Early discoveries in the penicillin series

TIBS16-MAY1991

REFLECTIONS onBIOCHEMISTRY IN THE TEN YEARS that followed Alexander Fleming's historic obsero ratio# of the antibacterial properties of a culture of PeniclTlium notatum in 1928, only a few sporadic attempts were made to isolate the active component. Penicillin was shown to be an acidic substance that could be extracted into organic solvents only at low pH, but its instability to acid, alkali and heat hampered development of purification techniques. Solid material of reasonable purity was obtained only when Ernst Chain, working in Howard Florey's laboratory at Oxford, utilized the relatively new techniques of column chromatography and freeze drying. The curative effect of penicillin against bacterial infection in mice was then demonstrated. By 1941 an expanded Oxford team had, with great difficulty, prepared sufficient material to treat a few human patients z. The immense potential of the new antibiotic was then recognized, but wartime Britain could not provide the resources for large-scale production. The problem was solved by Anglo-American collaboration on a massive scale, but the results were not published until after the war. Two significant advances were initiated at the Northern Regional Laboratories, Peoria, lllinois3: (I) selection of a strain of Penicillium chrysogenum led to greatly increased titres and (2) replacement of surface culture of the mould by deep fermentation permitted the use of largecapacity fermenters. Between 1942 and 1945 the efforts of many chemists 4 in both Britain and the USA, with a vital contribution from Dorothy Hodgkin and other X-ray crystallographers, established the existence of five penicillins (Fig. I, structure I) in which different acyl side-chains (RCH2CO) were attached to a common bi-cyc!ic nucleus. In the antibiotic prepared in Britain, 'R' was mainly an alkyl or alkenyl group of four to six carbon atoms, whereas in the American product, derived from fermentation on corn

It is now 50 years since the therapeutic potential of penicillin was first demonstrated. This first antibiotic and the series of compounds derived from it have been of immense importance in modern medicine. This article descdbes the early search for more potent penicillin derivatives, culminating with the discovery of an intermediate of penicillin biosynthesis, 6-p-amino penicillanic acid (6-APA). A companion article in next month's TIBS will chart the subsequent exploitation of 6-APA and the preparation of a range of clinically important semi-synthetic penicillins. steep liquor, the main component was benzylpenicillin [I; R=Ph(phenyl)]. It was then found that addition of phenylacetic acid or certain of its derivatives to the nutrient medium greatly increased the yield of benzylpenicillin. Radioactive tracer studies established that the PhCH2CO (where Ph=phenyl) group was incorporated intact into the side chain. The five original penicillins showed broadly similar antibacterial properties, so it was largely on the basis of its greater suitability for production in bulk that benzylpenicillin (penicillin G) became the standard product for clinical use.

The search for diversity The success of phenylacetic acid in stimulating the production of benzylpenicillin, with near total suppression of the other 'natural' peni-

J. H. C. Nayler's addressfor correspondence is 31 SwanMill Gardens,Dorking,Surrey RH4 1PN,UK. © 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00

cillins, prompted attempts to prepare novel penicillins by supplementing the fermentation medium with appropriate 'precursors'. Many mono-substituted acetic acids were indeed accepted by the mould, and patents filed in the late 1940s record more than a hundred such 'biosynthetic' penicillins, of which about 20 were well characterized s. None was superior to benzyipenicillin in antibacterial activity, but one of them, phenoxymethylpenicillin (I, R=PhO), was later reinvestigated at the Biochemie laboratories in Austria and shown to be more suitable than benzylpenicillin for administration by mouth, on account of much greater resistance to degradation by stomach acid 6. This compound (penicillin V) thus became the second penicillin to achieve extensive clinical usage, specifically by oral administration.

(I)

X = RCH2CO X = H2NCH(CH2)3C'O

I

co2H CO2H

(3)

X = H2N - - ~

(4)

X=:H

(5)

