00:00:01 My first exposure to peptide chemistry was as a graduate student in Henry Rappaport's lab at UC Berkeley.

00:00:16 He had me make a simple peptide. I did it by solution phase method. This was in 1970.

00:00:22 And it took me three months to carry out this project.

00:00:25 I then went as a post-doc into C. H. Lee's lab in San Francisco, and I was first introduced to Merrifield's solid phase approach.

00:00:33 The enormous savings in time and energy and effort using this approach were evident from the very first synthesis I carried out.

00:00:41 I was able to carry out a much more complicated synthesis in a week, whereas before, with a simpler peptide, it took me three months.

00:00:50 So this was an eye-opener to me, and it made me realize the advances you could have by methodologies.

00:00:57 Even as a post-doc in C. H. Lee's lab, I was trying to make the method more efficient.

00:01:02 I was trying to use threaded glass tubes to compartmentalize the resin in order to carry out syntheses in parallel.

00:01:09 This didn't work. All I ended up with was a bunch of broken glass threaded tubes.

00:01:13 But it put in my mind the concept of, let's get there faster, let's get there more efficiently, and ultimately, let's go there farther.

00:01:21 I left Lee's lab, and after a short stint in New York City, I came to work with Richard Lerner at the Scripps Research Institute.

00:01:30 This was in 1980. The driving force for most inventions is getting there faster, and it usually falls on the individual.

00:01:38 In my case, I had five automated synthesizers, five technicians running them, and the limiting step in every study we did was the availability of the synthetic peptides.

00:01:49 Mario Gayson's publication in 1985 of the pin technology was really a prompt to say,

00:01:56 how can we get larger amounts of material, since Mario's procedure was small amounts on the end of pins, how can we get larger amounts of material?

00:02:04 That telescope me back to the time I was a postdoc and compartmentalizing of resins.

00:02:09 What we came up with in my lab was a means of using polypropylene mesh packets that were labeled to enable us to put many different peptide resins into one container.

00:02:21 This enabled us to carry out all the common steps at one time, and was a tremendous efficiency in terms of materials and time, and ultimately, of course, economics.

00:02:33 What we were hoping to do was enable us to move forward on these methodological studies, our own internal studies, collaborations, to break the necessity to have more peptides.

00:02:47 The feedback procedure enabled us to move 10 to 20 times faster and cheaper than existing methods.

00:02:54 We did it without automation, we did it with the same number of people, same amount of space, and very importantly for us initially, no automation.

00:03:02 So we were able to move our studies much more rapidly.

00:03:06 We could do studies that would certainly not have been economically feasible, studies on HPLC retention time, technological studies, studies involving receptor binding work for the opiates, and a whole range of studies.

00:03:19 So this opened up a means to get to individual peptides much more quickly.

00:03:25 Ultimately, the teabag is a very simple, practical means to compartmentalize resin.

00:03:34 This is what a teabag looks like that we use in the laboratory.

00:03:38 This holds about 50 milligrams of resin.

00:03:40 We'll make approximately 25 to 50 milligrams of crude peptide, and you can put a whole bunch of these together.

00:03:47 You can put literally hundreds of these in a single container.

00:03:50 All of the common steps in solid phase synthesis, the washings, the deprotections, the neutralizations, are all carried about individually.

00:03:58 Then you take the individual teabags, and you take them out, and they're numbered.

00:04:02 You put them in a particular amino acid that's activated that you want to couple next, and that's what you do.

00:04:08 You've sorted these out, and you're now coupling.

00:04:11 The only individual step in solid phase synthesis is the addition of the next amino acid.

00:04:16 So that's what we do.

00:04:21 This is a visual of a resin packet, also called a teabag.

00:04:26 What we'll do here is I'll describe the teabag, and then we'll give it a pause, and I'll just let you have a certain period of time on the tape that's blank.

00:04:38 This is a resin packet, also called a teabag.

00:04:41 The essential components of this are simply that the resin stay in the teabag.

00:04:46 The solvents can get in and out of the teabag, so good solvent flow in and out.

00:04:50 As you can see from this diagram, the front view, what you really need is what I've mentioned about the solvent in and out and the resin to stay in,

00:04:59 but you very importantly need a label that will stay indelible throughout the process of synthesis.

00:05:05 This is a very simple concept.

00:05:07 These are typically 1 inch by 1 1⁄4 inch in length and size, and typically 50 to 100 milligrams of resin is very standard.

00:05:15 As you can see from the side view, the resin's inside the packet, and we have seals all around, so if the resin doesn't come out, the solvents can get inside readily,

00:05:24 and that's the resin packet or the teabag.

