Welcome back, we will now talk about something
which is possibly the subject is about 30
years old. This is actually this whole area
is what is under the domain of called Synthetic
Biology.
Now, you know synthetic chemistry like synthetic
organic chemistry is basically making of new
entities, new molecules that is all about
synthetic chemistry; either you make a natural
new molecules or it could be some natural
products which is a existing in the plant
or other microorganisms; so that is synthetic
chemistry. So, synthetic like, our synthetic
textile and then you have the natural textile
which is cotton; synthetic text tiles are
polymer based ok.
Similarly, a question was raised that can
we have typical biological entities, can we
make them synthetically or artificially what
we can say in the lab to do reactions or to
do functions, which proteins are doing or
other biomolecules are doing. So, is it possible
to design our own catalytic system, which
are like enzymes. So, that was a one domain
of synthetic biology that you are trying to
make which are called synthetic enzymes in
order to catalyze a particular reaction.
Remember nature has given us this enzymes,
these proteins, which act as enzymes, how
many number of enzymes are there? It could
be a huge number that is true. What type of
reactions they catalyze? That also depends
on the nature of the enzyme, but I have given
you six classes of enzymes; oxidoreductase,
transferase, hydrolase, lyase then isomerase
and finally, ligase; that is the sixth one.
Now, when you isolate enzymes from natural
sources; that is what are enzymes; enzymes
cannot be isolated from unnatural sources.
So, natural sources you isolate an enzyme;
now these enzyme is actually tailor made by
nature to catalyze a particular reaction,
ok. Now organic chemistry were all the time
looking for catalyze for catalysis of their
reactions that they are doing ok. Now, number
of organic reactions are huge difficult type
of reactions, different type of reactions,
but the number of biochemical reactions may
be restricted. Actually it is restricted because
it depends on the number of enzymes that are
available.
In organic chemistry, I give you an example;
in organic chemistry there are some reactions
say this you talk about a reaction which is
very famous in organic chemistry calls the;
Diels called the Diels Alder reaction Diels
Alder reaction. So, what is made here; that
you have you make a cyclic compound the double
bond rearrange and you get this, this is also
called a 4 plus 2 cyclo addition reaction
ok.
Now, if I want to have a catalyst see this
Diels alder reaction; in order to take place,
you have to take these two systems one is
butadiene and another is ethylene. You have
to take in a flask, add the solvent and then
heat it; unless you heat it, this reaction
will not go; howevere there are some Diels
Alder reactions which are which leads lower
temperature, but a majority of them needs
refluxing in solvent high temperature is needed,
ok.
You know enzyme reactions; in enzyme reactions
are happening at what temperature? At the
biological temperature, what is a biological
temperature? So, if I think of a biological
temperature at the in a human system it is
about 37 degree centigrade. So, in our body
about 37 degree centigrade reactions are happening.
Now, for enzymes heating the heating has a
negative effect on the activity of the enzyme.
If you heat the enzyme, what happens? The
enzymes are actually folded in a three dimensional
network that we showed that is called the
tertiary structure. So, as we heat the enzyme
the conformation that is required to catalyze
the reaction, that changes and if that changes
the enzymes will ultimately loose the activity.
So, if you; so, people started thinking that
because enzymes can catalyze reactions at
room temperature or the biological temperature;
so basically 30 to 37 degree centigrade. So,
can we have now can we design an enzyme for
Diels Alder reaction if we can design an enzyme
and then synthesis that; then we will have
this reaction which will be catalyzed by the
enzyme. So, what a but unless see why synthetic
biology is required? Because the question
is whether there is any enzyme which does
this Diels Alder reaction or not.
Today, there are few enzymes available to
carry out the Diels Alder reaction, but may
be 20 years back; people thought that these
pericyclic reactions will not be catalyzed
by enzymes. So, they were after this type
of making biological systems; taking the help
of biology I will show you how you can make
those synthetic or artificial enzymes; enzyme
like molecules, you cannot call them enzymes
because enzymes are natural. So, they are
enzyme like molecules and how to then make
those artificial things to catalyze certain
reactions.
