Welcome back to this course on Organic Chemistry
In Biology and Drug design; last time if you
remember we were discussing.
The aspects; some of the aspects of synthetic
biology, specially we were interested to know
that whether enzyme like molecules can be
made to catalyze a particular reaction, ok.
I told you the limitations of the enzyme chemistry,
the limitation is basically the number of
enzymes which nature has provided us, that
is the limitations; because if there is an
organic reaction where there is no counter
part of an enzyme, then you do not have any
enzyme catalysis for that reaction, ok.
So, people have thought that once we know
the principle as the principle of enzyme catalyzed
reactions are known.
So, whether that principle can be adapted
and whether the biological system can be manipulated
to generate enzyme like molecules that can
catalyze, virtually any reaction provided
you know the mechanism of the reaction and
the transition state the geometry of the transition
state for that reaction, ok.
So, that was we started and we basically we
know that enzymes catalyze reaction by stabilizing
the transition state more than the substrate
or the product ok; that means, the transition
state geometry is complementary to the enzyme
active site.
This complementarity means electronic complimentarity
as well as steady complementarity.
Now there is in the biology there is one process
which guards us against infection that is
constantly acting as a surveillance mechanism
to protect us from environmental systems,
which are harmful to the body and this is
called what is called immunity, we have immunity.
So, long as we are we are living.
So, we have immunity that is guarding us against
infections.
Now immunity we know that basically the immunity
is a response that is done in the body, that
is created in the body and against some invading
organism or invading molecule, that needs
to be that is not known to the to the system;
that means, which is not self molecule.
So, it should be something which is foreign
particle and this foreign particle is known
as antigen.
And what happens in case of humoral immunity;
I told you humoral immunity is basically done
by mediated through the through proteins that
are flowing in the blood cell; because there
is another kind of immunity which is called
cellular immunity, which is which guards us
against as I said the infections the infections
foreign agent, but that is by cell mediated
mainly T cells that guard against us, but
this is humoral immunity, where there is antibodies
which are which are made depending on the
structure of the antigen.
And this antigen has the exact complementarity
to the this antibody or the antigen has complementary
to the antibody or vice versa, ok.
So, when the so, these fact was known and
sorry just a second and so, people thought
that whether this matching of a antigen antibody
whether that can be exploited to make enzyme
like molecules.
And the theory behind this is shown here;
I will just skip some slides yes.
See if you know the transition state of a
molecule and if this transition state, this
transition state will not be stable at all
not if this transition state is not stable
that is always the case, then what we can
do; we can make a molecule which is analogous
to the transition state because transition
states are transitory in nature, they cannot
be isolated.
Ok.
And so, you make an analogous molecule whose
geometry and electronic character is very
similar to the transition state for the reaction
and this analogue is stable; obviously, than
the transition state and this isolable can
be made and this is what is called a transition
state analogue, ok.
So, transition state analogues are basically
molecules which are electronically and sterically
very similar to the transition state of a
particular reaction ok.
So, what you do in this case to produce antibodies
which recognize this transition state analogue.
So, you basically write what is the transition
state analogue possible, make that molecule.
And then you have to because last time I said
that small molecules small molecules do not
elicit immune response because small molecules
are not captured by our immune system.
So, there is no immune response because they
are very small.
So, in order to have immune response for the
small molecule, what you do; you attach the
small molecule through a linker to a large
molecule say another a protein.
So, you have a transition state analogue protein
conjugate and then you inject that into an
animal like mice, and then what the mice will
develop antibody because they also have the
immune system like us; the mice will generate
antibody and these antibodies I told you that
is when the antibodies are generated they
are called polyclonal antibodies because they
recognize, because they recognize different
parts of the suppose this is your antigen
and where your transition state is attached
this is like the transition state analogue.
So, now, when the antibodies are generated,
so, some antibodies will bind here, some antibodies
will bind there, some antibodies may bind
there.
So, it just looks for the characteristic shape
of the surface and accordingly it generates
the antibody.
And if you are it can generate the antibody
for the transition state analogue also.
So, you what you have now is a collection
of different antibodies and these antibodies
one common thing is that, they go for the
same antigen but they bind at different sites
and they have these binding affinity are also
different.
These are called polyclonal antibodies, ok.
So, if you have polyclonal antibodies some
of the antibodies in this polyclone may be
recognizing your transition state; may be
recognizing your transition state.
Now remember this site where the antibody
binds, we have a name for that, that is called
epitope.
Epitope is the site where the antibody binds,
ok.
