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Sterol Biosynthesis: The Early Days
Biochemical and Biophysical Research Communications 292, 1129 –1138 (2002) doi:10.1006/bbrc.2001.2006, available online at http://www.idealibrary.com on
Sterol Biosynthesis: The Early Days John Warcup Cornforth 1 School of Chemistry, Physics and Environmental Science, University of Sussex, Falmer, Brighton BN1 9QJ, United Kingdom
Having chosen a title for this article, I need to define its meaning. The study of sterol biosynthesis continues to this day, but what were the early days? Arbitrarily, I have chosen to draw a line early in the 1960s. By then, the work that won Konrad Bloch and Feodor Lynen a joint Nobel Prize had been done: the pathway to the sterols was plain for all to see. A few gaps, a few unsuspected intermediates, and many unforeseen subtleties remained to be detected and explored, but I assign this work to a later period. Few scientific legends link organic chemistry and biochemistry so intimately as the story of sterol biosynthesis. The linkage originated from an understandable preference of chemists to work with distillable liquids and crystalline solids. Crystalline cholesterol has been known for two centuries, crystalline cholic acid for a century and a half; so they escaped Emil Fischer’s put-down: “Tierchemie ist Schmierchemie” (animal chemistry is the chemistry of smears). But they are both fairly large and complex molecules, and one has to admire the tenacity of the early chemists who tackled them. With few methods of separation, with tools that always destroyed what they examined (elementary analysis, chemical degradation) and with no guides except chemical identification of functional groups and empirical interpretation of sequential reactions, they arrived step by painful step at structures that were very nearly right. That cholesterol is C 27H 46O, that its functional groups are one hydroxyl and one double bond and therefore that it has four carbocyclic rings, that the hydroxyl and the double bond are in different but adjacent rings, that the ring structure in cholesterol and cholic acid is the same, that cholesterol has a branched side-chain of eight carbons shortened in cholic acid to a chain of five carbons terminating in the carboxyl group . . . all this and much more was won by the same laborious procedures extended over the span of a human life. The work won Nobel Prizes in chemistry for Heinrich Wieland in 1927 and for Adolf Windaus in 1928 for their studies on bile acids and sterols—and tangentially in 1923 for Fritz Pregl. From a long degradation of a bile acid, he had 1
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emerged with insufficient material for a conventional analysis. He preferred to invent microanalysis rather than to face the labour of repeating the sequence. These pioneering efforts had produced by 1928 a partial structure 1 for cholesterol and a similar structure for bile acids; but the conclusion, though justly honoured, was a little wide of the goal. An empirical rule, correlating the size of a ring with a property of the dicarboxylic acid obtained by oxidative ring-opening, had been applied without strict logic. It was truly prophetic that the clue leading to revision came, in 1932, from a physical method: the emerging technique of X-ray crystallography. Bernal’s evidence that the yeast sterol ergosterol had a long thin molecule, incompatible with the chunky structure 1, forced a reinterpretation and led very quickly to proposals of a structure (2, minus most of the stereochemical detail) now repeatedly confirmed by physical and by chemical evidence as the correct one. Good accounts of the structural problems and their solution are given by Lettre´ and Inhoffen (1) and by Fieser and Fieser (2). The early 1930s were a seminal time for biochemistry. In particular, isolation procedures coupled with quantitative bioassay had brought dramatic advances in the fields of vitamins and hormones, where measurements of biological response were not too difficult. As evidence mounted that the sex hormones and the antirachitic Vitamin D were steroids, the chemical fraternity promoted cholesterol from the status of a fascinating chemical puzzle to the roles of a raw material for chemical manipulation and an objective for chemical synthesis. And to biochemists, steroids became an important problem of biosynthesis. The known facts were few, and outnumbered by the speculations. Feeding studies had shown that laboratory animals, although they can absorb cholesterol from food, must also be able to synthesize it, and do not make it from the slightly different sterols of food plants. And there was one tantalizing finding from 1926: the acyclic C 30H 50 hydrocarbon squalene 3, abundant in shark liver oils, increased the cholesterol content of rats that fed on it. This experiment (3) was made before the structure of either compound was fully known, but it had been suggested (4) by perceived structural similarities, and in 1934 with knowledge of both structures Robert Robinson (5) proposed a
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squalene-to-cholesterol conversion that was right in principle, though wrong in detail (and characteristically, he had published a rudimentary form of this speculation two years earlier (6), before he could be sure of either structure!). Happily the arising need to explore the pathway of sterol biosynthesis coincided with the advent of compounds enriched in the naturally occurring heavy isotopes of hydrogen, carbon and oxygen. To label a hypothetical precursor of, e.g., cholesterol, to administer it to a living system, and to measure the proportion of isotope incorporated in sterol, was now possible and not so difficult as to discourage investigation. Around that time, Konrad Bloch and Feodor Lynen were studying in Mu¨ nchen for their first degrees. Lynen was to stay in Mu¨ nchen for the rest of his career (he married Heinrich Wieland’s daughter) but Bloch went first to Switzerland and then to Columbia University, New York, whither Rudolf Schoenheimer had migrated a little earlier. The Nazis enriched the science of many countries while damaging their own. But the first contribution of tracer methods to the problem of steroid biosynthesis still came from Mu¨ nchen. Robert Sonderhoff, a pupil of Wieland, used trideuterioacetic acid CD 3CO 2H to test Wieland’s theory that oxidation in vivo is often dehydrogenation, and what organism but brewer’s yeast would he use at that place and time? He and Heinz Thomas (7) incubated yeast aerobically with the labelled acetate and measured deuterium in the succinic and citric acids that were formed. They also looked, almost incidentally, at the total fat and found it quite rich in deuterium; but a surprise came when they isolated the unsaponifiable fraction, mostly ergosterol, of the fat. This weighed around one quarter of the total fat but contained more than half the deuterium: it was by far the most highly labelled component of the yeast. They drew the correct conclusion that acetate is di-
rectly involved in sterol biosynthesis (and that sterols are not constructed from fatty acids, as Windaus had once speculated). Deuterium content in those days was determined by burning the sample and measuring the density of the combustion water by methods as sensitive as the quantity available would allow. Measurement by stopwatch of the time taken by a standardsized drop to fall between two marks on a vertical column filled with 2-fluorotoluene became a popular technique. At Columbia Schoenheimer was pursuing the ground-breaking studies, based on feeding experimental animals with heavy water, so classically summed up in a little book (alas, his last) The dynamic state of body constituents (8). Bloch was much attracted by this work and he lost no time in joining Schoenheimer’s group after gaining his doctorate. With David Rittenberg he published a sequence of papers establishing deuterioacetate as a primary source of deuteriocholesterol in mice and rats. The first primitive degradation of a labelled cholesterol, a pyrolysis of cholesteryl chloride that Mauthner (9) had shown to separate the nucleus from the side chain, proved that label was present in both fragments. Bloch continued this work after his move to Chicago in 1946. The discovery of a mutant of Neurospora crassa that required acetate for growth permitted an experiment (10) with [ 13C]acetate indicating that all carbon in the sterol (ergosterol) was derived from acetate. But by this time, the 1940 discovery that slow neutrons will convert 14N nuclei into the long-lived radioactive 14C was beginning to provide commercial quantities of radioactive carbon. The great advantage of this material for studies of biosynthesis was the sensitivity with which it could be measured: usually, as barium carbonate spread on a disk, but sometimes as carbon dioxide in a proportional gas counter. As a weak
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beta-emitter it was also relatively safe to handle, so that elaborate chemistry could be used to prepare labelled substrates and to degrade labelled products. Best of all, long degradative sequences could be contemplated, since the inevitable attrition of material at each step could be countered by occasional controlled dilution with non-radioactive material. The first example in this genre was Bloch’s use of 14C-labelled cholesterol, synthesized in rat liver from acetates labelled in the methyl and in the carboxyl group, to examine the distribution of isotope in the side chain of the sterol (11). Windaus and Wieland had already provided, in their investigations of structure, the methods of stepwise degradation that allowed each of the eight carbons in the side chain (except for the then indistinguishable pair of methyl groups at one end) to be separated from the others and examined for radioactivity. The results were illuminating. When carboxyllabelled acetate was the precursor, only three of the eight side-chain carbons were radioactive. These radioactivities were equal. With methyl-labelled acetate, these three carbons were (almost) inactive and the remaining five carbons were all radioactive. This supplemented earlier work (12) in which the Mauthner pyrolysis had been used to analyse cholesterol generated from acetic acid labelled positionally with 13C and with 14C. Calculations of the relative amounts of each isotope in the derived cholesterol had indicated that fifteen of its carbons were derived from the methyl group and twelve from the carboxyl. The clear presumption was that each and every carbon in cholesterol could be designated m or c according to its origin from acetate methyl or carboxyl respectively. The resulting pattern in the side chain was as shown in 4.
