Skip to 0 minutes and 6 secondsDifferent antibiotics have different modes of action. This is because of their structures and degrees of affinity to certain target areas within bacterial cells. For example, antibiotics can inhibit protein or nucleic acid synthesis, or damage or disrupt the cell membrane. Alternatively, antibiotics like penicillin work by inhibiting the synthesis of the cell wall. The cells of humans and animals do not have cell walls, but this structure is critical for the life and survival of bacteria. The modes of action of both early sulfonamides and modern penicillins revolve around the concept of enzyme inhibition. Most enzymes will bind to a specific molecule called a substrate. The binding site on the substrate molecule must be complimentary to the active site of the enzyme.

Skip to 1 minute and 1 secondSulfonamides act to inhibit the synthesis of DNA within bacteria. This does not kill bacteria outright but inhibits the growth and multiplication of bacteria; giving the body's immune system time to fight the infection. As such, sulfonamides are called bacteriostatic agents, which inhibit growth of bacterial colonies. Penicillins and other beta-lactam antibiotics are bactericidal as they actively kill bacterial cells. One important sulfonamide is Prontosil, a red azo dye, which was found to be antibacterially effective in vivo (when tested in animals) but ineffective in vitro (when placed directly into a test tube of bacteria). This arises because Prontosil is converted into the active sulfonamide by bacteria present in the small intestine of mammals.

Skip to 1 minute and 52 secondsSulfanilamide is an effective antibiotic due its similar shape to para-aminobenzoic acid (or PABA). PABA is required for the production of tetrahydrofolate, which is needed for the synthesis of DNA in bacteria. The conversion of PABA into tetrahydrofolate is achieved by the enzyme DHPS (dihydroproteroate synthetase), which binds to the PABA molecule at its active site. As the structure of sulfanilamide is so similar to that of PABA the DHPS enzyme is not able to differentiate between the two; both have complimentary shapes to the active site in DHPS. So sulfanilamide can occupy the DHPS active site, preventing PABA from binding to DHPS and therefore preventing the production of bacterial tetrahydrofolate and DNA.

Skip to 2 minutes and 44 secondsAlthough discovered later on, penicillins have proved to be the preferred method of treatment. Their action leads directly to cell death, rather than just the containment strategy provided by sulfonamides. Unlike animal cells, bacteria possess cell walls. For gram-positive bacteria this is comprised of a thick peptidoglycan wall. In contrast, a thinner peptidoglycan layer, supported by an outer liposaccharide membrane, encapsulates gram-negative bacteria. Individual peptidoglycan molecules are long chain polymers made by linking together sugars and amino acids. When linked together many peptidoglycan chains can form a rigid wall which protects the bacteria from harmful chemicals and stops them swelling and bursting. The peptidoglycan wall is constantly being assembled and repaired.

Skip to 3 minutes and 36 secondsThis is accomplished using a transpeptidase enzyme, which is able to link amino acids at the end of two separate peptidoglycan chains, joining them together to form the strong peptidoglycan structure. The mechanism involves two nucleophilic acyl substitution reactions; the first reaction forms an intermediate ester, which reacts to form a new amide bond that links the peptidoglycan chains.

Skip to 4 minutes and 8 secondsPenicillin acts by inhibiting the transpeptidase enzyme - it mimics a peptidoglycan chain and an ester is formed that joins the penicillin to the enzyme. As the penicillin group is so large, it prevents attack of a nucleophile at the ester carbonyl and so the ester does not react with the second peptidoglycan chain. With the enzyme unable to form cross-links the peptidoglycan wall begins to degrade. No new peptidoglycan chains can be added to the cell wall and eventually the cell bursts.

The mode of action of penicillin

We have seen that the basic structure of penicillin consists of a β-lactam structure ring and an acylamino side chain. 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 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 general structure of penicillin

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.

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). Note that the C=O bond in the amide 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 as the oxygen atom can feed its lone pair into the adjacent carbonyl group.

The different reactivities of the three C=O bonds

Unfortunately, because of the high reactivity of the β-lactam ring, a penicillin can react with water under acidic conditions, to break the β-lactam ring, in a hydrolysis reaction. The reaction mechanism is a nucleophilic acyl substitution reaction.

Reaction of water with the beta-lactam ring in a hydrolysis reaction

Also, the acylamino side chain can help 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 is sometimes described as a ‘self-destruct’ mechanism.

The acylamino side chain can act as an internal nucleophile

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 accepts 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.

A comparison of penicillins G and V

Penicillin V contains an 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.

We have just covered some pretty challenging aspects on reactions mechanisms. Don’t worry if you have not grasped all of the fine details, 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.

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Exploring Everyday Chemistry

University of York