00:01:03 OK.

00:01:05 Stand up.

00:01:07 5, 4, 3.

00:01:12 Another more recent piece of work which also presented a real challenge in isolation was the work on Tunichrome B, which is a reducing blood pigment of the sea squirt.

00:01:29 How did you get into it and what was the real challenge there?

00:01:36 This is exposing us to a new experience.

00:01:41 It was brought to our attention in collaboration with Ken Kustin, who is a professor in inorganic chemistry.

00:01:47 He's a bioinorganic chemist at Brandeis University.

00:01:52 He brought the thing to us, so we started collaborating with him.

00:01:58 We got the tunicates. These are fist-sized animals coated in black tunic.

00:02:06 That's why it's called tunicate.

00:02:08 We brought them from Florida.

00:02:14 They were brought alive to our lab in lots of 1000.

00:02:22 The collectors would tell us which ship, on which plane it's been shipped onto.

00:02:32 Then we would go to La Guardia and then collect it and then soon come back here.

00:02:38 That would be 8 o'clock.

00:02:40 Then about 10 of us would be prepared.

00:02:43 Then we'd do it all on a conveyor belt and process the whole thing that night, ending about 1 o'clock.

00:02:52 You would already start the extraction process right there.

00:02:55 Yes, by sacrificing the animal and collecting the blood.

00:03:02 The problem here is when you have a tunicate and the blood starts bleeding in the air,

00:03:11 it is noticed that the color already changes.

00:03:15 Even Socrates, thousands of years ago, had noticed this.

00:03:20 A more serious scientific effort started in 1911 in the aquarium of Naples.

00:03:28 Since then, many places have been trying to isolate and identify this.

00:03:33 The main thing is because the tunicates are well known to accumulate vanadium or iron from the ocean.

00:03:43 They concentrate it about a million or ten million fold in the blood.

00:03:51 No one knows what the biochemical role of these vanadium trace elements are.

00:03:59 It has been found recently that the humans also contain vanadium in the liver and the blood.

00:04:07 No one knows what the biochemical role is.

00:04:11 I was very lucky because I had a marvelous German natural product chemist.

00:04:22 His name is Reimer Bruning and he is now an assistant professor in Hawaii.

00:04:27 He was the leader of this whole thing.

00:04:33 It took him four years of solid work until we got the structure.

00:04:40 But because of this, during the four years, he had no other publication.

00:04:46 The major breakthrough was several things.

00:04:51 For example, we found that the moment you slit the heart open and then collect the blood,

00:04:59 from there on, it had to be done under specially purified argon.

00:05:05 Plus, although it is an aquatic animal, total exclusion of humidity.

00:05:15 If you expose it to, I mean, except for the blood.

00:05:22 So the blood cells were also crushed in a special way and then processed.

00:05:29 All very special.

00:05:31 Also, we were lucky.

00:05:33 We had a new toy, isolation toy, which is called counter-current CPC.

00:05:43 That's a counter-current partition chromatography.

00:05:48 It's a new gadget, which is made in Japan.

00:05:52 I luckily happened to have a prototype in my lab.

00:05:56 So the blood was collected, processed, and in a glove box, etc., etc.,

00:06:03 even centrifuged under this argon,

00:06:07 and then put through this CPC, centrifugal partition chromatography,

00:06:13 and then finally purified.

00:06:16 Because if we derivatize it, we know it could be stabilized,

00:06:21 but then it will lose its activity.

00:06:23 That's the way we managed to get the structure.

00:06:27 So you oxidized it up until the very end, and then you derivatized it?

00:06:31 Yes, exactly.

00:06:33 Another thing is it's an odd case because there was no known biochemical activity of this.

00:06:39 We only knew we had to follow a pigment contained in the blood,

00:06:45 which had a certain absorption at 320 and 280, and it had to be very unstable.

00:06:53 Those are the two criteria.

00:06:56 Once we had that in a pure form, then of course we can derivatize it and go on the structure.

00:07:05 Now, okay.

00:07:07 The original blood is, I mean, coming out of the heart is...

00:07:10 It's green.

00:07:12 It's green before it hits the air.

00:07:14 No, no, no. It's, well, greenish-yellow.

00:07:18 Okay.

00:07:19 When it hits the air, it becomes brown, polymerizes, and I don't know,

00:07:23 it starts polymerizing and so on.

00:07:26 The structure of tunichrome turns out to contain a pyrogyl and three pyrogyls and so on,

00:07:33 and it's bound to be very air-unstable.

00:07:37 Now, since, okay, we publish the structure, then I really start to feel the following.

00:07:46 In this case in particular, before, once you isolate and publish, no, and deduce the structure,

00:07:56 it was regarded usually the end of that project,

00:08:00 and maybe if people wanted to synthesize this for confirming the structure, fine.

00:08:06 And some of these, of course, is a very intriguing target for total synthetic people.

00:08:14 We are also trying to synthesize this now.

00:08:16 But my attitude now is, okay, we got the structure.

00:08:22 Now it is only the beginning of the next step in the research, and what is its function?

00:08:30 How does it complex with vanadium, iron, and so on?

