 So, now we are going to invoke a different kind of an experiment to simplify the cross peak fine structure. And this is called as COSY 45 or one can also use what is called as the E COSY. And COSY 45 is simply a modification from the COSY experiment. The COSY experiment has normally this sort of a sequence here T1, T2 and the COSY 45 means this angle is 45 degree, this flip angle is 45 degrees. This is 90 degree and this angle is 45 degrees, you can also use a smaller one, you can also use a 30 degrees and 40 degrees or whatever, but a smaller angle not 90 degree. What is the result of this? The result of this is the intensities in the multiplets, intensities in the cross peak are altered, as a result what happens is you may not see all the components, you will see some components and therefore you will see a better fine structure. But the some intensities will go to 0, therefore the cancellations will be less and you will see a better fine structure in this cross peaks. Now you can see the number of components is almost reduced to half when we do this sort of an experiment there. So in this of course this will not be present there and this one this is a 1 prime, 2 prime peak here and these peaks are not present and you see this structure is reduced, this structure reduced number of components is reduced. You can compare this with the peak here, see the kind of a structure what we had in this here see these ones are more components here, but the many components are cancelled and when the components are cancelled you are not able to measure the coupling constants, but you need to get those coupling constants. So therefore you need to do extract those informations, now we see you can see more components here in this because the cancellations are reduced because the intensities of the components are altered by this use of this 45 degree flip angle for this pulse. Now you can see many more components here therefore you can do a simulation which will be which will allow you to extract this coupling constants. So once again you can go here and then of course you do not see the cross peaks here as expected. So similarly for the 1 prime to double prime peak, this is the 1 prime to double prime peak now you see this of course does not matter because of the 1 prime, 2 prime coupling is not there it is 0 and this structure remains the same. But most importantly you can see here the way these peaks which were earlier present in this area, this portion they have been they have been reduced to 0 intensity. Therefore you do not see these peaks there and you will see better resolution within the fine structure. You will see 8 components here instead of the 16 components you are seeing only 8 components and that is what is very clear. So once you have this 8 components so you can actually measure this individual coupling constants very clearly in this area in all the C2 prime and all this corresponds to the C2 prime endo geometry area. This is the C3 prime endo geometry area this one is little bit more complex but here it becomes much more clearer with regard to the components which are present there. So and this of course becomes similar to this here and the C2 prime endo geometry is very well characterized by this fine structure here. You can clearly see 4 here and 4 there and these will be the plus minus plus minus and then you have the plus minus plus minus here and these ones are extremely useful for calculating the coupling constants. So this I will demonstrate to you in certain other things and similarly this one is now for the 2 prime 3 prime and 2 double prime 3 prime peaks. These are for the 2 prime 3 prime peak and these are for the 2 double prime 3 prime cross peaks. Now these are for the COSY 90 these are not for the COSY 45 and in this case all the components present will not calculate this fine structure it can be calculated in the same way as I indicated to you for the 1 prime 2 prime and the 1 prime 2 double prime cross peaks taking the splitting patterns of the individual nuclear individual protons and combining them with the splitting of the other ones you will calculate the structures here. So you see there are many cancellations depending upon the sugar geometry you will have different kinds of fine structures here. So one has to simulate these and all the peak patterns have to match at the same time. So you want to choose a certain set of coupling constants you not only must match the 1 prime 2 prime 1 prime 2 double prime must also match the 2 prime 3 prime and 2 double prime 3 prime fine structures. Then only you can be confident that your coupling constants calculations are correct. This is the same thing 2 prime 3 prime peaks how these fine structures are present in the individual as a function of the pseudo rotation angle there. So this one cannot remember these ones but whenever there is one face with a particular situation then you must this forms a database. Using this database one can compare your experimental