Ok, in the last session, we have discussed
how to produce the monoclonal antibodies by
the hybridoma technology in connection with
the generation of abzymes, which are basically
antibody acting as enzymes, ok.
Now, I ended up by saying that as organic
chemist what we can do is, we can help in
designing of the hapten.
Because it is the hapten which will be attached
to the to the protein the carrier molecule,
and the hapten should have the should have
your epitope.
Epitope is required in the hapten.
Now, the initial what are the different strategies
to come out with the hapten design.
First strategy was really not transition state
analogue, because the simplest strategy is
if you know the transition state, make an
analogue of transition state attach it to
the protein generate the monoclonal antibodies,
but that was not the initial studies that
were made.
The initial study by the way this whole catalytic
antibody is discovered in the in the western
coast in the California particularly in an
institute called Scripps Research Institute,
where two scientists Richard Lerner and Peter
Schultz, they developed this technology the
catalytic antibody, ok.
So, the first examples were based on the,
were based on producing the catalytic site
in the antibody, because every the enzymes
have a have a catalytic site, ok.
And this catalytic site contains amino acid
residues that help in doing the reaction like
in chymotrypsin what you have seen there in
chemotropism and the catalytic site, there
is a catalytic triad, serine then histidine
and aspartate, ok.
So, if these three, if you can generate them
artificially at the proper position, then
you can make an antibody which looks like
which will catalyze like chymotrypsin.
But to generate three different amino acids
at particular at designed positions is really
difficult; is really difficult.
So, people went for actually producing the
active site amino acid just only one, but
which is involved in catalysis, ok.
So, the first technique that was used was
basically generating the catalytic site; catalytic
site which will catalyze the reaction, ok.
I think if I give an example, then it will
be much more clear.
See there is a molecule a nitro phenyl with
a fluorine at the benzylic carbon and then
there is a hydrogen.
And this hydrogen has been made quite acidic
because there is a carbonyl group attached
to it, ok.
We know that if I want to a make double bond
here, I will definitely add a base.
And if and the mechanism for that is that
the base abstracts the hydrogen and the CH
bond pair goes here and the fluoride leaves.
So, that is the simple mechanism.
So, it is a beta elimination reaction
Now, instead of base, now if I want suppose
this we have not specified R, because some
times what happens R may be some group which
is very sensitive to base.
So, you cannot use base in that case to induce
this elimination, but we know that enzymes
work at almost neutral pH, so that is the
advantage, another advantage of catalytic
antibody that instead of base which can do
this elimination, whether if you can generate
a catalytic antibody which will also do this
elimination process, then that will be very
useful because that will be under very mild
condition.
Of course, this is an example which is nothing
but they wanted to have a proof of concept
that yes this is possible.
You can generate the particular amino acid
responsible for catalysis at the catalytic
site which is the active site, ok.
Now, what was done that you know that if I
have a if thing that if the antigen which
is invading our system, suppose that has lot
of amino acids like lysine lot of amino acids
like lysine.
So, its surface will be full of lysine and
lysines will be positively charged, ok.
Now, if this is a antigen, now if an antibody
is generated against it; if an antibody is
generated against it, I can definitely assume
that the antibody must be such because the
antibody is recognizing this, the antibody
must be such that it has got lot of carboxylate
groups.
Because only then there will be an interaction
with the NH3 plus, because otherwise how there
will be binding between the or how there is
recognition between the antigen and the antibody.
So, if the antigen has lot of positive charge
on the surface, so definitely the antibody
will have lot of negative charge on its surface
when it binds.
Similarly, if the antigen has lot of aromatic
rings suppose, then the antibody will also
have aromatic rings because that will give
you pi-pi stacking interactions ok.
So, these are certain very simple things which
you can predict that this is my antibody that
will be looking like this depending on the
structure of the antigen.
Now, in this case remember the antigen what
you are using is basically your that reaction
substrate here.
What you want is to generate an antibody which
will have a, so basically what I want to generate
an antibody which is a protein, instead of
the base the protein has a base, the protein
has a base right at that position where the
hydrogen is residing.
If the protein has a base which is generated
here by manipulation, then the same reaction
will take place, because what you need is
a base very in proximity with the hydrogen,
if there is a basic group at in proximity
with the hydrogen.
And if this molecule, of course if this molecule
binds to this antibody that is also very important
some binding parameter has to be there, so
it binds and the base is right in front of
the hydrogen.
So, now the base will abstract the hydrogen
and the elimination process will take place.
