Chemical Glycobiology: Study of Glycans and the Immune System
Transcript of Part 2: Imaging the Glycome
00:00:06.26 So welcome to the second lecture in the series 00:00:09.00 called Chemical Glycobiology. 00:00:11.07 My name is Professor Carolyn Bertozzi. 00:00:13.26 I am in the department of Chemistry at UC Berkeley, 00:00:17.21 with also an appointment in Molecular and Cell Biology Department 00:00:21.11 and I am also an investigator of the Howard Hughes Medical Institute. 00:00:25.06 I.. This is the second part of a two part series. 00:00:28.23 The first part focused on the background and history 00:00:32.09 about the field of glycobiology and the structures of sugars. 00:00:36.18 And this part, which is part two, is going to focus more 00:00:40.26 on some recent research from my own laboratory at Berkeley 00:00:43.25 with the goal towards imaging the glycome. 00:00:46.18 And in fact, what you are looking at here is 00:00:49.07 a fluorescence image of a zebrafish 00:00:53.06 that's five days old in which some of the sugars have been tagged 00:00:57.24 with fluorescent molecules and this allows us to literally visualize those sugars 00:01:02.18 in a live organism. And so let me tell you about 00:01:06.15 why we are interested in that. 00:01:08.03 Okay, now the field of molecular imaging is certainly large 00:01:13.15 and expanding rapidly, and it's very important 00:01:16.28 both from the perspective of clinical medicine 00:01:19.15 as well as in the research laboratory. 00:01:22.15 So many of you might have found yourself lying on a table like this 00:01:26.14 at one point in your lives, as have I. 00:01:29.05 This is a magnetic resonance imaging scanner, or a MRI scanner 00:01:34.24 and what this scanner can do 00:01:36.26 is literally take a picture of molecules 00:01:39.29 inside our bodies without having to cut us open. 00:01:42.24 Which is very convenient for many of us. 00:01:44.26 And there are devices very similar to this one which allow 00:01:49.13 clinicians to look at other molecules in our bodies, 00:01:52.00 molecules that are radiolabeled for example. 00:01:54.15 And that is what this image is here. This is called a PET scan. 00:01:59.00 of a human body that has been injected with a radiolabeled version of glucose. 00:02:04.02 Now these are important tools for looking at molecules in clinical settings. 00:02:09.28 but we also like to look at molecules in research settings 00:02:12.16 particularly to understand what molecules are doing inside 00:02:16.02 living cells and nowadays we have some wonderfully advanced 00:02:20.06 fluorescence microscopy tools 00:02:22.10 that allow us not just to see the collections of molecules in cells, 00:02:26.12 but individual fluorescent molecules as well. 00:02:29.12 So it's a very exciting time to be doing molecular imaging in living systems. 00:02:34.24 Now, I mentioned in my first lecture that one of the most 00:02:38.29 intriguing aspects of glycobiology is the fact that 00:02:43.15 the glycome or the complete collection of glycans that a cell makes 00:02:48.09 is dynamic. It's not fixed. 00:02:50.19 So, the glycome, or the collection of sugars on the surface of a cell 00:02:55.21 when it's in one particular state 00:02:57.12 is different from the collection of sugars on that same cell 00:03:00.27 when the cell has transformed its state. 00:03:04.02 This is certainly the case when embryonic stem cells 00:03:07.11 choose a differentiation fate 00:03:10.00 and then become a differentiated cell, 00:03:12.09 like a muscle cell, or a neuron. If you look at the collection of 00:03:16.02 the glycans on the cell surface glycolipids and glycoproteins, 00:03:20.03 you'll find that the nature of that collection has changed 00:03:23.01 as the cell has undergone a differentiation process. 00:03:26.12 From the perspective of clinical medicine, 00:03:30.07 it's interesting to note that the glycome changes when healthy cells 00:03:34.18 transform and become cancer cells. 00:03:37.28 So, you can understand if the sugars on the surface of the cell 00:03:42.02 are changing in a manner that is characteristic of a cancerous state, 00:03:46.06 it would be very convenient if we could actually see 00:03:49.19 those sugars change in vivo, in living human beings. 00:03:54.10 Because that would offer the possibility of imaging those changes 00:03:58.15 and detecting cancers when they are at hopefully very early stages. 00:04:02.17 So there are many reasons why one would want to image the glycome, 00:04:07.11 to take a look at how those sugars are changing inside living subjects. 00:04:12.00 So we can understand how do the glycans relate to differentiation of cells 00:04:17.06 or to organogenesis. And can we detect changes in the glycome 00:04:22.20 that correlate with disease states. 00:04:24.23 So that we can detect those diseases at very early stages. 00:04:29.11 The challenge, of course, is to figure out how to tag these sugar molecules with 00:04:35.14 probes that we can actually visualize using imaging technologies. 00:04:40.16 So even in the research laboratory, this is very challenging. 00:04:43.22 The most commonly used imaging technique in research laboratories 00:04:48.05 is fluorescence imaging, or optical imaging. 00:04:51.02 And we don't even have a way to put fluorescent tags 00:04:55.02 on these sugars. So that is a challenge 00:04:57.24 that students and postdoctoral fellows 00:05:00.02 in my laboratory have taken on. 00:05:03.09 And it's a project we started working on back in the late 1990's. 00:05:07.08 So it's been progressing now for over ten years. 00:05:09.21 Just to give you a little bit of counterpoint, 00:05:12.17 people who study proteins using molecular imaging techniques, 00:05:16.22 have a wonderful arsenal of tools available to them, 00:05:20.18 which is why images like the one I showed on the previous slide 00:05:24.06 are so common and so familiar. 00:05:26.23 to most cell biologists. In fact, 00:05:28.15 most cell biologists probably spend a good portion of their day 00:05:31.22 looking at images similar to this one. 00:05:34.06 In this cell two different proteins have been labeled 00:05:37.14 with two different fluorescent dyes, 00:05:39.28 one green and one red. 00:05:40.24 And the protein that appears green has been labeled using a genetically encoded reporter. 00:05:47.03 So let me just show you what that reporter is. 00:05:49.15 It is called the green fluorescent protein, 00:05:52.19 abbreviated GFP, and it's one of the most important tools for fluorescence imaging 00:05:59.21 of proteins. The green fluorescent protein, because it's a protein that is encoded by a gene, 00:06:05.01 and it turns out that one can fuse the gene that encodes GFP 00:06:08.20 to the gene that encodes the protein that you are interested in imaging. 00:06:12.19 And now, when the GFP protein fusion is expressed, 00:06:17.14 basically your protein of interest is labeled with a fluorescent molecule. 00:06:21.08 The GFP has a chromophore inside that is fluorescent. 00:06:25.09 And this is such an important tool. It's revolutionized our abilities 00:06:31.08 to study proteins, not just in live cells, but even in transgenic organisms. 00:06:35.19 which we express these GFP-fusions by manipulating the genome 00:06:40.20 in the embryonic stem cell stage. 00:06:42.22 This is so important that it was recognized with the Nobel Prize in Chemistry 00:06:47.03 in the year 2008, and that prize was granted to these three scientists. 00:06:52.00 Very well deserved. Very exciting development 00:06:54.23 in the field of molecular imaging. 00:06:56.22 Now those of us who study glycobiology, of course, 00:07:01.09 would love it if we had a tool that was comparable 00:07:04.12 to the green fluorescent protein. 00:07:05.28 So that we could label our sugars with a fluorescent tag, 00:07:09.05 the way that people label proteins with this fluorescent tag. 00:07:12.16 But unfortunately the biosynthetic machinery, 00:07:17.17 by which sugars are built, doesn't really lend itself 00:07:21.02 to these genetically encoded reporters. 