1 00:00:00,500 --> 00:00:02,810 The following content is provided under a Creative 2 00:00:02,810 --> 00:00:04,380 Commons license. 3 00:00:04,380 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,010 continue to offer high-quality educational resources for free. 5 00:00:11,010 --> 00:00:13,670 To make a donation or view additional materials 6 00:00:13,670 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,800 at ocw.mit.edu. 8 00:00:25,800 --> 00:00:29,130 JOANNE STUBBE: So we're talking about cholesterol homeostasis. 9 00:00:29,130 --> 00:00:31,770 And I said at the very beginning, 10 00:00:31,770 --> 00:00:36,030 the first two lectures are going to be focused on the terpenome 11 00:00:36,030 --> 00:00:38,430 and how you make cholesterol. 12 00:00:38,430 --> 00:00:44,100 And in the beginning in Monday's lecture, 13 00:00:44,100 --> 00:00:46,170 we had gotten through the first few steps 14 00:00:46,170 --> 00:00:50,490 in cholesterol biosynthesis, starting with acetyl CoA. 15 00:00:50,490 --> 00:00:53,220 And we'd gotten up to the position where 16 00:00:53,220 --> 00:00:57,240 we had condensed three molecules of acetyl CoA using 17 00:00:57,240 --> 00:01:01,020 Claisen and aldol reactions to form this molecule, 18 00:01:01,020 --> 00:01:03,090 hydroxymethyl glutaryl CoA. 19 00:01:03,090 --> 00:01:05,069 And at the end of the last lecture, 20 00:01:05,069 --> 00:01:09,600 we were talking about HMG CoA reductase, which 21 00:01:09,600 --> 00:01:12,150 is abbreviated in the Brown and Goldstein papers 22 00:01:12,150 --> 00:01:16,170 like that, which requires two molecules. 23 00:01:16,170 --> 00:01:17,910 This should be "NADPH." 24 00:01:17,910 --> 00:01:20,970 I announced that before all of the notes. 25 00:01:20,970 --> 00:01:23,910 "NADH" should be replaced with "NADPH." 26 00:01:23,910 --> 00:01:26,340 We're doing biosynthesis. 27 00:01:26,340 --> 00:01:33,630 We talked about the mechanism of how you go from this CoA analog 28 00:01:33,630 --> 00:01:38,070 to a double reduction to form an alcohol. 29 00:01:38,070 --> 00:01:41,993 And the question is, why is that interesting and important? 30 00:01:41,993 --> 00:01:43,410 And it's interesting and important 31 00:01:43,410 --> 00:01:49,930 because it's the major target of $30 billion drugs, the statins, 32 00:01:49,930 --> 00:01:54,300 which specifically target HMG CoA reductase. 33 00:01:54,300 --> 00:01:56,205 And so what I want to do-- 34 00:01:56,205 --> 00:01:58,080 I'm not going to spend a lot of time on this, 35 00:01:58,080 --> 00:01:59,830 but I want to say a little bit about this, 36 00:01:59,830 --> 00:02:06,690 given its central role in many people's health nowadays. 37 00:02:06,690 --> 00:02:12,360 So how do these analogs end up working? 38 00:02:12,360 --> 00:02:16,680 And so we have an intermediate in this process, which 39 00:02:16,680 --> 00:02:20,890 I drew out in detail last time. 40 00:02:20,890 --> 00:02:22,860 I'm not going to draw it out again. 41 00:02:22,860 --> 00:02:25,050 So we're going to go through two reductions. 42 00:02:25,050 --> 00:02:29,880 The first reduction forms a thiohemiacetal, 43 00:02:29,880 --> 00:02:32,250 which then kicks out CoA to form the aldehyde, which 44 00:02:32,250 --> 00:02:33,360 then gets reduced again. 45 00:02:33,360 --> 00:02:35,050 I'm not going to write that out. 46 00:02:35,050 --> 00:02:37,620 This is the first intermediate you see. 47 00:02:37,620 --> 00:02:42,140 And what we'll see is the inhibitors 48 00:02:42,140 --> 00:02:45,590 all look like this intermediate. 49 00:02:45,590 --> 00:02:47,750 So when they look like the normal substrate 50 00:02:47,750 --> 00:02:50,900 or an intermediate along the pathway, 51 00:02:50,900 --> 00:02:53,420 those are called "competitive inhibitors." 52 00:02:53,420 --> 00:02:57,080 So these are competitive inhibitors. 53 00:02:57,080 --> 00:02:59,510 If you don't remember what a competitive inhibitor is 54 00:02:59,510 --> 00:03:01,120 or how it's described, you might want 55 00:03:01,120 --> 00:03:03,940 to go back and look it up in Voet 56 00:03:03,940 --> 00:03:08,000 or whatever your basic biochemistry textbook is. 57 00:03:08,000 --> 00:03:14,090 And so what we'll see is all of the compounds that are actually 58 00:03:14,090 --> 00:03:17,570 used clinically look like this. 59 00:03:20,540 --> 00:03:25,670 And so if you look at this, they're not exactly the same. 60 00:03:25,670 --> 00:03:30,870 But it's proposed to be a model of this particular intermediate 61 00:03:30,870 --> 00:03:31,710 in the reaction. 62 00:03:31,710 --> 00:03:35,000 So this is the thiohemiacetal. 63 00:03:35,000 --> 00:03:39,620 But here, I've drawn a lactone rather than an acid. 64 00:03:39,620 --> 00:03:42,350 And in general-- whoops. 65 00:03:42,350 --> 00:03:45,560 In general, lactones are actually 66 00:03:45,560 --> 00:03:48,830 given as the drug, rather than the acid. 67 00:03:48,830 --> 00:03:52,550 And does anybody have any clue as to why that might be true? 68 00:03:55,480 --> 00:03:59,830 Why would you want to use this molecule, as opposed 69 00:03:59,830 --> 00:04:07,145 to the ring-opened species, which would look like this? 70 00:04:07,145 --> 00:04:08,075 AUDIENCE: Uptake. 71 00:04:08,075 --> 00:04:10,387 JOANNE STUBBE: Pardon me? 72 00:04:10,387 --> 00:04:11,720 AUDIENCE: I said, uptake issues. 73 00:04:11,720 --> 00:04:11,960 JOANNE STUBBE: Yeah. 74 00:04:11,960 --> 00:04:13,190 So it's uptake issues. 75 00:04:13,190 --> 00:04:15,020 So what happens is you give this-- 76 00:04:15,020 --> 00:04:19,640 and you'll see this is true also in the last section 77 00:04:19,640 --> 00:04:20,899 in purines and pyrimidines. 78 00:04:20,899 --> 00:04:23,030 How you deliver the drug, it needs 79 00:04:23,030 --> 00:04:24,650 to be able to get across the membrane 80 00:04:24,650 --> 00:04:26,720 in an efficient fashion. 81 00:04:26,720 --> 00:04:30,380 And so they give them lactone, but the lactone rapidly 82 00:04:30,380 --> 00:04:34,120 ring-opens inside the cell. 83 00:04:34,120 --> 00:04:35,323 So that's the analog. 84 00:04:35,323 --> 00:04:36,990 I don't know if you can see it, but it's 85 00:04:36,990 --> 00:04:38,550 the same analog I've drawn up there 86 00:04:38,550 --> 00:04:41,280 and I drew last time on the board. 87 00:04:41,280 --> 00:04:43,080 And then these are the drugs. 88 00:04:43,080 --> 00:04:44,910 And so the drug-- 89 00:04:44,910 --> 00:04:47,920 I've written this part looks like this part. 90 00:04:47,920 --> 00:04:51,000 So this is where the competitive part comes from. 91 00:04:51,000 --> 00:04:52,410 And what's down here? 92 00:04:52,410 --> 00:04:56,040 Down here is, remember, the CoA. 93 00:04:56,040 --> 00:04:59,100 And down here in the drugs is stuff. 94 00:04:59,100 --> 00:05:03,540 And the key to this stuff is hydrophobicity. 95 00:05:03,540 --> 00:05:09,330 And so the key to almost all drugs is hydrophobicity. 96 00:05:09,330 --> 00:05:12,450 So it can't be so hydrophobic that it's insoluble. 97 00:05:12,450 --> 00:05:14,982 But lots of times, you have pockets 98 00:05:14,982 --> 00:05:16,690 that you can't see in protein structures. 99 00:05:16,690 --> 00:05:20,190 It inserts itself in, and you get a lot of binding energy. 100 00:05:20,190 --> 00:05:22,290 So if you look at these, these, again, 101 00:05:22,290 --> 00:05:24,750 are on the PowerPoint presentation. 102 00:05:24,750 --> 00:05:27,390 What you see here, these are all the ring-opened here 103 00:05:27,390 --> 00:05:29,100 and ring-closed. 104 00:05:29,100 --> 00:05:32,990 And then if you look down here, what you see is all the stuff. 105 00:05:32,990 --> 00:05:36,630 And you see the stuff is dramatically different. 106 00:05:36,630 --> 00:05:38,970 You can look at it, but each company 107 00:05:38,970 --> 00:05:45,040 has tried to get its cut of the $30 billion market. 108 00:05:45,040 --> 00:05:47,040 And what I'm going to show you on the next slide 109 00:05:47,040 --> 00:05:48,810 is this molecule. 110 00:05:48,810 --> 00:05:50,400 So again, the key thing is what's 111 00:05:50,400 --> 00:05:57,480 common to all of these inhibitors is this moiety. 112 00:05:57,480 --> 00:05:59,640 And when you look at the structures, 113 00:05:59,640 --> 00:06:02,100 you can see this guy. 114 00:06:02,100 --> 00:06:05,580 And then this guy will have slightly different orientations 115 00:06:05,580 --> 00:06:07,650 within the structures. 116 00:06:07,650 --> 00:06:12,270 So if you look at the structures of HMG CoA reductase, 117 00:06:12,270 --> 00:06:15,115 what you can see-- and if you pull this up and stare at it, 118 00:06:15,115 --> 00:06:15,990 it looks like a mess. 119 00:06:15,990 --> 00:06:18,870 But it really isn't a mess. 120 00:06:18,870 --> 00:06:20,730 If you look at this, you're going 121 00:06:20,730 --> 00:06:23,130 to see in all the structures, here's the carboxylate. 122 00:06:23,130 --> 00:06:24,900 Here's a carboxylate. 123 00:06:24,900 --> 00:06:26,910 And this is the hydroxyl group. 124 00:06:26,910 --> 00:06:30,660 So that's the key part over here, the carboxylate 125 00:06:30,660 --> 00:06:33,660 in the hydroxyl group. 