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Chemistry of Penicillin

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Chemistry of Penicillins

(Refer Slide Time: 00:30)

Welcome back, now we are going to take the individual antimicrobial compounds and
discuss their chemistry and biochemistry. We will just recapitulate; we started with these
compounds. This is the structure of trimethoprim, an antimalarial compound; this is para
amino phenyl sulphone; that is an anti-leprosy compound, this is sulphamethoxazole
which is an antibacterial compound.
You see here it is written bacteriostatic; that means, these are the antibacterial agents
which inhibit the cell growth. Why these are anti-bacteriostatic? Because these are the
compounds which stop the folic acid biosynthesis which is one carbon transfer; it is a
very important conversion that is uracil to thymine and that was ultimately the
consequence of perturbing the folic acid biosynthesis.
So, one of this compound is like sulphamethoxazole or this anti-leprosy compounds this
is little bit different because that inhibits the DHFR (dihydrofolate reductase) of
microorganisms; . And these two are nothing, but you can write them as SO2NR; so para
amino SO2 and then NR. It mimics the para aminobenzoic acid which is a component of

the folic acid. So, para aminobenzoic acid has to be incorporated into the dihydro
pteridine nucleus. If you remember, it is the 7, 8-dihydro pteridine that is formed.
So, that reacts with this para aminobenzoic acid and the enzyme that does that it is called
a pteroate synthase. Now, this pteroate synthase must be such that there is hydrogen
bonding; it goes to the active site of the enzyme and this is the NH2 which forms the
hydrogen bond with some amino acid here.
There is an aromatic amino acid somewhere here, it must be so that there is π-staking
interaction with this benzene ring. And then there must be some ionic interaction; that
means, there must be some sites like NH3 plus which forms an electrostatic interaction
here. So, for the para aminobenzoic sulphanomide, NH2 forms this hydrogen bond, this
is the aromatic ring and instead of this CO2 minus you have SO2 minus; so that forms a
ionic bond.
So, this is a very good competitive inhibitor of para aminobenzoic acid; these
compounds are also called antimetabolite. What is antimetabolite? Compounds which
interfere with the metabolic processes that are that are going inside the microorganism or
any species. And since this is now inhibiting the incorporation of para aminobenzoic
acid; that means, it disturbs the normal metabolic process; so initially this was
considered to be a very good antibiotic.
But today the bacteria has developed resistance against the sulphonamides by producing
more of para aminobenzoic acid and you know that in competitive inhibition, if you
increase the concentration of the substrate, you can overcome the inhibition; that is the
nature of competitive inhibition. So, now sulphonamides are virtually not working
against any microbial infection.

(Refer Slide Time: 04:31)

Now, let us come to the chemistry of penicillin. So, this is the structure of penicillin;
what is the pharmacophore of penicillin? Pharmacophore is the molecular entity present
in the entire molecule; it is the essential part of the molecule which is interacting with
some enzyme or any nucleic acid, whatever is the target.
So, basically what we are saying that is that if I have a big molecule like this and if I see
that only this part is the one which is interacting with the enzyme, then this part is what
is called the pharmacophore. So, the pharmacophore of penicillin is this part which is put
as light blue; so that part is the pharmacophore. Now, what is there? There are two rings
which are fused to one another.
Now, this is called a β-lactam ring which is fused with this thiazolidine ring; that is the
name of the nitrogen and sulfur containing heterocycle; then there is the substituent

amine here which is acylated. So, you have an acyl side chain here and you have a α-
carboxy group attached to the thiazolidine ring.