00:07:23.09 That's because sugars, unlike proteins, are not primary gene products. 00:07:28.21 Each of these complex glycans is not encoded by a single gene. 00:07:33.17 Rather, they are the products of a complex series of 00:07:38.26 metabolic steps inside the cell. 00:07:41.02 They are products of metabolism. 00:07:43.21 And so for that reason, we cannot use genetically encoded reporters like GFP 00:07:48.02 to introduce labels onto sugars. 00:07:50.17 So I drew this cartoon as kind of some comedy, because we would love 00:07:54.24 to be able to put a tag like GFP on the sugars, 00:07:57.18 but there is really no mechanism to do that using genetics. 00:08:01.11 So, in my laboratory, we have been focusing 00:08:04.21 on using the metabolic pathways of the cell. 00:08:08.22 to introduce fluorescent probes into the sugars 00:08:12.12 that are not the GFP but rather small molecule fluorescent reporters. 00:08:17.18 And let me show you schematically how we go about doing this. 00:08:21.22 It's a two-step process, and we call this metabolic labeling of glycans 00:08:27.14 with chemical reporters. 00:08:29.15 The first step is to feed cells a synthetic 00:08:33.14 monosaccharide building block 00:08:35.17 Now this synthetic sugar looks very much like one of the natural 00:08:40.08 monosaccharide building blocks that you might find 00:08:43.06 in foods that you eat, and I introduced those structures in my first lecture. 00:08:47.26 But the difference is that we have altered the structure 00:08:51.07 of this monosaccharide building block to introduce 00:08:54.22 a chemical functional group "X" 00:08:57.14 and "X" is just the generic for now, 00:08:59.08 and in a minute we will get to what that is. 00:09:00.23 But "X" is what we call the chemical reporter. 00:09:04.17 It's a reactive functional group 00:09:07.05 so that when the cell takes this monosaccharide 00:09:10.18 building block, and when the enzymes process it and integrate it into the glycans, 00:09:15.29 when the glycan appears on the cell surface, that reactive functional group 00:09:20.07 is now on display and can be used in a second step 00:09:24.09 which is a chemical reaction with a probe molecule. 00:09:27.25 Now the probe is a fluorescent dye, for example. 00:09:31.18 And that probe has its own chemical functional group, let's call it "Y". 00:09:35.10 just as a generic label. But "X" and "Y" have to be designed 00:09:40.15 so they react with each other to form a bond. 00:09:43.29 And once that bond is formed 00:09:45.17 now there is a fluorescent probe molecule 00:09:49.20 bound to a cell surface glycan. 00:09:53.07 And that probe now allows us to visualize all of the glycans 00:09:57.01 that possess this red monosaccharide 00:09:59.17 building block as one of their constituents. 00:10:01.29 So, this is a very straightforward schematic 00:10:05.19 in that what we need to do is feed the cell a modified sugar 00:10:09.15 and then do a chemical reaction on that sugar 00:10:12.27 once it's been displayed on the cell surface with a probe. 00:10:15.21 But it turned out that there was a very significant chemical challenge. 00:10:20.08 embedded in this problem because we have to select 00:10:24.03 the chemical reporter "X" and a complementary functional group "Y", 00:10:29.09 so that these two functional groups react 00:10:33.00 only with each other and not with anything else inside this cell. 00:10:38.25 And let me tell you, there is a lot of functionality inside this cell. 00:10:43.21 All of your proteins and your lipids, your nucleic acids, 00:10:47.15 metabolites, there's water, you know, there are a lot of chemical entities inside this cell 00:10:53.27 and "X" and "Y" have to be designed to avoid any side reaction 00:10:58.26 with those biological groups and still react only with each other. 00:11:03.14 So that was a pretty significant challenge 00:11:06.11 and we embodied that concept of that challenge 00:11:09.12 with this term "bioorthogonal". 00:11:12.29 So, what this means literally is not interacting with biology. 00:11:18.07 "X" and "Y" have to react mutually and selectively with each other. 00:11:22.10 They cannot react with anything 00:11:24.07 that's found in nature. And of course, they certainly cannot be harmful 00:11:28.09 to the biological system under study 00:11:30.25 otherwise that would be a disaster from the perspective of 00:11:34.10 imaging sugars in live animals. 00:11:36.15 So we spent many years trying to craft reactions that 00:11:42.24 basically fit this description where the two 00:11:45.27 reactive counterparts, X and Y, are bioorthogonal. 00:11:50.03 And to make a long story short, what we discovered is that 00:11:55.14 an ideal chemical reporter, the group X that we put inside the sugar 00:12:00.16 is the azide, which is defined as having 00:12:04.12 three nitrogen atoms linked to each other 00:12:06.10 in a manner that is shown here. 00:12:08.26 Now the azide has a number of properties that make it very well suited 00:12:14.04 for this particular application as a chemical reporter. 00:12:17.04 First and foremost, azides are not found in biological systems. 00:12:22.03 As far as we know chemists invented the azide. 00:12:26.06 We have not found it in nature. 00:12:27.25 Maybe nature overlooked this functional group 00:12:29.27 when she was creating all of the chemical 00:12:32.21 diversity that's found on our planet. 00:12:34.22 Furthermore, azides are essentially inert in biological systems. 00:12:39.29 There's really not much that they can react with 00:12:41.22 that is normally found in a biological environment. 00:12:45.22 Azides are very easy to attach to sugar molecules. 00:12:50.14 The chemistry is very simple. 00:12:52.05 And importantly, azides are small. 00:12:54.12 So the van der Wahl's radius of an azide is only about 2.4 Angstroms. 00:12:59.22 Which means that it is just a little bit larger than a methyl group. 00:13:03.08 And that's important because we are attaching an azide to a sugar 00:13:08.11 and then asking all of the enzymes 00:13:09.23 inside the cell to metabolize that sugar as if 00:13:12.17 it were a normal metabolic substrate. 00:13:15.14 And so we can't change the structure too much, 00:13:18.12 otherwise those enzymes might reject 00:13:21.06 that sugar as being too foreign, too unnatural. 00:13:23.25 But the azide is a pretty small modification. 00:13:26.19 We thought maybe we could sneak it through the door. 00:13:28.18 of some of those enzymes. 00:13:31.01 Let me just make a point because 00:13:33.08 I am asked this question frequently 00:13:34.20 amongst biologists. Many biologists are familiar with the word azide 00:13:40.08 in the context of sodium azide 00:13:42.13 and they know sodium azide to be a metabolic poison. 00:13:46.27 They often put a little bit of sodium azide into their buffers 00:13:51.01 to prevent bacterial growth. So they know that sodium azide is cytotoxic. 00:13:55.08 and therefore they ask me, "Aren't azides cytotoxic? 00:13:59.04 How can you put an azide on a sugar 00:14:00.18 and feed it to cells and feed it to organisms? Isn't that toxic?" 00:14:03.24 The answer is no. Sodium azide is a very different entity 00:14:08.11 from azides attached to a molecule. 00:14:11.14 So once the azide is attached to a molecule like a sugar, 00:14:14.19 it's totally harmless. By contrast, if azide is a salt like sodium azide, 00:14:20.02 you are right, then it is very toxic. 00:14:21.25 But we don't actually have to worry about that. 00:14:24.01 because there is no way in your body to convert a sugar azide 00:14:28.03 to something like sodium azide. 00:14:30.14 So, don't worry about that. 00:14:31.24 Okay. Now what's most important about the azide 00:14:35.16 is that the azide can undergo chemical 00:14:38.20 reactions with other functional groups 00:14:41.12 which remember in the previous slide I called those Y. 00:14:44.06 The other group is Y. This group is X. 00:14:46.18 There are several different Y groups that will react with azides. 