126 00:06:33,660 --> 00:06:37,210 There in the hemiacetal, the carboxylate's not there yet. 127 00:06:37,210 --> 00:06:40,380 So you can't put in the active species, or they turn over. 128 00:06:40,380 --> 00:06:43,470 So what you're doing is throwing in components 129 00:06:43,470 --> 00:06:45,300 to prevent turnover but to give you 130 00:06:45,300 --> 00:06:48,650 a feeling for where the different substrates bind. 131 00:06:48,650 --> 00:06:51,690 CoA is not attached to this moiety, 132 00:06:51,690 --> 00:06:55,170 but CoA is attached here. 133 00:06:55,170 --> 00:06:57,060 And I think what's unusual about this-- 134 00:06:57,060 --> 00:06:59,430 I don't know if you looked at any structures 135 00:06:59,430 --> 00:07:01,180 in the polyketide synthases. 136 00:07:01,180 --> 00:07:04,410 But you would think at the end of CoA, 137 00:07:04,410 --> 00:07:07,470 if you look at the structure, it has an adenine moiety on it. 138 00:07:07,470 --> 00:07:10,620 And you would think you would get a lot of binding energy 139 00:07:10,620 --> 00:07:13,080 onto this hydrophobic adenine moiety, which 140 00:07:13,080 --> 00:07:14,630 can also hydrogen-bond. 141 00:07:14,630 --> 00:07:16,950 And almost all structures to that part of the molecule 142 00:07:16,950 --> 00:07:18,570 never bind to the protein. 143 00:07:18,570 --> 00:07:20,320 It's stuck out in solution. 144 00:07:20,320 --> 00:07:23,190 So this is sort of typical. 145 00:07:23,190 --> 00:07:25,740 Why nature designed things this way, I don't know. 146 00:07:25,740 --> 00:07:26,970 She's got this huge-- 147 00:07:26,970 --> 00:07:29,585 she could have used a thiomethyl group, 148 00:07:29,585 --> 00:07:31,710 and the chemistry would have been exactly the same. 149 00:07:31,710 --> 00:07:35,140 But she uses this huge CoA moiety. 150 00:07:35,140 --> 00:07:37,560 And in most cases, you see the chain extended 151 00:07:37,560 --> 00:07:40,530 and the adenine on the outside. 152 00:07:40,530 --> 00:07:42,360 So if you look at that, the chemistry 153 00:07:42,360 --> 00:07:44,080 is going to come over here. 154 00:07:44,080 --> 00:07:46,980 And if you look at this yellow, yellow is sulfur. 155 00:07:46,980 --> 00:07:50,010 So that's where the sulfur would be connected 156 00:07:50,010 --> 00:07:52,440 to the hydroxymethyl glutarate. 157 00:07:52,440 --> 00:07:54,090 And then what do you see over here? 158 00:07:54,090 --> 00:07:55,380 Hopefully, you can see this. 159 00:07:55,380 --> 00:07:57,600 But this is another adenine ring. 160 00:07:57,600 --> 00:08:00,975 And then over here is the pyridine ring. 161 00:08:00,975 --> 00:08:02,350 And we went through the mechanism 162 00:08:02,350 --> 00:08:04,590 last time that transfers the hydride. 163 00:08:04,590 --> 00:08:08,360 So this green part is the redundant, 164 00:08:08,360 --> 00:08:15,090 and this part is a mimic of the hydroxymethyl glutaryl CoA. 165 00:08:15,090 --> 00:08:16,680 And in all cases-- 166 00:08:16,680 --> 00:08:19,260 we have hundreds of structures now-- 167 00:08:19,260 --> 00:08:22,890 what you will see if you look at the inhibitor binding 168 00:08:22,890 --> 00:08:26,280 is you have changes in conformation in this region. 169 00:08:26,280 --> 00:08:30,240 And the Km for binding of hydroxymethyl glutaryl CoA 170 00:08:30,240 --> 00:08:31,770 is 4 micromolar. 171 00:08:31,770 --> 00:08:33,809 But the KD for binding of the inhibitors 172 00:08:33,809 --> 00:08:36,480 is about a nanomolar. 173 00:08:36,480 --> 00:08:39,830 So you're gaining a lot by sticking this hydrophobic mess 174 00:08:39,830 --> 00:08:40,919 on. 175 00:08:40,919 --> 00:08:45,785 And what happens-- so here's, again, this hydrophobic mess. 176 00:08:45,785 --> 00:08:47,160 And what you can see, this is one 177 00:08:47,160 --> 00:08:49,980 of the analogs I had before. 178 00:08:49,980 --> 00:08:54,360 Again, hydroxymethyl glutarate-- the glutarate and the two 179 00:08:54,360 --> 00:08:56,520 carboxylates are there. 180 00:08:56,520 --> 00:08:59,940 And then they stick on something hydrophobic 181 00:08:59,940 --> 00:09:01,750 in this part of the molecule. 182 00:09:01,750 --> 00:09:04,440 And if you look at the conformation 183 00:09:04,440 --> 00:09:06,480 of these helices in this region-- 184 00:09:06,480 --> 00:09:09,270 and you really have to look at the three-dimensional structure 185 00:09:09,270 --> 00:09:11,190 to see something-- that's the region where 186 00:09:11,190 --> 00:09:12,440 you see changes in binding. 187 00:09:12,440 --> 00:09:16,650 So it's an induced fit mechanism of binding. 188 00:09:16,650 --> 00:09:21,000 And in fact, this induced fit occurs 189 00:09:21,000 --> 00:09:23,790 in all of the analogs that are looked at. 190 00:09:23,790 --> 00:09:26,408 And so if you look here-- 191 00:09:26,408 --> 00:09:27,450 and you can look at this. 192 00:09:27,450 --> 00:09:31,380 I would say take it home and spend some time looking at it. 193 00:09:31,380 --> 00:09:36,000 Again, this part of the molecule in all of these analogs 194 00:09:36,000 --> 00:09:37,380 is exactly the same. 195 00:09:37,380 --> 00:09:41,880 And what you had different is this hydrophobic mess 196 00:09:41,880 --> 00:09:44,970 in this part of the molecule and changes 197 00:09:44,970 --> 00:09:47,130 in this region of binding. 198 00:09:47,130 --> 00:09:50,010 And people are still working on it, trying to make-- 199 00:09:50,010 --> 00:09:52,020 usually, it's the first couple of drugs 200 00:09:52,020 --> 00:09:53,130 that make all the money. 201 00:09:53,130 --> 00:09:55,830 And if you're third or fourth, you don't make enough money. 202 00:09:55,830 --> 00:10:00,300 But there are lots of problems that keep coming up. 203 00:10:00,300 --> 00:10:02,340 And so people are still really heavily focused 204 00:10:02,340 --> 00:10:06,150 on trying to lower cholesterol levels. 205 00:10:06,150 --> 00:10:10,010 So HMG CoA reductase-- 206 00:10:10,010 --> 00:10:14,760 I told you last time, it's a huge protein, 880 amino acids. 207 00:10:14,760 --> 00:10:17,730 Half of it's stuck in the ER. 208 00:10:17,730 --> 00:10:21,300 You can cut off the-- the other half that is soluble 209 00:10:21,300 --> 00:10:23,580 is in the cytosol. 210 00:10:23,580 --> 00:10:25,110 And we're going to come back to this 211 00:10:25,110 --> 00:10:29,800 because this rate-limiting step plays a key role in sensing 212 00:10:29,800 --> 00:10:31,160 of cholesterol levels. 213 00:10:31,160 --> 00:10:35,280 So the end of lecture three and into lecture four, 214 00:10:35,280 --> 00:10:39,750 we're going to come back to HMG CoA reductase because 215 00:10:39,750 --> 00:10:43,410 of its central role in cholesterol homeostasis. 216 00:10:43,410 --> 00:10:44,940 So we're not here yet. 217 00:10:44,940 --> 00:10:50,910 Remember, the goal of the terpenome 218 00:10:50,910 --> 00:10:52,620 was to get to the building blocks. 219 00:10:52,620 --> 00:10:54,780 We still haven't gotten to the building blocks yet. 220 00:10:54,780 --> 00:10:56,650 What were the building blocks? 221 00:10:56,650 --> 00:11:00,300 Isopentenyl and dimethylallyl pyrophosphate. 222 00:11:00,300 --> 00:11:04,560 Remember, the common building block is an isoprene, 223 00:11:04,560 --> 00:11:07,200 but the isoprene is not the reactive species 224 00:11:07,200 --> 00:11:09,980 we needed to get it into some form 225 00:11:09,980 --> 00:11:11,730 where you can actually do chemistry on it. 226 00:11:11,730 --> 00:11:18,210 So our goal has been to get to IPP, and we are here. 227 00:11:18,210 --> 00:11:25,800 So the rest of the biosynthetic pathway to get to IPP 228 00:11:25,800 --> 00:11:28,470 is pretty straightforward. 229 00:11:28,470 --> 00:11:31,620 I'm not going to draw out the details at all. 230 00:11:31,620 --> 00:11:35,930 But we go from mevalonic acid. 231 00:11:35,930 --> 00:11:40,710 And so we use ATP and a kinase. 232 00:11:40,710 --> 00:11:47,760 And we use a second ATP and a kinase. 233 00:11:47,760 --> 00:11:53,220 And then we use a third ATP. 234 00:11:53,220 --> 00:11:56,820 And so if you look at the pathway here, what happens 235 00:11:56,820 --> 00:12:02,010 is you're phosphorylating the alcohol that we just 236 00:12:02,010 --> 00:12:05,160 created with HMG CoA reductase. 237 00:12:05,160 --> 00:12:07,680 So we're sticking a phosphate on. 238 00:12:07,680 --> 00:12:11,340 Another ATP sticks a second phosphate on. 239 00:12:11,340 --> 00:12:16,660 And in the end, we need to get to isopentenyl pyrophosphate. 240 00:12:16,660 --> 00:12:19,860 And so we have a third enzyme that 241 00:12:19,860 --> 00:12:25,380 is going to phosphorylate to facilitate, finally, conversion 242 00:12:25,380 --> 00:12:32,450 of the C6, three acetyl CoAs, into the C5 isopentenyl 243 00:12:32,450 --> 00:12:34,410 pyrophosphate. 244 00:12:34,410 --> 00:12:37,710 So if you look at what's going on in that reaction, 245 00:12:37,710 --> 00:12:39,330 we have a third ATP. 246 00:12:39,330 --> 00:12:53,250 And the ATP is used to phosphorylate the alcohol. 247 00:12:53,250 --> 00:12:56,010 So what we're doing basically is making it 248 00:12:56,010 --> 00:12:57,420 into a good leaving group. 249 00:13:00,060 --> 00:13:02,070 And we've got the two phosphates on there 250 00:13:02,070 --> 00:13:04,740 by the first two kinases. 