B lactam antibiotics

Question 1

Bacteria

Answer 1

Microorganisms, usually consisting of a single cell that can rapidly multiply under favourable conditions. Human cells are termed eukaryotic: they have a nucleus and DNA arranged in linear chromosomes. Bacteria, on the other hand, are prokaryotic cells. These cells are simpler in structure with no nucleus and a small amount of DNA in one circular chromosome. Bacteria come in three basic shapes: rod-shaped (bacilli), spherical (cocci), or helical (spirilla).

Bacteria live in every climate and location on earth. Some are airborne while others live in water or soil. Bacteria are hugely important: allowing plants to grow and aiding human and animal digestion.

Question 2

Gram-positive bacteria

Answer 2

They have a thick peptidoglycan cell wall

Question 3

Gram-negative bacteria

Answer 3

They have a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing lipopolysaccharide. There is a space between the cytoplasmic membrane and the outer membrane called the periplasmic space or periplasm. The periplasmic space contains the loose network of peptidoglycan chains referred to as the peptidoglycan layer.

Question 4

How is a Gram stain test done?

Answer 4

Crystal violet is used to stain a sample of bacteria. Gram-positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet. Gram-negative bacteria stain red, due to a thinner peptidoglycan wall, which does not retain the crystal violet during the decolouring process.

Question 5

Narrow spectrum and broad spectrum antibiotics

Answer 5

Antibiotics must permeate the bacterial cell wall to be effective. This is easier in gram-positive bacteria, due to the nature of the cell wall. This difference in cell wall structure can give rise to broad and narrow-spectrum antibiotics.

Narrow-spectrum antibiotics only work on a small subset of bacteria, for example only gram-positive, whereas broad-spectrum work on a much wider number of bacteria.

Gram-negative bacteria are more resistant to antibiotics than gram-positive bacteria, because they have a largely impermeable cell wall. The bacteria responsible for MRSA and acne are examples of gram-positive bacteria, whilst those responsible for Lyme disease and pneumonia are examples of gram-negative bacteria.

Positively charged molecules seemed to permeate the cell walls easier than neutral or negatively charged molecules.

Question 6

Penicillin

Answer 6

The first antibiotic was discovered in 1928 by Sir Alexander Fleming, a leading Scottish microbiologist and physician who had been studying bacteria and antiseptics for many years. Fleming’s discovery was purely serendipitous. Whilst experimenting with staphylococci bacteria, Fleming left some of his culture trays out in his laboratory before leaving for vacation. On his return he discovered mould growing on one of the cultures but more importantly that the bacteria next to the mould was dead. He determined the ‘bacteria-killing’ mould to be part of the penicillium family and named this ‘antibiotic’ penicillin. Fleming is quoted as saying:

Question 7

Penicillin structure

Answer 7

contains a characteristic β-lactam core: a highly-strained 4-membered amide ring. The ring is highly reactive and is crucial for antibiotic efficacy. Note that the picture below shows a generic penicillin structure and penicillin G. Penicillin can come in many forms, differing only through the “R group”.

Question 8

How does penicillin work?

Answer 8

By inhibiting bacterial cell wall synthesis and thereby stopping rapid cell growth.

Question 9

Peptidoglycan

Answer 9

The cell wall of most bacteria is made from a strong lattice of sugars and peptides.

Question 10

What is the structure of peptidoglycan?

Answer 10

It consists of polymer chains of alternating NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid) sugars. Peptide chains of three to five amino acids form links between the NAM sugars and between layers

Question 11

What does the enzyme transpeptidase, or penicillin-binding protein (PBP)

Answer 11

It catalyses the final step in the cell wall biosynthesis: transpeptidation. Transpeptidation is the cross linking of the peptidoglycan layers with short peptide chains. Penicillin irreversibly binds to the PBP enzyme, thus inhibiting its activity, stopping the production of peptidoglycan and leading to cell death. Bacterial cells are under high osmotic pressure, packed with proteins and small molecules on the inside whereas the environment outside the cell is dilute. Thus without the strength of peptidoglycan the cell walls rapidly burst.

