VOL. 53 NO. 10, OCT. 2000 THE JOURNAL OF pp.1003 - 1007

LEADING ARTICLE

The and Sir Edward Abraham: Recollections about a Great Scientist and His Part in the Discovery of These Antibiotics

Jeremy M. T. Hamilton-Miller Department of Medical Microbiology, Royal Free and University College Medical School, Royal Free Campus, London NW32PF, U.K. (Received for publication August 3, 2000)

Personal Memories of Oxford and Edward Abraham ambition. There was thus no need to do any more work except to fulfil his statutory duties to give 12 lectures each I had the great good fortune-that I did not fully year and to live within 6 miles of the centre of Oxford. appreciate at the time-of working as a post-doctoral Whenonce I offered to lend him mybicycle, he remarked Fellow for 3y years (between 1967 and 1971) at The Sir that he always walked everywhere, otherwise he would get William Dunn School of Pathology at Oxford University, back too quickly. in the very laboratories where and the * A few years after I had left Oxford, Abraham did me cephalosporins had been developed. At that time Sir the great honour of asking meto examine one his DPhil Edward was just Dr EP Abraham, Reader in Chemical students. Whenthe oral had been satisfactorily concluded I Pathology. The Professor of Pathology and Head of the casually asked the candidate why he had worked for three School at that time was Henry Harris, following in the line years on a topic that appeared to have no practical use of such famous namesas Dreyer and Florey. whatever. The facial expressions of those present madeit Although it is of course impossible to imagine what it quite clear that it was totally out of order to expect an was like working on the cephalosporins between 1949 and Oxford man to have done anything that could be thought of 1959, the atmosphere would probably have been similar as "applied science". I was never asked to be an examiner to that when I worked there. It was all very calm and again. unhurried, almost casual. The day started late by modern When I joined Abraham's unit, the only senior standards but continued into the evening, although on permanent memberof staff was Dr GuyNewton, whose Saturdays one could go home at teatime. There was always title was Senior Research Officer. There were three PhD a week's holiday at Easter and Christmas (other Public students, working respectively on purifying /Mactamase II Holidays were ignored), and the School closed down for (the Zn-containing enzyme) from Bacillus cereusl\ three weeks in August. The Oxford University of those days isolation of the bacilysin2), and the biosynthesis was thus a very different place from what it is now, and of C by protoplasts3). Abrahamalso usually had changed very little over half a century. This can be played host to a visiting Fellow from overseas-guests illustrated by the following: during the 1960s included John Kasik (mycobacterial * Abrahamtold me how he had returned to Oxford in /J-lactamase4)), Lee Sabath (Pseudomonas aeruginosa 1939 having completed a three year post-doctoral j6-lactamase5)) and Vladimir Betina (the antibiotic Fellowship abroad. It happened that his former supervisor cyanein6)). Abraham and Newton themselves were saw him in the street and said "Haven't seen you for a working on, amongst other things, the biosynthesis of while; have you had a cold?" cephalosporin C, chemical modification of the antibiotic * Oneof the Lecturers at the DunnSchool, a manwith a actinonin and the molecular changes that occurred when the brilliant recent career, decided he had achieved his /3-lactam ring of cephalosporins was broken, either 1004 THE JOURNAL OF ANTIBIOTICS OCT. 2000 chemically or by the action of /Mactamases. I became that microbiologists find of particular value. Although involved in the latter project, and whenit was solved7'8) we respecting his opinion, I regard it as unfortunate; he and moved on to study the comparative immunological Newtonmust have mademanyfascinating observations properties of cephalosporins and penicillins9). that they could not explain at the time during the 10 years Abrahamwas a quiet, almost shy man, from whomI, of cephalosporin development, and the non-recording of also an introvert, often had difficulty in extracting the help these must represent a loss to science. Ona personal basis, and advice I, as a microbiologist, needed to take forward I regret being allowed