00:08:37 And it turns out, after we got the structure, now my current group in this field is five.

00:08:45 One is doing synthesis.

00:08:48 One is in hematology.

00:08:50 One in biology.

00:08:52 Another one is inorganic chemist.

00:08:55 And also we were lucky to have a visiting professor.

00:09:00 He's a well-known, Simeon Pollack, a hematologist from Albert Einstein visiting us,

00:09:05 and he was on our team too.

00:09:07 In addition, we are working with the New England Aquarium, as well as with Ken Kustin,

00:09:15 and it's totally interdisciplinary now.

00:09:18 And the amazing thing that we have found, and this is what it tells,

00:09:24 we have recently started to believe that the vanadium and the tunichrome exist in different cells.

00:09:34 They are not in the same cell.

00:09:36 You see, the tunichrome blood cell, it's got several different cells, type of cells, blood cells,

00:09:46 and there is a way in which you can sort them out, separate them.

00:09:50 And then we found that at least most of the vanadium, most of the tunichrome, exists in different blood cells.

00:09:58 So, I mean, it's no wonder people, when they were trying to isolate this,

00:10:05 Lyma, of course, we didn't know this, but they were already grinding up the blood.

00:10:09 The moment you do that, you mix the vanadium, you mix the tunichrome,

00:10:14 and then they instantaneously form a complex, you see.

00:10:18 But now we are more careful, and we're starting to do experiments with the separated blood cells.

00:10:25 So this is a totally new area, and the only organic chemistry existing here is the synthesis we're trying to do.

00:10:33 I don't know what to define it, so we're all groping.

00:10:38 Do you search for individuals to work in this field, or do they come to you and say,

00:10:43 this is a problem I would like to...

00:10:45 Well, when someone joins my group, you see, for example, the hematology was done by a graduate student here.

00:10:57 He just thought it was intriguing, so together with a visiting professor, we started working with hematology.

00:11:04 We went to the medical school, started sorting out the blood cells and so on.

00:11:08 Inorganic chemist, she is a transfer student from another inorganic group.

00:11:13 Happily, she happened to be working with vanadium, so she's just right for that.

00:11:19 And so we're doing magnetic susceptibility measurements, and x-ray of some complexes, hematology, and also some ecology.

00:11:31 For example, we go to Woods Hole, collected the same species of tunicate,

00:11:38 and found that in one species existing on the northern side of the bay that doesn't contain tunicron,

00:11:45 those existing on the southern part of the bay contains it.

00:11:49 So we transfer these and find that the ones which come from the south do not make tunicron.

00:11:55 We don't know what this all is, so it's getting very interdisciplinary, branching out,

00:12:02 but eventually we want to find out what is at war, and what is vanadium doing it.

00:12:11 So the tunicron structure determination is really only the first step.

00:12:17 But because we have the structure, now we know a little bit more in concrete terms what we are after.

00:12:25 If we didn't know the tunicron structure, of course we would be totally lost.

00:12:29 So I think it's very important to have a structure that we can discuss and start from that,

00:12:38 and then go into biochemical problems.

00:12:41 And that's, I think, where we can contribute in science, people like ourselves.

00:12:47 Because I'm convinced that it is organic chemists who have the clearest view on what molecular structures are,

00:12:57 conformation, configuration, and so on, you see.

00:13:01 But we don't know where the exciting problems are,

00:13:05 and on the other hand, biochemists may have exciting problems, biologists certainly, of course,

00:13:11 but they don't even realize that this is an organic problem.

00:13:15 So that's where the interaction comes.

00:13:19 And then biologists, I'm sure that any biologist would have an exciting project that can be tackled by organic chemists.

00:13:28 This, however, has only become feasible during the past two, three years.

00:13:34 Like this tunicron mode of action studies, before that, it would have been totally impossible, I think.

00:13:40 But now we can start speaking about, if you have an active compound, how does it interact, you see.

00:13:46 I think that, in simple terms, the mode of action can be said, in most cases, in one sentence.

00:13:57 How does a small molecule interact with its receptor?

00:14:02 That's what the body's done, I think.

00:14:05 And you're certainly advancing in that area a great deal more.

00:14:09 I know another recent project in which you've done work is with mitomycin G, I guess.

00:14:21 C, sorry.

00:14:23 An anti-tumor drug, where you've done work similar to work that you had done earlier with benzopyrene.

00:14:31 I think that needs a little bit of exposition as well.

00:14:35 Yeah, this was done with Maria Tomas.

00:14:39 She's a professor in the chemistry department at Hunter College.

00:14:43 She was a Gilbert Stork graduate student, so she was a synthetic chemist, but now she's become a biochemist.

00:14:50 She is the foremost, I think, organic chemist working in the field of mitomycin.

00:15:00 Mitomycin is a popular clinical anti-tumor drug.

00:15:08 Again, it was not known why it's cytotoxic.

00:15:15 Together with Maria Tomas and using some specially developed spectroscopic techniques,

00:15:27 incidentally, this was done mostly on our side by Greg Verdine, who is an excellent graduate student.

