Scientist Stories: Morten Meldal, Inventing Click Chemistry

Morten Meldal, he is a native of Denmark and was born in Copenhagen in 1954. In 1986, he was awarded a PhD from DTU, the Technical University of Denmark, located in Lyngby, just a bit north of Copenhagen, and he's now active as professor at the University of Copenhagen. So by that, please join me in welcoming Dr. Meldal up on stage.

Dear colleagues, friends. Sorry. Dear colleagues, friends, ladies and gentlemen, I ride a leap from shoulders of giants, and I would like to start in reverse order of the talk we just heard by thanking all the giants that are behind the work that I'm going to talk about today. So my heroes, some of them, Klaus Bock here in the audience, together with Wüthrich on NMR structural studies on carbohydrates and peptides. Husken and Woodward and other people that are doing cycloaddition reactions. Prigogine, Buck, and Gray on quantum chemistry. Paulson, Lemieux, and I think that we should also mention Sheppard and Merrifield on the peptides and carbohydrates, and then Furka and Lam and many more on combinatorial chemistry. These are really the people that we built on in our research. So before I dive into the lecture, I would like to thank also the Nobel Foundation and the Nobel Prize Committee of Chemistry, as well as the Royal Swedish Academy of Sciences for this absolutely unique event. And for— I'm very appreciative and very thankful for being able to stand here today and very proud to share this moment with Carolyn Bertozzi and Barry Sharpless. I also thank all the colleagues at Carlsberg Laboratory and University of Copenhagen for all the things that they have done during the years in development, discovery, development, and application of this click chemistry. And finally, I need to, I want to express my thanks and love for my family who have supported me over the many years of science., and in particular my wife, Fitra Santillan, my son, Adjani, who is here today and is celebrating his birthday, my daughter, Anna, and Anna's mother, which is not here today, Sandra de Vaulieu. Finally, I would like to express the thanks to my late parents, who would be so proud to be here. And my perception of nature, and in particular the underlying chemistry, is a testimony to me of the absolute beauty and the complexity of the nature and our very existence. So nature has been with me since early childhood, and I'm very grateful to my parents for dragging me through forests and making collections of rocks, butterflies, insects, and so on, so that they actually spurred my interest for science. And later in life, I chose science as a subject for my education. And at a certain point, I had to choose between chemistry, physics, and computational sciences, and I chose, fortunately, chemistry, Because today, to me, chemistry is everything. There's nothing around us that is not chemistry. You are chemistry, I am chemistry, all our surroundings are chemistry. And in fact, we will need chemistry very much if we have to solve all the challenges that are ahead of us, the global challenges will need chemistry solutions, and we need our young people to learn chemistry from the very point. So from very young, we need to go through this process and learn about chemistry. So after completing my study, I went into carbohydrates and peptides, and I spent some time at the university, and then I ended up at Carlsberg Laboratory. And at Carlsberg Laboratory, I combined glycopeptides or peptides and glycans into glycopeptides in order to start to study glycoprotein functions. And during this process, I also established the SPOC Centre funded by the Danish National Research Foundation. And the reason was that there was a great need for quantitative chemical reactions at that point. So we had a great focus on QCT reactions, as we called them at the time. And this was within carbohydrates, peptides, and proteins using combinatorial chemistry.. And for those who don't know what combinatorial chemistry is, I would like to give a short explanation. So we have some small plastic beads that we use for combinatorial chemistry, and they can contain different starting points. As you can see, they are orange and green and purple. And now we can couple reagents with quantitative chemical reactions to these beads and form one combination. We can then mix these beads up into a complete mixture, and then we can redistribute them into these wells and couple a second reagent. Now, each of these small plastic beads is a container for biological assays, as well as they contain only one chemical compound. So if we have 3 Lego blocks and 3 steps of reaction here, we'll get 27 compounds, or 3 to the 3rd. If we have 5 steps and 5 compounds, we get 3,000 compounds, or 5 to the 5th. If we have 20 compounds and 5 steps, we'll get 3.2 million compounds. So you can see this is an exponentially growing process. Generating compounds at very large numbers. And we therefore needed these very perfect reactions in order to do that. So during this time, yeah, this is showing how we can do this in practice. The beads are in here, we do the coupling reactions, or mixing them, and we prepare them for another coupling reaction, and we take around this cycle many times.. At the end, we can take these beads into a biological assay with enzymes or binding biomolecules, and then we can identify the active compounds on those beads that are showing a sign of activity. So this is from the combinatorial chemistry side. Very important that we can identify those compounds here like needles in a haystack, and we're using mass spectrometry to do that work. But this is another