Another piece of work which led to no useful result (as far as I was concerned at least) was that which we carried out on blood anticoagulants. During my first year in Manchester I was asked by my friend C. H. Best of Toronto if I would have a look at heparin, which the Canadians were producing at their Connaught Laboratories. He sent over one of his technicians (Arthur Charles) with a modest supply of material so that we could do some work on it, but we had no time to do much with it before war broke out and Charles had to return to Canada. I would probably have paid no further attention to the problem, since I really was not very interested in carbohydrate chemistry, had it not been for the panic about spies and Nazi sympathisers which occurred following the German invasion of France. All German subjects, including many Jewish refugees, were suddenly rounded up and interned (quite arbitrarily as far as I could see). One victim of this was my valued colleague and friend Dr Anni Jacob. She was arrested, put for a time in Holloway Prison in London, and then moved to an internment camp at Port Erin, Isle of Man. This was a tragic waste of scientific talent at a time when this country needed all it could find, and was equally tragic from a human point of view, since Anni was one of those Germans who, although without any Jewish ancestry, nevertheless elected to leave her country rather than live there under Hitler. I had a long battle with the Home Office before I finally obtained her release, but, between times, in an effort to relieve the boredom of her stay at Port Erin, I arranged with the Director of the Marine Biological Station there that she could collect and process various types of seaweed, and, in collaboration with me in Manchester, look for possible blood anticoagulants in them (it was known that the Irish seaweed product carragheen had anticoagulant properties). Although we carried on some of this work for a short time after Anni was released, it really did not lead us anywhere and we abandoned it; but it probably helped her to retain her sanity during her internment. While we were still in Manchester Anni married Dr Juan Madinaveitia, who had been associated with me in Edinburgh, London and Manchester, and who was himself a refugee from the Franco regime in Spain. Madinaveitia subsequently joined I.C.I. (Pharmaceuticals) Ltd and he and Anni settled down happily in Cheshire and brought up a family.
Needless to say, I took part in the wartime Anglo-American cooperative research project on penicillin, but I was not involved in the early stages and our efforts beginning in, I think, 1943, were on a relatively modest scale. Looking back at it now, I find it rather amusing that, by showing that penicillin readily formed a sulphoxide, we did indeed establish that it was a true lactam; furthermore, our sulphoxide has, in recent years, sprung into prominence as a starting material for much synthetic work on beta-lactam antibiotics in general. I confess that I got little pleasure from our penicillin work, and that, I believe, for two reasons. In the first place I found the constant stream of research reports from all participants in the cooperative venture, amounting almost to a flood, very distracting, and, indeed, counterproductive in that they hampered the free development of my own ideas. Secondly -and this applied in variable degree to all our war work - I have always found it difficult to do good research unless the subject is one in which I have a strong personal interest. I think this latter point applies to many academic research scientists, and that is why they are usually not very efficient in industrial contract work. Indeed, my advice to an industrial firm with a research problem which it wishes to solve expeditiously, is to do it within the firm, taking external advice as appropriate but not to contract it out to a university. I know, of course, that in recent years several industrially oriented units for contract research have been set up in a number of universities; but these are quite different in outlook and staffing from normal university departments, and have had variable fortunes. I still think that the proper place for industrial research is in industry.
I have already explained how chance in the shape of George Barger introduced me to the vitamin field; from that introduction grew an interest in vitamins, and especially in the chemical reasons for their importance. These interests are at the heart of what most people would probably regard as my main contributions to research - the chemistry of vitamins, of nucleosides, nucleotides, coenzymes and nucleic acids. Such a view is entirely reasonable, since there can be little doubt that our nucleotide work and the establishment of the chemical structure of nucleic acids form the base on which molecular biology and modern genetics have developed in such spectacular fashion during the past quarter of a century. Yet, in addition, I have always had a deep interest in natural colouring matters - an interest triggered by my association with Robert Robinson in research on the beautiful red and blue colouring matters of flowers known by the generic name of anthocyanins. As a result, I have, during my career, done a good deal of work on natural colouring matters and especially on those remarkable pigments found in aphids, those well-known sucking insects which are the bane of many gardeners' lives; I shall recount something of that research later.
