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Module 1: Synthesis of Nucleobases, Nucleosides and Nucleotides

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Hello everybody and Welcome back! I will come back now to the nucleotide synthesis. So we have done nucleobases then, we have done nucleoside that is basis plus sugar base plus sugar. That is nucleoside and now comes the Nucleotide base plus sugar plus phosphate. Now here, I will not show individual nucleotide synthesis. We will straightaway go for the synthesis of a chain of DNA. So basically nucleotide synthesis includes this is one of the key aspects of the chemical biology and enormous contribution from organic chemistry that has enabled us to synthesize short stretches of DNA in the laboratory. So, small fragments of DNA cannot be obtained in the biological systems. In biological systems all we have long genes and the chromosomes to work in your laboratory to understand the role of specific sequences in for biological functions as well as for there are plenty of other applications that we will see just in this next module if you have a fragmented DNA. So synthesizing short stretches of DNA typically it can be any length maybe 5, 5 base pairs to up to 50. We can go even longer now it is base pairs DNA they are very important for working in the biological laboratory or chemistry laboratory. So we will see now how this DNA can be synthesized in the laboratory. And this is beautiful chemistry that has been developed by M. Caruthers, he is an American it is not the same Caruthers who wrote the organic chemistry book. This is Marvin Caruthers who is famous for the synthesis for developing a complete protocol for the synthesis of DNA stretches which is known as Oligonucleotides. So this is basically when you talk about DNA synthesis we basically mean Oligonucleotides which means the oligomers of the nucleotides. So that involves the synthesis of the nucleotides as well. So the chemistry is a little bit of modern organic chemistry and at an advanced level. So I will show you the individual steps just in a moment. But still, for this course, I am teaching this because in this synthesis lot of different aspects have been developed. And they are really interesting one fact is that that this whole of this chemistry is done inside a machine. So you do not have to go to a laboratory to your laboratory and mix things up in an into a round bottom flask or in a test tube and do the chemistry. So, everything is can be performed within a machine and that machine is programmed in a, with a computer. So just by putting the software on in the place you can comment and then the machine will do the, do every artwork for you so this whole chemistry involves 6 seven different steps that I will show now. But all of those steps happen one by one step by step commanded by the computer in this machine. Here you can see the machine. So this chemistry is known as the phosphoramidite chemistry. So as I mentioned the organic mechanisms or they are actually a little bit at an advanced level. But still, I would like to teach it even for the school children, just to show how chemistry can be done, inside a machine inside an automated machine that can perform by itself. So this is one of the machines that I am depicting here the machine is known as DNA synthesizer This is a typical model of DNA synthesizer which had bottles here you can see there are many bottles here that are attached there. And these are the bottles for reagents, the different reagents that are needed in every step. And there are also these are little bit larger bottles because you need them in, in milliliter quantity for the synthesis and then there are smaller ones this-this-this you can see they are very small actually for which you need the volume in the level of micromolar quantity. So we can do very fine chemistry here in the level of microlitre volume of material that can be or liquid can be used for the synthesis. These are the other reagent bottles which are a little bit larger which require a little bit more quantity than this but much less than this. And these all these bottles are connected to two channels here you can see the channels and those channels go all the way off here as well as there. And this is where your synthesis will happen actually. So this is the place where the reaction happens and these are the vessels they are called columns or called the cartridge basically they are the, I will show you what are these, these are basically cartridge which has some solid materials inside. So these are placed in this chamber and the Machine pumps of all the required reagents inadequate volume all the way to here and the chemistry happens here. And there is a display over here which can tell you what is happening, which step you are in or some monitoring steps are also there. So this is a beautiful invention beautiful discovery that allows you to do multi-steps organic reactions, very in a very clean manner all automated. You can simply press the start button on the computer