00:00:00 It was 10 to the 10th BC when it all began.

00:00:22 Water and the elements were formed.

00:00:32 Galaxies, stars, and our Earth were formed.

00:00:52 Four million years ago, life appeared.

00:00:57 In our lifetime, there have been remarkable advances in science and technology that have

00:01:02 profoundly changed our world.

00:01:05 We have looked to the ends of our universe and mapped the oceans.

00:01:10 We have advanced the laws of relativity and quantum theory and discovered additional elements

00:01:15 and subatomic particles.

00:01:17 In experimental chemistry and biology, we identified DNA as the fundamental genetic

00:01:22 material that directs the synthesis of protein.

00:01:26 Recently, we sequenced the human genome.

00:01:30 In peptide science, we now know the compositions and amino acid sequences of thousands of proteins.

00:01:37 We have determined many three-dimensional structures.

00:01:41 The field of proteomics is developing rapidly.

00:01:45 Peptide science can be divided into three broad areas, chemistry, physics, and biology.

00:01:52 They depend on each other and are complementary.

00:01:55 Our focus in this program will be on synthetic chemistry.

00:01:59 We have synthesized large numbers of naturally occurring peptide hormones and have designed

00:02:04 novel peptide structures that are effective therapeutics.

00:02:09 We can synthesize thousands or even millions of peptides in a single experiment, test them

00:02:14 for biological activity, and use this information for drug discovery.

00:02:19 In certain cases, we can produce multi-kilogram quantities of synthesized peptides.

00:02:26 All of this flows from discoveries that are part of our history.

00:02:36 None of this would be possible without the work of many people beginning exactly 100

00:02:41 years ago when the great organic chemist, Emil Fischer, synthesized the first peptide.

00:02:47 Fischer coined the term peptide and proposed the polyamide structure for proteins.

00:02:54 Whereas professional colleagues fear that a rational study of this class of compounds,

00:02:59 because of their complicated structure and their highly inconvenient physical characteristics,

00:03:05 would today still uncover insurmountable difficulties, other optimistically endowed observers, among

00:03:12 which I will count myself, are inclined to the view that an attempt should at least be

00:03:16 made to besiege this virgin fortress with all the expedience of the present, because

00:03:22 only through this hazardous affair can the limitations of the ability of our methods

00:03:27 be ascertained.

00:03:29 Of course, because no sequence was known at that time, Fischer could not synthesize a

00:03:33 natural protein.

00:03:35 Some believe that proteins were not single homogeneous compounds, but Fischer laid the

00:03:40 groundwork for achievements that came about 60 years later.

00:03:44 Fischer and other scientists such as Theodor Kutius, Emil Abderhalden, and Hermann Leuchts

00:03:49 were critically handicapped.

00:03:51 There were no reversible protecting groups for the alpha amine.

00:04:02 Fittingly, it was one of Fischer's former students, Max Bergmann, with his associate

00:04:06 Leonidas Servas, who solved the amino protecting group problem 30 years later.

00:04:12 However, the carbobenzoxy group can be removed by catalytic hydrogenolysis.

00:04:17 The discovery instantly transformed the field and is still used today.

00:04:22 In 1937, Josef Fruton joined Bergmann and used the carbobenzoxy method to synthesize

00:04:28 a large number of small free peptides.

00:04:31 This allowed him to determine the substrate specificity of proteolytic enzymes.

00:04:36 This was the first time synthetic peptides were used to solve an existing biochemical

00:04:41 problem.

00:04:42 It was a landmark achievement.

00:04:46 Miklosz Bodanski contributed to the field by dividing peptide synthesis into strategy

00:04:51 and tactics.

00:04:53 Strategy describes synthesis design, while tactics focuses on detailed chemical procedures.

00:05:00 In spite of this new approach, important developments in reversible N-alpha amine protection tactics

00:05:05 were slow to emerge.

00:05:08 In 1957, Louis Carpino, using modern physical organic chemistry principles, proposed the

00:05:14 tertiary butyl function for a urethane protecting group.

00:05:18 It was a better leaving group than benzyl and could be removed in mild acid.

00:05:23 This led to the development of the BAC group.

00:05:27 The next major development in amine protection occurred in 1972, again by Carpino, who introduced

00:05:33 the base labile acid stable FMOC group.

00:05:37 The FMOC group can be removed by beta elimination with piperidine.

00:05:42 In 1978, the Meyenhofer group and the Shepard group independently used FMOC protection in

00:05:48 all steps of a long synthesis.

00:05:51 The orthogonality of this base labile group and the acid labile side chain tert-butyl

00:05:56 group is an important advantage.

00:06:01 So I became involved with Hans and Xu Ducheng to prepare, by stepwise solid phase synthesis,

00:06:09 using the FMOC t-butyl strategy, making somatostatin, and it worked out extremely well.

00:06:14 The FMOC strategy also has the added advantage that you can monitor the deprotection of the

00:06:22 FMOC group at every stage using absorption methodologies.

00:06:31 Other N-alpha protecting groups have been introduced, including those removable under

00:06:35 neutral conditions by photolysis, dialysis, and solvolysis.

00:06:47 Vincent Duvigneau used sodium in liquid ammonia to remove benzyl groups instead of catalytic

00:06:52 hydrogenolysis, which cannot be used for sulfur-containing peptides.

00:06:58 It was later shown that catalytic hydrogenolysis in liquid ammonia can be used for sulfur-containing

00:07:04 peptides.

00:07:05 Hans had learned before he came to Roche that it was possible to use liquid ammonia to be

00:07:11 able to, with palladium black, to be able to catalytically hydrogenate, for example,

00:07:17 a carbobenzoxy group or a benzyl ester.

00:07:20 So he suggested to me that I use that methodology in the synthesis of somatostatin.

00:07:26 It was very straightforward.

00:07:30 In 1966, Shumpei Sakakibara made the important discovery that anhydrous liquid hydrogen fluoride

00:07:37 was a safe and powerful cleavage reagent.

00:07:41 Sakakibara was a student of the pioneering protein chemist Shiro Akabori.

00:07:46 At that time, I was working with one Korean post-doctor.

00:07:53 He just tried to put carbon dioxide peptide in HF, and he thought that nothing happened.

00:08:03 This was immediately found that carbon dioxide evolved from the HF solution, and the solution

00:08:12 gave us some amorphous white precipitate.

00:08:16 Amorphous oily materials come out from the solution.

00:08:21 Of course, the solution turned to pinky colors, I think due to some small metals.

00:08:33 We tried to recover the compound and found that no carbon dioxide remained in the structure.

00:08:41 At that time, I asked Dr. George Hess, this must be a very interesting reaction.

00:08:48 I asked him to continue this work besides studying the rate of the O2N or N2O shift

00:08:56 or cleavage of peptide bond by HF.

00:08:59 But he did not show any interest in that.

00:09:03 And he asked me if I'd like to do this work in Cornell.

00:09:08 Must do it on Sunday, not on working day.

00:09:12 In 1982, James Tam and Bill Heath found a way to minimize side reactions with this strong

00:09:18 anhydrous acid.

00:09:20 They used dimethyl sulfide as a base to reduce acid strength while retaining the ability

00:09:26 to cleave esters, ethers, and urethanes.

00:09:37 In 1884, Curtius was the first to report the activation of a protected amino acid.

00:09:43 He used the azide to form the peptide bond.

00:09:47 Joseph Ruttinger improved this method in 1961.

00:09:52 During the rapid growth period in peptide synthesis in the 1950s, Theodor Wieland first

00:09:57 introduced mixed anhydrides.

00:09:59 The anhydride method was followed by Wieland's use of thiophenyl esters as coupling reagents.

00:10:06 I derived from the Theodor Wieland School, world famous in peptide chemistry because

00:10:13 he among others dates back in his peptide chemistry developments to Emil Fischer's work

00:10:21 because Theodor Wieland is the son of the Nobel Laureate Heinrich Wieland and Heinrich

00:10:27 Wieland was the successor of Emil Fischer.

00:10:31 Theodor Wieland is world famous for his patience, his tolerance, his friendliness.

00:10:38 He never created enemies.

00:10:41 I don't know a single one.

00:10:45 So he is one of these characters which really combine the various religious beliefs in how

00:10:52 to deal with the human race, with human beings.

00:10:58 To me he was an ideal leader.

00:11:02 Robert Schweitzer soon proposed cyanomethyl esters and Bodansky then introduced nitrophenyl esters.

00:11:08 Others have introduced a variety of additional activated esters.

00:11:13 John Sheehan made one of the greatest advances in coupling reagents.

00:11:17 He utilized dicyclohexylcarbodiimide to close the beta-lactam in penicillin and extended

00:11:24 its application to the formation of the peptide bond.

00:11:28 Previously Todd Kenner and Korana had used carbodiimide successfully for dinucleotide

00:11:33 synthesis and had even made amide bonds but they did not apply it to peptides.

00:11:39 Murray Goodman tells the diimide story.

00:11:42 I began my work in the area of peptide chemistry in the laboratories of John Sheehan and that

00:11:51 was in late 1952.

00:11:54 John Sheehan was in England in 1951 and heard some wonderful stories about the use of carbodiimides

00:12:07 in the area of nucleotide synthesis in Cambridge in the laboratories of Professor Todd.

00:12:16 This was work carried out by H. Gobin Korana and it was truly a breakthrough in nucleotide synthesis.

00:12:28 We had a full and extensive discussion about these molecules and John Sheehan assigned

00:12:37 the synthesis of dicyclohexylcarbodiimide to George Hess and ditalylcarbodiimide to me.

00:12:46 We then undertook the synthesis of these molecules.

00:12:50 It turns out that the synthesis of ditalylcarbodiimide is much easier than the synthesis of dicyclohexylcarbodiimide.

00:12:58 I found that I could make this quite readily and prepare derivatives of protected amino acids and peptides,

00:13:09 most of which were crystalline and totally useless in the field of peptide chemistry

00:13:16 because the ditalylcarbodiimide rearranged rapidly to the acylureas,

00:13:22 which were stable crystalline and unreactive compounds.