to publish neither my finding that a what turned out to be a complex piece of chemistry. This certain isoxazolyl penicillin was extremely haemolytic nor was myownfault, as I was in awe of the great manand mymodification in 1968 of the Microtiter apparatus to reluctant to disturb him whenhe was shut away, as he often allow MICs to be determined (this technology was was, in his ownlittle study, which was the only private originally conceived and at that time was marketed solely room in the unit. Whenapproached, he always had an for immunological purposes). Abraham also strongly answer to myparticular problem of the moment.Onthe disapproved of putting the names of technical staff on other hand, GuyNewton was instantly accessible, as he did papers. This arose from an incident when, a technician not have a room of his own. He was always forthcoming having been cited as an author, someone from another and extremely helpful, going out of his way to give University inadvertently requested their services as a PhD assistance to anyone who asked. His sudden premature examiner. Abraham had found explaining the situation death during the Christmas holidays of 1969 was a great deeply embarrassing, and thus vowed never to let anything shock as well as a serious loss to science. similar happen again. What I found fascinating about Abraham, besides the Abrahamspoke nothing of his personal part in the vital part he played in the penicillin team and his leading development of penicillin. However, each summer he would role in the discovery of the cephalosporins, was his ability take us all out for an afternoon's punting trip, and he would to see so far ahead. The project he set meto work on very occasionally reminisce about those days. Two stories soon showed that the immediate product of aminolysis or especially stick in my mind, both concerning Norman enzymatic hydrolysis from a cephalosporin was short- Heatley. When Heatley had to fly to the USAduring the lived, the "cephalosporoate" decaying in a few minutes to war to brief his Americancollaborators on the culture of give fission products. Abraham immediately realized that Penicillium chrysogenum, he impregnated the inner seams this instability could be of fundamental interest to of his jacket with fungal spores, so that in the event of his immunologists, as the hapten created whena cephalosporin aeroplane being shot down there was a chance that the reacted with body protein would have only a short existence fungus could be recovered if his coat were found. Again, it before it changed into a different determinant. James is no secret that during the early days of the penicillin work Gowans, Professor of Immunology at the Dunn School, relations between Heatley and Chain broke down was fascinated by the possible implications of the existence completely. They refused even to be in the same laboratory of an unstable hapten, and would undoubtedly have worked unless it was absolutely necessary, and did not speak to on this problem had he not movedshortly afterwards to an each other at all. Whencommunication between them was Administrative post. Abraham also realized that the essential for the continuance of the project, Abraham had chemistry of the reaction we were studying would become to act as a go-between. much clearer if we applied the relatively new technique of Many feel that Heatley's contribution has not been nuclear magnetic resonance. This is howI came to observe emphasized sufficiently. For example, his ingenuity in the /3-lactamase mediated hydrolysis of a cephalosporin in inventing and developing the plate diffusion assay10) has an NMRspectrometer, all reagents being fully deuterated, had a permanent influence of the practice of medical in collaboration with Eva Richards, the wife of the microbiology. Heatley was an incredibly precise man; I inventor of the technique, Rex Richards. recall watching him during a very mundanejournal club Abraham's training as a chemist gave him an insight taking copious notes with a mappingpen in tiny, amazingly into the of micro-organisms that opened up neat writing on the inside of a used envelope that he had entirely new horizons. However, this precise discipline opened up. He perfected the mercury piston pipette, and also accounted for a refusal to allow the publication of once showed me a micropipette he had made from any phenomena that could not be fully explained, discarded