In Manchester we had laid the ground for our foray into the field of nucleotide coenzymes. We had developed new methods for nucleoside synthesis and for phosphorylation of nucleosides with dibenzyl phosphorochloridate, and had discovered a new method for phosphorylating amines using diesters of phosphorous acid and polyhalogen compounds. (This latter reaction was, in fact, accidentally discovered by F. R. Atherton when he tried to remove acid impurities from a solution of dibenzyl hydrogen phosphite in carbon tetrachloride by shaking it with ammonia; the whole mixture set to a semi-solid mass of dibenzyl phosphoramidate.) I need not elaborate in detail, but during the first few years in Cambridge we had effected the first of our coenzyme syntheses — that of adenosine triphosphate (ATP), the substance which is involved in phosphate transfer and acts as the necessary reservoir of energy for muscular activity in animals. We also not only settled the structure of the known natural nucleosides and nucleotides by synthesis, but established their stereochemical configuration as well.
Already in 1938 when I started work in this field I was of the opinion that nucleic acids might be involved in the transmission of hereditary characteristics as had been suggested by the earlier work of Griffith on pneumococcal transformation; Avery's demonstration in 1944 that deoxyribonucleic acid (DNA) was the transforming factor seemed to me to settle the issue. Curiously enough Avery's work - so beautiful and, to me, so convincing - did not convince everybody. In the summer of 1946 I attended a symposium on nucleic acids held in Cambridge by the Society of Experimental Biology to which I agreed to contribute a paper on 'The structure and synthesis of nucleotides'. At that symposium I remember a violent argument between E. Stedman, who stoutly maintained that histones and not nucleic acids were the carriers of hereditary characteristics, and a number of others and notably Caspersson who, like me (and with better evidence), was a proponent of nucleic acid. I think it was really at that meeting that I first met and talked with the main operators in the biology and biochemistry of nucleic acids, and heard from Astbury about his X-ray studies. My interest was aroused, and I began - again for the first time -seriously to consider the chemical structural problems presented by the two types of nucleic acid - ribonucleic (RNA) and deoxyribonucleic (DNA). The time was in any case propitious, because we were just on the point of completing our first ATP synthesis and we now knew sufficient about phosphates and their behaviour to make nucleic acids, chemically speaking, a bit less daunting than they were to most people at that time. So we began to think about the problem a bit, and to study the behaviour of simple nucleotides alongside our coenzyme work.
I was, of course, familiar with most of the chemical literature on nucleic acids and their component nucleotides. As a result of his work extending over many years, P. A. Levene had substantially clarified the structure of the simple nucleotides and nucleosides which can be obtained by hydrolysis, and deduced correctly that the nucleic acids were made up of nucleotides linked together in some way through phosphate residues. But he had, based on analytical methods we now know to have been inaccu
rate, reckoned that only two nucleic acids existed - one from plants (RNA) and one from animals (DNA) and that each was composed of four nucleotides present in equal amounts. More unfortunate still, he supported the idea that the nucleic acids might be simply tetranucleotides which formed colloidal aggregates in solution. From the moment I read his claims and views I found myself in total disagreement. For one thing, there was already some evidence to suggest molecular weights of 500 000 or more for DNA, and, in any case, its general properties suggested strongly that it was a macromolecular substance like protein or one of the polymeric materials studied by Staudinger, and even then appearing in commerce in the form of synthetic rubber and synthetic fibres. Nor could I accept the idea that we were dealing with polymerised tetranucleotide units; on the evidence available I had doubts about the constant composition of both types of nucleic acids and the claimed existence of only two acids. These views were, of course, fully confirmed during the years that followed, but belief in the so-called 'tetranucleotide hypothesis' was, in my view, largely responsible for the slowness with which biochemists and biologists came to realise the importance of the nucleic acids in hereditary transmission. I have sometimes wondered whether the ready acceptance of the tetranucleotide hypothesis by many biochemists was not, perhaps, due to their belief that proteins with their manifold properties would be found to be responsible for all life processes, and they accordingly felt no need to look any further!
