A Time to Remember

by Alexander Todd

  I suppose the main reason for my decision to leave the main brunt of work on polynucleotide synthesis and sequence determination to others was that I was still much involved in coenzyme synthesis, and in the development and refinement of procedures for polyphosphorylation which were a prerequisite for it. Furthermore, I was engaged on structural studies on vitamin B12 and in addition to a number of other smaller efforts I was already getting deeply involved in the fascinating problems presented by the remarkable colouring matters present in the haemolymph of aphids. Even although I had for those days a very large research school, there were limitations to what I could tackle!

  Following our synthesis of adenosine triphosphate (ATP) our next major triumph in the coenzyme field was the synthesis of flavin-adenine-dinucleotide (FAD) published with G. W. Kenner and S. M. H. Christie in 1952. In the years that followed a variety of other compounds of this type were synthesised including inter alia cozymase (nicotinamide-adenine-dinucleotide) and uridine-diphosphate-glucose. It was, indeed, for my work on phosphorylation and nucleotide coenzymes that I received the Nobel Prize for chemistry in 1957.

  Ever since my early work on vitamin B1 in Edinburgh, I retained an interest in the B group of water-soluble vitamins, and did indeed carry out work on some of them in Manchester. One of the most intriguing features of the group was its association with the anaemias. The picture was very confused, and it was not until the 1940s that it became clear that, although such members of the B group as folic acid were involved in nutritional anaemias, the factor involved in pernicious anaemia - the 'external factor' present in liver extracts - was still unisolated. I had not myself taken much interest in pernicious anaemia, and had confined my interest largely to the B vitamins involved in nutritional macrocytic anaemias, but my attention had been drawn to the problem by H. D. Dakin, when I visited him at his home near New York on my way to Pasadena with my wife in 1938. Dakin had been interested in the problem ever since Minot and Murphy in 1926 had shown that whole liver would cure pernicious anaemia, and he had been trying to isolate the material responsible from liver extracts. He had, however, like other workers, made very slow progress indeed since the only way one could test the material was on human patients. Such a test was bound to be inaccurate, but what was (from Dakin's point of view) worse, was that clear cut cases of pernicious anaemia were none too common and clinicians, not unnaturally perhaps, were more interested in curing their patients than in testing Dakin's extracts. When I returned to England I remember discussing the matter with the research group at Glaxo Laboratories Ltd; one of their number, E. Lester Smith, was determined to go ahead on liver extract, and we encouraged him to do so. He slogged on despite every kind of discouragement encountered in the course of testing on human patients and eventually, in 1948, only a very short time after Folkers and his group at the Merck Laboratories in the United States, he did indeed isolate the anti-pernicious anaemia factor Vitamin B12. These two nearly simultaneous isolations of the vitamin were quite independent of one another; but it is remarkable that they should have been so close, when we know that the American group were able to use a microbiological test, while Lester Smith had to go all the way with the much more difficult clinical test procedure. Since I had been associated with the Glaxo isolation work throughout its course, it was perhaps not surprising that I should have been asked if I would undertake a chemical study of it, while Dorothy Hodgkin studied the vitamin by the X-ray method. This I agreed to do, and with my friend and colleague A. W. Johnson we started work. It proved extremely difficult; for one thing we had, during the first year or two of our studies, extremely small amounts of vitamin available to us, and, even more importantly, the molecule proved to be one of almost fantastic complexity. We were able to settle some of its features and learn something about the central part of the molecule by hydrolytic and oxidative studies, but our major contribution lay, perhaps, in the fact that some of our degradation products materially helped Dorothy Hodgkin in her X-ray studies, which in 1955 finally gave the complete vitamin structure. Subsequently, with V. M. Clark, I carried out quite a bit of work on methods which might be applicable to the synthesis of the vitamin, but dropped them, partly because a total synthesis using them would have absorbed a greater part of our research effort than we wished to devote to it, and partly because R. B. Woodward, who had also taken up the synthesis, appeared to me to have a method more likely to succeed (as indeed, with the cooperation of Albert Eschenmoser and his group at Zurich, it ultimately did). A third major topic of research extending over more than twenty years in Cambridge concerned the colouring matters present in the haemolymph of insects belonging to the family Aphididae. My reasons for becoming involved in work on aphid pigments are rather interesting. When I was in Oxford with Robinson I did some work on the colouring matters present in the mycelia of some plant pathogenic fungi of the Helminthosporium group. These colouring matters were derivatives of anthraquinone, and, out of curiosity, I went through the literature and listed all the anthraquinones known to occur in nature together with their source and the pattern of substitution in them. It appeared to me that they seemed to fall very roughly into two groups according to their nuclear substitution - those from higher plants on the one hand, and those from fungi on the other. There were, however, two odd ones obtained from insects - carminic acid from cochineal, and kermesic acid from the oak chermes - which seemed to resemble the fungal anthraquinones rather than those from higher plants. This might not seem very remarkable, but I recalled that these insects belonged to the family Coccididae whose members are known to contain symbiotic fungi located in special cells called mycetomes. Accordingly, I found myself wondering whether it was the insect or the symbiotic fungi that produced the anthraquinone pigments, and I decided I would look into this when I had some time and opportunity. While in Edinburgh I tried to pursue the matter further. It was, of course, necessary for me to obtain supplies of living cochineal insects since examination of the cochineal of commerce would teach me nothing. I soon found, however, that the authorities were not at all enthusiastic about my importing the insects, and the project went into cold storage until 1939. In the early summer of that year, I drove with my wife and some friends from Manchester to Lake Bala in north Wales for a day's outing and we picnicked by the lake hard by a sizeable stand of foxgloves. As I lay dozing after lunch looking up at the foxgloves, I noticed that one of them had a heavy infestation of black aphids on one of the flower heads; and, I thought, ' aphids are zoologically very close to the coccids - perhaps they too have anthraquinones'. So I took a few aphids and rubbed them between my fingers; sure enough, my fingers were stained. But, oddly, the stain was at first yellowish and then after a short time became red (which was the colour expected of an anthraquinone). So I cut the whole head off the infected foxglove, took it back to Manchester, and had a look at the aphids. It was soon clear that the colouring matter in them was not an anthraquinone, and I confirmed the fact that the coloured substance in the insects did undergo a curious change of colour from a kind of khaki to red within a very short time of its extraction. I also went to the entomological literature and found that the Aphididae like the Coccididae contained symbiotic fungi. I decided there and then, that I would leave the Coccididae alone and look at the Aphididae to satisfy my curiosity both as to the nature of their colouring matters and their true origin, i.e. from insect or fungus. But I had to wait until the early summer of 1940 for the next aphid season; by then the war had started in earnest, and I had to put the matter to one side. I resolved to take it up again when the war was over, and so indeed I did.

