00:00:00 Okay. One of the first problems you worked on after coming to Columbia was the fluorescent

00:00:29 Y-base from yeast phenylalanine transfer RNA. And can you tell me a little bit about

00:00:35 how you got into that problem and what the challenges were?

00:00:38 Yes. Well, it was suggested by Charlie Cantor, who is now chairman of the human genetics

00:00:48 at our medical school. And he said that, I understand you do structures on this. I can

00:00:55 suggest you a very fascinating problem. And it is this fluorescent Y-base, which is contained

00:01:03 the position near the anticodon of phenylalanine tRNA. And because it's very unstable and it's

00:01:11 fluorescent and people have been trying to clarify the structure and why don't you start

00:01:18 doing it. And so this was done in collaboration with Bernie Weinstein and Dijon Gruenberger,

00:01:28 who are both now professors uptown. And Bernie Weinstein is now head of the cancer institute

00:01:36 there, I think. And so we collaborated with them. They got the yeast phenylalanine for

00:01:41 us. And the most challenging thing was its instability and it's extremely minuscule amount,

00:01:50 which is available. And it was known how to keep this Y-base off from the tRNA. But it

00:01:59 is only stable from between pH 3 to 10, only neutral conditions. And after lots of hard

00:02:07 work, we were able to purify for the first time, but the whole entire amount was only

00:02:14 about 300 micrograms. And I had also, it was the first real work I did when I came to Columbia

00:02:22 from Japan. So it was mostly done with the postdocs, which came from Japan with me. We

00:02:30 are not used to the American system too. And I remember we arrived at Columbia in the end

00:02:36 of August. And I think, well, the lab was quite not set up and we were, I was so glad

00:02:44 when we were able to carry out our first reflux about two months later. Anyhow, because of

00:02:54 the limited amount and at that time, the spectroscopy was not as vast as it was now. And we had

00:03:03 to do things extremely carefully and coupled with synthesis and made a slow step-wise progress

00:03:13 until we got the full structure. And I'm still proud of this because it's got a, it is still

00:03:20 the only known tricyclic nucleic acid base. And it had one chiral center and we carried

00:03:28 out a micro's analysis with 30 micrograms and isolated the compound and got the absolute

00:03:37 configuration. And when we first published this, many people were dubious about the results,

00:03:44 but next year we managed to synthesize it and prove that it was correct. So it's still

00:03:48 a very pleasant recollection for me.

00:03:55 Probably one of the most exciting areas that you've been working in and you've been working

00:04:00 on in the chemistry of vision for 10 or 12 years now. And the work goes on and I suspect,

00:04:12 well it would be nice if you'd tell me something about that work, how it got started and where

00:04:16 you're, you are today.

00:04:19 Yes, this was again, I forgot, I think it was early, around 73, 74. And there were at

00:04:28 that time two young colleagues of mine in the biology department. The names are Barry

00:04:35 Horny, who is now a professor in the, again, the biochemistry department at our place.

00:04:41 And the other guy was Tom Ebry, who is now a professor in biophysics at Champaign-Urbana.

00:04:48 And they said, told me, why don't you come and have a look in our labs? And it was the

00:04:53 first time I went into a dark room where they were carrying out some rhodopsin-related studies.

00:05:01 Incidentally, these two are one of the earlier workers in this field and they had, at that

00:05:08 time, published quite a few review articles. Barry Horny had a hypothesis in which he had

00:05:16 published with Martin Karplus and this hypothesis, correctness or not, could be checked by making

00:05:23 a synderic retinal analogue. So we started working on that and that was in 1975, our

00:05:31 first paper. And since then we've been in this field and currently about half of my

00:05:41 group, namely about 15 people, are in this group. And I think we, over the past 10 years

00:05:49 or so, we have now made about over 80 analogues of retinals. Our basic approach is the

00:05:57 following. Many people in my retinal group have synderic backgrounds and we do some

00:06:06 tailored synthesis and making retinal models, analogues, and then putting them into natural

00:06:14 rhodopsins. Incidentally, rhodopsin now, the family has grown quite big and generically

00:06:23 call them the retinal proteins. And this includes the visual pigments and then the proton

00:06:29 pumping protons, proteins. Also, I'll mention in a moment, the chlamydomonas receptor, which

00:06:38 is responsible for the phototactic action of chlamydomonas. And we call all of these

00:06:45 together retinal proteins. And they have a common basis for cyclic adenylate systems

00:06:53 and so it's attracting lots of attention. First of all, what is, in the case of vision,

00:07:04 how does it ends up, the protein gets modified. You see, in the protein, the retinal in the

00:07:14 rhodopsin case, it is 11-cis retinal, which is hooked to the protein through a protonated

00:07:22 shift base onto the lysine. And then upon light irradiation, the 11-cis isomerizes to

00:07:30 all trans. And then that sort of starts, triggers a change in the shape of the protein and it

00:07:38 initiates a cascade of enzymatic reactions, which ends up in a hydrolysis of cyclic GMP

00:07:46 to GMP. And at this stage, one photon of light is amplified by 10 to the 5th. And so

00:07:56 what is the mechanism, for example? And that is, she has many common bases with the cyclic

00:08:01 adenylate system. And we want to understand all of this more clearly on a molecular structure

00:08:07 basis. The same thing happens with bacterial rhodopsin, which is another retinal protein.