I have referred to the daunting nature of chemical studies in the nucleotide/nucleic acid field. At the time of which I am writing, and, indeed, throughout virtually all the work on nucleic acid components - and even on the basic purines and pyrimidines let alone their phosphorylated derivatives - poor solubility in organic solvents, difficulty of separation and purification, and lack of proper melting points or other reliable criteria of purity, made life very difficult for the chemist, and led to a lot of confusion and, indeed, errors in the literature. In those days we had only the early forms of chromatography available, and, apart from ultraviolet spectroscopy, and in the late stages of our work some minor applications of the newly developing technique of infra-red spectroscopy, we had to rely on the traditional methods of the organic chemist developed many years before for compounds quite different in type and in physical properties. Even in our own work we were led by such deficiencies into errors in the identification of simple nucleotides, at one point, by accepting the wholly invalid 'proof of the structure of benzylidene-adenosine due to Gulland, and had to do quite a bit of work before we realised the true position. As we went along several observations began to loom large in our thoughts. For one thing, we were struck by the astonishing difference in the ease with which RNA underwent hydrolysis as compared with DNA and probably related to this the ease with which one could purify deoxyribonucleotides as compared with ribonucleotides. To get a really pure specimen of yeast adenylic acid (adenosine 3'-phosphate) was a very difficult task indeed, since recrystallisation seemed often to operate in the reverse direction; this should have suggested phosphate migration to us as a possible reason, but I am afraid it did not immediately do so. What was, of course, clear, was that the difference between the stability of the two nucleic acids lay in the sugar portion of the molecule, and this also suggested to me that, while the stable DNA was involved in hereditary transmission, RNA probably had some quite different function in nature, where its impermanence would be an advantage. As far as we were concerned the final breakthrough came in 1949 when Waldo Cohn at Oak Ridge, Tennessee, applied ion-exchange chromatography to alkaline hydrolysates of yeast ribonucleic acid, and isolated, not just the nucleoside 3'-phosphates hitherto regarded on the basis of studies by Levene and others as the sole products, but also the 2'-phosphates. In Cambridge my colleague D. M. Brown drew my attention to some earlier and virtually unnoticed work by Fono on the hydrolysis of glycerophosphates, and suddenly the whole jigsaw fell into place, and we could explain all the hitherto puzzling facts about RNA and understand fully the differences in chemical behaviour between RNA and DNA. Moreover, we were able to establish conclusively by synthetic studies the reality of the alkaline degradation of RNA, first to four cyclic nucleotides and thence to an equilibrium mixture of the 2'- and 3'-phosphates, which undergo interconversion in an acid medium, and we were able to explain the nature of ribonuclease action and its significance for RNA structure. Having done this, Dan Brown and I were able to put forward definitive structures for the two types of nucleic acid as unbranched 3:5-linked polynucleotides in 1951 at the 75th Anniversary Meeting of the American Chemical Society (our detailed paper did not actually appear in print in the Journal of the Chemical Society until January 1952).