Now, that means, the what you have to do,
when you design the hapten, you have to think
that how can I generate a basic center at
the catalytic site.
How can I generate?
I already told you that if the antibody is
positively charged, then you get a negative
carboxylate in the antibody.
Now, here you want a basic group.
What is a basic group in a protein at biological
pH; that is also sometimes we think that the
basic groups are arginine, arginine, lysine,
etcetera.
But the problem with these that at the biological
pH lysine is present as a NH3 plus it is lone
pair is no longer available, arginine will
be present as the positively charged conjugate
acid.
So, they are not bases in the biological system
at the pH 7.2.
So, what are the bases then in biological
system?
A base is something which has got excess electron,
either the lone pair or it could be negatively
charged a like OH minus.
So, we have to think of which amino acid residue
exists as the negative charge and that is
nothing but the carboxylate, because it is
the carboxylic acid at biological pH glutamate
or aspartate present as the anion, so that
is your source of base, it is a carboxylate
ok.
So, if that means I have to generate a carboxylate
at the active site.
How can I do that?
If I have a hapten, see here it is written,
if I have a hapten like this where I put a
NHMe plus here, that means, the hapten have
a plus charge somewhere in the middle where
there was the hydrogen attached.
So, I have a positive charge here.
I keep this aromatic ring intact other things
more or less intact only I have to attach
the large protein because that you know that
you have to attach the carrier protein.
So, you make a this is up to this point is
your hapten and then you attach it to the
protein; this is the hapten.
In the hapten design, what you are you will
notice that this part I did not change.
Because why should I change?
Because these part will make a another aromatic
amino acid here which will recognize this,
this nitro phenyl group, and have a stabilizing
interaction by pi stacking.
And these NH plus NHMe plus will generate
definitely a carboxylate here because in order
to have this antigen antibody interaction,
ok, and exactly that was what was done.
So, this molecule was injected to mice, and
the anti body was isolated, the monoclone
antibody.
And each had an aromatic group here which
stabilize the nitro phenyl, and it had a carboxylic
group right where you wanted, and this then
underwent elimination reaction as desired.
So, this is one of the first example of catalytic
antibody, but again I repeat that this is
not the transition state analogue approach,
this is the an approach which directs the
generation of the catalytic residue at the
in the antibody.
Now, let us go to other examples, ok.
Here are the different strategies I just told
you that this is the first strategy; that
generation of catalytic residues.
I have given you generation of a carboxylate,
if some reaction if you want generation of
a positive charge something like NH3 plus
then you have to use a carboxylate in the
in the hapten, just the reverse way, ok.
Then now we will go to the I think this is
all detail, so I can we have already discussed
this.
So, we can skip it and go to the next one,
ok.
The next one is that what is the transition
state analogue approach.
And the first reaction to try that was tried
was the hydrolysis of say an ester.
Here this is X suppose this X is OMe say,
ok; alkaline hydrolysis of an ester.
Now, this is also called saponification hydrolysis,
we know that this is also called saponification
of ester.
Now, the mechanism of ester hydrolysis by
base is like this, suppose this is your base.
So, the base first attacks and forms what
is called a tetrahedral intermediate, ok,
this is a tetrahedral intermediate.
And this is your rate determining step, this
attack by the nucleophile on to it.
It is a bimolecular reaction, two molecules
are involved to form the intermediate via
the transition state.
And the next step is this O minus comes back
and this the leaving group leaves.
So, you get the hydrolyzed product, ok.
The hydrolyzed, this is Y, suppose the Y is
your OH minus then you get the carboxylic
acid, ok.
So, this is the mechanism, that means, hydrolysis
of an acyl system which is an ester goes through
a tetrahedral intermediate via a transition
state.
Now, as I said the intermediate is the one
closest to the transition state.
And so when I do the hapten design, I can
take the intermediate and see which molecule
resembles this, but it is stable, because
this is not stable nobody could isolate this.
You have to once it becomes O minus, it is
very transitory, this comes back and X leaves.
So, you cannot have this taken in a test tube
and seal it and that will remain forever that
is not true, this is also transitory, ok.
So, what you need is a stable molecule which
looks like this that means you should have
a molecule because this tetrahedral, it should
be tetrahedral not, number 2 it should have
a negative charge on the on the outward atoms
ok, and basically that is it.
So, what you need is your R group I said that
do not disturb the R group because if the
R group depending on the R group your binding
parameters are decided.
What you do is take a phos, this is called
phosphonate, sorry phosphonate, ok.