I can remember reading these papers with delight. Both George Popja´ k and I had joined the National Institute for Medical Research late in 1946. I continued to collaborate with Robert Robinson at Oxford on the total chemical synthesis (completed early in 1951) of the sterol nucleus, but Popja´ k was already interested in lipid biosynthesis and in the use of isotopic labels to investigate it. Gradually we began to concert experiments in which chemistry and biochemistry were complementary: a collaboration that was to last for more than twenty years. Our first project was to decipher a corresponding pattern of m and c carbons in the tetracyclic nucleus of cholesterol. This work, reported in part in 1953 (13) and finally in 1957 (14), completed the pattern shown in 5. Thus by the early 1950s it was clear that a pathway ran from acetate to cholesterol. Those years also saw a convergence leading to the identification of two more intermediates, and Konrad Bloch’s laboratory was the focal point of both developments. Chemists studying the terpenes, a large class of natural products including many essential oils and resins from plants, had noticed that the molecules could very often be dissected into five-carbon units of which isoprene 6, a compound obtainable by destructive distillation of rubber, could be regarded as the parent. The hypothesis (the isoprene rule) that terpenes were formed by association of similar units had proved useful in guiding chemists towards experimental proof of terpenoid structures, and it was developed particularly by Ruzicka who in 1939 shared a Nobel Prize in chemistry for, in part, his work on the higher terpenes. Squalene 3, as shown by the broken lines, can be dissected into six isoprene units and Channon’s evidence (3) and Robinson’s hypothesis (5) indicated a possible link with cholesterol.
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Bonner and Arreguin’s (15) work on rubber formation in guayule seedlings provided another possible link. When various pure compounds were added to the nutrient medium and any increase in the rubber content of the seedlings was measured by turbidimetry, only a few compounds were found effective; but these included acetate, acetoacetate, acetone, and senecioic acid 7 (3-methyl-2-butenoic acid, a natural product). The findings were strung together in a scheme of biosynthesis obviously inspired by the isoprene rule: two molecules of acetate condensed to form acetoacetate, which decarboxylated to acetone, which condensed with a third acetate molecule to yield senecioate, which was reduced to isoprene, which polymerized to rubber. Stimulation of growth is not of course equivalent to incorporation which was later (16) verified, for acetate only, by isotopic labelling. Still, the hypothesis could have predicted, in rubber made from carbon-labelled acetate, a pattern of m and c atoms similar to the one found by Bloch in the cholesterol side-chain. This result and the earlier speculations influenced Bloch to examine squalene as a possible intermediate between acetate and cholesterol. After unsuccessful experiments with sharks he and Langdon (17) tried feeding rats first with excess of squalene and then with labelled acetate. This procedure was adopted because very little squalene is normally present in animal tissues and it is turned over rapidly. The squalene recovered from the rats’ tissues was radioactive and, when fed to mice, generated radioactive cholesterol with notable efficiency. Popja´ k (18) extended the result by showing that squalene and cholesterol are formed concurrently from acetate in rat liver slices. I worked out a carbon-by-carbon degradation of the squalene molecule and together we showed (19) that the pattern of incorporation of acetate into squalene synthesized from radioactive acetate in minced rat liver was as in 8, in agreement with the distribution in the side-chain of cholesterol. Another link in the chain of evidence was already forming. The alcohol lanosterol (later and more pre-
cisely termed lanostadienol) from wool fat had attracted the attention of Wieland and Windaus. With thirty carbon atoms, the same as in squalene, it was technically a triterpene so its provenance from an animal source was remarkable. The structure proved even more difficult than that of cholesterol to derive by degradation but by 1951 it had been shown in Ruzicka’s (20) and Barton’s (21) laboratories to have a side-chain identical, except for the presence of one double bond, with that of cholesterol. The full structure 9a followed a year later (22): lanosterol is a 4,4,14trimethylsterol. The idea that lanosterol lay on the pathway between squalene and cholesterol was not easy to entertain on the basis of Robinson’s suggested mode of squalene cyclization, but the genius of Robert Woodward saw an alternative folding, leading directly to lanosterol. He and Bloch published in 1954 (the year of Bloch’s move to Harvard) both the hypothesis and its substantiation by evidence that C-13 is derived from the methyl group of acetate and not from the carboxyl group as the Robinson folding would require (23). The demonstrations in Bloch’s laboratory that lanosterol is formed from radioactive acetate in homogenates of rat liver (24), and that it is subsequently converted into radioactive cholesterol (25), came soon after. Woodward’s folding requires the rearrangement of at least one methyl group. Treatments of the cyclization process by Stork and Burgstahler (26) and by Eschenmoser, Ruzicka, Jeger and Arigoni (27), based on the stereochemistry of acid-catalysed alkene cyclizations, preferred a double methyl rearrangement as part of a concerted process leading from squalene to lanosterol (Scheme 1). With complete knowledge of the pattern of incorporation of acetate into cholesterol it became possible to show that the methyl group on C-13 in cholesterol arrived from C-14 by an intramolecular 1,2-shift (28) and that the methyl group on C-14 in lanosterol also arrived there by migration (29). Acetate 3 squalene 3 lanosterol 3 cholesterol had now become the perceived pathway. In addition, the transformation of squalene C 30H 50 into lanosterol C 30H 50O is formally an oxidation, and Tchen and Bloch (30) showed that
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SCHEME 1
molecular oxygen is the sole source of the hydroxyl group in lanosterol and that no stably bound hydrogen from the aqueous medium is incorporated during the process. Later the hypothetical hydroxyl cation, as shown in Scheme 1, was superseded when squalene epoxide was recognized as an intermediate. Most of the work that revealed the pathway from lanosterol to cholesterol was later than the period under review, but some important signposts were discerned in Bloch’s laboratory (31). Saponification of the livers and intestines of rats that had been injected with radioactive acetate and killed soon afterwards gave, along with lanosterol, minute amounts of a radioactive fraction that was provisionally identified by ingenious tracer experiments, often using inactive lanosterol as a carrier, as a 4,4-dimethylcholesta-8,24-dien-3-ol 9b. The assignment was confirmed by chemical synthesis of the 24,25-dihydro derivative (32). Thus the 14methyl group was indicated as the first of the three that must be removed on the way to cholesterol. The earliest experiments with labelled precursors were done with living animals. As the work proceeded, the liver was recognized as an active generator of cholesterol in vivo and the use of sliced or pulped liver tissue became popular. A major advance by Nancy Bucher (33) made active liver homogenates available and fractions could be separated from these by differ-
ential centrifugation. Formation of cholesterol from acetate required the presence of the 105,000g supernatant plus a particulate fraction. When air was replaced by nitrogen, squalene accumulated. During the early 1950s the slowest progress was made on the stages between acetate and squalene. Most of the intermediates postulated by Bonner and Arreguin gave positive but quantitatively disappointing results when labelled and tested (none of them is in fact a mainstream intermediate). The best of them was senecioic acid 7, which the rat incorporates into cholesterol more efficiently than acetate (34). Activation of acetate as the coenzyme A thiolester was already known, but there seems to have been a tacit assumption that other carboxylic acids would also be activated in vivo, and this led to a missed insight when labelled 3-hydroxy-3-methylglutarate 10 was tested. Enlightenment leading to unprecedented progress came from an entirely unexpected direction. In the Merck, Sharp and Dohme laboratories at Rahway, New Jersey, a team led by Karl Folkers was studying water-soluble accessory food factors. A counter-current distribution between water and diethyl ether of a fraction from dried “distillers’ solubles” (a by-product of industrial alcoholic fermentation) had been undertaken in order to demonstrate the presence of lipoic acid. Fractions were assayed against a strain
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of Lactobacillus acidophilus that normally requires acetate for growth. As well as lipoic acid, an acetatereplacing factor was found that was 1600 times as active as sodium acetate in promoting growth of the test organism. This new natural product was named first divalonic acid, then mevalonic acid since it proved to be a 3-methylvaleric acid derivative (35). An identical product was independently isolated by Tamura (36) from Aspergillus oryzae and named hiochic acid. The chemical structure is 11, though the absolute configuration came later (37). It was not difficult to synthesize, though optical resolution was harder (38), and versions labelled with 14C at the 1- and 2-positions were soon prepared in racemic form. A remarkable intuitive leap led Tavormina, Gibbs and Huff (1956) to try these labelled materials as precursors of cholesterol in rat liver homogenates (39). The results were spectacular and decisive. The 1- 14Clabelled mevalonate gave no incorporation of isotope into cholesterol but produced radioactive carbon dioxide. The 2- 14C-labelled mevalonate incorporated 43.4% of its radioactivity into sterol. If all the sterol was cholesterol and if (as was later found to be so) only one enantiomer of the mevalonate was used, this incorporation is about 104% of the figure now considered theoretically possible! Experimental uncertainties apart, the finding did indicate that the pathway from mevalonate to cholesterol was virtually an unbranched oneway street. I well remember the excitement, and explosion of research, that followed publication of these findings. The book (40) of the Ciba Foundation symposium in May 1958 reviews an extraordinary volume of work and much of it was based on the availability of labelled mevalonic acid. The pathway between acetate and mevalonate was soon traced out, principally by Rudney (41) and Lynen (42), using cell-free preparations from liver and yeast. Rudney studied the synthesis of 3-hydroxy-3methylglutarate 10 (HMG) from acetate and found that acetyl-CoA and acetoacetyl-CoA (but not acetoacetate) were true intermediates. The enzyme acetyl-CoA acetyltransferase (thiolase) catalysed this first, reversible, condensation in the synthesis. In preparations where thiolase was poisoned with iodoacetamide, for-