Now, this is the minimum; so for any penicillin, the variation that we see in different
penicillins; you know the names of different penicillins, ampicillin, amoxicillin,
cloxacillin, methicillin; whatever name any penicillin the difference is only in this R
group. So, you have different R groups and you get different types of penicillins. The
initial penicillin that was made was benzylpenicillin called penicillin G and another one

was called penicillin V that was phenoxymethylpenicillin; phenoxymethylpenicillin also
called penicillin V. In fact, penicillin G is still available in the market.
It is usually now prescribed for rheumatoid arthritis; usually for this, benzylpenicillin
was used. Otherwise, for other bacterial infections, respiratory infection usually this
penicillin G is not used. But historically these two compounds are important because the
first penicillins that were introduced into the market where this benzylpenicillin and this
penicillin V, penicillin G and penicillin V.
(Refer Slide Time: 07:25)

We will talk about this biosynthesis later on because we know that penicillin is made by
the microorganism that is why this is an antibiotic. Now, that means, the microorganism
has a biosynthetic machinery to make this penicillin; enzyme systems which starts form
building blocks and join them together and final get to the penicillin.
We will discuss this formation of penicillin in a much elaborate way, but here just to let
you know that it is a very simple, these three simple building blocks which ultimately
combine and make the penicillin; one is this variable amino acid, then this is cysteine
and this is valine. So, cysteine, valine and another amino acid which I am not naming,
actually this amino acid is alpha aminoadipic acid which is not a protein amino acid.

So, basically this α-aminoadipic acid combine with cysteine and that also combines with
valine to make what will be a tripeptide; tripeptide then cyclizes and you have this
penicillin molecule.
Remember, the α-aminoadipic acid it is the ε-carboxylic acid that reacts with cysteine
and cysteine reacts with valine to make a tripeptide which will be called A C V; Adipoyl
Cysteinyl Valine that cyclizes to give the penicillin. Now, another interesting aspect of
the structure of penicillin for which the activity is there is the reactivity of this β-lactam
ring.
Reactivity to what? Reactivity towards nucleophile because cyclic amide carbonyls are
susceptible to ring opening by hydrolysis; like if you take an amide RCONH2 and if you
heat it with alkali you get RCO2H or RCO2Na because you are using alkali say NaOH
and you get the ammonia. And if it is NHR; then you will get RNH2 the amine, but in
order to do this you have to heat it with alkali.
It does not work at room temperature or if you want to do it at room temperature; then
you have to wait for months and months so that the hydrolysis will take place. Now, why
amides are difficult to hydrolyze? Because of this resonance; where nitrogen lone pair is
resonating with the carbonyl; so you have a resonating structure where the nitrogen is
planer and this bond is also becoming very rigid; so that will not allow you allow the
bonds to rotate. This is exactly what happens in peptide bonds when we discuss the
structure of peptides; we have seen that the amide bond in this nitrogen carbon
framework has a significant amount of double bond character and that stops the rotation.
So, in the normal amides, because of this resonance, this carbon is losing the
electrophilicity. Because if there is a only carbonyl then the oxygen will pull the
electrons towards itself by electromeric effect and then this will have a positive charge,
but here nitrogen lone pair is there that will neutralize the positive charge on the carbon.
So, nucleophilic attack on the amide requires higher temperature or higher activation
energy. On the other hand, in penicillin what happens? This is the structure of penicillin;
see it is not a planar molecule, it is like this is a planar β-lactam ring; this 4-membered
ring is almost planar and then you have this 5-membered ring which is going upwards.
So, basically it is like an open book type. So, this is the 5-membered ring sulfur; this is
the carbonyl, this is the nitrogen.

So, it is like an open book type of or a butterfly type of structure. So, it is not a planar
molecule. In acyclic amides; this lone pair is entering into resonance with the carbonyl
that is actually not permissible in β-lactam. Because if the lone pair participates in
resonance then this angle becomes 120°; that means, the angles strain in a four
membered ring; the angle is virtually 90°.
So; that means, you have already deviated the angle quite much from 90°; but actually
the hybridization demands this angle to be 109.5°. So, if you have a resonance where the
angle is still increasing further here; so that will not be allowed. So, this will be flat and
it will be highly strained. So, this type of resonance is almost not possible and that is
spectroscopically reflected; if you take the IR spectroscopy, then you will see that if
there is resonance then the carbonyl stretching frequency will be low.
And if there is no resonance; the carbonyl stretching frequency will be higher. So, in
penicillin the carbonyl stretching frequency is about 1770 cm-1. On the other hand, in the
amide carbonyl it is around 1680 cm-1. So, that shows that it is a very highly strained
molecule and the carbonyl is virtually present as a separate carbonyl; there is no such
assistance from the nitrogen lone pair adjacent to it. So, that makes its carbon highly
electrophilic; that makes this carbon highly susceptible to nucleophilic attack. So, that is
one of the major reason why this molecule acts as an antibacterial agent.
(Refer Slide Time: 14:59)