00:14:50.27 And the first such group that we explored 00:14:54.03 is a phosphine. So a phosphine is what you get when you take a phosphorus atom 00:14:59.18 and you link three substituents to it like I've shown here. 00:15:03.25 Now back in the early part of the 20th century. 00:15:07.27 We are talking around 1915 to 1920, 00:15:11.12 a German chemist by the name of Hermann Staudinger 00:15:14.28 discovered that phosphines will react with azides to generate 00:15:20.17 a product in which there is a phosphorus-nitrogen bond 00:15:24.08 and he called this kind of an intermediate an aza-ylide. 00:15:27.19 It's a very interesting reaction in that these 00:15:30.22 two chemical groups join together and make this covalent 00:15:34.22 adduct. We were intrigued by this Staudinger chemistry 00:15:38.29 because both phosphines and azides are bioorthogonal 00:15:43.08 and their reaction is very selective. They are very non-toxic. 00:15:47.27 And it seemed to fulfill many of these criteria for bioorthogonality. 00:15:52.07 With one exception. It turns out that aza-ylides 00:15:55.16 are not particularly stable in water. 00:15:58.00 They tend to hydrolyze. 00:16:01.05 So the P-N bond gets cleaved. 00:16:03.00 And of course, if our goal is to use the chemistry 00:16:05.22 to attach a probe to a sugar in the living body 00:16:09.10 where we are surrounded by water 00:16:12.09 then that hydrolytic sensitivity would be a major problem for an aza-ylide. 00:16:17.01 So what we did was we modified this reaction in a fairly subtle way 00:16:22.19 where we attached to the phosphorus atom a benzene ring 00:16:26.18 that had this methoxycarbonyl group. 00:16:29.01 So now, once the aza-ylide was formed 00:16:32.18 faster than it could hydrolyze, this nitrogen atom 00:16:35.16 cyclized to this carbonyl, cleaved the ester bond, 00:16:39.18 and formed an amide. 00:16:41.29 And this cyclization was so fast that it didn't 00:16:45.04 even matter whether there was water around. 00:16:47.09 Now once this product was formed, 00:16:49.08 water could come along at its leisure 00:16:51.15 hydrolyzed the P-N bond and form this product, but now, because an amide 00:16:56.28 bond had formed during this intramolecular cyclization step 00:17:01.04 these two parts, one that came from the azide 00:17:04.00 the other attached to the phosphine 00:17:06.14 were still linked together through a very stable linkage. 00:17:09.09 So this is what we call the Staudinger ligation. 00:17:13.18 It's a reaction in which we take advantage of the classic Staudinger chemistry 00:17:19.02 in the first step but then we alter it to form a product 00:17:22.20 in which the two components are permanently 00:17:24.14 ligated together in a manner that is 00:17:26.08 very stable in a biological system. 00:17:29.03 And the Staudinger ligation was one of the first 00:17:32.06 chemical reactions that we used to try to image sugars in living systems. 00:17:38.01 So which sugars would you like to image? 00:17:41.24 Of course there are many interesting choices. 00:17:44.08 And as I introduced in my first lecture, 00:17:46.24 in vertebrates there are nine 00:17:49.07 fundamental monosaccharide building blocks 00:17:52.01 that are the constituents of our various glycans. 00:17:55.04 They are all interesting in their own right, 00:17:58.01 but when we were thinking about what sugar would we like to image, 00:18:01.10 we were naturally drawn to this monosaccharide, 00:18:05.24 called sialic acid. And as I mentioned in my first lecture, 00:18:09.21 sialic acid tends to appear in some very interesting circumstances 00:18:15.08 in vertebrate biological systems. 00:18:18.12 I mentioned for example, that sialic acid 00:18:21.01 is a component of the ligand for the selectins. 00:18:24.27 I also mentioned that sialic acid 00:18:27.01 is the structure that the influenza virus binds to. 00:18:30.18 Well, it turns out that sialic acid is a pretty busy monosaccharide. 00:18:36.20 because it also seems to be related to embryonic development 00:18:40.19 and to cancer. In fact, the glycomes of embryonic cells, 00:18:46.01 differentiating tissue, as well as cancer cells 00:18:49.14 have been studied in much detail 00:18:52.15 and it turns out that many of the glycans 00:18:54.25 found both embryonically and on cancers 00:18:57.22 have sialic acid as one of their constituents. 00:19:00.19 So what I have shown here are just three examples from a much larger set. 00:19:05.03 These are glycan structures that have been found to be highly elevated 00:19:10.25 on cancers compared to the normal healthy tissue counterpart. 00:19:14.14 And many of them are also developmentally regulated. 00:19:17.29 which is probably not an accident, because we know that often cancers 00:19:22.14 start to acquire properties that we 00:19:25.12 normally associate with embryonic tissue. 00:19:27.19 They've sort of lost their identity 00:19:30.11 and they are reverting to an embryonic state. 00:19:32.23 and that's reflected in their glycan structures. 00:19:35.27 So polysialic acid which is a homo-polymer 00:19:38.28 made up of repeating units of sialic acid 00:19:41.16 is highly abundant on a variety of tumors 00:19:44.13 that are derived from neural crest tissue. 00:19:46.16 Sialyl Lewis X, which I mentioned before 00:19:50.02 is part of the ligand for the selectins, 00:19:54.02 is also found in elevated levels on a variety of different epithelial cancers. 00:19:57.08 and blood cancers. And Sialyl Tn, a very simple disaccharide, 00:20:03.12 is actually not normally found on any healthy human tissue 00:20:07.12 but it appears on a variety of prostate cancers. 00:20:11.03 and we don't really know why that is, 00:20:12.29 but the fact that these structures appear in the context of malignancies 00:20:18.06 has suggested to many people that there might be 00:20:21.03 elevated levels of sialic acids on these tissues. 00:20:25.25 So if we could image the sialic acids, 00:20:28.05 maybe we could detect these tumors in living systems. 00:20:31.25 That was one of the motivations to study the sialic acids. 00:20:35.25 Okay. So then the question became, in our minds, 00:20:39.24 how do we put an azide into the sialic acids? 00:20:43.06 Well, we have to understand the fundamental metabolism 00:20:46.22 that produces sialic acid in our body. 00:20:50.05 And that's what is shown in this slide. 00:20:52.15 It all begins with this simple monosaccharide 00:20:56.23 building block called N-acetylmannosamine. 00:21:00.08 and we abbreviate that ManNAc for short. 00:21:03.14 So you eat ManNAc and in fact if you eat bread or drink beer, 00:21:09.13 any yeast product will have some ManNAc. 00:21:11.21 If you don't eat ManNAc, that's okay 00:21:14.15 because you are able to biosynthesize it from glucose 00:21:17.21 through other enzymatic steps that I haven't shown. 00:21:20.27 But in any event, once ManNAc enters your system, 00:21:23.19 it has basically one metabolic fate. 00:21:26.15 It is destined to be converted to sialic acid. 00:21:28.27 in your cells. And that happen through a series of enzymatic steps 00:21:34.18 and I am not going to go through all these details. 00:21:37.18 You can certainly look at the slide in detail if you are interested. 00:21:40.17 But it suffices to say, that you eat this sugar 00:21:44.00 and after all of these enzymes have acted on it 00:21:46.29 at the end of the day a sialic acid is made, 00:21:50.05 and it appears usually at the end of complex glycan chain 00:21:54.19 on the surface of your cell, either in a glycoproteins or glycolipids. 00:21:58.13 So we knew from the work of many labs 00:22:01.17 that this sugar gets converted to this sugar. 00:22:04.07 And I've shown the acetyl group in blue. 00:22:08.04 So that it is clear where that ends up in the biosynthetic product. 