00:08:14 And in this case, it is one of the simplest of the membrane proteins. And we don't understand

00:08:20 many things about the membrane proteins. It's very easy to grow. And also, many people are

00:08:28 studying this because upon irradiation of light, in this case, a proton is transported

00:08:36 through the membrane. So it is a simple system to study the transport of ions through the

00:08:44 membrane. So we make these over 80 or so and then fit them into the proteins. And then

00:08:54 of course, it alters their physical properties, biochemical properties, biological properties.

00:09:01 And so as a result of this, it ends up in many, many cases, collaboration with other

00:09:08 specific groups. And this has led us into a very interdisciplinary area. And as a result

00:09:22 of this, I have started working together with physicists and even some theoretical people

00:09:29 and many biophysicists, spectroscopists, very specialized spectroscopists, like picosecond

00:09:37 spectroscopists and so on. And I don't know whether this is healthy or not, but if we

00:09:44 count the groups with whom we are collaborating with, it's between 15 to 20 right now.

00:09:52 In this particular problem?

00:09:53 Yes. And my wife says I'm not only in this case, but in other cases, too. I mean, spread

00:10:01 out too much. And I started selling rotten fruit, she said.

00:10:08 This sort of is now a certainly common thread through a lot of your work in that you're

00:10:15 becoming more and more interdisciplinary.

00:10:19 I do enjoy it, but I cannot deny the fact that it's spreading out too much. And maybe

00:10:29 in some cases it's not healthy to spread out too much. On the other hand, for me, there

00:10:40 are so many exciting problems in the interdisciplinary field. And when people approach us and say

00:10:47 would you like to do this and so on, and then in many cases...

00:10:51 It's very enticing to do that.

00:10:53 Yes, and we can contribute to a certain extent. So it's a very difficult decision to make.

00:10:59 Yes.

00:11:00 Another thing is about the, I mean I've told you before, but so-called percentage. Because

00:11:10 it's interdisciplinary, you see, quite a proportion of the work does not terminate.

00:11:26 In other words, it doesn't end up in writing a paper. And maybe the batting average in

00:11:31 that sense is about 50%.

00:11:33 I see.

00:11:35 But so far I'm collaborating with many, many people, and I think almost without exception

00:11:42 it's a very pleasant collaboration.

00:11:47 Okay. I know another piece of work that was a collaborative effort in which you ended

00:11:56 up with at least determining what I thought was a very exciting structure was the work

00:12:00 on the red tide dinoflagellate and the structure of brevitoxin, first of brevitoxin B and then

00:12:07 later brevitoxin A. And maybe again you can tell me a little bit about that work.

00:12:13 Yes. It happens that, well this is the toxin which leads to the red tide in toxins which

00:12:24 we find in the Gulf of Mexico. And this toxin was known for about 20 years. And just by

00:12:33 accident it turns out that before, a few months before I was approached by this group in Texas

00:12:44 for collaboration, I was reading a thesis written by a biochemist, and I was in fact

00:12:51 a referee. And it was a very long thesis, about 150 pages or so. And it was dealing

00:12:59 with isolation, purification, toxicity, electrophysiological properties and so on, you see.

00:13:06 Now, I don't want to say anything negative, but a few months later it was brought into

00:13:13 our hands. And then, because we are professionals in isolation and so on, without boasting,

00:13:21 and it was given to a post-doc from Poland who had excellent hands, and he managed to

00:13:31 purify it in a very straightforward manner. And in about two months it fortunately crystallized

00:13:39 in the NMR tube. And then after that, two, three months later, we had the full structure.

00:13:44 This was Brevitoxin B?

00:13:46 That is Brevitoxin B, which means that, you see, this Brevitoxin, the presence of that

00:13:54 was known for years. But because it was not purified, the data coming out of this was

00:14:04 you were not sure, I mean, what it really meant, and quantitatively, of course.

00:14:11 But we must have been very lucky, somehow. Because there was another, I learned later

00:14:18 that Professor Shimizu at the University of Rhode Island, he is probably the number one

00:14:25 expert on these red type toxins. He's done an enormous amount of work on the other type

00:14:31 of red type toxins, and he was also on it. And somehow, it was not crystallizing in his

00:14:40 hands, so we must have been lucky. On the other hand, the other toxin you mentioned,

00:14:45 Brevitoxin A, this is the reverse. We tried to crystallize this for years, and then Shimizu

00:14:51 managed it, and he made the structure of that first.

00:14:55 You had also worked on the structure of A, but using different spectroscopic methods.

00:15:01 Yes, that's right. Because we couldn't get it into crystals, so I do have a tendency

00:15:09 maybe of overusing spectroscopy, and as a result, I have made quite a few mistakes structure-wise.

00:15:18 But my main interest is developing spectroscopic methods, and so we use this Brevitoxin A as

00:15:25 an ultimate example of what one can do with current day NMR and mass spectrometry.

00:15:33 I had two fantastic co-workers, one with Jan Pablak, the other one is Mike Tempesta.

00:15:46 It started at Santori Institute, which I will mention later, and then it just continued.