story. Of course, here at Carlsberg Laboratory, we did the discovery and the multifunctional molecules by click that I will describe. Click reactions, what is it actually, the click reaction? And then I have some applications I would like to present to you. During this very active period from '97 A lot of people were working in the lab on establishing enacylaminium reactions, amination reactions, and also we tried to make ketoalkynes for branching peptides and making disorder reactions on peptides. And this here is a method for peptide synthesis that we developed where we can synthesize extremely difficult peptides cannot be synthesised in any other way. It's based on the azide as a protecting group for amines and the acid chlorides for coupling. Very high activity of these compounds in the coupling reactions. So one of our very skilled PhD student, Christian Toner, who is here in the audience, had a project in which he had to take alkynes, make the cobalt acetylenes, and then react those with the acid chlorides so we get these acyl alkynes. That did not happen in his reactions. He fought a brave battle in order to make this reaction work over several months. Nothing would work. And the reason was that this irritating byproduct was formed all the time. It was such a pain. But when we looked in detail into this irritating byproduct, we found that this was actually the triazole. And it was formed quantitatively even in the presence of this very reactive acid chloride. And you can see there's no starting material left and everything is converted in a short period of time. So Christian investigated the compatibility of this reaction with all the peptide functionality that you have in proteins. And he presented this work at the APS in San Diego here. So thank you very much, Christian, for your work. Carolyn alluded to a universe I'll just talk about a toolbox, a toolbox that pharmaceutical industry is using with all these molecules that we make in pharmaceutical industry and the many problems that are associated with solvents, with excess reagents and so on in this toolbox to form the well-known drugs that we are taking today. Also in material sciences, you have these kind For example, in polymer synthesis, you have these kinds of tools that are not very clean and not very green chemistries. And then you have a very small toolbox today, as introduced by Carolyn already, which contains the CuOC reaction and Carolyn's two reactions that you see here. This little toolbox contains, as you also saw from Carolyn's slides, only 3 reactions, and it's 20 years ago we started this research, which means that it's extremely difficult to find reactions like this. So, we have this little toolbox that's completely orthogonal to the chemistry going on out here. And that means that we can take things from this big toolbox and combine it like molecular Lego. So I would like to show you what we can do with click chemistry in a putative example where we have formed this guy here. He is good to kill cells from the inside by apoptosis. Then we have this monoclonal antibody that can locate tumor tissue, and we have a cell-penetrating peptide that can take the other guys with them into the cell if they are connected. So now we take these 3 guys and we put them into a solution of copper(II) ions. And once they are in there, they find each other on the surface of the copper and they couple and make the triazole. So we can do that even in completely— with completely deprotected proteins and in water solutions. This is quite unique. And we get this guy here, and now this guy, he can find a cancer cell like this one, and he sits on the cancer cell for a while, and the cell-penetrating peptide does the work, carries them inside, and now the apoptosis can work, and if we are lucky, the cell is dead. So, click is good, and click is green. So click can be carried out in water, and click doesn't lose any of its material. If we are clicking these two guys together, you can see we can find all the atoms in the product. So atom economy is very important here. So this is a kind of reaction that you would really like in order to combine two large parts in a new pharmaceutical. But what is really click? We start out with an alkyne, and this is a big bag of electrons. It's full of electrons in orbitals or balloons or whatever you would call them. Here sitting on the surface of the alkyne. That's negative charge. It would love to see a copper(1) atom and bind it here to this orbital. That acidifies the alkyne out here, so the copper atom takes the place of the proton on the end of the alkyne. That's still not reactive enough, so we take another copper atom and take it in. Comes in there, sits on the electrons, and it peels off the electrons from the alkyne. So we get a positively charged alkyne, so to speak, with empty orbitals that are looking for electrons. And where can we get the electrons from? Well, one place we can get them from, which fits almost exactly with the faces of these balloons here, is the A-site, and here comes the A-site, and it fits exactly to form what we call a metallo-cycle. The metallo-cycle that loses first one copper atom to form a triazole and a second copper atom so that we get the triazole product that now connects this functionality with that functionality. I think this is what we got the Nobel Prize for. So the fact is that we can use this reaction to couple very complex molecules because this reaction is compatible with all the different stuff you can see here in these two parts and form a triazole very selectively. And now we have a peptide that has the functionality of some recognition