So, basically what we are trying to say here
is that, take a general case. I want to have
a reaction where there is substrate A, I want
to convert it into the product B. First, I
will search whether there is any enzyme which
does this reaction. Suppose there is no enzyme
available to do this reaction; actually many
of the organic reactions are not catalyzed
by enzymes that we normally do in the laboratory.
On the other hand, there are again different
types of enzymes are used in synthetic organic
chemistry to carry out certain transformations.
Now, suppose this A to B conversion there
is no enzyme available; if enzyme is available
then people were will not go after, go after
making new enzymes to catalyze this reaction.
Because always seen that enzymes have been
designed and ultimately made through evolution
which natured had this time to make the enzymes
perfect for certain reactions or selective
for certain reactions over millions and millions
of years, ok.
So, after several if you; if you think that
nature is a craftsman or craftswoman, then
you have this; so, he or she tried all these
things and ultimately came out with the best;
best possible protein which can act as the
enzyme, ok. So, again making same type of
enzyme, it will be very difficult to compete
with the natural system. So, usually the synthetic
biology people, they look for reactions where
no enzyme is known.
So, this A to B suppose there is no enzyme
that is known, but I want a catalyst enzyme,
like catalyst ok; not the there are different
catalysts in organic chemistry that are used
like transition metal based catalyst, Wilkinson’s
catalyst, raney nickel, palladium on charcoal;
all these are these are catalyzed some are
homogeneous some are heterogeneous, ok. We
are not talking about those type of catalysts,
we are talking about a catalyst which works
like whose mechanism of reaction is very similar
to the enzyme, ok. So, that was the desire
of organic chemist.
If that becomes successful, if the strategy
becomes successful, that you can catalyze
the reaction of A to B, then by following
the same strategy you can catalyze a reaction
from C to D. So, any reaction virtually if
you of course, that if is there if you can
design an enzyme like catalyst; then virtually
any reaction what are there in organic chemistry,
you can have a catalyst, ok. So, that is the
whole scenario; although we are far from that
yet to have catalyst for each reaction; that
is still there are some difficulties; I will
tell you what are the difficulties, but this
is the idea to have a catalyst to catalyze
a reaction where there is no enzyme is possible,
ok.
And if your strategy is correct, if your principle
is correct, then in theory you can catalyze
any reaction. Of course, there is one question
that is that will be asked that you have to
know; what is the mechanism of that organic
reaction. Unless you know the mechanism, you
will not be able to design an artificial catalyst
enzyme like catalyst. Why the mechanism is
required?
Because if you know the mechanism, then only
you know what is the transition state; what
is the transition state for the reaction.
In order to know the transition state you
need to know the mechanism of the reaction.
Like suppose there are reactions SN2 and SN1
reactions and the transition state for both
the reactions are different ok. So, unless
you know the mechanism you will not know the
transition state; so, that is important the
mechanism has to be established, ok.
Today with the advancement of spectroscopy
techniques, many of the mechanisms or organic
reactions have been unraveled ok. So, mechanism
is not a much way bigger problem these days;
so we know the mechanism. So, if you know
the mechanism we know the transition state
we can roughly draw the transition state.
Because transition state also very difficult
to pin point what is the structure of the
transition state.
Remember what is the transition state? where
bond formation and bond breakage they are
half way through; they are not complete, ok.
Now, some half way through means some may
be three fourth through; that means, the bond
breakage is more suppose and bond formation
is less. So, it is really very difficult to
know the actual structure of the transition
state, but what one can do that you take;
the if there is an intermediate involved in
the reaction, I told you last time that according
to Hammond’s postulate; transition state
structure resembles most the intermediate
for that reaction and that is simply because
the energy profile diagram is something like
this.
So, this is your intermediate and this is
your transition state. Now there are two transition
state for this reaction; however, always we
look for the slowest step of the reaction
which determines the rate of a reaction. So,
this has got an activation energy which is
much higher than the activation energy for
the second step and this is the intermediate.
Now; that means, we will be interested to
know what is the structure of this transition
state and this is closest in energy not with
the substrate, not with the product, but with
the intermediate.