00:15:53 Mike Tempesta started working on the Brevitoxin A structure at Columbia as well.

00:16:03 I think it's got 50 or so carbon atoms, 70 protons, and the nucleus of this is quite

00:16:16 different from Brevitoxin B. With the mass spec, which is done by Jan Pablak in a very

00:16:23 elegant way, I must say, coupled with the NMR, and we got the whole structure of this

00:16:30 compound using about a total of 6 milligrams, which I think was quite an achievement, except

00:16:37 that there was one misinterpretation of nuclear overhauls effects, and there was one method

00:16:43 group in which we had the configuration wrong, and it was published. It was announced at

00:16:49 the same meeting in which Shimizu gave his structure, which was done by X-ray, and he

00:16:56 first couldn't believe that we had the whole structure.

00:17:00 For me, this was, for us, it was maybe a happy ending, but I think this era has gone now.

00:17:10 We only do the things to prepare ourselves so that there are many, many cases in which

00:17:15 you cannot get things crystalline, especially when they are in the interdisciplinary area,

00:17:21 and the factors which are intimately related to the maintenance of life and so on, many

00:17:27 things are not crystalline, so this gives us good exposure, experience, and also forces

00:17:34 us to develop new methods. Of course, if one can do it by X-ray, there's no, I mean,

00:17:41 X-ray is by far quicker, more efficient, and less prone to error.

00:17:48 So, actually, the brevitoxin B, which you first isolated and crystallized, that was done by X-ray?

00:17:54 That was done by X-ray, and I'm glad at that time, because X-ray was done by John Clady,

00:17:59 brevitoxin A also was done by John Clady, but at that time, it was impossible to do it.

00:18:06 Any other way, right?

00:18:07 Yeah, any other way.

00:18:08 Yeah, no, it's an extremely complex structure.

00:18:10 Yeah, yeah.

00:18:16 Some of your more current work.

00:18:19 Can we do the current part now?

00:18:21 Sure.

00:18:22 I don't want to change things.

00:18:23 Yeah, anyway.

00:18:24 Okay, stand up.

00:18:31 Five, four, three.

00:18:36 Another more recent piece of work in which also presented a real challenge in isolation

00:18:47 was the work on tunichrome B, which is a reducing blood pigment of the sea squirt.

00:18:54 And how did you get into it, and what was the real challenge there?

00:19:01 This is exposing us to a new experience.

00:19:06 It has brought to our attention, it was in collaboration with Ken Kustin,

00:19:10 who is a professor in inorganic chemistry.

00:19:13 He's a bioinorganic chemist at Brandeis University.

00:19:17 And he brought the thing to us.

00:19:20 And then, so we started collaborating with him.

00:19:24 We got the tunicates.

00:19:26 These are fist-sized animals coated in black tunic.

00:19:32 That's why it's called tunicate.

00:19:34 And we brought them from, they were caught in Florida.

00:19:41 And then they are brought alive to our lab in lots of 1,000.

00:19:47 And the collectors would tell us which ship, on which plane it's been shipped onto.

00:19:58 And then we would go to La Guardia and then collect it, and then soon come back here.

00:20:03 That would be 8 o'clock.

00:20:05 And then about 10 of us would be prepared.

00:20:08 And then we'd do it all on a conveyor belt and process the whole thing that night,

00:20:15 ending about 1 o'clock.

00:20:18 You would already start the extraction process right there.

00:20:20 Yes, by sacrificing the animal and collecting the blood.

00:20:28 Now the problem here is when you have a tunicate and the blood,

00:20:34 when the blood starts bleeding in the air, it was noticed that the color already changes.

00:20:41 Even Socrates, thousands of years ago, had noticed this.

00:20:45 More serious scientific effort started in 1911 in the aquarium of Naples.

00:20:53 And since then, many places have been trying to isolate, identify this.

00:20:58 The main thing is because the tunicates are well known to accumulate vanadium or iron from the ocean.

00:21:09 And they concentrate it about a million or ten million fold in the blood.

00:21:16 And no one knows what the biochemical role of these vanadium trace elements are.

00:21:24 And it was being found recently that the humans also contain vanadium in the liver and the blood.

00:21:32 And no one knows what the biochemical role is.

00:21:36 And so I was very lucky because I had a marvelous German national product chemist.

00:21:48 His name is Reimer Bruning, and he's now an assistant professor in Hawaii.

00:21:53 And he was the leader of this whole thing.

00:21:58 And it took him four years, solid work, until we got the structure.

00:22:06 But because of this, during the four years, he had no other publication.

00:22:11 And the major, I think, breakthrough was several things.

00:22:17 For example, we found that the moment you slit the heart open and then collect the blood,

00:22:24 from there on, it had to be done under specially purified argon.

00:22:31 And plus that, although it's an aquatic animal, total exclusion of humidity.

00:22:42 And if you expose it to, I mean, except for the blood.

00:22:47 And so the blood cells were also crushed in a special way and then processed.

00:22:54 All very special.

00:22:56 And also we were lucky.

00:22:58 We had a new toy, isolation toy, which is called counter-current CPC.

00:23:08 That's a counter-current partition chromatography.