spectra with this and simulate this using this of course this software was written for simulation purposes and so all these data is already published in progress in NMR and those you can make use this as the database. You calculate this pattern and experimentally experimental spectra you can compare and you can estimate the sugar geometries. Now this is for the 2 double prime 3 prime now this is for the Cozy 45. In the Cozy 45 you can see how these ones will change. So here you see for the Cozy 45 to 2 prime 3 prime peak this actually looks much more simpler. In the 2 prime 3 prime for the 2 prime 3 prime coupling constant of course will be there because that is about 6 to 7 hertz. So you will see in the C 3 prime endo domain also you will see that all these cross peaks 2 prime 3 prime cross peaks will be present. 2 double prime 3 prime peaks will not be present but the 2 prime 3 prime peaks will be present. For the north region of course this will be present all of these will be present and you can see this calculation that how the pattern happens this is in the Cozy 45 and once you have that you can actually see how the pattern number of components is reduced these resolution will allow you to estimate the coupling constant. And this is for the 2 double prime 3 prime peak the 2 double prime 3 prime peak will not be present in the C 2 prime endo geometry here because this is 2 double prime 3 prime coupling is 0 at this in this domain in this whole area from here to here that coupling is 0 therefore you will not see this you will only see for these and this is for the Cozy 45 you can combine this with the Cozy 40 experiment by and large it is better to simulate these ones than the Cozy 90 because the number of components is relatively less therefore you can obtain better dispersion of the multiple components and calculate the structure. Now this is for the 3 prime 4 prime peak now the 3 prime 4 prime peak so you see in the C 2 prime endo geometry region 3.4 prime coupling is 0 in the C 2 prime endo geometry it is very strong in the C 3 prime endo geometry that is in the northern region 3 prime 4 prime peak is very large therefore you will see very strong cross peak this is for the Cozy 90 this is not for Cozy 45 and you will see only for these ones you will see a very strong peak and the fine structures here will depend upon 3 prime 4 prime coupling 3 prime 2 prime coupling and that is how the 2 couplings which are present you can calculate the fine structures based on the coupling constant values and of course when you are calculating the 4 primes one has to take care of the fine structure of the 4 prime proton as well the 4 prime proton has a fine structure because of the coupling with 4 prime 3 prime but it also has the fine structure with 4 prime 5 prime 5 double prime okay one such remember that here so 4 prime will have a fine structure due to this is a 3 prime 4 prime but it will also have couplings to 4 prime 5 prime 4 prime 5 prime 4 prime 5 prime then you have 4 prime 5 double prime okay so plus plus plus plus minus minus minus so this will be 4 prime 5 double prime so therefore this way this has 8 components here therefore this structure will be very complex so this structure will be complex along this axis all of that is not resolved and many of those ones will overlap finally you will see only this much the total width is sort of the sum of the various coupling constants there therefore within that you have this various fine structures plus minuses and you will only thing you can see is that the 3 prime 4 prime peaks are not present in the in this domain which is the C2 prime endo geometry okay now this is the cosy 45 experiment for this oligomer for the experimental spectrum for this oligomer now you can see here the fine structures of the one prime these are the top ones are the one prime two prime peaks the corresponding bottom ones are the one prime to double prime okay now there are two one prime two prime there are two nucleotides here and these ones are the corresponding one prime to double prime peaks this is the one prime to prime peak of one nucleotide this is the 1 prime to prime of another nucleotide and the corresponding two double primes are here okay imagine the centres so this and this former pair this and these former pair and similarly in this area there is an overlap of two nucleotides so there are two one prime two prime peaks here and correspondingly the two one prime to double prime peaks are present here But there is one more nucleotide here which is embedded into this area, this is quite a substantial overlap of this here. Here again there is a quite a substantial overlap of the 1 prime, 2 prime, 1 prime, 2 double prime peaks. This one is very clear, there is a 1 prime, 2 prime, 1 prime, 2 double prime, 1 prime, 2 prime, 1 prime, 2 double prime. Now you can see the better resolution because of the COSY 45. If you had taken this COSY 40 you would not have been able to just separate out these