We will slowly go back to that, but again before I go any further, I should tell you that
whatever mechanism that we are discussing today that this is the way the penicillins
work; that is what is called post facto analysis.
That means it is after the fact that penicillin is a very good antibiotic, it kills the
microbes; it kills the bacteria and once that is known at that time Fleming or Florey or
Chain or Dorothy Hodgkins or even Woodward, Robert Robinson; they did not know
how it is working only thing they knew that it was a very good antibacterial agent.
Of course that is what ultimately matters. Some people might say that I do not care how
does it work, but it is working nicely to cure me of the infection. However, science can’t
stop there, science has to progress you have to know how penicillins work. Because until
we know that we cannot device or rationally design new antibiotics based on the
mechanism of action of penicillin because today we are living in an era where most of
the earlier penicillins are not working.
So, now how to make new penicillins and new type of antibiotics that depends on our
understanding of the antibacterial activity of penicillin. Just to make it very clear that
whatever we are discussing now this is after the discovery of penicillin and after the
penicillin entered into the market. So, the mechanism was established. Now, what is the
mechanism? I told you one thing that penicillins works against the bacterial cell wall. So,
we have to now know what is a cell wall in bacteria; that is so unique for them.
Cell wall is a rigid wall that protects the cytosolic material inside the bacteria. Now,
there are two kinds of bacteria; one is gram positive, another is gram negative. So, gram
positive bacteria have a thick cell wall structure and it is made up of peptidoglycan; what
is a peptidoglycan? Peptidoglycan means you have peptide units as well as glycan units,
glycan means sugar units.
So, basically you have carbohydrate which is the part of the sugar and you have peptide.
So, that is why this is called peptidoglycan. In gram positive, you have the peptidoglycan
as the outer wall and then you have the membrane like we have; this lipid bilayer
membrane and in that membrane there will be definitely channels because bacteria has to
communicate with the outside world.

Some molecules will come inside; there are carrier molecules which will carry the
outside molecules that are needed. So it has to be controlled like ion channels. Bacteria
have these pores also; so through that pores, materials enter or go out, ok.
So, this is your peptidoglycan and that is quite thick in gram positive bacteria. In gram
negative bacteria what happens? First it has an outer membrane which is a lipid bilayer
and then there are these pores these are called porin channels. Then there is a much
thinner peptidoglycan that was earlier for the gram positive it is outside, then little bit
inner; that means, this is protected by a lipid bilayer membrane and then you have
another membrane, an inner membrane and then the cytosol.
So, there is a distinct difference and you can now say that penicillins work against the
gram positive very well because in the gram positive the peptidoglycan is exposed
outside. So, penicillin comes and destroys the peptidoglycan, but here the penicillin has
to initially traverse through barrier.
Here these spaces are by the way called periplasmic spaces and this spaces where the
peptidoglycan is there. So, then it has to come inside here and then act on the synthesis
of the peptidoglycan. So, it will be difficult to kill the gram negative bacteria by this type
of mechanism, but anyway let us see what the other problems in gram negative bacteria
are.
(Refer Slide Time: 20:27)

Gram negative bacteria have other sorts of problem; so, we will discuss that. Let us
inspect it at the molecular level the peptidoglycan. Here this is the schematic diagram
you see this is one; one sugar unit that is called NAM. What is NAM?
N-acetyl muramic acid is NAM. And the other is called NAG; NAG is N-acetyl
glucosamine. So, these are the two sugar units; they are joined one after another, NAM
then NAG, via glycosidic linkages. NAM, NAG; NAM, NAG NAM NAG; so this way
some chains of sugar are there, then another chain of sugar is there, then another chain of
sugar is there.
In each strand of sugar polymer; in the NAM (N-acetyl muramic acid), there is a peptide

