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Combinatorial Chemistry

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Welcome back to this course on Organic Chemistry in Biology and Drug Development.
In the last session, we were discussing about combinatorial chemistry which is a
technology that has been developed to produce a large library of compounds and then
testing it through high throughput screening.
(Refer Slide Time: 00:54)

And the whole exercise is done to have a quick access to the hit compound, so that the
hit compound can be found, which would pave the way for finding the lead compound;
and ultimately lead optimization. This process, usually in earlier days, it was taking lot of
time because the biological screening was taking a lot of time. So, even if you make few
compounds per day or per week that would have been sufficient for a testing within the
time framed by the biologist.
Now, because of the advent of high throughput screening, there is a demand that you
produce lot of compounds in a particular day or two. And then get it tested as quickly as
possible, because the whole idea of medicinal chemistry or drug development is that one
should really very quickly know what are the failure compounds which are going to fail

or which are failing in the high throughput screening and try to pick out the lead
compounds or the hit compounds as early as possible without wasting much time and
energy.
So, towards that end, this combinatorial chemistry was developed. This is basically a
synthetic technology in which a large library of compounds are made. Basically there are
two techniques, one is called parallel synthesis and the other is mix and split method. In
parallel synthesis, this is basically a 96 well plate, in each well there is a bead, there are
few beads, and then you have produced a particular type of compound, it’s not that in a
particular well you have a mixture of compounds. There are several beads.
(Refer Slide Time: 03:03)

Suppose if I take this groove, this groove will have ultimately in the final product which
is represented by Z1, but Z1 is basically a combination of X1-Y1, and then you are
adding this making the hydantoins, basically it is a two-step process. The first step is that
adding the Y1 to the X1 and then you did some reaction; you apply some reaction
condition like heating with hot 6M HCl, so that this is a particular type of combinatorial
chemistry where you are making hydantoins. And what I am trying to say that each well
has only a defined compound and you know what is the structure of that compound.

(Refer Slide Time: 04:05)

On the other hand, if you go to the other technology which is called mix and split, in this
case you get a much larger library of compounds, but the problem is that in a particular
well or if you are doing it in test tubes or your container, we will have beads where each
bead is connected to a particular compound, but you do not know the identity of the
compound. It is a collection of beads of different compounds. But only one thing you
know that each bead is only connected to one particular type of compound, but you do
not know which bead it is.
So, let us start from there where we ended. So, in this mix and split, you take the resin
bead attached with the functionality. And then you attach; you divided into three pots.
And in this pot, you add A; and in the other pot, you add B and in the third pot, you add
C. So, you have resin bead here attached to a resin bead B, and here resin bead is
attached to C.
But then you mix these two, take all of them together and then split. So, what will
happen? Each of these beads in a particular well or test tube, will have A, will have
attachment to B and it will have also attachment with C. So, that means, there are beads
which are attached to A, there are beads which are attached to B, there are beads which
are attached to C. Now, you basically you have distributed into 3 pots or 3 wells and
your adding D in the first pot, E in the second and F in the third.

So, in the process, you are getting here A-D, B-D and C-D. Here you will get A-E, B-E,
C-E and so on. So, in the third one, since you are adding F, so A-F, B-F and C-F; then
you again mix it and split. So, when you spilt your each well will have beads which are
connected to A-D, B-D, C-D, A-E, B-E, C-E as well as A-F, B-F and C-F. Now, what
you are doing you have again split it into 3, 3 pots and then add G. So, when in one pot
you are adding G, in the other pot you are adding H and in the third one you are adding I.
So, there will be 9 different compounds attached to the beads, attached to separate beads;
you must understand this that a particular bead will not have say A-D-G and also
attached to B-D- G, that will not happen; because you are covering the functionality
(whatever number of functional groups attached to the bead) as they are all attached to
either A or B or C, when you started the synthesis. So, each bead is connected to one
type of compound not a mixture of compounds.
So, now you have 9 compounds here attached to the beads; here also 9 compounds; and
here also 9 compounds. Now, you test these beads containing the compounds and see if
there is any bioactivity in any one of these wells or pots, wherever you are doing the
reaction. Suppose there is some activity shown by this cluster, the next thing is that you
do not know actually know which bead is connected to what, but what you know is that
out of these beads, at least some compounds are bioactive. So, the problem now is to
basically know what is the compound attached to a particular bead; see you can
individually separate these beads and also test its bioactivity.
And then suppose I get a bead which shows bioactivity, but I do not know what is
attached here, whether it is A-D-G or B-D-G or C-D-G; so, how to do that? Now, you
can say that I will take the bead, and then detach whatever compounds are there;
remember one bead does not have only one valency, they are polyvalent beads that
means, from one bead you can get several molecules of these, but all are same
compound; if it is A-D-G, then all are A-D-G here. So, there is no scrambling of the
structure of the compound that is attached to a particular bead. But the big question is
how to know, what is the compound that is attached to a particular bead; because these
beads are not colored, it is not that blue beads are always having ADG, red beads are
having other compounds, and each bead is of same colour and everything.