mation of HMG-CoA 12 from acetoacetyl-CoA and acetyl-CoA, with liberation of CoASH from the acetyl moiety, could be demonstrated. The condensing enzyme, hydroxymethylglutaryl-CoA synthase, from yeast is more stable than its highly labile analogue from liver. In a two-step reduction, the coenzyme NADPH reduces HMG-CoA to mevalonate 11 on an enzyme from yeast or liver. Notably, the acetoacetylthiolester used for sterol synthesis is not produced via a biotin-mediated carboxylation of acetyl-CoA and subsequent decarboxylative condensation with a second molecule of acetyl-CoA, as it is in fatty acid biosynthesis. Bloomfield and Bloch (43) found that biotindeficient yeast would synthesize sterol easily from acetate while fatty acid synthesis was greatly impaired. Senecioic acid 7, an important intermediate in the Bonner-Arreguin scheme for polyisoprenoid synthesis, was relegated to a side-line. Results from the laboratories of Coon (44), Rudney (41) and Lynen (42) showed that its coenzyme A thiolester, a product of leucine catabolism, can undergo carboxylation to 3-methylglutaconyl-CoA 13 which is hydrated enzymically to HMG-CoA. A cleavage enzyme splits HMG-CoA to acetyl-CoA and free acetoacetate, which in the systems used for sterol biosynthesis can be converted into its CoA thiolester and used by the condensing enzyme. This explains an early observation by Zabin and Bloch (45) that the amount of radioactive carbonate incorporated by rats into cholesterol increased fivefold when inactive isovalerate (3-methylbutanoate) was fed at the same time. Just as with senecioic acid, some chemically plausible schemes could be formulated if mevaldic acid 14, a putative intermediate in the reduction of HMG-CoA to mevalonate, was the true intermediate for squalene synthesis. It is easily synthesized, and systems from liver or yeast incorporated radioactivity from the labelled form into squalene and cholesterol. But Amdur, Rilling and Bloch showed in 1957 (46) that when mevalonate was converted into squalene too little hydrogen was lost from the 5 position of mevalonate for the process to proceed by way of mevaldate; and by 1958 Lynen and co-workers had other evidence (42) that mevalonate lies closer to squalene than does mevaldate. Later work showed that mevaldate is not even a normal intermediate. The primary reduction product of HMG-CoA is the hemithioacetal 15, which is further reduced on the same enzyme to mevalonate. Mevaldate can be reduced to mevalonate in liver or yeast, but the process is non-specific and converts both enantiomers. Mevalonate is easily converted into squalene in preparations of yeast, and all but the last of the intervening stages were soon identified in the laboratories of Lynen and Bloch. The discovery of preparations that needed only ATP and NADPH as co-factors led to a search for phosphorylated intermediates, facilitated by the availability of radio-labelled mevalonate and by the tech-
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nique of paper electrophoresis supplementing paper chromatography. Mevalonate-5-phosphate 16 (42) and subsequently mevalonate-5-diphosphate 17 (47) were soon identified and with difficulty synthesized (as racemates: the kinase producing 16 accepts only the natural 3R mevalonate). But the diphosphate still required ATP and NADPH for conversion into squalene and sterol. The next identifiable intermediate, isolated and synthesized in Lynen’s laboratory (48), was 3-methyl3-buten-1-yl diphosphate 18, known as isopentenyl pyrophosphate. Its formation used one equivalent of ATP, which is not again required in the biosynthesis. The carboxyl group was lost, as shown by absence of radioactivity in the product when 1- 14C-labelled mevalonate was the precursor. Because the process also occurred without loss of carbon-bound hydrogen and without acceptance of hydrogen from the medium, a concerted elimination of carbon dioxide and orthophosphate from a transient intermediate phosphorylated at position 3 was indicated. Later, when mevalonate labelled with 18 O at position 3 was used, 18O-labelled orthophosphate was formed along with unlabelled ADP. Chemical analogues of this elimination were known at the time, but no biochemical example. Isopentenyl pyrophosphate is formally an adduct of isoprene and pyrophosphoric acid and in preference to mevalonic acid it is to be regarded as the universal precursor of terpenoids. But it is by no means the last phosphorylated intermediate in the synthesis,and three more were quickly discovered in Lynen’s laboratory (48 –50). The two terminal isopropylidene groups in squalene 3, as distinct from the isopropenyl group in 18, suggested isomerization at some stage. Lynen looked for, and found, an isomerase catalysing the interconversion of 18 and the allylic 3-methyl-2-buten1-yl diphosphate 19, known as dimethylallyl pyrophosphate. The equilibrium 18 7 19 was around 9:1 in favour of 19. The isomerase was sensitive to poisoning by iodoacetamide and suitable yeast preparations would, when so poisoned, accumulate 18 when fed with mevalonate-5-phosphate. This finding, incidentally,
was another proof that 18 precedes 19 in the biosynthesis. Further steps in the biosynthesis were then uncovered using the pyrophosphates 18 and 19 as precursors, and a particle-free yeast extract. The fifteencarbon farnesyl pyrophosphate 20 was the principal product, but when excess of dimethylallyl pyrophosphate was present it was possible also to isolate the ten-carbon geranyl pyrophosphate 21. To obtain squalene from this system, supplementation with NADPH and with a particulate fraction from yeast was necessary. This wonderful piece of biochemistry from Lynen and his able colleagues provided a mechanism for polyisoprenoid assembly that stands in all essentials to this day. The process can be regarded as an
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addition of a carbocation to an alkene followed by elimination of a proton. Many chemical examples were already known, particularly in the petrochemical industry, but this was the first on an enzyme. The subtlety and economy of the enzymic process lie first in the use of allylic pyrophosphates—pyrophosphate anion is a much better “leaving group” than orthophosphate, and the adjacent double bond stabilizes the developing positive charge—and secondly in the specificity of the elimination. This generates a new allylic pyrophosphate ready to initiate addition, on the same enzyme, of another molecule of the “monomer,” the non-allylic isopentenyl pyrophosphate. Popja´ k was able to show (51) that the more difficult preparations from liver could perform the same sequence, from mevalonate to squalene. Formation of the C 30 squalene from two molecules of the C 15 farnesyl pyrophosphate requires a particulate fraction from yeast or liver supplemented only by NADPH. Many chemically reasonable mechanisms could be, and were, postulated for this “dimerization.” Most of them were negated by our discovery (52) that in the process one, and only one, hydrogen is removed from the oxygenated carbon of one, and only one, molecule of farnesyl pyrophosphate, and is replaced by one hydrogen from NADPH. This finding was to lead, later, to the establishment of presqualene pyrophosphate 22 as the last intermediate stage on the road to squalene. A chemist reflecting on the earlier efforts that defined the structure of the sterols might exclaim: “These were problems that we could now solve in a few days. They needed better tools, not this laborious blind groping. Pregl was right: it is better to improve your powers of analysis than to push existing techniques to the limit.” And now, forty years on, a biochemist might make similar comment on the work I have described here. I doubt that Konrad Bloch was affected at any time by this kind of thinking. I saw him as totally focussed on the problem: “how do living cells make sterols?” He always seemed content to use existing techniques; he did not modify or improve them but he used a wide range of them with virtuosity and he was a slave to none of them. A splendid deviser and planner of experiments, he used the tools that were to hand and his logic in interpreting results could seldom be ques-
tioned. He was the right man— unspecialized, clearsighted, undismayed by strange problems—to tackle a biosynthesis that began with familiar, aniondominated biochemistry and developed, after isopentenyl pyrophosphate, into the quite novel cationcontrolled alkylations and rearrangements leading to squalene and to lanosterol. I had feared when we first met that he might regard Popja´ k and me as interlopers in a field that he had made his own. Instead, he saw that further progress would need innovative chemistry. He welcomed our contribution and we were friends from that time on. As each twist and turn of the biosynthetic pathway was revealed, however clumsy and sometimes inconclusive the detective work may have been, all of us were entranced by the beauty of the unfolding story, beautiful beyond anything that we could (or, at the time, did!) imagine. And as the later investigations proceeded, using methods and facilities immensely more powerful than those that we had commanded, I was sometimes afraid that the subtlety and complexity would collapse into something simpler and less interesting. Nothing of the kind happened: as each remaining gap was filled in, the picture simply became more varied and richer in harmonious detail. As one who had synthesized cholesterol the hard way, I am now looking at a synthesis that I could never have planned, let alone executed. By revealing the beauty of her result, Nature has shown us chemists how much we still have to learn. REFERENCES
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¨ ber Sterine, Gallensa¨ u1. Lettre´ , H., and Inhoffen, H. H. (1936) U ren und verwandte Naturstoffe, Enke, Stuttgart. 2. Fieser, L. F., and Fieser, M. A. (1949) Natural Products Related to Phenanthrene, 3rd ed. Am. Chem. Soc. Monograph Series No. 70. Reinhold, New York. 3. Channon, H. J. (1929) Biological significance of the unsaponifiable matter of oils. I. Experiments with the unsaturated hydrocarbon squalene (spinacene). Biochem. J. 20, 400 – 408. 4. Heilbron, I. M., Kamm, E. D., and Owens, W. M. (1926) The unsaponifiable matter from the oils of elasmobranch fish. Part I. A contribution to the study of the constitution of squalene (spinacene). J. Chem. Soc., 1630 –1644. 5. Robinson, R. (1934) Structure of cholesterol. Chem. Ind., 1062– 1063.