which is attached to one of the hydroxy. So, the first amino acid is L-alanine, then D-
glutamic acid then L-lysine, then D-alanine. And actually there was a another D-alanine,

but by reaction while forming this a cell wall, one of the D-alanine goes out.
This is the structure of the cell wall when the matured cell wall is there. But we are
discussing that just before the formation of the matured cell wall; what is the status? The
status is that that all these peptide bonds are there and the amino acids are there including
the last D-alanine.
So, this is a pentapeptide; they are hanging from one strand. And in adjacent strand, there
is again those five amino acids which are hanging.
Now, each peptide has a side chain also which is the reactive arm. Like this one has a
side arm then there is another one, suppose here which has got a side arm and then there,
this has got a reactive side arm. Now; there will be a reaction between this strand and the
side arms. So, there will be a attachment like this; then there will be an attachment like
this.
If that happens, then the bacterial cell becomes very rigid. Suppose you have lot of
bamboo rods and then you have to put to make a fence. If you want to do that by only
putting these bamboo rods; the bamboo poles will not be sufficient to protect. So, in
order to make them very rigid, you do this type of cross-links to make it very strong.
That is to reinforce the structure. So, to reinforce the structure, this cross-link is required.
So, basically now what we have learnt? That there is this glycan units glycan polymer

that is NAG NAM NAG and NAM NAG; in each of these, NAM is attached to a
pentapeptide and then there is cross-link between the adjacent pentapeptides. So, we will
look very carefully at what are these reactions; how these cross-link is formed.
(Refer Slide Time: 25:20)

Here it is now written in little bit better way; let me write the structure of the sugar. So,
this is the glycoside linkage, this is N-acetyl glucosamine means NH. Glucosamine
means in glucose only one hydroxyl is replaced by an amine which is acetylated. You
know in glucose what happens? This is α then the next one is β, the third one is this is
number 1, this is number 2, this is 3rd, this is number 4 carbon OH is alpha and then you
have this CH2OH.
So, the glycoside linkage is made between C1 and C4; that means, this is attached to
another sugar. So, this is your NAG; N-acetyl glucosamine; and what is your NAM? The
NAM is like this, NHAc; N-acetyl that is there and this is attached to another sugar unit
via glycosidic linkage and here the OH is β. So, that is attached to a lactic acid this is
CH2OH.
So, this lactic acid moiety is attached by an ether linkage. So, this is your NAM and this
is your NAG; so this is how it is done. So, this is NAG attached to a NAM. NAM has a
carboxy which is attached via a lactate side chain and this carboxy is the one which is
used to make the peptide. So, it will be CO; then there will be alanine, then there will be

glutamic acid, then there will be lysine, then there will be alanine, there will be alanine. I
hope this is clear.
So, every NAM is attached to alanine glutamic acid lysine alanine alanine. Also look at
the configuration of this alanine this is L-alanine, this is D-glutamic acid; I told you that
in bacteria only, D-amino acids are present; where from this D amino acids come? By
isomerisation of the L-amino acids.
But this lysine side chain amine is attached to 5 glycine units. So, what is the end point
here? Is it the N-terminus or C-terminus that you have to be clear now; lysine ends up
with NH then you have a glycine; so NHCO glycine. Then glycine ends up with NH2 that
is attached to another glycines; so NHCO. If that be the case, you can say that it ends up
with an amine.
Lysine starts with NH. So, NHCO; then ultimately it does not end up with a CO2H, it
ends up with NH2 and then you have D-alanine. And in the adjacent NAM NAG; you
have the same chain alanine D-glutamic acid lysine that has got penta glycine; and then
D-alanine, D-alanine.
Now, the reaction that takes place in cross linking is that this side chain reacts with the
primary chain that is hanging here; these D-alanine D-alanine; again you have to be sure
that what ends up here; you started with NAM; NAM is this one. So, this is CO then NH.
So, alanine NH is on this side and CO2H on this side; that means, this will end up with a
CO2H.
So, this is a carboxy end here. In between D-alanine D-alanine, you have a CONH. So,
you have a D-alanine here that is the end point and this is the CO2H and this D-alanine is
attached to lysine.
So, now the reaction that takes place is basically that the glycine NH2 attacks this amide
bond and kicks out the last D-alanine. So, what will happen now? This is the chain that is
now cross-linked with this chain and the result is a new peptide bond between the two
peptide chains.