X ="PhCO

CI-12CO

Figure 1 Structures of some early penicillins and 6-APA. 195

TIBS 16-MAY1991

Beecham Research Laboratories at their research station at Brockham Park, Surrey, which had been formally opened the previous year, curiously enough, by Sir Alexander Fleming. Beecham were well established in overthe-counter remedies and toiletries, but the Brockham laboratories rewesented a new venture into prescription medicines. In 1956 the decision was made to enter the antibiotic field and, more specifically, on the advice of Ernst Chain who was engaged as a co~ultant, to attempt to develop new penicillins 9. George Rolinson was appointed to head a new Microbiology Department while Peter Doyle, head of the Chemistry Department, asked me to take charge of the chemical aspects of the new project. Chain proposed that p-aminobenzylpenicillin (3) be prepared by fermentation using p-aminophenylacetic acid The discoverers of 6.APA(from left to right): Peter Doyle, George Rolinson, Ralph Batchelor as a side-chain precursor, and then and John Naylor. modified by appropriate chemical reactions at the amino group in the same The relative stability of penicillin V at (cephalosporin P) was a steroid of little way that Newton and Abrahams had low pH can be attributed to the elec- interest, while another (cephalosporin modified penicillin N (2). Pending comtron-withdrawing property of the phen- C), when its structure was elucidated pletion of a microbiological pilot plant oxy group, which reduces the tendency some years later, became the progeni- at Brockham, George Rolinson and a of the side-chain carbonyl oxygen to tor of a second important series of young biochemist, Ralph Batchelor, attack the ~-lactam ring and initiate ~-lactam-containing antibiotics, the were seconded to Chain's department intramolecular rearrangement. The same cephalosporins. The third (:originally at the Istituto Superiore di Sanita in electronic effect was utilized by John called cephalosporin N) was found to Rome, which possessed a small fermenSheehan at Massachusetts Institute of be a penicillin (2) with an unusual side tation plant. There they prepared a Technology (MIT) when he chose peni- chain derived from aminoadipic acid, so quantity of p-aminobenzylpenicillin and cillin V as the target of the first rational it was renamed penicillin N. It was less sent it to Brockham where I and total synthesis of a penicillin7. The final active than penicillin G against Gram- another chemist, Harry Smith, used it to step in the synthesis was the closure of positive bacteria but more active prepare some 30 new p-substituted the []-lactam ring by means of a carbodi- against certain Gram-negative organ- benzylpenicillins. None of them imide, a reagent only recently introduced isms, especially strains of Salmonella. showed particularly interesting antibacinto organic chemistry for the for- Indeed, an American group that had iso- terial activity. mation of amide bonds under mild con- lated penicillin N independently under The work in Rome involved numerous ditions. Total synthesis, whether by this the name of synnematin B, used it in a trials to establish preferred fermenroute or by others developed later limited clinical trial against typhoid in tation conditions. Two assay prowould, in principle, permit the prepar- Mexican children. Penicillin N was cedures were used to determine the conation of penicillins with many kinds of never developed commercially, but it centration of penicillin produced, both side chain. Hence it should be much pro,Aded the first evidence that the in the presence of p-aminophenylacetic more versatile than precursor-assisted antibacterial spectrum of penicillins acid and in control experiments without fermentation, which is restricted to could be influenced significantly and in a side-chain precursor. The latter were derivatives of monosubstituted acetic a useful manner by the chemical nature expected to produce only small quanacids. However, total synthesis is econ- of the side chain. It was also interesting tities of several 'natural' penicillins (/), omically unattractive and, in practice, in that the primary amino group offered but it was with them that Rolinson and has resulted in virtually no additions to a convenient 'handle' for chemical Batchelor observed anomalous results. the range of known penicillins. For the modification under mild conditions. The strained, four-member~d ~lactam latter purpose, it was rapidly super- The Oxford workers s used it to prepare ring of penicillin behaves as an actiseded by the much cheaper and more several new penicillins by treatment vated derivative of a carboxylic acid convenient semi-synthetic approach to with acylating agents, isocyanates, etC., and reacts with hydroxylamine to produce be described later. in neutral aqueous solution. a hydroxamic acid, which can be estiMeanwhile, a sixth 'natural' penicillin mated colorimetrically by its reaction was discovered at Oxford by Abraham Beechamjoins the hunt with ferric ions. Application of this chemiand Newton, while investigating a My involvement with penicillins cal assay to the precursor-free brews trio of antibiotics from a strain of began in 1956. In 1948, as a newly grad- regularly gave higher results than microCephalosporium. One component uated organic chemist, I had joined biological plate assays. 40 ~..,6

TIBS16- MAY1991 UPOn returning to England early in 1957, Rolinson and Batchelor mentioned the anomaly to Peter Doyle and myself. The high chemical assays suggested to both of us that the precursorfree brews contained some component that was not an antibacterially active penicillin, but which nevertheless could react with hydroxylamine to produce a hydroxamic acid. This led us to speculate that it might be some hitherto unrecognized ~-lactam-containing intermediate on the biosynthetic pathway to penicillin, and Peter Doyle proposed structure (4) as a likely candidate, i.e. penicillin without an acyl side-chain.