  Among the large group of workers from overseas flooding into my laboratories in Cambridge once the war was over, was a remarkably able Canadian, S. F. MacDonald. He had spent some time with Hans Fischer in Munich, and was a porphyrin-chlorophyll expert doing excellent work in that field. He was a tremendous believer in the use of a hand spectroscope in studying the reactions of coloured compounds, and was immediately fascinated by my
account of the aphid colours. A. W. Johnson too was strongly attracted, because I had already got him interested in some other aspects of insect chemistry. So the three of us got to work, and, with a growing band of research students - mainly from overseas - proceeded to unravel the complex puzzle of the aphid pigments. It soon became clear that we had, by so doing, moved into a very large field which absorbed a big effort in manpower for some twenty years. I do not propose to discuss the work in detail. Suffice to say that in general the living dark coloured aphids (red, brown and black) contain a yellowish protoaphin converted on the death of the insects by enzyme action to a yellow xanthoaphin; this latter is unstable and undergoes conversion in solution successively to an orange chrysoaphin and finally to a deep red erythroaphin. All of these compounds are complex quinones of a type not hitherto found in nature; they are not anthra-quinones. The green aphids - e.g. Macrosiphium rosae, the 'green fly' of cultivated roses - do not, contrary to popular assumption, contain any chlorophyll derivatives; they owe their colour to a green quinone related in structure to the aphins. It is of interest that the green fungus (Peziza aeruginosa) found on rotting wood also contains a green pigment (xylindein) not very dissimilar in type from that in the green aphids. Much of the later definitive work on the structure of these pigments was carried out in the 1960s, my chief colleague in that phase being D. A. Cameron, now Professor of Organic Chemistry in Melbourne who is still carrying on work in this field (although aphids are not a common pest in Australia, which suffers more from the depredations of coccids!). I never thought when I began work with aphids that it would turn out to be such a monumental undertaking, but it did give a great deal of interest, and my final satisfaction came just about twenty years after I began it, when my young colleague Jonathan Banks established that the colouring matters are indeed produced in the mycetomes of the insects, i.e. they are probably of fungal origin. It would be nice to check the situation in the cochineal insects, and in the lac insects cultivated commercially in India; my guess is that their colouring matters also originate in their fungal symbionts.