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6. Robinson, R. (1932) Constitution of cholesterol. Nature, 540 – 541. 7. Sonderhoff, R., and Thomas, H. (1937) Die enzymatische Dehydrierung der Trideutero-essigsa¨ ure. Liebigs Ann. Chem. 530, 195–213. 8. Schoenheimer, R. (1942) The Dynamic State of Body Constituents, Harvard Univ. Press, Cambridge, MA. 9. Mauthner, J., and Suida, W. (1986) Beitra¨ ge zur Kenntniss der Cholesterins. III Abhandlung. Monatsh. Chem. 17, 29 – 49. 10. Ottke, R. C., Tatum, E. L., Zabin, I., and Bloch, K. (1951) Isotopic acetate and isovalerate in the synthesis of ergosterol by Neurospora. J. Biol. Chem. 189, 429 – 433. 11. Wu¨ rsch, J., Huang, R. L., and Bloch, K. (1952) The origin of the isoo¨ ctyl side chain of cholesterol. J. Biol. Chem. 195, 439 – 446. 12. Little, H. N., and Bloch, K. (1950) The utilization of acetic acid for the biological synthesis of cholesterol. J. Biol. Chem. 183, 33– 46. 13. Cornforth, J. W., Hunter, G. D., and Popja´ k, G. (1953) Studies of cholesterol biosynthesis. 1. A new chemical degradation of cholesterol. 2. Distribution of acetate carbon in the ring structure. Biochem. J. 54, 590 –597 and 597– 601. 14. Cornforth, J. W., Gore, I. Y., and Popja´ k, G. (1957) Studies of cholesterol biosynthesis. 4. Degradation of rings C and D. Biochem. J. 65, 94 –109. 15. Bonner, J., and Arreguin, B. (1949) Biochemistry of rubber formation in the guayule. I. Rubber formation from seedlings. Arch. Biochem. 21, 109 –124. 16. Arreguin, B., Bonner, J., and Wood, B. J. (1951) The mechanism of rubber formation in the guayule. III. Experiments with isotopic carbon. Arch. Biochem. Biophys. 31, 234 –247. 17. Langdon, R. G., and Bloch, K. The biosynthesis of squalene: The utilization of squalene in the biosynthesis of cholesterol. J. Biol. Chem. 200, 129 –134 and 135–144. 18. Popja´ k, G. (1954) Biosynthesis of squalene and cholesterol in vitro from acetate-1- 14C. Arch. Biochem. Biophys. 48, 102–106. 19. Cornforth, J. W., and Popja´ k, G. (1954) Studies on the biosynthesis of cholesterol. 3. Distribution of 14C in squalene biosynthesized from [Me- 14C]acetate. Biochem. J. 58, 403– 407. ¨ ber 20. Voser, W., Mijovic´ , M. V., Jeger, O., and Ruzicka, L. (1951) U der Vervollsta¨ ndigung der Teilformel des Lanostadienols. Helv. Chim. Acta 34, 1585–1596. 21. Barnes, C. S., Barton, D. H. R., Fawcett, J. S., Knight, S. K., McGhie, J. F., Pradhan, M. K., and Thomas, B. R. (1951) Chem. Ind. (London), 1067–1068. 22. Voser, W., Mijovic´ , M. V., Heusser, H., Jeger, O., and Ruzicka, L. ¨ ber Steroide und Sexualhormone. 186 Mitteilung. U ¨ ber (1952) U die Konstitution des Lanostadienols (Lanosterins) und seine Zugeho¨ rigkeit zu den Steroiden. Helv. Chim. Acta 35, 2414 – 2430. 23. Woodward, R. B., and Bloch, K. (1953) The cyclization of squalene in cholesterol synthesis. J. Am. Chem. Soc. 75, 2023– 2024. 24. Tchen, T. T., and Bloch, K. (1955) In vitro conversion of squalene to lanosterol and cholesterol. J. Am. Chem. Soc. 77, 6085– 6086. 25. Clayton, R. B., and Bloch, K. (1956) Biological synthesis of lanosterol and agnosterol. J. Biol. Chem. 218, 305–318. 26. Stork, G., and Burgstahler, A. W. (1955) The stereochemistry of polyene cyclization. J. Am. Chem. Soc. 77, 5068 –5077. 27. Eschenmoser, A., Ruzicka, L., Jeger, O., and Arigoni, D. (1955) Zur Kenntnis der Triterpene. 190 Mitteilung. Eine stereochemische Interpretation der biogenetische Isoprenregel bei der Triterpenen. Helv. Chim. Acta 38, 1890. 28. Cornforth, J. W., Cornforth, R. H., Pelter, A., Horning, M. G., and Popja´ k, G. (1958) Rearrangement of methyl groups in the
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enzymic cyclization of squalene to lanosterol. Proc. Chem. Soc., 112–113; and (1959) Studies on the biosynthesis of cholesterol. 7. Rearrangement of methyl groups during enzymic cyclization of squalene. Tetrahedron 5, 311–339. Maudgal, R. K., Tchen, T. T., and Bloch, K. (1958) 1,2-Methyl shifts in the cyclization of squalene to lanosterol. J. Am. Chem. Soc. 80, 2589 –2590. Tchen, T. T., and Bloch, K. (1956) The mechanism of cyclization of squalene to lanosterol. J. Am. Chem. Soc. 78, 1516 –1517. Gautschi, F., and Bloch, K. (1957) The structure of an intermediate in the biological demethylation of lanosterol. J. Am. Chem. Soc. 79, 684 – 689. Gautschi, F., and Bloch, K. (1958) Synthesis of isomeric 4,4dimethylcholestenols and identification of a lanosterol metabolite. J. Biol. Chem. 233, 1343–1347. Bucher, N. L. R. (1953) The formation of radioactive cholesterol and fatty acids from 14C-labelled acetate by rat liver homogenates. J. Am. Chem. Soc. 75, 498. Bloch, K., Clarke, L. C., and Harary, I. (1954) Utilization of branched-chain acids in cholesterol biosynthesis. J. Am. Chem. Soc. 76, 3859 –3860. Wolf, D. E., Hofmann, C. H., Aldrich, P. E., Skeggs, H. R., Wright, L. D., and Folkers, K. (1956) -Hydroxy--methyl-␦valerolactone (divalonic acid), a new biological factor. J. Am. Chem. Soc. 78, 4499. Tamura, G. (1956) Hiochic acid: A new growth factor for Lactobacillus homohiochi and L. heterohiochi. J. Gen. Appl. Microbiol. 2, 431– 434. Eberle, M., and Arigoni, D. (1960) Absolute Konfiguration des Mevalolactons. Helv. Chim. Acta 43, 1508 –1513. Shunk, C. H., Linn, B. O., Huff, J. W., Gilfillan, J. L., Skeggs, H. R., and Folkers, K. (1957) Resolution of DL-mevalonic acid and the synthesis and biological activities of DL-3-hydroxy-3methylglutaraldehydic acid. J. Am. Chem. Soc. 79, 3294 –3295. Tavormina, P. A., Gibbs, M. H., and Huff, J. W. (1956) The utilization of -hydroxy--methyl-␦-valerolactone in cholesterol biosynthesis. J. Am. Chem. Soc. 78, 4498 – 4499. Wolstenholme, G. E. W., and O’Connor, M. (Eds.) (1959) CIBA Foundation Symposium on the Biosynthesis of Terpenes and Sterols. Churchill, London. Rudney, H. (1959) The biosynthesis of -hydroxy--methylglutaryl coenzyme A and its conversion to mevalonic acid. Ibid., pp. 75–90. Lynen, F., Eggerer, H., Henning, U., Knappe, J., Kessel, I., and Ringelmann, E. (1959) New aspects of acetate incorporation into isoprenoid precursors. Ibid., pp. 95–116. Bloomfield, D. K., and Bloch, K. (1960) Formation of ⌬ 9unsaturated fatty acids. J. Biol. Chem. 235, 337–345. Coon, M. J., Kupiecki, F. P., Dekker, E. E., Schlesinger, M. J., and del Campillo, A. (1959) The enzymic synthesis of branchedchain acids. In CIBA Foundation Symposium on the Biosynthesis of Terpenes and Sterols. Wolstenholme, G. E. W., and O’Connor, M. (Eds.), pp. 62–72. Churchill, London. Zabin, I., and Bloch, K. (1951) The utilization of isovaleric acid in cholesterol synthesis. J. Biol. Chem. 192, 267–273. Amdur, B. H., Rilling, H., and Bloch, K. (1957) The enzymatic conversion of mevalonic acid to squalene. J. Am. Chem. Soc. 79, 2646 –2647. Chaykin, S., Law, J., Phillips, A. H., Tchen, T. T., and Bloch, K. (1958) Phosphorylated intermediates in the synthesis of squalene. Proc. Natl. Acad. Sci. USA 44, 998 –1004. Lynen, F., Eggerer, H., Henning, U., and Kessel, I. (1958) Farnesyl-pyrophosphat und 3-methyl-⌬ 3-butenyl-1-pyrophos-
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phat, die biologischen Vorstufen des Squalens. Zur Biosynthese der Terpene III. Angew. Chem. 70, 738 –742. 49. Agranoff, B. W., Eggerer, H., Henning, U., and Lynen, F. (1959) Isopentenol pyrophosphate isomerase. J. Am. Chem. Soc. 81, 1254 –1255. 50. Lynen, F., Agranoff, B. W., Eggerer, H., Henning, U., and Mo¨ slein, E. M. (1959) ␥␥-Dimethyl-allyl-pyrophosphat und Geranyl-pyrophosphat, biologische Vorstufen des Squalens. Zur Biosynthese der Terpene, VI. Angew. Chem. 71, 657– 663.