00:13:26 George Hess proceeded very slowly and efficiently and produced dicyclohexylcarbodiimide,

00:13:34 a rather amorphous nondescript reagent,

00:13:41 which when added to a protected amino acid and then mixed with an amine component

00:13:50 produced an amide derivative, or if it was an amino ester, a peptide derivative, readily and in high yield.

00:13:58 George Hess's route to dicyclohexylcarbodiimide led to a revolution in peptide synthesis.

00:14:08 In recent years, a number of new activating reagents have been developed.

00:14:12 Bertrand Castro introduced BOP, a phosphonium salt, as a very reactive coupling reagent.

00:14:18 But it was enough to tweak our interest, so we decided to try using this BOP reagent for the cyclization,

00:14:24 and we got a big surprise that it was the most efficient reagent for the on-resin cyclization.

00:14:31 Dortoglu and later Knorr reported other reagents that replaced phosphonium derivatives with uronium salts.

00:14:38 N-carboxyanhydrides, or NCAs, were introduced by Leuchs and used to prepare polyalpha amino acids.

00:14:46 Denkwalter and Hirschman adapted NCAs to a stepwise procedure in aqueous solution under carefully controlled conditions.

00:14:55 Goodman and his co-workers obtained urethane-protected N-carboxyanhydrides, or UNCAs, in stable crystalline condition.

00:15:04 They are very reactive, even in hindered couplings.

00:15:14 In 1953, the first landmark synthesis of a biologically active peptide occurred when Duvignaux synthesized the nonapeptide oxytocin.

00:15:24 With associates Charlotte Ressler and Panagiotis Katsianis and others, Duvignaux drew on the large array of chemical methods available at the time.

00:15:32 One day I received a letter in the mail from Vincent Duvignaux inviting me to join him at Cornell Medical College

00:15:41 on his program on the structure activity studies of oxytocin and visopressin.

00:15:48 Well, the reason the fragment strategy was used, because that was all that was available at the time.

00:15:55 This was prior to the days of the Paralyte to Front Lester active ester synthesis.

00:16:00 The stepwise approach hadn't even come along.

00:16:03 And so they were dependent on using, for protection of the amino groups, carbon monoxy and protection of the arginine group, the tosyl.

00:16:13 And they had very limited availability of coupling methods.

00:16:18 So basically, you know, it was a trial and error of how to actually put the molecule together.

00:16:25 Starting with the tetrapeptide, then making the pentapeptide, then adding two more, and then finally coupling the seven and the two.

00:16:32 But the linchpin was actually the sodium liquid ammonia method.

00:16:38 The sodium liquid ammonia method had arisen from Duvignaux's work on insulin years earlier.

00:16:45 And it was the application of the sodium liquid ammonia method to the synthesis of oxytocin that really allowed Duvignaux to break through to accomplish the synthesis.

00:16:58 Buransky actually had been in Hungary working on oxytocin and working on active ester methods.

00:17:04 But in 1956, after the Hungarian Revolution, he came to Duvignaux's lab.

00:17:09 And Duvignaux, of course, was very happy to have a very experienced peptide chemist in his group.

00:17:14 And Buransky came with the active ester method.

00:17:18 And again, this, I think, was Duvignaux's strength, that he was able to capitalize on the talents of the people that came to work with him.

00:17:25 So he immediately saw the value of trying to make oxytocin using the active ester method.

00:17:31 I first became interested in peptide chemistry as a result of a phone call from Professor Vincent Duvignaux, who was a Nobel laureate,

00:17:42 who was the discoverer of oxytocin, did the total synthesis, the structure,

00:17:51 and actually put it in clinical medicine with his medical colleagues shortly thereafter.

00:17:58 And it's been used by literally hundreds of millions of women ever since.

00:18:03 And so I went to Duvignaux's laboratory.

00:18:05 I was very fortunate that I worked with Don Yamashiro, who was really an expert in the total synthesis of peptides at that time.

00:18:13 But it took several months just to synthesize acetone oxytocin in those days.

00:18:18 And so we worked out a very good synthesis, just so I could have large amounts for doing NMR and other physical studies,

00:18:24 infrared spectroscopy, UV, and so forth.

00:18:27 And we finally did solve the structure.

00:18:29 The oxytocin synthesis by Duvignaux was the forerunner of a flood of work on peptide hormones by peptide chemists.

00:18:37 These included vasopressin, again by Duvignaux in 1954, angiotensin by Riddle and Schweitzer in 1957, and alpha MSH by Boissonnaz in 1958.

00:18:50 That was the time when I was working with Robert Schweitzer.

00:18:54 Up in Zurich at the ETH, Robert Schweitzer has been a professor at the ETH and had a school,

00:19:02 one of the schools of peptide chemistry and peptide biology in Switzerland.

00:19:08 He was certainly one of the founders of peptide research.

00:19:13 He started, I think, his peptide research at CIPA Geigy.

00:19:19 You know, he was a kind of a pupil of Paul Carroll, who was a Nobel laureate.

00:19:26 And that's where he became interested in amino acids first.

00:19:31 And his first peptide was angiotensin, which he made, hypertensin called at the time.

00:19:37 And then at the beginning of the 60s, he synthesized ACTH 1-24 and soon afterwards ACTH 1-39,

00:19:46 which was the longest peptide at that time.

00:19:49 In the early 1960s, synthetic fragments of the ribonuclease S-peptide were reported independently by the Hoffman and Scaffoni groups.

00:19:57 Other notable synthetic achievements include gastrin by Kenner in 1965, secretin by Budansky and Andetti in 1966, and glucagon by Vinch in 1967.

00:20:10 By the mid-1960s, larger peptides were synthesized, such as ACTH 1-24 by Beiermann and by Schweitzer.

00:20:19 ACTH 1-39 was independently synthesized by both Schweitzer and Sieber and a team composed of the Bayeux, Kisfaluti and Medzarotsky groups.

00:20:29 When we finished the polyglutamic acid synthesis, then we switched a little bit and there came a good opportunity for cooperation in the peptide chemistry.

00:20:39 It means that two research groups dealing with peptide chemistry, one of the research institutes of the Pharmaceutical Research Institute led by Dr. Bayeux

00:20:49 and the other one from the Gedeon-Richter factory led by Dr. Kisfaluti, and myself from the Institute of Organic Chemistry with the leadership or organizer of the whole group is Professor Bruckner,

00:21:01 and we decided to synthesize the human ACTH.

00:21:04 It's just the classical route and the synthesis consisted of 132 chemical steps.

00:21:13 The race to synthesize insulin was quite remarkable.

00:21:17 This 51-residue, two-chain peptide was first sequenced by Friedrich Sanger in 1952.

00:21:24 The Katsayanis group in Pittsburgh first reported their synthesis in June of 1963, but their publication did not appear until 1964.

00:21:33 The Zahn group completed their synthesis in late 1963 and published their findings at the end of that same year.

00:21:41 The Chinese group had also been reporting progress for several years, but didn't publish their final synthesis of active insulin until 1965.

00:21:52 All three groups used standard solution fragment strategies, but with somewhat different tactics.

00:21:58 The difficult step in all three cases was to combine the separate purified A and B chains to give the correct three disulfide bonds.

00:22:07 This was the yield-limiting step.

00:22:12 I am Helmut Zahn. I am presently Professor Emeritus of our Rheinisch-Westfälische Technische Hochschule,

00:22:21 and we had the idea, let's synthesize tyrosine peptides from insulin to study the role of tyrosine for properties.

00:22:31 Johannes Meinhofer, who had spent a sabbatical year in the United States,

00:22:41 he had worked with Zahn with insulin, actually, using for the first time bifunctional reagents,

00:22:48 cross-linked reagents, and determined distances within insulin at a very early time, in 1958 or so.

00:22:57 So he went to America, also Eugen Schnabel went to America, both returned,

00:23:02 and then, I think, Johannes Meinhofer told Zahn, well, this making of small insulin peptides is not very useful,

00:23:13 we should really head for insulin chains.

00:23:15 Of course, we knew of the competition of Pano-Katianis in the United States and of the Chinese groups.

00:23:23 The decisive event was 60, a publication by Dixon and Wartelow,

00:23:28 who have shown that it is possible to synthesize insulin if you have a synthetic A-chain and you have a synthetic B-chain.

00:23:37 We completed the synthesis of A-chain and B-chain in 62, 63.

00:23:47 The joining of the two chains was accomplished towards the end, the very end of 63.

00:23:55 And then we had a big problem.

00:23:58 We could, we have made some preparation, but we didn't know the biological activity,

00:24:04 and without a biological activity, we could not publish.

00:24:08 And it was shortly before Christmas, and we telephoned to Düsseldorf,

00:24:12 and this man said, no, it's Christmas time, nobody in the laboratory,

00:24:17 all the glucose, the radioactive glucose is no longer available.

00:24:21 But there is one very ambitious professor, Pfeiffer, in Frankfurt.

00:24:25 I telephoned him, he didn't know me.

00:24:27 Ja, wir machen das.

00:24:29 And then Meinhofer went by night train with a sample, two samples, to Frankfurt.

00:24:36 And Pfeiffer personally, his good friend, he no longer is, he passed away.

00:24:41 It was a pity.

00:24:43 And each night, they made the test of the two samples.

00:24:47 And Meinhofer came back, and he came, was I convinced, and he came in my room and said,

00:24:55 Professor Sahn, what is your suggestion?

00:24:58 How much activity have we in our preparation?

00:25:03 I said, if we have one, if we have 0.1, I would be happy,

00:25:08 because this was some of the data which, he said, 1%.

00:25:13 So the short account was quickly written up and submitted,

00:25:17 and yet, and still appeared in the December issue, 1963.

00:25:25 By then, the competitors, Katsianis, came out with their first paper beginning of 1964.

00:25:32 So it was really a very close run.

00:25:35 Yu Kang Du, one of the original young members of the insulin group in Shanghai,

00:25:40 summarizes the early work of these Chinese laboratories.

00:25:44 I worked on the insulin project at the beginning from 1958.

00:25:53 Just a graduate student came from Peking University.

00:25:59 At that time, the government asked people to do a big job.

00:26:05 So what should we do? What topics?

00:26:10 For example, some high blood pressure, or cancer, or synthesize, or something.