See basically what we are doing phosphonate
is, when a phosphorus is directly attached
to a carbon that is what is called let us
just remove Y and X.
We just say that this is minus and that is
your say O OR right now, right OR.
So, this is a stable molecule this is a phosphonate
molecule.
Now, look at the structure of the tetrahedral
intermediate and the phosphonate; instead
of carbon what you have is a phosphorous,
but this is tetrahedral.
And this has also got a negative charge, this
is like the carbon CO minus, you have phosphorous
O minus.
So, instead of carbon, you have just a phosphorous
is just slightly bulkier then carbon that
is the only difference.
So, what you need to do, you take an example,
let us see.
So, this is A, this is A, where ester this
is the ester, ok.
This is the ester functionality and you want
to hydrolyze it, this ester and the normal
is saponification OH minus.
So, this is the tetrahedral intermediate,
and this tetrahedral intermediate then it
comes here, and this group leaves; this groups
leaves here, ok.
So, you get the carboxylic acid and the corresponding
phenol.
In this case, the phenol the alcohol part
is the phenol ok, so that is the mechanism.
So, this is your intermediate.
So, what will be your hapten, then the hapten
is this part you do not change, again I repeat
this part remains the same.
This is now instead of carbon you take a phosphonate;
P double bond O, O minus and the other part
remaining the same.
Only thing through this R you attach it to
a linker and then the protein; you attach
it to a linker, and then that goes to the
protein, ok.
So, this is the hapten, it resembles perfectly
the that intermediate and the intermediate
is close to the transition state.
So, if you can generate an antibody which
recognizes this, then that is going to catalyze
the hydrolysis of this compound, ok, so that
is very simple, and they have done that, ok.
Similarly, if you want to hydrolyze an amide;
just like all those proteases do amide.
Again you can do it with alkali.
So, if you add alkali again you have that
tetrahedral intermediate, and then the tetrahedral
intermediate collapses, and this goes out,
and you get the carboxylate and the nitro
amine, ok.
You want a catalytic antibody to catalyze
this reaction.
So, now you generate the hapten.
Now, here is instead of carbon you have nitrogen.
So, this is nitrogen, now this phosphorous.
It should have a double bond O better, I write
it whatever is written is the resonance form,
but I just resonance hybrid form, but I am
not I write the full whatever.
So, double bond O, O minus and then this whole
nitro group is there, ok; via this group you
attach it to the protein, and then generate
the antibodies.
So, this is the this is what is called phosphor
phosphoramidite, this type of phosphorous
compounds, ok.
So, this is very simple, of course, the reactions
are not very difficult, the reactions are
basically your trying to synthesize generate
catalytic antibodies for hydrolysis of amide,
for hydrolysis of ester.
They have done also hydrolysis of carbonate,
all these things they have done.
But this is important you might say that sir
these reactions are quite easy, but as I said
in the actual case you might have certain
reactions, certain groups which react under
these conditions.
This is very mild when you use the catalytic
antibody.
And the other thing is that this will because
these are enzyme like reactions, their turnover
number will also be very high unlike the basic
conditions, the saponification, the turn over
number is also very high because their enzyme
like catalysts, ok.
Now, we come to a more difficult reaction,
ok.
This is you know some of the amino acids as
I said some of the amino acids are essential,
amino acids some are non-essential amino acids.
Essential amino acids means you have to give
them from outside, ok.
Now, the some of the microorganisms, they
can make one particular amino acid like phenylalanine
which we cannot make, phenylalanine has to
come from outside, ok.
Now, the biosynthesis of phenylalanine is
little complex, but we have taken only some
intermediate steps where phenylalanine, biosynthesis
is involved, ok.
Now, this is a molecule which is called chorismic,
OH the spelling is wrong, chorismic acid,
chorismic acid, ok.
So, via some biosynthetic path way this molecule
is generated.
And this chorismic acid, by an enzyme called
chorismate; chorismate mutase, it is telled
chrosmic mutase.
But actually chorismate because all these
remember all these acids are actually present
in the carboxylate form that is how you always
have glutamate aspartate.
So, this will be chorismate mutase; mutase
basically an isomerization reaction which
where the skeleton changes, isomerization
means molecular formula of this is same as
that.
So, it must be a kind of a rearrangement reaction,
ok.
So, that isomerization reaction is done by
chorismate mutase, mutase means where the
skeleton changes, ok.
The skeleton looks entirely different what
is there.
Now, if you look at this reaction, if you
want to do it chemically, you can do it chemically,
but you have to heat it on a very high temperature.