51. Popja´ k, G. (1959) The biosynthesis of derivatives of allylic alcohols from [2- 14C]mevalonate in liver enzyme preparations and their relation to the synthesis of squalene. Tetrahedron Lett. No. 19, 19 –28. 52. Popja´ k, G., Goodman, De, W. S., Cornforth, J. W., Cornforth, R. H., and Ryhage, R. (1961) Studies on the biosynthesis of cholesterol. XV. Mechanism of squalene biosynthesis from farnesyl pyrophosphate and from mevalonate. J. Biol. Chem. 236, 1934 –1947.
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Sterol Biosynthesis: The Early Days John Warcup Cornforth 1 School of Chemistry, Physics and Environmental Science, University of Sussex, Falmer, Brighton BN1 9QJ, United Kingdom
Having chosen a title for this article, I need to define its meaning. The study of sterol biosynthesis continues to this day, but what were the early days? Arbitrarily, I have chosen to draw a line early in the 1960s. By then, the work that won Konrad Bloch and Feodor Lynen a joint Nobel Prize had been done: the pathway to the sterols was plain for all to see. A few gaps, a few unsuspected intermediates, and many unforeseen subtleties remained to be detected and explored, but I assign this work to a later period. Few scientific legends link organic chemistry and biochemistry so intimately as the story of sterol biosynthesis. The linkage originated from an understandable preference of chemists to work with distillable liquids and crystalline solids. Crystalline cholesterol has been known for two centuries, crystalline cholic acid for a century and a half; so they escaped Emil Fischer’s put-down: “Tierchemie ist Schmierchemie” (animal chemistry is the chemistry of smears). But they are both fairly large and complex molecules, and one has to admire the tenacity of the early chemists who tackled them. With few methods of separation, with tools that always destroyed what they examined (elementary analysis, chemical degradation) and with no guides except chemical identification of functional groups and empirical interpretation of sequential reactions, they arrived step by painful step at structures that were very nearly right. That cholesterol is C 27H 46O, that its functional groups are one hydroxyl and one double bond and therefore that it has four carbocyclic rings, that the hydroxyl and the double bond are in different but adjacent rings, that the ring structure in cholesterol and cholic acid is the same, that cholesterol has a branched side-chain of eight carbons shortened in cholic acid to a chain of five carbons terminating in the carboxyl group . . . all this and much more was won by the same laborious procedures extended over the span of a human life. The work won Nobel Prizes in chemistry for Heinrich Wieland in 1927 and for Adolf Windaus in 1928 for their studies on bile acids and sterols—and tangentially in 1923 for Fritz Pregl. From a long degradation of a bile acid, he had 1
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emerged with insufficient material for a conventional analysis. He preferred to invent microanalysis rather than to face the labour of repeating the sequence. These pioneering efforts had produced by 1928 a partial structure 1 for cholesterol and a similar structure for bile acids; but the conclusion, though justly honoured, was a little wide of the goal. An empirical rule, correlating the size of a ring with a property of the dicarboxylic acid obtained by oxidative ring-opening, had been applied without strict logic. It was truly prophetic that the clue leading to revision came, in 1932, from a physical method: the emerging technique of X-ray crystallography. Bernal’s evidence that the yeast sterol ergosterol had a long thin molecule, incompatible with the chunky structure 1, forced a reinterpretation and led very quickly to proposals of a structure (2, minus most of the stereochemical detail) now repeatedly confirmed by physical and by chemical evidence as the correct one. Good accounts of the structural problems and their solution are given by Lettre´ and Inhoffen (1) and by Fieser and Fieser (2). The early 1930s were a seminal time for biochemistry. In particular, isolation procedures coupled with quantitative bioassay had brought dramatic advances in the fields of vitamins and hormones, where measurements of biological response were not too difficult. As evidence mounted that the sex hormones and the antirachitic Vitamin D were steroids, the chemical fraternity promoted cholesterol from the status of a fascinating chemical puzzle to the roles of a raw material for chemical manipulation and an objective for chemical synthesis. And to biochemists, steroids became an important problem of biosynthesis. The known facts were few, and outnumbered by the speculations. Feeding studies had shown that laboratory animals, although they can absorb cholesterol from food, must also be able to synthesize it, and do not make it from the slightly different sterols of food plants. And there was one tantalizing finding from 1926: the acyclic C 30H 50 hydrocarbon squalene 3, abundant in shark liver oils, increased the cholesterol content of rats that fed on it. This experiment (3) was made before the structure of either compound was fully known, but it had been suggested (4) by perceived structural similarities, and in 1934 with knowledge of both structures Robert Robinson (5) proposed a
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squalene-to-cholesterol conversion that was right in principle, though wrong in detail (and characteristically, he had published a rudimentary form of this speculation two years earlier (6), before he could be sure of either structure!). Happily the arising need to explore the pathway of sterol biosynthesis coincided with the advent of compounds enriched in the naturally occurring heavy isotopes of hydrogen, carbon and oxygen. To label a hypothetical precursor of, e.g., cholesterol, to administer it to a living system, and to measure the proportion of isotope incorporated in sterol, was now possible and not so difficult as to discourage investigation. Around that time, Konrad Bloch and Feodor Lynen were studying in Mu¨ nchen for their first degrees. Lynen was to stay in Mu¨ nchen for the rest of his career (he married Heinrich Wieland’s daughter) but Bloch went first to Switzerland and then to Columbia University, New York, whither Rudolf Schoenheimer had migrated a little earlier. The Nazis enriched the science of many countries while damaging their own. But the first contribution of tracer methods to the problem of steroid biosynthesis still came from Mu¨ nchen. Robert Sonderhoff, a pupil of Wieland, used trideuterioacetic acid CD 3CO 2H to test Wieland’s theory that oxidation in vivo is often dehydrogenation, and what organism but brewer’s yeast would he use at that place and time? He and Heinz Thomas (7) incubated yeast aerobically with the labelled acetate and measured deuterium in the succinic and citric acids that were formed. They also looked, almost incidentally, at the total fat and found it quite rich in deuterium; but a surprise came when they isolated the unsaponifiable fraction, mostly ergosterol, of the fat. This weighed around one quarter of the total fat but contained more than half the deuterium: it was by far the most highly labelled component of the yeast. They drew the correct conclusion that acetate is di-
rectly involved in sterol biosynthesis (and that sterols are not constructed from fatty acids, as Windaus had once speculated). Deuterium content in those days was determined by burning the sample and measuring the density of the combustion water by methods as sensitive as the quantity available would allow. Measurement by stopwatch of the time taken by a standardsized drop to fall between two marks on a vertical column filled with 2-fluorotoluene became a popular technique. At Columbia Schoenheimer was pursuing the ground-breaking studies, based on feeding experimental animals with heavy water, so classically summed up in a little book (alas, his last) The dynamic state of body constituents (8). Bloch was much attracted by this work and he lost no time in joining Schoenheimer’s group after gaining his doctorate. With David Rittenberg he published a sequence of papers establishing deuterioacetate as a primary source of deuteriocholesterol in mice and rats. The first primitive degradation of a labelled cholesterol, a pyrolysis of cholesteryl chloride that Mauthner (9) had shown to separate the nucleus from the side chain, proved that label was present in both fragments. Bloch continued this work after his move to Chicago in 1946. The discovery of a mutant of Neurospora crassa that required acetate for growth permitted an experiment (10) with [ 13C]acetate indicating that all carbon in the sterol (ergosterol) was derived from acetate. But by this time, the 1940 discovery that slow neutrons will convert 14N nuclei into the long-lived radioactive 14C was beginning to provide commercial quantities of radioactive carbon. The great advantage of this material for studies of biosynthesis was the sensitivity with which it could be measured: usually, as barium carbonate spread on a disk, but sometimes as carbon dioxide in a proportional gas counter. As a weak
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beta-emitter it was also relatively safe to handle, so that elaborate chemistry could be used to prepare labelled substrates and to degrade labelled products. Best of all, long degradative sequences could be contemplated, since the inevitable attrition of material at each step could be countered by occasional controlled dilution with non-radioactive material. The first example in this genre was Bloch’s use of 14C-labelled cholesterol, synthesized in rat liver from acetates labelled in the methyl and in the carboxyl group, to examine the distribution of isotope in the side chain of the sterol (11). Windaus and Wieland had already provided, in their investigations of structure, the methods of stepwise degradation that allowed each of the eight carbons in the side chain (except for the then indistinguishable pair of methyl groups at one end) to be separated from the others and examined for radioactivity. The results were illuminating. When carboxyllabelled acetate was the precursor, only three of the eight side-chain carbons were radioactive. These radioactivities were equal. With methyl-labelled acetate, these three carbons were (almost) inactive and the remaining five carbons were all radioactive. This supplemented earlier work (12) in which the Mauthner pyrolysis had been used to analyse cholesterol generated from acetic acid labelled positionally with 13C and with 14C. Calculations of the relative amounts of each isotope in the derived cholesterol had indicated that fifteen of its carbons were derived from the methyl group and twelve from the carboxyl. The clear presumption was that each and every carbon in cholesterol could be designated m or c according to its origin from acetate methyl or carboxyl respectively. The resulting pattern in the side chain was as shown in 4.