00:23:13 It was a new gadget which was made in Japan, and I luckily happened to have a prototype in my lab.

00:23:21 And so the blood was collected, processed, and in a glove box, etc., etc., even centrifuged under this argon.

00:23:32 And then put through the CPC, centrifugal partition chromatography.

00:23:38 And then finally purified.

00:23:41 And because if we derivatize it, we know it could be stabilized.

00:23:46 But then it can lose its activities.

00:23:49 And that's the way we managed to get the structure.

00:23:53 So you assayed it up until the very end and then you derivatized it and then did spectra?

00:23:57 Yes, exactly.

00:23:58 Another thing is it's an odd case because there was no known biochemical activity on this.

00:24:04 We only knew we had to follow a pigment contained in the blood which had a certain absorption at 320 and 280.

00:24:16 And it had to be very unstable.

00:24:18 Those are the two criteria.

00:24:21 And once we had that in a pure form, then of course we can derivatize it and build a structure.

00:24:30 Now, okay.

00:24:32 Now the original blood is, I mean, coming out of the heart is...

00:24:35 It's green.

00:24:36 It's green before it hits the air.

00:24:39 No, no, no. It's, well, greenish yellow.

00:24:43 Okay.

00:24:44 When it hits the air, it becomes brown, polymerizes, and I don't know.

00:24:48 I see.

00:24:49 It starts polymerizing and so on.

00:24:51 The structure of tunichrome turns out to be contains a pyrrhogorole and three pyrrhogoroles and so on.

00:24:58 And it's bound to be very air unstable.

00:25:01 Right.

00:25:02 Now, since, okay, we published the structure, then I really start to feel the following.

00:25:12 In this case in particular, before, once you isolate and publish, no, and deduce the structure,

00:25:21 it was regarded usually the end of that project and maybe if people wanted to synthesize this for confirming the structure, fine.

00:25:31 And some of these, of course, is a very intriguing target for total synthetic people.

00:25:38 Right.

00:25:39 We are also trying to synthesize this now.

00:25:41 But my attitude now is, okay, we got the structure.

00:25:47 Now it is only the beginning of the next step in the research.

00:25:53 And what is its function?

00:25:55 How does it complex with vanadium, iron, and so on?

00:26:03 And it turns out after we got the structure, now my current group in this field is five.

00:26:09 And one is doing synthesis.

00:26:12 One is in hematology.

00:26:15 One in biology.

00:26:17 Another one is inorganic chemist.

00:26:20 And also we were lucky to have a visiting professor.

00:26:26 He is a well-known, Simeon Polak, a hematologist from Albert Einstein visiting us.

00:26:31 And he was on our team, too.

00:26:33 In addition, we are working with the New England Aquarium as well as with Ken Kustin.

00:26:41 And it is totally interdisciplinary now.

00:26:44 And the amazing thing that we have found, and this is what it tells,

00:26:50 we have recently started to believe that the vanadium and the tunichrome exist in different cells.

00:27:00 They are not in the same cell.

00:27:02 You see the tunichrome blood cell, it has got several different cells, type of cells, blood cells.

00:27:12 And there is a way in which you can sort them out, separate them.

00:27:16 And then we found that at least most of the vanadium, most of the tunichrome, exists in different blood cells.

00:27:24 So, I mean, it's no wonder people, when they were trying to isolate this,

00:27:30 RIMA, of course, we didn't know this, but they were already grinding up the blood.

00:27:34 The moment you do that, you mix the vanadium, you mix the tunichrome,

00:27:39 and then they instantaneously form a complex, you see.

00:27:43 But now we are more careful, and we are starting to do experiments with the separated blood cells.

00:27:50 I see.

00:27:51 So this is a totally new area, and the only organic chemistry existing here is the synthesis we are trying to do.

00:27:58 The rest is, I don't know how to define it, so we are all groping.

00:28:03 Do you search for individuals to work in this field, or do they come to you and say,

00:28:08 this is a problem I would like to, I mean, people outside of organic chemistry?

00:28:14 For example, the hematology was done by a graduate student here,

00:28:21 and he just thought it was intriguing, so together with a visiting professor,

00:28:27 we started working the hematology, and we went to the medical school,

00:28:30 started sorting out the blood cells and so on.

00:28:33 Inorganic chemist, she is a transfer student from another inorganic group.

00:28:38 Happily, she happened to be working in Vanadium, so she was just right for that.

00:28:44 And so we are doing, well, magnetic susceptibility measurements,

00:28:49 and X-ray of some complexes, hematology, and also some ecology.

00:28:56 And, for example, we go to Woods Hole, collected the same species of tunicate,

00:29:03 and found that in one species existing on the northern side of the bay,

00:29:08 that doesn't contain tunicron, those existing on the southern part of the bay contains it.

00:29:14 So we transfer these and find that the ones which come from the south do not make tunicron.

00:29:20 We don't know what this all is.

00:29:22 So it's getting very interdisciplinary, branching out,

00:29:27 but eventually we want to find out what is it for.

00:29:32 And what is Vanadium doing?

00:29:35 So the tunicron structure determination is really only the first step.

00:29:43 But because we have the structure, now we know a little bit more in concrete terms what we are after.