00:05:32 Okay, we'll cut here.

00:05:35 Whoever's editing this, this is a visual of the process of making peptides using the resin packet or teabag approach,

00:05:43 and I will talk through this and then again leave a pause.

00:05:48 I'll begin talking now.

00:05:50 This is a diagram of how the teabag approach is used to carry out peptide synthesis.

00:05:56 As you can see, the bottle on the top right with the green cap and the yellow packets in it illustrates what we do with the teabag approach.

00:06:05 In the particular bottle, there are between 10 and 100 or 500 individual teabags, and these will represent individual peptides being prepared,

00:06:13 and steps 1, 2, and 3 and 4 are the processes and all the steps involved in making an individual peptide,

00:06:22 but they are also all the steps in making the peptides up until the point where you get to step 3, where the orange box is,

00:06:30 where you separate the packets out and couple those to the individual amino acids of interest to the particular peptide.

00:06:37 So, in fact, what you do is all the teabags are in a particular bottle.

00:06:42 You carry out the deprotection steps, the wash steps, the neutralization steps,

00:06:46 and once the growing peptide on the resin in the teabag has been neutralized,

00:06:53 you then separate that out and add it to the individual bottles shown next to the orange box with a particular activated amino acid of interest.

00:07:01 Once that is coupled for a particular length of time, then you combine the bags again into the same bottle and repeat the process,

00:07:09 and that's the arrow to the right.

00:07:11 This has enabled us to go through all the steps up to 30 or 40 residue peptides quite readily, 100 to 200 or 300 at a time.

00:07:19 It's a tremendous savings in time, efficiency, and cost.

00:07:25 Break.

00:07:29 So the process enables us to make protected peptide resins in great numbers.

00:07:34 The next step in the difficulty is cleavage of the peptide.

00:07:37 This is typically done, or was done at the time, in liquid hydrogen fluoride.

00:07:42 The apparatuses that were available at the time could cleave one peptide resin at a time.

00:07:48 If we could make 500 peptide resins in a month, then we were greatly, again, limited by the number of peptides we could cleave,

00:07:54 and so we came up with a multiple cleavage apparatus that initially was 25 different HF vessels in one container

00:08:03 and ultimately became 120 individual resin packets.

00:08:07 So this has enabled us to get very rapidly into individual peptides, large numbers, purity just the same as if you were making individual resins,

00:08:16 and it was quite successful and enabled us to jump into studies that were simply not economically feasible before.

00:08:22 The cost was reduced by approximately 50-fold from what was available before.

00:08:28 And again, this enabled us to jump into studies that were just not practical before.

00:08:32 This all follows on Bruce Merrifield's initial solid phase approach.

00:08:37 One of the benefits of that is the phenomenal speed in which you can get to peptides, especially longer peptides,

00:08:44 which were very, very difficult to make at the time.

00:08:47 Now, what we found with the ability to make hundreds or 500 or 1,000 peptides in a year,

00:08:55 or before we were able to attempt that, was we were able to get these into the hands of other researchers in our own studies,

00:09:01 but very rapidly the number of peptides that we could make became a limiting factor again.

00:09:08 So early on in the early, mid-80s, we jumped into making mixtures of peptides, and this was the next step.

00:09:15 The individual peptides we could make were a very, very tiny fraction of the possible peptides that one would conceive of or would need,

00:09:24 and so then we'd get into using mixtures and a variety of procedures to define these individual compounds within the mixtures that were active.

00:09:33 So the mixtures we made initially were a hexapeptide library, and we made these,

00:09:38 and this was 64 million individual hexapeptides used as mixture format,

00:09:43 and we used a variety of deconvolution procedures, including an iterative approach, a positional scan approach,

00:09:49 both of which enabled us to very rapidly get information from extremely large libraries of compounds.

00:09:56 We now have three different peptides have been into clinical trials using these methods.

00:10:02 These methods are simple procedures that enabled the individual researcher and those in my lab to move forward more quickly

00:10:09 where it was really not a competitive situation for us before.

00:10:13 The instrumentation, the economics, the time frames were not ones that we could work with,

00:10:18 but with the TBAG procedure and with mixtures and positional scan and deconvolution methods we developed,

00:10:24 we can now work in studies that involve immunology, T cell development for vaccines,

00:10:31 individual compounds for receptor binding, especially in the opiates.

00:10:35 We've worked with a wide range of assay types from antibacterial to individual receptor binding to enzymes.

00:10:42 We've worked with whole tissues.