and go home. And the machine will do all the work for you. So you can all you have to do to tell the Machine that sees that the desired sequence or your DNA that you want to synthesize and then it will do it automatically. So there are too many machines here. These machines are very much commercially used by the pharmaceutical industries as well as the biochemistry industries which supply and the oligonucleotides to customers. These basically synthesize a lot of DNA for different purposes which we need for our laboratories so we can purchase from them. Many numbers of machines connected to the computer as you can see, so all automated. So now, the synthesis here, the synthesis protocol involves a solid phase synthesis that is what I have written here DNA synthesis is done in a solid phase force for immediate chemistry. Phosphoramidite is the name of the reagent that you use for the nucleus or it is kind of a derivative of the nucleus. You do not have to remember or you do not have to even understand much of the mechanism that I will show. But still, it is I think it is interesting to see how stepwise chemistry can be done and how clean are they. And the whole chemistry is done not in a solution phase but in a solid phase. So the solid powder is packed inside this cartridge. So if you do, solution-phase chemistry, why not this is happening in solution? So, if you try to do solution-phase chemistry in a round bottom flask in your laboratory, or in a test tube, so for example, let us take a target first. For example, I want to synthesize a random DNA sequence A T G C. It is ATGC and AT, 5 prime, 3 prime. This is my target that is what I want to synthesize. Now, if you want to synthesize in solution what you will do first you will take A. So this is your A basically there will be a hydroxyl group here. And here you will see you will require a DMT that you will see protection would be needed because you do not want to touch here 5 prime. This is your A+ you have to take your T maybe this is OH or it can be another protection group whatever chemistry you do. I will show you the problem here. So and of course you have to synthesize the phosphate so what you need a phosphate and then an X whatever that can be it can be a living group so because you need a phosphate. I am writing p4 phosphate and X for a living group. So the essential chemistry is this will go and X will be eliminated. Let us say this chemistry works although it is very hard to find do direct chemistry like this. But let us say this works. So if you do this chemistry then what you get. You get A T sequence I am not right drawing the phosphate so a phosphate T basically in solution phase you get your desired A T so first is a tea you get your A T. What else do you get? One A may react with another A that will give you A, A. One T may react with the other T that will give you T, T plus. You will have unreacted T I am writing hydroxyl group free because of this plus unreacted A, not all reactions are complete. So if even if you use when you use these reagents after the end of the reactions they cannot complete all of them will not be consumed. So chances are high that some of these materials will, will remain as in free form even after the reactions. So after the first very first step what you get you to get 1 2 3 4 5 components in your solution. Out of this, only this one you desire. So if you want to get this in the pure form you have to separate all others. That is again that is a difficulty or you can move on to the second step second cycle. And then use G. G OH means I am writing because since the 5 prime is reacting always I am writing OH for 5 prime positions, GOH. Of course, the G means the whole of G. Here only the replacement will be. In the second phase what will happen? You will have A T G your desired compound plus here A A G plus T T G Plus G may react with itself you will have G, G you had some unreacted T left over here. That can react now with this G in the second step. So we will have T G you had unreacted A which may again react with G. So we will have G A and then again unreacted G will be there. So after the second step, you have multiple, so many numbers of products are formed out of them. Only this is your desired compound. So if you carry on, if you, like this, if you want to synthesize just 6 membered rings a 6 membered DNA then, the number of products would DNA products would be so high that you cannot find even your desired component. Or else you will need purification in each step which is really a painful job. So when you want to synthesize 50 more long DNA 50 equally bases long DNA then obviously you can see the problem in the solution phase. It is very, very hard to do in the solution phase rather if you choose to do it in a solid phase. Solid-phase is like this. Solid-phase synthesis means you have solid support. In this case, we use glass support. So this is a glass basically CPG is called the glass basically glass powder and that is connected with a functional group. Let us say it is connected with Ted O