00:29:51 If we didn't know the tunicron structure, of course, we would be totally lost.

00:29:55 So I think it's very important to have a structure that we can discuss and start from that,

00:30:03 and then go into biochemical problems.

00:30:06 And that's, I think, where we can contribute in science, people like ourselves.

00:30:12 Because I'm convinced that it is organic chemists who have the clearest view

00:30:20 on what molecular structures are, confirmation, configuration, and so on, you see.

00:30:26 But we don't know where the exciting problems are.

00:30:30 And on the other hand, biochemists may have exciting problems,

00:30:34 biologists certainly, of course, but they don't even realize that this is an organic problem.

00:30:40 So that's where the interaction comes.

00:30:44 And then biologists, I'm sure that any biologist would have an exciting project

00:30:49 that can be tackled by organic chemists.

00:30:53 This, however, has only become feasible during the past two, three years.

00:30:59 Like this tunicron mode of action studies, before that, it would have been totally impossible, I think.

00:31:05 But now we can start speaking about, if you have an active compound, how does it interact, you see.

00:31:12 I think that, in simple terms, the mode of action can be said, in most cases, in one sentence.

00:31:22 How does a small molecule interact with its receptor?

00:31:27 That's what it boils down to, I think.

00:31:30 And you're certainly advancing in that area a great deal more.

00:31:34 I know another recent project in which you've done work is with mitomycin G, I guess, C, sorry,

00:31:48 an anti-tumor drug, where you've done work similar to work that you had done earlier with benzopyrene.

00:31:56 And I think that needs a little bit of exposition as well.

00:32:01 Yeah, this was done with Maria Tomas.

00:32:04 And she's a professor in the chemistry department at Hunter College.

00:32:08 And she was a Gilbert Stork graduate student.

00:32:12 So she was a synthetic chemist, but now she's become a biochemist.

00:32:16 And she is the foremost, I think, organic chemist working in the field of mitomycin.

00:32:25 And mitomycin is a popular clinical anti-tumor drug.

00:32:33 And, again, it was not known how, why is it cytotoxic.

00:32:40 And together with Maria Tomas and using some specially developed spectroscopic techniques,

00:32:52 incidentally, this was done mostly on our side by Greg Verdine, who is an excellent graduate student.

00:33:00 And we managed to fish out the most important compound was mitomycin bridging across the two DNA strands,

00:33:15 the cross-linking so-called, besides many other things, many other adducts.

00:33:20 What specific spectroscopic techniques are you using for this?

00:33:24 Well, one is, we call it second derivative difference, FTIR, and second derivative difference, UV.

00:33:39 What it amounts to is second derivatization of curves has been a well-known technique.

00:33:46 But, strangely enough, it has not been used by the organic chemist.

00:33:52 And if you take an infrared band and do a second derivative, a broad band will sharpen.

00:34:01 Not only that, a tiny shoulder will appear as a nice sharp peak, so that you get more characteristic frequencies out of this.

00:34:11 And so what we did in this case of an adduct was, suppose this is the structure.

00:34:17 This is a mitomycin moiety and this is the DNA moiety, the base.

00:34:23 And the DNA base can be attached to this mitomycin moiety through this finger, this finger, this finger, this finger.

00:34:30 Now, when you're dealing with microgram quantities, it's very difficult to do this by NMR.

00:34:37 Which finger is this attached to?

00:34:39 In particular, in the nucleic acid basis, it's impossible because there are not enough protons.

00:34:46 There's only one proton on this set.

00:34:48 So, what we did was, and it's the same in UV and in IR, the difference, the second derivative difference technique is the following.

00:34:59 We take the infrared or UV of this whole adduct, and from this, we subtract this one.

00:35:07 So, we're left with a different spectrum, which is this thing.

00:35:11 And then we compare the residual spectrum with this nucleic acid base.

00:35:18 In this case, it turns out to be deoxyguanosine, which is methylated here, methylated here, methylated here.

00:35:25 I see.

00:35:26 And see which it resembles.

00:35:27 And there's no question it resembles, in this case, the two amino groups.

00:35:32 We got the same consistent results, both from infrared and UV.

00:35:38 And it's a micro technique, of course.

00:35:41 And I think that was the main tool.

00:35:44 But it had to be developed again because it was so challenging.

00:35:48 And it forced us to develop this method.

00:35:52 And we are now going to use this for some other things I will mention later.

00:35:57 Now, anyhow, we managed to fish out this cross-linked DNA adduct.

00:36:04 And with Maria, also, we have come out with a logical mechanism.

00:36:12 And now we're trying to prove this mechanism and see what is the real mode of action.

00:36:19 But again, step one, we got the structure.

00:36:22 And it's the same in the benzylpyrin DNA adduct case, also.

00:36:26 Okay.

00:36:29 I won't go into detail, but I think this difference FTIR technique, we are using a lot, in particular, in proteins.

00:36:41 And I think there's a tremendous future.

00:36:45 And I will just mention verbally, there's a paper coming out in JACS, which we did with the late Laura Eisenstein.

00:36:55 I don't know. Did I tell you about her?

00:36:57 She was from the University of Illinois.

00:36:59 We talked about her at one time.

00:37:01 Yes.

00:37:02 Well, this is a technique started by her group.