00:15:36 And we managed to fish out the most important compound was mitomycin bridging across the two DNA strands,

00:15:50 the cross-linking so-called, besides many other things, many other adducts.

00:15:55 What specific spectroscopic techniques are you using for this?

00:15:59 Well, one is, we call it second derivative difference, FTIR, and second derivative difference, UV.

00:16:14 What it amounts to is second derivatization of curves has been a well-known technique,

00:16:21 but strangely enough, it has not been used by the organic chemists.

00:16:27 If you take an infrared band and do a second derivative, a broad band will sharpen,

00:16:36 not only that, a tiny shoulder will appear as a nice sharp peak,

00:16:40 so that you get more characteristic frequencies out of this.

00:16:45 And so what we did in this case of an adduct was, suppose this is the structure.

00:16:52 This is a mitomycin moiety and this is the DNA moiety, the base.

00:16:57 And the DNA base can be attached to this mitomycin moiety through this finger, this finger, this finger, this finger.

00:17:05 Now, when you're dealing with microgram quantities, it's very difficult to do this by NMR.

00:17:11 Which finger is this attached to?

00:17:13 In particular, in the nucleic acid basis, it's impossible because there are not enough protons.

00:17:20 There's only one proton on this set.

00:17:23 So what we did was, and it's the same in UV and in IR, the difference,

00:17:30 the second derivative difference technique is the following.

00:17:34 We take the infrared or UV of this whole adduct, and from this, we subtract this one.

00:17:42 So we're left with a different spectrum, which is this thing.

00:17:46 And then we compare the residual spectrum with this nucleic acid base.

00:17:53 In this case, it turns out to be deoxyguanosine, which is methylated here, methylated here, methylated here,

00:18:00 and see which it resembles.

00:18:02 And there's no question it resembles, in this case, the two amino groups.

00:18:07 We got to the same consistent results, both from infrared and UV.

00:18:13 And it's a microtechnique, of course, and I think that was the main tool.

00:18:19 But it had to be developed, again, because it was so challenging.

00:18:23 And it forced us to develop this method.

00:18:27 And we are now going to use this for some other things I will mention later.

00:18:32 Now, anyhow, we managed to fish out this cross-linked DNA adduct.

00:18:39 And with Maria, also, we have come out with a logical mechanism.

00:18:46 And now we're trying to prove this mechanism and see what is the real mode of action.

00:18:54 But again, step one, we got the structure.

00:18:56 The same in the benzopyrene DNA adduct case, also.

00:19:06 I won't go into detail, but I think this difference FTIR technique,

00:19:12 we are using a lot, in particular, in proteins now.

00:19:17 And I think there's a tremendous future.

00:19:20 And I will just mention, verbally, there's a paper coming out in JACS,

00:19:25 which we did with the late Laura Eisenstein.

00:19:29 I don't know. Did I tell you about her? Probably not.

00:19:32 She was in the University of Illinois. I think we talked about her one time.

00:19:36 Yes. Well, this is a technique started by her group.

00:19:42 And I was collaborating with her.

00:19:45 And then she had this tragic death.

00:19:48 And since then, NIH has been generous enough to continue to support this.

00:19:54 And since her death, I think we have about six, seven papers,

00:20:01 which have been published or coming out.

00:20:04 And the most, I think, it's got a tremendous future

00:20:09 because the paper which is coming out now leads to the following conclusion.

00:20:17 We did it on bacteriorhodopsin, which has a molecular weight of about 29,000.

00:20:22 And we can follow the protonation, deprotonation of one amino acid residue,

00:20:29 in this case tyrosine or glutamic acid or aspartic acid, you see.

00:20:34 And this technique has been, it can be done in several,

00:20:41 a few other places in this country, at Ken Rothschild at Boston.

00:20:46 And there's a group in Germany which does this type of thing as well.

00:20:51 But these are physical chemists.

00:20:53 And we are now trying to use this same technique to follow organic reactions time-wise.

00:21:01 For example, I don't know whether this is going to work, but we're trying to do this now.

00:21:07 Mitomycin, for example, you see.

00:21:10 We come up with a reaction mechanism sequence.

00:21:15 And then we're going to mix that with DNA.

00:21:19 Or to put it more simply, if you have an enzyme and you have a substrate

00:21:26 and you want to follow how the substrate changes,

00:21:29 so we put it in an infrared cell and then because it's Fourier transformed,

00:21:36 you can measure, I mean, a pulse, three pulses every second or so.

00:21:42 So you accumulate enough and say five seconds later you take another infrared.

00:21:48 Five minutes, five seconds later, another infrared.

00:21:51 And then you just take the difference, difference, difference.

00:21:54 And the nice thing about this is the big molecule which does not change its infrared is nullified.

00:22:01 Only the changes appear.

00:22:03 So I think it's going to be a potentially extremely powerful method of following enzymatic reactions.

00:22:10 Or you can say about this squalene cyclization, for example.

00:22:15 Is that concerted or stepwise?

00:22:18 And maybe, at least theoretically, it's quite possible to follow these kind of things.

00:22:23 And that's what we're trying to do now.

00:22:25 Yeah, as long as you get the right time frame.