just consider kind of a DMT. Now, this is solid so you treat now this with the first nucleobase was eh-eh-OH-O. So, instead of I am straight ahead taking OH here because I have to deprotect DMT otherwise again, it would record an additional step. So this is connected this functional group is connected to the glass solid support now with a proper reagent. You can do this reaction and you can have A here with the OH free at the 3 prime ends. And what else you can have? You can have the unreacted of this. But this is now in liquid form. So after doing the reaction if you wash away all the liquid and only get the solid then all you have here is this plus, of course, the glass which did not react, the unreacted glass. And now I will show when we actually do the cycle of the DNA that we actually make this one inactive so that it does not react in the further step. Now in the second cycle, so this is the first cycle, the second cycle is you add the T, do the same thing, you get A T OH free and of course, all the unreacted liquid form can be washed away. So if you go like this and obtain your solid all the time, the chances of getting the side products of undesired products, smaller ones, are very, very less. So the productivity of the synthesis would be much higher if you do the solid phase synthesis because you are doing it in a located place. And you can remove the solid thing out of the reagents which are in the liquid form. So that is what the beauty of this synthesis, that this particular the phosphor emanate chemistry works on, solid support and it is solid-phase DNA chemistry.  So now if you look at the biology of how these nucleotides are synthesized, how this phosphate is synthesized, how that 5 prime position and the 3 prime positions of a nucleobase react? B1 B2 OH. In the biological cells, it is a spontaneous process and this reaction happens in the presence of an enzyme that we will see in vivid details in the just in the next module. So how it happens is that it has the Triphosphate O P P P I am not writing the phosphate. This is Triphosphate which is called the, in general, they are known as DNTP. Deoxyribose in its nucleobase T is Triphosphate for example ATP deoxyribose ATP is a form of triphosphate if there is adenine here is called adenosine triphosphate. So, in our body we have triphosphates we have a nucleobase triphosphates, they are available. And this reaction is catalyzed by the enzyme polymerase we will see just in the next module reacts with the phosphate eliminates diphosphate. And that will give you the nucleic acids phosphate so, B2 B1 OH that is how you get it in the biological cells. Now you try to do this reaction in the laboratory you will not succeed. It is very hard to do this reaction without the use of the enzyme. And therefore, we had to develop, people had to develop for a very long time there are many different methods that make changes to these variations to make this reaction happen. So in the solid phase for phosphoramidite chemistry, we will see that what is the reagent that we use instead of that triphosphate is this nucleobase analog that is used. This is the base where they are 5 prime is protected with a DMT and the 3 prime is protected with this which has a phosphorus group. And this is because this chemistry works well. It is only for the laboratory and this, after a lot of research and a lot of modulations, people have come with the idea that this molecule works best, so far. So here I will show you the first cycle of how it works and then go to the individual steps when you do it in the machine in the DNA synthesizer. So, the first thing that let us start with here is your solid support CPG and you have already done a little bit of synthesis. And now we are trying to add more nucleobases here. So the first step is that you deprotect the DMT out of this. And convert it this DMT is converted into the free hydroxyl group here. That is the step called Deprotection. So this is attached to the solid glass and then the reagent the second nucleus that you wish to add will pour in will be pumped into the solid. And then, it will react this OH is free and it can act as a nucleophile so this who can react with this phosphate. Before that this molecule is stable so you need to activate it. And that is done with activating reagent like this. So it will activate and then this OH will react to this phosphorus clipping this out. This is the coupling means you are joining to the solid phase with the new nucleobase. This is coupled together. So if you turn this as B3 let us say, here is your B3, so this should be B3. Your B3 is now attached to the solid phase along with the others. So coupling is done, your essential part is done here. And now, if you see as I have talked about that, you will have the unreacted material here the unreacted solid which did not couple will be leftover. And if you leave it as it is then it will react in the next phase. We do not want that. So, therefore, the unreacted thing is capped and gone away once that is that unreacted part is