scraps of glass and rubber tubing. Heannounced notwithstanding the fact that the biological literature is with great pride "this delivers precisely 1.32juY\ I did not full of observations without adequate explanations, ask him what he intended to use it for, but I dare say he VOL.53 NO. 10 THE JOURNAL OF ANTIBIOTICS 1005 found a purpose for it. C.acremoniumproved to be a remarkably prolific fungus. Abraham had signed the Official Secrets Act during the The first antibacterial compounds to be isolated and war when he was working on penicillin, and this had, identified, the cephalosporin P family, were hydrophobic amongother things, a lasting effect on the way he gave or and active against Gram-positive only. They proved showed papers to his colleagues. Long after he stopped to have a tetracyclic triterpene skeleton, related to fusidic handling secrets he used to slide documents over a desk, acid, that was discovered subsequently, and helvolic acid, usually face down, instead of handing them out openly. As that had been previously reported from the DunnSchool. he soon discovered that I found it quite easy to read script The chemistry and properties of this class of antibiotics that is upside down, he could not help being instinctively have been well summarised by Godtfredsen12). slightly suspicious when I was on the other side of a table Cephalosporin P could probably have accounted for the on which he had papers, however innocent their content activity against staphylococci and streptococci reported by was. He was highly amused once whena visitor from the Brotzu, but does not explain the action of the culture USSRat the height of the Cold War flew into a rage when filtrates against Gram-negative organisms. he caught sight of a document labelled Top Secret lying on Abraham and Newton next found a hydrophilic a bench when he was being shown round the Laboratory, antibiotic that was labile to penicillinase and gave a thinking this was a trap designed to discredit him. In fact, characteristic penillic acid when acidified. It was first the documentwas manyyears old, and it was there purely namedcephalosporin N, as it was active against Gram- by chance; all its contents had become commonknowledge. negative species, but this was changed to penicillin N when Another encounter that Abraham had with the USSR its chemical structure was elucidated13). The sidechain of caused him slightly less pleasure: on a lecture tour he was penicillin N, derived from a-aminoadipic acid, is charged at told that royalties were due to him for the manufacture of physiological pH values, that explains whyits properties cephalosporins in that country, but that the accumulated are different from those of other biosynthetic roubles could be spent only in the USSR. He remarked with {e.g. penicillins G, V, O, K and F), in which the sidechain is a wry smile that all he could have bought was a balalaika. not ionized. Penicillin Nwas difficult to purify, but in the early to mid 1950s was regarded as a potentially extremely valuable antibiotic due to its activity against Gram-negative Howthe Cephalosporins were Developed pathogens, many of which were resistant or had acquired resistance to the few antibacterial agents clinically available Brotzu decided in 1945 to test the sea water from at that time. Penicillin N was given an approved name around a sewage outflow pipe close to the University (adicillin) and a few clinical trials were done, but when of Cagliari in order to explain the apparent absence of cases ampicillin came on the market production was of typhoid (endemic in Sardinia at that time) arising discontinued. from bathing in the sea and eating shellfish harvested It was during the studies on penicillin Nthat the crucial in the vicinity. Among the organisms he isolated was experiment was carried out in which cephalosporin C was a filamentous fungus identified as Cephalosporium discovered, by a chance observation. Abraham and acremonium(now renamed Acremoniumchrysogenum) that Newtonneeded to knowthe precise molecular weight of when grown in his laboratory inhibited several important penicillin N; as the antibiotic was only available as a crude Gram-positive and Gram-negative pathogens. Brotzu used solution containing manyimpurities, their strategy was to crude culture filtrates from this fungus to treat localized convert penicillin Ninto its isomeric penillic acid, that