and some cell penetration. But there's one problem, and that's a redox issue. The problem is that the copper(I) actually very easily becomes copper(II). And this is a reaction that we can actually— we cannot prevent it, but we can sort of help out by reducing back copper(II) to copper(I) here. This is fine if we have unsensitive molecular structures, but as soon as we have highly susceptible functional groups as in macromolecules, this is really an important issue. You have to get rid of all the reactive oxygen species, as they are called. That's these guys here. They can damage your protein completely. And the way to do that is to avoid oxygen, avoid redox cycles, and use protective ligands for your copper(I) catalyst. Once you do that, you can actually do with 0.01% of copper(I). So it's a real true catalytic reaction. And I would like to present some of Christian's work where he has seen if we can use this to do inhibitors for enzymes, use this click reaction. So we are having these combinatorial libraries of beads. On these beads, we have synthesized by the combinatorial method I showed you. A very large number of compounds which have variations in these 5 positions. And what we are looking at here is a sand fly that transmits Leishmania mexicana, something that you die from. So if we can prevent that, that would be a very good thing. And we can now look at the beads And because we have here substrate which is fluorescent, when we remove that part over here, we can look at enzymes that cleave this substrate when this is not an inhibitor. But if it is an inhibitor, we cannot cleave this substrate, which is attached to the same reactor, the same small microparticle. And here's one that is an active inhibitor in the presence of all the inactive molecules here. So Christian isolated a number of those and found the best one. And this is the best one, 870 nanomolar inhibitor. We weren't very excited about that because this looks like we have much better inhibition. So when we looked at this, We thought maybe this leucine actually is the one that's binding in the first site just on top of the active cysteine in this protease CP28. So Christian synthesized the same structure as you have up here, but now with the mass spacer that we use for analytical purposes here. And now we get a very decent inhibitor of 76 nanomolar. So we thought it would bind like this with the triazole just on top of the active cysteine, but in reality, it bound like this on a much larger surface and interacted very strongly with the entire binding site here, and that's how this inhibitor actually works. That was the first example. The second example I will show you is on the melanocortin receptors. These are very important receptors, and we found out already in the beginning of the click era that we could do microcyclizations to maintain structure in peptides and proteins. So here we have a cyclization to give us a 21-membered ring structure, and this ring structure happened to present these three pharmacophores in just the right way for interaction with the melanocortin receptors. So we have here the 3 pharmacophores, and they interact with these 5 receptors that are very important receptors. They are involved in obesity, in growth and puberty, in sexual dysfunction, memory, pigmentation, and many more. Here you see one of the receptors in a membrane, in a lipid membrane of a cell. And if we now activate with a ligand here from the outside of the cell, we get a response that is transmitted to the nucleus to express proteins. And that is a function of the receptor. So here we have the decoupled protein sitting on the inside of the membrane sending the signal to the nucleus. When we come with a ligand up here in the top, these two helices will change conformation and transmit the signal. So we tried to see what is it that happens with this transmission. So we did these 17 compounds with different components here on these positions. And we could do that very readily because we had the click reaction to form the cycle. So now we are binding the ligands up here in the top, and that affects this conformation of these two helices, sending a signal to the nucleus. And then we, instead of the proteins that normally are expressed there, we express the green fluorescent protein, which gives us green cells, and we can measure the amount of activation of the receptor by the fluorescence intensity. So that is what you see here with increasing amounts of activation reagents. You can see that the cells become more and more fluorescent. And we get these kind of curves of activity out of it. And the difference between these curves and these two is 300, which means that we were able to get a 300-fold activity on the MC4 receptor compared to the MC3 and MC5 receptors. And this MC4 receptor is the one that is involved in obesity. So we would really like to see if we can use that as a starting point for development of medicinal products towards obesity. In our third example, we are talking about the guy you see here. This is a Japanese horseshoe crab. It's a very special creature. It has been on the Earth for 400 million years, and the reason is the innate immune system that this fellow has. It's a blue blood that encapsulates the bacteria and kills it with these antimicrobial compounds that you see here. This is beta hairpins that are linked together with two disulfide bridges. So what we did was to take this compound and then substitute the two disulfides with triazoles. So we were able to cyclize this in two positions with completely unprotected peptides at the same time. Getting these structures very cleanly, and we could