251 00:13:04,740 --> 00:13:06,220 And so now what we want to do-- 252 00:13:06,220 --> 00:13:07,530 I forgot a methyl group here. 253 00:13:10,540 --> 00:13:15,400 And so now what we've done by phosphorylating this is we've 254 00:13:15,400 --> 00:13:18,005 activated this for a decarboxylative elimination 255 00:13:18,005 --> 00:13:18,505 reaction. 256 00:13:23,110 --> 00:13:24,910 And so now, where are we? 257 00:13:24,910 --> 00:13:32,710 We're now finally at isopentenyl pyrophosphate. 258 00:13:32,710 --> 00:13:35,350 So we've gotten to our C5. 259 00:13:35,350 --> 00:13:39,760 And the key thing, remember, is we started with acetyl CoA. 260 00:13:39,760 --> 00:13:44,440 During this reaction, we lose CO2. 261 00:13:44,440 --> 00:13:48,670 And that's why we've gone from a C6 to a C5 262 00:13:48,670 --> 00:13:49,630 during this reaction. 263 00:13:49,630 --> 00:13:51,010 We lose that. 264 00:13:51,010 --> 00:13:53,360 And again, we see ATP used over and over again. 265 00:13:53,360 --> 00:13:55,510 And GTP, you see the same thing. 266 00:13:55,510 --> 00:13:59,110 It's used to make things into better leaving groups 267 00:13:59,110 --> 00:14:02,140 and facilitate the overall chemistry. 268 00:14:02,140 --> 00:14:05,200 So I'm not going to talk again about the details of any 269 00:14:05,200 --> 00:14:07,123 of these steps. 270 00:14:07,123 --> 00:14:08,540 The steps are all straightforward. 271 00:14:08,540 --> 00:14:10,630 You've seen these steps in primary metabolism 272 00:14:10,630 --> 00:14:13,480 with the role of ATP over and over again. 273 00:14:13,480 --> 00:14:18,040 But the key thing we want to talk about is the terpenome. 274 00:14:18,040 --> 00:14:20,530 And to get to the terpenome, we needed 275 00:14:20,530 --> 00:14:25,270 to get to isopentenyl pyrophosphate and dimethylallyl 276 00:14:25,270 --> 00:14:29,680 pyrophosphate so that we can look 277 00:14:29,680 --> 00:14:35,010 at the new way of forming carbon-carbon bonds with C5 278 00:14:35,010 --> 00:14:37,030 units. 279 00:14:37,030 --> 00:14:40,840 So we've gotten through the first few steps. 280 00:14:40,840 --> 00:14:43,090 That's what I call the "initiation process." 281 00:14:43,090 --> 00:14:48,460 We started with acetyl CoA and got to IPP, 282 00:14:48,460 --> 00:14:52,213 dimethylallyl pyrophosphate. 283 00:14:52,213 --> 00:14:54,130 If you look at these hydrogens, hopefully, you 284 00:14:54,130 --> 00:14:56,860 know allylic hydrogens are moderately acidic. 285 00:14:56,860 --> 00:15:01,870 And there's an isomerase that can convert this molecule 286 00:15:01,870 --> 00:15:03,650 into this molecule. 287 00:15:03,650 --> 00:15:06,790 And so this is dimethylallyl pyrophosphate. 288 00:15:06,790 --> 00:15:12,010 And we're into the second part of the biosynthetic pathway 289 00:15:12,010 --> 00:15:12,950 for cholesterol. 290 00:15:12,950 --> 00:15:14,320 So we're through the initiation. 291 00:15:14,320 --> 00:15:16,210 We've got our building blocks. 292 00:15:16,210 --> 00:15:20,830 Now, what we want to do is do the elongation step. 293 00:15:20,830 --> 00:15:22,870 And so now we're going to use this. 294 00:15:22,870 --> 00:15:30,670 So IPP-- we're now going to look at the elongation reactions. 295 00:15:30,670 --> 00:15:33,730 And I guess I'll use a second board over here. 296 00:15:33,730 --> 00:15:36,370 And so let me do it over here, and then I'll do the next one. 297 00:15:36,370 --> 00:15:45,783 And so what we want to do then is take C5 plus C5. 298 00:15:49,850 --> 00:15:53,200 And we're going to look at this reaction in detail. 299 00:15:53,200 --> 00:15:58,450 And the enzyme that's going to do this 300 00:15:58,450 --> 00:16:04,180 is called FPP synthase, farnesyl pyrophosphate synthase. 301 00:16:04,180 --> 00:16:06,190 I'll write that down in a minute. 302 00:16:06,190 --> 00:16:08,440 And you form C10. 303 00:16:11,170 --> 00:16:15,770 And C10-- then this is the same enzyme. 304 00:16:15,770 --> 00:16:18,790 So it's also FPP synthase. 305 00:16:18,790 --> 00:16:22,140 FPP is the product, farnesyl pyrophosphate. 306 00:16:22,140 --> 00:16:27,970 And IPP gives us C15. 307 00:16:27,970 --> 00:16:31,000 So this is a major elongation reaction. 308 00:16:31,000 --> 00:16:36,310 And what I want you to see is that this C15, three C5s stuck 309 00:16:36,310 --> 00:16:38,500 together, is linear. 310 00:16:38,500 --> 00:16:43,900 And if you go back and you look at your notes from last time, 311 00:16:43,900 --> 00:16:46,600 we talked about isopernoids and terpenoids. 312 00:16:46,600 --> 00:16:48,850 And you can make linear molecules 313 00:16:48,850 --> 00:16:51,940 that can go from a couple of units, 314 00:16:51,940 --> 00:16:54,670 like geranyl, geranyl, C10-- 315 00:16:54,670 --> 00:16:56,470 sorry-- geranyl pyrophosphate. 316 00:16:56,470 --> 00:16:57,700 That's C10. 317 00:16:57,700 --> 00:17:01,210 That's called a "monoterpene." 318 00:17:01,210 --> 00:17:06,490 And you can add another C5, isopentenyl pyrophosphate. 319 00:17:06,490 --> 00:17:07,930 That's a C15. 320 00:17:07,930 --> 00:17:10,579 That's called a "sesquiterpene." 321 00:17:10,579 --> 00:17:12,430 "Sesqui" comes from 1 and 1/2. 322 00:17:12,430 --> 00:17:15,599 So what you'll see in the next thing, if you add another five, 323 00:17:15,599 --> 00:17:16,839 you have a C20. 324 00:17:16,839 --> 00:17:18,400 That's a diterpene. 325 00:17:18,400 --> 00:17:21,880 And if you go to a C30, that's a triterpene. 326 00:17:21,880 --> 00:17:24,710 You can google it, but the nomenclature's complicated. 327 00:17:24,710 --> 00:17:28,930 But that's where they come from is the different C5 units. 328 00:17:28,930 --> 00:17:35,740 So really, this chemistry is the basis for all the reactions 329 00:17:35,740 --> 00:17:37,288 in the terpenome. 330 00:17:37,288 --> 00:17:39,580 So what we're going to do is go through that chemistry. 331 00:17:39,580 --> 00:17:42,250 How do you form a new carbon-carbon bond 332 00:17:42,250 --> 00:17:43,640 using these building blocks? 333 00:17:43,640 --> 00:17:45,550 What are the general principles? 334 00:17:45,550 --> 00:17:47,650 And I showed you the hundreds of different kinds 335 00:17:47,650 --> 00:17:49,630 of natural products that you can find 336 00:17:49,630 --> 00:17:52,350 in humans and plants and bacteria all over the place. 337 00:17:52,350 --> 00:17:53,980 They play an incredibly important role 338 00:17:53,980 --> 00:17:56,920 in primary and secondary metabolism. 339 00:17:56,920 --> 00:17:59,680 And what we're going to look at is the general way 340 00:17:59,680 --> 00:18:02,440 that these carbon-carbon bonds are made. 341 00:18:06,030 --> 00:18:06,760 All right. 342 00:18:06,760 --> 00:18:11,770 So again, let me stress that this is linear. 343 00:18:11,770 --> 00:18:14,800 And you'll see that when we actually look at this. 344 00:18:14,800 --> 00:18:20,500 So FPP-- let me write this down. 345 00:18:20,500 --> 00:18:25,390 So it's farnesyl PP, pyrophosphate. 346 00:18:25,390 --> 00:18:27,640 We talked about this last time. 347 00:18:27,640 --> 00:18:30,690 It's a central player in many, many, many reactions, 348 00:18:30,690 --> 00:18:32,770 and it's a C15. 349 00:18:32,770 --> 00:18:37,750 So the farnesyl pyrophosphate synthase 350 00:18:37,750 --> 00:18:41,650 was the first enzyme characterized 351 00:18:41,650 --> 00:18:45,400 for parental transfer reactions, for these C5-forming reactions. 352 00:18:45,400 --> 00:18:50,320 It's been studied extremely extensively by Dale Poulter's 353 00:18:50,320 --> 00:18:53,050 lab at Utah, and it's served as a paradigm, 354 00:18:53,050 --> 00:18:55,880 really, for thinking about all of the biochemistry. 355 00:18:55,880 --> 00:18:59,336 Did any of you guys ever hear of Saul Winstein? 356 00:18:59,336 --> 00:18:59,836 No. 357 00:18:59,836 --> 00:19:01,380 It shows how old I am. 358 00:19:01,380 --> 00:19:05,100 Anyhow, Saul Winstein was a faculty member 359 00:19:05,100 --> 00:19:09,360 at UCLA many years ago, probably in the 1970s. 360 00:19:09,360 --> 00:19:12,810 But if you've taken 5.43, hopefully, they still 361 00:19:12,810 --> 00:19:13,920 talk about-- 362 00:19:13,920 --> 00:19:17,310 or what's the advanced physical organic chemistry 363 00:19:17,310 --> 00:19:18,360 course you guys take? 364 00:19:18,360 --> 00:19:19,980 Any of you'd had that? 365 00:19:19,980 --> 00:19:22,370 Anyhow, you've had-- have you heard about Saul? 366 00:19:22,370 --> 00:19:24,780 You've never heard of Saul-- bad, bad. 367 00:19:24,780 --> 00:19:27,590 Anyhow, he's the one that figured out 368 00:19:27,590 --> 00:19:31,860 how to think about classical and non-classical carbocations. 369 00:19:31,860 --> 00:19:33,370 And Dale Poulter worked from him. 370 00:19:33,370 --> 00:19:36,420 Dale Poulter moved into enzymatic reaction systems 371 00:19:36,420 --> 00:19:39,270 and really sort of unravelled how these things work. 372 00:19:39,270 --> 00:19:41,880 And the paradigm I'm going to give you-- every enzyme's 373 00:19:41,880 --> 00:19:42,493 different. 374 00:19:42,493 --> 00:19:44,160 But the paradigm I'm going to give you I 375 00:19:44,160 --> 00:19:46,740 really think came partially founded 376 00:19:46,740 --> 00:19:50,520 on the physical organic chemistry and from Dale's lab. 377 00:19:50,520 --> 00:19:53,170 So these are pretty important contributions. 