00:37:08 And I was collaborating with her.

00:37:10 And then she had this tragic death.

00:37:14 And since then, NIH has been generous enough to continue to support this.

00:37:20 And since her death, I think we have about six, seven papers, which have been published or coming out.

00:37:30 And I think it's got a tremendous future, because the paper which is coming out now leads to the following conclusion.

00:37:43 We did it on bacterial rhodopsin, which has a molecular weight of about 29,000.

00:37:49 And we can follow the deprotonation of one amino acid residue.

00:37:55 In this case, tyrosine, or glutamic acid, or aspartic acid, you see.

00:38:00 And this technique has been, it can be done in several, a few other places in this country.

00:38:09 At Ken Rothschild at Boston, and there's a group in Germany which does this type of thing as well.

00:38:16 But these are physical chemists.

00:38:18 And we are now trying to use the same technique to follow organic reactions, time-wise.

00:38:27 For example, I don't know whether this is going to work, but we're trying to do this now.

00:38:32 Mitomycin, for example, you see.

00:38:35 We come up with a reaction mechanism sequence.

00:38:40 And then we're going to mix that with DNA.

00:38:45 Or to put it more simply, if you have an enzyme, and you have a substrate,

00:38:51 and you want to follow how the substrate changes.

00:38:55 So we put it in an infrared cell, and then because it's Fourier transformed,

00:39:01 you can measure, I mean, a pulse, three pulses every second or so.

00:39:08 So you accumulate enough, and say five seconds later, you take another infrared.

00:39:13 Five seconds later, another infrared.

00:39:16 And then you just take the difference, difference, difference.

00:39:19 And the nice thing about this is the big molecule, which does not change its infrared, is nullified.

00:39:26 Only the changes appear.

00:39:28 So I think it's going to be a potentially extremely powerful method of following enzymatic reactions.

00:39:35 Or you can say about this squalene cyclization, for example.

00:39:40 Is that concerted or stepwise?

00:39:43 And maybe, at least theoretically, it's quite possible to follow those kind of things.

00:39:48 And that's what we're trying to do now.

00:39:50 Yeah, as long as you get the right time frame.

00:39:58 Would you like some coffee?

00:40:11 Four, three.

00:40:17 Now for the big, sort of big general questions.

00:40:25 First, what are the important problems for you right now?

00:40:29 We've talked about the kinds of work you're doing, the isolations, the, where are your groups going?

00:40:37 You have a big group in vision.

00:40:40 You've got people working on the tunichromes.

00:40:43 What other kinds of things are you involved in at this particular point?

00:40:53 I think, although I'm not a, most, many of our problems are getting involved more and more with proteins, I think.

00:41:04 And I don't want to use big words, but the rhodopsin also, it's the interaction between a receptor protein and a small molecule, retinal, you see.

00:41:16 And there are a couple of works we are doing at the Suntory Institute, which is also, again, interaction between small molecules and big receptor molecules.

00:41:28 So that's one big area we are moving into, whether we like it or not.

00:41:37 And we're trying to do this by trying to understand it, clarify the things on a microscopic structural basis.

00:41:47 And another thing is totally unrelated, but a micro-scale determination of glucosidic linkages of oligosaccharides.

00:42:01 And that I'm facing quite a bit of emphasis.

00:42:08 And this, shall I just?

00:42:12 Sure.

00:42:14 You see, this is at least our goal.

00:42:18 You know, the molecular biology has advanced dramatically in recent years because micro-analytical methods for determining the structures of nucleic acids and proteins have been worked out routinely, routine methods.

00:42:35 But this is not necessarily the case in oligosaccharides or polysaccharides.

00:42:40 And this is the last nasty area which is left.

00:42:44 And it's becoming more and more important because of cell walls and then glycoproteins in general, again, related to proteins.

00:42:54 And the methods which have been used is more or less classical.

00:42:59 Not only that, one had to have reference sugars.

00:43:04 And we, the past five years or four years, we've been concentrating, focusing our attention to develop a micro-method which is more or less totally based on circled dichroism, the coupled oscillator method.

00:43:20 And trying to develop a micro-scale structure determination for determining the glucosidic linkages of oligosaccharides on a scale that one cannot use NMR.

00:43:34 And I can say that we have finished clarifying the ground rules, principles, which are operating.

00:43:45 And during this process we have found some, I think, very interesting, some interesting results which I'll mention in a moment.

00:43:54 And now we have, we are going into the practical application stage.

00:43:58 We have started to work on gangliosides and so on.

00:44:01 We want to come out with a simple method, as simple as possible, and general so that we don't have to have reference compounds.

00:44:14 Just some physical constants, which is in this case circled dichroism, which you can read from a table or look at the spectrum.

00:44:24 And one important principle that we have found is that the additivity relation.

00:44:35 You have many series of complex compounds in a compound.

00:44:40 It has many chromophores and it will give rise to a very complex circled dichroism curve.

00:44:46 And we have found and proven that these can be dissected into pairwise interaction.

00:44:57 And then the whole CD curve can be reconstructed from this pairwise interaction.

00:45:04 And this has been a principle, for example, advocated by Kaufman many years ago.

00:45:11 But this was in the D-line era.