00:26:22 At last, they finished this argument,

00:26:28 decided to do the synthesis of protein.

00:26:36 But the first protein known the structure only when it's insulin.

00:26:46 So we chose the insulin to synthesize.

00:26:49 Should we say, Shanghai Institute of Biochemistry,

00:26:53 there were two groups called a combination of H&B chain,

00:26:59 from H&B chain and the B-chain synthesizing.

00:27:04 And the H-chain synthesizing is collaboration by Peking University and the Organic Chemistry Institute.

00:27:14 The team, we are very young.

00:27:18 All of us are less than 30 years old.

00:27:21 I think I was 28 years old.

00:27:25 My child is only one year old.

00:27:28 And my colleagues, their children are also one or two years old.

00:27:35 So the children left in Beijing or in other places,

00:27:41 and only ourselves in Shanghai.

00:27:45 We work till morning, until night, and also in the weekend.

00:27:51 We never have a relaxing weekend.

00:27:56 So every day we work very hard.

00:27:59 We hope to get the first synthesis of insulin in the world.

00:28:06 Talking about the major problems for the synthesis of insulin,

00:28:12 in my opinion, I think it is the combination of peptide chains.

00:28:20 Du carried out 600 experiments to find the best conditions to refold native A and B chains.

00:28:27 One of the keys to success was operating at 4 degrees centigrade.

00:28:32 In 1966, Arnold Margolin showed that the A and B chains could be synthesized by solid phase methods,

00:28:38 and when oxidized by Du's method, gave rise to insulin activity similar to that from the other syntheses.

00:28:45 It seemed to me, and to Bruce, that the way to do the problem was to synthesize the B chain,

00:28:52 synthesize the A chain, purify them as well as they could be purified,

00:28:56 and then try to recombine them by methods that were similar to what had been done elsewhere.

00:29:02 I synthesized the B chain first because it was a longer chain.

00:29:06 That had to do with Bruce's conviction that a long peptide could be made,

00:29:12 and I think in part his impatience to get the longer peptide made first.

00:29:18 The B chain had 30 residues, the A chain had 21.

00:29:22 That was actually fortunate because the B chain was easier to synthesize, as it turned out,

00:29:26 and made the B chain, struggled with the A chain, finally was able to make a decent A chain,

00:29:32 and then was able to recombine them.

00:29:35 What you could do was take natural chains, treat them the same way,

00:29:39 and show that the synthetic products were identical to natural chains.

00:29:42 That was the best you're going to get.

00:29:44 If you change the stoichiometry of the reaction,

00:29:47 for example, you had more B chain than A chain, or more A chain than B chain,

00:29:50 or anyway, you change the stoichiometry in some way,

00:29:53 and then you monitored the yield based on the change in stoichiometry,

00:29:57 you could perhaps get a better recombination yield.

00:30:01 That was what the Chinese under Liu Kang did,

00:30:05 and they did very good, careful analytical work

00:30:09 to show what the optimum chain proportions might be,

00:30:14 and so we used that method.

00:30:16 The yields were comparable to yields that had been got by classical non-solid phase techniques.

00:30:24 A few years later, Sieber's group completed a total synthesis of insulin

00:30:28 by selective formation of the three disulfides.

00:30:39 This produced crystalline human insulin of full biological activity.

00:30:47 Margolin believed that insulin is synthesized in vivo as a single chain,

00:30:51 and after folding and disulfide formation was cleaved into two chains.

00:30:56 Well, biosynthesis of insulin at that time was thought to occur in two chains.

00:31:02 There was evidence that the A and the B chain could profitably be looked at as one chain.

00:31:08 At exactly that time, Dorothy Hodgkin's group had solved the X-ray crystal structure of insulin,

00:31:15 and it turned out that indeed that was correct.

00:31:18 The B chain was close to the C terminal of the A chain.

00:31:25 So I thought, why not synthesize it that way,

00:31:28 and why bother to synthesize it in two chains since it was so hard to recombine the chains?

00:31:34 It might be better to just synthesize it in one chain anyway.

00:31:37 I thought that if I synthesized the two chains, separated them by methionine,

00:31:41 then cleaved the two chains, reoxidized them as a single chain project,

00:31:47 cleaved the two chains with cyanogen bromide,

00:31:51 I would end up with a C terminal residue that I could remove with a peptidase or some other method

00:31:58 and end up with my two insulin chains and not have to worry about the recombination,

00:32:03 which was still the big problem.

00:32:05 I did some work on that and actually synthesized an insulin

00:32:09 that had very close biological activity to the insulin I'd already synthesized.

00:32:13 While I was at the NIH, Don Steiner in Chicago showed that insulin was made in a single chain,

00:32:19 but that the linker protein was a 33 amino acid connecting peptide,

00:32:24 which somehow facilitated single chain approximation of insulin,

00:32:28 presumably as disulfide bonds were formed,

00:32:31 and then the connecting peptide was cleaved out by a series of enzymatic reactions to form insulin.

00:32:38 The Yamaihara group in Japan achieved a synthesis of pro-insulin.

00:32:42 This showed that insulin was folded and oxidized in high yield while in a single chain.

00:32:49 The insulin story was a landmark chapter in the history of peptide chemistry.

00:32:54 The research on insulin has continued up to the present day.

00:32:58 We very quickly recognized that we didn't want a monomeric insulin,

00:33:02 we wanted a rapidly dissociating hexamer,

00:33:05 because that would provide us the commercial stability so that it could be used in the marketplace,

00:33:11 but also the ability to mix it with long-acting insulins.

00:33:15 So it actually comes in two forms.

00:33:17 One is as the fast-acting insulin just for mealtime use,

00:33:21 but for those who also want to take it simultaneous to their long-acting evening insulin,

00:33:27 then they have a premix with what is known as NPH insulin.

00:33:33 And what we demonstrated was that by inverting what naturally is the proline-lysine sequence

00:33:39 in the C-terminus of the B-chain to lysine-proline,

00:33:42 something that occurs in the IGF-1 molecule, a homologous peptide,

00:33:48 that we were able to provide an insulin that did not self-associate to the propensity that human insulin did.

00:33:55 That gave you a more precise ability to administer the insulin

00:34:00 so that you had a quicker on rate and a quicker off rate.

00:34:03 And what has been shown in large-scale clinical studies and in practice

00:34:08 is that you can get improvements in hemoglobin A1c, glucose control.

00:34:13 You can get an appreciable reduction in the incidence of hypoglycemia,

00:34:19 as well as the added convenience of using the insulin at the time of the meal

00:34:23 as opposed to having to dose 30 to 45 minutes before the meal.

00:34:28 So it's clearly shown that it is a more efficacious insulin.

00:34:32 It's a more convenient insulin.

00:34:35 And today it's becoming the preferred insulin for treating insulin-dependent diabetes.

00:34:48 Rüdinger in 1958, with the encouragement of Frentisek Schorm,

00:34:52 director of the Institute of Chemistry in Prague,

00:34:55 organized the first European peptide symposium.

00:34:58 Rüdinger extended invitations to all leading peptide scientists in Europe.

00:35:03 Twelve European peptide chemists attended

00:35:06 to discuss the advancements and challenges within the field.

00:35:10 The symposium naturally centered on synthetic chemistry,

00:35:13 the central concern at the time.

00:35:16 In September 2000, the 26th symposium, organized by Jean Martinez,

00:35:21 was held in Montpellier.

00:35:23 These biennial meetings now host over 1,000 scientists.

00:35:28 In 1968, the American Peptide Symposium

00:35:32 was founded by Saul Landy and Boris Weinstein.

00:35:36 In the early 1990s, Japan, China, and Australia

00:35:40 each organized peptide societies.

00:35:43 These societies also organized periodic international peptide symposia.

00:35:48 At a Federation meeting in the spring of 1962,

00:35:58 Bruce Merrifield reported the general principle

00:36:01 of carrying out peptide synthesis on a solid support.

00:36:05 I was making these small phenepheptides,

00:36:09 and even though you could make them, it took a very long time,

00:36:13 the yields were very low, even for a small peptide,

00:36:17 and very time-consuming.

00:36:19 So as an amateur in the field, I thought,

00:36:23 well, there really ought to be some way to do this better,

00:36:27 some better way to make peptides than the classical methods

00:36:32 that everybody's using.

00:36:34 And I thought about it for some long time,

00:36:37 and then suddenly one night, I believe,

00:36:41 I had an idea, and it just came to me,

00:36:44 of how we might go about that.

00:36:47 And the idea was to make use of an insoluble, solid support

00:36:52 to hold the peptide chain while you're synthesizing it,

00:36:57 while it's growing in length.

00:37:00 And that was the fundamental idea.

00:37:04 It looked like if you could make it work,

00:37:08 and of course I had no idea whether you could or not,

00:37:11 that it would be simpler in the first place,

00:37:15 it would be more rapid,

00:37:17 and you might get better yields than you do by the standard method.

00:37:22 So that was the origin of the idea.

00:37:25 He figured out there ought to be a better way of doing it,

00:37:27 and sat down and wrote out the whole idea of solid-phase synthesis

00:37:31 in his lab notebook.

00:37:33 And I thought about it a day or two,

00:37:36 and then I had to go to the boss, to Dr. Woolley,

00:37:42 and we happened to be riding up to the fourth floor on the elevator.

00:37:47 So I told him in just a matter of 30 seconds or something

00:37:51 what the idea was, and what did he think about it.

00:37:54 And he got off and left. He didn't say anything.

00:37:57 Well, I wasn't too encouraged by that.

00:38:00 The next day, he came into my lab.

00:38:03 He says, you know, that's a pretty good idea.

00:38:06 Maybe you ought to work on that.

00:38:08 I said, well, great.

00:38:10 You know, if I could take off two or three months,

00:38:13 I could get this to work, or find out if it'll work.

00:38:18 And he said, okay. He thought that was reasonable.

00:38:21 And I went from there.

00:38:25 The difference is it wasn't three months, it was three years.

00:38:30 There were a lot of variables in this simple idea

00:38:35 that you had to work out,

00:38:37 and they all had to be worked out at the same time.