If the epitope happens to be present in the
transition state analogue, then you will get
an antibody generated against it, ok.
Now the next target or the task is to separate
this polyclonal set where few of the antibodies
may be recognizing the transition state and
you are only interested in this one, because
that is the one which will catalyze your reaction
because that is recognizing your transition
state analogue; that means, that will recognize
your transition state; that means, that is
going to catalyze your reaction because the
transition state energy will be lowered by
stabilization with the like the antigen antibody
interaction..
So, what you need to do; out of this collection
of antibodies polyclonal antibodies you have
to separate, you have to separate the separate
individual antibodies specially you are interested
you are not interested in antibodies which
binds to the protein molecule, you are only
interested in the antibody that binds to the
transition state analogue because that is
going to be your catalyst.
So, now, but this will be monoclonal antibody
because that goes only to one site they are
binding in the same site with same efficiency,
ok.
So, these are called monoclonal antibodies.
So, I think I have a definition here polyclonal
and monoclonal antibody.
This is the one antibodies are produced by
a single clone of B cells, clone means virtually
nothing, but xerox copies of of something
ok.
So, you have a xerox copy of your whatever
mark sheet or certificate.
So, those collection of xerox copies of the
same mark sheet are what is what are called
clones, ok.
Now if you have say mark sheet for the higher
secondary exam and this and your plus 10 exam
and if you have mixed up all these mark sheets
then you have a polyclone, ok.
You have a polyclone the more number of exam
mark sheets are mixed together the more polyclonal
the system is, ok.
So, from that system of polyclones you have
to now separates; a the certificates basically
the Xerox copies which are the exact certificate
or exact xerox copy of the original mark sheet
of our class 12.
So, that is what is the monoclonal.
So, you want a monoclonal set which is only;
that means, they are all same type; they are
the they are all same.
So, they are produced by same B cells because
there are different types of B cells one type
of B cell will produce one set of antibodies
another type of B cell will produce another
set of antibodies; and then there are different
kinds of B cells.
So, they will all produce different sets of
antibodies and these different sets of antibodies
ultimately their target is that whatever is
the invading organism, but they are polyclonal.
So, now, you have to separate it into monoclone.
So, that is the challenge.
Now there is a technology which is called
hybridoma technology, that hybridoma technology
allows us to separate or to isolate monoclonal
antibodies from a polyclonal set, ok.
And this is a very important development in
molecular biology, this hybridoma technology,
this is very important tool in biochemistry,
molecular biology and also in medicine because
if you want targeted drug delivery then what
you do; you attach a monoclonal antibody which
targets something, some particular cell which
you are interested to destroy and so, it will
go and bind to that cell and then the drug
is released and the cell dies that is one
aspect.
Another aspect is diagnostic tool.
If you want to really know the presence of
certain things in the blood, in tidy, in minute
amount but still you can use monoclonal antibodies
which recognize and changes there are techniques
which changes color.
So, those are diagnostic tools, ok.
Now let us a just give a brief glipmse of
; again just a quick review of these definitions.
Hapten is the small molecule; that means,
basically the transition state analogue, which
is which itself cannot elicit immune response
but when attached to a large molecule like
a protein can generate antibodies which recognize
the small molecule; that means, the transition
state analogue then that will be called a
hapten.
An epitope is what I said is the site where
the antibodies bind or this is also known
as antigenic determinant and the third term
is abzymes; abzymes is the actually the whatever
we are describing now abzymes are these enzymes
which are also called catalytic antibodies;
that name has come from antibody as enzymes.
So, that is why the name is abzymes but it
is also known as initially it was known as
catalytic antibodies, but to abbreviate it
you have now the name given to abzymes; antibody
as enzymes.
We have done this monoclonal and polyclonal
antibodies, next is that how do you really
now isolate the monoclonal antibodies.
Possibly before we go into that we just see,
what is the structure of the antibodies.
The structure of the antibodies is shown here
antibodies are basically is a tetrameric protein
molecule, they are proteins they are made
up of amino acids.
So, it is a Y shape kind of thing, but you
see there is one bigger chain that is called
the heavy chain and this is called the light
chain; this is the light chain.
And these two chains are attached by disulphide
bonds and this part this red part is what
is the antigen binding site.
So, the antigen actually binds here and this
is also the variable part, because different
antigens are different structures different
characteristics.
So, the most variable part is this is this
antigen binding site which is at the top,
ok.
So, this is just a cartoon picture of what
is the shape of an antibody, ok.
Now, let us come to that, how do isolate the
monoclonal antibodies.
I think I will just write in pens.