S PA,

pa Ch

The systematic name for structure (4) is 6-[$.amino-penicillanic acid, conveniently contracted to 6-APA. Doyle and I immediately recognized that if, in the absence of a side-chain precursor, the mould indeed excreted 6-APA it should be possible to convert the latter into an active penicillin by simple chemical acylation of the amino group. We were, of course, fully familiar with appropriate procedures, because we were already engaged in similar acylations of p.aminobenzylpenicillin (3). in this way, we hoped not only to detect the suspected 6-APA, but also to prepare from it a greatly extended range of new penicillins. Chemical acylation would be capable of introducing any kind of side chain and not just the monosubstituted acetic acids, which were all the enzyme in the mould appeared to accept. In May 1957, filtered liquor from a Penicillium chrysogenum fermentation, carded out in the absence of an added side-chain precursor, was treated at room temperature with phenylacetyl chloride in the presence of sodium bicarbonate, whereupon a marked increase in the potency of the solution was observed. The effect was seen more clearly when the natural penicillins were removed by solvent extraction at low pH before carrying out the phenylacetylation. Under these conditions, the active material produced appeared to be a single antibiotic that was sensitive to penicillinase and was indistinguishable by paper chromatography from benzylpenicillin (I, R=Ph). Use of phenoxyacetyl chloride instead of phenylacetyl chloride gave a different antibiotic that was chromatographically indistinguishable from penicillin V (1, R=PhO) and had :ne characteristic stability of the latter in acid.

However, when the fermentation liquor. was treated with penicillinase before acylation, there was no antibacterial activity. All the above reaction-s were carried out with very dilute and crude solutions, with no reaction products being isolated. In these circumstances the technique of paper strip chromatography proved invaluable. Small quantities of solution were applied at one end of a paper strip and, alter development in a suitable solvent system, the strip was laid on a seeded agar plate. On incubation, visible zones of inhibition of bacterial growth appeared in the agar in characteristic positions. The supposed 6-APA gave no such zone if untreated, but could be visualized if the strip was sprayed with phenylacetyl chloride before 'plating out'. By June 1957 we were confident that we had indeed detected 6-APA.Over the next few weeks both dilute and partially concentrated brews were acylated to give various semi-synthetic penicillins, including types such as phenylpenicillin (5), which could not have been prepared by earlier methods. Partial purification was achieved by solvent extraction at low pH, but the products remained very crude and only a rather rudimentary screen against a limited range of bacteria was considered practicable. Clearly the next task was to isolate 6-APAin pure form. Unlike most penicillins, 6-APA is a zwitterion, so the normal procedure of solvent extraction at low pH was not applicable. After trying numerous methods, isolation was achieved by initial concentration of a clarified broth followed by adsorption and elution from a suitable ion-exchange resin. If desired, further purification could be effected by chromatography on cellulose. A final

concentration, followed by acidification to the point of minimum solubility (pH 4.3) gave crystals of pure 6-APA. Elemental analysis and the classical methods of chemical derivatization and degradation finally confirmed structure (4) early in 1958. On 2 August 1957 Beecham filed a British Provisional Patent Specification to protect our discovery. When completed in July 1958 the patent contained a description of pure 6-APA and the preparation-from it of 23 reasonably pure penicillins. Nine of these were novel m'bstances and details of their anti-bacterial activity were given. Corresponding patents were filed in all major countries, and before the end of 1958 the unexamined patent had been published in Belgium. A short paper ~° describing the detection of 6-APA, the properties of the pure isolated substance, and the chemical evidence for its structure was published on 24 January 1959.

Ilefemmces 1 Reming, A. (1929) Brit. J. Exp. Path. 10, 226-236 2 Abraham, E. P., Chain, E., Retcher,C. M., Gardner,A. D., Heatley, N. G., Jennings, M. A. and Rorey, H. W. (1941) Lancet ii, 177-188 3 Moyer,A. J. and Coghill, R. D. (1946) J. Bact. 51, 57-78, 79-93 4 Clarke, H. T., Johnson,J. R. and Robinson, R. (eds) (1949) The Chemistry of Penicillin, Princeton UniversityPress 5 Behrens, O. K., Corse, J., Jones, R. G,, Kleidener, E. C., Soper, Q. F., Van Abeele, F. R. and Whitehead,C. W. (1948) J. BioL Chem. 175, 793-8O9 6 Brandl, E. and Margreiter, H. (1954) ()st. Chem. Ztg. 55, 11-21 7 Sheehan,J. C. and Henery-Logan,K. R. (1957) J. Amer. Chem. Soc: 79,1262-1263 8 Newton, G. G. R and Abraham, E. R (1954) Biochem. J. 58, 103-111 9 Lazell, H. G. (1975) From Pills to Penicillin: The Beecham Story, Heinemann 10 Batchelor, R R., Doyle, F. R, Nayler,J. H. C. and Rolinson, G. N. (1959) Nature, 183, 257-258

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