00:38:40 In other words, you had to have a solid,

00:38:43 mainly a resin support.

00:38:46 You had to have a way to attach the chain to it,

00:38:50 coupling reactions,

00:38:53 deprotection steps,

00:38:55 and finally removal of the peptide from the resin

00:38:58 at the end of the synthesis.

00:39:00 So each one of those turned out to be a much bigger problem

00:39:04 than I had anticipated.

00:39:07 And sometimes you could get one of them to work fine.

00:39:11 You may get a good resin,

00:39:13 but your coupling reaction was no good.

00:39:16 So it didn't work. The overall synthesis didn't work.

00:39:19 They had to all come together at the same time.

00:39:22 I started out with cellulose.

00:39:26 And the reason was I knew that cellulose

00:39:30 had been used to fractionate proteins.

00:39:33 So I thought, well, there must be room in this cellulose

00:39:37 to handle a material of that size.

00:39:41 So I started it, and I got a little way.

00:39:45 I made a dipeptide,

00:39:47 but it was much too fragile for the chemistry I was using.

00:39:50 That's another example of I had a support,

00:39:53 but I didn't have the right chemistry.

00:39:56 And so I had to give that up.

00:39:58 It didn't work well.

00:40:00 I tried other things like polyacrylamide,

00:40:03 methyl methacrylate,

00:40:07 even porous glass,

00:40:09 which is a good hard solid,

00:40:11 although not really a solid in the other sense.

00:40:15 And there were probably six or eight

00:40:21 resins or solids that I tried to use.

00:40:24 I tried to use the sulfonated polystyrene resins

00:40:30 that are used for amino acid and protein fractionation.

00:40:35 And I knew about them because I'd used them before

00:40:38 for that purpose.

00:40:39 So I was clearly influenced by that.

00:40:43 A lot of trouble.

00:40:44 A lot of trouble because they were highly substituted.

00:40:47 You had too many functional groups on them,

00:40:51 and you could do something with it,

00:40:54 but not very well.

00:40:56 And this is where all my three years went,

00:40:59 doing this sort of thing.

00:41:01 Then I got a hold from Dow Chemical,

00:41:04 a sample of a polystyrene cross-linked

00:41:08 with a little bit of divinyl benzene.

00:41:11 This is a starting material for their ion exchange resins.

00:41:15 They sent me some,

00:41:18 and of course you have to have a functional group on it.

00:41:21 So I decided, among other things, finally,

00:41:24 that a chloromethylation reaction

00:41:28 would give you a substituted benzyl chloride,

00:41:33 which is quite a reactive substance.

00:41:36 And having made that,

00:41:38 then I could take the amino acid

00:41:40 that I wanted to attach

00:41:42 and form a benzyl ester

00:41:45 of the carboxyl group.

00:41:47 Now that was the stable covalent bond,

00:41:52 which was holding the pheftide to the resin.

00:41:55 Then the question is,

00:41:57 how do you protect the amino end?

00:41:59 And I, of course, used the carbobenzoxy group.

00:42:02 It was too acid stable and caused side reactions.

00:42:06 Then I went to the new VOC group

00:42:09 because it could be removed

00:42:11 under mild acidic conditions.

00:42:14 The activating reagent was also very important.

00:42:18 The activesters, mixed anhydrides, and azides

00:42:22 were not fully satisfactory.

00:42:25 The best activating reagent

00:42:27 was dicyclohexylcarbodiimide.

00:42:30 It gave rapid reactions and went in very high yield.

00:42:35 To synthesize a longer pheftide,

00:42:37 the sequence of deprotection, neutralization, and coupling

00:42:42 was repeated over and over

00:42:44 for each subsequent amino acid.

00:42:47 Purification at every step

00:42:50 was by rapid and simple but thorough washing

00:42:53 to remove excess reagents and byproducts.

00:42:58 The last step was cleavage of the pheftide

00:43:01 from the solid support.

00:43:03 Hydrogen bromide and later hydrogen fluoride

00:43:07 were the best strong acid reagents.

00:43:10 A final selective purification procedure

00:43:13 was then necessary.

00:43:16 There were really three advantages.

00:43:18 One was the feed.

00:43:20 One was that you don't have to transfer the material

00:43:25 from one container to another

00:43:27 as you do in an ordinary synthesis.

00:43:31 Once you put it inside a reaction vessel,

00:43:33 it just stays there until the pheftide is completed.

00:43:37 So that's an advantage.

00:43:39 And maybe the most important is

00:43:43 at every intermediate stage of the synthesis,

00:43:47 every time you add an amino acid,

00:43:49 you just purify by simple,

00:43:53 thorough washing with solvent

00:43:56 and don't have to go through

00:43:59 chromatographic purification or crystallization,

00:44:04 which are good but very time-consuming.

00:44:07 And for a long pheftide,

00:44:10 and very few people working on it,

00:44:13 it becomes tedious and time-consuming.

00:44:17 It takes very long.

00:44:19 And so I think that's the other main advantage.

00:44:22 Initially, I had envisioned a flow system

00:44:25 for the synthesis using a pump.

00:44:28 I loaded resin into a tube

00:44:31 with a glass filter at the bottom

00:44:33 and a solvent inlet at the top.

00:44:36 This was a very crude system

00:44:38 and did not work well at all,

00:44:40 partly because the resin became compressed

00:44:43 and flugged the filter.

00:44:45 So I went to a vat process in a closed vessel

00:44:50 with a glass filter at the bottom

00:44:52 and a rocker to mix the reagents.

00:44:55 After about three years,

00:44:58 I had made a model tetra-pheftide.

00:45:02 And this worked really quite well by then.

00:45:06 At this point, I needed a name for the new process

00:45:10 and settled on solid-phase pheftide synthesis.

00:45:14 And at that time, I gave a favor

00:45:18 down at Atlantic City at the Federation meeting.

00:45:21 There was a reporter there that thought it was great.

00:45:24 There were two professional expert pheftide chemists

00:45:28 right down the front row.

00:45:30 Their reaction was not all that good.

00:45:33 They were skeptical,

00:45:35 and what I found over the years after that

00:45:37 is the vest of the pheftide chemists

00:45:41 were very reluctant to accept this idea.

00:45:44 There was a tremendous skepticism

00:45:46 among organic chemists

00:45:48 about the use of solid-phase chemistry

00:45:50 because intermediates were not being isolated

00:45:56 and their structure determined.

00:45:58 I wrote a full paper then for 1963

00:46:01 and described how to do it,

00:46:03 what I thought were the limitations.

00:46:05 There were a lot, and I knew about a lot of them.

00:46:08 And I mentioned those,

00:46:10 that you have to worry about these various things.

00:46:13 For example, the reactions have to go very close to 100%.

00:46:17 If they don't, you'll end up with

00:46:19 what we now call deletion pheftides,

00:46:21 where you'll have a missing link in your pheftide.

00:46:25 And I knew about that,

00:46:27 and we were very much concerned about it,

00:46:29 even in the first paper that we wrote.

00:46:32 The best example I have is the work that John Stewart did.

00:46:37 In this next year, I was able to synthesize

00:46:40 25 bradykinin analogs using solid-phase,

00:46:44 three in one year to 25 in a year.

00:46:47 In recent years,

00:46:48 Stewart has made effective antagonists of bradykinin

00:46:51 that are expected to become important drugs.

00:46:54 In 1964, Garland Marshall joined Merrifield

00:46:58 as his first graduate student.

00:47:00 He synthesized the hypertensive octopeptide angiotensin,

00:47:03 which extended the solid-phase method

00:47:05 to include new amino acids.

00:47:08 He was the first to realize

00:47:09 that a fragment synthesis was possible by this method.

00:47:12 Marshall felt strongly about the generality

00:47:15 of the solid-phase synthesis principle.

00:47:17 Bruce always said that,

00:47:21 you know, we're going to do,

00:47:23 this is solid-phase peptide synthesis.

00:47:26 And I used to say,

00:47:28 but Bruce, it's solid-phase synthesis.

00:47:31 You can do anything with this.

00:47:34 And he says, of course.

00:47:36 And it turns out that I actually made

00:47:38 the first dinucleotide

00:47:40 on the solid support in Bruce's lab.

00:47:42 I made DTT in his lab.

00:47:45 But it was very clear to me from the beginning,

00:47:47 and I think just as clear to Bruce,

00:47:49 that this methodology was applicable

00:47:51 to chemistry in general.

00:47:54 And in fact, I really,

00:47:56 I think the first science paper review he wrote,

00:48:00 I got him to generalize the scheme

00:48:04 to make it a polymeric protecting group

00:48:06 and generalize the monomer units

00:48:09 because he never wanted to

00:48:14 overemphasize or over-claim anything.

00:48:19 Maurice Manning was among the many peptide chemists

00:48:22 who soon used the new synthetic method.

00:48:24 I quite honestly have to say that

00:48:26 had it not been for the solid-phase method,

00:48:28 I would have given up peptide chemistry

00:48:31 because the classical method of peptide chemistry

00:48:34 in those days was very time-consuming,

00:48:38 I mean, it could take you anywhere

00:48:40 from six months to a year

00:48:42 to make an oxytocin analog by the classical methods.

00:48:46 But once the solid-phase method came along,

00:48:48 and I could see, you know,

00:48:50 while others were doing,

00:48:52 Bruce in his lab and John making,

00:48:54 this was fantastic,

00:48:55 but once we got going,

00:48:57 especially down in Toledo,

00:48:59 one person could make 30 analogs a year.

00:49:02 So you can see the increased efficiency

00:49:05 that the solid-phase method brought along.

00:49:07 It was phenomenal.

00:49:08 Very quickly, we developed methods

00:49:10 and other people developed methods

00:49:12 to make larger quantities of peptides,

00:49:14 which were not possible at that time

00:49:16 except for extended periods of time.

00:49:18 The effort that would be required

00:49:20 required extensive periods of time

00:49:22 to get that much by solution methods.

00:49:24 So I think it's very important for Bruce

00:49:26 to realize that, you know,

00:49:28 the farsighted people in the field

00:49:30 saw the significance of his work.

