Penny le Couteur & Jay Burreson

by Napoleon's Buttons: How 17 Molecules Changed History

  Ehrlich’s first success was with a dye called trypan red I, which acted very much as he had hoped against trypanosomes—a protozoic parasite—in laboratory mice. Unfortunately it was not effective against the type of trypanosome responsible for the human disease known as African sleeping sickness, which Ehrlich had hoped to cure.

  Undeterred, Ehrlich continued. He had shown that his method could work, and he knew it was only a matter of finding a suitable magic bullet for the right disease. He began investigating syphilis, an affliction caused by a corkscrew-shaped bacterium known as a spirochete. Theories of how syphilis came to Europe abound; one of the most widely acknowledged was that it returned from the New World with Columbus’s sailors. A form of “leprosy” reported in Europe before Columbus’s time, however, was known to be highly contagious and venereally spread. Like syphilis it also sometimes responded to treatment with mercury. None of these observations fit what we know about leprosy, and it is possible that what was described was actually syphilis.

  By the time Ehrlich began looking for a magic bullet against this bacterium, mercury cures had been claimed for syphilis for over four hundred years. Yet mercury could hardly be considered a magic bullet for syphilis, as it often killed its patients. Victims died of heart failure, dehydration, and suffocation during the process of being heated in an oven while breathing mercury fumes. If one survived this procedure, typical symptoms of mercury poisoning—loss of hair and teeth, uncontrollable drooling, anemia, depression, and kidney and liver failure—took their toll.

  In 1909, after testing 605 different chemicals, Ehrlich finally found a compound that was both reasonably effective and reasonably safe. “Number 606,” an arsenic-containing aromatic compound, proved active against the syphilis spirochete. Hoechst Dyeworks—the company Ehrlich collaborated with—marketed this compound in 1910, under the name salvarsan. Compared with the torture of the mercury remedy, the new treatment was a great improvement. Despite some toxic side effects and the fact that it did not always cure syphilitic patients even after a number of treatments, salvarsan greatly reduced the incidence of the disease wherever it was used. For Hoechst Dyeworks it proved extremely profitable, providing the capital to diversify into other pharmaceuticals.

  After the achievement of salvarsan, chemists sought further magic bullets by testing tens of thousands of compounds for their effect on microorganisms, then making slight changes to chemical structures and testing again. There were no successes. It seemed as if the promise of what Ehrlich had termed “chemotherapy” would not live up to expectations. But then in the early 1930s Gerhard Dogmak, a doctor working with the IG Farben research group, decided to use a dye called prontosil red to treat his daughter, who was desperately ill with a streptococcal infection contracted from a simple pinprick. He had been experimenting with prontosil red at the IG Farben laboratory, and though it had shown no activity against bacteria grown in laboratory cultures, it did inhibit the growth of streptococci in laboratory mice. No doubt deciding he had nothing to lose, Dogmak gave his daughter an oral dose of the still-experimental dye. Her recovery was fast and complete.

  It was at first assumed that the dye action—the actual staining of the cells—was responsible for the antibacterial properties of prontosil red. But researchers soon realized that antibacterial effects had nothing to do with dye action. In the human body the prontosil red molecule breaks down to produce sulfanilamide, and it is the sulfanilamide that has the antibiotic effect.

  This, of course, was why prontosil red had been inactive in test tubes (in vitro) but not in live animals (in vivo). Sulfanilamide was found to be effective against many diseases other than streptococcal infections, including pneumonia, scarlet fever, and gonorrhea. Having recognized sulfanilamide as an antibacterial agent, chemists quickly started to synthesize similar compounds, hoping that slight modifications of the molecular structure would increase effectiveness and lessen any side effects. The knowledge that prontosil red was not the active molecule was extremely important. As can be seen from the structures, prontosil red is a more complicated molecule than sulfanilamide and it is more difficult to synthesize and to modify.