I can remember reading these papers with delight. Both George Popja´ k and I had joined the National Institute for Medical Research late in 1946. I continued to collaborate with Robert Robinson at Oxford on the total chemical synthesis (completed early in 1951) of the sterol nucleus, but Popja´ k was already interested in lipid biosynthesis and in the use of isotopic labels to investigate it. Gradually we began to concert experiments in which chemistry and biochemistry were complementary: a collaboration that was to last for more than twenty years. Our first project was to decipher a corresponding pattern of m and c carbons in the tetracyclic nucleus of cholesterol. This work, reported in part in 1953 (13) and finally in 1957 (14), completed the pattern shown in 5. Thus by the early 1950s it was clear that a pathway ran from acetate to cholesterol. Those years also saw a convergence leading to the identification of two more intermediates, and Konrad Bloch’s laboratory was the focal point of both developments. Chemists studying the terpenes, a large class of natural products including many essential oils and resins from plants, had noticed that the molecules could very often be dissected into five-carbon units of which isoprene 6, a compound obtainable by destructive distillation of rubber, could be regarded as the parent. The hypothesis (the isoprene rule) that terpenes were formed by association of similar units had proved useful in guiding chemists towards experimental proof of terpenoid structures, and it was developed particularly by Ruzicka who in 1939 shared a Nobel Prize in chemistry for, in part, his work on the higher terpenes. Squalene 3, as shown by the broken lines, can be dissected into six isoprene units and Channon’s evidence (3) and Robinson’s hypothesis (5) indicated a possible link with cholesterol.
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Bonner and Arreguin’s (15) work on rubber formation in guayule seedlings provided another possible link. When various pure compounds were added to the nutrient medium and any increase in the rubber content of the seedlings was measured by turbidimetry, only a few compounds were found effective; but these included acetate, acetoacetate, acetone, and senecioic acid 7 (3-methyl-2-butenoic acid, a natural product). The findings were strung together in a scheme of biosynthesis obviously inspired by the isoprene rule: two molecules of acetate condensed to form acetoacetate, which decarboxylated to acetone, which condensed with a third acetate molecule to yield senecioate, which was reduced to isoprene, which polymerized to rubber. Stimulation of growth is not of course equivalent to incorporation which was later (16) verified, for acetate only, by isotopic labelling. Still, the hypothesis could have predicted, in rubber made from carbon-labelled acetate, a pattern of m and c atoms similar to the one found by Bloch in the cholesterol side-chain. This result and the earlier speculations influenced Bloch to examine squalene as a possible intermediate between acetate and cholesterol. After unsuccessful experiments with sharks he and Langdon (17) tried feeding rats first with excess of squalene and then with labelled acetate. This procedure was adopted because very little squalene is normally present in animal tissues and it is turned over rapidly. The squalene recovered from the rats’ tissues was radioactive and, when fed to mice, generated radioactive cholesterol with notable efficiency. Popja´ k (18) extended the result by showing that squalene and cholesterol are formed concurrently from acetate in rat liver slices. I worked out a carbon-by-carbon degradation of the squalene molecule and together we showed (19) that the pattern of incorporation of acetate into squalene synthesized from radioactive acetate in minced rat liver was as in 8, in agreement with the distribution in the side-chain of cholesterol. Another link in the chain of evidence was already forming. The alcohol lanosterol (later and more pre-
cisely termed lanostadienol) from wool fat had attracted the attention of Wieland and Windaus. With thirty carbon atoms, the same as in squalene, it was technically a triterpene so its provenance from an animal source was remarkable. The structure proved even more difficult than that of cholesterol to derive by degradation but by 1951 it had been shown in Ruzicka’s (20) and Barton’s (21) laboratories to have a side-chain identical, except for the presence of one double bond, with that of cholesterol. The full structure 9a followed a year later (22): lanosterol is a 4,4,14trimethylsterol. The idea that lanosterol lay on the pathway between squalene and cholesterol was not easy to entertain on the basis of Robinson’s suggested mode of squalene cyclization, but the genius of Robert Woodward saw an alternative folding, leading directly to lanosterol. He and Bloch published in 1954 (the year of Bloch’s move to Harvard) both the hypothesis and its substantiation by evidence that C-13 is derived from the methyl group of acetate and not from the carboxyl group as the Robinson folding would require (23). The demonstrations in Bloch’s laboratory that lanosterol is formed from radioactive acetate in homogenates of rat liver (24), and that it is subsequently converted into radioactive cholesterol (25), came soon after. Woodward’s folding requires the rearrangement of at least one methyl group. Treatments of the cyclization process by Stork and Burgstahler (26) and by Eschenmoser, Ruzicka, Jeger and Arigoni (27), based on the stereochemistry of acid-catalysed alkene cyclizations, preferred a double methyl rearrangement as part of a concerted process leading from squalene to lanosterol (Scheme 1). With complete knowledge of the pattern of incorporation of acetate into cholesterol it became possible to show that the methyl group on C-13 in cholesterol arrived from C-14 by an intramolecular 1,2-shift (28) and that the methyl group on C-14 in lanosterol also arrived there by migration (29). Acetate 3 squalene 3 lanosterol 3 cholesterol had now become the perceived pathway. In addition, the transformation of squalene C 30H 50 into lanosterol C 30H 50O is formally an oxidation, and Tchen and Bloch (30) showed that
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SCHEME 1
molecular oxygen is the sole source of the hydroxyl group in lanosterol and that no stably bound hydrogen from the aqueous medium is incorporated during the process. Later the hypothetical hydroxyl cation, as shown in Scheme 1, was superseded when squalene epoxide was recognized as an intermediate. Most of the work that revealed the pathway from lanosterol to cholesterol was later than the period under review, but some important signposts were discerned in Bloch’s laboratory (31). Saponification of the livers and intestines of rats that had been injected with radioactive acetate and killed soon afterwards gave, along with lanosterol, minute amounts of a radioactive fraction that was provisionally identified by ingenious tracer experiments, often using inactive lanosterol as a carrier, as a 4,4-dimethylcholesta-8,24-dien-3-ol 9b. The assignment was confirmed by chemical synthesis of the 24,25-dihydro derivative (32). Thus the 14methyl group was indicated as the first of the three that must be removed on the way to cholesterol. The earliest experiments with labelled precursors were done with living animals. As the work proceeded, the liver was recognized as an active generator of cholesterol in vivo and the use of sliced or pulped liver tissue became popular. A major advance by Nancy Bucher (33) made active liver homogenates available and fractions could be separated from these by differ-
ential centrifugation. Formation of cholesterol from acetate required the presence of the 105,000g supernatant plus a particulate fraction. When air was replaced by nitrogen, squalene accumulated. During the early 1950s the slowest progress was made on the stages between acetate and squalene. Most of the intermediates postulated by Bonner and Arreguin gave positive but quantitatively disappointing results when labelled and tested (none of them is in fact a mainstream intermediate). The best of them was senecioic acid 7, which the rat incorporates into cholesterol more efficiently than acetate (34). Activation of acetate as the coenzyme A thiolester was already known, but there seems to have been a tacit assumption that other carboxylic acids would also be activated in vivo, and this led to a missed insight when labelled 3-hydroxy-3-methylglutarate 10 was tested. Enlightenment leading to unprecedented progress came from an entirely unexpected direction. In the Merck, Sharp and Dohme laboratories at Rahway, New Jersey, a team led by Karl Folkers was studying water-soluble accessory food factors. A counter-current distribution between water and diethyl ether of a fraction from dried “distillers’ solubles” (a by-product of industrial alcoholic fermentation) had been undertaken in order to demonstrate the presence of lipoic acid. Fractions were assayed against a strain