So, what we have; we have a challenge now
that is antibody we have antibodies which
are polyclonal antibodies; antibodies are
made I told you by the B cells; ok, by the
B cells; antibodies are produced by the B
cells.
Now different when you inject the transition
state analogue coupled with the protein and
then inject it into the mice, antibodies are
generated.
You do not isolate the antibodies because
they will be very small in number what you
isolate is the antibody producing cells, which
produces the antibodies the antibody producing
cells and they are the B cells.
So, you isolate B cells now when you isolate
these B cells, you have different types of
B cells because they are they are making polyclonal
antibodies.
So, some B cells produce one type of antibodies
and there will be some B cells which will
make another type of antibodies.
So, these are the different B cells, ok.
Now these B cells what I said that, they make
polyclonal antibodies; polyclonal antibodies.
Now unfortunately these B cells are very short
living, short life; they have extremely short
life.
So, if you isolate them they are going to
die; you cannot really multiply them and unless
you multiply them, you will not get a supply
of the antibodies.
Remember each of these B cells are producing
antibodies, but if you want to get a supply
of antibodies you have to culture them, you
have to grow them, but unfortunately they
have a short life and they die very quickly.
So, now, what you have to do?
You have to make them kind of immortal, means,
so, that they do not die ok.
You have to make them immortal and to do that
what you take the help of something which
is extremely dreadful, if it is present in
the body, but that we will see the application
of those type of cells.
See when there is a we know that the cancer
cells which are also like myeloma cells, they
are they cannot be destroyed very easily they
are they always replicate they are virtually
immortal; unless you bombard them with UV
light or some chemotherapy or radiotherapy,
otherwise cancer cells are immortal they will
grow continuously; that is the problem.
So, that is called the this malignancy they
grow they have a they grow continuously they
are immortal basically.
So, now, what you do?
You take a cancer cell and what you do, take
all these cells and these collection of this
cancer cells which are these are also called
myeloma say myeloma cells I think the spelling
is like this myeloma cells.
So, when you take all these together and put
lot of pressure in a solvent in say polyethylene
glycol then what happens?
Like if you take two blocks of ice and then
press them and release the two blocks of ice
become one, this is the process called regelation
and that you have read in physics, high school
physics.
So, here very similar thing happens; if you
take all these cells and then force them then
what will happen?
They will fuse with one another.
So, what you will get is the cancer cells
which are fused, I think I have to take different
colors; see some are fused with these red
ones.
So, you have a cell now which will look like
this, this is your cell; part of it is cancer
cell part of it is here what is that; part
of it is your that B cells, ok.
So, now you have this.
So, your cell will look like this, again it
is another B cell which is fused to the cancer
cell and I will just write the third one also.
So, that will also fuse with these sorry,
I think this is the same color; just a second,
the pen which color it is that this is the
color ok.
So, you have now the other cells ok.
So, you have now these cells which are fused
the B cells fused with the myeloma cells,
which are basically cancer cells and these
cells are what are called hybridoma cells
because they are hybrid cells.
You have a mixed you have a hybridoma cells
ok.
Interestingly because these hybridoma cells
have the cancer cells in it so, they are basically
now immortal, they are basically immortal.
But remember when I do this fusion, some of
the cells may not fuse at all because there
is no guarantee that all cells will fuse,
ok.
So, some will be the just the myeloma cells
nothing else and if there are B cells, B cells
which are not fused they will die automatically.
So, you do not have to do anything.
So, now, what you do; you culture this in
a plate and in a special medium which is called
HAT medium (Hypoxanthine Aminopterin and Thymidine).
It is a special medium; this medium does not
allow only cancer cells to grow.
So, it must be having some antitumor agent
here, ok.
So, these does not allow these only cancer
cells to grow, but this allows the other cells
where there is this cancer cell plus the B
cells they will grow here, ok.
So, interesting; so, some of the cells.
So, basically this will grow here, this will
grow here that will grow here ok, but the
cancer cells cannot grow, they cannot grow,
this is called HAT medium.
So, once you have that we are not going into
very details you have a polyclonal you have
a polyclonal set here, because some of the
cancer cells are fused with this one some
will be the blue one, some will be the red
one.
So, now it is your now there is a process
by which you can separate this antibody producing
cells, hybridoma cells, monoclonal antibody
producing hybridoma cells.
I can talk about the principle behind this.
The principle is that once these cells grow
you take these cells collection of polyclonal
antibodies and then you do dilution; that
means, you suppose you have 100 cells in a
test tube, these are polyclonal, but if you
dilute double dilution.