00:45:14 And it was almost impossible to prove or disprove at that age, at that time.

00:45:20 And we have shown that you can do it, pairwise addition, additivity relation holds in all CD curves, I think.

00:45:28 But that's the basis.

00:45:30 Anyhow, it's getting less and less so-called organic chemistry.

00:45:39 But the basic techniques, of course, we are operating as an organic chemist group.

00:45:45 Working, trying to find out how mode of action mostly are of these life science related factors.

00:45:58 And the real structure determination, per se, of a small molecule has usually become quite routine.

00:46:06 And that is not the challenging thing.

00:46:08 The challenging factor is once you determine a structure, how does it react?

00:46:14 Of course, there is also a challenge at the other end for you, at least, and that's the isolation.

00:46:20 That's never become sort of generalized.

00:46:22 That's another thing.

00:46:24 I mean, people tend to regard isolation as it's not my business and so on.

00:46:31 And even if it's a painstaking isolation which has taken us three, four years, there are two things.

00:46:40 One is many factors which make isolation difficult.

00:46:44 One of the major factors is an assay.

00:46:47 When you're dealing with these life science related factors, it means that these factors have to have a unique assay.

00:47:00 It's not general screening, the factors we are looking after.

00:47:04 And many times, this assay is the bottleneck.

00:47:11 The next one is the stability and instability.

00:47:14 The next one, also a big important factor, is the availability of starting material.

00:47:19 And I know this is too much, maybe exaggerating, but unless it presents a challenging problem in isolation,

00:47:31 the problem itself may not be that exciting.

00:47:37 I mean, it's a little bit extreme, but that's the way I feel.

00:47:49 Where do you think organic chemistry, and in particular natural products chemistry, is headed?

00:47:57 Where do you think we'll be going in the next ten years?

00:48:02 Well, I really think it should go, and it is heading towards clarifying these exciting problems which lie in the interdisciplinary area.

00:48:14 But we have to maintain ourselves as organic chemists and not to get lost.

00:48:19 There's no sense in organic chemists becoming biologists.

00:48:23 We have to operate and stay as an organic chemist, but work on these interdisciplinary problems using our techniques.

00:48:31 And in that sense, it's only the organic chemists who can do this structural elucidation of interactions between small molecules and macromolecules on a molecular structure basis.

00:48:51 One thing that occurred to me while you were talking was that there could be a fear that we would become a service organization for biologists and biochemists.

00:49:04 Well, I think if the organic chemist is confident...

00:49:09 I mean, I admit it, you can call yourself technicians, but I don't mind being called technician myself.

00:49:18 So far as you're not told by the other people what to do, then you're a technician.

00:49:23 But if you know what you're going to do, and they do not know, and you do it on your initiative, which is always the case, then I don't care what it is.

00:49:32 You've mentioned Centauri Institute a couple of times, and I know in 1979 you became a director at the Centauri Institute.

00:49:43 Maybe you could tell me a little bit about the Institute and its mode of action and the kinds of things they're interested in.

00:49:49 The proper name is Centauri Institute for Bio-Organic Research, and it is almost totally supported by Centauri Company, the brewery whisky-making company.

00:50:09 The president is Saji, he's an organic chemist.

00:50:14 He wanted to become a professor in a university, at Osaka University, but unfortunately his elder brother died, so he had to succeed his family business.

00:50:25 So he has a nostalgia for doing basic research.

00:50:30 Then right after the war, in 1947, I think, he set up the so-called Institute for Food Science.

00:50:37 This is when the food situation in Japan was very poor.

00:50:41 Then it went on until several directors and so on.

00:50:47 Then when the previous director was about to retire, my high school friend who was a member of the board, Seno, came to me and asked whether I'd be interested in becoming a director.

00:50:59 I said, well, I'd like to give it a try.

00:51:04 At that time, President Saji's understanding, they put in a tremendous amount of money into this institute and built it to what it is now, which I'll explain to you in a moment.

00:51:17 First of all, they increased the faculty, I mean the staff members, quite a bit.

00:51:30 Currently, it is roughly about 30 permanent staff.

00:51:36 In addition to that, I started this post-doc system.

00:51:43 I will mention that in a moment.

00:51:45 Then we bought many advanced equipment.

00:51:52 I think it is now one of the best equipped institutes for the type of work I'm doing in the world.

00:52:02 It's a small institute, only a total of 36 or so.

00:52:06 We have this 500 MHz, 360 MHz, 300 MHz, and the 100 MHz instrument.

00:52:16 Not only do we have it, but they are all maintained in top condition.

00:52:21 That's the big difference.

00:52:23 There are many places that have the instrument, but it's not running.

00:52:26 All of these instruments, not only that, the mass spec, FTIR, CD, and then many other things.

00:52:32 Plus that, we have no restriction in what topics to work on.

00:52:39 Before I go into the topics, let me just briefly explain about this post-doc system.

00:52:46 Where is the home of the institute now?

00:52:48 It's between Osaka and Kyoto, a little bit south of Kyoto.

00:52:54 The post-doc system.

00:52:59 I have become very sarcastic against the Japanese government.

00:53:05 I even recently wrote in an editorial that the Japanese Ministry of Education should withdraw itself from all universities.