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of Lactobacillus acidophilus that normally requires acetate for growth. As well as lipoic acid, an acetatereplacing factor was found that was 1600 times as active as sodium acetate in promoting growth of the test organism. This new natural product was named first divalonic acid, then mevalonic acid since it proved to be a 3-methylvaleric acid derivative (35). An identical product was independently isolated by Tamura (36) from Aspergillus oryzae and named hiochic acid. The chemical structure is 11, though the absolute configuration came later (37). It was not difficult to synthesize, though optical resolution was harder (38), and versions labelled with 14C at the 1- and 2-positions were soon prepared in racemic form. A remarkable intuitive leap led Tavormina, Gibbs and Huff (1956) to try these labelled materials as precursors of cholesterol in rat liver homogenates (39). The results were spectacular and decisive. The 1- 14Clabelled mevalonate gave no incorporation of isotope into cholesterol but produced radioactive carbon dioxide. The 2- 14C-labelled mevalonate incorporated 43.4% of its radioactivity into sterol. If all the sterol was cholesterol and if (as was later found to be so) only one enantiomer of the mevalonate was used, this incorporation is about 104% of the figure now considered theoretically possible! Experimental uncertainties apart, the finding did indicate that the pathway from mevalonate to cholesterol was virtually an unbranched oneway street. I well remember the excitement, and explosion of research, that followed publication of these findings. The book (40) of the Ciba Foundation symposium in May 1958 reviews an extraordinary volume of work and much of it was based on the availability of labelled mevalonic acid. The pathway between acetate and mevalonate was soon traced out, principally by Rudney (41) and Lynen (42), using cell-free preparations from liver and yeast. Rudney studied the synthesis of 3-hydroxy-3methylglutarate 10 (HMG) from acetate and found that acetyl-CoA and acetoacetyl-CoA (but not acetoacetate) were true intermediates. The enzyme acetyl-CoA acetyltransferase (thiolase) catalysed this first, reversible, condensation in the synthesis. In preparations where thiolase was poisoned with iodoacetamide, for-
mation of HMG-CoA 12 from acetoacetyl-CoA and acetyl-CoA, with liberation of CoASH from the acetyl moiety, could be demonstrated. The condensing enzyme, hydroxymethylglutaryl-CoA synthase, from yeast is more stable than its highly labile analogue from liver. In a two-step reduction, the coenzyme NADPH reduces HMG-CoA to mevalonate 11 on an enzyme from yeast or liver. Notably, the acetoacetylthiolester used for sterol synthesis is not produced via a biotin-mediated carboxylation of acetyl-CoA and subsequent decarboxylative condensation with a second molecule of acetyl-CoA, as it is in fatty acid biosynthesis. Bloomfield and Bloch (43) found that biotindeficient yeast would synthesize sterol easily from acetate while fatty acid synthesis was greatly impaired. Senecioic acid 7, an important intermediate in the Bonner-Arreguin scheme for polyisoprenoid synthesis, was relegated to a side-line. Results from the laboratories of Coon (44), Rudney (41) and Lynen (42) showed that its coenzyme A thiolester, a product of leucine catabolism, can undergo carboxylation to 3-methylglutaconyl-CoA 13 which is hydrated enzymically to HMG-CoA. A cleavage enzyme splits HMG-CoA to acetyl-CoA and free acetoacetate, which in the systems used for sterol biosynthesis can be converted into its CoA thiolester and used by the condensing enzyme. This explains an early observation by Zabin and Bloch (45) that the amount of radioactive carbonate incorporated by rats into cholesterol increased fivefold when inactive isovalerate (3-methylbutanoate) was fed at the same time. Just as with senecioic acid, some chemically plausible schemes could be formulated if mevaldic acid 14, a putative intermediate in the reduction of HMG-CoA to mevalonate, was the true intermediate for squalene synthesis. It is easily synthesized, and systems from liver or yeast incorporated radioactivity from the labelled form into squalene and cholesterol. But Amdur, Rilling and Bloch showed in 1957 (46) that when mevalonate was converted into squalene too little hydrogen was lost from the 5 position of mevalonate for the process to proceed by way of mevaldate; and by 1958 Lynen and co-workers had other evidence (42) that mevalonate lies closer to squalene than does mevaldate. Later work showed that mevaldate is not even a normal intermediate. The primary reduction product of HMG-CoA is the hemithioacetal 15, which is further reduced on the same enzyme to mevalonate. Mevaldate can be reduced to mevalonate in liver or yeast, but the process is non-specific and converts both enantiomers. Mevalonate is easily converted into squalene in preparations of yeast, and all but the last of the intervening stages were soon identified in the laboratories of Lynen and Bloch. The discovery of preparations that needed only ATP and NADPH as co-factors led to a search for phosphorylated intermediates, facilitated by the availability of radio-labelled mevalonate and by the tech-
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nique of paper electrophoresis supplementing paper chromatography. Mevalonate-5-phosphate 16 (42) and subsequently mevalonate-5-diphosphate 17 (47) were soon identified and with difficulty synthesized (as racemates: the kinase producing 16 accepts only the natural 3R mevalonate). But the diphosphate still required ATP and NADPH for conversion into squalene and sterol. The next identifiable intermediate, isolated and synthesized in Lynen’s laboratory (48), was 3-methyl3-buten-1-yl diphosphate 18, known as isopentenyl pyrophosphate. Its formation used one equivalent of ATP, which is not again required in the biosynthesis. The carboxyl group was lost, as shown by absence of radioactivity in the product when 1- 14C-labelled mevalonate was the precursor. Because the process also occurred without loss of carbon-bound hydrogen and without acceptance of hydrogen from the medium, a concerted elimination of carbon dioxide and orthophosphate from a transient intermediate phosphorylated at position 3 was indicated. Later, when mevalonate labelled with 18 O at position 3 was used, 18O-labelled orthophosphate was formed along with unlabelled ADP. Chemical analogues of this elimination were known at the time, but no biochemical example. Isopentenyl pyrophosphate is formally an adduct of isoprene and pyrophosphoric acid and in preference to mevalonic acid it is to be regarded as the universal precursor of terpenoids. But it is by no means the last phosphorylated intermediate in the synthesis,and three more were quickly discovered in Lynen’s laboratory (48 –50). The two terminal isopropylidene groups in squalene 3, as distinct from the isopropenyl group in 18, suggested isomerization at some stage. Lynen looked for, and found, an isomerase catalysing the interconversion of 18 and the allylic 3-methyl-2-buten1-yl diphosphate 19, known as dimethylallyl pyrophosphate. The equilibrium 18 7 19 was around 9:1 in favour of 19. The isomerase was sensitive to poisoning by iodoacetamide and suitable yeast preparations would, when so poisoned, accumulate 18 when fed with mevalonate-5-phosphate. This finding, incidentally,
was another proof that 18 precedes 19 in the biosynthesis. Further steps in the biosynthesis were then uncovered using the pyrophosphates 18 and 19 as precursors, and a particle-free yeast extract. The fifteencarbon farnesyl pyrophosphate 20 was the principal product, but when excess of dimethylallyl pyrophosphate was present it was possible also to isolate the ten-carbon geranyl pyrophosphate 21. To obtain squalene from this system, supplementation with NADPH and with a particulate fraction from yeast was necessary. This wonderful piece of biochemistry from Lynen and his able colleagues provided a mechanism for polyisoprenoid assembly that stands in all essentials to this day. The process can be regarded as an