00:22:11.28 And the reason that I highlight that acetyl group 00:22:14.12 is because we know from the work of several groups 00:22:17.27 that you can make subtle modifications 00:22:20.22 to the structure at this position without 00:22:23.14 significantly harming the efficiency of 00:22:26.13 any of these enzymatic steps, 00:22:28.06 which is really quite remarkable if you think about it. 00:22:30.23 Normally we think of enzymes as being very particular 00:22:34.18 for their substrates, but in this pathway 00:22:37.26 there is a little bit of leeway and you 00:22:39.00 can modify this side chain a little bit. 00:22:41.26 So knowing that, it was clear to us that this was a site 00:22:46.08 that we might be able to attach an azide 00:22:48.16 without confusing the enzymes in the cell. 00:22:52.01 And that's exactly what we did. 00:22:54.27 So by chemical synthesis we generate this unnatural version 00:22:59.04 of ManNAc, which has an azide. 00:23:01.21 So as an abbreviation we call this compound ManNAz, 00:23:06.04 where it is the azide version of ManNAc. 00:23:09.10 And we wanted to get ManNAz into cells, so to do that we 00:23:15.12 block each of the hydroxyl groups, which are characteristic of sugar molecules 00:23:19.29 with acetyl esters. Those are protecting groups. 00:23:24.13 So once the acetyl groups are in place the sugar is now sufficiently lipophilic 00:23:29.21 that it will penetrate through the cell's membrane, and once it is inside the cell, 00:23:35.09 we have non-specific esterases that simply cleave 00:23:38.25 these acetyl groups off and throw them away. 00:23:40.22 And then the sugar is ready for metabolism. 00:23:43.05 So the enzymes start processing the sugar 00:23:46.11 and after several hours what we found is that, 00:23:48.15 lo and behold, some fraction of the sialic acid residues 00:23:52.07 on these cells had been replaced with an azido analog. 00:23:56.23 So in other words, all we do is add this compound to cell culture media, 00:24:01.16 and the cells do all the hard work 00:24:03.05 and they produce this azido-sialic acid on their cell surface glycans. 00:24:08.01 Remarkably the cells don't seem particularly 00:24:11.28 offended by this transformation. 00:24:13.26 Some fraction of their sialic acids 00:24:16.18 have been replaced with this unnatural variant 00:24:18.29 and that seems fine with them. 00:24:21.16 But it's very convenient for us 00:24:22.28 because now we can use the azide to do chemistry. 00:24:26.18 And so by introducing a phosphine reagent, 00:24:29.05 that's been linked to a fluorescent probe, we 00:24:32.13 do a Staudinger ligation between the phosphine and the azide. 00:24:35.10 now there's a covalent bond between the probe molecule and the sugar. 00:24:40.22 So wherever there is a sialic acid, 00:24:42.16 we can now visualize it 00:24:44.06 using fluorescence microscopy focusing on the probe. 00:24:47.12 And so in this manner we have been able to image 00:24:50.17 sialic acids on a variety of interesting cell types. 00:24:54.01 And sialic acid is not the only sugar 00:24:56.29 that we can see using the azide as a chemical reporter. 00:25:00.20 So, just as we can introduce the azide into sialic acid, 00:25:05.07 by feeding the cells ManNAz. 00:25:08.09 It turns out we can introduce an azide into fucose 00:25:11.22 simply by feeding the cells this 6-azido fucose derivative 00:25:16.23 that has acetyl esters protecting its hydroxy groups. 00:25:19.26 And likewise, we can introduce azides into N-acetylgalactosamine 00:25:25.18 or GalNAc, by feeding cells this modified form of GalNAc, 00:25:30.18 that has an azide on its acetyl group. We call this GalNAz. 00:25:34.13 analogous to ManNAz, and I actually will be coming back to this sugar 00:25:38.20 GalNAz towards the end of the lecture. 00:25:41.10 We also put quite a bit of time into developing probes 00:25:45.16 that we can use to visualize these sugars. 00:25:49.10 Now the Staudinger ligation is wonderfully selective 00:25:51.24 in that it allows us to form a covalent bond between the probe and the sugar. 00:25:57.06 But more than that we can exploit elements 00:25:59.24 of that ligation chemistry in order to design what we call smart probes. 00:26:05.11 So these are probes that are actually invisible 00:26:08.06 until they find an azido sugar 00:26:11.28 form that bond through the Staudinger ligation, 00:26:14.13 and then they become fluorescent. 00:26:16.04 So they basically swim around in the system, 00:26:18.11 they look for azides and as soon as they find them, 00:26:20.26 they make a bond and light up, and you don't see them otherwise. 00:26:23.29 This allows us to do fluorescence imaging 00:26:26.01 with very good signal above background. 00:26:28.23 The way we do this is to exploit the fact that 00:26:32.19 during the course of the Staudinger ligation, 00:26:34.11 this ester bond will be cleaved. 00:26:36.18 In the previous slides I had simply a methyl ester at this position. 00:26:41.18 where methanol was released during the reaction 00:26:44.28 which is not particularly remarkable. 00:26:46.25 But, one could replace that methyl ester 00:26:49.00 with an ester linkage to a fluorescence quencher. 00:26:52.23 And if there is a fluorescent molecule bound elsewhere, 00:26:55.16 that fluorophore will be quenched 00:26:57.19 by the quencher in this initial molecule. 00:27:01.01 But as soon as this probe finds an azide, 00:27:03.21 located within a sialic acid, let's say, or another sugar on the cell, 00:27:08.02 the phosphine reacts with the azide 00:27:10.14 the Staudinger ligation unfolds, 00:27:13.01 the ester is cleaved, the quencher is released, 00:27:16.12 now that fluorophore is activated. 00:27:19.18 So only once the fluorophore finds its target 00:27:22.09 and reacts, only then do you see the fluorescence. 00:27:25.14 Otherwise it's invisible. 00:27:26.27 Let me show you an actual chemical structure of a probe 00:27:29.25 that we prepared that follows precisely this mechanism. 00:27:35.08 This is a molecule that we call affectionately "QPhos", 00:27:40.06 which stands for quenched phosphine. 00:27:41.22 It's got three parts to it: 00:27:44.20 so over here, this green part is a very well known 00:27:48.08 fluorescent molecule called fluorescein. 00:27:50.07 It's a green fluorescent dye commonly used in the research laboratory. 00:27:55.22 In the middle in black is a phosphine that's all set up for a Staudinger ligation. 00:28:01.29 You can see that on this benzene ring there is an ester group. 00:28:06.05 That's positioned for that intramolecular reaction 00:28:09.14 of the aza-ylide intermediate. 00:28:11.06 And then finally over here, the blue part 00:28:14.03 is a well-known fluorescence quencher, 00:28:16.23 called Disperse Red 1. 00:28:18.28 And as long as this Disperse Red 1 is linked to this ester 00:28:22.15 it will quench the fluorescence of the fluorescein molecule. 00:28:25.26 So let me show you some images that we took using QPhos 00:28:30.07 to detect sialic acid on cancer cells in culture. 00:28:35.12 So in this experiment what we did was we grew some cells 00:28:40.12 called HeLa cells. These are human cervical epithelial cancer cells 00:28:45.22 that you can grow in a test tube. And while those cells were growing, 00:28:49.13 we fed them ManNAz in the media. 00:28:53.03 We just added it to the cell culture media, 00:28:55.08 they took it up, they digested it, and they put azides into their sialic acids. 00:29:00.13 Then, after a few days of treatment with ManNAz, 00:29:03.09 we reacted those cells with QPhos. 00:29:06.22 And then after a couple of hours we took a fluorescence image of those cells. 00:29:11.13 The cells were also stained with a blue fluorescent dye 00:29:15.03 that is specific for the nucleus. 00:29:16.17 That just helps you to orient exactly where the cells are positioned 00:29:20.27 since each one has one nucleus. 