So, if you know the some intermediates can
be isolated, their existence can be proven,
their structure can be predicted that this
is the structure of the intermediate. So,
if you know the structure of the intermediate;
you can approximate it that this will be the
very similar the transition state will be
very similar to the structure of the intermediate,
ok; that is known.
Now, how to develop an enzyme like catalyst?
The principle is very simple that you know
that enzyme catalysis in the enzyme catalysis
substrate binds to the enzyme, then it goes
to the transition state; the transition state
is also bound to the enzyme and then it goes
to the product; the product was initially
bound to the enzyme then it is released. Now,
out of this three species, I told you the
very first day of enzyme catalysis, that which
one has the highest affinity for the enzyme;
that means, the enzyme offers stability to
either the substrate, to the transition state
as well as to the product.
But it offers least stability to the product
because product has to be released very quickly
so that new enzyme molecules are made. So,
then out of these, transition state is the
one which is stabilized most because that
will lower the activation energy. So, out
of these three species, transition state will
be the will be will be the highest will be
the most stabilized by the enzyme. That means,
the enzyme active site; you can always start
elaborating it the transition state has a
structure which is geometrically and electronically
complementary to the structure of the active
site in the enzyme, ok. So, transition state;
so what you need is basically A to B again
the reaction you have a transition state.
So, what you need is that you should make
a protein like molecule and that should have;
that should have some site which is electronically
and geometrically complementary to the transition
state.
So, suppose this is the active site of the
transition state, the substrate goes and binds
then it becomes the transition state and it
is stabilized by different types of weak interactions,
ok. Now the question is how to create? So,
the problem boils down to creation of the
active site in the enzyme, ok.
Now, what is the technique that is people
have adapted is that, we know that if a foreign
particle enters into our body; foreign particle
or foreign organism enters into our body something
is generated in the body flow, it in the blood
stream, and that is what are called antibodies.
So, when some molecule comes and invades and
it is in the blood circulation; then what
happens? Some again some molecules are made
by our body system, it immediately recognize
that something foreign has entered in my body;
so there is something what is called immune
response.
So, our immune response then immediately makes
takes some time and then it makes the what
are called antibodies. What are these antibodies?
The antibodies are the one which recognizes
the invading molecule; which recognizes the
invading molecule; that means, the; that antibody
has a structure which is complementary to
the invading molecule structure, ok; otherwise
how it recognizes the invading molecules.
So, basically if the invading molecule suppose
looks like this, then the this is remember
this is called the antigen (the invading foreign
molecule). Now, if you can generate antibody
against this. So, the antibody will be we
have a geometry which will definitely look
like something like this and then in order
to have the complementarity against the antigen,
ok.
So, basically what you what I said? That if
some molecule enters into the body then what
we see? There is an immune response and some
proteins which are antibodies also called
immunoglobulins; these antibodies are generated
which have complementary structure to the
antigen, ok. Now, suppose this antigen I make
an antigen which is which looks like the transition
state of a reaction.
So, my antigen is basically the transition
state, but remember again I said the transition
states are very unstable, transitory in nature.
So, better this is approximated to the intermediate
structure because intermediate lies in the
energy minima. See, if you go to the physical chemistry
site now; so what are isolable here is this
one, this one and that one.
This is the intermediate; that means, intermediate
has stability. So, you can make something
which looks like the intermediate. So, now,
suppose you have the intermediate in hand.
So, what I should do? I should inject the
intermediate into say mice suppose I will
not inject into a human body, that will not
be allowed ethically not correct.
So, you inject into the mice and then what
will happen? I will expect that there will
be immune response in the mice and if there
is immune response, then antibodies will be
generated. And these antibodies, if antibodies
are generated, then these antibodies are what?
These antibodies will recognize this intermediate
because this is your antigen now; this is
your antigen. So, that will be recognized
by the antibodies and then that will bind
to this intermediate.
Then there are complex biological processes,
immunological processes which ultimately destroys
the antigen, ok. So, now you come back again,
I want to have a catalyst to do the reaction;
to catalyze the reaction A to B; I know the
mechanism of this reaction. This mechanism
has a intermediate produced in between the
substrate and the product; I know the structure
of the intermediate.