378 00:19:53,170 --> 00:20:00,510 And what we'll see is this is called a "type I synthase." 379 00:20:00,510 --> 00:20:03,400 And if you read the assigned reading, 380 00:20:03,400 --> 00:20:05,610 you'll see there are type II synthases. 381 00:20:05,610 --> 00:20:09,900 So there's more than one structure 382 00:20:09,900 --> 00:20:12,060 of the enzymes involved in these systems. 383 00:20:12,060 --> 00:20:16,050 We're going to specifically focus in class 384 00:20:16,050 --> 00:20:17,820 on the type I synthase. 385 00:20:17,820 --> 00:20:20,685 And it's basically an alpha helical bundle. 386 00:20:26,490 --> 00:20:30,110 And I think on the next-- if I could remember what I have. 387 00:20:30,110 --> 00:20:30,630 Yeah. 388 00:20:30,630 --> 00:20:32,490 So this was taken out of the article 389 00:20:32,490 --> 00:20:35,100 you are supposed to read. 390 00:20:35,100 --> 00:20:37,260 And so this is FPP synthase. 391 00:20:37,260 --> 00:20:39,630 This is a monomer, but it's a dimer. 392 00:20:39,630 --> 00:20:46,350 And all I want you to see if you take the 30,000-foot view, 393 00:20:46,350 --> 00:20:48,150 there are five helices here. 394 00:20:48,150 --> 00:20:50,130 They're in red. 395 00:20:50,130 --> 00:20:53,740 If you look at this long helix, it's everywhere. 396 00:20:53,740 --> 00:20:56,820 Everything's a little bit juggled around. 397 00:20:56,820 --> 00:20:58,695 You can see you have a couple blues here 398 00:20:58,695 --> 00:20:59,820 and a couple of blues here. 399 00:20:59,820 --> 00:21:02,920 So they're structurally homologous to each other. 400 00:21:02,920 --> 00:21:05,490 And I think what's most remarkable about this-- 401 00:21:05,490 --> 00:21:11,760 so FPP synthase takes two C5s, makes a C10-- this is a C15. 402 00:21:11,760 --> 00:21:14,010 So it's a linear. 403 00:21:14,010 --> 00:21:16,830 Squalene synthase, which we'll look at in a minute, 404 00:21:16,830 --> 00:21:20,670 takes two C15s and makes a C30, the precursor 405 00:21:20,670 --> 00:21:23,310 to making the ring structure for cholesterol. 406 00:21:23,310 --> 00:21:26,640 But these two guys in the middle, which look-- 407 00:21:26,640 --> 00:21:28,440 and this is linear, as well. 408 00:21:28,440 --> 00:21:31,290 These two guys in the middle, which look remarkably similar-- 409 00:21:31,290 --> 00:21:36,605 actually, structurally, if you superimpose the structures, 410 00:21:36,605 --> 00:21:37,605 they're really similar-- 411 00:21:40,200 --> 00:21:41,940 form cyclic terpenes. 412 00:21:41,940 --> 00:21:43,830 So they form cyclic sesquiterpenes. 413 00:21:43,830 --> 00:21:45,750 I'll show you this in a minute. 414 00:21:45,750 --> 00:21:47,940 And they use FPP. 415 00:21:47,940 --> 00:21:51,780 So here, all of these enzymes use FPP. 416 00:21:51,780 --> 00:21:57,220 And they all look alike sort of from the 30,000-foot point 417 00:21:57,220 --> 00:21:57,720 of view. 418 00:21:57,720 --> 00:21:59,220 And the question is then, how do you 419 00:21:59,220 --> 00:22:03,870 control what the chemistry is in the active site? 420 00:22:03,870 --> 00:22:06,340 So they have homologous structure. 421 00:22:06,340 --> 00:22:08,850 So these are all structurally homologous. 422 00:22:08,850 --> 00:22:13,140 Another thing that you need to remember about these systems 423 00:22:13,140 --> 00:22:20,970 is that they have similar metal binding motifs. 424 00:22:25,710 --> 00:22:30,270 Now, if you look at the reaction of IPP, 425 00:22:30,270 --> 00:22:34,060 if you think about this, this isn't what PP looks like. 426 00:22:34,060 --> 00:22:36,100 Does everybody know what PP looks like? 427 00:22:36,100 --> 00:22:38,100 Hopefully, you've seen this over and over again. 428 00:22:38,100 --> 00:22:41,180 What would the metal be or metals? 429 00:22:41,180 --> 00:22:42,160 AUDIENCE: Magnesium. 430 00:22:42,160 --> 00:22:42,993 JOANNE STUBBE: Yeah. 431 00:22:42,993 --> 00:22:46,470 So whenever you have pyrophosphates or ATPs or GTPs, 432 00:22:46,470 --> 00:22:47,730 you always have magnesium. 433 00:22:47,730 --> 00:22:50,940 Magnesium plays a central role in everything in biology. 434 00:22:50,940 --> 00:22:53,280 And we can never look at magnesium 435 00:22:53,280 --> 00:22:55,470 because the ligands are fast-changing. 436 00:22:55,470 --> 00:22:57,250 It moves around all over the place. 437 00:22:57,250 --> 00:23:00,060 So it's hard to freeze out and understand 438 00:23:00,060 --> 00:23:01,380 the function of magnesium. 439 00:23:01,380 --> 00:23:06,000 But it turns out most of these proteins 440 00:23:06,000 --> 00:23:08,550 require three magnesiums. 441 00:23:08,550 --> 00:23:11,490 And as with many metal-based reactions, 442 00:23:11,490 --> 00:23:14,220 if you line things up, you really 443 00:23:14,220 --> 00:23:16,080 don't find that much sequence homology. 444 00:23:16,080 --> 00:23:18,510 But if you know where to look, you find sequence homology 445 00:23:18,510 --> 00:23:20,160 around where the metals bind. 446 00:23:20,160 --> 00:23:25,260 So what you see in the case of the linear farnesyl 447 00:23:25,260 --> 00:23:30,750 pyrophosphate, you see a DDXXD motif. 448 00:23:34,440 --> 00:23:37,950 And you find that in almost all of these enzymes. 449 00:23:37,950 --> 00:23:39,560 And if you go to the terpenoids-- 450 00:23:39,560 --> 00:23:41,730 so the non-linear ones-- 451 00:23:41,730 --> 00:23:43,950 you see a D. 452 00:23:43,950 --> 00:23:46,470 Again, I don't expect you to remember something like this. 453 00:23:46,470 --> 00:23:50,400 But I do expect you to remember that these metal 454 00:23:50,400 --> 00:23:54,480 motifs, once you know how to think about something, 455 00:23:54,480 --> 00:23:56,850 are actually very helpful in trying 456 00:23:56,850 --> 00:24:00,170 to define the function of an unknown open reading frame, 457 00:24:00,170 --> 00:24:01,750 if you know how to look. 458 00:24:01,750 --> 00:24:07,580 And more than 50% of all annotated genes 459 00:24:07,580 --> 00:24:10,340 code for proteins we have no idea what they do. 460 00:24:10,340 --> 00:24:13,190 So looking at these kinds of motifs 461 00:24:13,190 --> 00:24:15,598 can actually be quite informative. 462 00:24:15,598 --> 00:24:18,140 So then what you need to do to really understand what they're 463 00:24:18,140 --> 00:24:20,415 doing is dive in and look at where 464 00:24:20,415 --> 00:24:22,040 the metals bind, if you're lucky enough 465 00:24:22,040 --> 00:24:26,940 to be able to get a structure with the metals bound. 466 00:24:26,940 --> 00:24:32,240 So we have alpha helical motifs and metal binding motifs. 467 00:24:32,240 --> 00:24:34,670 And then the other kind of motif that I think 468 00:24:34,670 --> 00:24:38,900 is really interesting for the linear system-- 469 00:24:38,900 --> 00:24:41,900 so that's farnesyl pyrophosphate-- 470 00:24:41,900 --> 00:24:43,720 is how you control chain length. 471 00:24:46,250 --> 00:24:51,120 So FPP synthase-- that's what we're talking about-- 472 00:24:51,120 --> 00:24:52,640 is a dimer. 473 00:24:52,640 --> 00:24:57,560 And the metal binding motifs sit up there. 474 00:24:57,560 --> 00:25:00,530 So the metal binding motifs sit in the top, the way 475 00:25:00,530 --> 00:25:01,430 I've drawn this here. 476 00:25:01,430 --> 00:25:03,713 So you have metals. 477 00:25:03,713 --> 00:25:05,255 There are thought to be three metals. 478 00:25:08,270 --> 00:25:11,360 And then we're building C5, C5, C5. 479 00:25:11,360 --> 00:25:12,440 Where does the chain go? 480 00:25:15,110 --> 00:25:17,270 And what you'll see is there is a cone 481 00:25:17,270 --> 00:25:20,990 shape which migrates towards the bottom of the structure. 482 00:25:20,990 --> 00:25:23,880 I'll show you a picture of this in a minute. 483 00:25:23,880 --> 00:25:27,320 And so then the question is, what controls chain length? 484 00:25:27,320 --> 00:25:32,930 So if you end up looking at the structure, what you see 485 00:25:32,930 --> 00:25:34,900 is a phenylalanine. 486 00:25:34,900 --> 00:25:37,070 And the phenylalanine is a molecular doorstop. 487 00:25:42,410 --> 00:25:46,430 So the chain is extending, because we're 488 00:25:46,430 --> 00:25:50,120 going from C5 to C10 to C15. 489 00:25:50,120 --> 00:25:52,280 Why don't we go to C50? 490 00:25:52,280 --> 00:25:55,370 And I showed you in the first lecture dolichol 491 00:25:55,370 --> 00:25:59,330 and lipid II have C20s, C55s. 492 00:25:59,330 --> 00:26:01,190 How do you control the chain length? 493 00:26:01,190 --> 00:26:02,690 So that's an interesting question 494 00:26:02,690 --> 00:26:05,630 in polymer biochemistry. 495 00:26:05,630 --> 00:26:08,660 Here, we control it by a molecular doorstop. 496 00:26:08,660 --> 00:26:13,580 So if I replace the phenylalanine with an alanine, 497 00:26:13,580 --> 00:26:14,360 what might happen? 498 00:26:19,750 --> 00:26:20,250 Go. 499 00:26:20,250 --> 00:26:21,300 AUDIENCE: You'd have longer. 500 00:26:21,300 --> 00:26:21,640 JOANNE STUBBE: Yeah. 501 00:26:21,640 --> 00:26:22,770 So they made up to-- 502 00:26:22,770 --> 00:26:23,580 I can't remember. 503 00:26:23,580 --> 00:26:25,247 I haven't read the paper in a long time. 504 00:26:25,247 --> 00:26:27,540 But they can make C50-mers. 505 00:26:27,540 --> 00:26:30,883 So they can actually see, and they're not uniform. 