00:53:19 We should set up a new ministry.

00:53:22 The reason is, I don't think there is a single PhD in the whole Ministry of Education.

00:53:28 Yet, they are in control from kindergarten all the way up to graduate schools.

00:53:33 There is no way that these people, conceptually or knowledge-wise, can compete with the ideas coming out from the scientists at NIH, NSF, all the research councils, and so on.

00:53:51 For years, the Japanese professors have been screaming, we want a post-doc system, we want a post-doc system.

00:53:59 There is no way they can understand this, because none of them know what research is.

00:54:05 The advantage of having a post-doc is, of course, for the people in this country or Europe, it's common knowledge.

00:54:13 If you want to go into a new field, you can get a post-doc from that group, and then you can immediately go into a new field.

00:54:20 But in Japan, it's not so.

00:54:22 The professor or the assistant professor or the assistant has to start that field themselves.

00:54:28 So, the starting base is quite different.

00:54:31 Anyhow, it's good for mixing blood, and makes it more fluid, dynamic.

00:54:37 The Japanese tend to be on a small island, they're all solidified, they're all glued to their own places.

00:54:42 I mean, it's a horrible situation.

00:54:46 So, the president had a good understanding, and this is the advantage of not being government.

00:54:53 I don't like bureaucrats at all.

00:54:56 So, he let us do whatever we wanted.

00:55:00 Fortunately, we have extremely good post-docs.

00:55:05 And from this country, I can mention, we've had from Harvard, Yale, Columbia, UCLA, Caltech, Kansas, all these places.

00:55:16 And we've also, from mainland China, Korea, and European countries.

00:55:24 And it's been quite successful.

00:55:27 And, of course, we had lots of difficulty, just to give you some idea, to tell you about the mentality of the local Japanese police.

00:55:35 And when we had a poll, and the mainland Chinese, the police would come once in a while and check, are they okay?

00:55:45 Unfortunately, right in front of the institute is a fire station.

00:55:49 There's no fire because it's a very peaceful residential area, so they're bored, they don't know what to do.

00:55:55 So, they come once in a while across the street to check what's going on at our institute, and are those people from eastern or the Chinese okay?

00:56:05 Then, my deputy director, who is a woman, Yoko Naira, she asked, why do you keep on checking those people?

00:56:13 It has become very unpleasant.

00:56:15 They said, well, once they want to emigrate or leave the country, we are here to protect them.

00:56:27 That's the excuse they made.

00:56:29 But, anyhow, Japan is still, it's undeveloped, I would say.

00:56:36 It's not even developing in that sense.

00:56:38 Anyhow, I should calm down now.

00:56:42 The postdoc system is going fine.

00:56:45 How many postdocs will you have in a given year?

00:56:48 Six.

00:56:49 Six postdocs.

00:56:50 So, it's a very competitive kind of...

00:56:52 Well, there's some waiting list.

00:56:54 I see.

00:56:55 Yes.

00:56:56 It's lucky.

00:56:58 But, you see, the postdocs come.

00:57:00 I mean, in Europe and this country, it's fine.

00:57:03 But, when they come to a country in which there has been no formal postdoc system, it's difficult for the receiving side also.

00:57:14 Most of the staff members are postdocs.

00:57:17 They have postdoc experience.

00:57:18 But, it's different when you are on the receiving side.

00:57:22 It took some time for me to clarify the situation in terms to make it clear that the postdocs come to do their postdoc as a stepping stone for the future, which is quite a different situation in Japan.

00:57:42 You don't worry too much about your job because the professors usually arrange it.

00:57:48 But, in this country and European countries, when you're interviewed for a job, you have to describe what you did for your research and so on in the postdoctoral years as well as your doctorate.

00:58:02 So, it's quite different.

00:58:04 Anyhow, the topics, there's no restriction.

00:58:10 So, if I mention it in one word, we're trying to do topics, research topics, which cannot be handled by a single group at the university.

00:58:25 But, we have to be very selective because, after all, the total membership is only 36.

00:58:33 And, about 10 or 12 of these are senior people.

00:58:38 So, they are all associate professor level people, you see.

00:58:42 And, they only have one assistant or they work together with one postdoc.

00:58:47 So, we have, I call this the pair system.

00:58:51 That's the way I started, that pair A and pair B and pair C, they could collaborate and overlapping where they can.

00:59:01 And then, thus, maintain some fluidity in the collaboration within the institute.

00:59:13 So, there's a whole series of different interests.

00:59:16 And, you try to get to a lot of people.

00:59:19 Yes.

00:59:20 But, now, it's eight years now and it's somewhat changing.

00:59:25 And, we start and get some structures, three or four big groups within.

00:59:31 But, still, the basic unit is senior, junior or a permanent staff and a postdoc.

00:59:38 Postdocs, of course, we do not consider as juniors.

00:59:40 These are colleagues.

00:59:43 Also, recently, we have employed on a permanent staff an Irish, which is, again, not so usual in Japan.

00:59:51 Yes.

00:59:52 He came as a postdoc and he fitted in nicely.

00:59:56 So, we have employed him as a permanent.

00:59:59 And, he's in charge and handles our mass spectroscopy.