Now, we have to basically deconvolute; you have a bead, which is attached to a
compound and that is showing some bioactivity. Now, the task is how to know what
compound is attached to the bead? One way is that you break this bond between the bead

and the terminal end and see the sequence of these different entities A-D-G or it is B-D-
G, you can check that.

But if these are some compounds which are not very easy to do the sequencing; you
know sequencing can be done usually on peptides and as well as for nucleic acids, these
are easier ones that you can do using Edman degradation or Sangers method as needed.
For other compounds, if it has different types of entities A, B, C are different entities,
then it will be very difficult to really know what is the structure of the compound that is
attached to a particular bead. So, how to know the structure of the compound attached to
a bead? There is a technique which is called tagging technique, I will show you what is
that.
(Refer Slide Time: 10:38)

This is the bead to start with, I said that these are polyvalent that means, there are lot of
binding sites here, where I can add A, B or C that is my reacting partner. So, when I have
this bead, I have the bead attached to a linker and that linker is distributed in two
channels, at one site, you can do one type of reaction and in the other site, you can do the
synthesis that you are interested in.

Maybe an example will demonstrate it and it will be easier to understand what I am
saying about the tagging method. So, you have a bead, you can forget about the linker for
the time being. So, the bead has reactive sites in one reactive site, you are adding A, B,
C, D, E, F and on the other site you are adding something which is easy to sequence.
Suppose, I have separated the beads into two aliquots; I am interested in making
peptides. So, in one aliquot I added glycine; so, the glycine gets attached at the synthesis
site. And at the same time I add some bases, in this case, it is CACATG. So, I add a base,
but for understanding you can say that I add something which is denoted as P1, here. And
here on the other aliquot, I add methionine, a different amino acid, and I add another set
of bases, which is denoted by P2. So, what will happen that this bead which now can be
represented that it has got glycine here and on the other side, because I am adding
glycine and then also I am adding this P1, which is a combination of bases, the bases that
are present in DNA.
So, glycine and on the other valent hand you are having this P1 and in this other aliquot,
you have methionine and here you are adding the P2 (another collection of bases). So,
then again you mix and split, if you mix and split and then suppose I add again glycine
here, so, what I will get? I have two containers. So, when I mix and split, so here the
beads will have both the characteristic this as well as that, because I have mixed it and
then splitted it.
So, when I added glycine what I will get? Remember whenever I add glycine, I have the
same set of bases P1. So, this bead will have glycine and then if I have added another
glycine and on the other side at the same time, I add this P1; so P1 will be attached to P1. I
also have methionine here so methionine and that will be attached to glycine and in the
tagging site that is my P2, and then P2 will be attached to P1, so this is the scenario. Now,
suppose I stop at the dipeptide. I see that one bead is giving some activity, then I am

interested what I have added here; whether it is methionine-glycine or it is glycine-
glycine. I know that if it is methionine-glycine, I will have P2-P1; and if it is glycine-
glycine, I will have P1-P1.

So, I make the complementary base sequence and see which gets attached to this base
sequence represented by P2 and P1; we call that primer. What is primer? Primer is that if
you have a sequence of bases you already know that and if you give the complementary

base, then they are going to go and hybridize. So, if it is P1-P1, you make a set of primer
which is complementary to P1-P1 and another set of primer which is complementary to
P2-P1.
So, now you see that which primer is actually giving the hybridization and then if you
see that they are complementary to P2-P1 and hybridizing with that bead tagging site,
then you know that it must be having methionine glycine. And once you know that then
you can separately make methionine glycine and do the bioassay. Now you know that the
first amino acid has to be methionine, so you can take methionine and then other amino
acids you can vary, and then optimize the hit.
So, basically you are synthesizing your compounds, at the same time you are adding a
tagging entity. In this case, our example was a particular type of base sequence, because
the base sequence is easy to detect by synthesis of the corresponding primers and see
whether it is hybridizing or not.
So, from that hybridization result, we can tell that what is the tagging code; it is P1 P2-P1
or it is P1-P1; and then you can actually detect the contents of the other test tubes; you are
adding again methionine. So, it will be here, it will be glycine and then methionine, so
that will be P1-P2 and the other cases P2-P2 that means, when the bead is connected to
glycine I know that the it is attached P1 on the tagging site; and when I added the
methionine, so that P1 will be attached to P2, because with methionine I add P2.
And then whenever there is glycine-methionine, then the tagging sequence will be P1-P2;
and whenever it is methionine-methionine, the tagging sequence will be P2-P2. So, you
can get four different tagging sequence attached to the bead, and from the tagging
sequence you can tell what is the peptide sequence in the desired compound.