00:29:22.29 So, what you can see is that each cell 00:29:25.17 is surrounded by a nice bright green outline. 00:29:29.15 Those are the sialic acids on the 00:29:32.05 membrane associated glycoproteins and glycolipids. 00:29:35.09 of the cell and they are nicely lit up. 00:29:37.25 We are actually imaging the sugars. 00:29:39.26 You might see a little bit of fluorescence inside the cells as well. 00:29:43.16 That's basically part of the secretory pathway, 00:29:47.23 the Golgi compartment for example. 00:29:49.01 And then there's a little bit of staining of 00:29:51.14 little vesicles throughout the cells as well. 00:29:53.05 That's because after we react sialic acids on the membrane 00:29:57.17 some of those sialic acids end up internalized 00:30:00.21 into vesicles because the membranes are constantly being 00:30:03.26 engulfed and recycled and turned over. 00:30:05.29 They are going back into the late part of the Golgi compartment 00:30:08.23 and then coming back to the membrane. 00:30:10.09 There's a lot going on in fact during the course of reaction with QPhos. 00:30:14.26 Now we had some issues once we started 00:30:17.29 doing these kinds of experiments. 00:30:19.07 For example, we realized that in order to get this nice bright staining 00:30:23.24 of the sialic acids, we had to react the cells with QPhos for several hours. 00:30:28.27 Now that's not a problem for imaging the sugars on cultured cells, 00:30:34.17 because the cells are in a flask with the reagents 00:30:37.26 and they can sit in the incubator for several hours. 00:30:40.12 and just let the reaction unfold. 00:30:42.09 The reason we had to let the reaction go for several hours 00:30:45.15 as opposed to several minutes 00:30:47.02 or several seconds is that it turns out the Staudinger ligation reaction 00:30:51.02 is intrinsically rather slow. It just takes that long for the reaction to proceed. 00:30:57.24 And although that was not too problematic 00:31:00.21 for imaging experiments on cultured cells, 00:31:03.25 we worried that that could be a problem 00:31:06.23 for imaging sugars in living animals. 00:31:10.09 because of course a living animal is not at equilibrium. 00:31:14.10 It is not cells in a flask sitting in an incubator for many hours. 00:31:17.26 Animals have an active metabolism. 00:31:21.04 As soon as you introduce reagents into their bodies, 00:31:24.19 their bodies are working on clearing those reagents right out again. 00:31:27.29 And that clearance process can be very rapid for certain molecules 00:31:32.18 and certain animals. In fact, when we started looking into the Staudinger ligation 00:31:38.06 as a tool for imaging sugars in laboratory mice 00:31:41.23 we immediately ran into this problem. 00:31:44.29 So we injected mice with ManNAz 00:31:48.08 and we found that in the animal 00:31:52.03 the cells were perfectly willing to take up this sugar 00:31:54.23 and convert it to the azido-sialic acid, 00:31:58.01 that was no problem. 00:31:59.21 And it seemed harmless to the mice. 00:32:01.17 So there was no obvious detriment to replacing 00:32:04.09 some fraction of the sialic acids with the azido version. 00:32:08.00 The problem came when we then injected those same mice 00:32:11.26 with probe molecules bound to a 00:32:15.09 phosphine for the Staudinger ligation. 00:32:17.04 Because what we discovered is that the 00:32:19.24 reaction between the phosphine and the azide 00:32:21.26 was just too slow compared to the rate of metabolic clearance 00:32:25.23 of the probe out of the animal's body. 00:32:28.02 So we could only detect a little bit of this product 00:32:31.26 just on certain organs and tissues 00:32:34.00 The organs and tissues that were the most accessible 00:32:36.22 to the reagents after we injected them 00:32:38.14 or the organs and tissues that had really, really high levels 00:32:41.23 of the sialic acid we were trying to image. 00:32:45.07 And that was a little frustrating, 00:32:47.00 but it basically pointed to a very important element 00:32:50.12 of this experiment, which we really hadn't considered at the very outset. 00:32:55.07 You know, ten years ago. 00:32:56.04 Which is that the kinetics of the reaction 00:32:59.05 between the azide and the probe molecule 00:33:01.16 are very, very important for imaging in living animals. 00:33:06.02 That reaction has got to be fast enough 00:33:08.29 so that the probe can find the azide and react 00:33:12.11 more quickly than it is cleared out of the body. 00:33:14.21 And after many years of experimentation, 00:33:17.21 we finally concluded that the Staudinger ligation was just too slow. 00:33:23.11 Its kinetics were too slow compared to its rate of metabolism. 00:33:27.22 And it probably didn't help the situation that phosphines, in general, 00:33:32.25 can be oxidized in the mammalian liver 00:33:35.15 by cytochrome P450 enzymes. 00:33:37.05 which convert the phosphine to a phosphine oxide 00:33:42.08 and once that occurs, now this product 00:33:45.08 is no longer active in a Staudinger ligation. 00:33:47.08 So, undoubtedly, liver metabolism of the phosphine 00:33:51.19 contributed to its rapid clearance rate 00:33:54.23 which just out competed the intrinsically slow kinetics of the reaction. 00:33:58.23 So the bottom-line is that the Staudinger ligation 00:34:02.04 was great for in vitro imaging on cells in culture. 00:34:06.00 Not so great for in vivo imaging in live organisms 00:34:10.00 that have the ability to clear reagents rapidly out of the system. 00:34:13.20 So at that point we shifted our attention 00:34:17.19 to a different kind of chemistry that azides can undergo. 00:34:21.16 It's another chemistry that has its origins in the last century 00:34:26.03 in the hands of a German chemist. 00:34:27.28 In this case, Rolf Huisgen, Professor at University of Munich. 00:34:31.26 who back in the 1950s discovered that azides 00:34:35.26 can react with alkynes, undergo a 1,3-dipolar 00:34:41.07 cycloaddition reaction whose mechanism is shown here, 00:34:44.19 to form a cyclo adduct product called a triazole. 00:34:49.00 Now like the Staudinger chemistry, 00:34:52.04 this Huisgen cycloaddition chemistry 00:34:55.02 is bioorthogonal in that alkynes and azides 00:35:00.08 both are not found in biological systems, 00:35:03.04 at least not in most biological systems 00:35:05.06 and they react with each other very selectively 00:35:08.25 without any unwanted side reactions in the living system. 00:35:13.19 So we thought this would be another very promising avenue to explore. 00:35:16.25 The problem is that the classic Huisgen reaction 00:35:21.09 as described with a standard linear alkyne 00:35:24.27 is also very slow. In fact, even slower 00:35:28.11 than the Staudinger chemistry with phosphines. 00:35:30.28 So slow, that when people perform 00:35:34.22 these reactions in a research laboratory, 00:35:37.04 they usually have to apply elevated temperatures 00:35:40.03 or elevated pressures in order to get the reaction 00:35:43.08 to proceed to completion at a reasonable rate. 00:35:45.26 And when I say elevated temperatures, 00:35:47.22 I mean above 100 degrees centigrade for example. 00:35:51.19 So refluxing toluene. Hotter than the boiling temperature of water. 00:35:56.27 Obviously we can't do that to cells 00:36:00.14 and certainly not to living organisms. 00:36:02.14 But we started thinking about ways that we might be able to 00:36:05.18 accelerate the rate of this chemistry. 00:36:08.21 It turns out, back in the 1960s, 00:36:11.19 there was a publication by Wittig and Krebs 00:36:14.25 in which they reported that phenylazide reacts with 00:36:20.08 cyclooctyne which has a triple bond crimped into an eight-member ring 00:36:26.03 to form the triazole product very rapidly. 00:36:30.16 Now my graduate students were reading this paper. 00:36:33.25 It was published in German, 00:36:35.29 and unfortunately at that time we did not have 00:36:38.25 any coworkers in the laboratory whose German was good enough 00:36:41.18 to really understand the paper in its fine detail. 