  Between 1935 and 1946 more than five thousand variations of the sulfanilamide molecule were made. A number of them proved superior to sulfanilamide, whose side effects can include allergic response—rashes and fever—and kidney damage. The best results from varying the sulfanilamide structure were obtained when one of the hydrogen atoms of the SO2NH2 was replaced with another group.

  The resulting molecules are all part of the family of antibiotic drugs known collectively as sulfanilamides or sulfa drugs. A few of the many examples are

  Sulfapyridine—used for pneumonia

  Sulfathiazole—used for gastrointestinal infections

  Sulfacetamide—used for urinary tract infections

  Sulfa drugs were soon being described as wonder drugs and miracle cures. While such descriptives may seem unduly exaggerated nowadays, when numerous effective treatments against bacteria are available, the results obtained from these compounds in the early decades of the twentieth century appeared to be extraordinary. For example, after the introduction of sulfanilamides, the number of deaths from pneumonia dropped by twenty-five thousand a year in the United States alone.

  In World War I, between 1914 and 1918, death from wound infection was as likely as death from injury on the battlefields of Europe. The major problem in the trenches and in any army hospital was a form of gangrene known as gas gangrene. Caused by a very virulent species of the Clostridium bacteria, the same genus responsible for the deadly botulism food poisoning, gas gangrene usually developed in deep wounds, typical of injuries from bombs and artillery where tissue was pierced or crushed. In the absence of oxygen, these bacteria multiply quickly. A brown foul-smelling pus is exuded, and gases from bacterial toxins bubble to the skin’s surface, causing a distinctive stench.

  Before the development of antibiotics there was only one treatment for gas gangrene—amputation of the infected limb above the site of infection, in the hope of removing all the gangrenous tissue. If amputation was not possible, death was inevitable. During World War II, thanks to antibiotics such as sulfapyridine and sulfathiazole—both effective against gangrene—thousands of injured were spared disfiguring amputations, not to mention death.

  We now know that the effectiveness of these compounds against bacterial infection has to do with the size and shape of the sulfanilamide molecule preventing bacteria from making an essential nutrient, folic acid. Folic acid, one of the B vitamins, is required for human cell growth. It is widely distributed in foods, such as leafy vegetables (hence the word folic from foliage), liver, cauliflower, yeast, wheat, and beef. Our bodies do not manufacture folic acid, so it is essential that we take it in with what we eat. Some bacteria, on the other hand, do not require supplemental folic acid, as they are able to make their own.

  The folic acid molecule is fairly large and looks complicated:

  Folic acid with the middle portion from the p-aminobenzoic acid molecule outlined

  Consider just the part of its structure shown inside the outlined box in the structure above. This middle portion of the folic acid molecule is derived (in bacteria that make their own folic acid) from a smaller molecule, p-aminobenzoic acid. p-Aminobenzoic acid is thus an essential nutrient for these microorganisms.

  The chemical structures of p-aminobenzoic acid and sulfanilamide are remarkably similar in shape and size, and it is this similarity that accounts for the antimicrobial activity of sulfanilamide. The lengths (as indicated by the square brackets) of each of these molecules measured from the hydrogen of the NH2 group to the doubly bonded oxygen atom are within 3 percent of each other. As well they have almost the same width.

  The bacterial enzymes involved in synthesizing folic acid appear to be unable to distinguish between the molecules of p-aminobenzoic acid that they need and the look-alike sulfanilamide molecules. Bacteria will thus unsuccessfully attempt to use
sulfanilamide instead of p-aminobenzoic acid—and ultimately die because they are unable to make enough folic acid. We, relying on folic acid absorbed from our food, are not negatively affected by the action of sulfanilamide.

  Technically, sulfanilamide-based sulfa drugs are not true antibiotics. Antibiotics are properly defined as “substances of microbial origin that in very small amounts have antimicrobial activity.” Sulfanilamide is not derived from a living cell. It is man-made and is properly classified as an antimetabolite, a chemical that inhibits the growth of microbes. But the term antibiotic is now commonly used for all substances, natural or artificial, that kill bacteria.

