00:49 do this better, some better way to make peptides than the classical methods that everybody

00:00:55 is using.

00:00:57 I thought about it for some long time, and then suddenly one night, I believe, I had

00:01:04 an idea, and it just came to me, of how we might go about that.

00:01:09 The idea was to make use of an insoluble solid support to hold the peptide chain while you're

00:01:18 synthesizing it, while it's growing in length.

00:01:23 That was the fundamental idea.

00:01:27 It looked like, if you could make it work, and of course I had no idea whether you could

00:01:32 or not, that it would be simpler in the first place, it would be more rapid, and you might

00:01:40 get better yields than you do by the standard method.

00:01:44 So that was the origin of the idea, and I thought about it a day or two, and then I

00:01:52 had to go to the boss, to Dr. Woolley, and we happened to be riding up to the fourth

00:01:58 floor on the elevator.

00:02:01 So I told him in just a matter of 30 seconds or something what the idea was, and what did

00:02:07 he think about it.

00:02:08 And he got off and left, he didn't say anything.

00:02:11 Well, I wasn't too encouraged by that.

00:02:13 The next day he came into my lab, he says, you know, that's a pretty good idea, maybe

00:02:20 you ought to work on that.

00:02:22 I said, well, great, you know, if I could take off two or three months, I could get

00:02:28 this to work, or find out if it'll work.

00:02:32 And he said, okay, he thought that was reasonable, and I went from there.

00:02:39 The difference is, it wasn't three months, it was three years.

00:02:43 And there were a lot of variables in this simple idea that you had to work out, and

00:02:51 they all had to be worked out at the same time.

00:02:54 In other words, you had to have a solid, mainly a resin support, you had to have a way to

00:03:01 attach the chain to it, coupling reactions, deprotection steps, and finally removal of

00:03:10 the peptide from the resin at the end of the synthesis.

00:03:14 So each one of those turned out to be a much bigger problem than I had anticipated, and

00:03:22 sometimes you could get one of them to work fine, you may get a good resin, but your coupling

00:03:28 reaction was no good.

00:03:29 So it didn't work, the overall synthesis didn't work.

00:03:33 They had to all come together at the same time.

00:03:36 I started out with cellulose, and the reason was, I knew that cellulose had been used to

00:03:45 fractionate proteins.

00:03:47 So I thought, well, there must be room in this cellulose to handle a material of that

00:03:54 size.

00:03:55 So I started it, and I got a little way, I made a dipeptide, but it was much too fragile

00:04:02 for the chemistry I was using.

00:04:04 That's another example of, I had a support, but I didn't have the right chemistry.

00:04:10 Then I got a hold from Dow Chemical, a sample of a polystyrene cross-linked with a little

00:04:18 bit of divinyl benzene.

00:04:20 This is the starting material for their ion exchange resins.

00:04:24 They sent me some, and of course you have to have a functional group on it.

00:04:31 So I decided, among other things, finally, that a chloromethylation reaction would give

00:04:38 you a substituted benzyl chloride, which is quite a reactive substance.

00:04:46 Having made that, then I could take the amino acid that I wanted to attach and form a benzyl

00:04:53 ester of the carboxyl group.

00:04:56 Now that was the stable covalent bond, covalent bond, which was holding the peptide to the

00:05:04 resin.

00:05:05 To synthesize a longer peptide, the sequence of deprotection, neutralization, and coupling

00:05:11 was repeated over and over for each subsequent amino acid.

00:05:17 Purification at every step was by rapid and simple but thorough washing to remove excess

00:05:24 reagents and bifuronics.

00:05:27 The last step was cleavage of the peptide from the solid support.

00:05:33 Hydrogen bromide and later hydrogen fluoride were the best strong acid reagents.

00:05:39 A final selective purification procedure was then necessary.

00:05:46 There were really three advantages.

00:05:48 One was the feed.

00:05:50 One was that you don't have to transfer the material from one container to another

00:05:57 as you do in an ordinary synthesis.

00:06:01 Once you put it inside a reaction vessel, it just stays there until the peptide is completed.

00:06:06 So that's an advantage.

00:06:11 Maybe the most important is at every intermediate stage of the synthesis, every time you add

00:06:17 an amino acid, you just purify by simple, thorough washing with solvent and don't

00:06:26 have to go through a chromatographic purification or a crystallization, which are good but very

00:06:35 time consuming.

00:06:36 After about three years, I had made a model tetrapeptide, and this worked really quite

00:06:44 well by then.