And a reaction that happens in organic chemistry,
we do it like this that this one goes here,
this comes there and that goes here, ok.
This is an example of what is called a 
sigmatropic reaction.
And specifically this is called a [3,3] sigmatropic
reaction why [3,3] because the bond that is
broken entirely is this one means why which
detaches the sigma bond that is broken when
you are talking about the pi bonds the sigmatropic;
that means, while looking at the sigma bond
which is broken.
If you look at all these, the sigma bond that
is broken is this one, and then this will
be one and that will be one and you would
number it on both ways.
So, this becomes 3 and this is also 1, and
that is 2 and that is 3.
So, this becomes ultimately the 3 and 3 carbons
are combining with each other, so that is
why called this is called a sigmatropic, [3,3]
sigmatropic shift, ok.
Now, this is the mechanism of course, this
is not the this is not the actual geometry
through which the reaction takes place.
The actual geometry is shown here.
It is little this the shape of this ring is
like this is like a kind of a boat kind of
thing inverted boat, ok.
And then you have this oxygen, you have that
carbon, then the double bond, and this CO2
minus, and here is double bond that means,
this is the same molecule.
But actually this is not a geometry when the
reaction takes place the molecule takes up
this geometry.
And then here what is what you can argue that
suppose this 3 and 3 are quite far apart,
how are they reacting with each other.
But in this geometry you can there is no such
problem because I can draw it here this, let
us see.
So, this is the O then that double bond and
this is the CO2 minus, and here you have a
CO2 minus.
So, when the reaction takes place, the arrow
goes like this, you have a transition state
which will look like a chair form, which will
look like a chair it is like this, which looks
like a chair.
I think if it is let us see in the next slide.
This diagram is not very well done.
I think I will try to see to show you that
how it looks like a chair.
It is actually the problem is; eraser, yes,
let us see the eraser; that is something.
So, this is the your this is the oxygen I
think better, remove it here, this is the
double bond, this is the CO2 minus, and your
earlier there was a double bond here and a
double bond there, and there was OH here,
ok.
So, now, the reaction goes by this, this comes
here, this goes there, that goes there, ok.
So, what you have is oxygen, then this, then
you have this, this something like this, it
is close to a chair.
If you look at this position, more or less
like a chair.
So, it is a chair like transition state which
is the intermediate; chair like transition
state that is involved in this process and
that is true for all [3,3] sigmatropic reactions
they go by chair like transition state they
are Cope, there are reactions which are Claisen
rearrangement that is a [3,3] sigmatropic
shift, ok.
Now, earlier people thought that is this is
a class of pericyclic reactions this occurs
either by a heat or by light we know all these.
But now earlier people believed that pericyclic
reactions cannot be catalyzed.
They are unaffected by; they are unaffected
by catalysts, ok.
But here is a catalyst here is an enzyme chorismate
mutase which does the rearrangement very fast
at room temperature you do not have to heat
it, ok.
So, that actually broke the myth that pericyclic
reactions can be catalyzed.
How the enzymes are catalyzing it?
Basically the enzyme helps the molecule to
adopt a structure, a conformation through
which the reaction takes place.
The enzyme helps the molecule to bind, adopt
a conformation through which the reaction
takes place, ok.
So, that is now if you want to make an make
a catalytic antibody to have this reaction
catalyzed, what you need to do is make a transition
state like molecule which is stable like this.
See this is the transition state what which
we are talking about and this is your there
is a OH here earlier, remember there is a
OH here in chorismic acid, so that OH via
that OH you attach it to the to the linker.
The linker remember has a diazo compound and
through the diazo compound you attach the
protein, because diazonium salts are prone
to attack and the diazo compound leaves.
So, by that you can attach your protein part,
ok.
So, basically what you have done?
You have made a molecule which looks like
the transition state, but this is stable;
you synthesize this molecule and via this
OH, you attach it to the protein, now you
generate catalytic antibody and that antibody
has been it is very successful that antibody
catalyzed this chorismate mutase reaction.
Chorismate going to brief prephinate; sorry,
hey what happened just a second prephenate,
this is called prephenic acid.
This is the intermediate for the phenylalanine
synthesis the so this is very critical step,
but this essential amino acid, so this has
to come from outside.
The question is how the plants or the microorganisms
make phenylalanine?
So, this is the route.
So, via the chorismic acid goes to the prephenic
acid, then there is a decarboxylative I can
show you.