  Although sulfa drugs were not the very first synthetic antibiotic—that honor belongs to Ehrlich’s syphilis-fighting molecule salvarsan—they were the first group of compounds that had widespread use in the fight against bacterial infection. Not only did they save the lives of hundreds of thousands of wounded soldiers and pneumonia victims, they were also responsible for an astounding drop in deaths of women in childbirth, because the streptococcus bacteria that cause puerperal or childbed fever also proved susceptible to sulfa drugs. More recently, however, the use of sulfa drugs has decreased worldwide, for a number of reasons: concern over their long-term side effects, the evolution of sulfanilamide-resistant bacteria, and the development of newer and more powerful antibiotics.

  PENICILLINS

  The earliest true antibiotics, from the penicillin family, are still in widespread use today. In 1877, Louis Pasteur was the first to demonstrate that one microorganism could be used to kill another. Pasteur showed that the growth of a strain of anthrax in urine could be prevented by the addition of some common bacteria. Subsequently Joseph Lister, having convinced the world of medicine of the value of phenol as an antiseptic, investigated the properties of molds, supposedly curing a persistent abscess in one of his patients with a compress soaked in a Penicillium-mold extract.

  Despite these positive results, further investigation of the curative properties of molds was sporadic until 1928, when a Scottish physician named Alexander Fleming, working at St. Mary’s Hospital Medical School of London University, discovered that a mold of the Penicillium family had contaminated cultures of the staphylococci bacteria he was studying. He noted that a colony of the mold became transparent and disintegrated (undergoing what is called lysis). Unlike others before him Fleming was intrigued enough to follow through with further experimentation. He assumed that some compound produced by the mold was responsible for the antibiotic effect on the staphylococcus bacteria, and his tests confirmed this. A filtered broth, made from cultured samples of what we now know was Penicillium notatum, proved remarkably effective in laboratory tests against staphylococci grown in glass dishes. Even if the mold extract was diluted eight hundred times, it was still active against the bacterial cells. Moreover mice, injected with the substance that Fleming was now calling penicillin, showed no toxic effects. Unlike phenol, penicillin was nonirritating and could be applied directly to infected tissues. It also seemed to be a more powerful bacterial inhibitor than phenol. It was active against many bacteria species, including those causing meningitis, gonorrhea, and streptococcal infections like strep throat.

  Although Fleming published his results in a medical journal, they aroused little interest. His penicillin broth was very dilute, and his attempts to isolate the active ingredient were not successful; we now know that penicillin is easily inactivated by many common laboratory chemicals, and by solvents and heat.

  Penicillin did not undergo clinical trials for more than a decade, during which time sulfanilamides became the major weapon against bacterial infections. In 1939 the success of sulfa drugs encouraged a group of chemists, microbiologists, and physicians at Oxford University to start working on a method to produce and isolate penicillin. The first clinical trial with crude penicillin was in 1941. Sadly, the results were much like the old punch line “The treatment was a success, but the patient died.” Intravenous penicillin treatment was given to one patient, a policeman suffering from both severe staphylococcal and streptococcal infections. After twenty-four hours an improvement was noted; five days later his fever was gone and his infection was clearing. But by then all of the penicillin available—about a teaspoon of the unrefined extract—had been used up. The man’s infection was still virulent. It expanded unchecked, and he soon died. A second patient also died. In a third trial, however, enough penicillin had been produced to completely eliminate a streptococcal infection in a fifteen-year-old boy. After that success penicillin cured staphylococcal blood poisoning in another child, and the Oxford group knew they had a winner. Penicillin proved active against a range of bacteria, and it had no harsh side effects, such as the kidney toxicity that had been reported with sulfanilamides. Later studies indicated that some penicillins inhibit the growth of streptococci at a dilution of one to fifty million, an amazingly small concentration.