Question 12

Peptide chains

Answer 12

The constituent amino acids in the linking peptide chains vary depending on the type of bacteria. However, peptidoglycan is one of the most prevalent sources of D-amino acids. You will recall that amino acids are chiral and thus can exist in two enantiomeric forms: D and L. The 3D structure of penicillin mimics the structure of D-alanine

The amide of D-alanine binds to a serine residue in the enzyme binding site, thus cleaving the amide bond. This opens up D-alanine to react with another amino acid to form a peptide chain. Penicillin reacts in the same way, with the amide of the β-lactam ring reacting with the same serine and cleaving. However, this reaction is irreversible and penicillin now blocks the enzyme binding site.

Question 13

The basic structure of penicillin

Answer 13

β-lactam ring and an acylamino side chain (RCONH–)

Question 14

Why is the β-lactam ring important? in penicillin

Answer 14

It is 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

Question 15

Why is the carboxylic acid group important? in penicillin

Answer 15

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

Question 16

What is the β-lactam Carbonyl?

Answer 16

it is the is the most electrophilic site. Of the three C=O bonds in penicillin, the β-lactam carbonyl is the most electrophilic.

Question 17

What part of the penicillin structure is not susceptible to nucleophilic attack?

Answer 17

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.

Question 18

What happens if the β-lactam ring is hydrolysed?

Answer 18

It makes it Inactive.

Question 19

How is the β-lactam ring hydrolysed?

Answer 19

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.

Question 20

What can the acylamino side chain do to aid the ring opening of the β-lactam ring?

Answer 20

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.

Question 21

Why have researchers altered the
Altering the Acylamino Side-chain?

Answer 21

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.

Question 22

How is penicillin G different to V?

Answer 22

Penicillin G is more prone to ‘self-destruct’ than penicillin V.

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.

Question 23

SAR

Answer 23

structure-activity relationship

Question 24

What is the penam ring system?

Answer 24

It is essential for activity. The penam ring is the name given to the β-lactam-thiazolidine fused ring system.

Question 25

What is the only part of penicillin that can be changed?

Answer 25

Only the ring side chain at position 6 could be changed and efficacy retained.

Question 26

The differences of bioavailability of penicillin types?

Answer 26

Penicillin G, the first penicillin to be isolated, is easily metabolised by gastric acid. Its bioavailability after oral administration is <30% and can only be administered by IV. Variants of penicillin G have greater bioavailability. For example, penicillin V is more acid-stable with a bioavailability of 60%–70%. Amoxicillin is better still with an oral bioavailability of 74%–92%:

Question 27

Why does changing the R group affect metabolism?

Answer 27

Penicillin G degrades in acidic media (stomach) by opening the β-lactam ring, through attack of the R group amide. Electron withdrawing substituents in the R group position, reduce the electron density on R group carbonyl thus reducing attack of the β-lactam ring and improving the molecule’s metabolic stability.

Question 28

The structural modification that enhances Penicillin V’s acid stability and oral bioavailability?

Answer 28

Replacing Penicillin G’s aromatic benzyl side chain with an aliphatic phenoxy methyl side chain.

Question 29

β-lactamase (Penicillinase)

Answer 29

The second main resistance mechanism for penicillins is to interact with the enzyme β-lactamase (also called penicillinase). Penicillinase has a similar active site as PBPs. A serine residue binds to penicillin, but instead of irreversibly binding, as in PBPs, penicillinase releases the antibiotic in an inactive form thus killing their antibiotic efficacy. Other β-lactamases use a zinc ion instead of a serine amino acid to inactivate the drug.

Question 30

How is steric hindrance involved in B lactamase resistance?

Answer 30

The penicillinase-resistant antibiotics have large bulky groups attached to the β-lactam ring. These groups are deliberate: sterically blocking the attack of the β-lactam by the penicillinase enzyme. The sterically bulky substituent must be directly attached to the amide in order to be inactive against penicillinase.