Suppose this is glycine that has got NH2 and then you have D-alanine D-alanine. So, D-
alanine then CO, then NH, then D-alanine and this was going to the top attached to a

NAM and now the reaction is basically this NH2 is attacking here that goes out; that
makes a cross-link. What is the nature of this reaction how can you classify this reaction?

This is nothing, but look at this D-alanine this was connected to another amino acid D-
alanine earlier. Now what you are doing? You are replacing this D-alanine by glycine.

So, this can be called a transpeptidation reaction; that means, your peptide was having
another amino acid that is replaced by a new amino acid.
So, what is transpeptidation? That if you have a peptide like this and if you have another
amino acid and if you get A-C plus B that is a transpeptidation; these are all amino acids.
So, this peptide bond is replaced by a new peptide bond; so that is why this is called
transpeptidase. So, there is an enzyme to do this; otherwise this reaction will not take
place, this enzyme is a serine based enzyme.
So, there is an enzyme it is written here enzyme OH. So, initially enzyme OH like
chymotrypsin what happens when you hydrolyze a peptide bond; you attack the peptide
bond with the serine OH, replace at that time you break that other amino acid and then
this serine is released again when the water comes and attacks. So, in this case; the
enzyme attacks the carbonyl forming the tetrahedral intermediate, the D-alanine leaves
and then instead of water now the adjacent glycine amine attacks and releases the serine
OH.
(Refer Slide Time: 33:09)

I can show it again in an even better way that basically what we are talking about
CONH; D-alanine and this is D-alanine and on this side there is this glycine NH2. So,
initially the enzyme which is having a serine OH that attacks here; forms the tetrahedral
intermediate; so D-alanine CO minus and then NH then D-alanine and this is the serine
enzyme. Now what will happen? So, there must be some oxyanion hole here which will
stabilize this O minus.
Now, this comes back; that goes out; so D-alanine has gone out. So, D-alanine and then
CO and that is attached to the serine and now the glycine which was waiting for this to
happen, comes attacks breaks this bond. So, you get the link between the glycine and the
D-alanine. Look at the work load on the bacteria; the work load on the bacteria is that it
has to synthesize the NAM, it has to synthesis the NAG and then combine NAM and
NAG form the glycoside bond and make the polymer, make another set of polymer.
All these rods which are made up of NAM NAG; NAM NAG; then it have to form the
peptides. And this is the last reaction in the synthesis of the bacterial cell wall; so the last
reaction is this transpeptidation reaction that is somehow stopped by penicillin.
So, how penicillin works? It stops the enzyme transpeptidase from working, but its a
beautiful way to really harass the bacteria. Because you are not killing the bacteria or
stopping the bacteria from the very beginning; you have done enormous amount of work;
you have prepared for an exam reading 24 hours a day and on the final day you could not
answer anything because the questions are so hard.
So, basically that is the case; the bacteria have worked to make all the cell walls; all the
components assembled together; but stopped in the last stage. Like in the marriage
ceremony, if the priest does not arrive the ceremony cannot take place. So, it is basically
there at the terminal point you are stopping the bacteria from forming the cell wall. And
if that cross-link does not happen; the cell wall is very fragile, water will now go through
the cell wall inside the cell and then the cell burst open and that is what is called lysis.