00:49:36 When I wrote the first paper in 1963,

00:49:42 I pointed out that this methodology

00:49:45 ought to lend itself well to automation.

00:49:48 And so we were thinking about it

00:49:51 right from the beginning.

00:49:52 Then after I got the method working,

00:49:55 Dr. Wooley and I got an NIH supplement

00:50:00 to our grant to build a machine.

00:50:04 So with that in hand,

00:50:05 I went in and talked to John Stewart,

00:50:07 the colleague here,

00:50:09 and invited him to work with me

00:50:11 on the construction of a synthesizer.

00:50:15 And he was very enthusiastic

00:50:17 and said yes, he'd like to do that.

00:50:19 So that was the beginning.

00:50:21 And then he laid out,

00:50:23 he was good at electronics,

00:50:25 and he laid out the wiring diagram

00:50:28 in some detail.

00:50:30 I spent more time on the mechanics,

00:50:34 the plumbing system,

00:50:36 and the chemistry.

00:50:37 And he spent more on the

00:50:39 electrical end of things.

00:50:41 What you can see is a reaction vessel,

00:50:45 which we already had in the manual method.

00:50:48 And that's shown here.

00:50:50 And it has the resin in it

00:50:52 with the pheptide.

00:50:55 Over here is a pump to deliver solvents

00:50:58 to the reaction vessel.

00:51:01 Over here is a rotary selector valve

00:51:04 to pick the right solvent

00:51:06 and the right reagent at the right time.

00:51:08 And there is another rotary valve

00:51:12 to select the right amino acid

00:51:14 at the right time.

00:51:16 The key to the success of the instrument

00:51:19 was a rotary selector valve.

00:51:22 And this is quite commonplace today

00:51:24 for HPLC solvent selection

00:51:27 or other purposes.

00:51:30 However, that was a totally new thing

00:51:32 in those days.

00:51:33 The guy who ran that was an expert

00:51:36 Swedish machinist named Nils Jornberg.

00:51:39 And he designed and built

00:51:41 a fantastic all-teflon

00:51:43 rotary selector valve.

00:51:46 We can have six amino acids lined up.

00:51:49 This will pick one at a time

00:51:51 and feed them into this cycle,

00:51:53 which then goes into the synthesis.

00:51:56 And the solvents are removed by a vacuum.

00:51:58 So that's the basic...

00:52:01 And this whole thing shakes, of course.

00:52:03 So that's the mechanical end.

00:52:05 Over here is the programmer,

00:52:07 which controls the events over there.

00:52:10 We have a set of timers

00:52:12 and this tenor drum.

00:52:17 It's an electromechanical device.

00:52:20 And it operates...

00:52:21 You set pins in it

00:52:23 to make your program.

00:52:25 Then it operates switches.

00:52:27 The switches operate the events over there.

00:52:30 When this goes around 100 steps,

00:52:33 then you will have added one amino acid

00:52:36 to the peptide chain.

00:52:37 So this is our synthesizer

00:52:39 and it works quite well.

00:52:43 It's the thing that Merck-Goude used

00:52:45 for the ribonuclease synthesis.

00:52:50 The first synthesizer that I know of

00:52:54 besides ours,

00:52:56 was by Art Robinson,

00:52:58 who was a student,

00:52:59 who came to our lab

00:53:01 to learn peptide chemistry.

00:53:03 And he went home and built a machine.

00:53:05 Right after that,

00:53:06 Victor Hruby,

00:53:07 he used to be across the street

00:53:08 with D'Avigno,

00:53:09 he came over

00:53:10 and we gave him some blueprints,

00:53:12 talked to him about it.

00:53:14 And he went back to Arizona,

00:53:17 University of Arizona,

00:53:18 and he built a machine.

00:53:21 I went to the University of Arizona.

00:53:23 I asked Professor Merrifield

00:53:25 if I should build an instrument

00:53:28 because I wanted to be able

00:53:29 to make peptides quickly

00:53:30 because I wanted to do NMR studies

00:53:32 and to put deuterated amino acids

00:53:34 into peptides

00:53:35 so that I could make the assignments

00:53:37 properly and so forth.

00:53:38 And he said,

00:53:39 sure, just come over anytime you want to

00:53:41 and I'll help you

00:53:42 to get ready to do this.

00:53:44 So I went and talked to him a few times

00:53:47 when I was still a postdoc

00:53:49 with Professor D'Avigno.

00:53:50 And then when I went to Arizona

00:53:52 about three months or four months

00:53:53 after I went there,

00:53:54 I went and visited Bruce

00:53:56 and spent almost, I think,

00:53:58 three or four days with him

00:53:59 in which he explained

00:54:00 all of the ways

00:54:01 in which a solid phase instrument worked.

00:54:04 He was very anxious for other people

00:54:05 to try his method

00:54:07 and he was extremely helpful.

00:54:10 He gave me the blueprints, actually,

00:54:12 for his first machine,

00:54:14 I think before it was published

00:54:16 and so forth

00:54:17 so we could use these things

00:54:19 and show them to our,

00:54:20 I wasn't an electronic technician

00:54:22 or anything,

00:54:23 so we could show these things

00:54:24 to our electronic people

00:54:25 at the University of Arizona

00:54:27 so they could help us build this instrument,

00:54:29 which I did in 1968, 69.

00:54:41 In Copenhagen,

00:54:42 Kai Brunfeld built an early machine

00:54:44 that became the first

00:54:45 commercial synthesizer.

00:54:47 Schwartz Biochemical Company

00:54:48 in New York produced it.

00:54:50 The Beckman Synthesizer

00:54:52 was probably the most successful

00:54:53 commercial machine

00:54:54 among the many built

00:54:56 between 1968 and 1972.

00:54:59 Bob Hodges,

00:55:00 while in Merrifield's lab,

00:55:01 added a monitoring system

00:55:03 to their Beckman Synthesizer.

00:55:05 The monitor automatically performed

00:55:07 a picrate titration

00:55:08 after each coupling

00:55:10 using the assay

00:55:11 developed by Balls-Giessen.

00:55:13 If significant amounts

00:55:14 of uncoupled amino groups

00:55:16 were still present,

00:55:17 the machine would go on hold

00:55:19 until the problem was corrected.

00:55:21 Eventually,

00:55:22 this instrument

00:55:23 laid the groundwork

00:55:24 for later commercial machines.

00:55:31 All these were

00:55:32 discontinuous batch machines

00:55:33 like the original.

00:55:39 Soon,

00:55:40 continuous flow machines

00:55:41 were constructed.

00:55:42 Initially,

00:55:43 they had the same problems

00:55:44 that Merrifield encountered

00:55:46 due to resin compression.

00:55:48 But Shepard and Atherton

00:55:49 overcame this difficulty

00:55:51 by putting their polyamide resin

00:55:53 inside a rigid porous zeolite cage.

00:55:55 They built

00:55:56 the photometric monitoring system

00:55:58 to estimate the extent

00:55:59 of coupling and deprotection.

00:56:01 It was based on the uptake

00:56:02 or release of the FMOC group.

00:56:04 This instrument

00:56:05 was marketed by Pharmacia

00:56:07 and by Millipore

00:56:08 and became quite popular.

00:56:10 I think the commercial instruments,

00:56:11 some of them,

00:56:12 have been a great benefit.

00:56:13 They're much more sophisticated

00:56:15 than this.

00:56:16 They have computer controls

00:56:18 and even some are robots.

00:56:23 And the robot

00:56:24 will make large numbers

00:56:26 essentially simultaneously.

00:56:36 With the availability

00:56:37 of the new solid phase method

00:56:39 and the automated synthesizer,

00:56:41 it was possible

00:56:42 to undertake the synthesis

00:56:43 of much larger

00:56:44 and more complicated molecules.

00:56:47 We wanted to test

00:56:48 the limits of our methodology.

00:56:51 And we knew

00:56:54 we could make peptides

00:56:55 of 20 or 30 amino acids.

00:56:57 Could you make something

00:56:58 over 100?

00:56:59 Well, we picked ribonuclease.

00:57:01 In a sense,

00:57:02 it's kind of a Rockefeller enzyme.

00:57:05 It was discovered here

00:57:07 by DeVos

00:57:08 and crystallized by Kunitz

00:57:10 and the molecular weight

00:57:13 Rotan worked on.

00:57:15 Morenstein did the sequence of it.

00:57:19 When Bernd Gutter

00:57:20 joined the Merrifield group,

00:57:22 he quickly put

00:57:23 the new technique to use.

00:57:25 In 1968,

00:57:26 he undertook

00:57:27 the total synthesis

00:57:28 of ribonuclease A.

00:57:30 He successfully synthesized

00:57:32 this 124 residue protein.

00:57:35 His ribonuclease A

00:57:36 had a specific activity

00:57:38 of 80%.

00:57:40 It had the correct amino acid composition,

00:57:42 substrate specificity,

00:57:44 and antibody specificity.

00:57:47 I started out manually.

00:57:49 I later switched

00:57:50 to this homemade automate

00:57:52 that was put

00:57:53 into the Rockefeller machine shop.

00:57:55 And I think,

00:57:58 I couldn't tell,

00:57:59 but I think it was something like

00:58:01 two months for the synthesis

00:58:03 and then maybe

00:58:06 twice or three times

00:58:07 this time for the purification.

00:58:10 Considering that

00:58:12 the chemistry of solid phase,

00:58:15 of the solid phase method,

00:58:17 mainly coupling procedures,

00:58:19 protecting groups,

00:58:20 and purification procedures,

00:58:22 and the instrumentation

00:58:25 were, of course,

00:58:26 not as advanced as they are now.

00:58:28 I think,

00:58:29 considering this,

00:58:30 I think

00:58:32 that it was quite remarkable

00:58:34 that we finally had a material

00:58:36 with almost full enzymatic activity.

00:58:39 This work showed

00:58:40 that a real protein

00:58:41 with true enzymatic activity

00:58:43 could be assembled

00:58:44 from simple amino acid derivatives.

00:58:46 It confirmed Anfinsen's hypothesis

00:58:49 that the primary structure of a protein

00:58:51 can determine its tertiary structure.

00:58:54 With this achievement,

00:58:55 Emil Fischer's dream

00:58:56 of protein synthesis

00:58:57 was finally realized.