00:36:44.06 but all of us read this sentence 00:36:46.08 and recognized three words: phenylazide, cyclooctyne, and explosion. 00:36:53.02 So as far as we could see, Wittig and Krebs 00:36:57.14 had combined these two reagents 00:36:59.12 and they formed a product like an explosion. 00:37:02.11 which suggested to us that this must be a pretty fast reaction. 00:37:05.17 Keep in mind however, that what Wittig and Krebs did 00:37:09.05 is to react neat cyclooctyne, 00:37:12.03 which is a liquid, with neat phenylazide with no solvent. 00:37:17.05 So these were very concentrated reagents that reacted like an explosion. 00:37:20.09 And we figured, if we dissolve these reagents at lower concentrations 00:37:25.08 and prototypical solvents, maybe the reaction 00:37:28.07 will be very nicely controlled, 00:37:29.19 hopefully still very rapid at room temperature. 00:37:32.13 Now why you might ask does cyclooctyne 00:37:36.06 react with an azide like an explosion whereas 00:37:39.00 linear alkynes are so slow that you have to 00:37:42.17 heat them up above 100 degrees? 00:37:44.05 That can be understood by looking at what happens 00:37:47.27 to an alkyne when you force it into an eight membered ring. 00:37:51.13 Now normally, alkynes are linear. 00:37:54.12 That is their preferred geometry. 00:37:56.08 And when an alkyne reacts with an azide, this bond angle 00:38:00.28 is going from 180 degrees down to 120 degrees in the triazole product. 00:38:07.02 So that bond deformation costs a lot of energy 00:38:11.01 in the transition state for the reaction. 00:38:12.29 That's why we have to heat it up. 00:38:14.24 To give some of that energy to get over that activation barrier. 00:38:17.23 By contrast, if the alkyne is embedded in an eight membered ring 00:38:23.04 now the bond angle is bent to 160 degrees 00:38:27.25 It's not ideal, in fact, it costs a lot of strain 00:38:31.19 to bend a triple bond to 160 degrees 00:38:35.16 But the upshot is that now the difference 00:38:38.13 between 160 degrees and 120 degrees is quite a bit smaller. 00:38:42.06 It doesn't cost as much energy to go 00:38:44.09 from this structure through a transition state 00:38:46.22 to get to this product. 00:38:48.03 So our hope was that by straining the starting material 00:38:52.12 and bending the bond angle so it's closer 00:38:54.25 to the structure of the transition state 00:38:56.17 we would lower the activation barrier 00:38:58.24 so that the reaction can proceed at room temperature. 00:39:01.13 And that's exactly what we observed when we chemically synthesized 00:39:05.17 a whole family of cyclooctynes in the laboratory. 00:39:09.16 So let me just quickly summarize some kinetic comparisons 00:39:14.10 that we performed using a variety of synthetic reagents. 00:39:19.10 And so each of these compounds is a cyclooctyne 00:39:22.16 of some sort in some cases with a heteroatom in the ring 00:39:26.20 with various different substituents at different sites 00:39:29.12 and we made these compounds for a variety of reasons 00:39:32.21 that I won't go into. 00:39:33.13 but you'll see in some cases there were sp2 hybridized carbons 00:39:37.11 elsewhere in the ring. Our hope was that we could increase the strain 00:39:41.15 energy a little bit more by putting 00:39:43.16 sp2 hybridized carbons in there. 00:39:46.01 And some of these reagents 00:39:47.03 have electronegative fluorine substituents. 00:39:49.27 which we also though might accelerate the reaction 00:39:53.01 based on some frontier molecular orbital arguments. 00:39:56.12 Also shown among this collection is a phosphine reagent 00:40:00.17 which is set up to a Staudinger ligation. 00:40:03.03 What we did is we took each of these compounds. 00:40:06.07 We reacted each compound with benzyl azide 00:40:09.27 in a test tube. And then we measured the 00:40:12.12 second order rate constant of the reaction. 00:40:14.26 And in blue are shown basically the relative values that we measured. 00:40:19.24 So the bottom-line is that we found a compound 00:40:24.03 that reacts very rapidly with azides in a test tube. 00:40:28.15 This is a di-fluorinated cyclooctyne 00:40:32.00 that we have given the abbreviation DIFO. 00:40:34.22 So that's stands for Di-Fluoro Cyclo Octyne. 00:40:39.00 DIFO. And the relative rate of reaction of DIFO 00:40:42.26 compared to some other compounds you'll see, 00:40:45.17 for example a compound without the fluorine atoms. 00:40:48.02 is very high. So we can accelerate the rate 75 fold 00:40:52.10 just by putting some fluorine atoms here 00:40:54.29 compared to some parent compounds. 00:40:57.08 Importantly, if you compare the relative rate of reactivity 00:41:00.08 between DIFO compared to a Staudinger ligation phosphine, 00:41:03.06 you will see that it is a good 15 times faster. 00:41:06.20 And so we felt that where the Staudinger ligation 00:41:09.26 failed in vivo because of its slow kinetics. 00:41:13.02 we might find success now with DIFO 00:41:16.11 as a reactive counterpart to the azide. 00:41:20.01 So we tested that concept in a variety of imaging experiments 00:41:25.15 starting simply with cultured HeLa cells. 00:41:28.15 So remember, in a previous slide, I showed you 00:41:31.02 some images of the sialic acids on HeLa cells 00:41:34.26 where we reacted the azides with QPhos. 00:41:37.19 This is now a very similar experiment but 00:41:41.10 once we put the azides into the sialic acids 00:41:43.24 this time we react the azides with this compound. 00:41:48.05 So we call this DIFO-488. 00:41:51.00 Down here is the DIFO part, the di-fluoro cyclooctyne, 00:41:55.23 The green part of the molecule is a commercial 00:41:58.22 fluorescent dye called Alexafluor 488. 00:42:01.23 It's a bit like fluorescein in its spectral properties. 00:42:05.02 That is why we call this DIFO-488. 00:42:07.22 So what you are looking at here are the cultured HeLa cells. 00:42:11.19 They have been fed ManNAz to put azides into the sialic acids. 00:42:16.05 Then they were reacted with DIFO-488 00:42:19.03 and you can see that the sialic acids are shown in these nice 00:42:22.02 bright green halos around each cell. 00:42:24.10 The membranes are nicely lit up. 00:42:26.20 Those are the sialic acids. 00:42:28.06 This is a control experiment 00:42:30.12 in which instead of feeding the cells ManNAz, 00:42:33.27 we now fed them the natural sugar, ManNAc. 00:42:36.28 There are no azides in these cells. 00:42:39.20 We also reacted them with DIFO-488. 00:42:43.08 And now you don't see any green labeling of the membranes. 00:42:47.01 That is because there are no azides for DIFO to react with. 00:42:50.09 That's a testament to the selectivity of DIFO. 00:42:54.06 It only reacts with azides. 00:42:55.26 If there are no azides around, there is nothing for it to react with. 00:42:58.14 You don't see the sugars. 00:43:00.11 But the most important number on this slide 00:43:03.02 is this one right here. 00:43:05.09 Now we can see a beautiful image like this 00:43:07.17 by reacting the cells for less than 1 minute with DIFO-488. 00:43:13.02 Whereas in the previous experiment with QPhos, 00:43:16.10 the Staudinger ligation reagent, 00:43:18.20 we had to react the cells for over two hours. 00:43:22.02 If we can get this kind of an image within a minute, 00:43:25.22 I think now we can image the sugars in living animals. 00:43:29.28 This reaction is fast enough, so we hoped. 00:43:32.24 So, what animal model would one want to use to image the sugars? 00:43:38.17 Mice are obviously a very attractive model because one can 00:43:42.17 study a variety of human diseases in that model organism 00:43:46.04 including cancer. However, mice are not ideal for optical imaging, 00:43:50.15 because, as you know they are not transparent. 00:43:53.18 They are opaque. 00:43:54.20 One can do fluorescence imaging inside a mouse, 00:43:57.15 but it requires a specific kind of dye, generally a near infrared dye. 00:44:02.06 and it's a more complicated experiment. 