Decarboxylative dehydration, but here OH is
a bad leaving group, so is has to be made
to a phosphate, so that leaves and that gives
you a compound which is this one CO-CO2H keto
acids or alpha keto acids, when we will read
the coenzyme some more coenzyme chemistry
we will see that this alpha keto acids can
be converted to amino acids, so that is the
story of phenylalanine and that has come because
of this catalytic antibody.
The last reaction is what I said that some
of the reactions which are disfavored reactions
which does not happen in standard organic
reactions which the that you can force by
catalytic antibody you can force the reaction
to follow a disfavored path way.
How?
So, let us take this example, this is a phenyl
with a with a some substituent then CH2-CH
2 and an epoxide and then the epoxide is connected
ultimately to a hydroxyl group.
Now, we here if you add some acid, so this
will be protonated the epoxide and as soon
as its protonated, because epoxides are strained
molecule they will open, ok; how they can
open?
Their their opening is assisted by this adjacent
nucleophile which is OH ok, so basically I
can write it here.
So, now you have two modes of reopening; one
is this can attack here that opens up or the
other is this can attack at this carbon and
this opens up ok, so these are the two possibilities.
The one is called 5-exo-tet.
What is tet?
Tet means you are attacking at a tetrahedral
carbon, because these are tetrahedral carbon
sp3 hybrized, so tet.
Why it is called 5-exo which is 5-exo?
That means, when it is attacking that one,
what you are making is a 5 member ring, so
that is 5.
And your whole epoxide is outside the ring
that is being formed, so that is why that
is called exo, so this is 5-exo-tet.
The other possibility where the lone pair
attacks this carbon, now this whole this bond
containing the epoxide becomes a part of the
ring, so this will be called endo, but again
you are attacking to a tetrahedral carbon,
but this is called as you are making a 6 member
ring, this is called 6-endo-tet ok; so this
is 6-endo-tet product, this is 5-exo-tet product.
Now, according to according to Sir Jack Baldwin;
this there are some rules called Baldwin’s
rules and if you do this reaction, you will
see that this is the product which is formed
because of the, because Baldwin’s rules
says that 5-exo-tet is favored over the 5-exo-tet
is favored and five 6-endo-tet is disfavored,
ok.
So, in the in the test tube if you do this
reaction with the chemical reagent is acid,
say like some Lewis acid you will get these
product as the major, but if you want these
acids if these your target is to make this
compound, then the question is how to make
a disfavored reaction favored, ok.
So, what was done is that when these compound
when this is attacking in the disfavored reaction
which is the endo, when it is attacking.
So, part of it is now broken part of this
CO bond; so this will be delta minus and this
oxygen will be delta plus, so the intermediate
or the transition state may be I can draw
the transition state, so that is delta minus
and then what you have is oxygen, these are
these are half bonds; ok, these are not formed
fully and this is the aromatic ring and that
will be delta plus, so that will be the if
you can mimic this with a molecule then that
will be your hapten, and that and with that
hapten if you can generate a catalytic antibody
and give it to this system, then that will
do these reaction, then that will do the 5;
6-endo-tet, ok.
So, what actually resembles this compound,
because this is not stable, so finally they
come out with a molecule which is an N-oxide
molecule.
Now, look at this molecule and try to check
here, see what you need is a is what is a
carbon here which is it may not be carbon,
you can have different atoms, but it should
be attached to a kind of a negatively charged
oxygen, so that what you have.
Instead of the carbon you a have nitrogen
here and you have a O minus here, ok.
But what you needed is a delta plus center
at the next carbon that means here, you want
a delta plus here and you want a minus here.
Now, because this nitrogen is positively charged
N-oxide; so why inductive effect this will
generate a delta plus at this position.
So, now if you look at this one and that one,
they are they resemble each other by see this
is tetrahedral, that is tetrahedral, this
has got a negative charge, this is the negative
charge and this is the positive charge and
this positive charge is generated by inductive
effect and this R is utilized to attach the
protein, ok.
So, now if you can generate a catalytic antibody
against this and that will be will act as
a catalyst to do to do a disfavored reaction
and ultimately you get this product.
In fact, this was done and this was a very
classic example and it showed the power of
catalytic antibody to do unfavored reactions;
there are many more examples of unfavored
reactions by using catalytic antibody approach.
We do not know any other approach by which
you can get a disfavored reaction into a favored
process, ok.
So, I think that ends this aspect of synthetic
biology that is the abzyme chemistry; or the
catalytic antibody chemistry, ok.
Thank you.