So, if I take the intermediate; intermediate
have some stability that is ok. If I take
the intermediate and inject into the mice
then what should happen? What I will expect?
I will expect the because it is a foreign
particle, so antibodies will be generated.
And if antibodies if I isolate this antibodies;
these antibodies are the ones which recognize
which will recognize the antigen; in this
case the antigen is the intermediate, ok.
So, then this antibodies will be my catalyst;
so if I add these antibodies to this A. So,
now, the transition state from A to B because
the intermediate is stabilized, the transition
state looks very similar to the intermediate
according to Hammond postulate; so the antibodies
will now stabilize the transition state. If
the transition state is stabilized; that means,
the activation energy will go down and; that
means, now the transition state will be easily
converted into the product B. So, that is
the whole idea of this topic of synthetic
biology. Synthetic biology have other domains;
we are just concentrating on how to make artificial
enzymes or enzyme like systems; it is through
our immunological response.
The whole basis is that you have to use the
immuno response that is available in a living
system like mice or human because our immune
system protects us from the infection or invading
agents from the outside, ok. Now there are
certain problems, so we will have to now discuss
the, we will discuss what are antibodies;
how they look like.
And then we will also talk about does any
foreign molecule generate antibodies in our
system or not, ok? So, these are very important
because some antigens or some molecules; if
I inject into the body, may not may or may
not generate antibodies. The classic example
is that when we become sick what we do? We
take a medicine, what are medicines? These
are small molecules; you will never find a
medicine the molecular weight is very large;
that is not possible.
So, what you do you take only small molecules
and if I take the small molecules; what happens
I if there is immunity developed against that
small molecule then the drug will not work
drug is a small molecule then the drug will
not work, ok. So; that means, antibodies are
not generated against small molecules. So,
that is a stumbling block here; that although
the theory looks very good that I should take
the inhibitor injected into the mice, isolate
the antibodies that should catalyze the reaction,
but antibodies are not generated against small
molecules.
When you talk about these reactions, this
reactions are very basically reactions between
small molecules not involving any large molecules.
So, that we have to talk; that means, small
molecules are not immunogenic. What is immunogenicity?
That if some molecules fail to develop immune
response, then that is not immunogenic. And
if the invading organism a invading molecule
generates immune response inside the body;
a biological term is elicits.
If an; if a small molecule elicits enzymes;
that means, a generates a generates a antibodies,
then that those molecules are called antigens,
ok. So, what the small molecules does in the
body? The small molecules sometimes gives
response which are called allergic response.
Some molecules see like many of the small
molecules when it goes into the body which
starts leasing or some skin rashes has come
up these are allergic reactions. Like some
penicillins some people are allergic to penicillins,
but nobody is will be nobody will be; will
develop an immune response against this small
molecules; so, small molecules are not immunogenic.
So, forget about isolating antibodies by injecting
small molecules. So, you have to do some other
strategy ok.
I think antibodies we have already told you
that antibodies are basically generated against
invading. It could be organisms, it could
be virus, it could be large molecules; antibodies
are generated and this is the process is called
the immune response, ok. Now, there are some
biological things here that there are two
types of immunity; one is called cellular
immunity which guards against the virally
infected cells or fungi, parasites and foreign
tissues. So, when these this is called cellular;
so some immunity are through cellular processes.
Our cells that are presents inside the body
specially that is what is called the T cells
or specifically they are called T lymphocyte
cells. These T cells because it is T stands
for because they are generated in the thymus.
And this T cells through a cellular immunology
processes they guard against the virally infected
cells; that means, the cells where some virus
has entered or some fungi or parasites or
tissue ok; so there is no that is one type.
The other type is humoral immunity. Humoral
means humor means fluid the body fluid is
called humor, ok.
This humoral antibody if the humoral immunity
is basically; this is the most effective against
bacterial infections and cellular phases of
viral infections. But basically what you know
that there are two types; one is cellular
immunity and the other is humoral immunity.
When there is humoral immunity that actually
is mediated by antibodies or what I called
that they are also called immunoglobulins,
ok.
So, we are interested in the second type of
immunity; the humoral immunity, ok. Antibodies
are produced; so, who produces the antibodies?