00:10:44 We've even gone directly into animals with libraries numbering in the hundreds of thousands of compounds per mixture in animals,

00:10:51 and we're able to deconvolute that very rapidly.

00:10:54 So these methods are all based on the need to move forward more quickly, the need to be competitive, the need to cut costs,

00:11:01 and this is the power of the solid phase approach Bruce came up with,

00:11:06 and these are the driving forces for any individual scientist to move forward

00:11:10 and to do things in a manner that is less expensive and can move faster

00:11:16 and ultimately move farther in science and development and discovery.

00:11:24 My involvement in peptide chemistry began in 1970 as a graduate student with Henry Rappaport at Berkeley.

00:11:30 He had me make my solid, I'm sorry, my solution phase methods.

00:11:34 Let's start over.

00:11:36 Over again, do again, do over.

00:11:38 My involvement in peptide chemistry began in 1970 as a graduate student with Henry Rappaport at University of California at Berkeley.

00:11:45 He had me make a short peptide sequence by solution phase methods,

00:11:49 and that process took me approximately three months.

00:11:52 Two or three years later, I was a postdoc in C.H. Lee's lab in San Francisco.

00:11:56 That's when I was first exposed to Bruce Merrifield's solid phase approach.

00:12:00 I was absolutely stunned by the efficiency of this method, the time saving, and ultimately the integrity and purity of the material I made.

00:12:09 And so this was my initial exposure to solid phase methods.

00:12:13 One thing I noticed very early on was the need to make more and more individual peptides.

00:12:19 We got into this in C.H. Lee's lab where I made 30 different analogs,

00:12:23 all of which were buried in the C-terminal end, which is the growing end of the peptide chain.

00:12:29 And I realized that if I could compartmentalize the resin,

00:12:33 and I used pretty glass containers at the time, I could capitalize on this.

00:12:38 It turned out that pretty glass didn't work. I just ended up with a whole range of broken pretty glass containers.

00:12:43 But this put back in my mind the need to go faster in terms of individual compounds.

00:12:49 I ended up in Richard Lerner's group in La Jolla, California in 1980,

00:12:56 and we were making very large numbers of peptides at the time,

00:12:59 very large was 500 a year, with five different synthesizers.

00:13:03 And Mario Ghesin's paper came out in 1984 using the PIN approach.

00:13:07 This prompted our desire to have larger amounts of peptides,

00:13:11 and we came up with a concept called the T-bag approach, or the resin packet approach.

00:13:17 And this enabled us to move very much more quickly to make individual peptides

00:13:22 because we could capitalize on the commonality of all the wash steps, the neutralization steps, the deprotection steps.

00:13:28 And the only individual steps where the resin packets became separate

00:13:31 was in the addition of the next individual specific amino acid particular to that particular given peptide.

00:13:38 So this enabled us to go approximately 10 or 20 times faster than the existing method,

00:13:43 and very importantly, 50 times less expensive.

00:13:46 So this enabled us to jump into a whole range of studies that were simply economically not possible previously.

00:13:52 So again, it was necessity as a mother of invention that enabled us to move more quickly, more efficiently,

00:13:57 with the same lab, the same people, and very importantly, the same economics.

00:14:02 What we found very quickly, however, was that the number of peptides we needed, individual peptides, was still the limiting factor.

00:14:10 And then we jumped into a procedure using mixture-based combinatorial libraries,

00:14:15 which got an enormous number of compounds.

00:14:18 The initial positional scan mixture-based library work was a hexapeptide,

00:14:24 and this is 64 million hexapeptides with just the L-amino acids.

00:14:28 With that, we were able to find new opiate compounds, new antibacterials, and a number of other compounds.

00:14:35 So this concept worked very, very well.

00:14:38 And what we were doing then is putting large numbers of very carefully controlled and segmented mixtures into an assay system,

00:14:47 and the assay system would pick up the individual activity of particular compounds.

00:14:51 That would be the signal, and based on the information and the formatting that we had,

00:14:55 we were able to decipher the individual compounds very, very quickly.

00:14:59 These methods have been used for peptides now.

00:15:01 They're used for peptidomimetics, as well as individual heterocycles and classic molecules of that type.

00:15:09 So these procedures are all based on earlier foundation of Bruce Merrifield's work in solid phase,

00:15:15 and they enable us to jump forward much more quickly,

00:15:18 and none of these would have been possible with existing methods that were available at the time.

00:15:22 All of these are simple, direct procedures that enable us to go into studies

00:15:27 and compete with others where it was simply not possible before.