then, with the help of Charlotte Gottfressen, who is here in the audience, analyze that these are actually forming these kind of cannonballs, where two of these go together, hiding away all the lipophilic stuff and being positively charged on the surface. So we have these cannonballs and a membrane that is negatively charged, The positive charge on the cannonball locates them on the surface of the cell, and we get a pore, and through this pore, all the contents of the cell will pour out until there's nothing left in the cell and it dies. So that is how this works, and we tried it out on different bacteria to see if our compound was just as active as the natural one. Here's the result. E. coli, Staphylococcus epidermidis, and Salmonella typhimurium, and Bacillus subtilis. Our compound was just as active as the natural antibiotic in 3 of the organisms here. In the 4th one, that had obviously developed a kind of resistance to this antibiotic. There was no activity. But our compounds were very active, probably because the bacillus had developed some sort of disulfide reductase elimination of this antimicrobial peptide. So CLIC is good also for antimicrobial peptides. And in the last example I will show you today, we use this for the clicking of two halves of a protease. Proteases are notoriously difficult to express, and they are difficult to express both because they usually can process themselves, so they will kill themselves during the expression process, and also they will kill the host cell because it will start hydrolysing all the proteins that are inside the cell. So this is a real difficulty, and usually you have to do great protective measures in order to get that expression to go. So the unhappy coli here, we had to do something. So we divided the protease in two in a loop, and we have two halves of the protease now that are expressed. And we express them with the so-called amber stop codon suppression technology where we get an alkyne and an A-site incorporated in the protein.. And now we can just ask the coli, is it happy? It was very happy. So we got large amounts of these two building blocks and we could connect them. Here you have the positions of the alkyne on the A-side. We could connect these two like that. And now they are just forming the structure in solution without connection. Then we could do the click reaction here by adding a copper(1) ion. It comes in and forms the triazole, and now we have a fully active protease, actually a little more active than the natural protease, as you can see. So this is on hydrolysis of this fluorescence quench substrate. So we are able to use this click chemistry for all sorts of bridging purposes. We can do protein structures, simply replacing disulfide bonds also in completely unnatural proteins. So there's a lot of opportunity for these click reactions in the future. So, on this note, I would like to come with some important last words. I'd like to emphasize The importance of serendipity in research, as you have seen, that was quite important. And I think that all great discoveries really have an element of serendipity in them. Because if it was obvious, you would already have done it. And the other thing is that great discoveries are not done behind the office desks They are done in the laboratory. Having an idea, making very creative experiments, having a keen observational eye on what's actually going on, you'll see that there's always something odd happening because nature is very, very complex. Yeah, and I think that could actually be an inspiration to make funding in a different way. Maybe we could do funding so that the researcher was free to decide exactly where to go at any moment, simply by looking at the performance over the last couple of years and see if this is worth funding into the future. That would be much, much better and much, much cheaper than the way we are doing it today. So the Nobel Prize in Chemistry is important More important than ever, it inspires, justifies, and provides direction in this challenged world. Chemistry is omnipresent. In fact, it's everything, as I said, it's you and me, and everything else in this world. And chemistry is more than an education, in my opinion. I think we should try to make chemistry a general education for the young people. In particular, I think that we have the possibility now to take our young people and teach them chemistry at a very early point because we have unique abilities to do animation, to show them a world of images of chemistry at a very early point where they can actually store these images and become excellent students when they come to university. As it is now, we have to start from scratch, from zero, when they come to the university, and then we very rapidly increase the learning curve, but it would be perfect if they had some visual understanding of chemistry when they start. So, with that, I would like to end this and thank you very much for your attention. And also I would like to thank all my colleagues. In particular, I would like to thank Christian Thorne, Claus Bock, who was my mentor, and Michael Bolts, who invited me to the University of Copenhagen and opened up all the opportunities there. I'd also like to thank the Danish National Research Foundation and Carlsberg Laboratory and University of Copenhagen for the support over the years. And not the least, I want to thank all my colleagues. All of these people have had something to do with the development of click chemistry in our laboratories. And my family, Pietra Marie Santillère, Gianni, Oliver Santillère-Miltail, Anna Davoglio-Miltail, and Sandra Davoglio. Thank you for your attention.