It produced by B cells or typically called
B lymphocytes ok; B cells which are actually
matured in the bone marrow; which are present
in the bone marrow. These B cells are the
ones which generates the antibodies because
somebody has to; it is the cell which makes
the proteins, the enzymes all these are all
proteins are made by the cells ok. I also
already told that what is immune response;
it is triggered by foreign molecules and this
is called antigen.
Foreign molecule is often a protein it could
be a big protein or it could be a carbohydrate
which are; that means, large macromolecules
are they can only act as an antigen; small
molecules cannot, ok. Small molecules can
invade our immunological machinery because
they are very small; they are not noticed,
ok.
So, the question big question is, what to
do how to generate antibodies against the
small molecule? So, that is a big challenge.
Number 2 is that there is there is another
problem that is called if you have some big
molecules entering into the body.
Suppose, this is the shape of the big molecule
entering into the body; so now, by some process,
signal will go to the B cells that something
has come and this is the shape of this shape
and structure of what is the big molecule,
ok.
Then these B cells starts producing this immunoglobulins
or antibodies. So, when they make the antibodies;
these antibodies are, some antibodies what
they will do? They can bind at this position;
there will be some antibodies all are against
this antigen remember this is the antigen
this is the big molecule, but there are different
types of antibodies some are going at different.
So, these are going at different sites at
different sites they are going and binding.
So, they are attacking the same molecule,
but targeting different sites. So; that means,
what you have? You have a collection of antibodies;
different types of antibodies. Suppose I have
different balls; so a red balls, 5 red balls,
4 blue balls all these say 7 green balls all
these balls are like footballs. Suppose, you
can play football with any one of these, but
what we say that you have a collection of
polyclonal balls means you have a collection
of poly different types of balls, but everything
is targeted towards playing football.
Similarly, here these antibodies they are
also targeting the same molecule, same antigen
molecule, but at different sites. So, what
is basically if you isolate the, if you take
the blood and try to isolate the antibodies
against a particular antigen; you will see
that it has got a variety of antibodies variety.
Their common; their commonality is that all
of them are attacking the same antigen ok,
but at different sites and also their efficiency
of binding will also be different.
So, this is what are called polyclonal antibodies;
polyclonal antibodies are antibodies that
are secreted by different B cells, B cell
lineages within the body, ok. So, we have
different types of B cells; one B cell will
generate antibodies which go to a particular
site, some B cells will make another sets
of antibodies which goes to other sites of
the antigen. So, different types of B cells,
they will make a set of polyclonal antibodies,
ok.
So, now, what you do? The strategy is like
this that we want to make antibodies against
the inhibitor, but we know that the inhibitor
does not generate any antibodies because it
is a small intermediate its a intermediate
not inhibitor this intermediate is a small
molecule, you cannot generate antibodies.
So, to generate antibodies, what you do? You
attach it the intermediate structure whatever
it is into a large molecule, ok.
So, you make a something like this; so you
conjugate the intermediate into a large molecule.
If you do that, now as I said always now this
is only one system and antibodies will be
generated at different generated which recognizes
different sites. Suppose that some antibodies
may be there which generates this sites, some
antibodies will be there which generates this
site.
But suppose there is some antibodies which
generates and which is which recognized the
intermediate that which is part of the whole
system, ok. Then these are the antibodies
you can separate and that will now stabilize
the intermediate; that means, in turn that
will stabilize the transition state. So, the
theory is again not very complicated if you
want to generate the antibodies against small
molecule; so what you do?
The small molecule has to be attached to a
big partner a macromolecule like a protein
another protein, you attach that. And then
this whole thing you can inject and you get
a set of polyclonal antibodies and if you
are lucky that some antibodies are there which
recognizes the intermediate structure of this
whole ensemble.
Then if you can isolate those antibodies,
they are going to catalyze your reaction because
they only recognize binds to the intermediate
part and finally, that will be the catalyst,
ok. So, this is the situation; now the challenge
is whatever; some definitions you should also
know see, what are monoclonal antibodies,
I have already told. They are antibodies that
are made by the same type of B cells same
type of B cells making only one type of antibody.