So, you have 50 cells and then you have 25
cells.
So, and then you every time you culture them.
So, at some point of time, you will get a
cell which is only particular type of B cells,
once you have that they are the monoclonal
antibody producing hybridoma cells.
Basically it is a high dilution technique
and ultimately you can separate these hybridoma
cells into monoclonal sets.
Remember if each hybridoma cell is immortal
because they are fused with the cancer cells.
So, they have the property of the cancer cells
at the same time, they will produce the antibody
which will bind to the antigen that you have
targeted, ok.
So, this is very interesting that is what
how do you separate this monoclonal antibody
producing.
Now suppose ultimately you get a monoclonal
antibody which recognizes or which binds to
your the transition state analogue; which
recognizes the transition state analogue.
If that be the case, then you take this hybridoma
producing these hybridoma cells, the advantage
is that you can always grow the cells whenever
you want.
You grow these cells and you can get grams
of this monoclonal antibodies, the antibodies
you want to produce antibodies; who produces
the antibodies; earlier the B cells, these
B cells are fused with cancer cells thus making
them immortal.
So, you keep it in the fridge, whenever you
require you take a specimen of this hybridoma
cells, do fermentation and then isolate because
they will generate the antibodies and you
can isolate the antibodies.
And these antibodies are the ones which will
catalyze the reaction that you are talking
about, ok.
I hope the principle is clear because upto
this part is biology but then we will go to
the actual chemical reactions that have been
catalyzed by or some examples we will describe
which are catalyzed by this by this technology
by abzymes, ok.
Now I told you in the when this I started
this, I told you something that this catalytic
antibody discovery is extremely important
in the sense that virtually any reaction can
be catalyzed but there is one rider that the
mechanism has to be known; but there is another
interesting part of it those who have studied
organic chemistry, they know that many of
the reactions are called regiospecific reaction,
stereoselective reactions, or chemoselective
reactions.
There are different kinds of reactions which
have a particular selectivity.
There are different types of selectivity;
regioselective, stereoselective or chemoselective
all these type of things are there.
Now there is something some reactions are
actually against the rules of selectivity.
So, those reactions are called disfavored
reactions; that means, if you try to do a
reaction forming a cycle and suppose it is
a 6 versus 5, there is a possibility of a
six-membered ring formation and a five-membered
ring formation.
Usually we think that 6 is more stable.
So, six-membered ring should be formed, but
the six-membered may be disfavored; disfavored
means the orbitals are not aligned to do a
bond forming reaction to lead to a six-membered
ring, ok.
So, there are certain reactions which are
called disfavored reactions and this catalytic
antibody technology can be used even to catalyze
disfavored reactions which were otherwise
not possible, ok.
So, that is the various advantages of this.
And again I say that catalytic once you strike
the a catalytic antibody, if you have isolated
the monoclonal antibody producing the catalytic
antibody then you are through, because that
is immortal and you can make grams of the
stuff whenever you want that, ok.
And these enzymes a these are not enzymes
they are catalytic antibodies acting as a
enzymes (abzymes), these abzymes actually
have much better bench stability or shelf
life than your other enzymes.
The shelf life is quite high, ok.
So, you do not have take extreme precaution
to preserve the catalytic activity of this
of these abzymes, ok.
Now the question is how do you generate; how
to generate the catalytic antibodies or how
do you design catalytic antibodies to catalyze
certain reactions, ok.
Generation I have told you that you have to
generally you make the transition state analogue
which are stable, attach it to a protein and
this protein is actually called the carrier
molecule but remember this there is a also
a linker that needs to be used.
This is the transition state analogue; usually
you should use a linker because if you if
the transition state analogue is embedded
right on the surface, then it may not be noticed
by the surveilling system, your immune response.
See something is trying to see what has come
whenever some foreign particle comes, our
system wants to see the just hover around
the surface and then see what are the different
geometries, what are the different amino acid
residues on the surface and accordingly information
is passed on to the B cells and the B cells
make the antibodies.
So, what happens, if your protein and transition
state analogue is embedded here, so, it may
the surveillance system may miss the transition
state analogue because it is right on the
surface.
So, it may not find this interesting that
this has no characteristic surface here but
if it is extended by a linker, then this will
definitely find something here that oh!
There is something here.
So, now, it may be a characteristic epitope.
So, see it basically you have to design a
hapten and that should have an epitope.
If it the; if the hapten does not have any
epitope, then no antibody will be generated
against the hapten; again I repeat what is
hapten; hapten is your that.