506 00:26:30,883 --> 00:26:31,800 So that's a key thing. 507 00:26:31,800 --> 00:26:34,180 You want them to be uniform. 508 00:26:34,180 --> 00:26:35,520 So if you look-- 509 00:26:35,520 --> 00:26:37,650 I think in the next has a picture of this. 510 00:26:37,650 --> 00:26:41,730 Again, this is graphics from really quite some time 511 00:26:41,730 --> 00:26:43,200 ago now, 1996. 512 00:26:43,200 --> 00:26:44,850 So the picture's not very good. 513 00:26:44,850 --> 00:26:47,310 But you can see sort of the tunnel, 514 00:26:47,310 --> 00:26:50,165 and the metal binding sites are actually up there. 515 00:26:50,165 --> 00:26:51,540 And if you look at the structure, 516 00:26:51,540 --> 00:26:54,210 you can see the phenylalanine. 517 00:26:54,210 --> 00:26:59,280 So that's what we know sort of about the type I synthases. 518 00:26:59,280 --> 00:27:06,000 They're involved in making the C15 farnesyl pyrophosphate. 519 00:27:06,000 --> 00:27:08,217 So now we want to look at the chemistry, what's 520 00:27:08,217 --> 00:27:09,300 going on in the chemistry. 521 00:27:09,300 --> 00:27:12,480 And can we make a generalization about how 522 00:27:12,480 --> 00:27:17,370 this chemistry is used to put all C5 units together? 523 00:27:17,370 --> 00:27:20,860 So that's what I want to focus on next. 524 00:27:24,120 --> 00:27:24,620 Whoops. 525 00:27:31,460 --> 00:27:34,750 So what I'm going to now look at are the proposed mechanisms. 526 00:27:39,330 --> 00:27:42,540 And I'm not really going to go into much detail. 527 00:27:42,540 --> 00:27:45,480 I'm going to give you a generic overview 528 00:27:45,480 --> 00:27:48,120 of the things you need to remember if you encounter 529 00:27:48,120 --> 00:27:50,220 something like this. 530 00:27:50,220 --> 00:27:52,110 The first guess would be a mechanism 531 00:27:52,110 --> 00:27:54,330 similar to the one I'm proposing now, 532 00:27:54,330 --> 00:27:56,370 but then you have to look at it in more detail 533 00:27:56,370 --> 00:27:59,030 to figure out what's really going on. 534 00:27:59,030 --> 00:28:00,030 So what do we have? 535 00:28:03,470 --> 00:28:06,760 We have dimethylallyl pyrophosphate. 536 00:28:06,760 --> 00:28:08,938 And I have a cartoon for you to look at there, 537 00:28:08,938 --> 00:28:11,230 but I'm going to draw it differently than this cartoon. 538 00:28:11,230 --> 00:28:14,560 But you can just watch me because, again, the key thing 539 00:28:14,560 --> 00:28:17,050 is thinking about how you form the carbon-carbon bond 540 00:28:17,050 --> 00:28:19,780 and what's going on in these reactions. 541 00:28:19,780 --> 00:28:23,350 So we just looked at the pyrophosphate. 542 00:28:23,350 --> 00:28:27,760 And if you look over there, what do you need? 543 00:28:27,760 --> 00:28:31,330 You need to have a bunch of metals bound. 544 00:28:31,330 --> 00:28:34,390 And recently-- this is a fairly old paper. 545 00:28:34,390 --> 00:28:35,530 They have better papers. 546 00:28:35,530 --> 00:28:37,892 I think I took all the pictures out, 547 00:28:37,892 --> 00:28:39,850 because it's hard to see things without looking 548 00:28:39,850 --> 00:28:41,260 at it in detail. 549 00:28:41,260 --> 00:28:44,080 But in fact, the magnesiums are interacting 550 00:28:44,080 --> 00:28:47,470 with the pyrophosphate and adjacent to the pyrophosphate. 551 00:28:47,470 --> 00:28:50,500 And it's clear they play a key role in catalysis. 552 00:28:50,500 --> 00:28:52,660 But whether they move during the transformation, 553 00:28:52,660 --> 00:28:55,690 again, I think we just don't know that much at this stage. 554 00:28:55,690 --> 00:29:00,010 It's hard to trap it in an informative state, 555 00:29:00,010 --> 00:29:03,400 like it is with all crystallography. 556 00:29:03,400 --> 00:29:06,590 So here's dimethylallyl pyrophosphate. 557 00:29:06,590 --> 00:29:08,860 Here's isopentenyl pyrophosphate, 558 00:29:08,860 --> 00:29:11,380 the two guys we were after. 559 00:29:11,380 --> 00:29:16,480 And the first step in all of these reactions is ionization. 560 00:29:16,480 --> 00:29:19,960 So this is an unusual reaction in biochemistry. 561 00:29:19,960 --> 00:29:22,960 There are almost no examples of carbocation 562 00:29:22,960 --> 00:29:25,220 in biological transformations. 563 00:29:25,220 --> 00:29:28,570 This is one of the few places where you see this. 564 00:29:28,570 --> 00:29:34,270 So this is the ionization step. 565 00:29:34,270 --> 00:29:35,890 And all of the reactions we are going 566 00:29:35,890 --> 00:29:39,710 to be looking at involve ionization, 567 00:29:39,710 --> 00:29:42,490 but other kinds of chemistry can also 568 00:29:42,490 --> 00:29:46,500 happen that we're not going to discuss. 569 00:29:46,500 --> 00:29:47,680 So what have we generated? 570 00:29:47,680 --> 00:29:51,040 We generated an allylic cation. 571 00:29:51,040 --> 00:29:54,180 And what we also have is we lost pyrophosphate. 572 00:29:58,120 --> 00:29:59,620 And I'm being sloppy. 573 00:29:59,620 --> 00:30:05,080 I'm not drawing out how these are interacting with metals, 574 00:30:05,080 --> 00:30:07,903 but the charges are pretty much neutralized in some form 575 00:30:07,903 --> 00:30:09,320 that we don't know the details of. 576 00:30:09,320 --> 00:30:12,470 So you can't forget about the charges. 577 00:30:12,470 --> 00:30:18,790 And so we can just put down magnesium 3+. 578 00:30:18,790 --> 00:30:20,740 And then what we want to do, we want 579 00:30:20,740 --> 00:30:22,375 to make a carbon-carbon bond. 580 00:30:24,900 --> 00:30:25,400 Whoops. 581 00:30:25,400 --> 00:30:26,590 Let me get this right. 582 00:30:29,160 --> 00:30:31,390 If I make a mistake on the board-- like sometimes, 583 00:30:31,390 --> 00:30:33,790 I always get mixed up with four or five carbons-- 584 00:30:33,790 --> 00:30:35,248 raise your hand and say, you've got 585 00:30:35,248 --> 00:30:36,760 the wrong number of carbons. 586 00:30:36,760 --> 00:30:37,360 You tell me. 587 00:30:37,360 --> 00:30:39,880 You be the cops. 588 00:30:39,880 --> 00:30:41,830 So what we're going to do now is we're 589 00:30:41,830 --> 00:30:44,440 ready to form a carbon-carbon bond. 590 00:30:44,440 --> 00:30:47,530 And we're going to be forming a new carbocation. 591 00:30:47,530 --> 00:30:49,960 Hopefully, you remember from introductory chemistry 592 00:30:49,960 --> 00:30:53,320 that carbocations that are tertiary are more stable. 593 00:30:53,320 --> 00:30:55,810 And when you look at terpene types of chemistry, 594 00:30:55,810 --> 00:30:58,210 you see tertiary carbocations used 595 00:30:58,210 --> 00:30:59,920 over and over and over again. 596 00:30:59,920 --> 00:31:05,920 That being said, I'm putting brackets around this 597 00:31:05,920 --> 00:31:08,830 because despite the fact that I draw this intermediate, 598 00:31:08,830 --> 00:31:11,590 no one's ever seen it in the enzymatic reaction using 599 00:31:11,590 --> 00:31:12,810 the normal substrates. 600 00:31:12,810 --> 00:31:15,280 So you have to play games to study mechanism, 601 00:31:15,280 --> 00:31:18,470 just like you have to do in organic chemistry. 602 00:31:18,470 --> 00:31:21,580 So what happens now is you're set up 603 00:31:21,580 --> 00:31:23,440 to form the carbon-carbon bond, which 604 00:31:23,440 --> 00:31:25,900 has been the goal of what we've been 605 00:31:25,900 --> 00:31:30,280 trying to do in the first couple of lectures. 606 00:31:30,280 --> 00:31:32,200 And so what do you generate? 607 00:31:32,200 --> 00:31:37,150 You generate the new carbon-carbon bond, 608 00:31:37,150 --> 00:31:41,770 which is the skeleton for geranyl pyrophosphate. 609 00:31:41,770 --> 00:31:45,760 You generated a new carbocation, and it's 610 00:31:45,760 --> 00:31:47,638 a tertiary carbocation. 611 00:31:52,840 --> 00:31:56,950 And our pyrophosphate is still sitting in the active site. 612 00:32:04,810 --> 00:32:07,090 And so now what we're ready to do 613 00:32:07,090 --> 00:32:11,718 is we're going to form our C10, geranyl pyrophosphate. 614 00:32:11,718 --> 00:32:13,510 And we'll see one of the types of reactions 615 00:32:13,510 --> 00:32:16,390 that you see over and over again when 616 00:32:16,390 --> 00:32:20,140 you make carbon-carbon bonds is loss of a proton. 617 00:32:20,140 --> 00:32:22,420 And that gives you the C10, which 618 00:32:22,420 --> 00:32:24,160 is these two things stuck together, which 619 00:32:24,160 --> 00:32:28,360 is a monoterpene, which is called "geranyl pyrophosphate." 620 00:32:28,360 --> 00:32:30,160 So what's interesting about this-- 621 00:32:30,160 --> 00:32:32,890 and I think this is sort of something that's pretty 622 00:32:32,890 --> 00:32:33,490 general-- 623 00:32:33,490 --> 00:32:35,470 is the pyrophosphate in the active site. 624 00:32:35,470 --> 00:32:39,100 If you look in the active sites, they're amazingly hydrophobic. 625 00:32:39,100 --> 00:32:43,860 And the pyrophosphate in some way stereospecifically-- 626 00:32:43,860 --> 00:32:45,610 I haven't drawn the stereochemistry here-- 627 00:32:45,610 --> 00:32:50,530 removes the HR proton to generate the olefin. 628 00:32:50,530 --> 00:32:54,460 So what you've now generated is geranyl pyrophosphate. 