00:44:04.19 And for a first attempt at in vivo optical imaging, 00:44:08.23 we thought we would use a model organism 00:44:11.02 that is a little friendlier to an optical microscope. 00:44:14.04 And the obvious choice was the zebrafish. 00:44:18.03 So zebrafish are translucent. 00:44:20.09 You can see right through them 00:44:21.24 and monitor their organs in vivo. 00:44:24.10 in the live animal. Also, zebrafish 00:44:27.16 are a very attractive model for vertebrate developmental studies. 00:44:31.29 They have all the same organs and parts that we have. 00:44:35.23 They are vertebrates. And their embryonic 00:44:38.04 developmental program has been very well characterized. 00:44:42.10 So we know what zebrafish embryos 00:44:43.28 should look like at virtually all stages of their development. 00:44:48.00 We also know that there are changes in the glycome. 00:44:51.29 that correspond with embryonic development. 00:44:55.05 And so we though this was a very nice organism 00:44:57.06 to first of all test our chemical tools, 00:44:59.25 but second of all we might be able to address 00:45:02.22 some very interesting fundamental questions 00:45:04.25 of how the glycome changes during development. 00:45:07.15 And learn something about the field of glycobiology. 00:45:10.06 So, we started with some proof of concept experiments. 00:45:14.09 in which we focused not on sialic acid 00:45:17.06 but rather on a sugar called N-acetylgalactosamine. 00:45:22.05 abbreviated GalNAc for short. 00:45:24.12 The reason we were so interested 00:45:26.11 in GalNAc is because, as I mentioned in my first lecture, 00:45:30.17 it's the conserved core residue in a family 00:45:34.09 of O-linked glycans that are associated 00:45:37.14 with glycoproteins from the mucin family. 00:45:41.10 Mucins are a general class of glycoproteins 00:45:44.29 that are characterized by having dense clusters 00:45:47.29 of these O-linked glycans along the polypeptide backbone. 00:45:52.10 They're involved in cell adhesion, 00:45:54.10 regulating cell-cell interactions, and they are known 00:45:57.24 to be both developmentally regulated 00:45:59.15 and sometimes upregulated during malignant transformation. 00:46:03.13 So the mucins are an interesting class of glycoproteins. 00:46:07.13 They all have a GalNAc residue at their core position. 00:46:10.28 So we thought if we could introduce an azide 00:46:13.03 into this GalNAc residue perhaps we could image the mucins 00:46:18.20 in vivo. And the way we do that is to 00:46:21.23 simply feed cells, or in this case, zebrafish embryos, 00:46:25.23 This azido-acetyl derivative that we call GalNAz 00:46:30.14 once again, the hydroxy groups are protected with acetyl esters. 00:46:34.13 So that the compound can penetrate cell membranes 00:46:37.08 and inside the cell the acetyl groups are removed by esterases. 00:46:41.07 in vivo. Okay. So this was one of our first experiments 00:46:46.00 we performed to simply ask the question will zebrafish embryos 00:46:50.02 metabolize GalNAz and introduce it into the mucins, 00:46:54.09 and if so, can we then tag the azides 00:46:57.05 with DIFO reagents and image that sugar in vivo. 00:47:01.27 So in this experiment we fertilized zebrafish embryos in a test tube 00:47:06.05 and we added GalNAz to the culture media 00:47:09.27 and just let them develop over the course 00:47:11.28 of several days and then after about five days 00:47:15.11 we took the five day old zebrafish larvae 00:47:19.23 and we reacted it with DIFO linked to a fluorescent dye, 00:47:23.23 in this case it was DIFO-488, 00:47:25.24 the same compound I showed on the previous slide 00:47:28.06 and then we put those labeled zebrafish in a fluorescence microscope 00:47:33.00 and we took a picture. So what you are 00:47:35.14 looking at here is the labeled zebrafish 00:47:39.03 five days old, treated just as shown 00:47:41.14 in this schematic. And wherever you see any brightness 00:47:44.27 basically you are looking at the DIFO label 00:47:48.09 which has been covalently attached to the azido sugar. 00:47:52.00 There's also a five day old zebrafish in this panel. 00:47:55.01 You can't see it, I realize. 00:47:56.25 The reason is that the only difference 00:47:58.24 is that this zebrafish embryo developed in the presence of 00:48:01.28 the natural sugar GalNAc, which has no azides. 00:48:05.16 So when we reacted this five day old zebrafish with DIFO-488, 00:48:10.06 there was nothing for the dye to react with 00:48:12.08 so you don't see any fluorescence. 00:48:13.14 Again it testifies to the exquisite selectivity of DIFO 00:48:18.13 for the azide. So basically this experiment 00:48:21.28 demonstrated to us that we can in fact image 00:48:25.03 those GalNAz residues in a live zebrafish. 00:48:29.20 And I should point out that these zebrafish 00:48:32.10 look perfectly normal, perfectly healthy 00:48:34.16 and happy, despite that fact that some fraction 00:48:37.11 of their GalNAc residues were replaced with GalNAz, 00:48:40.14 and that some fraction of those GalNAz residues 00:48:43.29 were chemically reacted with the DIFO reagent. 00:48:47.10 These zebrafish were then released back into their tanks, 00:48:50.17 and they go on to live a normal life as far as we can see. 00:48:53.22 So it doesn't appear to be harmful to the organism 00:48:56.06 which is important if the goal is to learn something 00:48:58.16 about the organism's fundamental biology. 00:49:01.16 We can also do experiments 00:49:03.18 where we probe for changes in the glycome, 00:49:06.03 as a function of time 00:49:07.27 which again, is one of the most interesting 00:49:10.01 aspects of the field of glycobiology. 00:49:11.27 For example, here is an experiment in which we took 00:49:16.02 a zebrafish embryo fertilized in culture 00:49:18.15 We add to the culture media GalNAz, 00:49:21.22 and we let those embryos develop in the presence of the azido sugar 00:49:25.17 Now, at a given time point, 00:49:28.01 we will take those embryos out of the media 00:49:30.15 rinse them off, and react them 00:49:33.11 with DIFO conjugated to a red fluorescent dye. 00:49:36.26 In this case it was Alexafluor 647. 00:49:40.09 Now some glycoproteins, those that were labeled 00:49:44.08 with azides now appear red, 00:49:46.16 but we can take those same embryos 00:49:47.25 that are carrying a fluorescent probe 00:49:49.21 and just put them back in media 00:49:51.09 and let them continue their developmental program. 00:49:54.04 What we do first is a 10 minute reaction 00:49:56.19 with a reagent called TCEP, 00:49:58.16 this is tricarboxydiethylphosphine. 00:50:01.05 What this reagent does is it reduces any 00:50:05.01 unreacted azides to the corresponding amine. 00:50:07.11 And incidentally the chemistry by which TCEP 00:50:10.17 reduces those unreacted azides 00:50:12.16 is classic Staudinger chemistry. 00:50:15.10 We are able to take advantage of that. 00:50:17.25 So we just destroy any unreacted azides. 00:50:20.08 We take these red labeled embryos, 00:50:22.16 put them back into the media containing GalNAz, 00:50:26.03 They continue to develop. 00:50:27.06 They take up more GalNAz as they are doing so. 00:50:29.21 And they'll integrate GalNAz into the next 00:50:32.26 wave of mucin glycoproteins. 00:50:35.06 And now we can take the embryos out 00:50:37.18 and label them again, this time with DIFO 00:50:40.03 conjugated to a green fluorescent dye. 00:50:42.16 So now, at the end of all of this, 00:50:44.29 there are two fluorescent dyes on the zebrafish. 00:50:48.13 The green dye reflect these azides that are 00:50:52.13 in glycoproteins biosynthesized most recently. 00:50:55.11 Whereas the red dye reflects the older population 00:50:58.18 of glycoproteins that were introduced earlier in development. 00:51:02.17 So this allows us to separate old glycoproteins from new glycoproteins 00:51:07.18 based on the color that they appear in the fluorescence microscope. 