Penicillin: Discovery and Chemistry

On a September morning in 1928, a Scottish bacteriologist came back from holiday to a laboratory bench cluttered with old culture plates. One of them had been spoiled by a blob of mould. Around it, the colonies of bacteria had died. Many scientists would have thrown the plate away. Alexander Fleming noticed something that would eventually save hundreds of millions of lives. But the story of penicillin isn’t the story of one lucky observation. It took more than a decade, a team of determined chemists, a war and a mouldy cantaloupe melon to turn Fleming’s plate into a medicine.

The observation (1928)

Alexander Fleming worked at St Mary’s Hospital in London, studying Staphylococcus bacteria, which cause boils, wound infections and blood poisoning. He grew them on shallow dishes of nutrient jelly (agar plates).

When he returned from his summer holiday in September 1928, he found that one plate had been contaminated by a mould. Around the mould was a clear ring where the bacterial colonies had dissolved. Something from the mould was killing the bacteria.

Fleming identified the mould as a species of Penicillium and named the active substance penicillin. He showed that a liquid “mould juice” from the culture:

• killed many kinds of disease-causing bacteria, including those behind pneumonia, gonorrhoea and meningitis;
• was not toxic to white blood cells or to laboratory animals.

The circumstances were remarkably lucky. The mould spores probably drifted up from a laboratory below, where mycologists were studying moulds; an unusually cool spell of weather let the mould grow first, before warmer weather allowed the bacteria to grow around it.

The idea stalls (1929–1938)

Fleming published his findings in 1929. But penicillin proved frustrating:

• it was produced in tiny amounts;
• it was chemically unstable, losing activity within days;
• Fleming and his assistants couldn’t purify or concentrate it;
• it seemed to act slowly and disappear quickly from the body.

Fleming mentioned it as a possible way to disinfect wounds or to select particular bacteria in the lab, but he didn’t pursue it as a drug for internal use. For about a decade, penicillin was largely forgotten.

The Oxford team (1939–1941)

In 1939, at the University of Oxford, the Australian pathologist Howard Florey and the German-born biochemist Ernst Chain, a refugee from Nazi Germany, began a systematic study of natural antibacterial substances. Chain came across Fleming’s paper. Penicillin looked promising, and nobody else was working on it.

The team faced a serious chemical problem: how to extract penicillin from the mould broth without destroying it. Norman Heatley, a biochemist with great practical skill, devised the solution:

• he found that penicillin could be moved between water and an organic solvent by changing the pH: in acidic conditions, penicillin (a weak acid) is uncharged and dissolves in the solvent; in neutral conditions, it becomes an ion and moves back into water (see pKa);
• he built a counter-current extraction apparatus from glass tubing, milk churns, bedpans and bathtubs to do this continuously;
• with too little money for proper equipment, the lab grew mould in hundreds of improvised vessels, and employed a team of young women, nicknamed the “penicillin girls”, to tend them.

The mouse experiment

On 25 May 1940, the team injected eight mice with a lethal dose of Streptococcus bacteria. Four were then given penicillin. By the next morning, the four untreated mice were dead; the four treated mice were alive. It was a dramatic result.

The first patient

In early 1941, Albert Alexander, an Oxford policeman, was dying from a severe infection that had spread from a scratch. Given penicillin, he improved remarkably within days. But the team didn’t have enough: they even recovered penicillin from his urine to reuse it. When the supply ran out, the infection returned and he died. The lesson was clear: penicillin worked, but it had to be made on an industrial scale.

Mass production (1941–1944)

Wartime Britain couldn’t provide the industrial capacity, so in 1941 Florey and Heatley flew to the United States. There, at a government agricultural research laboratory in Peoria, Illinois, scientists made key advances:

• Corn steep liquor, a by-product of maize processing, greatly increased the yield when added to the growth medium.
• A search for better mould strains turned up a high-yielding one, Penicillium chrysogenum, growing on a mouldy cantaloupe melon from a Peoria market. Its descendants, improved by mutation with X-rays and ultraviolet light, became the ancestors of most industrial penicillin strains.
• Pharmaceutical companies developed deep-tank fermentation, growing the mould in huge stirred and aerated vessels instead of shallow trays.

Production soared. By D-Day in June 1944, enough penicillin was available to treat Allied casualties, saving many soldiers from infected wounds and pneumonia.

In 1945, Fleming, Florey and Chain shared the Nobel Prize in Physiology or Medicine. Heatley, whose extraction method made it all possible, was honoured much later, receiving an honorary medical doctorate from Oxford in 1990.

Solving the structure (1945)

Chemists argued about penicillin’s structure. The key disagreement was whether it contained a β-lactam ring, a strained four-membered ring containing nitrogen that many chemists considered too unstable to exist in a natural product.

In 1945, Dorothy Crowfoot Hodgkin at Oxford, using X-ray crystallography on crystals of penicillin salts, proved that the β-lactam ring was there. It was one of the first times X-ray crystallography had been used to determine the structure of a complex molecule whose structure chemists couldn’t establish by other means. Hodgkin went on to solve the structures of vitamin B12 and insulin, and received the 1964 Nobel Prize in Chemistry.

The penicillin core consists of:

• a β-lactam ring (four atoms: three carbons and a nitrogen, with a C=O);
• fused to a five-membered thiazolidine ring containing sulfur;
• a variable side chain attached through an amide group, which differs between types of penicillin.

Why the structure works

The β-lactam ring is highly strained: its bond angles are squeezed to about 90°, far from their preferred values, and the fused ring prevents the normal stabilisation of its amide bond. That makes the ring’s carbonyl carbon unusually reactive.

Penicillin’s shape mimics the end of the peptide that bacteria cross-link when building their cell walls. The bacterial enzyme that does this cross-linking attacks the β-lactam ring, the ring springs open, and penicillin becomes permanently attached to the enzyme’s active site. Without cross-links, the bacterial wall is weak, and the bacteria burst. Human cells have no such walls, which is why penicillin is so selective. See how antibiotics work for the details.

After the war: new penicillins

• In 1957, John Sheehan at MIT achieved the first total chemical synthesis of penicillin V, but fermentation remained far cheaper for production.
• In 1958–1959, scientists at Beecham in Britain isolated 6-aminopenicillanic acid (6-APA), the penicillin core without a side chain. Chemists could now attach new side chains, creating semi-synthetic penicillins such as ampicillin, amoxicillin and methicillin, with wider activity or resistance to bacterial enzymes.

A warning from the start

In his 1945 Nobel lecture, Fleming warned that bacteria could become resistant if penicillin was misused, for example by taking too little. He was right. Bacteria soon evolved β-lactamase enzymes that break the β-lactam ring. Antibiotic resistance is now one of the world’s major health challenges.

Key takeaways

• 1928: Alexander Fleming noticed that Penicillium mould killed bacteria, but couldn’t purify the unstable penicillin.
• 1939–1941: Florey, Chain and Heatley at Oxford purified it using pH-controlled solvent extraction, proved it worked in mice and tested it in the first patient.
• 1941–1944: US work with corn steep liquor, a strain from a mouldy cantaloupe and deep-tank fermentation made mass production possible.
• 1945: Dorothy Hodgkin used X-ray crystallography to prove the β-lactam ring, whose strain explains how penicillin works.
• Semi-synthetic penicillins followed, and so did resistance, as Fleming had warned. For more stories from chemistry’s past, browse the history shelf.