capped, it is dead. It cannot react again. So after that what is left you get the free form the same thing without the impurity without the other solid impurity. Next, if you see here, you are in your phosphate the phosphorus is pentavalent and that has an oxidation charge of plus 5 on the phosphorous. Here if you see that we have started with the plus 3 oxidation state on the phosphorous. Here also you have a plus 3 oxidation state of phosphorous. So we need to oxidize this phosphorous further into the plus 5 states that are called the oxidation. What it does is that it adds double bond oxygen here. So you now have a pentavalent phosphorous. And then you can continue with the cycle. You can still see your phosphorous is not really ready you still have a short tail and that we actually can eliminate in the last stage that I will show. So I will take the individual step one by one and then you can see how it over how it happens. So first let us start with the first nucleobase attached with the glass. This is your CPG solid, it is basically, solid glass that is connected via a linker. And that linker is connected with the forced sugar. This is the first base, base 1. And 5 prime position is protected with DMT. So, the first step is that you have to eliminate the DMT. And that is done by using a very weak acid that is trifluoroacetic acid or trichloroacetic acid just a little bit of 3% very dilute solution of this, it will eliminate the DMT, will make a free hydroxyl group. Again you do not have to remember all the mechanisms, but I am just trying to the mechanism is shown here but I will not explain it in great detail because I want to show you the exact processes first. That is the essence of it. Usually, I teach this at an advanced level so that is why I want to explain the mechanism in detail. So you get a free hydroxyl group here and your DMT will be eliminated in the form of a carbo-cation and this has a beautiful color. This has an orange to purple color and that is actually a way I will show you here, to understand whether your reaction is happening or not. Here once it happens here, you can see the outlet chamber when the liquid is going out of it, you can see the beautiful orange color that is passing through the channel. So that proves that your DMT has been deprotected; it is kind of monitoring the reaction actually. DMT will be out and you will have a free hydroxyl group. This is the most crucial step actually and now you have this free hydroxyl group with the solid phase attached base one. Now you add your pump in your phosphoramidite this is base two with the DMT protection of the 5 prime ends. And this molecule is pretty stable and therefore is less reactive. So we have to make it a little bit reactive so that this hydroxyl group reacts there acts as a good nucleophile. What we do is we use a little bit of Tetragyl. This acts as a catalyst here what the Tetrogyl does is that Tetrogyl is just a little bit more acidic compared to this nitrogen. So this nitrogen group here is a little bit acidic compared to this nitrogen. And therefore it provides a proton here. The proton from the tetralogy is shifted to this nitrogen. And then, you have the NH plus in this position. And Tetragyl becomes n minus which can be stabilized by the internal resonance. So essentially you have this form and this becomes a good leaving group. So now the hydroxyl group can react here eliminating this. And you have this compound here not the compound with the solid phase with your base one and this is your base two plus you have the solid with the base one. I should draw this is base one and your free hydroxyl here. This, this, this, this, this whole thing which did not react, unreacted this, this is your CPG, unreacted this. Now you have to make this inert otherwise it will react in the next phase. So the first thing that we do is wash away all the liquid out and write. So all these unreacted of the starting material this is gone; all the byproduct that is in liquid form are all gone. Tetragyl is gone, all in the liquid phase are gone. What will remain is only this and this, the unreacted one. Now we need to cap this. We need to make this inactive. So that is called the capping stage. So this is will be capped is basically a stratification reaction you will form an Ester here. Once you form the Ester is very unreactive it will be dead. It will not react in future steps. So all you have now is this with your base 1 and base 2. Now if we want to stop here also what are the other steps that are remaining? If you want to stop right here, if you want to continue, you can move on just like the two steps that I have just described. So now here as I was saying that it has a +3 oxidation state so it needs oxidation which is done usually by iodine in the presence of water and pyridine. All it does it includes double-bonded oxygen in this position. This is base 1, this is base 2, OR and this OR means the protection groups. So you have basically 5 plus 5 oxidation state phosphate ready. So we, you have still had this tail.