It was now clear that both types of nucleic acids were unbranched linear 3': 5'-linked polynucleotides, and that individual members differed from one another in molecular size and in the sequence of nucleotides present in them. Accordingly, methods both for synthesis and for sequence determination would in due course become important. We did indeed carry out some work on stepwise degradation of polynucleotides by chemical means, but it quickly became clear that such methods would be tedious in the extreme, and probably inaccurate. It seemed to me that little real progress was likely to be made until enzymes could be found which would chop up polynucleotide chains in a manner analogous to the specific enzymes which were known to attack polypeptides, and which were being used with such success in the case of insulin and other proteins by F. Sanger. Very few nucleotidases of such a nature were known at this time, and it seemed to me wisest to leave the further development of sequence determination to men like Fred Sanger, or to one of the numerous young men who were going out from my laboratory to build up new research groups in the nucleotide field in many countries. On the side of synthesis A. M. Michelson and I showed that the synthesis of oligonucleotides was quite feasible by synthesising dithymidine-3':5'-dinucleotide. I confess that I have never been much attracted to the kind of repetitive procedures involved in synthesising either polynucleotides or polypeptides, so I left further developments in synthesis to my 'offspring' including such men as A. M. Michelson, F. Cramer, H. G. Khorana and C. B. Reese. The spectactular results which later emerged culminating in Khorana's synthesis of a gene are well known. There still remain serious problems to be solved, however, even in the oligonucleotide field; especially is this true of the ribonucleotides, where the presence of cis-vicinal hydroxyl groups in the ribose residue presents a difficult problem for the synthetic chemist.
In the course of our studies on methods for the phosphorylation of nucleosides (i.e. nucleotide synthesis) we developed and used as the method of choice what has come to be known as the 'triester procedure', i.e. one in which one uses an active diester of, for example, phosphorochloridic acid as phosphorylating agent and subsequently removes selectively one or two ester groups from the resulting triester to give either a di-or a monoester of phosphoric acid. Although demonstrated by us to give good results in synthesising a dinucleotide, it appeared for some years to be superseded by other quicker but less selective routes based on direct diester formation. I find it sometimes a little difficult not to say 'I told you so!' when I see the triester procedure now being belatedly adopted for oligonucleotide syntheses as the method of choice where selectivity as well as yield is of importance.
The double helical conformation of DNA was advanced by Watson and Crick about two years after our clarification of the chemical structure which made it possible. I and my colleagues played no part in the development of the Watson-Crick model, largely because our interests at the time were essentially chemical and we really gave little thought to the physical conformation of the polynucleotide molecules in nature. A secondary reason may have been the almost total lack of contact between physics and chemistry in Cambridge - a lack of contact which is all too common in universities. It is true that before Watson and Crick were allowed to publish their paper Sir Lawrence Bragg, who was head of the Cavendish Laboratory, insisted that D. M. Brown and I should
approve their model (which we did!). The reason for this insistence (which is mentioned in Watson's book The Double Helix but with no explanation) lay in the fact that only a year or two earlier Pauling had published the alpha-helical structure for a protein. Pauling sent copies of his manuscript both to Bragg and myself, and I well remember Bragg coming over to see me in the chemical laboratory (for the first time since my arrival in Cambridge) and asking me how Pauling could have chosen the alpha-helix from among three structures all equally possible on the basis of X-ray evidence, and all of which he (Bragg) had indicated in a paper with Perutz and Kendrew. He was quite shattered when I pointed out that any competent organic chemist, given the X-ray evidence, would unhesitatingly have chosen the alpha-helix. It was a direct consequence of this that he decided that no nucleic acid structure based on X-ray evidence would go out from his laboratory without it first being approved by me!