00:59:00 At this same time,

00:59:01 the exploratory research group

00:59:03 of Merck, Sharp, and Dohm

00:59:05 under Robert Denkerwalter

00:59:06 and Ralph Hirschman,

00:59:07 with the strong support

00:59:08 of Max Tischler,

00:59:09 undertook the synthesis

00:59:11 of ribonuclease S-protein 21 to 124

00:59:14 by using fragment synthesis

00:59:16 in solution.

00:59:17 After combination with S-peptide,

00:59:19 they obtained some enzymatic activity.

00:59:22 It really grew out of

00:59:25 us trying to prove

00:59:28 that the methods

00:59:29 that we were working on

00:59:31 actually could be applied

00:59:32 to making really big peptides

00:59:35 and something that had

00:59:38 a real activity,

00:59:42 something that would bring us

00:59:44 close to the machinery of life.

00:59:47 We had several major groups

00:59:50 of chemists under Ralph

00:59:53 setting up a strategy.

00:59:55 This was not the same

00:59:57 as the linear strategy

00:59:59 that Merrifield moved towards.

01:00:04 It was a complex strategy

01:00:07 of building up pieces

01:00:09 of the enzyme.

01:00:11 Ralph was a key element

01:00:13 in bringing together

01:00:15 all the strategies

01:00:17 that we executed.

01:00:19 I think there was

01:00:21 a great deal of excitement

01:00:23 and recognition

01:00:24 that we were taking

01:00:27 an important step,

01:00:28 that we'd moved from one stage

01:00:31 in chemical synthesis

01:00:32 to another.

01:00:35 Even the highest levels

01:00:37 of management were really pleased

01:00:41 that we were doing

01:00:42 something important.

01:00:43 I have a picture

01:00:44 of all the people

01:00:45 that were participants.

01:00:48 I think when we pulled

01:00:50 the group all together

01:00:52 in a room,

01:00:53 I think it was close

01:00:54 to 50 people.

01:00:55 Ralph Hirschman

01:00:57 and Bob Dankwalter

01:00:59 did hide somewhat

01:01:02 the amount of effort

01:01:04 that actually went into this.

01:01:06 The president of the company

01:01:08 at our celebration

01:01:10 was actually quite surprised

01:01:13 at how many people

01:01:15 had actually been involved

01:01:17 in carrying out this synthesis.

01:01:21 But he recognized

01:01:25 the importance.

01:01:28 About 10 years later,

01:01:29 Yajima and Fuji

01:01:30 also prepared ribonuclease A

01:01:32 by solution fragment synthesis.

01:01:35 They obtained

01:01:36 a fully active enzyme

01:01:37 after purifying

01:01:38 the crude product

01:01:39 by affinity chromatography.

01:01:42 The solid phase approach

01:01:43 enabled more detailed studies

01:01:45 of the enzymatic properties

01:01:47 of ribonuclease A.

01:01:49 Bernd Gutte, Michael Lin,

01:01:51 and later Bob Hodges

01:01:52 showed that the carboxyl

01:01:54 terminal synthetic

01:01:55 tetradecapeptide

01:01:56 could be used

01:01:57 to activate

01:01:58 shortened ribonuclease 1 to 118.

01:02:01 They studied the role

01:02:02 of different residues

01:02:03 at the carboxyl terminus,

01:02:06 much as Hoffman

01:02:07 and Scaffoni had done

01:02:08 with the S-peptide

01:02:09 at the amino terminus.

01:02:12 They also constructed

01:02:13 the first non-covalently bound

01:02:15 three-component protein

01:02:17 consisting of ribonuclease

01:02:19 S-peptide 1 to 20,

01:02:21 C-peptide 111 to 124,

01:02:24 and a core protein

01:02:25 21 to 118.

01:02:27 The complex had

01:02:28 good enzymatic activity.

01:02:31 In 1974,

01:02:33 Gutte undertook

01:02:34 a de novo synthesis

01:02:35 of a shortened protein

01:02:36 with ribonuclease specificity.

01:02:42 Ribonuclease synthesis,

01:02:44 again, was the nucleus

01:02:46 and a model of the X-ray structure

01:02:49 of ribonuclease

01:02:50 that was one flight up

01:02:51 from Bruce's lab

01:02:54 in Professor Moore's department.

01:02:57 They had this ribonuclease model,

01:02:59 and in the night hours

01:03:00 when nobody was around,

01:03:02 I started to play around with it

01:03:05 and to sort of construct

01:03:08 from this complete model

01:03:10 of the ribonuclease

01:03:11 a mini-ribonuclease.

01:03:14 The result was that

01:03:16 one could do this.

01:03:17 It had still some activity.

01:03:19 And then from this arose the idea

01:03:21 that it may be possible

01:03:23 to even come up

01:03:25 with completely new proteins,

01:03:27 sort of de novo designed proteins.

01:03:29 You know, this knowledge

01:03:30 that an enzyme

01:03:32 doesn't have to be

01:03:34 over 100 residues,

01:03:35 but it can be maybe 60 residues

01:03:37 and still be active,

01:03:38 and the fact that you could

01:03:40 maybe design

01:03:42 with a certain probability

01:03:44 a helix or a beta sheet,

01:03:46 is sort of where the pieces

01:03:48 are put together

01:03:49 and to try to make some new proteins.

01:03:52 This is considered to be

01:03:53 the first de novo synthesis

01:03:55 of a designed protein.

01:03:57 Examples of proteins synthesized

01:03:59 by the solid phase method include

01:04:01 inhibin,

01:04:03 HIV protease,

01:04:05 chaperonin-10,

01:04:07 migration inhibitor factor,

01:04:09 leukemia protease,

01:04:11 integrin,

01:04:13 and colony stimulating factor.

01:04:15 Sakakibara realized

01:04:17 that the main obstacle

01:04:18 to the synthesis of proteins

01:04:20 by solution methods

01:04:21 was the insolubility

01:04:22 of large protected intermediates.

01:04:25 He set out to devise

01:04:26 better solvent mixtures.

01:04:28 The best solvent mixture

01:04:29 used chloroform

01:04:30 with trifluoroethanol,

01:04:32 hexafluoroisopropanol,

01:04:34 or phenol.

01:04:35 Virtually all of the several hundred

01:04:37 medium and large peptides examined

01:04:39 were now soluble.

01:04:41 Examples of their successful

01:04:42 protein synthesis

01:04:43 include human midkind

01:04:45 and the 238 residue precursor

01:04:48 of the green fluorescent protein.

01:04:57 The simultaneous multiple synthesis

01:04:59 of peptides

01:05:00 was anticipated

01:05:01 by a number of laboratories,

01:05:03 but it remained for Gason and Houghton

01:05:04 to independently achieve this goal.

01:05:07 Gason used multiple plastic pins

01:05:09 to build a large set of peptides,

01:05:11 while Houghton used

01:05:12 small porous plastic bags.

01:05:16 Mario Gason's paper came out in 1984

01:05:19 using the pin approach.

01:05:20 This prompted our desire

01:05:22 to have larger amounts of peptides,

01:05:24 and we came up with the concept

01:05:26 called the tea bag approach

01:05:28 or the present packet approach.

01:05:30 And this enabled us to move

01:05:31 very much more quickly

01:05:33 to make individual peptides

01:05:35 because we could capitalize

01:05:36 on the commonality

01:05:37 of all the wash steps,

01:05:39 the neutralization steps,

01:05:40 the deprotection steps,

01:05:41 and the only individual steps

01:05:42 where the resin packets

01:05:43 became separate

01:05:44 was in the addition

01:05:45 of the next individual

01:05:47 specific amino acid

01:05:48 for that particular given peptide.

01:05:51 So this enabled us

01:05:52 to go approximately

01:05:54 10 to 20 times faster

01:05:55 than the existing method,

01:05:56 and very importantly

01:05:57 50 times less expensive.

01:05:59 So this enabled us

01:06:00 to jump into a whole range of studies

01:06:02 that were simply economically

01:06:03 not possible previously.

01:06:05 So early on,

01:06:06 in the early mid-80s,

01:06:08 we jumped into making

01:06:09 mixtures of peptides,

01:06:11 and this was the next step.

01:06:13 What we found very quickly,

01:06:15 however,

01:06:16 was that the number of peptides

01:06:17 we needed,

01:06:18 individual peptides,

01:06:19 was still the limiting factor.

01:06:21 And then we jumped into

01:06:22 a procedure using

01:06:24 mixture-based combinatorial libraries

01:06:26 which got an enormous number

01:06:28 of compounds.

01:06:29 The initial positional scan

01:06:32 mixture-based library work

01:06:34 was a hexapeptide,

01:06:35 and this is 64 million hexapeptides

01:06:37 with just the L-amino acids.

01:06:39 So these procedures

01:06:40 are all based

01:06:41 on earlier foundation

01:06:43 of Bruce Merrifield's work

01:06:45 in solid phase,

01:06:46 and they enabled us

01:06:47 to jump forward much more quickly,

01:06:48 and none of these

01:06:49 would have been possible

01:06:50 with the existing methods

01:06:51 that were available at the time.

01:06:53 Fodor and his collaborators

01:06:55 used photolithography

01:06:56 to produce all sequences

01:06:58 of a set of amino acids

01:06:59 in a given peptide,

01:07:01 each with a defined address

01:07:03 on a plate.

01:07:05 In 1990,

01:07:06 Furka reported the design

01:07:08 of the divide-couple-and-mix strategy

01:07:10 for the combinatorial synthesis

01:07:12 on resin beads

01:07:13 of a mixture containing

01:07:14 all possible combinations

01:07:16 of a given size

01:07:17 and fixed number

01:07:18 of amino acids.

01:07:20 A hexapeptide library

01:07:21 containing all combinations

01:07:23 of just seven amino acids

01:07:25 gives rise to over

01:07:26 100,000 peptides.

01:07:28 At about the same time,

01:07:30 Kit Lam independently devised

01:07:32 a similar split-synthesis strategy

01:07:34 to solve the problem

01:07:35 of making combinatorial libraries

01:07:37 containing equimolar amounts

01:07:39 of peptides.

