Mode of Action of Penicillin

We have seen that antibiotics, like penicillin, stop the growth of the outer casing of the bacteria, which is called a cell wall. Just like the walls of a house, without a strong cell wall, the bacterium collapses. You may find this analogy useful in helping to understand the mode of Action of penicillin. Bacteria have a very different type of cell wall to what we find in human cells – we can compare the different walls to an insulated double brick house in cold climates versus a single layer of wood in a timber house in warmer climates.

In people, cells are protected by being surrounded by other cells inside our bodies (like the timber house), while bacteria are exposed out in the environment and need stronger cell walls (like the insulated double brick house). In this analogy, the antibiotics are designed to attack and destroy the bricks, but not touch the timber, so they kill the bacteria, but not us.

The β-lactam Ring is Key for Antibiotic Activity

We have seen that the basic structure of penicillin consists of a β-lactam ring and an acylamino side chain (RCONH–). Based on the mode of action, the β-lactam ring is clearly crucial for its biological activity – the carbon atom in the C=O of the lactam is particularly electrophilic (δ+) and the adjacent thiazolidine ring confers further strain on the β-lactam ring, making it even more reactive to nucleophilic attack (try making a model of the ring system and you will see how strained the β-lactam ring is).

The carboxylic acid group is also important – this is normally deprotonated within the body and the negatively charged carboxylate ion (RCO₂^–) binds to a positively charged amino acid within the active site of the transpeptidase enzyme.

The β-lactam Carbonyl is the Most Electrophilic Site

At the top of the β-lactam ring, a cis–arrangement of hydrogens (both on the same side of the ring) is required for the biological activity, as is an acylamino side chain at the ‘top left’. This acts as an electron-withdrawing group (the nitrogen atom accepts electron density from the β-lactam carbonyl making it an even stronger electrophile). Of the three C=O bonds in penicillin, the β-lactam carbonyl is the most electrophilic. The C=O bond in the acylamino side-chain is not susceptible to nucleophilic attack, because, as is typical of amides, the nitrogen atom can feed its lone pair into the carbonyl group which makes it a weaker electrophile. Similarly, the C=O bond in the carboxylic acid side-chain, or the carboxylate ion, is not susceptible to nucleophilic attack because the oxygen atom can feed its lone pair into the adjacent carbonyl group. (The wavy lines indicate only a partial structure is shown.)

Hydrolysis of the β-lactam Ring in Penicillin Makes it Inactive

Unfortunately, because of the high reactivity of the β-lactam ring, penicillin can react with water under acidic conditions (as found in the stomach), to break the β-lactam ring, in a hydrolysis reaction. The reaction mechanism is a nucleophilic acyl substitution reaction, forming a penicilloic acid, which does not have the desired antibiotic activity – the penicillin is rendered useless.

The Acylamino Side-chain Can Open the β-lactam Ring

Also, the acylamino side-chain can aid the ring-opening of the β-lactam ring. It can act as an internal nucleophile and attack the β-lactam carbonyl forming a very strained intermediate that then opens to break the β-lactam ring. This makes penicillin inactive and is sometimes described as a ‘self-destruct’ mechanism.

Altering the Acylamino Side-chain To Make a More Stable Penicillin

To reduce or stop the involvement of the acylamino side chain, and self-destruction, researchers have placed an electron-withdrawing substituent within the side-chain. This group likes to accept electrons and this makes the amide carbonyl group a weaker nucleophile, which is less likely to react with the β-lactam carbonyl. For example, penicillin G is more prone to ‘self-destruct’ than penicillin V, or phenoxymethylpenicillin.

Penicillin V contains electronegative oxygen in the PhO substituent, which draws the electron density away from the amide carbonyl group and so reduces its tendency to act as a nucleophile and react with the β-lactam ring. This has important consequences. While penicillin V is stable enough to survive the acidic aqueous conditions in the stomach and so can be taken as a tablet (which is typically preferred by patients), penicillin G does not and so needs to be administered by injection.

We have just covered some pretty challenging aspects of reaction mechanisms. Don’t worry if you have not grasped all of the fine details, you will still be able to make good progress. The key points to take on board are the importance of the β-lactam ring for biological activity and that the reactivity of the ring is affected by the groups that surround it within the penicillin.

Sounding Out Drug Design

We have seen that the 3-dimensional shape of penicillin is key to its biological activity, ensuring that it can dock into an enzyme (like a key fitting into a lock). The same is true for anticancer agents and researchers at York are investigating whether using 3D sound and visuals will allow scientists to design effective anticancer drugs quickly and more efficiently. The ‘Sonicules’ project models the interaction of a drug molecule with an enzyme and displays the interaction as sound, alongside visual representations. The software has been designed that portrays drug design in a more game-like way – members of the public can have a go at a computer game to help understand how drugs are designed and how sound helps this process.