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addition of a carbocation to an alkene followed by elimination of a proton. Many chemical examples were already known, particularly in the petrochemical industry, but this was the first on an enzyme. The subtlety and economy of the enzymic process lie first in the use of allylic pyrophosphates—pyrophosphate anion is a much better “leaving group” than orthophosphate, and the adjacent double bond stabilizes the developing positive charge—and secondly in the specificity of the elimination. This generates a new allylic pyrophosphate ready to initiate addition, on the same enzyme, of another molecule of the “monomer,” the non-allylic isopentenyl pyrophosphate. Popja´ k was able to show (51) that the more difficult preparations from liver could perform the same sequence, from mevalonate to squalene. Formation of the C 30 squalene from two molecules of the C 15 farnesyl pyrophosphate requires a particulate fraction from yeast or liver supplemented only by NADPH. Many chemically reasonable mechanisms could be, and were, postulated for this “dimerization.” Most of them were negated by our discovery (52) that in the process one, and only one, hydrogen is removed from the oxygenated carbon of one, and only one, molecule of farnesyl pyrophosphate, and is replaced by one hydrogen from NADPH. This finding was to lead, later, to the establishment of presqualene pyrophosphate 22 as the last intermediate stage on the road to squalene. A chemist reflecting on the earlier efforts that defined the structure of the sterols might exclaim: “These were problems that we could now solve in a few days. They needed better tools, not this laborious blind groping. Pregl was right: it is better to improve your powers of analysis than to push existing techniques to the limit.” And now, forty years on, a biochemist might make similar comment on the work I have described here. I doubt that Konrad Bloch was affected at any time by this kind of thinking. I saw him as totally focussed on the problem: “how do living cells make sterols?” He always seemed content to use existing techniques; he did not modify or improve them but he used a wide range of them with virtuosity and he was a slave to none of them. A splendid deviser and planner of experiments, he used the tools that were to hand and his logic in interpreting results could seldom be ques-
tioned. He was the right man— unspecialized, clearsighted, undismayed by strange problems—to tackle a biosynthesis that began with familiar, aniondominated biochemistry and developed, after isopentenyl pyrophosphate, into the quite novel cationcontrolled alkylations and rearrangements leading to squalene and to lanosterol. I had feared when we first met that he might regard Popja´ k and me as interlopers in a field that he had made his own. Instead, he saw that further progress would need innovative chemistry. He welcomed our contribution and we were friends from that time on. As each twist and turn of the biosynthetic pathway was revealed, however clumsy and sometimes inconclusive the detective work may have been, all of us were entranced by the beauty of the unfolding story, beautiful beyond anything that we could (or, at the time, did!) imagine. And as the later investigations proceeded, using methods and facilities immensely more powerful than those that we had commanded, I was sometimes afraid that the subtlety and complexity would collapse into something simpler and less interesting. Nothing of the kind happened: as each remaining gap was filled in, the picture simply became more varied and richer in harmonious detail. As one who had synthesized cholesterol the hard way, I am now looking at a synthesis that I could never have planned, let alone executed. By revealing the beauty of her result, Nature has shown us chemists how much we still have to learn. REFERENCES
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¨ ber Sterine, Gallensa¨ u1. Lettre´ , H., and Inhoffen, H. H. (1936) U ren und verwandte Naturstoffe, Enke, Stuttgart. 2. Fieser, L. F., and Fieser, M. A. (1949) Natural Products Related to Phenanthrene, 3rd ed. Am. Chem. Soc. Monograph Series No. 70. Reinhold, New York. 3. Channon, H. J. (1929) Biological significance of the unsaponifiable matter of oils. I. Experiments with the unsaturated hydrocarbon squalene (spinacene). Biochem. J. 20, 400 – 408. 4. Heilbron, I. M., Kamm, E. D., and Owens, W. M. (1926) The unsaponifiable matter from the oils of elasmobranch fish. Part I. A contribution to the study of the constitution of squalene (spinacene). J. Chem. Soc., 1630 –1644. 5. Robinson, R. (1934) Structure of cholesterol. Chem. Ind., 1062– 1063.
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6. Robinson, R. (1932) Constitution of cholesterol. Nature, 540 – 541. 7. Sonderhoff, R., and Thomas, H. (1937) Die enzymatische Dehydrierung der Trideutero-essigsa¨ ure. Liebigs Ann. Chem. 530, 195–213. 8. Schoenheimer, R. (1942) The Dynamic State of Body Constituents, Harvard Univ. Press, Cambridge, MA. 9. Mauthner, J., and Suida, W. (1986) Beitra¨ ge zur Kenntniss der Cholesterins. III Abhandlung. Monatsh. Chem. 17, 29 – 49. 10. Ottke, R. C., Tatum, E. L., Zabin, I., and Bloch, K. (1951) Isotopic acetate and isovalerate in the synthesis of ergosterol by Neurospora. J. Biol. Chem. 189, 429 – 433. 11. Wu¨ rsch, J., Huang, R. L., and Bloch, K. (1952) The origin of the isoo¨ ctyl side chain of cholesterol. J. Biol. Chem. 195, 439 – 446. 12. Little, H. N., and Bloch, K. (1950) The utilization of acetic acid for the biological synthesis of cholesterol. J. Biol. Chem. 183, 33– 46. 13. Cornforth, J. W., Hunter, G. D., and Popja´ k, G. (1953) Studies of cholesterol biosynthesis. 1. A new chemical degradation of cholesterol. 2. Distribution of acetate carbon in the ring structure. Biochem. J. 54, 590 –597 and 597– 601. 14. Cornforth, J. W., Gore, I. Y., and Popja´ k, G. (1957) Studies of cholesterol biosynthesis. 4. Degradation of rings C and D. Biochem. J. 65, 94 –109. 15. Bonner, J., and Arreguin, B. (1949) Biochemistry of rubber formation in the guayule. I. Rubber formation from seedlings. Arch. Biochem. 21, 109 –124. 16. Arreguin, B., Bonner, J., and Wood, B. J. (1951) The mechanism of rubber formation in the guayule. III. Experiments with isotopic carbon. Arch. Biochem. Biophys. 31, 234 –247. 17. Langdon, R. G., and Bloch, K. The biosynthesis of squalene: The utilization of squalene in the biosynthesis of cholesterol. J. Biol. Chem. 200, 129 –134 and 135–144. 18. Popja´ k, G. (1954) Biosynthesis of squalene and cholesterol in vitro from acetate-1- 14C. Arch. Biochem. Biophys. 48, 102–106. 19. Cornforth, J. W., and Popja´ k, G. (1954) Studies on the biosynthesis of cholesterol. 3. Distribution of 14C in squalene biosynthesized from [Me- 14C]acetate. Biochem. J. 58, 403– 407. ¨ ber 20. Voser, W., Mijovic´ , M. V., Jeger, O., and Ruzicka, L. (1951) U der Vervollsta¨ ndigung der Teilformel des Lanostadienols. Helv. Chim. Acta 34, 1585–1596. 21. Barnes, C. S., Barton, D. H. R., Fawcett, J. S., Knight, S. K., McGhie, J. F., Pradhan, M. K., and Thomas, B. R. (1951) Chem. Ind. (London), 1067–1068. 22. Voser, W., Mijovic´ , M. V., Heusser, H., Jeger, O., and Ruzicka, L. ¨ ber Steroide und Sexualhormone. 186 Mitteilung. U ¨ ber (1952) U die Konstitution des Lanostadienols (Lanosterins) und seine Zugeho¨ rigkeit zu den Steroiden. Helv. Chim. Acta 35, 2414 – 2430. 23. Woodward, R. B., and Bloch, K. (1953) The cyclization of squalene in cholesterol synthesis. J. Am. Chem. Soc. 75, 2023– 2024. 24. Tchen, T. T., and Bloch, K. (1955) In vitro conversion of squalene to lanosterol and cholesterol. J. Am. Chem. Soc. 77, 6085– 6086. 25. Clayton, R. B., and Bloch, K. (1956) Biological synthesis of lanosterol and agnosterol. J. Biol. Chem. 218, 305–318. 26. Stork, G., and Burgstahler, A. W. (1955) The stereochemistry of polyene cyclization. J. Am. Chem. Soc. 77, 5068 –5077. 27. Eschenmoser, A., Ruzicka, L., Jeger, O., and Arigoni, D. (1955) Zur Kenntnis der Triterpene. 190 Mitteilung. Eine stereochemische Interpretation der biogenetische Isoprenregel bei der Triterpenen. Helv. Chim. Acta 38, 1890. 28. Cornforth, J. W., Cornforth, R. H., Pelter, A., Horning, M. G., and Popja´ k, G. (1958) Rearrangement of methyl groups in the
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phat, die biologischen Vorstufen des Squalens. Zur Biosynthese der Terpene III. Angew. Chem. 70, 738 –742. 49. Agranoff, B. W., Eggerer, H., Henning, U., and Lynen, F. (1959) Isopentenol pyrophosphate isomerase. J. Am. Chem. Soc. 81, 1254 –1255. 50. Lynen, F., Agranoff, B. W., Eggerer, H., Henning, U., and Mo¨ slein, E. M. (1959) ␥␥-Dimethyl-allyl-pyrophosphat und Geranyl-pyrophosphat, biologische Vorstufen des Squalens. Zur Biosynthese der Terpene, VI. Angew. Chem. 71, 657– 663.
51. Popja´ k, G. (1959) The biosynthesis of derivatives of allylic alcohols from [2- 14C]mevalonate in liver enzyme preparations and their relation to the synthesis of squalene. Tetrahedron Lett. No. 19, 19 –28. 52. Popja´ k, G., Goodman, De, W. S., Cornforth, J. W., Cornforth, R. H., and Ryhage, R. (1961) Studies on the biosynthesis of cholesterol. XV. Mechanism of squalene biosynthesis from farnesyl pyrophosphate and from mevalonate. J. Biol. Chem. 236, 1934 –1947.
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