ones components very clearly. So now you can use this to calculate. So once again here also you can see 2 here, 1 here and 1 there. This is 1, this is 2 and here there are 2. Now notice here this fellow, the 1 prime, 2 double prime peak is on the top, 1 prime, 2 prime is down. And this is because of the terminal one something is, this is identified and this pattern is very characteristic of the 1 prime, 2 double prime peak. You can see that all in all of these places. But there is an extensive overlap of peaks here. Now we will see, we will have to use different tricks to separate this out. We will see what to do there but I just indicate you do the experimental example. Now this is an example which clearly shows using COSY 45 how one can identify the coupling constants. So you see the fine structures here. What is present along this axis is the 1 prime multiplied, this is 1 prime. The 4 components you have the 1 prime, 2 prime coupling and 1 prime, 2 double prime coupling. And what is this peak? This peak is my 1 prime, 2 prime peak. This is my 1 prime, 2 double prime peak. So you can see here the fine structure. There are 8 components, there are 8 components here, also 8 components here depending on the coupling constants what we have there and the various coupling constants are indicated here. So 2 prime, 2 double prime coupling indicated here and then the 1 prime, 2 prime coupling indicated here. You measure the 1 prime, 2 prime, 2 double prime coupling along this axis and measure the 1 prime, 2 prime coupling along this axis or 1 prime, 2 double prime coupling along this axis. Also in this one you can measure the 1 prime, 2 prime coupling which is 9.5 hertz. This is the larger. And then this is the same 14.1 is the 2 prime to double prime coupling is the same. So therefore, you can determine 1 prime, 2 double prime coupling, 1 prime, 2 prime coupling and the 2 prime, 2 double prime coupling, all the 3 couplings one can measure from this 2 cross peaks. So this is illustrated in this experimental spectrum. This is the very beautiful spectrum and you can actually see the fine structures in these ones. Okay, now this is another experimental spectrum and then the simulated spectrum shown here for this one particular cross peak. The same spectrum which I showed you earlier, the big one and one particular cross peak of that one is, this is the A5 nucleotide, A5 nucleotide that is this one here, this is the phi and the 1 prime to double prime coupling of that. You see here this is the experimental spectrum and this is the simulated spectrum and overlay on this perfectly overlapping and when you do that of course you can get the coupling constants very clearly. Now this is a complex here with some drug and one can study what happens when the drug is binding whether there is a change in the sugar geometry as a result of the binding of a particular drug and so on. So this is a particular application to demonstrate that there is a change in the coupling constant. The sugar geometry actually changes from C2 prime endo to C3 prime endo when this happens there. Okay, now here are the simulations of the same spectrum which I showed earlier. So you remember this overlapping areas here? You remember the overlapping? This was the way which was so many nucleotides are getting overlapped there. In the COSY 45 experimental spectrum there are 3 nucleotides here, 3 nucleotides there like C1, C13 and this contains both of the H2 prime, H2 double prime cross peaks. All of them are present in this area. Now one could simulate this, this could only by simulation you can extract this coupling constant. You vary this different parameters there, how many coupling constants are present? You can actually vary this coupling constants and simulate this to match this perfectly. So this is the wonderful demonstration of how to extract the coupling constants by simulations. And these are the little simpler ones, you have the T8 and T7, there are 2 nucleotides here, these are the 1 prime, 2 prime peaks and then this is the T7 experimental spectrum and the simulated spectrum and these are identical and you can confidently determine the coupling constants. Now here there is an overlap of C1 and C13 here, 2 1 prime, 2 prime peaks are overlapping in this area and at, see you look at that the way the chemical shifts are and the coupling constants are, the patterns are looking different. So this one of course now we simulate it perfectly to get this coupling constants from there. Okay, now that is so much for using the COSY and the COSY 45 but now you get into even more difficulties of course that is that the simulation was done fine but can we do something more? Well so we said that we have this coupling information, a redundant coupling information along both the frequency axis. We have the coupling information along the F2 axis as well as the H1 axis. For example the H1 prime coupling is present along H1 prime H2 prime coupling is present along with the 2 axis and