  The researches on the aphid pigments had their lighter side. If one wanted to get hold of the protoaphins, the insects had to be alive and undamaged; death or injury seemed to liberate an enzyme which at once set in train the series of changes leading to erythroaphins. In the case of Aphis fabae, the 'black fly' of cultivated beans (to get them we used to search for a badly infested field, and then pay the farmer to let us cut off the tops of infected plants) we used to place the aphid-infested bean-tops on horizontal shelves in dustbins laid on their side, with a piece of butter muslin stretched across the open end instead of the lid. We then placed a bright light in front of this peculiar piece of equipment; the insects, being phototropic, thereupon withdrew their probosces from the bean plants, walked on to the butter muslin and, hey presto! we had a supply of live undamaged insects. In the case of such insects as the willow aphid Tuberolachnus salignus, which lives on twigs and branches rather than on leaves, we had another technique. Aphids, although they can walk quite happily on paper, are completely unable to do so on cellophane; we therefore used to put sloping cellophane sheets under infested twigs then tapped the latter gently. This apparently annoyed the aphids who pulled out their probosces whereupon they fell on to the cellophane and were duly collected. My wife has vivid memories of the early summer of 1948 when we had a heavy infestation of the cherry aphid (Myzus cerasi) on a double row of ornamental cherry trees bordering the avenue outside our house. The infestation coincided with the first visit of an Australian cricket side to England after the war. As it happened the gang of research students whom I sent out to climb and 'delouse' the trees were Australians; I understand that they used to descend from their perch in the trees every half-hour or so, to have some tea and keep up with the Test match on the radio! For the aphid work we needed very large amounts of insects, and so used to collect them by the kilogram. In a poor aphid year we sometimes had a good deal of difficulty in getting particular species. I recall that in one year we put an advertisement in the local newspaper asking people to let us know if they came across aphid infestations, so that we could collect the insects. We only did it once; no-one gave us any information, but we had a flood of envelopes from helpful people enclosing a leaf with two or three aphids on it, and about twice as many crawling all over the outside of the envelope. I don't think the postal authorities thought much of the result either.

  6. The post-war years. Involvement in science policy and international activities

  As far as we in Cambridge were concerned, the war effectively ended with the collapse of Germany in May 1945. True, the struggle with Japan continued, but it was rather remote from us. Although air-raids from Germany had ceased, we were, until the end of the war in Europe, subject to the continued threat of flying bombs and the more dangerous rocket-propelled V-2s. With that menace removed, we seemed effectively at peace, although all or most of our wartime restrictions continued and life was certainly austere. In the autumn of 1945 I received an invitation from the Swiss Chemical Society to visit Switzerland in early December, and lecture in Zurich and Basle on my researches on Cannabis. This was my first trip abroad since the outbreak of war, and I was very excited about it. I reported at Northolt airport on a bitterly cold morning to fly in a Swissair DC-3 to Zurich. Fog delayed our departure for an hour or two, much to the pilot's concern - he was clearly afraid that if he didn't get to Zurich before dark he might not be able to land there. However, we finally took off, and when we arrived over Zurich it was already dark but clear and frosty. Looking down from the plane on a city blazing with lights and flashing advertisements, I could hardly believe it - so accustomed had we become to the gloom of the blackout in Britain. Once landed, the shock was even greater. I find it hard to express my feelings at seeing again well-lit streets and shops, with glittering snow all around - there were sweet stalls, and you could even buy bananas in the greengrocers' shops! I had almost forgotten what the world had been like before 1939! But it was a magnificent feeling — this was peace, and soon all Europe would be like this again (although, in fact, it was to take longer than I expected). It was a most successful visit; my lectures went well, and I was able to renew my contacts with Professors Reichstein, Ruzicka and Karrer. I also, of course, made touch with my old research colleagues from London and Manchester - Marguerite Steiger now running her family's pharmaceutical business in Zurich, and Hans Waldmann with Hoffmann La Roche in Basle. I also renewed acquaintance with two old Oxford friends - Emil Schlittler and Rudolf Morf; the latter I was to see much of in later years through our joint concern with the International Union of Pure and Applied Chemistry, of which he became Secretary General in 1955 in succession to Professor Delaby of Paris.