And that is the these are called monoclonal
antibodies; that means, if I have those red
balls blue balls and green balls if I separate
only the red balls from here; then this red
balls are all identical; so that is a that
is a monoclonal set. Similarly, I have different
sets of antibodies; some goes and binds here,
some goes binds here some here some in the
intermediate, ok.
So, this polyclonal antibody set, I have to
have some mechanism to separate it into monoclonal
antibody which recognizes only one site of
this whole thing, ok. And this monoclonal
antibodies come from only a particular type
of B cells, other type of B cells we generate
other type of antibodies, ok. So, now just
to summarize that in order to create an artificial
enzyme; then what you do?
You draw the mechanism of the reaction, draw
the intermediate and if you have a similar
structure like intermediate, or if the intermediate
is quite stable; then you attach it to a large
molecule which is by the way called the carrier
molecule. Because that is carrying the intermediate
along with it and then you develop polyclonal
antibodies against this whole system and then
your task is basically separate this polyclonal
into monoclonal antibodies and so that this
monoclonal set recognizes the inhibitor this
intermediate, ok.
So, this separation is what is required and
this attachment is required; conjugation bioconjugation
it is called and now what is the name of this;
there are certain I think the next page will
have that this.
I already told single clone of B cells will
produce only one type; only one site, only
one site. Now, these sites there suppose see
why antibodies are not generated against small
molecules, but it is generated against large
molecules. Because large molecules have characteristics
surface they have different geometries at
the substrates which can be noticeable, ok;
but if you have a small molecule you can consider
to a biological system they are considered
as tiny dots. So, if there are tiny dots there
is no characteristic feature of the surface
of the large molecule.
If that is not present then for antibodies
to bind that will be difficult. So, you should
have some characteristic features in the surface
like this is a bent one. So, now, you can
have a complementary structure for that; you
can have a complementary structure for this
site also ok. That is why large molecules
are recognized and not the small molecules
because small molecules are look basically
tiny dots; they do not have any structure
structural pattern.
Now, these sites where antibodies binds, these
are called epitope. Epitope are the; are the
sites because not all portion antibodies will
bind; there are specific sites in the antigen
where antibody binds. So, these specific sites
are called epitopes and this small molecule
which is attached to the large carrier molecule,
and you are developing antibodies against
the small molecule, ok.
And those small molecule will be called is
called hapten. Hapten are basically small
molecules which mimics the intermediate and
when it attached to the carrier molecule;
it develops antibodies which recognizes the
small molecule. This small molecule is called
in biology it is called hapten.
I think every time I think it is there hapten;
where is hapten the definition was there somewhere
here.
Yes, haptens are small molecules that elicit
immune response only when attached to a large
carrier such as a protein. The carrier may
be the one that also does; does that actually
creates that itself can create immune response
and it can create immune response even when
attached to the with the small molecule ok.
So, that is hapten; it is clear what is hapten.
And then epitope I already told, but there
is a another name for epitope that is called
antigenic determinant; is the part of the
antigen that is recognized by the immune systems
specifically by the antibodies the B cells
or the T cells.
And this epitope is the specific part actually
it is written piece that is also of the antigen
to which antibody binds; I think for us that
is ok. Now the things are little bit easier
means as we proceeded. So, basically what
you have? You have to have a intermediate
attached to a carrier molecule and then isolate
the monoclonal antibody from the polyclonal
system. And this monoclonal antibody should
recognize should have a binding affinity to
the intermediate, then that will become a
catalyst, ok.
Because catalyst lowers the stabilizes the
transition state and transition state mimics
the intermediate. Now interestingly when Linus
Pauling who had received two noble prizes,
the first one was basically the structure
of proteins the alpha helical structure of
proteins and he did the structure of insulin.
So, at that time he studied many enzyme reactions
protein, how they catalyze and he predicted
at that time he had a very famous book called
Nature of Chemical Bond.
This volume was the same volume who gave the
hybridization theory. So, at that time he
said that enzyme stabilize the transition
state and one day may come that people can
develop enzyme like systems which recognize
the transition state and then it will catalyze
reactions. So, that will open up new avenues
for organic synthesis ok. I think.
Thank you.