(Refer Slide Time: 18:58)

There are different tagging techniques. I will not go into very details, but I will show
another tagging technique. And instead of having this nucleotide bases, you can have
other types of tagging systems. Like in this case it is said that resin bead.
In resin bead, you know that there is a synthesis site, where you do the synthesis and
there is a tagging site, where you do the tagging. So, whenever you add one component,
you have to add the corresponding tag. Now, this tag earlier I told you about the base
nucleotide bases, someone has devised this that whenever there is a synthesis done, on
the other tagging site, you are adding this nitrobenzyloxycarbonyl attached to a linker
that is attached to a substituted aromatic moiety. Again I repeat, the bead has synthesis
sites and bead has tagging sites.
One more important point is that the synthesis site is not just one, there may be several
synthesis sites and there may be more tagging sites also. Basically when you do the
synthesis, you maintain the concentration at such a level, so that the synthesis sites are
more or less covered. Leaving the tagging sites, tagging sites are also reactive entities.
So, we have to be careful that the tagging site are left free.
Suppose, I put A1 in the tagging site, I put this via ortho-nitro benzyloxycarbonyl group.
This ortho-nitro benzyloxycarbonyl is also photo labile. So, if you shine light here, this
goes off and carbon dioxide is liberated, then you are generating aryl halide. So, this is

the mechanism that forms the aldehyde and this loses the carbon dioxide and finally, this
aryl tag comes out as the benzyl system CH2OH.
So, basically there is a linker which is photo labile group and then you have this aromatic
ring Ar1. Now, there are other tagging sites here and there are also synthesis site here. So,
you maintain it in such a way that all the synthesis sites are blocked with A1.
You maintain such a concentration that some of the tagging sites are still vacant. So,
whenever you are adding A1, you are adding this benzyloxycarbonyl with Ar at the
terminal end with a different Ar1 aromatic aryl ring; some of the tagging sites still vacant.
So, when you do the next reaction, which means, in the first reaction you are adding A1
and you are also adding Ar1, Ar1 means via this benzyloxycarbonyl.
And then you are adding now A2; so, when you add A2, you add Ar2. So, A2 will be
attached to A1 and this Ar1 actually ends there, because Ar does not react with another
Ar. So, because of some of the tagging sites are now vacant, so now this will be
connected to Ar2. Still some tagging sites are left vacant; it is just a calibration of the
concentration that will work here, so this is a little tricky that you maintain the
concentration in such a way that your synthesis sites are all or filled up, but the tagging
reactive sites are still free.
So, one site is occupied by Ar1 via this benzyloxycarbonyl, and then the next adjacent
site you put a Ar2, when you do the second reaction. And if you do a third reaction you
can consider Ar3, because still some tagging sites are left empty, so you can put Ar3.
Now, after everything is done, you shine light and when you shine light your
benzyloxycarbonyl falls off.
So, what you will get is the Ar1-CH2OH, Ar2-CH2OH, then you will get Ar3-CH2OH.
Now, you do a HPLC, what will happen? Each tag is different, so each tag will show its
profile in the chromatogram. And depending on the number of tags or number of peaks
corresponding to different tags, you can identify what is the sequence of these entities
A1, A2, A3.

(Refer Slide Time: 26:16)

Whenever you add glycine, you have these tagging sites, so you add T1. So, basically
you are adding glycine, so you are adding T1. These beads are distributed suppose in
three pots. So, in the first bead you are adding glycine and you are adding a tag T1.
In the second container, you are adding alanine and you are adding the tag, tag means
representing the aromatic ring, because ortho-nitro benzyloxycarbonyl is common and
then the linker is also common. So, the substitution pattern is different in the aromatic
ring, so you add T2. And then here you add suppose serine and when you add serine, you
do not need to add another tag T3, but you can add T1 and T2 both.
So, what will happen here the bead will have glycine and then T1, and this bead will have
alanine and T2, and in case of serine you have T1, T2.
Now, suppose I stop here, I mix the beads, and then I distribute. So, I can now mix it and
then split it into 3, and then I add another glycine. So, in this pot, because I have mixed
all these, so it will have glycine and whenever I add glycine, I add T1. So, whenever there
is glycine, it will only have T1. I repeat, each pot now will have all the 3 components.
So, I will have alanine here and this alanine will be attached to glycine, but T2 is already
attached to the alanine bead. Whenever I add glycine I add T1, so that will have T2 and
T1. And then I have the third one that is serine. Serine already has T1 as well as T2. Now,
I have added glycine, I add only T1.