629 00:33:04,490 --> 00:33:10,430 So here's C10, and this is geranyl pyrophosphate. 630 00:33:10,430 --> 00:33:15,740 Let me also put brackets around this intermediate. 631 00:33:15,740 --> 00:33:18,830 Again, we haven't seen this intermediate. 632 00:33:18,830 --> 00:33:20,540 And how do we know this is true? 633 00:33:20,540 --> 00:33:22,850 Because we know a lot from Winstein and Brown 634 00:33:22,850 --> 00:33:24,440 about carbocation chemistry. 635 00:33:24,440 --> 00:33:27,020 And people have been really creative in figuring out 636 00:33:27,020 --> 00:33:30,290 how to show that this model is in fact correct. 637 00:33:30,290 --> 00:33:34,880 Hopefully, I have C10 there. 638 00:33:34,880 --> 00:33:38,030 And so this is an intermediate because we're still 639 00:33:38,030 --> 00:33:38,720 going to go on. 640 00:33:38,720 --> 00:33:40,340 The enzyme doesn't stop at C10. 641 00:33:40,340 --> 00:33:43,820 It adds another isopentenyl pyrophosphate. 642 00:33:43,820 --> 00:33:47,420 So if you want to think about how nature might design that, 643 00:33:47,420 --> 00:33:50,150 if you look at this molecule and you 644 00:33:50,150 --> 00:33:53,420 look at this part of the molecule 645 00:33:53,420 --> 00:33:56,330 and replace it with an R group-- 646 00:33:56,330 --> 00:33:57,320 so we have an R here. 647 00:33:57,320 --> 00:33:58,850 What does this look like? 648 00:33:58,850 --> 00:34:04,270 It looks just like dimethylallyl pyrophosphate. 649 00:34:04,270 --> 00:34:06,180 But we need to put the R group somewhere. 650 00:34:06,180 --> 00:34:09,980 So in the case of FPP synthase, we're going down the tunnel. 651 00:34:09,980 --> 00:34:12,010 So we're getting it out of the way. 652 00:34:12,010 --> 00:34:14,719 But we're going to do the same chemistry that we just 653 00:34:14,719 --> 00:34:19,280 did over again, and we just replaced a methyl 654 00:34:19,280 --> 00:34:21,710 with an R group. 655 00:34:21,710 --> 00:34:24,139 So that's the basic chemistry. 656 00:34:24,139 --> 00:34:26,960 It's pretty straightforward, the only chemistry 657 00:34:26,960 --> 00:34:29,800 that I'm aware of in biological systems that 658 00:34:29,800 --> 00:34:31,760 involves carbocations. 659 00:34:31,760 --> 00:34:35,210 These are special carbocations. 660 00:34:35,210 --> 00:34:37,239 That is, they're, in general, stabilized. 661 00:34:37,239 --> 00:34:38,510 They're allylic. 662 00:34:38,510 --> 00:34:42,500 Or in many cases, they can be tertiary. 663 00:34:42,500 --> 00:34:44,233 So let's emphasize that. 664 00:34:44,233 --> 00:34:46,400 Again, if you don't remember your organic chemistry, 665 00:34:46,400 --> 00:34:51,230 you should go back and look up the sections on carbocations. 666 00:34:51,230 --> 00:34:54,139 So I told you the farnesyl pyrophosphate is 667 00:34:54,139 --> 00:34:57,410 sort of central to many things. 668 00:34:57,410 --> 00:34:59,930 And farnesyl, in this case-- 669 00:34:59,930 --> 00:35:02,588 I'm not going to draw out farnesyl pyrophosphate. 670 00:35:02,588 --> 00:35:03,630 The chemistry's the same. 671 00:35:03,630 --> 00:35:06,360 You can repeat it yourself. 672 00:35:06,360 --> 00:35:09,170 But here is our farnesyl pyrophosphate, 673 00:35:09,170 --> 00:35:12,340 but look what it can form. 674 00:35:12,340 --> 00:35:14,410 Remember, you saw all those smells. 675 00:35:14,410 --> 00:35:18,760 If you break a pine needle, you have pinene. 676 00:35:18,760 --> 00:35:22,990 What you see is this one intermediate 677 00:35:22,990 --> 00:35:26,840 can form all of these compounds. 678 00:35:26,840 --> 00:35:28,660 So the question is-- with an enzyme 679 00:35:28,660 --> 00:35:30,790 that looks just like farnesyl pyrophosphate 680 00:35:30,790 --> 00:35:32,710 in three-dimensional structure. 681 00:35:32,710 --> 00:35:34,780 So that's sort of amazing. 682 00:35:34,780 --> 00:35:38,680 And what you're doing here is taking a linear molecule. 683 00:35:38,680 --> 00:35:41,770 And in this particular case-- and I'm not going to talk about 684 00:35:41,770 --> 00:35:47,170 this slide in detail, but I will talk about one case in detail-- 685 00:35:47,170 --> 00:35:50,980 what you're now doing is getting it to do alternative chemistry. 686 00:35:50,980 --> 00:35:55,660 So how would you design the active site of your enzyme 687 00:35:55,660 --> 00:36:00,370 to end up doing that, to use the same chemistry, ionization? 688 00:36:00,370 --> 00:36:03,220 And then you have to do cyclization and loss 689 00:36:03,220 --> 00:36:05,410 of a proton or whatever. 690 00:36:05,410 --> 00:36:07,430 How does nature design all of this? 691 00:36:07,430 --> 00:36:10,420 So once we get through this set of lectures, 692 00:36:10,420 --> 00:36:12,910 I would suggest this would be something you 693 00:36:12,910 --> 00:36:14,440 could go back and practice on. 694 00:36:14,440 --> 00:36:16,050 How do we get to all these guys? 695 00:36:16,050 --> 00:36:17,800 I'm going to show you one example of that. 696 00:36:17,800 --> 00:36:19,425 I'm not going to go through this slide. 697 00:36:19,425 --> 00:36:21,190 It's way too complicated, but I think 698 00:36:21,190 --> 00:36:25,420 it shows you sort of the amazing diversity of the terpenome, 699 00:36:25,420 --> 00:36:28,710 using farnesyl pyrophosphate. 700 00:36:28,710 --> 00:36:34,300 So what I want to do is give you an overview of the rules. 701 00:36:34,300 --> 00:36:37,060 And then I'll go through one specific example. 702 00:36:37,060 --> 00:36:47,030 So let's make general mechanistic comments. 703 00:36:47,030 --> 00:36:51,140 And in the original, version of the PowerPoint, 704 00:36:51,140 --> 00:36:53,240 this slide wasn't in there. 705 00:36:53,240 --> 00:36:58,610 Anyhow, the first thing is you've already seen up here, 706 00:36:58,610 --> 00:37:02,830 and this is going to be common, is you lose a proton. 707 00:37:02,830 --> 00:37:06,080 So the first step is ionization. 708 00:37:06,080 --> 00:37:10,200 So ionization happens in almost all these reactions. 709 00:37:10,200 --> 00:37:13,790 There are exceptions to this, but most first steps 710 00:37:13,790 --> 00:37:16,040 are ionization. 711 00:37:16,040 --> 00:37:19,670 The second step can involve proton loss. 712 00:37:22,452 --> 00:37:24,410 And I'm going to write down what the steps are, 713 00:37:24,410 --> 00:37:28,760 and then we'll come back and look at a specific example. 714 00:37:28,760 --> 00:37:31,340 And we're going to see this in cholesterol. 715 00:37:31,340 --> 00:37:33,680 One can have with carbocations-- if you go back 716 00:37:33,680 --> 00:37:36,170 and you think about what you learned if you've had 717 00:37:36,170 --> 00:37:38,900 the second semester of organic. 718 00:37:38,900 --> 00:37:42,260 With carbocations, you can do hydride transfers. 719 00:37:42,260 --> 00:37:47,970 So that's a hydrogen with a pair of electrons. 720 00:37:47,970 --> 00:37:51,260 We can have hydride transfers. 721 00:37:51,260 --> 00:37:52,600 We're also going to see-- 722 00:37:52,600 --> 00:37:57,440 and both of these are key in cholesterol biosynthesis. 723 00:37:57,440 --> 00:38:00,440 We can have methyl anion transfers. 724 00:38:07,090 --> 00:38:09,800 And the other thing is these reactions 725 00:38:09,800 --> 00:38:11,900 all go stereospecifically. 726 00:38:15,410 --> 00:38:16,790 And that's one thing. 727 00:38:16,790 --> 00:38:19,720 If you become an enzymologist, you realize that's what's cool. 728 00:38:19,720 --> 00:38:21,470 That's why you have such big huge enzymes, 729 00:38:21,470 --> 00:38:23,870 so they can control the stereochemistry of everything. 730 00:38:23,870 --> 00:38:26,055 So they do everything with 100% EE. 731 00:38:26,055 --> 00:38:28,430 And they don't have to worry about it like chemists worry 732 00:38:28,430 --> 00:38:30,950 about it, but they pay a price. 733 00:38:30,950 --> 00:38:34,130 They have a big huge protein. 734 00:38:34,130 --> 00:38:36,620 The third thing-- and this is going to become important. 735 00:38:36,620 --> 00:38:40,430 It was just important in the slide I showed you previously-- 736 00:38:40,430 --> 00:38:42,065 we're going to see cyclizations. 737 00:38:44,600 --> 00:38:51,600 And cyclizations require, in general, 738 00:38:51,600 --> 00:38:56,040 protonation of an olefin-- 739 00:38:56,040 --> 00:38:58,200 I'll give you an example of that-- 740 00:38:58,200 --> 00:39:05,460 or protonation of an epoxide. 741 00:39:05,460 --> 00:39:08,520 So in some way, you're going to have to do some more 742 00:39:08,520 --> 00:39:11,160 chemistry to get your olefin. 743 00:39:11,160 --> 00:39:14,130 Everybody know what an epoxide is? 744 00:39:14,130 --> 00:39:16,380 So we're converting an olefin into an epoxide. 745 00:39:16,380 --> 00:39:18,172 We're going to protonate it, and then we're 746 00:39:18,172 --> 00:39:20,550 going to do cyclizations. 747 00:39:20,550 --> 00:39:25,050 And the third general type of reactions is water addition. 748 00:39:28,560 --> 00:39:30,480 So if you have a carbocation sitting around. 749 00:39:30,480 --> 00:39:34,635 You add water, bang, you have a reaction and form an alcohol. 750 00:39:40,200 --> 00:39:42,293 So the other generalizations I want 751 00:39:42,293 --> 00:39:43,710 to make-- so that's the chemistry. 