When I saw the Watson-Crick model that day in their laboratory, I at once recognised that, by a brilliant imaginative jump, they had not only solved the basic problem of a self-replicating molecule, but had thereby opened the way to a new world in genetics. Maybe it is a pity that the physicists and the chemists were not closer at that time, but, even if they had been, we might at best have enabled the physicists to make the imaginative jump a year or so earlier, but probably not much more. Arising from our synthetic studies on simple nucleotides, I had long since learned that the nucleosides were effectively flat, and their stereochemistry indicated that, linked together by phosphate residues, they must form some kind of helical structure. We also knew, from X-ray studies of some of our materials by W. Cochrane and his group, that nucleosides and, indeed, their parent pyrimidines and purines were strongly hydrogen bonded. I recall telling Astbury of these views as early as 1947. I knew, of course, that the DNA molecule must contain some kind of code if it were to transmit hereditary characteristics, but, save in a very desultory way, I never considered the matter very seriously. Thus, although well aware of Chargaff's analytical findings in 1950 and 1951, I never gave any serious thought to their possible significance as part of a physical arrangement of DNA which could provide the basis of the genetic code. All of which is just an illustration of the way in which scientists are very often blind to matters which happen to lie outside their own specific field of interest. A further striking example of this last point is to be found in our work on organic phosphates, a facet of our nucleotide coenzyme studies. One of the problems we had to face quite early in our work aimed at coenzymes, most of which were unsymmetrical pyro- or triphosphates, was that the initial phosphorylation of a nucleoside by our normal method (using dibenzyl phosphorochloridate) gave rise to a triester, from which one esterifying group had to be selectively removed under very mild conditions which would not damage other parts of the molecule. One of our most successful devices for this purpose was to make use of (a) the electrophilic character of the CH2-grouping in the benzyl residue and (b) the fact that diesters of phosphoric acid are strong acids, i.e. they have very stable anions. Thus, when a triester of phosphoric acid containing a benzyl (or, for that matter, an allyl) group is treated with a nucleophile such as a tertiary base, or an anion such as chloride or iodide, the nucleophile attaches itself to the benzyl or allyl group liberating the anion of a diester of phosphoric acid. The more powerful the nucleophile and the stronger the diester of phosphoric acid produced in the reaction the better it goes. Now, of course, ethylenic compounds are nucleophilic, but rather weakly so, and accordingly were not of any value to us in the nucleotide work. But, around 1952, my colleague, F. R. Atherton (now with Roche Products Ltd and one of the world's experts on organic phosphates) decided to explore their use in this reaction. In the course of his work he found that geranyl diethyl phosphate was quite stable, but geranyl diphenyl phosphate was unstable and underwent cyclisation to give a mixture of cyclic terpenes, diphenyl phosphoric acid (a very strong acid) being expelled. What had happened was, of course, that the isolated double bond in the geranyl residue was sufficiently nucleophilic to attack the allylic carbon intramolecularly. This was quite interesting, and was in accord with expectations. What we failed to realise was that he had, in fact, discovered the way in which nature makes carbon-carbon bonds! Our interest lay in the behaviour of the phosphate - not the geranyl residue. During the 1950s there was an increasing interest in the mechanism by which nature synthesises compounds like terpenoids and steroids containing recurring 'isoprene units' in their carbon skeleton, starting from acetate. The discovery of mevalonic acid as an intermediate by Karl Folkers and his group in 1956 gave a big fillip to research in this area, and several of my friends -J. W. Cornforth and G. Popjak in England, Konrad Bloch in the United States and Feodor Lynen in Germany - were deeply involved. I paid no more than passing attention to this field, being absorbed in nucleotide coenzyme studies as well as work on vitamin B12 and aphid colouring matters. In August 1958, however, I went on holiday to Lugano with my family and one sunny morning, having just swum in the lake, I was sitting on the hotel terrace with my wife having a Campari when a somewhat decrepit car drew up close by and much to our astonishment the Bloch family emerged from it. Greetings having been exchanged, they joined us on the terrace for a gossip. While thus engaged Konrad said he thought it might interest me to know that there appeared to be a phosphate group in the precursor of the terpenes, which was produced in nature from mevalonic acid. I said to Konrad 'I'm not surprised, but I would bet that the intermediate will be a pyrophosphate' and left the matter at that. It was only later that I remembered Atherton's work, and realised that it really held the key to the problem. Subsequently, of course, the intermediate was identified as iso-pentenyl pyrophosphate and this led to the beautiful work on terpenoid and steroid biosynthesis carried out by Bloch, Cornforth and Popjak and by Lynen.
A Time to Remember Page 10