was infections such as abscesses and systemic infections such as easy to isolate and purify, and determine the molecular typhoid and brucellosis. Having published his findings in weight of the latter. This involved acidification of an impure the one and only number of a journal specifically created antibiotic solution that had been concentrated, and for this purpose11\ he sought advice as to howmore could separation of various componentsin the resultant mixture be found out about his discovery, as he was aware that on an ion exchange column monitored by following the facilities to do this did not exist locally. Through the good ultraviolet absorption of the fractions. After the desired offices of a British Public Health doctor whomBrotzu had product, the penillic acid of penicillin N, had been eluted met after the war, a culture was sent to the Dunn School the columnwasfor somereason kept running, revealing a of Pathology at Oxford, where asked later peak14). This turned out to be cephalosporin C, that Abraham and Newton to investigate its antibiotic was present in a very small amount and that had too low an properties. intrinsic antibacterial activity to have been recognized by 1006 THE JOURNAL OF ANTIBIOTICS OCT. 2000 bioassay in the original culture filtrate. In due course as a crucial stage in this is the involvement of an Abrahamand Newtondiscovered that cephalosporin C "expandase" enzyme that converts penicillin N into differed markedly from penicillin N, being stable to deacetoxycephalosporin C, precisely analogous to the penicillinase and to acid, not forming a penillic acid and chemical reaction mentioned above. absorbing ultraviolet light. Its most exciting property at that time (the mid 1950s) was its consistent activity against S. aureus. The latter species, in the form of the "hospital Afterthoughts staph, phage type 80/81" was in those days widespread in hospitals all over the world and had become very difficult Abraham wrote in 197020): "some of the problems which (in some cases impossible) to treat. Florey encouraged had to be faced in the development of the cephalosporins Abraham's wish to switch emphasis from penicillin N to seemed at the time to be so formidable that one wondered cephalosporin C, which now became the focal point of whether their solution would be possible". It was entirely research. Further work was made easier by the isolation of due to his enthusiasm, flair and industry, together with a mutant of C.acremoniumthat produced larger amounts of Newton's meticulous experimental technique and attention cephalosporin C. In due course, after much painstaking to detail, that the project was carried through successfully. labour, Newton and Abrahamdeduced that cephalosporin In addition, as usual, in terms of luck, fortune favoured the C consisted of a fused dihydrothiazine//3-lactam nucleus brave. with an a-aminoadipoyl sidechain at position 7 and an It is nonetheless fascinating to speculate what might have acetoxymethyl group at C-315). The details of this structure happened had one of the numerous twists and turns gone were disputed at the time, doubts being expressed in another way. Would the cephalosporins ever have been particular that the conjugated double bond system present discovered had, for example, Brotzu not gone fishing for would have an absorption maximumat 260 nm. fungi in the Mediterranean, or had he not met Dr Blyth By now it was 1959, and workers at Beecham Research Brooke, or had the experiment to isolate penicillin N's Laboratories had created the semi-synthetic penicillins by penillic acid been terminated as soon as this compoundhad chemical modification of the penicillin nucleus 6APA16). been obtained? The latter was available either by removal of the Given the soil screening programmes that were being 6-sidechain of a natural penicillin using the enzyme actively pursued up until the end of the 1970s, and bearing penicillin acylase or by growing P. chrysogenum in the in mind that several genera of bacteria (e.g. Streptomyces absence of sidechain precursors. However, neither of these spp., Azotobacter spp., Lysobacter spp., Xanthomonasspp., routes could be used to makethe cephalosporin nucleus Flavobacterium spp.) as well as fungi produce cephem 7ACA, without which semisynthetic derivatives could not antibiotics (e.g. cephamycins and cephabacins as well as be made. Abrahamand Newton were thus compelled