Now, if you stop at the stage one, we have mixed it and I want to know the compound
attached to each bead; whether it is attached to glycine, whether it is attached to serine or
whether it is attached to alanine. How do you decide? You separate the bead and then
strip off this by shining light, so T1, T2s will fall off, containing different aromatic rings.
Now, you push it into the HPLC, you get a chromatogram and then you match with your
reference one that where the retention times of each of these tags are there. Now suppose
I stopped at the first stage; question is whether the bead is attached to glycine or it is
attached to alanine or it attached to serine, how do I know? I just strip off this T1, T2 all
these tags and then push it into the HPLC or GC.
I will get the different peaks corresponding to this T1, T2, T3. And what happens that if
the bead is attached to only glycine, I will see only T1, because whenever I added glycine
I add only T1. If I see that there is T2 coming out as demonstrated by HPLC, then I know
that the bead is attached to alanine. If I see that both T1 and T2 are coming, then I know
that it is serine.
So, basically this process allows you to combine these T1, T2 and T1 plus T2; you are not
adding another extra T3. So; that means if you want to discriminate between 3 substrates,
in this case glycine, alanine and serine, you need two tags. One separate tag for glycine,
a separate tag for alanine and a combined tag for the serine.
So, if you carry on the synthesis of glycine, you introduce T3 another tag. So, whenever
you are adding the second glycine you add T3, when you are adding the second alanine
your adding T4, when you are adding the serine you are adding T3 plus T4. And if you
make a tripeptide, then when you add glycine you add T5, when you add alanine you get
you add T6, and when you add serine, T5 plus T6 ok, so that means, you have made
9 compounds, but you have used only 6 tags; you are not using 9 tags; because you have
a combination of tags. So, now suppose I see that in the bead I strip off, the tags attached
are here T1, T2 and suppose I also see T3 and I also see T4. I stop at the T3 and T4.
I take the HPLC chromatogram and what I see that I could see T1, I could see T2, I will
see T3 and I will see T4. So, if I see T1 and both T2, because they are involved in only the
first step of the synthesis that means, it must be serine as the first amino acid. Because, if
the first amino acid had been glycine I would not have seen the T2. If I only see T2, then I

know that the first amino acid is alanine, but I am seeing T1 plus T2 if that be the case
that means, the first amino acid is serine. What about the second amino acid, the second
amino acid I will see what are by tags T3 or T4 or a both T3 and T4. So, here I am seeing
both T3 and T4 if that be the case that means, the second one is also serine, so that means
I am having serine-serine linkage.
(Refer Slide Time: 34:48)

And so now I can develop a problem like this, suppose I see T1 I do not see T2, I see T3, I
am making the tripeptide now; and then I see T5 and T6. So, what is the sequence of the
amino acids? In the first one I am seeing only the T1, now T1 is given only when the
glycine is there. And then T3 second one, the possibilities are that I will see only T3 or I
will see only T4 or I will see T3 plus T4. Here I see only T3, so it must be glycine.
And then I see T5 and T6 together, if I see both together that is the third step that means,
it is serine, so that is the sequence of the compound attached to a particular bead. So, this
techniques actually simplifies if you increase the number of amino acids, but
proportionately you are not increasing the number of tags. Basically it is like a binary
system that if you have 0 and 1, you can get a combination out of only 0, 1.
On the other hand, suppose if you have T1, T2, T3, T4, T5, T6, then what will be the
sequence of your peptide it will be T4, T5, T6. So, I get T1 plus T2 that means it is serine; I
get T3 plus T4 that means this is also serine; and I get T5 plus T6 that means that is also
serine so that is the peptide tripeptide.

There are other methods of doing this; I just mentioned two methods, one is this primer
based method where you add the DNA base sequences attached as the tagging entity.
And then finally, you add the primer and then see which primer is hybridizing with the
nucleotide sequence that is attached to a bead as the tag.
And the second one you are taking a photo labile, ortho-nitro benzyloxycarbonyl
attached through a linker to an aromatic ring, these aromatic rings are differently
substituted. So, they will have different retention times, so that acts as T1, T2, T3, T4, T5,
T6 and the trick is that you use a two tags for three components that reduces the number
of tags, because after all that will be expensive. If you need the similar number of tags,
so that will be economically more expensive than if you reduce the number of tags.
Next we will go to some medicinal aspects, but before that we have now discussed the
drug discovery process, in general what is done, what are the different steps, how you do
that; and the second thing that we did is the combinatorial chemistry that is the
requirement for the day that how to get a large library of compounds, and then get it
tested very quickly. And also how to know the structure of the compound that you are
generating which is attached to a particular bead.
Thank you.