01:07:40 In 1987,

01:07:42 when I became

01:07:43 a clinical fellow

01:07:44 in medical oncology

01:07:46 at the Arizona Cancer Center,

01:07:48 at that time,

01:07:49 I started to work

01:07:51 on generating

01:07:52 a large peptide library,

01:07:55 a solution phase peptide library

01:07:57 from which I was hoping

01:07:59 to be able to isolate

01:08:00 peptides that bind

01:08:01 to the specific

01:08:02 cell surface etiotypes

01:08:03 of the lymphoma cell.

01:08:05 So that really began

01:08:07 my work

01:08:08 on combinatorial chemistry.

01:08:10 For the experiment,

01:08:11 what we did is to immobilize

01:08:12 a specific anti-peptide

01:08:14 monoclonal antibody

01:08:15 on a solid support

01:08:17 to form an affinity column.

01:08:19 Then we used

01:08:21 a solid phase peptide synthesis

01:08:23 approach to generate

01:08:24 a solution peptide library.

01:08:28 And then we

01:08:30 poured these solution peptide libraries

01:08:32 over the monoclonal antibody column.

01:08:36 Using this approach,

01:08:37 we were able to

01:08:39 spike the peptide library

01:08:41 of a specific peptide first,

01:08:43 and then use the column

01:08:45 to retrieve that peptide back.

01:08:47 However, we were unable

01:08:49 to use the same approach

01:08:52 to isolate a peptide

01:08:55 from the library

01:08:57 that binds to this antibody.

01:09:01 And I think the problem

01:09:03 we were encountering

01:09:04 was that we did not have

01:09:05 an equal molar ratio

01:09:07 of peptides.

01:09:08 In other words,

01:09:09 some peptides have

01:09:10 very large quantities

01:09:11 and some have

01:09:12 very little quantity.

01:09:14 So the one that

01:09:15 the antibody binds to

01:09:17 may not be present

01:09:18 in sufficient quantity

01:09:19 for us to retrieve it.

01:09:21 And so this became

01:09:22 a big problem for us.

01:09:24 And one night,

01:09:26 in 1989,

01:09:28 while I was

01:09:30 sitting in my rocking chair

01:09:31 trying to figure out

01:09:32 how to solve this problem,

01:09:33 and suddenly I had an Eureka.

01:09:35 And I figured out that

01:09:37 there's an approach

01:09:38 to generate

01:09:41 equal molar ratio of peptides.

01:09:43 So what we call

01:09:44 the split synthesis approach.

01:09:46 And that is to

01:09:47 split the resin speed

01:09:48 into equal volumes

01:09:52 in multiple fractions,

01:09:53 and then each of the fractions

01:09:54 will react with

01:09:55 a specific amino acid.

01:09:57 And then we can mix

01:09:58 all the resin speeds

01:10:02 together again

01:10:03 after the first coupling,

01:10:04 and then we split them again

01:10:05 in the subsequent steps,

01:10:06 and so on and so forth.

01:10:08 So after several cycles

01:10:10 of coupling with

01:10:11 splitting into

01:10:13 20 different amino acids,

01:10:15 we were able to generate

01:10:16 a pentapeptide library

01:10:18 with 3.2 million

01:10:19 possible permutations.

01:10:21 Lam contributed

01:10:22 a major insight

01:10:23 when he realized

01:10:24 that in such a mixture,

01:10:25 each individual bead

01:10:26 contained multiple copies

01:10:27 of only a single

01:10:29 peptide sequence.

01:10:31 About 30 minutes after

01:10:33 that I figured out

01:10:35 that the split synthesis

01:10:36 approach will work,

01:10:39 I have a second Eureka.

01:10:40 And that one is

01:10:41 I suddenly realized that

01:10:44 using the split synthesis

01:10:45 approach to generate

01:10:47 this library,

01:10:48 each bead will contain

01:10:49 one single peptide.

01:10:50 That is what we call

01:10:51 the one bead,

01:10:52 one peptide concept.

01:10:54 And I guess perhaps

01:10:56 the reason I can figure it out

01:10:58 was because of my

01:10:59 immunology background,

01:11:00 and I was thinking that,

01:11:01 you know, that the way

01:11:03 we were doing this

01:11:06 split synthesis approach

01:11:07 and each bead turned out

01:11:10 to have different

01:11:11 individual peptides,

01:11:12 and then we can use

01:11:13 a immunological approach

01:11:15 to actually do the screening

01:11:17 able to retrieve

01:11:19 the peptide that may be

01:11:21 of biological interest.

01:11:22 And then we decided

01:11:24 to proceed and embark

01:11:27 on a series of experiments

01:11:29 and able to reduce it

01:11:31 to practice and a proof

01:11:33 of concept of this approach

01:11:35 within nine months.

01:11:37 And we first presented

01:11:39 our work at the

01:11:40 American Peptide Society

01:11:41 meeting at Boston

01:11:43 in June of 91,

01:11:45 and then we also published

01:11:46 a paper about this technique

01:11:48 in December 91 in Nature.

01:11:50 An array of peptide beads

01:11:52 could be screened

01:11:53 by a suitable binding assay.

01:11:55 The fluorescent beads

01:11:57 are separated,

01:11:58 micro-sequenced,

01:11:59 and synthesized.

01:12:00 Over the last 10 years,

01:12:02 we have applied the

01:12:04 one bead, one compound

01:12:05 combinatorial library approach

01:12:07 to many areas of research.

01:12:09 We used multiple

01:12:11 different biological targets

01:12:12 which include monoclonal antibodies,

01:12:14 protein kinases,

01:12:15 cell surface receptors,

01:12:17 bacteria, etc.

01:12:20 And I'm very pleased

01:12:21 to say that the peptide chemists

01:12:25 are really the pioneers

01:12:26 in the field of

01:12:27 combinatorial chemistry,

01:12:29 starting with Dr. Bruce Merrifield

01:12:32 who invented the

01:12:34 solid phase techniques

01:12:35 back about 40 years ago.

01:12:39 Well, I had written

01:12:41 in more than one review article

01:12:44 that I think solid phase synthesis

01:12:48 would be a goldmine, I said,

01:12:52 for organic chemists

01:12:54 who would use it to facilitate

01:12:57 and direct their syntheses.

01:12:59 It took another 25 years

01:13:01 before major developments

01:13:03 along these lines

01:13:04 in organic synthesis

01:13:05 were accomplished.

01:13:06 An early example

01:13:07 is a benzodiazepine library.

01:13:11 In recent years,

01:13:12 K.C. Nicolaou has applied

01:13:14 solid phase combinatorial

01:13:16 synthesis techniques

01:13:17 to the synthesis

01:13:18 of more structurally complex

01:13:20 natural product-based libraries.

01:13:23 He points out,

01:13:24 Merrifield's pioneering work

01:13:25 in solid phase chemistry

01:13:27 revolutionized the field

01:13:28 of peptide synthesis.

01:13:30 The same philosophy

01:13:31 of solid phase chemistry

01:13:33 is now being implemented

01:13:34 in the latest revolution

01:13:36 in organic synthesis.

01:13:43 Dan Kemp devised a thiol capture

01:13:48 followed by an intramolecular

01:13:50 first-order reaction

01:13:51 to link two peptides

01:13:53 at high effective concentration.

01:13:56 In the early 50s,

01:13:57 Wieland's work

01:13:58 showing that thioacids

01:13:59 and thiophenyl esters

01:14:01 are highly activated

01:14:02 was the forerunner

01:14:03 of the field now called

01:14:04 peptide ligation.

01:14:06 Some 30 years later,

01:14:08 Blake reacted

01:14:09 a minimally protected

01:14:10 peptide thioacid

01:14:11 with silver ion

01:14:12 and a second peptide

01:14:14 to give a new amide bond.

01:14:17 In 1994,

01:14:18 both the Kent and Tam groups

01:14:20 independently used

01:14:21 thioester chemistry

01:14:23 to effect a fragment coupling

01:14:24 between two fully

01:14:25 unprotected peptides.

01:14:28 The method departs

01:14:29 from the concept

01:14:30 introduced by Bergman

01:14:31 60 years earlier

01:14:33 because it is conducted

01:14:34 in water

01:14:35 without an added

01:14:36 coupling reagent

01:14:37 and does not use

01:14:38 protecting groups

01:14:39 even for cis or lyse residues.

01:14:42 In the simplest term,

01:14:44 peptide ligation

01:14:46 is a controlled

01:14:48 chemical reaction

01:14:50 to couple two molecules together

01:14:53 to form a specific bond

01:14:56 at a specific site.

01:14:58 In this case,

01:15:00 at least one of the molecules

01:15:02 is a peptide

01:15:04 or a protein

01:15:05 in its native state

01:15:07 and free of protecting groups

01:15:09 and a bond being formed

01:15:11 is a peptide

01:15:13 or amide bond.

01:15:14 It is a conceptually

01:15:17 different method

01:15:21 than the conventional

01:15:24 peptide synthesis methods

01:15:27 because peptides

01:15:30 are proteins

01:15:31 in their native states

01:15:32 contain many functional groups

01:15:34 such as amines

01:15:36 and carboxylic acids.

01:15:39 It is difficult

01:15:40 to form such a

01:15:42 specific peptide bond

01:15:44 between two free peptides

01:15:46 in the presence of so many

01:15:47 reactive functional groups.

01:15:51 The conventional methods

01:15:53 require protecting groups,

01:15:56 coupling reagents,

01:15:57 and conditions

01:16:00 in organic solvents.

01:16:03 But such strategy

01:16:04 also requires

01:16:07 the repetitive process

01:16:09 of the protection,

01:16:10 which is labor intensive

01:16:13 and sometimes inefficient

01:16:15 and often provides

01:16:17 low yields.

01:16:20 Peptide ligation

01:16:22 does not need any of these

01:16:27 to achieve the same result

01:16:30 of a peptide or amide bond.

01:16:33 What is so unique

01:16:34 about peptide ligation

01:16:35 is that it can accomplish

01:16:38 what the conventional methods do

01:16:40 without the protecting groups

01:16:43 and coupling reagents.