752 00:39:43,710 --> 00:39:46,380 We're going to see this chemistry play out 753 00:39:46,380 --> 00:39:49,530 over and over again because I've selected examples 754 00:39:49,530 --> 00:39:51,790 of this for you to look at. 755 00:39:51,790 --> 00:39:53,640 But it's quite common. 756 00:39:53,640 --> 00:39:56,580 The second thing besides these mechanistic issues 757 00:39:56,580 --> 00:40:01,800 is, how do you distinguish between linear versus cyclic? 758 00:40:04,390 --> 00:40:06,570 And you've already seen the strategy 759 00:40:06,570 --> 00:40:08,790 with farnesyl pyrophosphate. 760 00:40:08,790 --> 00:40:10,890 You really sort of have a tiny little cavity 761 00:40:10,890 --> 00:40:13,260 where the IPP and the dimethylallyl 762 00:40:13,260 --> 00:40:17,280 pyrophosphate bind, and then you have a long tunnel. 763 00:40:17,280 --> 00:40:21,810 What do you have in the case of cyclic terpenes, which 764 00:40:21,810 --> 00:40:25,770 you saw in the previous slide to this one? 765 00:40:25,770 --> 00:40:32,170 And the key thing is the shape of the active site. 766 00:40:34,970 --> 00:40:36,680 And what you will see if you look 767 00:40:36,680 --> 00:40:39,680 at a lot of these active sites is, in general, 768 00:40:39,680 --> 00:40:43,430 they're very hydrophobic. 769 00:40:43,430 --> 00:40:44,933 Why is that true? 770 00:40:44,933 --> 00:40:47,350 So somehow, you've got to take care of the pyrophosphates. 771 00:40:47,350 --> 00:40:49,100 But they're very hydrophobic because we're 772 00:40:49,100 --> 00:40:53,150 dealing with these hydrocarbons, which are hydrophobic. 773 00:40:53,150 --> 00:40:54,830 So the question then is, can you take 774 00:40:54,830 --> 00:40:58,310 this farnesyl pyrophosphate and fold it? 775 00:40:58,310 --> 00:41:00,730 And folding it in different ways-- 776 00:41:00,730 --> 00:41:03,540 if we go back to the last-- 777 00:41:03,540 --> 00:41:04,110 whoops. 778 00:41:04,110 --> 00:41:07,050 If we go back to the last slide, if you look at it here, 779 00:41:07,050 --> 00:41:12,180 for example, and you ionize here to form a carbocation, 780 00:41:12,180 --> 00:41:15,180 you can have a cis or a trans carbocation. 781 00:41:15,180 --> 00:41:18,570 And that then can lead to further types of chemistry, 782 00:41:18,570 --> 00:41:21,240 where you form different kinds of ring structures. 783 00:41:21,240 --> 00:41:25,600 So it really is all about folding in the active site 784 00:41:25,600 --> 00:41:26,340 of the enzyme. 785 00:41:26,340 --> 00:41:29,310 So the active site is the key to determine 786 00:41:29,310 --> 00:41:31,200 which of these many kinds of things that 787 00:41:31,200 --> 00:41:33,600 can happen that if you did this in solution, 788 00:41:33,600 --> 00:41:36,360 you might actually get a mixture of all 789 00:41:36,360 --> 00:41:38,610 of these kinds of things. 790 00:41:38,610 --> 00:41:43,050 So the key then is hydrophobic and the shape 791 00:41:43,050 --> 00:41:44,190 of the active site. 792 00:41:44,190 --> 00:41:48,660 And then another key thing is I'm going to show you that 793 00:41:48,660 --> 00:41:50,730 in many of these reactions, you go through-- 794 00:41:50,730 --> 00:41:52,170 like we saw up there-- 795 00:41:52,170 --> 00:41:54,930 these carbocation intermediates. 796 00:41:54,930 --> 00:41:56,940 Well, there might be three different carbocation 797 00:41:56,940 --> 00:41:58,560 intermediates you could go through. 798 00:41:58,560 --> 00:42:00,240 How do you decide? 799 00:42:00,240 --> 00:42:01,740 How do you decide-- 800 00:42:01,740 --> 00:42:06,750 how did enzymes evolve to give you specific carbocation 801 00:42:06,750 --> 00:42:07,380 intermediates? 802 00:42:07,380 --> 00:42:10,800 How might you stabilize a carbocation intermediate? 803 00:42:10,800 --> 00:42:13,440 Anybody got any ideas? 804 00:42:13,440 --> 00:42:15,690 What would you expect to find in the active site then? 805 00:42:15,690 --> 00:42:18,480 I'm going to show you on the next slide, which 806 00:42:18,480 --> 00:42:23,470 is sort of a generic active site of a terpene that can cyclize. 807 00:42:26,350 --> 00:42:27,280 Any guesses? 808 00:42:27,280 --> 00:42:29,101 How would you stabilize a carbocation? 809 00:42:34,392 --> 00:42:35,840 AUDIENCE: Negative mixtures. 810 00:42:35,840 --> 00:42:36,673 JOANNE STUBBE: Yeah. 811 00:42:36,673 --> 00:42:38,920 So one way-- you might have an aspartate. 812 00:42:38,920 --> 00:42:40,710 Nature doesn't do that. 813 00:42:40,710 --> 00:42:43,300 So that might-- well, the problem is if you do that 814 00:42:43,300 --> 00:42:45,010 and you form a covalent bond, that's 815 00:42:45,010 --> 00:42:46,790 the end of your reaction. 816 00:42:46,790 --> 00:42:47,290 So 817 00:42:47,290 --> 00:42:50,037 So how you do this is I don't think 818 00:42:50,037 --> 00:42:51,370 we really totally understand it. 819 00:42:51,370 --> 00:42:53,320 But how else could you stabilize it? 820 00:42:53,320 --> 00:42:54,430 Anybody else? 821 00:42:54,430 --> 00:42:56,920 What did you learn about weak non-covalent interactions 822 00:42:56,920 --> 00:42:59,950 in biochemistry that could help us? 823 00:42:59,950 --> 00:43:02,530 Everybody hates waiting on covalent interactions, the key 824 00:43:02,530 --> 00:43:03,760 to everything-- 825 00:43:03,760 --> 00:43:06,015 key to everything in how enzymes function. 826 00:43:06,015 --> 00:43:08,140 AUDIENCE: You could just have something [INAUDIBLE] 827 00:43:08,140 --> 00:43:09,090 in general. 828 00:43:09,090 --> 00:43:10,720 JOANNE STUBBE: But electron-rich-- 829 00:43:10,720 --> 00:43:13,095 but that would be doing-- that's what she was suggesting. 830 00:43:13,095 --> 00:43:16,210 You have a carboxylate, an aspartate or a glutamate. 831 00:43:16,210 --> 00:43:19,540 Then you would form a bond, and then you would be stuck. 832 00:43:19,540 --> 00:43:24,020 So the way nature actually does this is she uses aromatics. 833 00:43:27,740 --> 00:43:31,430 And it was discovered maybe about 15 years ago 834 00:43:31,430 --> 00:43:35,530 that you can have an aromatic whatever. 835 00:43:35,530 --> 00:43:38,960 And you have some kind of a cation. 836 00:43:38,960 --> 00:43:44,750 So this is called a "pi cation interaction." 837 00:43:44,750 --> 00:43:47,780 Usually, the pi cation interactions are with metals. 838 00:43:47,780 --> 00:43:49,610 But here, we have a carbocation. 839 00:43:49,610 --> 00:43:52,280 So the model is that you might find 840 00:43:52,280 --> 00:43:58,070 in the active site tryptophans, tyrosines, phenylalanines. 841 00:43:58,070 --> 00:44:00,710 And so these become really key. 842 00:44:00,710 --> 00:44:04,370 And in fact, if you look at an active site-- 843 00:44:04,370 --> 00:44:07,610 so I don't even remember which enzyme this is. 844 00:44:07,610 --> 00:44:09,470 And somebody was trying to study something, 845 00:44:09,470 --> 00:44:13,340 and they have a small inhibitor in the active site. 846 00:44:13,340 --> 00:44:16,220 But you notice you don't have a long site where 847 00:44:16,220 --> 00:44:18,140 this chain can extend. 848 00:44:18,140 --> 00:44:21,440 What you've done is constrained the active site much more, 849 00:44:21,440 --> 00:44:24,740 and that shape is going to be key to the many different 850 00:44:24,740 --> 00:44:26,090 reactions you could have. 851 00:44:26,090 --> 00:44:27,710 And then if you look carefully, you 852 00:44:27,710 --> 00:44:29,030 can't really think about this. 853 00:44:29,030 --> 00:44:31,970 But you have phenylalanine, tyrosine, tryptophan, 854 00:44:31,970 --> 00:44:33,830 and another tyrosine in the active site. 855 00:44:33,830 --> 00:44:37,730 And that's what you see in many of these protein structures 856 00:44:37,730 --> 00:44:38,240 all over. 857 00:44:38,240 --> 00:44:41,390 Again, we have FPP synthase, which has this thing. 858 00:44:41,390 --> 00:44:43,490 And then we have these terpene cyclases, 859 00:44:43,490 --> 00:44:44,960 which have this thing. 860 00:44:44,960 --> 00:44:46,820 And each one of them is different. 861 00:44:46,820 --> 00:44:51,590 And so the difference is related to the shape. 862 00:44:51,590 --> 00:44:54,710 And it's proposed that this stabilizes this interaction. 863 00:44:54,710 --> 00:44:57,350 It's been challenging to show this chemically, 864 00:44:57,350 --> 00:45:00,230 but these interactions are worth quite a bit. 865 00:45:00,230 --> 00:45:02,300 These are also hard to measure, but it's 866 00:45:02,300 --> 00:45:06,050 something that was discovered and now has been actually 867 00:45:06,050 --> 00:45:07,923 widely observed. 868 00:45:07,923 --> 00:45:10,340 And the other thing I want to mention about these enzymes, 869 00:45:10,340 --> 00:45:14,720 which I think is interesting and distinct from other enzymes 870 00:45:14,720 --> 00:45:18,470 that you've encountered, is that, in general, they're 871 00:45:18,470 --> 00:45:20,760 really not very specific. 872 00:45:20,760 --> 00:45:23,630 So if you start looking at these-- 873 00:45:23,630 --> 00:45:24,230 look at this. 874 00:45:24,230 --> 00:45:28,910 How could you make one cation here versus the three others? 