to cephalosporins), it seems quite likely that cephalosporins use chemical methods in order to perform the tricky would have been discovered eventually. However, even had operation of removing the 7-sidechain from cephalosporin this been the case, it is doubtful whether they would have C while maintaining the integrity of the nucleus. This was reached the pre-eminent position they occupy today, for successfully achieved, albeit in very small yield, by acid which we have to thank chiefly Sir Edward Abraham. hydrolysis; the resulting traces of 7ACAwere acylated on chromatography paper with phenylacetyl chloride to yield References the highly microbiologically active cephalosporin analogue (cephaloram) of penicillin G17). Shortly afterwards, workers 1) Kuwabara, S.: Purification and properties of two extracellular /3-lactamases from Bacillus cereus 569-H. at Lilly Research Laboratories used nitrosyl chloride to Biochem. J. 118: 457-465, 1970 remove the sidechain of cephalosporin C in 40%yield, 2) Walker, J. E. & Abraham, E. P.: Isolation ofbacilysin and this figure has been improved to 90% by later and a new aminoacid from culture filtrates of Bacillus developments. Finally, it was found possible to expand the subtilis. Biochem. J. 118: 557-561, 1970 5-membered thiazolidine ring in penicillins, via a 3) Fawcett, P. K.; P. B. Loder, M. J. Duncan, T. J. Beesley & E. P. Abraham: Formation and properties of sulphoxide, to the 6-membered dihydrothiazine ring, protoplasts from antibiotic-producing strains of leading to a cephalosporin directly without the need to Penicillium chrysogenum and Cephalosporium isolate 7ACA18). Although Abraham was not involved in acremonium. J. Gen. Microbiol. 79: 293-309, 1973 4) Kasik, J. E. & L. Peacham: Properties of /3-lactamases this particular work, the reaction fits in neatly with his produced by three species of mycobacteria. Biochem. J. continuing interest in the biosynthesis of cephalosporins19), 107: 675-682, 1968 VOL.53 NO. 10 THE JOURNAL OF ANTIBIOTICS 1007 Sabath, L. D.; M. Jago & E. P. Abraham: structure and some derivatives of cephalosporin N. Cephalosporinase and penicillinase activities of a /3- Biochem. J. 58: 103-111, 1954 lactamase from Pseudomonaspyocyanea. Biochem. J. 14) Newton, G. G. E & E. P. Abraham: Isolation of 96: 739-752, 1965 cephalosporin G, penicillin-like antibiotic containing Betina, V; J. Fuska, A. Kjaer, M. Kutkova, P. Nemec D-a-aminoadipic acid. Biochem. J. 62: 651-658, 1956 &R. H. Shapiro: Production of cyanein by Penicillium 15) Abraham, E. P. & G. G. E Newton: The structure of simplicissum. J. Antibiotics 19: 1 15-1 17, 1966 cephalosporin C. Biochem. J. 79: 377-393, 1961 Hamilton-Miller, J. M. T.; G. G. E Newton & E. P. 16) Rolinson, G. N.; E R. Batchelor, D. Butterworth, Abraham: Products of aminolysis and enzymic J. Cameron Wood, M. Cole, G. C. Eustace, M. V hydrolysis of the cephalosporins. Biochem. J. 1 16: 371- Hart, M. Richards & E. B. Chain: Formation of 384, 1970 6-aminopenicillanic acid from penicillin by enzymatic Hamilton-Miller, J. M. T; E. Richards & E. P. hydrolysis. Nature 187: 236-237, 1960 Abraham: Changes in proton magnetic resonance 17) Loder, B.; G. G. E Newton & E. P. Abraham: The spectra during aminolysis and enzymic hydrolysis of cephalosporin C nucleus (7-aminocephalosporanic acid) cephalosporins. Biochem. J. 1 16: 385-395, 1970 and some of its derivatives. Biochem. J. 79: 408-416, Hamilton-Miller, J. M. T. & E. P. Abraham: 1961 Specificities of haemagglutinating antibodies evoked 18) Morin, R. B.; B. G. Jackson, R. A. Muller, E. R. by members of the cephalosporin C family and Lavagnino, W. B. Scanlon & S. L. Andrews: benzylpenicillin. Biochem. J. 123: 183-190, 1971 Chemistry of cephalosporin antibiotics. II. Chemical Heatley, N. G.: Method for assay of penicillin. correlation of penicillin and cephalosporin antibiotics. J. Biochem. 1 38: 61-65, 1944 Amer. Chem. Soc. 85: 1896-1897, 1963 Brotzu, G.: Ricerche su di un nuovo antibiotico. Lav. 19) Perry, D.; E. P. Abraham & I E. Baldwin: Factors 1st. Ig. Cagliari, 1-ll, 1948 affecting the isopenicillin N synthetase reaction. Godtfredsen, W. O.: Fusidic acid and some related Biochem. J. 255: 345-351, 1988 antibiotics. Leo Laboratories, Copenhagen. 1 967 20) Abraham, E. P.: Reflections on the development of the Newton, G. G. E & E. P. Abraham: The degradation, cephalosporins. Giorn. Ital. Chimioter. 17: 4-13, 1970