01:16:45 With the purpose of producing

01:16:47 much larger proteins,

01:16:48 a semi-synthetic procedure

01:16:50 has been devised independently

01:16:51 at Rockefeller University

01:16:53 by Muir and Cole

01:16:54 and at New England Biolabs

01:16:56 by Evans, Benner, and Zhu.

01:16:58 The process uses

01:16:59 synthetic peptides

01:17:00 and large proteins

01:17:02 produced by molecular

01:17:03 biological procedures.

01:17:05 These peptides

01:17:06 are then ligated

01:17:07 to the proteins

01:17:08 using a method called

01:17:09 entine-mediated protein ligation.

01:17:12 Proteins as large

01:17:13 as 600 residues

01:17:14 have been produced

01:17:15 in this way.

01:17:17 Currently,

01:17:18 there is no upper limit in sight.

01:17:28 The value of peptides as drugs

01:17:30 is still an open question

01:17:31 that generates vigorous debate.

01:17:34 Eberle expressed

01:17:35 the following opinion.

01:18:00 The same or similar peptides

01:18:02 were done by Sandoz.

01:18:04 They also had calcitonin.

01:18:06 Sandoz produced

01:18:07 the salmon calcitonin

01:18:09 whereas the SIBA

01:18:10 had the human calcitonin.

01:18:12 And when SIBA and Sandoz

01:18:14 merged

01:18:16 some, I think,

01:18:18 eight or so years ago,

01:18:20 then, of course,

01:18:22 they could continue

01:18:24 with their work

01:18:26 in the same way

01:18:28 or they could continue

01:18:30 with a better one

01:18:31 which was the salmon calcitonin.

01:18:33 So what I say is

01:18:35 many peptides have been produced

01:18:37 by these companies

01:18:38 and are still being produced

01:18:40 and sold.

01:18:41 Of course,

01:18:43 in the last ten years,

01:18:45 the peptides

01:18:47 were no more so much

01:18:49 in the focus of management.

01:18:51 So management decided

01:18:53 to use approaches

01:18:55 as they are used

01:18:56 in other countries

01:18:57 namely small molecules.

01:18:59 So peptides served merely

01:19:01 as a model to develop

01:19:03 the assays and so on.

01:19:05 And the small molecules

01:19:07 have become the predominant

01:19:09 topics for these

01:19:11 companies.

01:19:13 And so the big peptide groups

01:19:15 like the ones of SIBA

01:19:17 with many peptide chemists

01:19:19 have slowly

01:19:21 shrunk a little bit

01:19:23 to perhaps

01:19:25 a few, a handful of peptide chemists

01:19:27 left at Novartis

01:19:29 at the present time.

01:19:30 I also believe that

01:19:32 as soon as

01:19:34 new galenic

01:19:36 models are being

01:19:38 developed to apply peptides

01:19:40 through the intestinal wall,

01:19:42 peptides

01:19:44 may become

01:19:46 more important again

01:19:48 as drugs.

01:19:49 The current situation

01:19:50 has been evaluated

01:19:51 by Teresa Kubiak.

01:19:53 1987, so there was

01:19:55 the HIV protease

01:19:57 inhibitors project,

01:19:59 renin inhibitors project,

01:20:01 the growth hormone releasing

01:20:03 factor analog project,

01:20:05 and that was the one that I was

01:20:07 working on.

01:20:08 And our hope was

01:20:10 to develop some of those

01:20:12 peptide leads into drugs.

01:20:14 And to our disappointment,

01:20:16 even though we were very successful

01:20:18 scientifically, the compounds

01:20:20 that we

01:20:22 chose for development failed

01:20:24 because we couldn't formulate them.

01:20:26 So there was

01:20:28 such an anti-peptide climate

01:20:30 then, no one within

01:20:32 an industrial

01:20:34 setting wanted to even hear

01:20:36 the word peptide. The same happened

01:20:38 with the Hoffman-Leroy group

01:20:40 and also

01:20:42 similar projects with different peptide

01:20:44 targets at Merck and other companies.

01:20:46 But now, the year is

01:20:48 2000, and it's so pleasing

01:20:50 to see that peptides are coming back

01:20:52 even though they are coming back

01:20:54 in a different capacity.

01:20:56 There is such an enormous explosion

01:20:58 of information coming

01:21:00 from various genome

01:21:02 projects. C. elegans,

01:21:04 drosophila, human genome

01:21:06 projects. And

01:21:08 so many novel

01:21:10 GPCRs have been identified

01:21:12 for which the native

01:21:14 ligands are not known.

01:21:16 And the vast majority

01:21:18 of those receptors are believed to be

01:21:20 peptide receptors.

01:21:22 Those might be even new subtypes

01:21:24 of the known receptors

01:21:26 or a completely novel

01:21:28 one for which

01:21:30 the ligands must be discovered

01:21:32 yet. So why are

01:21:34 peptides needed in order

01:21:36 to find the function of a peptide?

01:21:38 The first of the

01:21:40 receptor, the first step is to match it

01:21:42 with its native ligand.

01:21:44 And since the ligands are not

01:21:46 known, so combinatorial peptide

01:21:48 libraries are very important.

01:21:50 So it's the solid phase

01:21:52 by which those peptides are

01:21:54 made. Not only that,

01:21:56 there are so many

01:21:58 new peptides predicted from

01:22:00 the precursor genes, again,

01:22:02 coming from the cDNA sequences

01:22:04 from the genome projects.

01:22:06 Recently, during a round table discussion,

01:22:16 several leaders in the field predicted

01:22:18 some of the likely directions of research

01:22:20 and potential findings in peptide

01:22:22 science.

01:22:24 According to Arno Spatola,

01:22:26 but synthetic chemists need

01:22:28 to consider not only new strategies

01:22:30 of pro-drugs, but must also

01:22:32 be well informed about alternate

01:22:34 delivery methods and the special

01:22:36 requirements that accompany

01:22:38 these modes.

01:22:40 Murray Goodman comments on the

01:22:42 following. We now

01:22:44 enter an era of molecular diversity,

01:22:46 which includes combinatorial

01:22:48 syntheses of libraries,

01:22:50 de novo design of protein

01:22:52 mimetics, and the synthesis

01:22:54 of dendrimers and other

01:22:56 macro structures.

01:22:58 According to Daniel Weber,

01:23:00 traditional organic chemists

01:23:02 are learning to use solid phase

01:23:04 chemistry, and peptide chemists

01:23:06 are broadening the scope of their

01:23:08 reaction base well beyond

01:23:10 the formation of amide

01:23:12 bonds. Charles Deaber

01:23:14 extends these ideas.

01:23:16 The array of biophysical techniques

01:23:18 for structure deduction and their

01:23:20 capabilities have

01:23:22 been vastly improving and expanding.

01:23:24 It was apparent

01:23:26 that the established techniques,

01:23:28 including circular dichroism,

01:23:30 NMR, X-ray crystallography,

01:23:32 fluorescence,

01:23:34 and mass spectrometry,

01:23:36 along with computational chemistry

01:23:38 and several developing techniques,

01:23:40 are being put to novel and important

01:23:42 uses.

01:23:44 The importance of physical chemical

01:23:46 methods was extended by Robert Hodges.

01:23:48 Understanding protein folding

01:23:50 and protein stability is critical

01:23:52 in the prediction of protein structure.

01:23:54 It is obvious that

01:23:56 even with the massive expansion

01:23:58 of partial biology,

01:24:00 that is, NMR, spectroscopy,

01:24:02 and X-ray crystallography,

01:24:04 which is taking place around the world,

01:24:06 we will not keep pace

01:24:08 with the hundreds of thousands of new protein

01:24:10 sequences available

01:24:12 from the Human Genome Project.

01:24:14 But Robin Offred points out,

01:24:16 don't let the size

01:24:18 and number of these potential

01:24:20 targets daunt you.

01:24:22 I have two take-home messages to put to you.

01:24:24 The first is that

01:24:26 whatever the size of the protein concerned,

01:24:28 the largest things that we will normally

01:24:30 need to synthesize will be the functional

01:24:32 domains. These are

01:24:34 typically around 200 residues

01:24:36 each. I would say

01:24:38 to any younger scientists, or even

01:24:40 older ones, who are wondering whether

01:24:42 they should remain in or enter this field

01:24:44 that, if this sort of thing interests

01:24:46 you at all, stay with it.

01:24:48 Peptide scientists, with the ability

01:24:50 to have total control over structure,

01:24:52 which is the hallmark of what we do,

01:24:54 are uniquely placed to exploit

01:24:56 to the full the fantastic situation

01:24:58 which is developing around us.

01:25:00 The ball is in your court.

01:25:04 Tom Muir continues along these lines.

01:25:06 The field of chemical ligation

01:25:08 has so blossomed in recent years

01:25:10 that I would submit that the routine application

01:25:12 of organic chemistry to the

01:25:14 synthesis of large proteins is actually

01:25:16 a reality.

01:25:18 As the last speaker on the panel,

01:25:20 Victor Ruby offers the final summary

01:25:22 of the future of peptide science.

01:25:24 Essentially,

01:25:26 the human genome and its use

01:25:28 in applications is up for grabs,

01:25:30 and those of us interested in

01:25:32 ligand-slash-drug design

01:25:34 have enormous opportunities to make

01:25:36 seminal contributions.

01:25:38 The wave of the future will be collaboration,

01:25:40 so that the structural,

01:25:42 chemical, biological,

01:25:44 and behavioral effects of our designed

01:25:46 ligands and drugs can be more

01:25:48 rapidly designed and evaluated.

01:25:52 Bruce Merrifield closed the meeting

01:25:54 with the comments.

01:25:56 It is clear that the peptide field

01:25:58 is alive and well.

01:26:00 We can't predict the next new discoveries

01:26:02 in this field, but we can

01:26:04 all be sure that many exciting

01:26:06 developments lie ahead.

01:26:22 In my laboratory,

01:26:50 I formed a bond with you.

01:26:54 In my laboratory,

01:26:56 my peptide dreams came true.

01:27:00 Take it from an old pro,

01:27:02 she may have a cyst for you.

01:27:06 And in your laboratory,

01:27:10 your dreams will, too.