875 00:45:28,910 --> 00:45:32,240 If you start looking at how to get to these cyclized products, 876 00:45:32,240 --> 00:45:34,550 you say, how the heck did nature ever do that? 877 00:45:34,550 --> 00:45:37,320 There's no way you could guess at what the product would be, 878 00:45:37,320 --> 00:45:38,580 in my opinion. 879 00:45:38,580 --> 00:45:41,987 So what happens is these enzymes actually 880 00:45:41,987 --> 00:45:44,570 when you start looking-- we have good analytical methods-- are 881 00:45:44,570 --> 00:45:46,460 really promiscuous. 882 00:45:46,460 --> 00:45:48,560 So they might produce a predominant product, 883 00:45:48,560 --> 00:45:51,110 but they always produce a bunch-- 884 00:45:51,110 --> 00:45:54,860 1%, 5%, sometimes even more-- 885 00:45:54,860 --> 00:45:57,420 of other products. 886 00:45:57,420 --> 00:45:59,570 And I think if you look at the chemistry 887 00:45:59,570 --> 00:46:02,330 that we've been talking about, basically, all 888 00:46:02,330 --> 00:46:05,900 of this sort of makes sense. 889 00:46:05,900 --> 00:46:08,540 So what I want to do now is give you 890 00:46:08,540 --> 00:46:13,040 an example of all of these reactions in one case. 891 00:46:13,040 --> 00:46:16,040 And this case, I guess I didn't write down the references. 892 00:46:16,040 --> 00:46:18,050 But I took it out of the literature. 893 00:46:18,050 --> 00:46:20,490 It's from David Christianson's lab. 894 00:46:20,490 --> 00:46:22,882 So here, we have farnesyl pyrophosphate, 895 00:46:22,882 --> 00:46:24,590 and here's the product we want to get to. 896 00:46:24,590 --> 00:46:28,100 So you'll have something like this 897 00:46:28,100 --> 00:46:31,040 on a problem set that I'm going to ask you, 898 00:46:31,040 --> 00:46:32,180 and it will be simple. 899 00:46:32,180 --> 00:46:34,222 I won't give you something that's so hard to see. 900 00:46:34,222 --> 00:46:35,840 But for me, lots of times, when you 901 00:46:35,840 --> 00:46:38,538 look at these rearrangements, it's easier if you make models. 902 00:46:38,538 --> 00:46:40,580 I don't know if anybody ever uses models anymore. 903 00:46:40,580 --> 00:46:43,430 I still use models, because you have to bend things 904 00:46:43,430 --> 00:46:45,230 in the right way to see what's possible 905 00:46:45,230 --> 00:46:47,480 and if the orbital's overlapping in the right way. 906 00:46:47,480 --> 00:46:50,600 You've got to really think about the stereochemistry. 907 00:46:50,600 --> 00:46:52,440 So what do we have here? 908 00:46:52,440 --> 00:46:54,470 So the first step is ionization. 909 00:46:54,470 --> 00:46:56,930 So we would form an allylic cation here. 910 00:46:56,930 --> 00:47:02,220 That's what we just did over here, which I hid. 911 00:47:02,220 --> 00:47:05,300 So that's what we just did over here. 912 00:47:05,300 --> 00:47:06,690 Oh, we didn't do it over here. 913 00:47:06,690 --> 00:47:11,990 Here-- over here, we formed this allylic cation. 914 00:47:11,990 --> 00:47:14,750 And once you do this, then they didn't 915 00:47:14,750 --> 00:47:15,920 show you that intermediate. 916 00:47:15,920 --> 00:47:17,250 They went on to the next step. 917 00:47:17,250 --> 00:47:19,940 So once you generate a cation there, 918 00:47:19,940 --> 00:47:24,710 they drew the conformation such that this thing could cyclize. 919 00:47:24,710 --> 00:47:29,450 But when you cyclize, you have electron deficiency 920 00:47:29,450 --> 00:47:30,740 at this carbon. 921 00:47:30,740 --> 00:47:33,200 So you have a second carbocation. 922 00:47:33,200 --> 00:47:37,680 This is not allylic, but it's a tertiary carbocation. 923 00:47:37,680 --> 00:47:40,610 So now the question is, what can happen? 924 00:47:40,610 --> 00:47:43,790 And again, you've got to keep your eye on what your goal is 925 00:47:43,790 --> 00:47:44,870 way down at the end. 926 00:47:44,870 --> 00:47:47,780 And you could probably draw more than one mechanism 927 00:47:47,780 --> 00:47:49,370 to get from A to B. And then you have 928 00:47:49,370 --> 00:47:51,500 to figure out experiments of how you would test it 929 00:47:51,500 --> 00:47:54,320 if you really care about that. 930 00:47:54,320 --> 00:47:55,970 So what happens here? 931 00:47:55,970 --> 00:47:58,100 You're losing a proton. 932 00:47:58,100 --> 00:48:01,170 And again, the pyrophosphate is acting as a general base 933 00:48:01,170 --> 00:48:01,670 catalyst. 934 00:48:01,670 --> 00:48:04,070 So that's exactly what happens in the case 935 00:48:04,070 --> 00:48:05,900 or what's proposed to happen in the case 936 00:48:05,900 --> 00:48:08,120 of farnesyl pyrophosphate. 937 00:48:08,120 --> 00:48:09,920 So you generate this species. 938 00:48:09,920 --> 00:48:11,120 Well, this might be stable. 939 00:48:11,120 --> 00:48:12,710 You might actually be able to isolate 940 00:48:12,710 --> 00:48:15,230 that as an intermediate along the reaction pathway. 941 00:48:15,230 --> 00:48:17,600 But we know in the end, we end up 942 00:48:17,600 --> 00:48:21,350 with two six-membered rings with this stereochemistry 943 00:48:21,350 --> 00:48:23,750 and with methyl groups in certain places. 944 00:48:23,750 --> 00:48:28,070 And so then you have to think about how can we get there. 945 00:48:28,070 --> 00:48:32,720 So remember that I told you that terpenoids do cyclizations. 946 00:48:32,720 --> 00:48:36,230 And one way they can do it is to protonate the olefin. 947 00:48:36,230 --> 00:48:40,130 So here, there might be a group in the active site. 948 00:48:40,130 --> 00:48:42,920 Maybe it's the phosphate that would help facilitate. 949 00:48:42,920 --> 00:48:45,710 You've just used it as a general base catalyst. 950 00:48:45,710 --> 00:48:47,270 Now, it's got a proton. 951 00:48:47,270 --> 00:48:50,600 It could now function as a general acid catalyst. 952 00:48:50,600 --> 00:48:53,780 You could protonate this position 953 00:48:53,780 --> 00:49:00,050 and now form two six-membered rings and a new carbocation. 954 00:49:00,050 --> 00:49:04,280 So in general, the nomenclature is when you draw these things, 955 00:49:04,280 --> 00:49:07,310 if you have a stick like that, that means 956 00:49:07,310 --> 00:49:09,920 you've got a methyl group. 957 00:49:09,920 --> 00:49:11,420 If you want to put a hydrogen there, 958 00:49:11,420 --> 00:49:12,890 you put a hydrogen on it. 959 00:49:12,890 --> 00:49:15,860 So if there's nothing there because CH3 takes up more room 960 00:49:15,860 --> 00:49:17,890 and they become very complicated to draw, 961 00:49:17,890 --> 00:49:19,530 the methyl group has methane. 962 00:49:19,530 --> 00:49:21,410 And the hydrogen, you put on. 963 00:49:21,410 --> 00:49:24,020 So you can distinguish one from the other. 964 00:49:24,020 --> 00:49:28,190 So now what happens is remember, one of the mechanisms 965 00:49:28,190 --> 00:49:30,510 I told you is hydride transfer. 966 00:49:30,510 --> 00:49:33,110 And again, I think looking at the stereochemistry 967 00:49:33,110 --> 00:49:35,580 of these systems helps see how this could happen. 968 00:49:35,580 --> 00:49:37,620 But these are all stereospecific. 969 00:49:37,620 --> 00:49:41,030 So you have hydride transfer from this position 970 00:49:41,030 --> 00:49:42,680 to this position. 971 00:49:42,680 --> 00:49:44,300 And when you have hydride, a hydrogen 972 00:49:44,300 --> 00:49:46,940 with a pair of electrons, what you have left 973 00:49:46,940 --> 00:49:50,240 is a new tertiary carbocation. 974 00:49:50,240 --> 00:49:52,340 And this new tertiary carbocation-- 975 00:49:52,340 --> 00:49:55,190 let me see what's going on-- is now-- 976 00:49:55,190 --> 00:49:56,870 in the end, we get a methyl group here. 977 00:49:56,870 --> 00:49:58,550 We have no methyl group there. 978 00:49:58,550 --> 00:50:03,080 Now, we have a CH3- group migrating. 979 00:50:03,080 --> 00:50:08,070 And that's the third method that I described. 980 00:50:08,070 --> 00:50:13,760 So the CH3- group migrates, giving you a new carbocation. 981 00:50:13,760 --> 00:50:17,210 And then the last step is, again, loss of a proton. 982 00:50:17,210 --> 00:50:18,570 So here's an example. 983 00:50:18,570 --> 00:50:24,230 This is a complex example, but there are 70,000 of these guys. 984 00:50:24,230 --> 00:50:27,650 So these are sort of the general rules. 985 00:50:27,650 --> 00:50:30,740 Nature has figured out how to make all these different kinds 986 00:50:30,740 --> 00:50:34,150 of natural products. 987 00:50:34,150 --> 00:50:36,710 So what I want to do now-- so those 988 00:50:36,710 --> 00:50:39,940 are the general overview of how these systems work. 989 00:50:39,940 --> 00:50:40,730 What am I doing? 990 00:50:40,730 --> 00:50:42,500 Oh, I'm sorry. 991 00:50:42,500 --> 00:50:43,460 I get so lost. 992 00:50:43,460 --> 00:50:45,440 Anyhow, I wanted to get through cholesterol. 993 00:50:45,440 --> 00:50:46,732 But next time, we'll come back. 994 00:50:46,732 --> 00:50:48,320 And in the very beginning, we're going 995 00:50:48,320 --> 00:50:53,060 to see how we take C15s to go to C30s 996 00:50:53,060 --> 00:50:56,060 and then how you cyclize this in the most, in my opinion, 997 00:50:56,060 --> 00:50:58,160 amazing reaction in biology-- 998 00:50:58,160 --> 00:51:02,030 other than ribonucleotide reductase, anyhow. 999 00:51:02,030 --> 00:51:04,630 See you next-- see you Friday.