(Refer Slide Time: 36:25)

Now, the question is why penicillin will stop this? Why will it stop this cell wall
formation at the terminal stage? What is the similarity of the reaction or the structure?
But before that I may mention that this is the structure that I gave you; that lysine,
connected to five glycine units; this is usually present in gram positive bacteria; in gram
negative bacteria like E. coli, this lysine is replaced by an amino acid which is called
diaminopimelic acid; Pimelic acid is 7-carbon dicarboxylic acid HO2C-(CH2)5-CO2H.
So, you have CO2H; NH2 and then all these and then another NH2 and CO2H. So, this
diaminopimelic acid you might see in different books. The mechanism is same in that
case the amine side chain of the diaminopimelic acid will react with that. And
incidentally this is the meso diaminopimelic acid because this has got two stereogenic
centers; if there is opposite configuration that becomes meso. It is the meso
diaminopimelic acid in E. coli that replaces the lysine.
This is normally written in the medicinal chemistry books; where the mechanism
remains the same. Now, let us see what is it is there; a meso diaminopimelic acid or
lysine any one of this, then penta glycine has to be attached; it is through the glycine side
chain.

(Refer Slide Time: 38:15)

This is the mechanism which I have already discussed. Penicillin is an inhibitor of this
transpeptidase cross-linking reaction. If this is the transpeptidase enzyme, then this is the
D-alanine D-alanine remember this is D; so, that has to be of configuration R; so 1, 2, 3.

So, that is R and this is also 1, 2, 3 but the methyl is α; so this is also R. So, this is the D-
alanine and D-alanine; so what is the reaction? Why this D-alanine is binding to the

enzyme; there must be positively charged species like lysine. So, that forms the salt
bridge.
So, this goes and binds and then this serine serine OH attacks, forms the acyl-enzyme
complex and now, the glycine NH2 attacks and forms the cross-link. In case of penicillin,
it has been found that instead of D-alanine; D-alanine penicillin comes; bacteria thinks
that penicillin is my substrate and penicillin has got CO2 minus that binds with the lysine
and this β-lactam moiety is a very reactive ring which can open up immediately.
So, serine OH attacks and via the formation of the tetrahedral intermediate, it goes back
and kicks out this. So, this is the situation now; so the serine is acylated by the penicillin
and now the serine can be regenerated provided water comes and breaks this bond, so
that the enzyme is freed again but this is a very slow reaction.
If it is a very slow reaction by the time this hydrolysis happens, the bacteria cell is
already lysed. Bacterial cell will not wait for such long time that the enzyme is free

again. So, basically it is an active site directed irreversible inhibition. What is active sight
directed? That it goes straight away and attacks the enzyme and stays there.
Formation of covalent bond means it is a irreversible inhibition and then after the
reaction, no further reaction happens to produce a more reactive species. So, this is
nothing, but an active site directed irreversible inhibition ASDII. So, this is the
mechanism; again this is written here; water attacking; this is blocked; water cannot
come and hydrolyze this one, ok. So, that is the mechanism of penicillin.
(Refer Slide Time: 41:33)

Now, the last thing that is remaining is why penicillin is so selective; why bacteria thinks
that this is my substrate. Now, compare the two structures; this is again another way of
writing D-alanine D-alanine. See this is the methyl; you see this has to be R
configuration because the methyl is α. So, it has to be R now; so that is D and this is also
D. So, I have written this structure D-alanine D-alanine in this fashion.
And look at the structure of penicillin; see this is same; if you look at the back bone only,
you will see the similarity of penicillin and D-alanine D-alanine. So; that means,
penicillin mimics D-alanine D-alanine in structure; it is only D D; it is not L L.
If bacteria had used L L, penicillin would have been ineffective; this streochemistry is
also matching; only thing penicillin has some extra portion. So, it is even better that it is
a conformationally constrained system which resembles D-alanine D-alanine. Of course,

D-alanine D-alanine must be having different types of confirmation, but in one
confirmation you see that it resembles the penicillin.
So, now in short what is the mechanism of reaction of penicillin? It is a transpeptidase
inhibitor. What is transpeptidase? Transpeptidase is an indispensible enzyme to make
cell wall. What it does? The terminal step again I repeat now the bacteria has made these
cell wall premature cell wall, it is like making premature or immature mRNA.
And then suppose you stop now the splicing with the spliceosome; so your mRNA will
be ineffective. So, similarly the bacteria makes everything except the cross-linking. So,
the terminal-cross linking is inhibited and that is how penicillin works. I think that is all
for this session; we will talk about the bacterial resistance in the next session.
Thank you.