the chemical shifts may overlap. Therefore what we do is, here we use a decoupling technique, decoupling technique this is constant time, constant time COSY. We had discussed this during the methods, so what happens is this will result in decoupling along F1 axis. Therefore in this now you do not see the splitting along the F1 axis at all. You only have couplings along the F2 axis, along the F, this is the F2 axis, H1 prime is the F2 axis, this is my F1 axis if I am looking at that. Therefore you do not see the splitting along this axis, therefore this simplifies the cross peak fine structures much more as a result of which you can measure this coupling also relatively better and if there are overlaps of the peaks that also will get resolved. These are the simulations for the different sugar geometries how it happens and once again for the 1 prime 2 prime there will be no peak here because this is the C3 prime endo geometry for the 9, these are C3 prime endo domain and 1 prime 2 prime coupling is 0 there you will not see as you start increasing it starts picking up numbers and you will see the highest intensities in this area for this particular peak 1 prime 2 prime and once again it will be 0 here and this is for the 1 prime 2 double prime. Once again this one is smaller here because this coupling constant is not that large as compared to the 1 prime 2 prime coupling but you can see the 4 components little bit better here because the relative intensities are different and that will allow you to measure the individual coupling constants in this because what you see here is the fine structure of the H1 prime alone or on this axis you do not have the H1 prime has is what it is a doublet of a doublet it has 2 couplings 1 prime to 2 prime and 1 prime to 2 double prime. So, you can see the 4 components here although there is some cancellation in the middle in this area that is why the central peaks have lower intensity but nonetheless you can actually calculate measure the separations you get an idea as to what they will be they may not be precise but you will get the precise values after the simulations. Now, this is for the 2 prime 3 prime in the same area for the same for entire region of the sugar geometries. So, these form a database all of these simulations for my database for the this is again F1 decoupled cosy this is constant time cosy constant time cosy or this is also called as F1 decoupled cosy. So, you will only see the fine structure of the what is present along this axis you would not see the cross fine structure here. So, this will show the fine structure of the 2 prime proton the 2 prime proton has what couplings it has a 2 prime 3 prime 1 prime 2 prime and 2 prime 2 double prime. So, it has those 3 coupling constants there and this will be 8 components there but of course all of them are not sufficiently resolved. So, but you can see 4 components there this ones and so you can see better representation of this more components here actually you see more components there same as to here it should be 8 components you see actually see 8 of them there because the relative intensities they you can see this separate them better here but the intensities become more than of course they tend to merge and then you will start seeing this sort of a pattern. Now, this is the same spectrum which I showed you earlier of the same molecule which was let me show you corresponding one I will go back and show you that this is constant time cosy this spectrum is of the same one as this one this is the same region it is the same region of the same molecule with the constant time cosy see now you can see all of them are so well resolved the ones which are present here every one of them is 1 1 1 1 line. So, 2 prime 2 double prime correspond you can also figure out which you are which 2 are in the line. So, this corresponds to this this corresponds to this. So, you can see you can draw the lines now you see here that there are 2 overlapping which were they are very complex now you see here there are 2 nucleotides here G2 and G12 and they are both the 2 prime 2 double prime they are both present here they are all overlapping and the G9 2 prime and 2 double prime peaks are also overlapping there and we since we removed the coupling constant along this axis they have become now one line here and one line there. So, similarly here for the G12 one line there one line down another G2 one line here one line there. So, each one of them is giving you 1 1 line. So, this is 1 line here 1 line there and the correspondingly 1 line here 1 line there and similarly this one 1 line here 1 line here 1 line here and 1 line here. So, that will allow you to figure out how many peaks are overlapping. So, even in such a complex situation by doing this constant time cosy you are able to separate out these various components then you can actually simulate these to calculate the coupling constants. Now, this is the 2 double 2 prime 2 double prime 3 prime cross peak