00:06:47 At this point, I needed a name for the new process and settled on solid phase peptide

00:06:53 synthesis.

00:06:55 And at that time, I gave a favor down at Atlantic City at the Federation meeting.

00:07:02 There was a reporter there that thought it was great.

00:07:05 There were two professional expert peptide chemists right down the front row.

00:07:11 Their reaction was not all that good.

00:07:13 They were skeptical, and what I found over the years after that is the vest of the peptide

00:07:20 chemists were very reluctant to accept this idea.

00:07:24 I wrote a full paper then for 63 and described how to do it, what I thought were the limitations.

00:07:32 There were a lot, and I knew about a lot of them.

00:07:34 And I mentioned those, that you have to worry about these various things.

00:07:39 For example, the reaction has to go very close to 100%.

00:07:44 If they don't, you'll end up with what we now call deletion peptides, where you'll

00:07:49 have a missing link in your peptide.

00:07:52 And I knew about that, and we were very much concerned about it, even in the first paper

00:07:58 that we wrote.

00:07:59 When I wrote the first paper in 1963, I pointed out that this methodology ought to lend itself

00:08:06 well to automation.

00:08:09 So we were thinking about it right from the beginning.

00:08:12 Then after I got the method working, Dr. Woolley and I got an NIH supplement to our grant to

00:08:21 build a machine.

00:08:24 So with that in hand, I went in and talked to John Stewart, the colleague here, and invited

00:08:30 him to work with me on the construction of a synthesizer.

00:08:35 And he was very enthusiastic and said yes, he'd like to do that.

00:08:38 So that was the beginning.

00:08:42 What you can see is a reaction vessel, which we already had in the manual method.

00:08:48 And that's shown here, and it has the resin in it with the peptide.

00:08:56 Over here is a pump to deliver solvents to the reaction vessel.

00:09:01 Over here is a rotary selector valve to pick the right solvent and the right reagent at

00:09:07 the right time.

00:09:09 And there is another rotary valve to select the right amino acid at the right time.

00:09:16 We can have six amino acids lined up, and this will pick one at a time and feed them

00:09:21 into this cycle, which then goes into the synthesis.

00:09:25 And the solvents are removed by a vacuum.

00:09:28 So that's the basic, and this whole thing shakes, of course.

00:09:32 So that's the mechanical end.

00:09:35 Over here is the programmer, which controls the events over there.

00:09:40 We have a set of timers, and this is a tenor drum.

00:09:46 It's an electromechanical device, and you set pins in it to make your program.

00:09:55 Then it operates switches, and the switches operate the events over there.

00:10:00 When this goes around a hundred steps, then you will have added one amino acid to the

00:10:06 peptide chain.

00:10:07 So this is our synthesizer, and it works quite well.

00:10:12 It's the thing that Merck-Gouda used for the ribonuclease synthesis.

00:10:20 With the availability of the new solid phase method and the automated synthesizer, it was

00:10:25 possible to undertake the synthesis of a much larger and more complicated molecule.

00:10:31 We wanted to test the limits of our methodology.

00:10:36 And we knew we could make peptides of 20 or 30 amino acids.

00:10:41 Could you make something over 100?

00:10:43 Well, we picked ribonuclease.

00:10:45 In a sense, it's kind of a Rockefeller enzyme.

00:10:49 It was discovered here by DeVos and crystallized by Kunitz, and the molecular weight Rotan

00:10:57 worked on.

00:11:00 Morenstein did the sequence of it.

00:11:03 When Bernd Gouda joined the Merrifield group, he quickly put the new technique to use.

00:11:09 In 1968, he undertook the total synthesis of ribonuclease A. He successfully synthesized

00:11:16 this 124 residue protein.

00:11:19 This work showed that a real protein with true enzymatic activity could be assembled

00:11:23 from simple amino acid derivatives.

00:11:26 Well, I had written in more than one review article that I think solid phase synthesis

00:11:36 would be a goldmine, I said, for organic chemists who would use it to facilitate and direct

00:11:44 their syntheses.

00:11:46 Merrifield's pioneering work in solid phase chemistry revolutionized the field of peptide synthesis.

00:11:52 The same philosophy of solid phase chemistry is now being implemented in the latest revolution

00:11:57 in organic synthesis.

00:12:00 It is clear that the peptide field is alive and well.

00:12:04 We can't predict the next new discoveries in this field, but we can all be sure that

00:12:09 Many exciting developments lie ahead.