DISCOVERY OF PENICILLIN

Bhagyashree Bachhav Shalaka Khobragade Pranjal Deshmukh Harini Krishnan Dhanashree Mahadik Shail Panchamia

Institute of Chemical Technology

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SERENDIPITY
Penicillin was discovered by bacteriologist Alexander

Fleming who observed that a plate culture of Staphylococcus had been contaminated by a blue-green mold and that colonies of bacteria adjacent to the mold were being dissolved. Curious, he grew the mold in a pure culture and found that it produced a substance that killed a number of diseasecausing bacteria. Naming the substance penicillin, Dr. Fleming in 1929 published the results of his investigations, noting that his discovery might have therapeutic value if it could be produced in quantity. Hodgkin used x-rays to find the structural layouts of atoms and the overall molecular shape of over 100 molecules including penicillin. His discovery of the molecular layout of penicillin helped lead scientists to develop other antibiotics.
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RING AND NUMBERING SYSTEMS OF B-LACTAM ANTIBIOTICS

Structure
Penicillin molecules all contain a highly

strained 4-membered -lactam ring fused to a 5-membered thiazolidene ring.

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 Bond angles: 90 instead of 120

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This prevents the overlap of the nitrogen lone

pair with the adjacent carbonyl system, and thus the -lactam carbonyl is much more electrophilic than a normal amide, and is therefore susceptible to nucleophilic attack.
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SAR

4 1

Changing one or more of these results in a loss of activity The bicyclic ring system containing the lactam cis relationship between the two hydrogens at positions 5 and 6 a free 2-carboxylate a 6-amide.
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Chemical Instabilities

B-lactamase hydrolysis Acid hydrolysis Nucleophilic attack

Elimination and Acid Hydrolysis

How Penicillin Works to Kill Bacteria

Bacterial cell walls are made of peptidoglycan. In

order to form a rigid structure, the polysaccharide chains (glycans) are linked together by peptide crosslinks. This peptide ends in two D-Alanine (D-Ala) residues. It is further modified by attaching an additional peptide to the middle of the first one creating a branched structure. Finally the peptide of one of the polysaccharide molecules is attached to another to form the crosslink. The reaction is a transpeptidase reaction.

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Almost all bacteria have cell walls and they have transpeptidase enzymes that catalyze this reaction. The activity of this enzyme is inhibited by penicillins or -lactams.

The mechanism of inhibition is well known. The -lactam region of the drug resembles the D-Ala-D-Ala end of the peptide to which the transpeptidase enzyme binds.
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 The business part of the molecule is the -lactam

moiety and the "R" represents various groups that can be bound to create different penicillin drugs. The structure of D-Ala-D-Ala is shown below. The structures of several different transpeptidases have been solved. The enzymes are usually called penicillin-binding proteins or PBP's. Most bacteria have several related versions of PDB genes but all of the enzymes are inhibited by -lactams.

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 The enzyme is inactive because the drug

binds very tightly to the active site and blocks the reaction. That's how penicillin works.

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How structure is modified to improve potency and reduce instabilities?

Stability towards B-lactamase is influenced by bulk in acyl group attached to the primary amine. The differential steric hindrance plays an important role in resistance to enzymatic degradation.

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 it was discovered that penicillin derivatives containing bulky groups on the side chains were poor substrates for -lactamases. But such additional bulk could also make the

penicillins less efficacious as substrates for the target transpeptidase enzyme. Thus, a great deal of work needed to be done to find the appropriate blocking groups that would prevent binding with the -lactamase enzyme while enabling interaction with the target enzyme.
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Individual Penicillins
Benzylpenicillin (Pen G):

1.

First penicillin used but very unstable in acidic conditions

Phenoxymethyl Penicillin (Pen V): 1. More acid stable than benzylpenicillin 2. Caused by the electronegative oxygen

3.

atom in the C-7 amide side chain inhibiting participation in -lactam bond hydrolysis. Pen V was the first oral penicillin, giving higher and more prolonged blood levels than Pen G.
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Ampicillin:

- Benzylpenicillin analogue in which one of the hydrogen atoms of the side chain methylene has been replaced with a primary amino group. - Acid stability: electron withdrawing character of the protonated primary amine group, reduces participation in hydrolysis of the lactam bond as well as to the comparative difficulty of bringing together H3O+ into the vicinity of the protonated amino group.
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Bacampicillin:
A prodrug of Ampicillin. Ampicillin - reasonable absorption of 30-55% via oral route. Its oral bioavailability can be significantly improved using

prodrug approach. Ampicillin is present in zwitterionic form, which further limits its oral absorption. Therefore to prevent the ionization carboxylic group of ampicillin is esterified increasing the prodrugs logP value. This enhances the prodrugs membrane permeability and hence contributes to its nearly complete absorption when administrated orally, preventing excess of unabsorbed ampicillin from remaining in intestinal lumen, which can affect the natural microflora of the GIT and hence causing diarrhea.

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 After its absorption, Bacampicillin ester drug

is first hydrolysed by esterase to produce ethanol and CO2, followed by spontaneous hydrolysis to release ampicillin and acetaldehyde. Ampicillin has lower lipid solubility than Bacampicillin because of the presence of a carboxylic functional group in its chemical structure, which is significantly ionized at intestinal pH.
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Clavulanic Acid:
It is a potent inhibitor of bacterial -lactamase inhibitor. Clavulanic acid is a lactam, which when given in

combination with penicillin is preferentially taken up by -lactamase and hydrolysed. It is used to overcome resistance in bacteria that secrete -lactamase, which otherwise inactivates most penicillins. In its most common form, the potassium salt potassium clavulanate is combined with: Amoxicillin (co-amoxiclav , trade names: Augmentin, Synulox [veterinary], and others) or ticarcillin (co-ticarclav, trade name Timentin).
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 Enzyme lactamase is a serine protease and can hydrolyse lactams such as penicillin antibiotics.

Clavulanic acid is a lactam, which when given in combination with penicillin is preferentially taken up by lactamase and hydrolysed. However during the process clavulanic acid undergoes a cleavage, leading to the formation of a Michael acceptor which subsequently alkylates a nucleophilic group on lactamase, causing irreversible inhibition.
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Thank you!!

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