region this is the extremely complex area extremely complex area look at the overlaps here. Therefore, this you could not have analyzed without the F1 decoupling this is again a constant time cosy this is again a constant time cosy or you are also called as F1 decoupling cosy and you see this area. So, how complex it is if you had the coupling constant information here this would have been impossible to analyze if you had the coupling information along this axis as well you would not have been able to separate out all of these components. So, now we can see clearly 3 lines 1 2 3 and that is a blow up here. So, similarly there are 2 lines here the T7 T8 we are so close and similarly C1 C3 these ones are again G2 G12 which you saw earlier also in the case of 1 prime 2 prime 2 double prime as well. So, therefore, while now by doing this you are able to figure out the individual patterns chemical shifts and the individual coupling constants. You have to be able to measure all of these coupling constants at the same time then only we will have the confidence in those ones. Now, you see these are the simulations having obtained those experimental spectra now to generate where there are overlaps you will have to resolve to the simulations. So, you have this experimental spectrum here the 1 prime 2 prime 2 double prime are overlapping here in this and that one is distinctly seen this was of the G6 residue which was there below and 1 prime 2 prime 2 double prime and this is the 2 prime 3 prime of the same residue. So, using the same coupling constants you must be able to fit all the cross peak fine structures the 1 prime 2 prime 2 double prime. So, you have the 2 prime here the 2 double prime here 2 prime there 2 double prime there and then you measure this fit the all the coupling constants and get the fine structure this is the 2 prime 3 prime. Now, in this case experimental and simulated once again you perfectly generate the simulations to get this individual 2 nucleotides overlapping even so you could get the coupling because you have one line each along both along the vertical along this axis and you have the fine structures along this axis therefore you can measure this coupling constants quite clearly. So, you can see here this one and this one and this one and this one these two belong to G2 top and belongs to G2 and these one belongs to G12. So, therefore you can actually measure this and separate the coupling constants. That is so much for the sugar geometry. Now, we turn to the one of the torsion angles along the backbone and that is epsilon torsion angle the C3 prime O3 prime torsion angle in the along the backbone and this actually couple is quite restricted how do we know this is restricted you do determine this by there is no measurable parameter here to find out this coupling constant NMR parameter although you can sometimes you can use the P31 spectra and here you also determine this by energy calculations you calculate the energy of the system for a particular dinucleotide and what are the favorable energies what is the most stable state it turns out that when you do this that you have the lowest energy for this epsilon torsion angle is around 270 degree and here you have a very nice minimum here with very small energy and as you go anywhere you go away from there the energy goes up very rapidly if the energy is like this is very unlikely that these will get populated in any normal situations. Therefore, by and large when you are actually calculating you generally consider only this value or this value for the epsilon torsion angle when you build the models for the structure calculations then you will have to use this sort of constraints you therefore what constraints you use first of all you have the assignments and then you have energy values here for the epsilon torsion angle and the sugar geometry determined from the coupling constants that is that forms the input for the structure calculations and we have not yet come to the distances we will use the distances that will form a particular part from the nosy is the ultimate finally for to calculate the structure because you have the various inter proton distances which we measured and that is the one which we are going to use for structure calculations this will form I guess we will go that into the next class. So, here so let me summarize once more we had the first of all we obtained the assignments using the nosy and based to 1 prime connectivities based to 2 prime to double prime connectivities and then from 1 prime to 2 prime to double prime connectivities and then we analyze in the cosy spectrum the fine structures in the cross peaks of 1 of the sugar ring 1 prime 2 prime to double prime 2 prime 3 prime 3 prime 4 prime and we saw how there is a distinct pattern of cross peaks depending upon the sugar geometry with that we get a well idea about the sugar geometry constraints this we have to use for the structure calculation which we will take up in the next class.