  At this time the chemical structure of penicillin was not yet known, and so it was not possible to make it synthetically. Penicillin still had to be extracted from molds, and the production of large amounts was a challenge for microbiologists and bacteriologists rather than chemists. The U.S. Department of Agriculture laboratory in Peoria, Illinois, had expertise in growing microorganisms and became the center of a massive research program. By July 1943 American pharmaceutical companies were producing 800 million units of the new antibiotic. One year later the monthly production topped 130 billion units.

  It has been estimated that during World War II a thousand chemists in thirty-nine laboratories in the United States and in Britain worked on the problems associated with establishing the chemical structure of and finding a way to synthesize penicillin. Finally, in 1946, the structure was determined, although it was successfully synthesized only in 1957.

  The structure of penicillin may not be as large or look as complicated a molecule as others we have discussed, but for chemists it is a most unusual molecule in that it contains a four-membered ring, known in this case as the β-lactam ring.

  The structure of the penicillin G molecule. The arrow indicates the four-membered β-lactam ring.

  Molecules with four-membered rings do exist in nature, but they are not common. Chemists can make such compounds, but it can be quite difficult. The reason is that the angles in a four-membered ring—a square—are 90 degrees, while normally the preferred bond angles for single-bonded carbon and nitrogen atoms are near 109 degrees. For a double-bonded carbon atom, the preferred bond angle is around 120 degrees.

  The single-bonded carbon and nitrogen atoms are three-dimensionally arranged in space, while the carbon double-bonded to an oxygen atom is in the same plane.

  In organic compounds a four-membered ring is not flat; it buckles slightly, but even this cannot reduce what chemists call ring strain, an instability that results mainly from atoms being forced to have bond angles too different from the preferred bond angle. But it is precisely this instability of the four-membered ring that accounts for the antibiotic activity of penicillin molecules. Bacteria have cell walls and produce an enzyme that is essential for cell wall formation. In the presence of this enzyme, the β-lactam ring of the penicillin molecule splits open, relieving ring strain. In the process an OH group on the bacterial enzyme is acylated (the same type of reaction that converted salicylic acid into aspirin). In this acylation reaction penicillin attaches the ring-opened molecule to the bacterial enzyme. Note that the five-membered ring is still intact, but the four-membered ring has opened up.

  The penicillin molecule attaches to the bacterial enzyme in this acylation reaction.

  This acylation deactivates the cell wall-forming enzyme. Without the ability to build cell walls, the growth of new bacteria in an organism is inhibited. Animal cells have a cell membrane rather than a cell wall and so do not have the same wall-forming enzyme as these bacteria. We are therefore not affected by the acylation reaction with the penicillin molecule.

  The instability of the four
-membered β-lactam ring of penicillin is also the reason that penicillins, unlike sulfa drugs, need to be stored at low temperatures. Once the ring opens—a process accelerated by heat—the molecule is no longer an effective antibiotic. Bacteria themselves seem to have discovered the secret of ring opening. Penicillin-resistant strains have developed a further enzyme that breaks open the β-lactam ring of penicillin before it has a chance to deactivate the enzyme responsible for cell wall formation.

  The structure of the penicillin molecule shown below is that of penicillin G, first produced from mold in 1940 and still widely used. Many other penicillin molecules have been isolated from molds, and a number have been synthesized chemically from the naturally occurring versions of this antibiotic. The structures of different penicillins vary only in the part of the molecule circled below.

  Penicillin G. The variable part of the molecule is circled.

  Ampicillin, a synthetic penicillin effective against bacteria that are resistant to penicillin G, is only slightly different. It has an extra NH2 group attached.

  Ampicillin

  The side group in amoxicillin, today one of the most widely prescribed drugs in the United States, is very similar to ampicillin but with an extra OH. The side group can be very simple, as in penicillin O, or more complicated, as in cloxacillin.