00:51:11.07 So that kind of an experiment is what led to images like this one 00:51:16.13 which I showed on the very first slide of this second lecture. 00:51:19.13 So this is the head of a 5 day old zebrafish 00:51:24.19 that has been labeled with GalNAz, 00:51:26.24 followed by three different colored fluorescent dyes 00:51:30.11 at three different time points. 00:51:31.17 In this particular image the newest glycoproteins 00:51:35.16 are appearing red, and you can see there's 00:51:37.11 a concentration of those glycoproteins 00:51:39.16 here in the olfactory organs, 00:51:41.22 which are basically the nostrils of the zebrafish. 00:51:45.05 Whereas older populations of glycoproteins appear either blue or green. 00:51:51.03 And you can see that the blue and the green 00:51:52.21 have quite a different distribution from the red. 00:51:55.10 And by looking at how these different colors 00:51:57.14 have moved around as a function of time, 00:51:59.26 we can learn something about the dynamics of the glycome. 00:52:04.02 At least from the perspective of these mucin glycoproteins 00:52:06.27 that we have labeled with GalNAz. 00:52:09.00 In fact one can use a confocal microscope to walk around 00:52:14.05 the labeled organism cell by cell 00:52:15.29 and take a very close look at what has happened 00:52:19.04 to the mucins as a function of time during the development. 00:52:22.21 Just for example, here is a panel of epithelial cells 00:52:26.16 and you can see that each epithelial cell looks as if it has 00:52:29.12 a red labeled membrane with blue and green puncta inside the cell. 00:52:34.26 Which is exactly what we would expect, 00:52:36.27 because in this experiment the red labeling 00:52:40.13 reflects the newest population of glycoproteins. 00:52:43.23 Those are the glycoproteins that just 00:52:45.18 emerged on the plasma membrane 00:52:47.08 with azides ready for us to label. 00:52:49.13 By contrast, older populations of glycoproteins 00:52:52.29 that were biosynthesized many hours before 00:52:55.15 we took this picture, they've already been recycled. 00:52:58.08 by endocytic vesicles in the membrane. 00:53:01.18 And that is why they appear inside the cell. 00:53:04.09 This is a structure that was rather dramatic 00:53:07.09 that appeared at around the 72 hour mark post fertilization. 00:53:12.11 What you are looking at here is a red projection 00:53:14.12 that is basically coming out of the screen 00:53:16.15 at you from the junction of three epithelial cells. 00:53:20.25 That red structure is a mechanosensory hair structure. 00:53:24.22 These are structures that protrude from the side 00:53:27.23 of the head of the embryo, 00:53:29.09 as you can see in this larger scale view. 00:53:32.06 These are structures that 00:53:33.19 the zebrafish can use to sense the flow of current in its environment. 00:53:36.28 for example. 00:53:38.20 And the fact that we captured this structure so dramatically in red 00:53:41.21 tells us first of all that it must contain these mucin like glycoproteins 00:53:47.29 otherwise we wouldn't be able to see it 00:53:49.23 because that is what we are imaging. 00:53:51.29 Secondly, that the majority of those glycoproteins 00:53:54.24 were formed within the window between 00:53:56.23 72 and 73 hours post-fertilization. 00:53:59.27 Which would be a very difficult window to capture 00:54:02.18 using other classic imaging technologies. 00:54:06.01 So these are the kinds of studies that are basically opening the window 00:54:10.01 through the vision of sugars into developmental biology. 00:54:14.10 And I think this is a very interesting 00:54:16.04 future application of the technology. 00:54:17.27 So what should you take home from this second lecture 00:54:23.02 in this series. First of all, there is a chemical lesson here, 00:54:26.11 which really has nothing to do with sugars. 00:54:28.15 It's the fact that, you know, we've taken advantage 00:54:31.13 of some very classic chemical tools. 00:54:34.08 These are chemical reaction that were first reported 00:54:37.20 as early as 1915. That is almost one hundred years ago. 00:54:41.15 And these old classic chemistries: the Staudinger chemistry 00:54:44.29 and the Huisgen cycloaddition chemistry 00:54:46.23 we have found to be real gems for modern applications in biology. 00:54:52.22 So by digging into the old chemical literature, 00:54:55.29 one can find some incredible tools 00:54:58.17 to bring forth into the modern era of biological research. 00:55:02.09 And that's very exciting for someone who was trained as a chemist 00:55:05.15 and at heart considers herself a chemist. 00:55:08.17 Secondly, you should remember the azide. 00:55:11.11 The azide has phenomenal properties 00:55:15.00 that make it so well suited for these applications 00:55:18.15 as chemical reporters. 00:55:19.25 And we think that the azide 00:55:21.13 has a lot of potential not just for imaging sugars, 00:55:24.09 the way that we do it, but for imaging 00:55:26.11 all kinds of interesting biomolecules. 00:55:28.26 including proteins, lipids, nucleic acids, 00:55:32.12 metabolites, things that are not necessarily encoded in the genome, 00:55:37.08 for which the GFP and other genetic reporters are really not well suited. 00:55:43.11 Third we now have shown that metabolic labeling with azido sugars 00:55:48.22 allows you to image the glycans in living systems. 00:55:51.23 And we think this will allow scientists 00:55:53.29 to study glycobiology using all of the hardware of molecular imaging 00:55:59.28 that so far has focused primarily on the study of proteins. 00:56:04.11 And finally, where we are going with this technology in my laboratory 00:56:10.04 is to continue our studies of developmental biologies 00:56:12.19 until we can understand how the glycome contributes 00:56:15.11 to some of the early cell fate decision making processes 00:56:18.12 during the embryogenic program. 00:56:20.10 For example, how does the glycome change 00:56:22.18 when pluripotent embryonic stem cells are just starting 00:56:27.12 to choose their ultimate differentiation fate. 00:56:30.06 We are hoping that we can capture a snapshot 00:56:32.22 of that moment from the perspective of the glycome. 00:56:35.20 And then finally, you know, one of our original motivations 00:56:38.27 was to develop these tools in a manner that 00:56:41.22 could be clinically useful. So we are hoping that 00:56:44.29 we can develop the chemical reagents 00:56:47.09 the azido-sugars in such a manner that you could 00:56:50.18 actually introduce these reagents into human subjects. 00:56:54.01 And use them to detect tumors at early stages 00:56:57.06 by virtue of their changing glycome. 00:57:00.01 So stay tuned, and hopefully we will have a lot more to say 00:57:03.02 about that in the future. 00:57:04.20 Finally I'd like to acknowledge the students 00:57:08.26 and postdoctoral fellows in my laboratory. 00:57:10.21 What you are seeing on this slide 00:57:12.17 is a snapshot of my laboratory at this particular moment in time 00:57:16.21 where I am showing you all of the students 00:57:19.02 and postdocs who are presently in the lab 00:57:20.25 although I have listed just a small list of alumni 00:57:24.10 whose work I alluded to during this lecture. 00:57:27.27 Most of their names were actually mentioned on the slides 00:57:30.19 along with reference to publications if you are interested in 00:57:33.10 digging into this in more gory detail. 00:57:36.07 But it should be said that all of the work that I presented 00:57:39.04 from my laboratory is in fact the hands-on research 00:57:43.10 of my students and postdoctoral fellows and I am 00:57:46.27 largely the mouthpiece that has the privilege of sharing this with the world. 00:57:50.29 So, thank you very much for tuning in, and I hope you enjoyed learning a little bit 00:57:55.04 about chemical glycobiology.